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Clinical Trial
. 2015 Feb 20;33(6):540-9.
doi: 10.1200/JCO.2014.56.2025. Epub 2014 Aug 25.

Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor

James N Kochenderfer  1 , Mark E Dudley  2 , Sadik H Kassim  2 , Robert P T Somerville  2 , Robert O Carpenter  2 , Maryalice Stetler-Stevenson  2 , James C Yang  2 , Giao Q Phan  2 , Marybeth S Hughes  2 , Richard M Sherry  2 , Mark Raffeld  2 , Steven Feldman  2 , Lily Lu  2 , Yong F Li  2 , Lien T Ngo  2 , Andre Goy  2 , Tatyana Feldman  2 , David E Spaner  2 , Michael L Wang  2 , Clara C Chen  2 , Sarah M Kranick  2 , Avindra Nath  2 , Debbie-Ann N Nathan  2 , Kathleen E Morton  2 , Mary Ann Toomey  2 , Steven A Rosenberg  2
Affiliations
Clinical Trial

Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor

James N Kochenderfer et al. J Clin Oncol. .

Abstract

Purpose: T cells can be genetically modified to express an anti-CD19 chimeric antigen receptor (CAR). We assessed the safety and efficacy of administering autologous anti-CD19 CAR T cells to patients with advanced CD19(+) B-cell malignancies.

Patients and methods: We treated 15 patients with advanced B-cell malignancies. Nine patients had diffuse large B-cell lymphoma (DLBCL), two had indolent lymphomas, and four had chronic lymphocytic leukemia. Patients received a conditioning chemotherapy regimen of cyclophosphamide and fludarabine followed by a single infusion of anti-CD19 CAR T cells.

Results: Of 15 patients, eight achieved complete remissions (CRs), four achieved partial remissions, one had stable lymphoma, and two were not evaluable for response. CRs were obtained by four of seven evaluable patients with chemotherapy-refractory DLBCL; three of these four CRs are ongoing, with durations ranging from 9 to 22 months. Acute toxicities including fever, hypotension, delirium, and other neurologic toxicities occurred in some patients after infusion of anti-CD19 CAR T cells; these toxicities resolved within 3 weeks after cell infusion. One patient died suddenly as a result of an unknown cause 16 days after cell infusion. CAR T cells were detected in the blood of patients at peak levels, ranging from nine to 777 CAR-positive T cells/μL.

Conclusion: This is the first report to our knowledge of successful treatment of DLBCL with anti-CD19 CAR T cells. These results demonstrate the feasibility and effectiveness of treating chemotherapy-refractory B-cell malignancies with anti-CD19 CAR T cells. The numerous remissions obtained provide strong support for further development of this approach.

Trial registration: ClinicalTrials.gov NCT00924326.

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Conflict of interest statement

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

Figures

Fig 1.
Fig 1.
Anti-CD19 chimeric antigen receptor (CAR) design and function. (A) Schematic of anti-CD19 CAR. Single-chain (sc) Fv region that recognizes CD19 was derived from FMC63 monoclonal antibody. CAR contained CD28 costimulatory domain and T-cell receptor (TCR) –ζ T-cell activation domain. (B) Anti-CD19 CAR T cells were produced by activating peripheral-blood mononuclear cells (PBMCs) with anti-CD3 antibody OKT3 on day 0 and transducing T cells on day 2. Cells were ready for infusion on day 10. (C) CAR expression on T-cell surface of infused cells of patient No. 1 was detected with anti-Fab antibodies. Isotype control staining of same T cells is also shown. Plots are gated on live CD3+ lymphocytes. (D) Plots show isotype control staining and CD45RA versus CCR7 staining of CD3+ CAR positive–infused cells of patient No. 1. (E) Anti-CD19 CAR-transduced T cells of patient No. 1 were cultured for 4 hours with either CD19-K562 cells expressing CD19 or nerve growth factor receptor (NGFR) –K562 cells not expressing CD19. CAR T cells upregulated CD107a, indicating degranulation, in CD19-specific manner. Plots gated on live CD3+ lymphocytes. Anti-CD19 CAR T cells of patient No. 1 were cultured for 6 hours with CD19-K562 or NGFR-K562 cells, and intracellular cytokine staining for (F) interferon gamma (IFNγ), (G) tumor necrosis factor (TNF), and (H) interleukin-2 (IL-2) was performed. CAR T cells produced cytokines in CD19-specific manner. Plots gated on CD3+ lymphocytes. For (E) to (H), experiments were performed on T cells at time of infusion into patient No. 1. LTR, long terminal repeat.
Fig 2.
Fig 2.
Complete remissions (CRs) of chemotherapy-refractory large-cell lymphomas in patients receiving anti-CD19 chimeric antigen receptor T cells. (A) Positron emission tomography (PET)/computed tomography (CT) scans show CR of chemotherapy-refractory primary mediastinal B-cell lymphoma (PMBCL) in patient No. 2. (B) PET/CT scans demonstrate CR of lymphoma in patient No. 8 who had chemotherapy-refractory PMBCL with extensive liver involvement. (C) PET/CT images show CR of diffuse large B-cell lymphoma, not otherwise specified, in patient No. 14, who had extensive splenic lymphoma.
Fig 3.
Fig 3.
Chimeric antigen receptor (CAR) –expressing T cells were detected in regressing lymph node mass of patient with chronic lymphocytic leukemia (CLL). (A) Computed tomography scans show regression of large cervical lymph node mass in patient No. 13. (B) Fine-needle aspiration of lymph node mass shown in (A) was performed 19 days after infusion of anti-CD19 CAR T cells. Aspirated cells were analyzed by flow cytometry with CAR-specific monoclonal antibody. Among lymphoid cells from mass, 70% were T cells, and 31% of T cells expressed anti-CD19 CAR. Plot gated on CD3+ lymphocytes. (C) Before treatment, flow cytometry of blood of patient No. 3 revealed large population of CLL cells as indicated by aberrant CD19+CD5+ phenotype; 91% of pretreatment blood B cells were CLL cells. (D) Ten weeks after treatment, all B cells were absent from blood of patient No. 3, as shown by complete lack of CD19+ cells. CD20+ and CD22+ cells were also absent, which confirmed lack of B cells. (E) One year after treatment, recovering B cells with normal CD19+CD5− phenotype were detected in blood of patient No. 3. (F) Polyclonality of recovering B cells was confirmed by kappa/lambda staining on CD19+ population from (E). In (C), (D), and (E), plots gated on lymphocytes.
Fig 4.
Fig 4.
Variable levels of anti-CD19 chimeric antigen receptor (CAR) T cells were detected in blood of patients. CAR-positive cells were detected in blood of all patients after infusion by quantitative polymerase chain reaction. Graphs show absolute number of total peripheral-blood mononuclear cells (PBMC) containing CAR gene. Peak levels of CAR-positive cells varied considerably from patient to patient, so patients were divided into three groups: patients with (A) peak blood CAR-positive cell levels of 115 to 777/μL, (B) blood CAR-positive cell levels of 35 to 86/μL, and (C) blood CAR-positive cell levels of 9 to 33/μL. No matter what peak CAR-positive cell level was, number of CAR-positive cells in blood followed similar pattern, with blood CAR-positive cell numbers increasing to peak between day 7 and 17 after infusion, followed by rapid decrease in CAR-positive cells. Low numbers of CAR-positive cells persisted for variable lengths of time. Note that CAR-positive cell curve for patient No. 4 ends abruptly, because she died 16 days after infusion of CAR T cells.
Fig 5.
Fig 5.
Most blood chimeric antigen receptor (CAR) –positive T cells expressed CD8, and CAR-positive T cells acquired more differentiated phenotype after infusion. (A) Example of anti-CAR antibody staining is shown. Plot gated on live lymphocytes. Lymphocytes were from blood of patient No. 14 and were collected 7 days after CAR T-cell infusion at time of peak number of blood CAR-positive cells. (B) At time of peak blood CAR-positive T cell levels, majority of CAR-positive T cells were CD8+ in 12 of 15 patients. Example of CD4 and CD8 staining at time of peak blood CAR-positive cells is shown. Plot gated on live CD3+ CAR-positive lymphocytes from patient No. 14. (C) Mean percentage of CD3+CD4+ CAR-positive lymphocytes expressing CCR7+CD45RA− central memory phenotype dropped from time of infusion to time of peak CAR-positive cell blood levels. (D) Mean percentage of CD3+CD8+ CAR-positive lymphocytes expressing CCR7+CD45RA− central memory phenotype dropped from time of infusion to time of peak CAR-positive cell blood levels. (E) Mean percentage of CD3+CD8+ CAR-positive lymphocytes expressing CCR7−CD45RA+ effector memory RA phenotype increased from time of infusion to time of peak CAR-positive cell blood levels. (F) Mean percentage of CD3+CD8+ CAR-positive lymphocytes expressing CD57 increased from time of infusion to time of peak CAR-positive cell blood levels. In (C), (D), (E), and (F), results from all 15 patients studied are included in all groups. All P values from two-tailed paired t tests comparing two groups; error bars represent SEMs.
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