Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells

Abstract

Adoptive immunotherapy with T cells expressing a tumor-specific chimeric T-cell receptor is a promising approach to cancer therapy that has not previously been explored for the treatment of lymphoma in human subjects. We report the results of a proof-of-concept clinical trial in which patients with relapsed or refractory indolent B-cell lymphoma or mantle cell lymphoma were treated with autologous T cells genetically modified by electroporation with a vector plasmid encoding a CD20-specific chimeric T-cell receptor and neomycin resistance gene. Transfected cells were immunophenotypically similar to CD8(+) effector cells and showed CD20-specific cytotoxicity in vitro. Seven patients received a total of 20 T-cell infusions, with minimal toxicities. Modified T cells persisted in vivo 1 to 3 weeks in the first 3 patients, who received T cells produced by limiting dilution methods, but persisted 5 to 9 weeks in the next 4 patients who received T cells produced in bulk cultures followed by 14 days of low-dose subcutaneous interleukin-2 (IL-2) injections. Of the 7 treated patients, 2 maintained a previous complete response, 1 achieved a partial response, and 4 had stable disease. These results show the safety, feasibility, and potential antitumor activity of adoptive T-cell therapy using this approach. This trial was registered at www.clinicaltrials.gov as #NCT00012207.

Figures

Figure 1

Schema of clinical protocol.

Figure 2

Expression of the CD20-specific cTCR. (A) Schematic diagram of the CD20-specific scFvFc:ζ chimeric T-cell receptor cDNA plasmid. (B) A representative Western blot analysis of cTCR expression performed using whole-cell lysates of preinfusion T cells from patient B, probed with mouse anti–human CD3ζ monoclonal Ab. Negative control was parental PBMCs, and positive control was transfected Jurkat cell line. A 21-kDa band corresponding to the endogenous CD3ζ chain and a 66-kDa band representing the expected cTCR protein were detected. The intermediate bands indicate degradation products or truncated forms of the cTCR.

Figure 3

Clonality of T cells produced by limiting dilution and in bulk culture. T-cell clonality was determined by flow cytometric T-cell receptor (TCR) Vβ spectratyping (top) and by PCR amplification of clonal V-J rearrangements at the TCRγ locus (bottom). Representative results for T cells produced by limiting dilution (A) and in bulk culture (B) are shown. (A) T cells produced by limiting dilution (patient B), showing clonal expression of Vβ17 in 98% of CD8+ T cells by Vβ spectratyping (top; ■) and showing 2 predominant TCRγ rearrangements (bottom). Because each T-cell clone can rearrange one or both of its TCRγ alleles, the 2 PCR products could represent either 1 T-cell clone with biallelic TCRγ rearrangements or 2 singly rearranged clones, although the single predominant Vβ17 clone identified by spectratyping would favor a single doubly rearranged clone. (B) T cells produced in bulk culture (patient G) showing oligoclonal Vβ expression in CD8+ T cells (16% Vβ16; 9% Vβ7.1; 3% each Vβ3, Vβ13.2 and Vβ17; 2% each Vβ1 and Vβ13.1; and 1% each Vβ5.1, Vβ13.6, Vβ21.3, and Vβ23) and 7 distinct TCRγ rearrangements by PCR (bottom; ■) that could correspond to between 4 and 7 different T-cell clones, depending on the number of singly and doubly rearranged clones (see Table S1). The □ in both top panels represent the average expression levels for each Vβ chain in normal polyclonal T-cell populations.

Figure 4

Growth curves of genetically modified T cells. Patient PBMCs were transfected with the scFvFc:ζ plasmid by electroporation after stimulation with OKT3. For patients A, B, and D, populations of G418-resistant T cells were generated by limiting dilution, and T-cell cultures exhibiting the most favorable cytotoxicity and growth profiles were selected for expansion to therapeutic numbers. For patients F through I, G418-resistant cells were grown as bulk cultures.

Figure 5

Immunophenotypes of infused T cells. The phenotypes shown were determined using multicolor flow cytometry and are expressed in terms of percentage of the population of infused cells. (A) CD8+ versus CD4+ cells, (B) cells with a CD8+ effector T-cell phenotype (CD8+/CD62L−/CCR7−/CD45RA−/CD127−), (C) cells with CD8+ central memory (CD62L+/CCR7+/CD45RA−/CD127+) versus effector memory (CD62L−/CCR7−/CD45RA−/CD127+) T-cell phenotypes, (D) cells with CD4+ central memory versus effector memory T-cell phenotypes, and (E) cells with a regulatory T-cell (Treg) phenotype (CD4+/CD25+/FoxP3+) are shown.

Figure 6

Cytotoxicity of modified T cells. Standard chromium release assays were performed using preinfusion-modified T cells at 8 to 12 days after restimulation, using the following MHC-mismatched target cells: EL4-CD20, a murine T-cell lymphoma line transfected to express the human CD20 molecule (----), the parental EL4 nontransfected CD20− line (…), and the Daudi CD20+ Burkitt lymphoma cell line (—), at the E:T ratios shown. The calculated specific cytolysis values are displayed as percentages. (A) The CD20-specific cytotoxicity of the reinfused T cells. Data shown represent the mean combined data from all treated patients (± 1 SD). Triplicate assays were performed for each patient. (B) CD20 expression of the target cell lines of EL4, EL4-CD20, and Daudi, as determined by flow cytometry using PE-labeled mouse anti–human CD20 Ab.

Figure 7

In vivo persistence of modified T cells. Genomic DNA was isolated from patient PBMCs collected at serial time points after T-cell infusions and used for quantitative real-time PCR using one primer within the human CD3ζ gene and the other from the adjacent CD4 transmembrane region in the scFvFc:ζ plasmid. The copy number of scFvFc:ζ-specific DNA based on quantitative reverse transcription-PCR results for all treated patients is shown. Arrows denote T-cell infusions, and horizontal black bars indicate the period of subcutaneous IL-2 injections for patients F, G, H, and I. Modified T cells were detectable for 12, 5, 21, 63, 63, 35, and 65 days, respectively, in the 7 patients.