A safe and potent anti-CD19 CAR T cell therapy

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.

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.

Fig. 1 |. CD19-BBz(86)-transduced CAR T cells have lower cytokine production and higher antiapoptotic molecule expression.

a, Schematic of the recombinant lentiviral vectors encoding the prototype anti-CD19 CAR (CD19-BBz(71); also termed CTL019) or variants generated in this study. Coexpression of tEGFR was facilitated by the internal ribosome entry site (IRES), and expression of the construct was under the control of the elongation factor (EF) 1α promoter. CD8(H/TM), CD8α hinge and transmembrane domains. b, Predicted tertiary structures of the CD19-BBz CAR variants in the PyMOL molecular graphics program. αCD19, anti-CD19 scFv; EC, extracellular domain; IC, intracellular domain; TM, transmembrane domain. c, Different levels of cytokines secreted by CD19-BBz-variant-transduced CAR T cells. Human T cells isolated from donor peripheral blood mononuclear cells (PBMCs) were stimulated with Dynabeads Human T Activator CD3/CD28 and then transduced with vectors encoding the indicated CD19-BBz variants. One week after transduction, CAR T cells were co-cultured with irradiated CD19-expressing K562 cells and the culture medium was collected for ELISA. Data are presented as the mean ± s.d. Experiments were repeated with T cells derived from four donors. A two-tailed, unpaired two-sample t test was used for statistical analysis; *P < 0.003, CD19-BBz(86) versus CD19-BBz(71). d, qRT–PCR analysis of the mRNA levels of apoptotic or antiapoptotic molecules in CD19-BBz-variant-transduced CAR T cells after co-culture with irradiated CD19-K562 cells. Data from one of three repeated experiments are presented as the mean ± s.d. Experiments were repeated with T cells derived from three donors. A two-tailed, unpaired two-sample t test was used for statistical analysis; *P < 0.002, CD19-BBz(86) versus CD19-BBz(71). e,f, Groups of 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 mock (PBS). e, Weight change in tumor-bearing mice after CAR T cell transfer. The weight for each mouse was normalized to the starting weight before CAR T cell transfer (n = 10 mice). Data are presented as the mean ± s.d. A multiple t test was used for statistical analysis; NS, not significant. f, Percentage of surviving tumor-bearing mice after i.p. injection with CAR T cells (n = 10 mice). A log-rank Mantel–Cox test was used for statistical analysis. Data are presented from one of two independent experiments with similar results. g, Serum human 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 mock. Sixty hours after CAR T cell injection, mice were bled and sera were isolated for determination of the serum concentrations of the indicated cytokines by ELISA (n = 6 for the control 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.

Fig. 2 |. Patients with refractory or relapsed lymphoma achieved durable remission after CD19-BBz(86) CAR T cell treatment.

a, Representative PET–CT scans of six patients before and after CAR T cell treatment showing durable complete remission. Red arrows indicate sites of lymphoma. Top, PET–CT scans taken before CAR T cell therapy; middle, PET–CT scans taken approximately 1 month after (days 27–35) CAR T cell therapy; bottom, PET–CT scans at the most recent follow-up (day 237 for patient BZ015, day 242 for patient BZ016, day 209 for patient BZ019, day 129 for patient BZ020, day 203 for patient BZ021 and day 119 for patient BZ025). Scale bars, 10 cm. b, CT scans showing long-term remission in patient BZ004 treated with CD19-BBz(86) CAR T cells at a dose of 3 × 10⁶ cells. Circles indicate sites of lymphoma. Scale bars, 10 cm. c, Treatment response of each patient after CD19-BBz(86) CAR T cell treatment and the duration of response. Ongoing remission is marked by a black arrow. Patient number and dose of CD19-BBz(86) CAR T cells are shown to the left of the y axis. CR, complete remission; PR, partial remission; SD, stable disease; PD, progressive disease.

Fig. 3 |. No significant elevation in serum cytokine levels after CD19-BBz(86) CAR T cell infusion.

a, Serum kinetics of a panel of cytokines in 11 patients who received infusions with high doses of CD19-BBz(86) CAR T cells (total of 2–4 × 10⁸ CAR T cells per patient), as determined by Luminex multiplex assay (R&D Systems). Of the 11 patients, 6 achieved complete remission, 2 achieved partial remission and 3 had progressive disease. Horizontal lines denote median values. b, Serum levels of CRP in patients who achieved remission (complete or partial; n = 8) or had progressive disease (n = 3) after CAR T cell infusion. ‘−1 to 0’, 1 d before or the same day as CAR T cell infusion. Horizontal lines denote median values. c, Serum granzyme levels were significantly higher in patients who achieved complete or partial remission (n = 8) than in patients who had progressive disease (n = 3) 1 week after CAR T cell infusion. P values were calculated by two-tailed Student’s t tests. Horizontal lines denote median values. d, Flow cytometry analysis showing in vivo expansion of tEGFR⁺ CD19-BBz(86) CAR T cells in the peripheral blood of representative patient BZ014, who achieved a PR.