Abstract
While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19− disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials (NCT02588456 and NCT02650414) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.
Similar content being viewed by others
Data availability
All requests for raw and analyzed preclinical data and materials will be promptly reviewed by the University of Pennsylvania to determine if they are subject to intellectual property or confidentiality obligations. Patient-related data not included in the paper were generated as part of clinical trials and may be subject to patient confidentiality. Any data and materials that can be shared will be released via a material transfer agreement. Sequences for the m971 CAR are publicly available under patent no. PCT/US2013/060332. Sequences for the CD22-f2 CAR are the private property of Novartis. The raw data for Supplementary Figs. 1, 2d–l, 3d–f and 4d–l are located in the Supplementary Dataset. Source data are provided with this paper.
References
Geyer, M. B. et al. Overall survival among older US adults with ALL remains low despite modest improvement since 1980: SEER analysis. Blood 129, 1878–1881 (2017).
Ma, H., Sun, H. & Sun, X. Survival improvement by decade of patients aged 0–14 years with acute lymphoblastic leukemia: a SEER analysis. Sci. Rep. 4, 4227 (2014).
Maude, S. L. et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N. Engl. J. Med. 378, 439–448 (2018).
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, 224ra225 (2014).
Gardner, R. A. et al. Intent-to-treat leukemia remission by CD19 CAR T cells of defined formulation and dose in children and young adults. Blood 129, 3322–3331 (2017).
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).
Grupp, S. A. et al. Chimeric antigen receptor–modified T cells for acute lymphoid leukemia. N. Engl. J. Med. 368, 1509–1518 (2013).
Hay, K. A. et al. Factors associated with durable EFS in adult B-cell ALL patients achieving MRD-negative CR after CD19 CAR T-cell therapy. Blood 133, 1652–1663 (2019).
Orlando, E. J. et al. Genetic mechanisms of target antigen loss in CAR19 therapy of acute lymphoblastic leukemia. Nat. Med. 24, 1504–1506 (2018).
Ruella, M. et al. Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell. Nat. Med. 24, 1499–1503 (2018).
Sotillo, E. et al. Convergence of acquired mutations and alternative splicing of CD19 enables resistance to CART-19 immunotherapy. Cancer Discov. 5, 1282–1295 (2015).
Hamieh, M. et al. CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape. Nature 568, 112–116 (2019).
Fry, T. J. et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nat. Med. 24, 20–28 (2018).
Shah, N. N. et al. CD4/CD8 T-cell selection affects chimeric antigen receptor (CAR) T-cell potency and toxicity: updated results from a phase I anti-CD22 CAR T-cell trial. J. Clin. Oncol. 38, 1938–1950 (2020).
Xiao, X., Ho, M., Zhu, Z., Pastan, I. & Dimitrov, D. S. Identification and characterization of fully human anti-CD22 monoclonal antibodies. MAbs 1, 297–303 (2009).
Haso, W. et al. Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia. Blood 121, 1165–1174 (2013).
Hudecek, M. et al. The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol. Res. 3, 125–135 (2015).
Jonnalagadda, M. et al. Chimeric antigen receptors with mutated IgG4 Fc spacer avoid Fc receptor binding and improve T cell persistence and antitumor efficacy. Mol. Ther. 23, 757–768 (2015).
Alabanza, L. et al. Function of novel anti-CD19 chimeric antigen receptors with human variable regions is affected by hinge and transmembrane domains. Mol. Ther. 25, 2452–2465 (2017).
Ying, Z. et al. A safe and potent anti-CD19 CAR T cell therapy. Nat. Med. 25, 947–953 (2019).
Richman, S. A. et al. High-affinity GD2-specific CAR T cells induce fatal encephalitis in a preclinical neuroblastoma model. Cancer Immunol. Res. 6, 36–46 (2018).
Maude, S. L. et al. Chimeric antigen receptor T cells for sustained remissions in leukemia. N. Engl. J. Med. 371, 1507–1517 (2014).
Kalos, M. et al. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci. Transl. Med. 3, 95ra73 (2011).
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).
Porter, D., Frey, N., Wood, P. A., Weng, Y. & Grupp, S. A. Grading of cytokine release syndrome associated with the CAR T cell therapy tisagenlecleucel. J. Hematol. Oncol. 11, 35 (2018).
Hudson, P. J. & Kortt, A. A. High avidity scFv multimers; diabodies and triabodies. J. Immunol. Methods 231, 177–189 (1999).
Schirrmann, T. et al. Oligomeric forms of single chain immunoglobulin (scIgG). MAbs 2, 73–76 (2010).
Colomb, F. et al. Galectin-9 mediates HIV transcription by inducing TCR-dependent ERK signaling. Front. Immunol. 10, 267 (2019).
Liu, D., Peterson, M. E. & Long, E. O. The adaptor protein Crk controls activation and inhibition of natural killer cells. Immunity 36, 600–611 (2012).
Xiong, W. et al. Immunological synapse predicts effectiveness of chimeric antigen receptor cells. Mol. Ther. 26, 963–975 (2018).
Dustin, M. L. & Depoil, D. New insights into the T cell synapse from single molecule techniques. Nat. Rev. Immunol. 11, 672–684 (2011).
Frigault, M. J. et al. Identification of chimeric antigen receptors that mediate constitutive or inducible proliferation of T cells. Cancer Immunol. Res. 3, 356–367 (2015).
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).
Lynn, R. C. et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 576, 293–300 (2019).
Schritt, D. et al. Repertoire Builder: high-throughput structural modeling of B and T cell receptors. Mol. Syst. Des. Eng. 4, 761–768 (2019).
Lis, M. et al. Bridging the gap between single-template and fragment based protein structure modeling using Spanner. Immunome Res. 7, 1–8 (2011).
Perisic, O., Webb, P. A., Holliger, P., Winter, G. & Williams, R. L. Crystal structure of a diabody, a bivalent antibody fragment. Structure 2, 1217–1226 (1994).
Liu, D. et al. Integrin-dependent organization and bidirectional vesicular traffic at cytotoxic immune synapses. Immunity 31, 99–109 (2009).
Ahmed, F., Friend, S., George, T. C., Barteneva, N. & Lieberman, J. Numbers matter: quantitative and dynamic analysis of the formation of an immunological synapse using imaging flow cytometry. J. Immunol. Methods 347, 79–86 (2009).
Barrett, D. M. et al. Noninvasive bioluminescent imaging of primary patient acute lymphoblastic leukemia: a strategy for preclinical modeling. Blood 118, e112–e117 (2011).
Acknowledgements
We thank F. Chen and N. Koterba for technical assistance with the cytokine quantification assays and J. Schug for assistance with the RNA sequencing. The pediatric trial was supported by the CHOP Immunotherapy Frontier Program, in addition to support from the Emily Whitehead Foundation, V Foundation and Curing Kids Cancer (S.A.G.). The preclinical research was supported by the Society of Immunotherapy for Cancer Holbrook Kohrt Immunotherapy Translational Fellowship (N.S.); a Breakthrough Bike Challenge Buz Cooper Scholarship (N.S.); Stand Up To Cancer (SU2C) Innovative Research Grant no. SU2C-AACR-IRG 12-17 (D.M.B.; SU2C is a program of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the Scientific Partner of SU2C); National Institutes of Health grant no. R01GM104867 (J.K.B.); a Cancer Research Institute Irvington Fellowship (N.H.R.); National Heart, Lung, and Blood Institute grant no. HL125018 and National Institute of Allergy and Infectious Diseases grant nos. AI124769, AI129594 and AI130197 (D.L.); Japan Agency for Medical Research and Development grant no. P20am0101108 (D.M.S.); NCI grant no. K08CA194256 (S.G.); an American Society of Hematology Scholar Award, NCI grant nos. 1K99CA212302 and R00CA212302 (M.R.); University of Pennsylvania-Novartis Alliance (S.G. and C.H.J.); and NCI grant nos. 1P01CA214278 and R01CA226983 (C.H.J.).
Author information
Authors and Affiliations
Contributions
N.S., B.E., D.M.B., O.S., P.R., K.D.C., Y.G.L., R.P., I.C., A.S., S.L.H., A.P., L.Z., L.P., B.G., M. Ramones, D.A.C., J.P., S.F.L., N.H.R., J.K.B., F.C., M.D., M.A.M., T.L., D.L., D.M.S., C.H.J., S.L.M., S.G. and M. Ruella designed, performed and oversaw the research. X.M.L. performed the biostatistical analysis on the RNA sequencing. N.V.F. was principal investigator of the adult clinical trial. S.A.G. was principal investigator of the pediatric clinical trial. R.M.Y. and J.L.B. provided significant intellectual contribution to the design and research. N.S., S.G. and M. Ruella wrote the manuscript. All authors reviewed the manuscript.
Corresponding authors
Ethics declarations
Competing interests
M.R. and S.G. hold patents related to CART22. C.H.J. has received grant support from Novartis and has patents related to CAR therapy with royalties paid from Novartis to the University of Pennsylvania. C.H.J. is also a scientific founder and holds equity in Tmunity Therapeutics. S.A.G. has received support from Novartis, Servier and Kite and serves as a consultant, member of the scientific advisory board or study steering committee for Novartis, Cellectis, Adaptimmune, Eureka, TCR2, Juno, GlaxoSmithKline, Vertex, Cure Genetics, Humanigen and Roche. B.E., L.Z., L.P., A.P., B.G., M. Ramones and J.B. are employees of Novartis. S.F.L. has received grant support from Novartis, Tmunity and Cabaletta and has patents related to CAR therapy with royalties paid from Novartis to the University of Pennsylvania; he has acted as a consultant for Kite/Gilead. S.L.M. has served as a member of advisory boards or steering committees for Novartis and Kite. All other authors declare no competing interests.
Additional information
Peer review information Nature Medicine thanks Aude Chapuis, Kristen Hege, Qian Wu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 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
Extended Data Fig. 1
CONSORT structure of clinical trials of CD22 CAR T cells in adults and children.
Extended Data Fig. 2 Concentration of serum cytokines in patients treated with CAR22-long T cells.
Adult patient 1 had grade 3 CRS with mean GCSF, IL-6 and MCP-1 greater than all other patients (denotated with an *), and significantly higher levels of IL-10, IFNγ, IL-1Ra, IL-2R, IL-8, MIP-1α and VEGF than at least 3 other patients.
Extended Data Fig. 3 Re-expansion of CART19 cells in patients treated with CART22.
a–d, CART19 and CART22 composition in apheresis and CART22 products in a, pediatric patient #2 and b, adult patient #2, and expansion of CART19 and CART22 in c, pediatric patient #2 and d, adult patients #2 over time after infusion of CART22. e-i, Evaluation of peripheral blood from adult patient 2 at peak CART19 expansion (day 21). CD3 + peripheral blood mononuclear cells (PBMCs) from a healthy donor were used as either e, negative, untransduced control or f, CAR19 and CAR22 dual-engineered positive control. g, CD3 + PBMCs, h, CD3 + CD4 + PBMCs, and i, CD3 + CD8 + PBMCs from adult patient 2.
Extended Data Fig. 4 CART22 phenotypes over time.
a, Cell growth during T cell engineering. Expression of b, PD-1, c, LAG3, and d, Tim3. *P < 0.05, **P < 0.001, ***P < 0.0001, ****P < 0.00001. Statistics reflect differences between CAR22-short and long T cells. Statistics reflect differences between CAR22-short and long T cells.
Extended Data Fig. 5
a, b, Fluorescent microscopy of T cells engineered with GFP-linked a, CAR19-short or b, CAR19-long constructs. c-f, CAR22-short T cells form more effective immune synapses. c, % of cells forming CART:tumor cell immune conjugates after 5 and 15 minutes of co-culture. d, phosphorylated CD3ζ (three independent experiments, n = 820 measurements per group) and e, phosphorylated Zap70 (n = 180 measurements per group) in CAR T cells engaged with Nalm6. f, Quantification of CAR T cell mobility in mice engrafted with Nalm6, as measured by % of cells moving within an intravital imaging field (n = 5 per group).
Extended Data Fig. 6 CART22-short cells are primed for activation.
a, Difference in phospho-peptide quantity in stimulated CAR22-short compared to CAR22-long T cells. Proteins with >1.5-fold difference are shown. b, Upregulated transcriptional programs in CAR22-short compared to CAR22-long T cells than have been stimulated by CD22-coated beads for 18 hours. c, Volcano plot of differentially expressed transcripts in CAR22-short compared to CAR22-long T cells.
Extended Data Fig. 7
a, Measurement of activation-induced cell death by Annexin-V expression. Anti-tumor activity of an in vitro b, treatment or c, stress model of T cells bearing either a short or long CAR composed of the m971 scFv with the CD28 co-stimulatory domain. *P < 0.05, **P < 0.001, ***P < 0.0001, ****P < 0.00001. Statistics reflect differences between CAR22-short and long T cells.
Extended Data Fig. 8 Functional characterization of CAR33-enginereed T cells.
a, Growth of CD33 + Molm14 acute myeloid leukemia cells and b, CAR33-bearing T cells during in vitro co-culture. Quantification of c, IFNγ, d, IL-2 and e, TNFα by CART33 cells during an in vitro co-culture with Molm14 cells. *P < 0.05, **P < 0.001, ***P < 0.0001, ****P < 0.00001. Statistics reflect differences between CAR33-short and long T cells.
Extended Data Fig. 9 Functional characteristics of CAR19-engineered T cells.
Anti-tumor activity in an in vitro a, treatment or b, stress model of T cells bearing either a short or long CD19-targeted CAR. c, Anti-tumor effect of long or short CART19 cells in a xenograft model of Nalm6 ALL (representative of two individual experiments, n=5 mice per condition; see Supplementary Figure 3 for individual animal responses from this experiment and the replicate experiments). Quantification of d, IFNγ, e, IL-2 and f, TNFα by CART33 cells during an in vitro co-culture with Molm14 cells. *P<0.05, **P<0.001, ***P<0.0001, ****P<0.00001. Statistics reflect differences between CAR19-short and long T cells.
Extended Data Fig. 10 Anti-tumor activity of all four CD22 CAR T cells in a xenograft model of SEM ALL.
n = 5 mice per condition; see Supplementary Figure 5 for individual animal responses.
Supplementary information
Supplementary Information
Supplementary Figs. 1–6, NCT02588456 Clinical Protocol, NCT02650414 Clinical Protocol, NCT02650414 DSMP.
Supplementary Table 1
Additional clinical data for NCT02588456 and NCT02650414.
Supplementary Table 2
Full toxicity reporting.
Supplementary Data 1
Source data for supplementary figures.
Source data
Source Data Fig. 3
Statistical source data.
Source Data Fig. 4
Statistical source data.
Source Data Fig. 5
Statistical source data.
Source Data Extended Data Fig. 4
Statistical source data.
Source Data Extended Data Fig. 5
Statistical source data.
Source Data Extended Data Fig. 6
Statistical source data.
Source Data Extended Data Fig. 7
Statistical source data.
Source Data Extended Data Fig. 8
Statistical source data.
Source Data Extended Data Fig. 9
Statistical source data.
Source Data Extended Data Fig. 10
Statistical source data.
Rights and permissions
About this article
Cite this article
Singh, N., Frey, N.V., Engels, B. et al. Antigen-independent activation enhances the efficacy of 4-1BB-costimulated CD22 CAR T cells. Nat Med 27, 842–850 (2021). https://doi.org/10.1038/s41591-021-01326-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41591-021-01326-5
- Springer Nature America, Inc.
This article is cited by
-
Gene editing technology to improve antitumor T-cell functions in adoptive immunotherapy
Inflammation and Regeneration (2024)
-
CD28 hinge used in chimeric antigen receptor (CAR) T-cells exhibits local structure and conformational exchange amidst global disorder
Communications Biology (2024)
-
Comparative performance of scFv-based anti-BCMA CAR formats for improved T cell therapy in multiple myeloma
Cancer Immunology, Immunotherapy (2024)
-
Characterization of chimeric antigen receptor modified T cells expressing scFv-IL-13Rα2 after radiolabeling with 89Zirconium oxine for PET imaging
Journal of Translational Medicine (2023)
-
Safety and efficacy of co-administration of CD19 and CD22 CAR-T cells in children with B-ALL relapse after CD19 CAR-T therapy
Journal of Translational Medicine (2023)