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.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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.
Lee, D. W. et al. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet 385, 517–528 (2015).
Turtle, C. J. et al. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J. Clin. Invest 126, 2123–2138 (2016).
Gardner, R. A. et al. Intent to treat leukemia remission by CD19CAR T cells of defined formulation and dose in children and young adults. Blood 129, 3322–3330 (2017).
Maude, S. L. et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N. Engl. J. Med. 378, 439–448 (2018).
Park, J. H. et al. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N. Engl. J. Med. 378, 449–459 (2018).
Qin, H. et al. Novel CD19/CD22 bicistronic chimeric antigen receptors outperform single or bivalent cars in eradicating CD19+CD22+, CD19- and CD22- Pre-B Leukemia. Blood 130, 810 (2017).
Hudecek, M. et al. Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells. Clin. Cancer Res 19, 3153–3164 (2013).
Milone, M. C. et al. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol. Ther. 17, 1453–1464 (2009).
Schmid, D. A. et al. Evidence for a TCR affinity threshold delimiting maximal CD8 T cell function. J. Immunol. 184, 4936–4946 (2010).
Thomas, S. et al. Human T cells expressing affinity-matured TCR display accelerated responses but fail to recognize low density of MHC-peptide antigen. Blood 118, 319–329 (2011).
Chmielewski, M., Hombach, A., Heuser, C., Adams, G. P. & Abken, H. T cell activation by antibody-like immunoreceptors: increase in affinity of the single-chain fragment domain above threshold does not increase T cell activation against antigen-positive target cells but decreases selectivity. J. Immunol. 173, 7647–7653 (2004).
Imai, C. et al. Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia 18, 676–684 (2004).
Kowolik, C. M. et al. CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells. Cancer Res. 66, 10995–11004 (2006).
Lee, D. W. et al. Current concepts in the diagnosis and management of cytokine release syndrome. Blood 124, 188–195 (2014).
Teachey, D. T. et al. Identification of predictive biomarkers for cytokine release syndrome after chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia. Cancer Discov. 6, 664–679 (2016).
Maude, S. L. et al. Chimeric antigen receptor t cells for sustained remissions in leukemia. N. Engl. J. Med. 371, 1507–1517 (2014).
Liu, X. et al. Affinity-tuned ErbB2 or EGFR chimeric antigen receptor T cells exhibit an increased therapeutic index against tumors in mice. Cancer Res. 75, 3596–3607 (2015).
Park, S. et al. Micromolar affinity CAR T cells to ICAM-1 achieves rapid tumor elimination while avoiding systemic toxicity. Sci. Rep. 7, 70 (2017).
Davila, M. L. et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci. Transl. Med. 6, 224ra25 (2014).
Maude, S. L. et al. Sustained remissions with CD19-specific chimeric antigen receptor (CAR)-modified T cells in children with relapsed/refractory ALL. J. Clin. Oncol. 34, 3011 (2016).
Mueller, K. T. et al. Cellular kinetics of CTL019 in relapsed/refractory B-cell acute lymphoblastic leukemia and chronic lymphocytic leukemia. Blood 130, 2317–2325 (2017).
Long, A. H. et al. 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat. Med. 21, 581–590 (2015).
Brentjens, R. J. et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci. Transl. Med. 5, 177ra38 (2013).
Orlando, E. J. et al. Genetic mechanisms of target antigen loss in CAR19 therapy of acute lymphoblastic leukemia. Nat. Med. 24, 1504–1506 (2018).
Almåsbak, H. et al. Inclusion of an IgG1-Fc spacer abrogates efficacy of CD19 CAR T cells in a xenograft mouse model. Gene. Ther. 22, 391–403 (2015).
Zola, H. et al. Preparation and characterization of a chimeric CD19 monoclonal antibody. Immunol. Cell Biol. 69, 411–422 (1991).
Donnelly, M. L. et al. The ‘cleavage’ activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring ‘2A-like’ sequences. J. Gen. Virol. 82, 1027–1041 (2001).
Niesen, F. H., Berglund, H. & Vedadi, M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat. Protoc. 2, 2212–2221 (2007).
Lugthart, G. et al. Simultaneous generation of multivirus-specific and regulatory T cells for adoptive immunotherapy. J. Immunother. 35, 42–53 (2012).
Ricciardelli, I. et al. Towards gene therapy for EBV-associated posttransplant lymphoma with genetically modified EBV-specific cytotoxic T cells. Blood 124, 2514 (2014).
Dull, T. et al. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471 (1998).
Kim, S. et al. Strelka2: fast and accurate calling of germline and somatic variants. Nat. Methods 15, 591–594 (2018).
Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strainw1118; iso-2; iso-3. Fly. (Austin) 6, 80–92 (2012).
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.
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.
Peer review information: Saheli Sadanand was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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.
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.
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
About this article
Cite this article
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
Annual Review of Pharmacology and Toxicology (2021)
Development and functional characterization of novel fully human anti‐CD19 chimeric antigen receptors for T‐cell therapy
Journal of Cellular Physiology (2021)
Journal of Biological Chemistry (2021)
Communications in Nonlinear Science and Numerical Simulation (2021)