Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Clinical Trial
. 2008 Sep 15;112(6):2261-71.
doi: 10.1182/blood-2007-12-128843. Epub 2008 May 28.

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

Brian G Till  1 , Michael C Jensen, Jinjuan Wang, Eric Y Chen, Brent L Wood, Harvey A Greisman, Xiaojun Qian, Scott E James, Andrew Raubitschek, Stephen J Forman, Ajay K Gopal, John M Pagel, Catherine G Lindgren, Philip D Greenberg, Stanley R Riddell, Oliver W Press
Affiliations
Clinical Trial

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

Brian G Till et al. Blood. .

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Schema of clinical protocol.
Figure 2
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
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
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
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
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
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.
See this image and copyright information in PMC

Comment in

  • Test-driving CARs.
    Cooper LJ. Cooper LJ. Blood. 2008 Sep 15;112(6):2172-3. doi: 10.1182/blood-2008-06-163006. Blood. 2008. PMID: 18779394 No abstract available.

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. McLaughlin P, Grillo-Lopez AJ, Link BK, et al. Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program. J Clin Oncol. 1998;16:2825–2833. - PubMed
    1. Foran JM, Rohatiner AZ, Cunningham D, et al. European phase II study of rituximab (chimeric anti-CD20 monoclonal antibody) for patients with newly diagnosed mantle-cell lymphoma and previously treated mantle-cell lymphoma, immunocytoma, and small B-cell lymphocytic lymphoma. J Clin Oncol. 2000;18:317–324. - PubMed
    1. Czuczman MS, Grillo-Lopez AJ, White CA, et al. Treatment of patients with low-grade B-cell lymphoma with the combination of chimeric anti-CD20 monoclonal antibody and CHOP chemotherapy. J Clin Oncol. 1999;17:268–276. - PubMed
    1. Forstpointner R, Dreyling M, Repp R, et al. The addition of rituximab to a combination of fludarabine, cyclophosphamide, mitoxantrone (FCM) significantly increases the response rate and prolongs survival as compared with FCM alone in patients with relapsed and refractory follicular and mantle cell lymphomas: results of a prospective randomized study of the German Low-Grade Lymphoma Study Group. Blood. 2004;104:3064–3071. - PubMed
    1. Howard OM, Gribben JG, Neuberg DS, et al. Rituximab and CHOP induction therapy for newly diagnosed mantle-cell lymphoma: molecular complete responses are not predictive of progression-free survival. J Clin Oncol. 2002;20:1288–1294. - PubMed

Publication types

Associated data