Open access Original research 41BB-­based and CD28-­based CD123-­ J Immunother Cancer: first published as 10.1136/jitc-2020-000845 on 10 June 2020. Downloaded from http://jitc.bmj.com/ on February 5, 2021 by guest. Protected by copyright. redirected T-­cells ablate human normal hematopoiesis in vivo Matteo Libero Baroni,1 Diego Sanchez Martinez,1 Francisco Gutierrez Aguera,1 Heleia Roca Ho,1 Maria Castella,1 Samanta Romina Zanetti ‍ ‍ ,1 Talia Velasco Hernandez ‍ ‍ ,1 Rafael Diaz de la Guardia,1 Julio Castaño,1 Eduardo Anguita,2 Susana Vives,3 Josep Nomdedeu,4 Helene Lapillone,5 Anne E Bras,6 Vincent H J van der Velden,6 Jordi Junca,1,3 Pedro Marin,7 Alex Bataller ‍ ‍ ,7 Jordi Esteve,7 Binje Vick,8 Irmela Jeremias,8,9 Angel Lopez,10 Marc Sorigue,1,3 Clara Bueno,1,11 Pablo Menendez1,11,12 To cite: Baroni ML, Sanchez Abstract results from the uncontrolled accumulation Martinez D, Gutierrez Aguera F, Background Acute myeloid leukemia (AML) is a of differentiation-­ defective hematopoietic et al. 41BB-­based and CD28-­ hematopoietic malignancy which is biologically, based CD123-­redirected stem/progenitor cells (HSPCs) or imma- phenotypically and genetically very heterogeneous. ture myeloid cells.1 2 AML is one of the most T-­cells ablate human normal Outcome of patients with AML remains dismal, highlighting common hematopoietic malignances, and hematopoiesis in vivo. Journal the need for improved, less toxic therapies. Chimeric for ImmunoTherapy of Cancer its incidence increases with age.3 4 Intensive 2020;8:e000845. doi:10.1136/ antigen receptor T-­cell (CART) immunotherapies for patients with refractory or relapse (R/R) AML are chemotherapy combos based on nucleoside jitc-2020-000845 challenging because of the absence of a universal pan-­ analogs plus anthracyclines remain the stan- AML target antigen and the shared expression of target dard front-­line treatment of AML,5 followed ►► Additional material is published online only. To view, antigens with normal hematopoietic stem/progenitor cells by allogeneic HSPC transplant (allo-­HSCT), please visit the journal online (HSPCs), which may lead to life-­threating on-­target/off-­ based on patients’ eligibility, to consolidate (http://​dx.​doi.o​ rg/​10.​1136/​jitc-​ tumor cytotoxicity. CD33-­redirected and CD123-­redirected complete remission (CR) and to prevent 2020-0​ 00845). CARTs for AML are in advanced preclinical and clinical relapse.6 However, with the exception of a development, and they exhibit robust antileukemic activity. few molecular subgroups (the ‘so-­ called’ Accepted 07 May 2020 However, preclinical and clinical controversy exists on low-­risk AMLs), relapses are common after whether such CARTs are myeloablative. consolidation therapy and/or allo-­ HSCT. Methods We set out to comparatively characterize in vitro and in vivo the efficacy and safety of 41BB-­based Chemotherapy-­ related toxicity, refracto- and CD28-­based CARCD123. We analyzed 97 diagnostic riness, and failure to eradicate leukemia-­ and relapse AML primary samples to investigate whether initiating cells are the major mechanisms CD123 is a suitable immunotherapeutic target, and underlying AML progression and relapse.7–10 we used several xenograft models and in vitro assays Unfortunately, improved AML treatments to assess the myeloablative potential of our second-­ have only experienced minor developments generation CD123 CARTs. over the last four decades, and current 5-­year Results Here, we show that CD123 represents a bona event-­free survival (EFS) remains ~20% in fide target for AML and show that both 41BB-­based and adults and <70% in children,11 12 highlighting CD28-­based CD123 CARTs are very efficient in eliminating the desperate need for safer and more effi- both AML cell lines and primary cells in vitro and in vivo. However, both 41BB-­based and CD28-­based CD123 cient therapeutics. CARTs ablate normal human hematopoiesis and prevent Immunotherapy has generated unprec- © Author(s) (or their the establishment of de novo hematopoietic reconstitution edented expectations in cancer treatment. employer(s)) 2020. Re-­use by targeting both immature and myeloid HSPCs. In AML, both CD33- and CD123-­ specific permitted under CC BY-­NC. No Conclusions This study calls for caution when clinically antibody-­drug conjugates have been used for commercial re-­use. See rights implementing CD123 CARTs, encouraging its preferential combination therapy with standard chemo- and permissions. Published by BMJ. use as a bridge to allo-­HSCT in patients with R/R AML. therapy with improved EFS,13 14 and Bispe- For numbered affiliations see cific T-­cell Engagers (BiTEs) for CD33 and end of article. Dual-­Affinity Retargeting (DART) antibodies Background for CD123 are being clinically assayed.15 16 Correspondence to Dr Pablo Menendez; Acute myeloid leukemia (AML) is a biolog- Adoptive cellular immunotherapy based on ​pmenendez@​carrerasresearch.​ ically, phenotypically and genetically very the engineering of human chimeric antigen org heterogeneous malignant disease which receptor T-­cells (CARTs) redirected against Baroni ML, et al. J Immunother Cancer 2020;8:e000845. doi:10.1136/jitc-2020-000845 1 Open access cell surface tumor antigens has shown robust clinical with vesicular stomatitis virus-­G (VSV-­G) were generated J Immunother Cancer: first published as 10.1136/jitc-2020-000845 on 10 June 2020. Downloaded from http://jitc.bmj.com/ on February 5, 2021 by guest. Protected by copyright. responses in patients with B-­cell malignances, thanks to using HEK 293T cells with a standard polyethylenimine the high efficacy, specificity and persistence of CARTs.17–19 (PEI) transfection protocol. For each production, plasmid However, the clinical implementation of AML-­ specific transfection was carried out using a 3:1 PEI:DNA ratio and safe CARTs for patients with refractory or relapse using 16 µg transfer vector, 16 µg of pSPAX2, and 8 µg (R/R) AML is still awaiting. Strategies targeting AML VSV-­G per plate, and viral particles were concentrated by using CARTs have proven more challenging than in B-­lin- ultracentrifugation as previously described.32 Peripheral eage malignancies because of two reasons: (1) the lack blood mononuclear cells (PBMCs) were isolated from of a universal pan-­AML target antigen due to the large buffy coats from healthy volunteers by Ficoll-­Hypaque disease heterogeneity, which hampers clinical implemen- gradient centrifugation. Buffy coats were obtained from tation, since a wide range of CARs would be needed to the Barcelona Blood and Tissue Bank (BST) on institu- cover the different leukemic phenotypes; and (2) the tional review board (IRB) approval (HCB/2018/0030). shared expression of immature target antigens between T-­ cells were activated by plate-­ bound OKT3 and anti-­ normal HSPCs and myeloid blasts, which compromises CD28 antibodies (BD Biosciences) for 2 days in the pres- safety due to potential on-­target/off-­tumor cytotoxicity ence of interleukin (IL)-7 and IL-15 (10 ng/mL, Mitenyi against HSPCs, leading to fatal aplasia.20 21 Biotec).33 34 Surface expression of CAR123 was traced by Adoptive immunotherapy for AML is in advanced fluorescence-­activated cell sorting (FACS). CAR detection preclinical and clinical development using CD33-­ was confirmed by GFP expression and by using an Affi- redirected and CD123-­redirected CARs (CD123 CARTs), niPure F(ab')₂ Fragment Goat Anti-­Human IgG (H+L) and they exhibit robust antileukemic activity in vitro (Jackson ImmunoResearch). Activation and subsetting of and in vivo.22–25 However, controversy exists on whether lentivirally transduced T-­cells were confirmed by surface CD123-­directed and CD33-­directed CARTs are myeloab- staining with CD25/CD69 (data no shown) and CD3/ lative. Some groups raised safety concerns leading to CD4/CD8, respectively. the development of complex target antigen knockout in HSPC or T-­cell suicide strategies to circumvent such Immunophenotyping of healthy HSPCs, primary AML samples a toxicity.23 26 In contrast, other groups showed a safety and cell lines profile with limited on-­target/off-­tumor toxicity of such Diagnostic immunophenotyping data for the most CARs.22 27–31 Here, we set out to characterize in vitro and commonly expressed antigens in AML (CD123, CD33, in vivo the efficacy and the safety of 41BB-­costimulated CD13, CD34, CD15, c-­kit, and CD66) were obtained for and CD28-­costimulated CD123CARs, based on a clinically 97 patients diagnosed at local hospitals: Germans Trias relevant single chain variable fragment (scFv) from the i Pujol (Barcelona, Spain), Hospital Clínico (Madrid, CSL362 monoclonal antibody (MoAb). Analysis of a large cohort of diagnostic and relapse AML primary samples Spain), Hôpital Armand Trousseau (Paris, France) and revealed that CD123 is a suitable target for AML, and Santa Creu i San Pau (Barcelona, Spain). CD123 expres- CD123 CARTs were very efficient in vitro and in vivo in sion was also compared in diagnostic-­ relapsed paired eliminating both AML cell lines and primary cells, regard- samples (n=68 patients) and in paired bulk leukemia– less of the costimulation motif. However, clonogenic leukemia stem cells (LSCs) (n=37 patients).35 Cell lines assays and several xenograft models revealed that both were stained with CD123-­ APC, CD33-­ BV-421, CD14-­ 41BB-­costimulated and CD28-­costimulated CD123 CARTs PerCP-­Cy5.5 and CD19-­APC. The expression of CD123 strongly ablate normal human hematopoiesis by targeting and CD33 antigens was prospectively compared in CD34+ both HSCs and myeloid progenitors. This study calls for HSPCs derived from healthy cord blood (CB, n=22), mobi- caution when clinically implementing CD123 CARTs and lized peripheral blood (PB, n=10) and diagnostic primary highlights its preferential use as a bridge to allo-­HSCT in AML samples (n=24). For HSPC subsetting, CD34+ cells patients with R/R AML. were stained with CD34-­PE or CD34-­PE-­Cy7, CD133-­PE, CD19-­FITC, CD90-­APC, CD13-­PE-­Cy7 and CD71-­ APC-­Cy7, which allow for the identification and quan- Materials and methods tification of immature HSCs (CD34++CD133+CD90+), Car design and vectors, lentiviral production and T-cell myeloid progenitors (CD34+CD13++CD71−/low), eryth- transduction roid progenitors (CD34+CD71++CD13 low), and B-­ cell The anti-­CD123 scFV derived from the clinically tested progenitors (CD34+CD19+CD71−CD13−). Isotype-­ CSL362 MoAb was generated and cloned into the pCCL matched, non-­reactive fluorochrome-­conjugated MoAbs lentiviral-­ based second-­ generation CAR backbone were always used as a fluorescence reference. All anti- containing a human CD8 transmembrane domain, a bodies were purchased from Beckton Dickinson. Cells human costimulatory domain (either 41BB or CD28), were incubated with MoAbs (30 min at 4°C in the dark- CD3z endodomain, and a T2A–green fluorescence protein ness), then washed in phosphate-­buffered saline (PBS) (GFP) cassette. The pCCL vector expressing GFP alone and analyzed in a FACSCanto-­II flow cytometer equipped (MOCK vector) was used as a control. Chimeric antigen with FACSDiva software (Becton Dickinson).36–38 Deter- receptor (CAR)-­ expressing viral particles pseudotyped mination of antigen density for CD33 and CD123 was 2 Baroni ML, et al. J Immunother Cancer 2020;8:e000845. doi:10.1136/jitc-2020-000845 Open access performed using BDQuantibrite-­PE (Becton Dickinson) chimerism at sacrifice. Cells were stained with anti-­HLA. J Immunother Cancer: first published as 10.1136/jitc-2020-000845 on 10 June 2020. Downloaded from http://jitc.bmj.com/ on February 5, 2021 by guest. Protected by copyright. according to the manufacturer’s instructions. ABC-­PE and CD45-­BV450. Engrafted mice were assessed for multilineage engraftment using anti-­CD123-­APC for In vitro cytotoxicity assays and cytokine release myeloid cells, anti-­CD19-­BV421 for lymphoid cells, and determination anti-­CD34-­PE.Cy7 for immature cells. Human absolute The cell lines THP-1, MOLM-13 and 697 were purchased engraftment in PB and BM was quantified using BD from DSMZ (Germany) and expanded according to DSMZ Trucount tubes according to manufacturer’s instructions. recommendations. Primary AMLs and healthy CD34+ cells were obtained from the aforementioned hospitals and Statistical analysis the BST, respectively (IRB approval: HCB/2018/0030). For comparison of CD123 expression between paired Target cells were incubated with CAR123 or MOCK ‘diagnostic-­ relapse’ and ‘bulk leukemic cells-­ LSC’, T-­ cells at different effector:target (E:T) ratios for the the Mann-­Whitney U test was used. For differences in indicated time periods. CART-­mediated cytotoxicity was antigen density and engraftment among groups, a one-­ determined by analyzing the residual alive (7-­ AAD-) way analysis of variance test was used. For the remaining target cells at each time point and E:T ratio. For absolute comparisons, Student’s t-­test was used. All p values were cell counting, Trucount absolute count beads (Becton considered statistically significant at <0.05 (*). Dickinson) were used. Furthermore, FACS-­sorted CD3+ mature T-­cells from the bone marrow (BM) of CD123+ patients with AML were activated, transduced with Results CD123 CAR and tested against their autologous-­matched CD123 represents a bona fide immunotarget for AML CD123+ AML blasts. Target cells (1×105) were used for We first analyzed by FACS the expression levels of the all cytotoxicity assays unless stated otherwise. Table 1 most common diagnostic myeloid markers in a cohort shows the clinical–biological features of the CD123+ AML of 97 patients with AML at presentation. We found that samples used for in vitro experiments. The production CD123 was the most common and homogeneously of the proinflammatory cytokines IL-2, tumor necrosis expressed antigen (86.4%±26.8 of AML blasts) followed factor alpha (TNF-α) and interferon gamma (IFN-γ) was by CD33 (77.4%±32.1) (figure 1A). Important, in 82% assessed by ELISA (Human ELISA SET, BD Biosciences) of the patients with AML analyzed, >80% of the blasts using in vitro supernatants harvested at 16 hours post-­T-­ were CD123+, while only 66% of the patients showed cell exposure, and sera were collected from mice 10 days positivity for CD33 in >80% of the blasts (figure 1A). A after CART infusion. target antigen for immunotherapy in AML should ideally be absent in HSPCs. CD123 and CD33 are both partially Colony-forming unit (CFU) assays expressed in healthy CD34+ HSPCs,22 23 so we next quan- CB-­derived CD34+ cells were exposed for 24 hours to tified the density (molecules/cell) of both antigens in either CD123 CARTs or MOCK T-­cells (E:T 1:1) and then fresh primary AML blasts (n=24), healthy CB-­ derived plated (2×103) onto serum-­free methylcellulose H4435 (n=22) and healthy mobilized PB-­derived CD34+ HSPCs (Stem Cell Technologies). CFUs were then counted and (n=10). Of note, 67% (16/24) of patients with AML scored after 12–14 days following standard procedures. displayed levels of CD123 significantly higher than those found in both CB-­derived and PB-­derived CD34+ HSPCs, In vivo xenograft models for AML, HSPCs and CARTs while only 41% (10/24) of patients with AML displayed Non-­obese diabetic-­Cg-­Prkdcscid Il2rgtm1Wjl/SzJ (NSG) levels of CD33 that segregate them from CB-­derived and mice (8–12 weeks old, Jackson Laboratory) were bred and PB-­derived CD34+ HSPCs (figure 1B). This suggests that housed under pathogen-­free conditions in the animal CD123 represents, a priori, a less myeloablative target facility of the Barcelona Biomedical Research Park. In than CD33. Of note, analysis of paired diagnostic (DX)-­ experiments addressing CAR123 efficacy, mice were relapse (RX) AML samples revealed that CD123 expres- intravenously transplanted with 0.25×106 Luc-­mCherry-­ sion is maintained at relapse, and in AML-­LSC (identified expressing patient-­ derived xenograft AML cells (PDX- as CD34+CD38−)35 (figure 1C), reinforcing CD123 as a 579)39 5 days before intravenous infusion of 3×106 of bona fide immunotarget for R/R AML. either 41BB-­CD123 or CD28-­CD123 CARTs derived from healthy PBMCs. Tumor burden was monitored at the 41BB-based and CD28-based CD123 CARTs efficiently indicated time points by bioluminescence (BLI) using eliminate AML primary cells in vitro and in vivo the Xenogen in vivo imaging system (IVIS) 50 Imaging We next designed second-­ generation 41BB-­ based and System (Perkin Elmer).32 In experiments addressing CD28-­ based CD123CARs coupled in-­ frame with GFP the myeloablative effect of CAR123, CD34+ HSPCs through a T2A sequence (figure 1D and online supple- (0.1×106) were intra-­BM transplanted in sublethally irra- mentary figure S1A). The expression of both 41BB-­CD123 diated (2 Gy) NSG mice, followed by intravenous infu- and CD28-­CD123 CAR in T-­cells was confirmed through sion of 3×106 of 41BB-­CD123, CD28-­CD123 CARTs or codetection of scFv and GFP (figure 1E and online supple- MOCK T-­cells either 1 day or 6 weeks after CD34+ trans- mentary figure S1B) and did not affect the CD4:CD8 ratio plantation. BM and PB were FACS-­analyzed for human (figure 1F). Importantly, activated (CD69+CD25+) T-­cells Baroni ML, et al. J Immunother Cancer 2020;8:e000845. doi:10.1136/jitc-2020-000845 3 Open access J Immunother Cancer: first published as 10.1136/jitc-2020-000845 on 10 June 2020. Downloaded from http://jitc.bmj.com/ on February 5, 2021 by guest. Protected by copyright. Table 1 Biological and cytogenetic-­molecular characteristics of blasts from diagnostic patients with AML Age Patient ID Diagnostic Cytogenetics Molecular (years) Gender Blasts (%) Cd123 (%) Use 14 085 AML 46,XY NPM1+, FLT3-­ 42 M 87 80 AG density ITD 14 176 AML-­M1 46,XX Normal 44 F 83 75 AG density ELISA 14 093 AML-­M4 46,XX NPM1MUT, FLT3-­ 52 F 81 86 AG density ITD 14 184 AML-­M1 46,XX, t(8;21) AML1-­ETO 14 F 90 84 AG density 14 268 AML 46,XY, inv(16) NPM1MUT, FLT3-­ 69 M 95 92 AG density ITD Autologous 14 269 AML-­M5 47,XX,+8 Normal 64 F 92 90 AG density ELISA 14 266 AML 46,XX,t(8;21) AML1-­ETO, 48 F 90 92 AG density FLT3-­ITD+ ELISA 14 123 AML 46,XY,t(3;3) Normal 28 M 42 77 AG density 14 185 AML 46,XY,inv(16) Normal 8 M 77 72 AG density 14 143  AML-­M1 46,XY Normal 43 M 90 90 AG density 14 156 AML 46,XY,11q23 MLL-­AF6 1 M 88 100 AG density 14 144 AML 46,XY,del(7)(q22) Normal 61 M 88 98 AG density 14 141 AML-­M5 46,XX, t(8;21) AML1-­ETO 39 F 83 82 AG density 14 091 AML-­M4 47,XX,+8 Normal 61 F 95 80 AG density 14 272 AML 46,XX NPM1MUT, FLT3-­ 44 F 73 88 Autologous ITD 14 274 AML 46,XX, t(8;21) AML1-­ETO 13 F 85 75 Autologous ABT3974 AML 46,XY,+9, inv(16) CEBPA, FLT3-­ 37 M 91 87 AG density (p13;q22), der(17)t(11;17) TKD, WT1 (q13;q25) ABT5270 AML 46,XY NPM1, 55 M 74 77 AG density DNMT3A, IDH1 ABT4435 AML 46,XX IDH2, DNMT3A 70 F 24 96 AG density ABT8326 sAML 44–45X-,Y, der(3),del(7) TET2, CALR 61 M 6 56 AG density (q22),der(8),add(12) (p13),−18,add(21)(q28) ABT7693 AML 46,XY NPM1, IDH1, 45 M 80 99 AG density PTPN11 ABT4470 AML 47,XX NPM1, IDH1, 72 F 71 76 AG density BCOR ABT5718 AML 46,XX CEBPAbi, 52 F 34 65 AG density DNMT3A, TET2 ABT8597 AML NA NPM1, IDH1, 69 F 71 100 AG density NRAS ABT3906 AML NA NPM1, FLT3-­ 40 M 93 77 AG density ITDHIGH, IDH1 ABT4685 tAML 46,XX CEBPAbi, TET2, 67 F 49 93 AG density WT1 AG, antigen; AML, acute myeloid leukemia; F, female; M, male; sAML, secondary acute myeloid leukemia; tAML, therapy-­related acute myeloid leukemia . continuously expanded ~50-­ fold over a 10-­ day period, We then tested the functionality of our 41BB-­CD123 similar to MOCK T-­cells (figure 1G), demonstrating that and CD28-­CD123 CARs in vitro and in vivo (figure 2 redirecting T-­cells against CD123 does not hamper T-­cell and online supplementary figure S1, S2). In vitro, both expansion. 41BB-­CD123 (figure 2A) and CD28-­ CD123 (online 4 Baroni ML, et al. J Immunother Cancer 2020;8:e000845. doi:10.1136/jitc-2020-000845 Open access J Immunother Cancer: first published as 10.1136/jitc-2020-000845 on 10 June 2020. Downloaded from http://jitc.bmj.com/ on February 5, 2021 by guest. Protected by copyright. Baroni M.L et al. Fig.1 A B CD123 CD33 ** % positve blasts at diagnosis 100 * *** ** 42000 45000 80 35000 37500 no AG molecules/cell no AG molecules/cell 28000 30000 21000 22500 60 14000 15000 7000 40 5000 10000 4000 7500 20 3000 5000 2000 1000 2500 0 0 0 CD33 CD15 CD34 CD13 CD123 CD6 6 c-kit AML ___________ CB PB AML ___________ CB PB mean 77.41 22.01 33.18 67.29 86.38 56.10 75.70 (16/24~67%) CD34+ HSPC CD34+ HSPC (10/24~41%) SD 32.08 30.21 29.45 33.85 26.79 43.66 36.28 Pts with > 80% 66% 8,5% 13% 53% 82% 50% 66% positive blasts C E Primary T-cells F CAR123 CAR123(GFP GATED) CAR123 Primary AML samples 59±19% 105 105 105 105 105 CD123-MFI(MEDIAN) 100 CD123-MFI(MEDIAN) 26±16% 100 CAR scFv 100 100 105 100 105 CD4 CD3 100 105 MOCK MOCK(GFP GATED) 103 103 MOCK 105 60±19% 105 105 100 100 32±17% 100 105 100 100 105 -103 GFP -103 100 105 CD8 GFP DX RX BLASTS LSC G 80 D no of T-cells *106 60 41BBz _ _ _ _ _ CD123 scFv 40 EF-1α CD8TM 4-1BB CD3ζ GFP 20 CAR123 MOCK 0 0 2 4 6 8 10 Days post transduction Figure 1 Expression of CD123 in AML and design, detection and expansion CD123 CARTs. (A) Immunophenotyping of the indicated diagnostic myeloid markers in a cohort of 97 patients with AML at presentation. Each dot denotes an individual patient. Red circles identify patients with >80% of blasts positive for the indicated marker. (B) Comparative antigen density (measured as antigen molecules/cell) for CD123 and CD33 in primary AML samples (n=24), CB-­derived (n=22) and PB-­derived (n=10) CD34+ cells from healthy donors. AML blasts were identified as 7AAD−CD3−CD45+/lowCD123+CD33+. One-­way analysis of variance; *p<0.05, **p<0.01, ***p<0.001. (C) Comparison of CD123 expression in 68 paired diagnostic-­relapse AML samples (left panel) and in bulk tumor versus AML-­LSC (n=37, right panel).33 (D) Scheme of the CD123 CAR structure. (E) CAR detection in primary T-­cells using an antihuman IgG F(ab')2 antibody and GFP. (F) Successful CAR123 transduction and detection in CD4+ and CD8+ T-­cells (n=3). (G) Robust expansion of activated T-­cells transduced with either MOCK (black line) or CAR123 (red line) (n=3). AML, acute myeloid leukemia; CAR, chimeric antigen receptor; CART, chimeric antigen receptor T-­cell; CB, cord blood; DX, diagnostic; GFP, green fluorescence protein; LSC, leukemia stem cell; PB, peripheral blood; RX,relapse. supplementary figure S1C) CARTs, but not MOCK (figure 2C). Patient-­derived CD123 CARTs were success- T-­cells, specifically eliminated the CD123+ AML cell lines fully generated from magnetic-­ activated cell sorting THP1 and MOLM13 in an E:T ratio-­dependent manner (MACS)-­sorted CD3+ T-­cells (>95% purity) and specifi- (online supplementary figure S2) while sparing the cally eliminated autologous patient-­ matched CD123+ CD123− B-­ALL cell line 697. In fact, CD123+ AML cells AML blasts (figure 2D). Important, both CD123 CARTs barely survived exposure to CD123 CARTs in a 48-­hour produced high levels of the proinflammatory cytokines absolute number assay at a 1:1 E:T ratio (figure 2B and online supplementary figure S1C). We then examined in IL-2, TNF-α, and IFN-γ on coculture with both AML cell an autologous setting whether CD3+ T-­cells deriving from lines (figure 2E and online supplementary figure S1D) patients with AML can be isolated, modified to express and primary blasts (figure 2F), confirming their robust CD123 CAR, expanded and used as cytotoxic effector cells cytotoxicity. Baroni ML, et al. J Immunother Cancer 2020;8:e000845. doi:10.1136/jitc-2020-000845 5 Open access J Immunother Cancer: first published as 10.1136/jitc-2020-000845 on 10 June 2020. Downloaded from http://jitc.bmj.com/ on February 5, 2021 by guest. Protected by copyright. Figure 2 41BB-­CD123 CARTs specifically target and eliminate CD123+ AML cells in vitro and in vivo. (A) Surface expression of CD123 (red) in THP-1, MOLM-13 and 697 cell lines. (B) Absolute counts of alive residual target cells measured by FACS in 48-­hour cytotoxicity assays at 1:1 E:T ratio (n=3). Data are presented as mean±SEM; *p<0.05, **p<0.01, ***p<0.001. (C) Graphical cartoon of the experimental design for autologous cytotoxic assays. Normal CD3+ T-­cells were FACS-­purified from the BM of patients with AML (n=3), infected with CD123 CAR, expanded, and exposed to autologous total PBMCs (1:1 E:T). Residual CD123+ blasts were quantified 48 hours post-­41BB-­CD123 CART exposure. (D) Left: representative FACS analysis of the cytotoxicity assay. T-­cells are shown in black and CD123+ blasts in blue. Right: absolute counts of alive AML blasts in 48-­hour cytotoxicity assays at 1:1 E:T ratio (n=3). (E,F) ELISA showing robust secretion of proinflammatory cytokines by 41BB-­CD123 CARTs after exposure to CD123+ cell lines (E) and AML primary blasts (F) for 16 hours at 1:2 E:T ratio (n=3). (G) Experimental design to assess in vivo the efficacy of both 41BB-­based and CD28-­based CD123 CAR. NSG mice were intravenously injected with 2.5×105 Luc-­expressing xenograft AML cells (PDX-579) followed 5 days after by a single intravenous injection of 3×106 CD123 CARTs (either 41BB or CD28) generated from healthy PBMCs. Tumor burden was monitored every 7–10 days by BLI using IVIS imaging. (H) IVIS imaging of tumor burden monitored by BLI at the indicated time points. (I) Left: total radiance quantification (p/s/cm2/sr) at the indicated time points for 41BB-­CD123 CARTs, CD28-­CD123 CARTs and untreated mice. *P<0.05. Right: absolute counts of residual AML cells in PB and BM at endpoint. (J) T-­cell persistence in PB and BM at endpoint. (K) In vivo quantification by ELISA of IFN-γ in PB sera collected in the acute phase (10 days post-­CART infusion). *p<0.05. AML, acute myeloid leukemia; BLI, bioluminescence; BM,bone marrow; CART, chimeric antigen receptor Tcell; E:T, effector:target; FACS, fluorescence-­activated cell sorting; IFN-γ, interferon gamma; IVIS, in vivo imaging system; ns, non-­significant; NSG, non-­obese diabetic-­Cg-­Prkdcscid Il2rgtm1Wjl/SzJ; PB, peripheral blood; PBMC, peripheral blood mononuclear cell. 6 Baroni ML, et al. J Immunother Cancer 2020;8:e000845. doi:10.1136/jitc-2020-000845 Open access We next compared the cytotoxic activity of 41BB-­CD123 hematopoiesis by transplanting sublethally irradiated J Immunother Cancer: first published as 10.1136/jitc-2020-000845 on 10 June 2020. Downloaded from http://jitc.bmj.com/ on February 5, 2021 by guest. Protected by copyright. and CD28-­ CD123 CARTs in vivo using Luc-­ expressing NSG mice with CD34+ HSPC and either 41BB-­ CD123, CD123+ AML xenograft (figure 2G). NSG mice were CD28-­ CD123 CARTs or MOCK T-­ cells 1 day after transplanted with 0.25×106 Luc-­expressing AML PDX cells (figure 3H). Long-­ term multilineage human engraft- 5 days prior to intravenous infusion of 3×106 41BB-­CD123 ment was found in both PB and BM in MOCK T-­cell-­ or CD28-­CD123 CARTs, and leukemia establishment was treated mice; however, human hematopoiesis was barely followed up weekly by BLI until disease signs were evident reconstituted in both 41BB-­ CD123 and CD28-­ CD123 (figure 2H). While control mice increasingly showed CART-­treated mice (figure 3I), indicating that both 41BB-­ aggressive disease and disseminated leukemia, CD123 CD123 and CD28-­CD123 CARTs prevent healthy hemato- CART-­ treated mice showed extensive disease control poietic reconstitution. across the experiment, regardless of the costimulation Finally, to further characterize the myeloablative effects domain used (figure 2H1). Of note, T-­cells persisted in of CD123 CARTs, we exposed total CD34+ HSPCs to either PB and BM at sacrifice, although at higher levels in 41BB-­ CD123 CARTs or MOCK T-­cells for 48 hours at 1:1 E:T CD123 CART-­ treated mice, in line with the reported ratio, and quantified afterwards whether the myeloab- longer persistence/effector function of 41BB-­ costimu- lative effects were CD34+ subset-­ specific (figures 3A, lated CARTs (figure 2J).40 Similarly, both CD123 CARTs, 4A). We found a significant loss of both immature/early especially 41BB-­ costimulated CD123 CARTs produced HSPCs (CD34 ++CD133+CD90+) and myeloid progen- high levels of the IFN-γ in vivo (figure 2K). Collectively, itors (CD34 +CD13+CD71 low), while B-­cell progenitors both 41BB-­CD123 and CD28-­CD123 CARTs have similarly (CD34 +CD19+CD13-­CD71−) and erythroid progenitors potent and specific antileukemic activity against AML (CD34+CD71+CD13 low) were unaffected by CD123 cells in vitro and in vivo. CARTs exposure (figure 4A). Of note, CD123 CART-­ mediated cytotoxicity correlated well with the expression On-target/off-tumor targeting of immature HSPCs and myeloid levels of CD123 in the different CD34+ subsets (figure 4C). progenitors render both 41BB-CD123 and CD28-CD123 CARTS Collectively, our results suggest that CD123 CARTs ablate severely myeloablative in vitro and in vivo human hematopoiesis by targeting both early/immature There is controversy on whether CD123-­redirected T-­cells HSPCs and myeloid progenitors. are myeloablative. To prospectively assess the potential myelotoxicity of CD123 CARTs, we first addressed in vitro whether exposure to CD123 CARTs hampers the viability Discussion and clonogenic capacity of CD34+ HSPCs (figure 3A). AML is a very heterogeneous stem cell malignant disease As compared with MOCK T-­ cells, both CD123 CARTs characterized by the progressive acquisition of (epi) induced a massive reduction in CD34+ cell counts in genetic alterations resulting in a clonal rapid expan- a 72 hours (E:T ratio 2:1) assay (figure 3B). Similarly, sion of differentiation-­defective HSPC in BM and PB.1 CD34+ HSPCs pre-­exposed to either CD123 CARTs (E:T Unfortunately, the prognosis of AML remains unfavor- ratio 1:1) for only 24 hours showed 50%–80% reduction able, especially in patients >60 years old, due to common in their clonogenic capacity (figure 3D). relapses, disease refractoriness and treatment-­ related We next assessed the myeloablative potential of both toxicities.41 Unfortunately, improved AML treatments 41BB-­CD123 and CD28-­ CD123 CARTs in vivo using have only experienced minor developments over the last xenograft models of human hematopoietic reconstitu- four decades, reinforcing the high-­demand for new ther- tion. In an initial set of experiments, sublethally irradi- apeutics with improved efficacy and reduced toxicity.20 23 ated NSG mice were reconstituted with 0.1×106 CD34+ In this context, the undisputable clinical improvements HSPCs, and 6 weeks later, when human multilineage of cancer immunotherapy have not gone unnoticed in engraftment was established, mice received 3×106 AML and undoubtedly represent the great hope of the 41BB-­CD123, CD28-­ CD123 CARTs or MOCK T-­ cells next decade in the treatment of AML. In fact, immuno- (figure 3E). Human engraftment was biweekly analyzed therapeutic targeting in AML is already well advanced in PB (figure 3F) and BM (figure 3G) over 6 weeks. in clinical trials using MoAb, antibody–drug conjugates, MOCK T-­cell-­treated mice consistently showed increased BiTEs, DARTs and CAR T-­ cell therapies against CD33 myeloid (HLA-­ABC +CD45+CD123+CD33+), B-­lymphoid and CD123.13–16 20 22 29 However, clinical progress and (HLA-­ABC+CD45+CD123 CD19+) and immature (HLA-­ regulatory approval of such immunotherapies have been ABC+CD45+CD34+) hematopoietic engraftment than hampered by the challenge to find a specific and safe that observed on the day of CART infusion. In contrast, targetable surface antigen.41 42 both 41BB-­CD123 and CD28-­CD123 CART-­treated mice CD33 and CD123 are the most extensively explored showed an impaired multilineage engraftment in both antigens for AML and blastic plasmocytoid dendritic PB and BM (figure 3F). However, in this xenograft model cell neoplasm immunotherapy.42 43 In this study, we of existing hematopoiesis, CD28-­ based CD123 CARTs aimed to better characterize the suitability of CD33 and proved less myeloablative than 41BB-­CD123 CARTs. CD123 in a large cohort of diagnostic and relapse AML Next, we assessed the capacity of both CD123 CARTs primary samples. We show that CD123 represents a bona in preventing de novo establishment of normal fide target for AML with a potentially safer profile than Baroni ML, et al. J Immunother Cancer 2020;8:e000845. doi:10.1136/jitc-2020-000845 7 Open access J Immunother Cancer: first published as 10.1136/jitc-2020-000845 on 10 June 2020. Downloaded from http://jitc.bmj.com/ on February 5, 2021 by guest. Protected by copyright. Baroni M.L et al. Fig.3 A B C D 3.0× 10 4 ** MOCK CAR123 140 *** absolute no of alive CD34+ n° of CFU/ 2*103 CD34+ Cytotoxicity 120 CD3 HSPCs 24 or 72 h exposure 100 + CFU scoring 2.0× 10 4 CARTs 80 GEMM Subsetting GFP GM 60 1.0× 10 4 G 40 M E 20 CD3 0 0 MOCK 41BBz CD28z MOCK 41BBz CD28z CD34 E F PB Myeloid graft B-Lymphoid graft Immature graft 40 41BBz 41BBz 41BBz № CD45+/CD123+ cells/ul 1500 CD28z CD28z 90 CD28z CD123 № CD45+/CD19+ cells/ul MOCK CD123 № CD45+/CD34+ cells/ul MOCK MOCK CD45 IRRADIATION 2Gy 30 + 41BBz/CD28z/MOCK SACRIFICE CB-CD34+ 1000 CD19 60 CD19 20 CD34 WEEKS 500 30 -6 0 6 10 6 WEEKS Bi-weekly bleeding 0 * 0 * * 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Weeks post CAR infusion Weeks post CAR infusion Weeks post CAR infusion G BM H Myeloid graft B-Lymphoid graft Immature graft № CD45+/CD123+ cells/5000 beads № CD45+/CD34+ cells/5000 beads № CD45+/CD19+ cells/5000 beads ∆ =+6,2 ∆ =-0,2 ∆ =+2,3 ∆ =+3,6 ∆ =-7,9 ∆ =+1,7 3.0× 10 4 7.5× 10 5 1.5× 10 5 ∆ =+3,1 ∆ =-12,4 ∆ =-2,0 IRRADIATION 2Gy + CB-CD34+ SACRIFICE 41BBz/CD28z/MOCK 2.0× 10 4 5.0× 10 5 1.0× 10 5 WEEKS 1.0× 10 4 2.5× 10 5 5.0× 10 4 0 DAY+1 6 6 WEEKS 0 0 _______ PRE _______ POST PRE POST _______ PRE POST 0 _______ PRE _______ POST PRE _______ POST PRE POST _______ PRE _______ POST PRE _______ POST PRE POST MOCK 41BBz CD28z MOCK 41BBz CD28z MOCK 41BBz CD28z I J BM PB Myeloid graft B-Lymphoid graft Immature graft Myeloid graft B-Lymphoid graft Immature graft № CD45+/CD123+ cells/5000 beads № CD45+/CD19+ cells/5000 beads * * *** № CD45+/CD34+ cells/5000 beads 2.0 * 40 * 12 p=0,12 1.0× 10 3 4.4× 10 3 1.8× 10 3 № CD45+/CD123+ cells/ul № CD45+/CD19+ cells/ul № CD45+/CD34+ cells/ul 1.5 30 7.5× 10 2 3.3× 10 3 8 1.2× 10 3 1.0 20 5.0× 10 2 2.2× 10 3 4 6.0× 10 2 0.5 10 2.5× 10 2 1.1× 10 3 0.0 0 0 0 0 0 MOCK 41BBz CD28z MOCK 41BBz CD28z MOCK 41BBz CD28z MOCK 41BBz CD28z MOCK 41BBz CD28z MOCK 41BBz CD28z Figure 3 Both 41BB-­CD123 and CD28-­CD123 CARTs eliminate healthy CD34+ HSPCs in vitro and in vivo. (A) Experimental scheme for in vitro assessment of CAR123 cytotoxicity on healthy CD34+ cells. (B) Representative FACS showing the residual CD34+ HSPCs (red) after exposure to CAR123 CARTs or MOCK T- cells for 72 hours at an E:T ratio of 2:1. (C) Absolute quantification of remaining alive CD34+ cells after exposure to either 41BB-­CD123 or CD28-­CD123 CARTS (72 hours, E:T 2:1). (D) Clonogenic assays performed with residual alive CD34+ HSPCs after 24 hours of coincubation with either 41BB-­CD123 CARTs, CD28-­CD123 CARTs or MOCK T-­cells (E:T 1:1) (n=3 donors). (E) Schematic representation of the in vivo experimental plan. CD34+ cells were intra-­BM transplanted into NSG mice, and 6 weeks later, the level of human engraftment was assessed by FACS analysis in PB and BM. Mice then received 3×106 of either CD123 CARTs (41BB or CD28) or MOCK T-­cells. PB bleedings were performed biweekly and PB/BM were analyzed at sacrifice (6 weeks after CART infusion). (F,G) Analysis of murine PB (F) and BM (G) multilineage reconstitution (CD19+ B lymphoid, CD123+ myeloid and CD34+ immature) at the indicated weeks post-­CART infusion. Final engraftment (POST) of myeloid, B lymphoid and immature HSPCs is presented as fold change in comparison to pre-­CART/MOCK infusion (PRE). (H) Schematic representation of the in vivo experimental plan. CD34+ cells were intra-­BM transplanted into NSG mice, followed the day after by infusion of either 3×106 CD123 CARTs (41BB or CD28) or MOCK T-­cells. Mice were sacrificed 6 weeks after and PB and BM were analyzed. (I,J) Analysis of murine PB (I) and BM (J) multilineage reconstitution (CD19+ B lymphoid, CD123+ myeloid and CD34+ immature) 6 weeks after CART infusion. *P<0.05, **P<0.01, ***P<0.001. BM, bone marrow; CART, chimeric antigen receptor T-­cell; E, erythroid colony-­forming unit; E:T, effector:target; FACS, fluorescence-­activated cell sorting; G, granulocytic colony-­forming unit; GEMM, granulocytic, erythroid, myelomonocytic colony-­forming unit; GM, granulomonocytic colony-­forming unit; HSPC, hematopoietic stem/progenitor cell; M, monocytic colony-­forming unit; NSG, non-­obese diabetic-­Cg-­Prkdcscid Il2rgtm1Wjl/SzJ; PB, peripheral blood. 8 Baroni ML, et al. J Immunother Cancer 2020;8:e000845. doi:10.1136/jitc-2020-000845 Open access for AML. More importantly, a target antigen for immu- J Immunother Cancer: first published as 10.1136/jitc-2020-000845 on 10 June 2020. Downloaded from http://jitc.bmj.com/ on February 5, 2021 by guest. Protected by copyright. notherapy in AML should ideally spare HSPCs. Previous studies about the expression of CD123 in CD34+ HSPCs have provided conflicting results based on the source of CD34+ cells and the MoAb used.20 22 29 Here, we demon- strate that CD123 is expressed in both CB-­derived and PB-­derived CD34+ HSPCs; however, in contrast to CD33, which discriminates more poorly AML from either CB-­de- rived or PB-­derived CD34+ HSPCs, two-­thirds of patients with AML express CD123 at levels significantly higher than those in healthy CD34+ cells, suggesting CD123 as a safer target than CD33 for AML. Extensive evidence supports CD123 targeting as a ther- apeutic approach for AML. First, major phenotypical (immature, granulocytic and monocytic) and cytogenetic (FLT3- and NPM1-­ mutated) AML subgroups express CD123.35 45 46 Second, CD123+ AML cells are capable of initiating leukemogenesis when transplanted in immuno- deficient mice, thus marking AML-­LSC.47–49 Third, the presence of CD34+ CD38 CD123+cells in AML at presen- tation is associated with lower disease-­free and overall survival and failure to achieve complete remission.50 51 Fourth, CD123 expression enhance AML cell prolifera- tion and induces downregulation of CXCR4, favoring the egress of BM AML-­LSCs into the circulation.52 Based on this background, we prompted to characterize and compare in vitro and in vivo the efficacy and safety profile of the second-­ generation 41BB-­ based and CD28-­ based CD123CARs derived from the clinically tested CSL362 humanized MoAb.14 53 Regardless of the costimulation motif, CD123 CARTs were very efficient in vitro and in vivo in eliminating both AML cell lines and primary cells, even at relatively low E:T ratios. Importantly, however, CD123 CARTs ablated existing normal human hema- topoiesis and prevented the establishment of de novo hematopoietic reconstitution by directly targeting both myeloid progenitors and early/immature HSPCs, with Figure 4 CAR123-­mediated cytotoxicity is CD34 subset specific. (A) Flow cytometry characterization of different subsequent functional consequences in all downstream subsets of CD34+ HSPCs post CD123 CART exposure. normal hematopoietic progenitors, rendering severe Left: identification of CD34+ HSPCs and CD123 CARTS. impairment of multilineage hematopoiesis in BM and Middle right: identification of CD90+ CD133+ early-­immature PB. This study adds information to the existing contro- CD34+ HSPCs (turquoise dots), CD13−CD71−CD19+ B-­ versy about the myeloablative potential of CD123 CARTs. lymphoid CD34+ HSPCs (green dots), CD13++CD71 dim Despite several reports showing a limited cytotoxic effect myeloid CD34+ HSPCs (gray dots) and CD13lowCD71++ on CD34+ HSPCs,22 25 29 our data support the work by erythroid CD34+ HSPCs (black dots). (B) Absolute Gill and coworkers who reported a myeloablative in vivo quantification by FACS of the different CD34+ subsets (as potential of 41BB-­based CD123 CARTs on CD34+ HSPCs. identified in A) on exposure to CD123 CARTS or MOCK T-­ cells (48 hours, E:T 1:1) (n=3). (C) MFI levels of CD123 in the The myeloablative effects observed here were not different CD34+ cell subsets (n=3). *P<0.05. CART, chimeric limited to the 41BB-­based CD123 CARTs but were simi- antigen receptor T-­cell; FACS, fluorescence-­activated cell larly observed with the CD28-­costimulated CD123CARTs. sorting; MFI, mean fluorescence intensity. Previous studies used different sources of CD34+ cells, different vector designs, and distinct CD123 scFvs. There- fore, current conflicting data may be attributed to distinct CD33. Not only is the most common and homogeneously vector architectures, CAR-­binding affinity, target density, expressed antigen in AML, but also its expression is also source of healthy CD34+ cells, or even experimental fully retained at relapse and in AML-­LSC. This lack of designs.31 The robust myeloablative effects reported in antigen plasticity, a phenomenon widely observed during this study calls for caution when clinically implementing the progression and relapse of acute leukemias,44 further CD123 CARTs. Unfortunately, however, immunother- strengthens the potential of CD123 as immunotarget apies for AML different from CARTs, such as DARTs or Baroni ML, et al. J Immunother Cancer 2020;8:e000845. doi:10.1136/jitc-2020-000845 9 Open access CD123-­directed MoAbs, resulted in limited clinical effi- were approved by the Animal Care Committee of The Barcelona Biomedical J Immunother Cancer: first published as 10.1136/jitc-2020-000845 on 10 June 2020. Downloaded from http://jitc.bmj.com/ on February 5, 2021 by guest. Protected by copyright. cacy unable to control the disease in the medium–long Research Park (HRH-17-0029-­P1). term.14 54 55 A potentially safer clinical approach to circum- Provenance and peer review Not commissioned; externally peer reviewed. vent myeloablation would be the use of potent CD123 Data availability statement All data relevant to the study are included in the CARTs to achieve CR followed by allo-­HSCT as a rescue article and uploaded as supplementary information. The datasets and materials generated in this study are available from the corresponding author on reasonable therapy. Finally, alternative sources of effector cells, such request. as natural killer (NK) cells, cytokine-­induced killer (CIK) Open access This is an open access article distributed in accordance with the cells or Vδ1 γδ T-­cells (DOTs) are being explored in order Creative Commons Attribution Non Commercial (CC BY-­NC 4.0) license, which to better control the in vivo persistence of CD123 CAR permits others to distribute, remix, adapt, build upon this work non-­commercially, bearing cells.56 57 and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-­commercial. See http://​creativecommons.​org/​licenses/​by-​nc/​4.​0/. Author affiliations 1 Biomedicine, Research Institute Against Leukemia Josep Carreras, Barcelona, ORCID iDs Catalunya, Spain Samanta Romina Zanetti http://​orcid.​org/​0000-​0002-​9052-​8751 2 Hematology and Hemotherapy Department, Hospital Clinico Universitario San Talia Velasco Hernandez http://​orcid.​org/​0000-​0003-​2183-​7443 Carlos Instituto Cardiovascular, Madrid, Comunidad de Madrid, Spain Alex Bataller http://​orcid.​org/​0000-​0002-​6085-​2745 3 Hematology Department, Hospital Universitari Germans Trias i Pujol, Badalona, Catalunya, Spain 4 Hematology Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Catalunya, Spain References 5 Centre de Recherce Saint-­Antoine, Armand-­Trousseau Childrens Hospital, Paris, 1 Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a Île-­de-­France, France hierarchy that originates from a primitive hematopoietic cell. Nat Med 6 Immunology Department, Erasmus Medical Center, Rotterdam, Zuid-­Holland, 1997;3:730–7. 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Leuk Lymphoma 2003;44:391–409. 11 Centro de investigación en Red-­Oncología, CIBERONC, Comunidad de Madrid, 6 Cornelissen JJ, Blaise D. Hematopoietic stem cell transplantation Madrid, Spain for patients with AML in first complete remission. Blood 12 Instituciò Catalana de Recerca i Estudis Avançats, ICREA, Barcelona, Catalunya, 2016;127:62–70. 7 Thol F, Schlenk RF, Heuser M, et al. How I treat refractory and early Spain relapsed acute myeloid leukemia. Blood 2015;126:319–27. 8 Bose P, Vachhani P, Cortes JE. Treatment of relapsed/refractory Correction notice Since the online publication of this article, it was noticed that acute myeloid leukemia. Curr Treat Options Oncol 2017;18:17. 'Samanta Romina Zanetti' was incorrectly spelt as 'Samanta Zanetti'. This error has 9 De Grandis M, Mancini SJ, Aurrand-­Lions M. In quest for leukemia been corrected. initiating cells in AML. Oncoscience 2018;5:9–10. 10 Guardia RDdela, González-­Silva L, López-­Millán B, et al. Bone AcknowledgmentsWe deeply thank Marixtell Vinyoles and Paolo Petazzi for their marrow clonogenic myeloid progenitors from NPM1-­Mutated AML technical support and Juan Jose Rodríguez-­Sevilla for critically reviewing the patients do not harbor the NPM1 mutation: implication for the Cell-­ manuscript. Of-­Origin of NPM1+ AML. Genes 2020;11:73. 11 Creutzig U, van den Heuvel-­Eibrink MM, Gibson B, et al. Diagnosis Authors’ contribution: MLB designed the experiments, analyzed/interpreted and management of acute myeloid leukemia in children and the data, and wrote the manuscript. DSM, FGA, HRH, MC, SZ, TVH, JC, and RDdlG adolescents: recommendations from an international expert panel. performed experiments. EA, SV, JN, HL, AEB, VHJvdV, JJ, PMa, AB, JE, AL, BV, IJ and Blood 2012;120:3187–205. MS provided clinical samples and biological data. CB designed the experiments, 12 Thein MS, Ershler WB, Jemal A, et al. Outcome of older patients with acute myeloid leukemia: an analysis of SEER data over 3 decades. interpreted the data, and financially supported the work. PMe conceived the study, Cancer 2013;119:2720–7. designed the experiments, wrote the manuscript, and financially supported the 13 Hills RK, Castaigne S, Appelbaum FR, et al. Addition of gemtuzumab work. ozogamicin to induction chemotherapy in adult patients with acute Funding We thank CERCA/Generalitat de Catalunya and Fundació Josep myeloid leukaemia: a meta-­analysis of individual patient data from randomised controlled trials. Lancet Oncol 2014;15:986–96. Carreras‐Obra Social la Caixa for their institutional support. PM acknowledges 14 Lee EM, Yee D, Busfield SJ, et al. Efficacy of an Fc-­modified anti-­ financial support from the European Research Council (CoG-2014–6 46 903, CD123 antibody (CSL362) combined with chemotherapy in xenograft PoC-2018–8 11 220), the Spanish Ministry of Economy and Competitiveness models of acute myelogenous leukemia in immunodeficient mice. (MINECO, SAF2016-80481-­R), the Spanish Cancer Research Association (AECC-­ Haematologica 2015;100:914–26. Semilla19), the Fundación Uno entre Cienmil, the Obra Social La Caixa (LCF/PR/ 15 Jitschin R, Saul D, Braun M, et al. CD33/CD3-­bispecific T-­cell HR19/52160011), the Leo Messi Foundation, the Banco Santander Foundation engaging (BiTE®) antibody construct targets monocytic AML and the “Heroes hasta la médula” initiative. CB was supported by the AECC and myeloid-­derived suppressor cells. J Immunother Cancer 2018;6:116. 16 Al-­Hussaini M, Rettig MP, Ritchey JK, et al. Targeting CD123 in acute the Health Institute Carlos III/FEDER (PI17/01028). MLB is supported by an FPI myeloid leukemia using a T-­cell-­directed dual-­affinity retargeting scholarship from MINECO. DSM is supported by a Sara Borrell fellowship from platform. Blood 2016;127:122–31. ISCIII. SZ and TVH are supported by Marie Sklodowska Curie Fellowships. PMa is an 17 Brentjens RJ, Rivière I, Park JH, et al. Safety and persistence of investigator of the Spanish Cell Therapy Network or TERCEL. adoptively transferred autologous CD19-­targeted T cells in patients with relapsed or chemotherapy refractory B-­cell leukemias. Blood Competing interests None declared. 2011;118:4817–28. Patient consent for publication Not required. 18 Lee DW, Kochenderfer JN, Stetler-­Stevenson M, et al. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic Ethics approval This study was approved by the institutional review board, leukaemia in children and young adults: a phase 1 dose-­escalation Barcelona Clinic Hospital Ethics Committee (HCB/2017/1056). 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J Immunother Cancer 2020;8:e000845. doi: 10.1136/jitc-2020-000845 Since the online publication of this article, it was noticed that 'Samanta Romina Zanetti' was incorrectly spelt as 'Samanta Zanetti'. This error has been corrected. Open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-­NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-­commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-­commercial. See: http://​creativecommons.​org/​licenses/​by-​nc/​4.​0/. © Author(s) (or their employer(s)) 2020. Re-­use permitted under CC BY-­NC. No commercial re-­use. See rights and permissions. Published by BMJ. J Immunother Cancer 2020;8:e000845corr1. doi:10.1136/jitc-2019-000845corr1 J Immunother Cancer 2020;8:e000845corr1. doi:10.1136/jitc-2019-000845corr1 1