Abstract
Chimeric antigen receptor (CAR)-modified T cells targeting CD19 demonstrate unparalleled responses in relapsed/refractory acute lymphoblastic leukemia (ALL)1,2,3,4,5, but toxicity, including cytokine-release syndrome (CRS) and neurotoxicity, limits broader application. Moreover, 40–60% of patients relapse owing to poor CAR T cell persistence or emergence of CD19− clones. Some factors, including the choice of single-chain spacer6 and extracellular7 and costimulatory domains8, have a profound effect on CAR T cell function and persistence. However, little is known about the impact of CAR binding affinity. There is evidence of a ceiling above which increased immunoreceptor affinity may adversely affect T cell responses9,10,11. We generated a novel CD19 CAR (CAT) with a lower affinity than FMC63, the high-affinity binder used in many clinical studies1,2,3,4. CAT CAR T cells showed increased proliferation and cytotoxicity in vitro and had enhanced proliferative and in vivo antitumor activity compared with FMC63 CAR T cells. In a clinical study (CARPALL, NCT02443831), 12/14 patients with relapsed/refractory pediatric B cell acute lymphoblastic leukemia treated with CAT CAR T cells achieved molecular remission. Persistence was demonstrated in 11 of 14 patients at last follow-up, with enhanced CAR T cell expansion compared with published data. Toxicity was low, with no severe CRS. One-year overall and event-free survival were 63% and 46%, respectively.
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Data availability
The whole-exome sequencing data files from the CARPALL study are available in controlled-access format from the European Genome-phenome Archive (http://www.ebi.ac.uk/ega; accession no. EGAS00001003733). Sequencing data requests will be reviewed by the Independent Data Monitoring Committee and Trial Management Group of the CARPALL study and may be subject to patient confidentiality. After approval, a data-access agreement with UCL will be required. All requests for raw and analyzed data and materials will be reviewed by UCL Business (UCLB) to verify whether the request is subject to any intellectual property or confidentiality obligations.
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Acknowledgements
This work was supported by Children with Cancer UK, Great Ormond St Children’s Charity, the JP Moulton Foundation and the National Institute for Health Research Biomedical Research Centres at Great Ormond Street Hospital for Children NHS Foundation Trust, University College London Hospital, and King’s Health Partners, as well as University College London. P.J.A. is a recipient of an NIHR Research Professorship, which also supported S.G. (grant code 514413). R.R. was supported by GOSH-CC (grant code 543539). F.C. was supported by the Stylian Petrov Foundation. M.P. is supported by the UK National Institute of Health Research University College London Hospital Biomedical Research Centre. F.F.’s group at King’s is supported by CRUK (grant code C604/A25135), the Experimental Cancer Medicine Centre (grant code C30122/A25150), and the NIHR Biomedical Research Centres (BRC) based at King’s Health Partners. The work carried out by S.G., A-M.K., R.R., S.J.A., J.C-C., F.C., B.P., K.V., J.Y., W.V., A.G., K.C., T.B., A.L. and A.H. was supported by Children with Cancer UK, Great Ormond St Children’s Charity and the JP Moulton Foundation (grant code 522356). P.W., L.M. and G.W-K.C. were supported by the European Union FP7 consortium ATECT (grant code 602239). We thank M. Brenner, J. Moppett and W. Qian for providing oversight of the study as the Independent Data Monitoring Committee, W. Qasim for technical support in GMP CAR T cell manufacture and M. Al-hajj and L. Stanczuk for multiplex cytokine analysis.
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Authors and Affiliations
Contributions
S.G. performed preclinical experiments, participated in writing study documentation, developed the manufacturing protocol and clinical assays, analyzed data and wrote the manuscript. A.M.K. designed, performed and analyzed pre-clinical experimental work. S.O. designed and performed surface plasmon resonance analysis, epitope mapping, thermal stability experiments and designed key reagents. G.W. carried out CAR T cell persistence analysis by qPCR and disease endpoint assessment by molecular PCR. J.B. analyzed exome sequencing data, and R.R. and J.C.-C. carried out clinical study assays, manufactured products and analyzed data. S.J.A. performed manufacturing scale ups, carried out clinical study assays and manufactured products. B.P., F.C., K.C. and K.V. wrote study documentation and provided trial management. J.Y. and W.V. carried out clinical study assays. P.A.W generated a key reagent. G.W.-K.C. and L.M. performed preclinical experimental work. A.G. participated in manufacturing products. D.P and J.C.-C., coordinated patient care and were responsible for data collection. G.L., J.S., O.C., A.L., R.C., K.R. and P.V. provided medical care and contributed to data collection for study patients. S.I. provided flow cytometry assessment of disease status. K.C.G. provided manufacturing and clinical assay expertise. G.A. was responsible for ATIMP storage and issue. M.F. and S.M. performed epitope mapping and biacore experiments. T.B. performed next generation exome sequencing. A.L. provided statistical analyses and wrote the manuscript. A.H provided statistical analyses and contributed to study design. F.F. manufactured lentiviral vector and contributed to study documentation. D.B., S.S., N.G., A.V. and P.V. contributed to study design, identified study patients and provided expertise in medical care for study patients. R.H. and R.W. were principal investigators for the study and provided medical care for study patients. M.A.P. conceived the idea, generated the CAR construct and participated in the design of experimental work. P.J.A. designed the experimental work, wrote study documentation, analyzed data, wrote the manuscript and was chief investigator of the study.
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Competing interests
S.G., A.M.K., L.M., G.W-K.C., M.A.P and P.J.A. have patent rights for CAT CAR in targeting CD19 (patent application, World Intellectual Property Organization, WO 2016/139487 Al) and may receive royalties from Autolus PLC, which has licensed the intellectual property and know-how from the CARPALL study. P.J.A. has research funding from bluebird bio Inc. O.C. and P.V. have received funding from Servier. P.V. has research funding from Bellicum Pharmaceuticals. F.F. has founder shares in Autolus PLC, and work in his laboratory is supported by Autolus funding. S.C.O. and M.A.P. are shareholders in and employees of Autolus PLC, which has licensed CAT CAR.
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Extended data
Extended Data Fig. 1 CD19 scFvs derived from different CD19 hybridomas show different kinetic binding properties.
Anti-CD19 scFvs were fused to mouse IgG2a-Fc in a SFG-eBFP γ-retroviral expression vector and HEK293T cells were transfected to generate secreted scFvs, which were then purified on a protein A column. a, Surface plasmon resonance sensograms of binding kinetics between CAT and FMC63 scFv-Fcs to CD19. b, Tabulated kinetic and equilibrium dissociation constants of the CD19 binders. Experiments were repeated independently twice with similar results.
Extended Data Fig. 2 CAT19 and FMC63 share similar biophysical properties, with the exception of binding kinetics.
a, Epitope mapping analysis of CAT and FMC63 binding to CD19 by flow cytometry following single-residue alanine scanning of CD19. b, Summary of CD19 loop residues involved in binding to CAT and FMC63. Specific mutants showing binding inhibition are highlighted in green. Mutated residues showing partial binding inhibition are highlighted in magenta. c, Differential scanning fluorimetry melting Temperature (Tm) analysis comparing CAT and FMC63. Tm was similar (55.1°C and 57.7°C, respectively) for CAT and FMC63. For experiments shown in a–c, anti-CD19 scFvs were fused to mouse IgG2a-Fc in a SFG-eBFP γ-retroviral expression vector and HEK293T cells were transfected to generate soluble protein, which was purified on a protein A column. d, Surface stability analysis comparing FMC63 and CAT CAR surface expression. T cells were transduced with bicistronic lentiviral vector constructs (see schematic). Relative CAR expression was detected independently of CAR affinity by flow cytometric staining with anti-V5-APC; mCherry fluorescence provided a measure of transduction efficiency. Experiments were repeated independently twice with similar results
Extended Data Fig. 3 In a xenograft NALM-6 model, CAT CAR T cells express similar levels of markers of activation and exhaustion (TIM3, LAG3, PD-1) to FMC63 CAR T cells, but a greater proportion express CD127 and Bcl-2 at the tumor site.
Mice were injected with 1×106 GFP+F-luc+ NALM-6 cells 24 h after sublethal irradiation and 7 d prior to T cell injection. Disease engraftment was assessed at day –1. Cohorts were randomized, and recipients with similar tumor burdens were distributed evenly across the groups prior to CAR T cell injection or nontransduced T cells as negative control. a, Tumor growth was evaluated using the IVIS imaging system. b, Blood NALM-6 cell absolute numbers are reduced in the cohort that received CAT CAR transduced T cells (n = 9) compared to FMC63 (n = 8). Data are mean ± s.d.; P = 0.001; two-sided Student’s t-test. c, There was no significant differences in median fluorescence intensity (MFI) of activation and exhaustion markers LAG-3, TIM-3 and PD-1, after gating on CAR+ cells. Data are mean ± s.d.; n = 9. d, Proportion of CD127-positive cells in BM was determined by flow cytometry after gating on CAR+ T cells. Data are mean ± s.d.; n = 9; ***P = 0.0007, two-sided Student’s t-test. e, Proportion of Bcl-2-positive cells in BM. Data are mean ± s.d.; n = 5 (FMC63 CAR); n = 9 (CAT CAR); ***P = 0.0004, two-sided Student’s t test. Experiments were repeated twice with similar results.
Extended Data Fig. 4 Screening, enrolment, treatment and follow-up of patients on the CARPALL study.
The progress of patients through the CARPALL study. 17 patients were registered and eligible for the study. It was possible to generate a product in 14 patients who received lymphodepletion and an infusion of CAR T cells. Two patients failed to respond within the first month, one died and one withdrew from the study. The remaining 12 patients were available for follow-up.
Extended Data Fig. 5 CAR T cell product phenotype and dose.
The CAR T cell product generated was assessed by flow cytometry for CAR T cell expression, and the percentage of viable CD3, CD4 and CD8 cells. In addition, viable CAR+CD45+CD3+ lymphocytes were stained for CD45RA and CCR7 to determine the proportion of CAR T cells in different T memory subsets (CD45RA+CCR7+ = T stem cell memory/naive; CD45RA–CCR7+ = T central memory; CD45RA–CCR7– = T effector memory; CD45RA+CCR7– = T effector memory re-expressing RA). Finally, viable CAR+CD45+CD3+ lymphocytes were stained for the activation and exhaustion markers PD-1 and TIM-3, and the proportion of CAR T cells expressing both of these markers was assessed. n = 14 products, except for exhaustion marker analysis in which n= 13 evaluable products. Lines represent median values. A target cryopreserved cell dose was 1.2 × 106 CAR T cells/kg, giving an infused dose of 106/kg, accounting for 20% cell loss during thawing.
Extended Data Fig. 6 Adverse events.
Frequency of adverse events noted post CAR T cell infusion, by grade and type of toxicity. Cytopenias were defined as reduced neutrophil or platelet count since B lymphocyte depletion was an expected consequence of CAR T cell therapy. B cell aplasia was defined as <5 B cells per µl blood post CAR T cell infusion. Hypogammaglobulinemia was defined as <3 g IgG per L blood.
Extended Data Fig. 7 Serum cytokine and CRP measurements.
Serum levels of IFN-γ, IL-6, IL-10 as well as CRP (a) as assessed by cytometric bead array during the first 14 d post CAR T cell infusion in all 14 patients; the y axis denotes serum level in pg/ml for cytokines and mg/L for CRP. Serum samples from 14 patients were also assessed by a 30-cytokine panel on a MagPix-Luminex platform. Maximal absolute values of analytes are given in b; lines represent median values. Maximal fold change relative to day 0 (that is, pre-CAR T cell infusion) is depicted in c, where red represents an increase and blue a decrease from baseline values.
Extended Data Fig. 8 CAR T cell persistence in bone marrow. Persistence of CAR T cells in the BM was assessed by flow cytometry as well as qPCR for a transgene-specific sequence post CAR T cell infusion.
a, Percentage of bone marrow CAR T cells in 13 evaluable patients. b, Absolute numbers of CAR T cells in the bone marrow in 13 evaluable patients. c, CAR T cell persistence in BM, as assessed by qPCR in 14 evaluable patients. d, Gating strategy used to identify singlet, viable, CD45+CD3+ lymphocytes populations. CAR expression was gated with reference to a healthy donor control. e, Two patients (CPL-10 and CPL-15) showed abrupt loss of detectable CAR T cells at 2 months and 7 d, respectively, post CAR T cell infusion. PBMCs taken from each patient (CPL-10 at 2 months and CPL-15 at 1 month post CAR T infusion) were stimulated twice with autologous irradiated CAR+ T cells. Cultured PBMCs were then incubated with 51Cr-labeled, autologous CAR+ T cells as well as CAR T cells at a range of E:T ratios in a standard 4-h chromium release assay. Specific lysis was calculated as described in Methods, data are mean ± s.d.
Extended Data Fig. 9 Summary of CAR T cell kinetic parameters as measured in peripheral blood by qPCR.
Cmax, maximum concentration; AUC, area under the curve; AUC (0 to 28) AUC from time zero to day 28; AUC (0 to t) AUC from time zero until last measurement; Time to Cmax is the time to reach peak CAR T cell concentration. CAR T cell persistence was defined as the median interval in days from infusion to first value <100 copies per µg DNA, or the last follow-up if this threshold level was not reached
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Ghorashian, S., Kramer, A.M., Onuoha, S. et al. Enhanced CAR T cell expansion and prolonged persistence in pediatric patients with ALL treated with a low-affinity CD19 CAR. Nat Med 25, 1408–1414 (2019). https://doi.org/10.1038/s41591-019-0549-5
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DOI: https://doi.org/10.1038/s41591-019-0549-5
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