Abstract
Anti-CD19 chimeric antigen receptor (CAR) T cell therapies can cause severe cytokine-release syndrome (CRS) and neurotoxicity, impeding their therapeutic application. Here we generated a new anti-CD19 CAR molecule (CD19-BBz(86)) derived from the CD19-BBz prototype bearing co-stimulatory 4-1BB and CD3ζ domains. We found that CD19-BBz(86) CAR T cells produced lower levels of cytokines, expressed higher levels of antiapoptotic molecules and proliferated more slowly than the prototype CD19-BBz CAR T cells, although they retained potent cytolytic activity. We performed a phase 1 trial of CD19-BBz(86) CAR T cell therapy in patients with B cell lymphoma (ClinicalTrials.gov identifier NCT02842138). Complete remission occurred in 6 of 11 patients (54.5%) who each received a dose of 2 × 108–4 × 108 CD19-BBz(86) CAR T cells. Notably, no neurological toxicity or CRS (greater than grade 1) occurred in any of the 25 patients treated. No significant elevation in serum cytokine levels after CAR T cell infusion was detected in the patients treated, including in those who achieved complete remission. CD19-BBz(86) CAR T cells persistently proliferated and differentiated into memory cells in vivo. Thus, therapy with the new CD19-BBz(86) CAR T cells produces a potent and durable antilymphoma response without causing neurotoxicity or severe CRS, representing a safe and potent anti-CD19 CAR T cell therapy.
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Acknowledgements
We would like to thank S. Yan, X. Wu, Y. Yan and members of the GMP-compliant CAR vector and CAR T cell manufacturing teams and the immunological monitoring team of Marino Biotechnology Corp. for their technical assistance. We would also like to thank Y. Wei and T. Liu for their valuable suggestions and help. This study was supported financially by the National Natural Science Foundation of China (No. 81600164, No. 81870154), Beijing Natural Science Foundation (No. 7172046), Beijing Municipal Administration of Hospitals Incubating Program (PX2017001), Capital’s Funds for Health Improvement and Research (No. 2018-1-2151), Beijing Municipal Administration of Hospitals’ Ascent Plan (No. DFL20151001), a generous donation from Y. L. Zhu and the Marino Biotechnology Corp.
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Z.Y., X.X., J. Zhu and S.-Y.C. designed the clinical trial; S.-Y.C., X.X. and X.F.H. designed the overall project. X.X., X.F.H., Y. Liu, X.K., X.G., H.L., T.Z., P.D., J. Zhang, Y.W., S.L., M.M., X.Y., L.F., S.W., S.L., H.S., G.W., S.-Y.C. and L.J. performed the experiments. Z.Y., J. Zhu, Y.S., N.D., Y. Lin, W.Z., Xiaopei Wang, N.L., M.T., Y.X., C.Z., W.L., L.D., S.G., L.P., Xuejuan Wang and N.Z. performed the clinical trial. S.-Y.C., X.X., X.F.H., Z.Y. and J. Zhu analyzed the results and wrote the manuscript.
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X.X., Y. Liu, X.G., H.L., T.Z., P.D., J. Zhang, Y.W., S.L., M.M., X.Y., L.F., S.W. and H.S. are employees of Marino Biotechnology Corp., whose potential product was studied in this work. S.-Y.C. is a consultant of Marino Biotechnology Corp. and a recipient of a research contract with the corporation.
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Extended data
Extended Data Fig. 1 In vitro and in vivo evaluation.
a, IL-6 levels in co-culture of monocytes and CD19-BBz-variant-transduced CAR T cells. Human T cells transduced with the indicated CD19-BBz variants were co-cultured with irradiated CD19-K562 cells in the presence of autologous monocytes (Mono) in a 24-well plate with or without a Corning Transwell (TW) to separate CAR T cells from monocytes. The culture medium was collected after 48 h of co-culture for analysis of IL-6 concentration by ELISA. Data are presented as the mean ± s.d. Experiments were repeated with four different donor-derived T cells (n = 4). A two-tailed, unpaired two-sample t test was used for statistical analysis. *P < 0.001, CD19-BBz(86) versus CD19-BBz(71). b, Proliferation of CD19-BBz-variant-transduced CAR T cells in co-culture with CD19-K562 cells. Human T cells were transduced with the indicated CD19-BBz variants and cultured for 1 week. tEGFR+ transduced CAR T cells were sorted by FACS and co-cultured with irradiated CD19-K562 cells. On the indicated days, T cells were counted, and the fold of T cell expansion is presented as the mean ± s.d. Experiments were repeated with three different donor-derived T cells (n = 3). c, [3H]thymidine incorporation assay to measure CD19-BBz-variant-transduced CAR T cell proliferation. Human T cells transduced with CD19-BBz variants were co-cultured with irradiated CD19-K562 cells in the presence of [3H]thymidine. Data are presented as the mean ± s.d. Experiments were repeated with four donor-derived T cells (n = 4). A two-tailed, unpaired two-sample t test was used for statistical analysis. *P < 0.001, CD19-BBz(86) versus CD19-BBz(71). d, Cytolytic activities of CD19-BBz-variant-transduced CAR T cells. CD19-BBz-variant-transduced CAR T cells were co-cultured with 51Cr-labeled CD19-K562 (left) or CD19+ Nalm-6 (right) cells in triplicate at the indicated E:T ratios. Cytotoxicity was measured by 51Cr release, and data are presented as the mean ± s.d. Experiments were repeated with three donor-derived T cells (n = 3). A two-tailed, unpaired two-sample t test was used for statistical analysis. NS, CD19-BBz(86) versus CD19-BBz(71). e, Annexin V expression in CD19-BBz-variant-transduced CAR T cells after co-culture with irradiated CD19-K562 cells or control K562 cells as detected by flow cytometry staining with anti-tEGFR and Annexin V. Data are presented as the mean ± s.d. Experiments were repeated with three donor-derived T cells (n = 3). A two-tailed, unpaired two-sample t test was used for statistical analysis. *P < 0.004, CD19-BBz(86) versus CD19-BBz(71). f, Serum mouse cytokine levels. SCID-beige mice were inoculated i.p. with 3 × 106 Raji cells followed by i.p. injection with 35 × 106 CD19-BBz(71) or CD19-BBz(86) CAR T cells or were mock treated. Sixty hours after CAR T cell injection, mice were bled and sera were isolated to determine the concentrations in serum of the indicated mouse cytokines by ELISA (n = 6 for the mock group, n = 12 for the CAR T cell groups). Data are presented as the mean ± s.d. A two-tailed, unpaired two-sample t test was used for statistical analysis. g, Absolute counts of intraperitoneal myeloid cell populations obtained by peritoneal lavage 60 h after injection of CAR T cells (i.p.). Data are presented as the mean ± s.d. (n = 4 for the mock group, n = 6 for the CAR T cell groups). A two-tailed, unpaired two-sample t test was used for statistical analysis. h, qRT–PCR analysis of mouse cytokine gene expression in intraperitoneal macrophages isolated from peritoneal lavage 60 h after injection of CAR T cells (i.p.). Data are presented as the mean ± s.d. (n = 4 for the mock group, n = 6 for the CAR T cell groups). A two-tailed, unpaired two-sample t test was used for statistical analysis. i, In vivo expansion of CAR T cells in tumor-bearing mice. Groups of NSG mice were inoculated intravenously (i.v.) with NALM-6 tumor cells followed by i.v. injection with CD19-BBz(71) or CD19-BBz(86) CAR T cells or mock T cells 4 d later. At days 7, 14 and 28 after CAR T cell injection, peripheral blood samples were collected for quantification of tEGFR+ CAR T cells in the blood. Data are presented as the mean ± s.d. (n = 4 for the mock group, n = 6 for the CAR T cell groups). A two-tailed, unpaired two-sample t test was used for statistical analysis.
Extended Data Fig. 2 Durable remission.
CT and PET–CT scans showing durable remission in patients BZ024 and BZ025 treated with CD19-BBz(86) CAR T cells. The arrow indicates sites of lymphoma. Scale bar, 10 cm.
Extended Data Fig. 3 Decrease in lymphocyte count after lymphodepletion chemotherapy in individual patients.
All patients were administrated 3-d lymphodepletion chemotherapy comprising fludarabine (25 mg m–2 on days 1–3) and cyclophosphamide (250 mg m–2 on days 1–3) before CAR T cell infusion. The time point of lymphodepletion was on the day of CAR T cell infusion (day 0) for all patients, except for patients BZ015 (day –1), BZ021 (day –2) and BZ026 (day –3). Each patient’s lymphocyte counts before and after lymphodepletion chemotherapy are connected by a line for pairwise comparison. The median blood lymphocyte count just before lymphodepletion chemotherapy was 1 × 109 cells L–1 (range of 0.49–1.73 × 109 cells L–1). The median lymphocyte count after chemotherapy on the day of CAR T cell infusion was 0.14 × 109 cells L–1 (range of 0.02–0.61 × 109 cells L–1).
Extended Data Fig. 4 Changes in blood IgA levels.
Changes in IgA levels were assessed after CD19-BBz(86) CAR T cell therapy in individual patients.
Extended Data Fig. 5 Changes in blood IgG levels.
Changes in IgG levels were assessed after CD19-BBz(86) CAR T cell therapy in individual patients.
Extended Data Fig. 6 Changes in blood IgM levels.
Changes in IgM levels were assessed after CD19-BBz(86) CAR T cell therapy in individual patients.
Extended Data Fig. 7 In vivo expansion and memory generation.
a, Flow cytometry analysis showing in vivo expansion of tEGFR+ CD19-BBz(86) CAR T cells in the peripheral blood of representative patients who achieved complete remission (patient BZ021) or had progressive disease (patient BZ013). b,c. qPCR showing in vivo CAR T cell expansion and persistence in the blood of patients who achieved complete or partial remission (patients BZ015, BZ016, BZ019, BZ020, BZ021, BZ024 and BZ025) (b) or had progressive disease (patients BZ017, BZ022 and BZ023) (c). d, Memory phenotype (CD45RA–CCR7+) of in vivo-expanded tEGFR+CD3+ CD19-BBz(86) CAR T cells and tEGFR–CD3+ normal T cells from six patients who had progressive disease (BZ013) or achieved partial remission (BZ014) or complete remission (BZ015, BZ019, BZ020 and BZ021). e, Percentage of CD45RA+ and CD45RA– subpopulations of in vivo-expanded tEGFR+CD3+ CD19-BBz(86) CAR T cells and tEGFR–CD3+ normal T cells from the six treated patients. P values were calculated from two-tailed Student’s t tests. Horizontal lines denote median values. f, Percentage of CD45RA+CCR7–, CD45RA+CCR7+, CD45RA–CCR7– and CD45RA–CCR7+ subpopulations of in vivo-expanded tEGFR+CD3+ CD19-BBz(86) CAR T cells and tEGFR–CD3+ normal T cells from the six treated patients. P values were calculated from two-tailed Student’s t tests. Horizontal lines denote median values. g–j, Detection of tEGFR+CD3+ CD19-BBz(86) CAR T cells in the peripheral blood of patient BZ015 on day 317 after cell infusion. g, Of the tEGFR+CD3+ CAR T cells, 96.7% were CD8+. h, CD19+ B cells were still depleted. i, 45.8% of the long-term, persistent tEGFR+CD3+ CD19-BBz(86) CAR T cells were CD45RO+CCR7+ central memory T cells in the peripheral blood of patient BZ015 on day 317 after cell infusion, while only 6.25% of the tEGFR–CD3+ normal T cells were CD45RO+CCR7+ central memory T cells. j, qPCR analyses in triplicate also showed long-term persistence of CD19-BBz(86) CAR T cells in peripheral blood.
Supplementary information
Supplementary Information
Supplementary Tables 1–13 and Supplementary Figures 1–16
Supplementary Data 1
CD19-BBz(71) DNA and amino acid sequences
Supplementary Data 2
CD19-BBz(75) DNA and amino acid sequences
Supplementary Data 3
CD19-BBz(82) DNA and amino acid sequences
Supplementary Data 4
CD19-BBz(86) DNA and amino acid sequences
Supplementary Data 5
CD19-BBz(96) DNA and amino acid sequences
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Ying, Z., Huang, X.F., Xiang, X. et al. A safe and potent anti-CD19 CAR T cell therapy. Nat Med 25, 947–953 (2019). https://doi.org/10.1038/s41591-019-0421-7
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DOI: https://doi.org/10.1038/s41591-019-0421-7
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