Open access Letter J Immunother Cancer: first published as 10.1136/jitc-2021-003644 on 14 December 2021. Downloaded from http://jitc.bmj.com/ on January 26, 2023 by guest. Protected by copyright. Factors associated with the clinical outcome of patients with relapsed/ refractory CD19+ acute lymphoblastic leukemia treated with ARI-­0001 CART19-­cell therapy Valentín Ortiz-­Maldonado ‍ ‍ ,1,2 Susana Rives,3,4 Marta Español-­Rego,5 Anna Alonso-­Saladrigues,3 Mercedes Montoro,1 Laura Magnano,1,2 Eva Giné,1,2,6 Mariona Pascal,2,5 Marina Díaz-­Beyá,1,2 Maria Castella,7 Albert Català,3,4,8 Anna Faura,3 Luis Gerardo Rodríguez-­Lobato,1,2 Aina Oliver-­Caldes ‍ ‍ ,1,2 Alexandra Martínez-­Roca,1 Montserrat Rovira,1,2 E Azucena González-­Navarro,5 Juan Ramón Ortega,5 Joan Cid,2,9 Miquel Lozano,2,9,10 Enric Garcia-­Rey,7 Sara Fernández,11 Pedro Castro,2,10,11 Iolanda Jordan,4,10,12 Neus Villamor,2,6,13 Marta Aymerich,2,13 Montserrat Torrebadell,4,8,14 Àngela Deyà,15 Carlos Fernández de Larrea,1,2,10 Daniel Benitez-­Ribas,2,5 Esteve Trias,16 Sara Varea,17 Gonzalo Calvo,16,17 Jordi Esteve,1,2,10,18 Alvaro Urbano-­Ispizua,2,10,18 Manel Juan ‍ ‍ ,2,5,10,19 Julio Delgado ‍ ‍ 1,2,6,10 To cite: Ortiz-­Maldonado V, The prognosis of patients with relapsed/ CUP. Detailed information on ARI-­0001 cell Rives S, Español-­Rego M, et al. refractory (R/R) acute lymphoblastic manufacturing, inclusion/exclusion criteria, Factors associated with the clinical outcome of patients leukemia (ALL) remains poor, particularly primary and secondary endpoints and assess- with relapsed/refractory for those relapsing after allogeneic hema- ment criteria can be found elsewhere.5 CD19+ acute lymphoblastic topoietic cell transplantation (alloHCT).1 Before ARI-­ 0001 cell infusion, patients leukemia treated with ARI-­0001 Novel agents such as inotuzumab ozogamicin received fludarabine at 30 mg/m2/day plus CART19-­cell therapy. Journal or blinatumomab achieve increased response cyclophosphamide at 300 mg/m2/day on for ImmunoTherapy of Cancer 2021;9:e003644. doi:10.1136/ rates, but these are generally transient unless days −6, –5, and −4 followed by ARI-­0001 cells. jitc-2021-003644 followed by alloHCT. Chimeric antigen The first 15 patients received a single intra- receptors (CAR) targeting CD19 have shown venous infusion of 0.5–1×106 ARI-­0001 cells/ ►► Additional supplemental promising results in R/R ALL, and one of kg (adults) or 5×106 ARI-­0001 cells/kg (chil- material is published online only. these products (tisagenlecleucel) has been dren) on day 0. The following 38 patients (23 To view, please visit the journal approved for the treatment of patients with from the CART19-­ BE-­ 01 trial and 15 from online (http://​dx.d​ oi.​org/​10.​ R/R ALL up to 25 years of age.2 the CUP) received 1×106 ARI-­0001 cells/kg 1136/​jitc-​2021-0​ 03644). In 2013, we designed our own CAR19 regardless of age: the first fraction (10%) Accepted 04 November 2021 construct, comprising a single chain variable on day 0, followed by the second (30%) and fraction sequence from the A3B1 hybridoma third (60%) fraction. The second fraction was and the 4-­ 1BB/CD3z signaling domains administered 24–48 hours after the first, and (online supplemental figure 1). After scaling the third 24–48 hours after the second, only if © Author(s) (or their employer(s)) 2021. Re-­use up both lentiviral and cell production,3 4 the the patient had no signs or symptoms of CRS permitted under CC BY-­NC. No Spanish Medicines Agency (AEMPS) approved (online supplemental figure 2). The reason commercial re-­use. See rights our product (ARI-­ 0001 cells) and the for this amendment to the protocol were and permissions. Published by CART19-­BE-­01 trial.5 In October 2019, once three cases of fatal toxicity (two patients, aged BMJ. the recruitment was completed, the AEMPS 11 and 19, who died of refractory CRS, and For numbered affiliations see end of article. approved a compassionate use program one patient, age 35, who died of pseudomem- (CUP) for patients fulfilling the same inclu- branous colitis as a complication of grade Correspondence to sion/exclusion criteria. The trial’s prelimi- 4 CRS).5 The entire patients’ disposition is Dr Julio Delgado; nary results were published elsewhere.5 Here, depicted in online supplemental figure 3. ​jdelgado@​clinic.c​ at we present the long-­term results of all consec- Adverse events and response rates are Dr Manel Juan; utive patients with R/R ALL recruited into presented with 95% exact Clopper-­Pearson ​mjuan@​clinic.c​ at the CART19-­BE-­01 trial and the subsequent CIs. The possible association between CRS Ortiz-­Maldonado V, et al. J Immunother Cancer 2021;9:e003644. doi:10.1136/jitc-2021-003644 1 Open access J Immunother Cancer: first published as 10.1136/jitc-2021-003644 on 14 December 2021. Downloaded from http://jitc.bmj.com/ on January 26, 2023 by guest. Protected by copyright. (all grades and grade ≥3), tumor burden at screening (>90% blasts in the BM) at study inclusion, and yet they (<5% vs ≥5% blasts in the bone marrow (BM)) and type of could receive therapy while avoiding irreversible toxicity. administration (single dose vs fractionated) was assessed The measurable residual disease (MRD)-­negative CR using Fisher’s exact test. We also analyzed the impact on rate was 88.6% (95% CI 77.0% to 95.7%) at day +28% and progression-­free survival (PFS) and overall survival (OS) 79.2% (95% CI 65.9% to 89.2%) at day +100. All three of the following variables: age (<25 vs ≥25 years), type of patients who received less than 1×106 ARI-­0001 cells/kg administration, tumor burden and loss of B-­cell aplasia due to toxicity achieved an MRD-­negative CR. All evalu- (BCA), the latter as a time-­dependent covariate. PFS/OS able patients (n=50) developed absolute BCA that lasted curves were plotted using the Kaplan-­Meier method for for a median of 4.2 months (95% CI 3.32 to 7.53 months). time-­fixed covariates and the Simon-­Makuch method for PFS was 50.9% (95% CI 38.4% to 67.4%) and 32.9% (95% BCA loss. Landmark analyses were performed to identify CI 20.6% to 52.6%) at one and 2 years, respectively, while the most appropriate timepoint for BCA loss. Univar- the 1-­year and 2-­year OS were 70.2% (95% CI 58.1% to iate Cox regression was used to evaluate the impact of 84.8%) and 53.9% (95% CI 40.5% to 71.8%). Progres- these covariates on PFS/OS, and those with Benjamini-­ sive disease has occurred in 27 (50.9%; 95% CI 36.8 to Hochberg adjusted p values lower than 0.1 were intro- 64.9%) patients at a median of 5.3 (range, 0.2–23.1) duced into a multivariate Cox regression. Schönfeld months. Tumor cells expressed CD19 in 24 (89%) of these residuals were used to check the proportional hazards relapses, while three (11%) were CD19-­ negative. ARI-­ assumption. The trial was registered at ​clinicaltrials.​gov 0001 cells served as a bridge to alloHCT in three (6%) (NCT03144583). patients. Second ARI-­0001 infusions were documented Fifty-­ three patients with R/R ALL received therapy in nine patients (three more than previously reported5: with ARI-­0001 cells: 38 in the context of the CART19-­ four due to CD19 +relapse and five in patients with early BE-­01 trial and 15 as part of the CUP. The median age BCA loss). These resulted in transient responses and brief was 30 years (range, 3–68), while 24 (45%) patients were periods of BCA, but one of these responses allowed the female. Baseline characteristics of the entire population patient to receive a second alloHCT. are displayed in online supplemental table 1). The data Subgroup analyses are depicted in figure 1 and online cut-­off date was March, 2021, when all infused patients supplemental table 2. By univariate analysis, only two vari- had a minimum follow-­up of 100 days or had experienced ables had a potential impact on PFS: tumor burden (<5% disease relapse or death before that date. vs ≥5% lymphoblasts in the BM at screening), with a 2-­year Patients received ARI-­0001 cells a median of 55.5 days PFS of 52.5% (95% CI 36.4% to 75.7%) vs 10.7% (95% CI (range, 27–216) after inclusion, and the median vein-­to-­ 2.1% to 54.4%) and an HR of 2.14 (95% CI 1.04 to 4.42) vein time (from apheresis to infusion) was 43 days (range, for patients with 5% or more blasts (adjusted p=0.077). 21–190). The original target dose was infused to all except On the other hand, loss of BCA had an HR of 4.41 3 (5.7%) patients who received 0.1–0.4×106 ARI-­0001 (95% CI 1.59 to 12.2), adjusted p=0.0172. Both variables cells/kg due to CRS. CRS was reported in 56.6% (95% (tumor burden and loss of BCA) were also confirmed in CI 42.3%–70.2%) of patients, being grade ≥3 in 11.3% the multivariate model, with an HR of 2.05 (95% CI 1.004 (95% CI 4.3% to 23%) and requiring treatment with to 4.17) for patients with 5% or more blasts at screening tocilizumab and steroids in 20.7% and 11.3% of patients, (p=0.0484) and an HR of 4.32 (95% CI 1.57 to 11.86) respectively. Patients with ≥5% lymphoblasts in the BM for patients with loss of BCA (p=0.0045). Regarding OS, had a higher incidence of CRS (any grade: 82% vs 39%, none of the covariates evaluated had sufficient impact to p=0.0022; grade ≥3: 27% vs 0%, p=0.0036) compared justify a multivariate analysis. Seeing that BCA loss had with those with <5% lymphoblasts. Moreover, the inci- such an impact on PFS, we performed a series of land- dence and severity of CRS was also associated with single mark analyses to identify the most appropriate cut-­off for dose versus fractionated administration of ARI-­0001 cells clinical practice. We chose 3 and 6 months as potential (any grade: 87% vs 45%, p=0.0064; grade ≥3: 27% vs 5%, landmark times because the median time to BCA loss was p=0.047). Neurotoxicity was observed in 13.2% (95% CI 4.2 months in our series. According to these analyses, the 5.5% to 25.3%) of patients, with one self-­limited grade ≥3 3-­month time point was the closest to statistical signif- occurrence (1.9%). No new second malignancies have icance (HR 1.83; 95% CI 0.82 to 4.11; p=0.15, (online been reported apart from a previously notified case of supplemental table 3). myelodysplasia (1/53; 1.9%).5 With more patients and longer follow-­up, we identi- The safety profile of ARI-­0001 cells was comparable to fied two covariates of potential predictive value: tumor that of similar products, with grade ≥3 CRS/neurotox- burden in the BM and loss of BCA. This contrasts with icity rates lower than 5%.2 6 Moreover, both fractionated our previous report, where this potential effect was not administration and tumor burden were significantly asso- fully evident.5 The adverse impact of tumor burden has ciated with the incidence of CRS, in keeping with similar been documented for other therapies, including alloHCT studies.7–10 Of note, two patients enrolled in the CUP and CART19-­cells,8 10 and is therefore not surprising. experienced grade ≥3 CRS with the first fraction (0.1×106 The importance of BCA loss is, on the other hand, more cells/kg), but successfully recovered after treatment with controversial. Our strategy was associated with a relatively tocilizumab. Both patients had a high tumor burden short in vivo ARI-­0001 cell survival (median 4.2 months) 2 Ortiz-­Maldonado V, et al. J Immunother Cancer 2021;9:e003644. doi:10.1136/jitc-2021-003644 Open access J Immunother Cancer: first published as 10.1136/jitc-2021-003644 on 14 December 2021. Downloaded from http://jitc.bmj.com/ on January 26, 2023 by guest. Protected by copyright. product regardless of age (tisagenlecleucel for younger patients; ARI-­0001 cells for older patients). Author affiliations 1 Department of Hematology, Hospital Clínic de Barcelona, Barcelona, Spain 2 Institut d’Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain 3 Department of Pediatric Hematology and Oncology, Hospital Sant Joan de Déu, Barcelona, Spain 4 Institut de Recerca Pediàtrica Sant Joan de Déu, Barcelona, Spain 5 Department of Immunology, Hospital Clínic de Barcelona, Barcelona, Spain 6 Centro de Investigación Biomédica en Red de Cáncer, Madrid, Spain 7 Banc de Sang i Teixits, Barcelona, Spain 8 Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, Spain 9 Apheresis & Cell Therapy Unit, Hospital Clínic de Barcelona, Barcelona, Spain 10 Department of Medicine, University of Barcelona, Barcelona, Spain 11 Medical Intensive Care Unit, Hospital Clínic de Barcelona, Barcelona, Spain 12 Pediatric Intensive Care Unit, Hospital Sant Joan de Déu, Barcelona, Spain 13 Hematopathology Unit, Hospital Clínic de Barcelona, Barcelona, Spain 14 Laboratory of Hematology, Hospital Sant Joan de Déu, Barcelona, Spain 15 Clinical Immunology and Primary Immunodeficiencies Unit, Hospital Sant Joan de Déu, Barcelona, Spain 16 Advanced Therapies Unit, Hospital Clínic de Barcelona, Barcelona, Spain 17 Department of Clinical Pharmacology, Hospital Clínic de Barcelona, Barcelona, Spain 18 Josep Carreras Leukemia Research Institute, Barcelona, Spain 19 Clinical Immunology Platform, Hospital Clínic-­Sant Joan de Déu, Barcelona, Spain Twitter Aina Oliver-­Caldes @ainitaoliver Acknowledgements We are very grateful to Prof. Josep Maria Ribera (Hospital Germans Trias i Pujol, Badalona), who critically read our manuscript. We thank the patients who participated in the study and their families, friends, caregivers and referring physicians. We are very grateful to Drs. Miguel Ángel Perales (Memorial Sloan Kettering Cancer Center, New York), Shannon Maude (Children’s Hospital of Figure 1 Progression-­free (PFS) and overall survival (OS) Philadelphia), Juan José Lasarte (Centro de Investigación Médica Aplicada, Navarra) of patients with relapsed/refractory acute lymphoblastic and Michael Schmitt (Universitäts Klinikum Heidelberg), members of the CART19-­ leukemia treated with ARI-­0001 cells. A, B show the PFS BE-­01 trial DSMB. We are also indebted to the study staff and health care providers (A) and OS (B) of the entire population. C–F depict the PFS at Hospitals Clínic and Sant Joan de Déu, specifically to Anna Boronat, Raquel of patients according to age (C) (red curve: <25 years; blue Martín-­Ibáñez, Unai Perpiñá, Josep M. Canals, Vanina Rodríguez and Guillermo curve: ≥25 years); type of administration (D) (red curve: single Suñé, who participated in the initial development of the construct; Judit Pich, dose; blue curve: fractionated); percentage of blasts in the Gabriela Recalde, Leticia Pereira and Joan Albert Arnáiz, from the Clinical Trials Unit; bone marrow (E) (red curve: <5%; blue curve: ≥5%); and loss Andrea Scalise, Joaquín Sáez-­Peñataro, Ferran Torres and Sandra Castaño, from the of B-­cell aplasia (F) (red curve: no; blue curve: yes). P values Department of Pharmacology; and Josep Maria Campistol, Antoni Castells, Aurea Mira, Manel del Castillo, Miquel Pons and Marc Roda as the main representatives refer to the Benjamini-­Hochberg-­adjusted univariate analysis. from both hospitals. Contributors VO-­M, SR, MJ and JD designed the clinical trial and wrote the paper; JE, GC and AU-­I provided significant contribution to the study planning and analysis; and most relapses had a CD19-­positive phenotype, all VO-­M, JE, LM, EG, MD-­B, MM, LGR-­L, AO-­C, AM-­R, MR, PC, SF and JD looked after in keeping with other studies.8 10 Consequently, further adult patients; SR, AA-­S, AC, AF and IJ looked after pediatric patients; DB-­R, EAG-­N, immune manipulation may be justified in patients with ME-­R, JRO, MP and MJ manufactured ARI-­0001 cells and monitored B-­cell aplasia; MC and MJ designed the CAR construct and produced the lentiviral vector; NV, MA a short-­lived BCA, perhaps less than 3 months, although and MT performed measurable residual disease tests; JC, EG-­R and ML performed this potential timepoint needs further validation. leukocytaphereses; JD performed the statistical analysis; ET, GC and SV coordinate In conclusion, both tumor burden and BCA loss the Advanced Therapies Unit at Hospital Clínic. All authors reviewed the study data, read and approved the manuscript. appeared to have a significant impact on PFS and could guide clinicians in the management of patients after cell Funding This study was funded by CatSalut, Projecte ARI and grants cofinanced by the Instituto de Salud Carlos III–Subdirección General de Evaluación y Fomento infusion. The validity of the fractionated cell administra- de la Investigación Sanitaria–and Fondo Europeo de Desarrollo Regional (FEDER) tion was also confirmed and will be explored in a phase PICI14/122, PI13/676, PIE13/33, PI18/775. VO-­M was a recipient of a research 2 trial. The results of the CART19-­BE-­01 trial led to the grant from FEHH and JD was a recipient of a research grant from the Generalitat de Catalunya (PERIS IPFE SLT006/17/301). approval by the AEMPS of ARI-­0001 cells for the treat- Competing interests VO-­M: Consultant or advisory role (Kite Gilead, Celgene, ment of patients with R/R ALL older than 25 years of age Novartis), travel grants (Kite Gilead, Celgene, Novartis, Roche, Takeda, Janssen), (Hospital Exemption). To the best of our knowledge, this honoraria (Kite Gilead). SR: Consultant or advisory role (Novartis, Jazz, Shire/ is the first purely academic CART19-­cell product approved Servier, Amgen, Celgene/Bristol-­Myers, Kite Gilead), travel grants (Novartis, Jazz, in any European country for any indication. This also Shire/Servier, Amgen, Celgene/Bristol-­Myers, Kite Gilead), honoraria (Novartis, Jazz, Shire/Servier, Amgen, Celgene/Bristol-­Myers, Kite Gilead). AA-­S: Consultant makes Spain the only country in Europe where patients or advisory role (Novartis), travel grants (Novartis), honoraria (Novartis). EG: with R/R ALL have at least one approved CART19-­cell Consultant or advisory role (Kite Gilead, Janssen, Genmab), research funding (Kite Ortiz-­Maldonado V, et al. J Immunother Cancer 2021;9:e003644. doi:10.1136/jitc-2021-003644 3 Open access J Immunother Cancer: first published as 10.1136/jitc-2021-003644 on 14 December 2021. Downloaded from http://jitc.bmj.com/ on January 26, 2023 by guest. Protected by copyright. Gilead, Janssen, Roche). MD-­B: Consultant or advisory role (Celgene, Novartis, and license their derivative works on different terms, provided the original work is Jazz, Astellas). AC: Consultant or advisory role (Novartis, Celgene), travel grants properly cited, appropriate credit is given, any changes made indicated, and the use (Novartis, Celgene), honoraria (Novartis, Celgene). AF: Consultant or advisory is non-­commercial. See http://​creativecommons.​org/​licenses/​by-​nc/​4.​0/. role (Novartis), travel grants (Novartis), honoraria (Novartis). LGR-­L: Travel grants (Kite Gilead, Amgen, Janssen). ML: Honoraria (Grifols, Fresenius Kabi), research ORCID iDs funding (Terumo BCT, Maco-­Pharma). AM-­R: Consultant or advisory role (Bristol Valentín Ortiz-­Maldonado http://​orcid.​org/​0000-​0003-​4699-​6862 Myers Squibb, Abbvie), travel grants (Kite Gilead, Roche, Takeda, Janssen, Abbvie), Aina Oliver-­Caldes http://​orcid.​org/​0000-​0002-​7921-​5420 honoraria (Abbvie). EG-­R: Honoraria (Novartis). PC: Consultant or advisory role Manel Juan http://​orcid.​org/​0000-​0002-​3064-​1648 (Kite Gilead, Celgene, Janssen), travel grants (Kite Gilead, MSD, Janssen). MT: Julio Delgado http://​orcid.​org/​0000-​0002-​5157-​4376 Consultant or advisory role (Novartis). CFDL: Consultant or advisory role (Janssen, Celgene/Bristol-­Myers, GSK), honoraria (Janssen, Celgene/Bristol-­Myers, Amgen, GSK), research funding (Janssen, Celgene/Bristol-­Myers, Amgen, Takeda). GC: Consultant or advisory role (Celgene, Novartis). JE: Consultant or advisory role REFERENCES (Abbvie, Novartis, Celgene, Astellas, Jazz, Daiichi Dankyo, Roche, Amgen, Pfizer), 1 Gökbuget N, Stanze D, Beck J, et al. Outcome of relapsed adult lymphoblastic leukemia depends on response to salvage travel grants (Celgene, Roche, Astellas, Daiichi Dankyo), research funding (Novartis, chemotherapy, prognostic factors, and performance of stem cell Celgene). AU-­I: Consultant or advisory role (Kite Gilead, Celgene/Bristol-­Myers, transplantation. Blood 2012;120:2032–41. Miltenyi), travel grants (Kite Gilead, Celgene/Bristol-­Myers). MJ: Consultant or 2 Maude SL, Laetsch TW, Buechner J, et al. Tisagenlecleucel in advisory role (Kite Gilead, Grifols), honoraria (Kite Gilead, Grifols). children and young adults with B-­cell lymphoblastic leukemia. N Engl J Med 2018;378:439–48. Patient consent for publication Not applicable. 3 Castella M, Boronat A, Martín-­Ibáñez R, et al. Development of Ethics approval The CART19-­BE-­01 clinical trial and subsequent compassionate a novel anti-­CD19 chimeric antigen receptor: a paradigm for an affordable CAR T cell production at academic institutions. Mol Ther use program were approved by the Spanish Medicines Agency (AEMPS) and both Methods Clin Dev 2019;12:134–44. Institutions’ Review Boards and Ethics Committees. All participants provided written 4 Castella M, Caballero-­Baños M, Ortiz-­Maldonado V, et al. Point-­ informed consent. of-­care CAR T-­cell production (ARI-­0001) using a closed semi-­ automatic bioreactor: experience from an academic phase I clinical Provenance and peer review Not commissioned; externally peer reviewed. trial. Front Immunol 2020;11:482. Data availability statement Not applicable (this trial started recruitment in July 5 Ortíz-­Maldonado V, Rives S, Castellà M, et al. CART19-­BE-­01: a 2017). multicenter trial of ari-­0001 cell therapy in patients with CD19+ relapsed/refractory malignancies. Mol Ther 2021;29:636–44. Supplemental material This content has been supplied by the author(s). It has 6 Sheth VS, Gauthier J. Taming the beast: CRS and ICANS after car not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been T-­cell therapy for all. Bone Marrow Transplant 2021;56:552–66. peer-­reviewed. Any opinions or recommendations discussed are solely those 7 Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and T cells for sustained remissions in leukemia. N Engl J Med responsibility arising from any reliance placed on the content. Where the content 2014;371:1507–17. 8 Park JH, Rivière I, Gonen M, et al. Long-­term follow-­up of CD19 includes any translated material, BMJ does not warrant the accuracy and reliability CAR therapy in acute lymphoblastic leukemia. N Engl J Med of the translations (including but not limited to local regulations, clinical guidelines, 2018;378:449–59. terminology, drug names and drug dosages), and is not responsible for any error 9 Frey NV, Shaw PA, Hexner EO, et al. Optimizing chimeric antigen and/or omissions arising from translation and adaptation or otherwise. receptor T-­cell therapy for adults with acute lymphoblastic leukemia. J Clin Oncol 2020;38:415–22. Open access This is an open access article distributed in accordance with the 10 Hay KA, Gauthier J, Hirayama AV, et al. Factors associated with Creative Commons Attribution Non Commercial (CC BY-­NC 4.0) license, which durable EFS in adult B-­cell all patients achieving MRD-­negative Cr permits others to distribute, remix, adapt, build upon this work non-­commercially, after CD19 CAR T-­cell therapy. Blood 2019;133:1652–63. 4 Ortiz-­Maldonado V, et al. J Immunother Cancer 2021;9:e003644. doi:10.1136/jitc-2021-003644 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer SUPPLEMENTARY APPENDIX Factors associated with the clinical outcome of patients with relapsed/refractory CD19+ acute lymphoblastic leukemia treated with ARI- 0001 CART19-cell therapy Valentín Ortíz-Maldonado et al. SUPPLEMENTARY TABLES Supplementary Table 1: Baseline characteristics of patients with relapsed- refractory acute lymphoblastic leukemia who received ARI-0001 cell therapy Characteristic n = 53 Age in years, median (range) 30 (3-68) Female sex, n (%) 24 (54) Presence of Philadelphia chromosome, n (%) 6 (11) Prior lines of therapy, median (range) 4 (2-8) Prior IO, n (%) 28 (53) Prior blinatumomab, n (%) 12 (23) Prior alloHCT, n (%) 42 (79) Measurable disease at screening, n (%) 42 (79) Extramedullary disease, n (%) 19 (36)  5% blasts in the bone marrow, n (%) 22 (42) CNS disease, n (%) 7 (13) Follow-up in months from infusion, median (range) 19 (3-40) Abbreviations: IO, inotuzumab ozogamicin; alloHCT, allogeneic hematopoietic cell transplantation; CNS, central nervous system. 1 Ortiz-Maldonado V, et al. J Immunother Cancer 2021; 9:e003644. doi: 10.1136/jitc-2021-003644 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer Supplementary Table 2: Subgroup analysis of efficacy of ARI-0001 cells in patients with acute lymphoblastic leukemia Population n Progression-free survival Overall survival Median (95% CI) Univariate HR Median (95% CI) Univariate HR 2-year rate (95% CI) (95% CI), p value* 2-year rate (95% CI) (95% CI), p value* Total 53 13.52 mo (7.14-NA) 29.2 mo (15.0-NA) 32.9% (20.6-52.6%) 53.9% (40.5-71.8%) Single dose 15 14.4 mo (6.94-NA) 29.2 mo (11.5-NA) 26.7% (11.5-61.7%) 53.3% (33.2-85.6%) Fractionated 38 13.5 mo (5.3-NA) 0.88 (0.41-1.86), p NA (14.5-NA) 0.91 (0.36-2.31), p 42.0% (27.5-64.2%) = 0.73 54.4% (38.4-77.1%) = 0.84 < 25 years 20 17.53 mo (13.52-NA) 29.2 mo (15.8-NA) 31.6% (14.5-68.9%) 57.2% (38.4-85.1%)  25 years 33 7.83 mo (5.23-NA) 1.34 (0.64-2.81), p NA (11.5-NA) 1.25 (0.52-3.01), p 37.6% (23-61.6%) = 0.59 53.4% (36.7-77.8%) = 0.84 < 5% blasts 31 NA (7.14-NA) NA (29.2-NA) in BM 52.5% (36.4-75.7%) 72% (56.2-92.2%)  5% blasts 22 9.38 mo (3.45-NA) 2.14 (1.04-4.42), p 15.4 mo (10.4-NA) 2.55 (1.06-6.17), p in BM 10.7% (2.1-54.4%) = 0.077 33.4% (17.6-63.5%) = 0.134 Loss of BCA 53 4.41 (1.59-12.21), p 1.28 (0.44-3.68), p = 0.0172 = 0.84 Abbreviations: BM, bone marrow; IO, inotuzumab ozogamicin; alloHCT, allogeneic hematopoietic cell transplantation; BCA, B-cell aplasia; CI, confidence interval; mo, months; NA, not available/not reached; HR, hazard ratio. *Adjusted according to Benjamini-Hochberg method 2 Ortiz-Maldonado V, et al. J Immunother Cancer 2021; 9:e003644. doi: 10.1136/jitc-2021-003644 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer Supplementary Table 3: Landmark analyses evaluating the impact of loss of B-cell aplasia at different time points Population n Progression-free survival Median (95% CI) Univariate HR 2-year rate (95% CI) (95% CI), p value Loss of BCA 15 11.4 (1.14-NE) 1.83 (0.82-4.11), p < 3 mo 22.5% (8.4-60.3%) = 0.15 Loss of BCA 30 15.5 mo (6.38-NE)  3 mo 49.3% (32.5-74.8%) Loss of BCA 17 17.1 (8.38-NE) 1.39 (0.45-4.28), p < 6 mo 43.5% (23.0-82.2%) = 0.56 Loss of BCA 15 NE (7.52-NE)  6 mo 55.3% (31.2-98.0%) Abbreviations: BCA, B-cell aplasia; CI, confidence interval; NE, not estimable; HR, hazard ratio. 3 Ortiz-Maldonado V, et al. J Immunother Cancer 2021; 9:e003644. doi: 10.1136/jitc-2021-003644 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer Ortiz-Maldonado V, et al. J Immunother Cancer 2021; 9:e003644. doi: 10.1136/jitc-2021-003644 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Immunother Cancer Supplementary Figure 3: Patients’ disposition Patients infused with ARI-0001 cells (n = 53) Single dose (n = 15, all Fractionated within the CART19-BE-01 administration (n = 38) trial) Pediatric patients, Adult patients, CART19-BE-01 trial, Compassionate use dose of 5 x106 dose of 1 x106 dose of 1 x106 program, dose of 1 x106 cells/kg (n = 4) cells/kg (n = 11)* cells/kg (n = 23) cells/kg¶ (n = 15, all adults) Pediatric Adult patients patients (n = 7) (n = 16)§ *The first patient ever treated with ARI-0001 cells only received 0.5 x106 cells/kg as mandated by the Spanish Medicines Agency. §One adult patient recruited into the CART19-BE-01 trial received 0.4 x106 cells/kg because of cytokine release syndrome after the second fraction. ¶Two adult patients recruited into the compassionate use program received 0.1 x106 cells/kg because of cytokine release syndrome after the first fraction. 5 Ortiz-Maldonado V, et al. J Immunother Cancer 2021; 9:e003644. doi: 10.1136/jitc-2021-003644