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Clinical Trial
. 2007 Oct 15;110(8):2838-45.
doi: 10.1182/blood-2007-05-091280. Epub 2007 Jul 3.

Complete responses of relapsed lymphoma following genetic modification of tumor-antigen presenting cells and T-lymphocyte transfer

Catherine M Bollard  1 , Stephen Gottschalk, Ann M Leen, Heidi Weiss, Karin C Straathof, George Carrum, Mariam Khalil, Meng-fen Wu, M Helen Huls, Chung-Che Chang, M Victoria Gresik, Adrian P Gee, Malcolm K Brenner, Cliona M Rooney, Helen E Heslop
Affiliations
Clinical Trial

"V体育ios版" Complete responses of relapsed lymphoma following genetic modification of tumor-antigen presenting cells and T-lymphocyte transfer

Catherine M Bollard et al. Blood. .

Abstract

Epstein-Barr virus (EBV)-associated tumors developing in immunocompetent individuals present a challenge to immunotherapy, since they lack expression of immunodominant viral antigens. However, the tumors consistently express viral proteins including LMP2, which are immunologically "weak" but may nonetheless be targets for immune T cells. We previously showed that a majority of cytotoxic T lymphocytes (CTLs) reactivated using EBV-transformed B-lymphoblastoid cells lines (LCLs) contained minor populations of LMP2-specific T cells and homed to tumor sites. However, they did not produce remissions in patients with bulky disease. We have now used gene transfer into antigen-presenting cells (APCs) to augment the expression and immunogenicity of LMP2. These modified APCs increased the frequency of LMP2-specific CTLs by up to 100-fold compared with unmodified LCL-APCs. The LMP2-specific population expanded and persisted in vivo without adverse effects. Nine of 10 patients treated in remission of high-risk disease remain in remission, and 5 of 6 patients with active relapsed disease had a tumor response, which was complete in 4 and sustained for more than 9 months. It is therefore possible to generate immune responses to weak tumor antigens by ex vivo genetic modification of APCs and the CTLs so produced can have substantial antitumor activity VSports手机版. This study is registered at http://www. cancer. gov/clinicaltrials (protocol IDs: BCM-H-9936, NCT00062868, NCT00070226). .

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Figure 1
Figure 1
LMP2-specific CTL lines derived from lymphoma patients contained functional LMP2-specific T-cell populations. This figure shows the LMP2-specific activity in a CTL line generated from a patient with relapsed lymphoma. The patient is representative of the 10 patients whose CTL lines recognized LMP2. To demonstrate cytolytic specificity of patient CTL lines in vitro, percentage specific 51Cr release was determined 6 hours after coincubation with HLA-matched fibroblasts transduced with Ad5LMP2 (■), or Ad5GFP (□), autologous LCLs (▴), and allogeneic LCLs (▵). The LMP2-specific CTL line from a representative patient shown in panel B showed killing of autologous LCLs and HLA-matched fibroblasts only if they were transduced with Ad5LMP2 (30% at an E/T ratio of 20:1). Killing was not due to adenovirus-directed CTLs, since fibroblasts infected with recombinant adenovirus encoding GFP were not recognized. There was no killing of HLA-mismatched LCLs, (A) or nontransduced fibroblasts (data not shown). Panel B shows the frequency of LMP2-specific T cells in the CTL line generated from the same patient (HLA A2;29/B13;27). CTLs were costained with PE-conjugated multimers CD8-FITC and CD3-PerCP. Multimers were as follows: LMP2-HLA-A*0201-LLW, HLA-A*0201-FLYALALLL, HLA A29-ILL, and EBNA 3C-HLA-B27-RRI. When compared with the EBV-LCL–activated CTL line, the LMP2-activated CTL line showed an increase in the frequency of T cells reactive with all 3 LMP2 tetramers, but decreased frequency in the EBNA3C tetramer reactivity (B). To assess the breadth and function of the LMP2-specific response, the CTLs were coincubated with overlapping LMP2 peptides and IFN-γ release in response to specific 15-mer and 8-mer peptides was measured in an ELISPOT assay. (C) This patient's polyclonal CTL line was then sorted for CD4+ and CD8+ T cells. Subsequently, recognition of LMP2 by these separated CD4+ and CD8+ T cells was determined in an IFN-γ ELISPOT assay using OKT3 blasts pulsed with either LMP2pepmix or CMVpepmix as the APCs (D). Error bars are SD.
Figure 2
Figure 2
The frequency of LMP2-specific T cells increased after infusion of polyclonal LMP2-specific CTLs. LMP2 multimer analysis was also used to compare the frequency of LMP2-specific CTLs before and after infusion. The average number of LMP2 and CMV multimer-reactive cells per 106 CD8+ T cells are shown for 1 patient before and after infusion (A). This patient was representative of the 10 patients who received CTLs where specific LMP2 epitopes had been characterized. In these 10 patients in whom the HLA-restricted LMP2 peptide(s) were available, peripheral blood T cells were incubated with LMP2pepmix, and the number of IFN-γ spot-forming cells per 2 × 105 mononuclear cells was measured (B). In the 6 patients in whom LMP2 peptides were not detected, peripheral blood T cells were incubated with autologous LCLs, and the number of IFN-γ spot-forming cells per 2 × 105 mononuclear cells was measured (C).
Figure 3
Figure 3
Induction of complete clinical response and LMP2-specific T-cell accumulation at tumor site after CTL infusion. A positron emission tomography (PET) scan demonstrating abnormal fluorodeoxyglucose (FDG) uptake in supraclavicular and para-aortic lymph nodes was observed before CTL infusion in a patient with NHL (pt 2). The follow-up scan 8 weeks after CTL infusion is reported as normal (A). Using immunohistochemistry, CD8+ infiltrating T cells were seen in lymph node biopsy after CTL infusion, which corresponded to a clearance of LMP2+ tumor cells (B). In addition, the percentage CD8+/LMP2 tetramer+ T cells in the lymph node and peripheral blood were compared after CTL infusion by flow cytometry (C). Images were acquired with an Olympus BX41 microscope (OlympusAmerica, Center Valley, PA) with a Plan Achromat 10×/0.25 NA oil objective lens (Olympus, Tokyo, Japan). Cells were stained with hematoxylin (Mayer)-eosin and also with CD8 monoclonal antibody (Dako, Carpinteria, CA) and LMP2 antibody (gift of Dr Friedrich A. Grässer, Institut für Mikrobiologie und Hygiene Abteilung Virologie, Homburg/Saar, Germany) and were used in immunoperoxidase protocol. Images were photographed with an Olympus Q-Color 5 digital color camera using FireWire technology and processed with Adobe Photoshop Elements 2.0 imaging software (Adobe Systems, San Jose, CA). Original magnification, ×10.
Figure 4
Figure 4
Induction of complete clinical response and T-cell accumulation at tumor site after CTL infusion. A PET scan demonstrating abnormal FDG uptake in the nasopharyngeal region was observed before CTL infusion in another patient (pt 4) with NK/T-cell lymphoma. The follow-up scan 8 weeks after CTL infusion was reported as normal (A). EBV DNA levels in this patient's (pt 4) PBMCs measured by quantitative real-time PCR before and after CTL infusion were plotted against the EBV(LCL)-specific T-cell response after infusion as measured by IFN-γ secretion in ELISPOT assay (B). Using immunohistochemistry, CD4+ infiltrating T cells were seen in a lymph node biopsy after CTL infusion, and corresponded to a clearance of LMP2-positive tumor cells in patient 4 (C). Images were acquired with an Olympus BX41 microscope, with a Plan Achromat 40×/0.65 NA oil objective lens (Olympus). Cells were stained with hematoxylin (Mayer)-eosin and CD4 monoclonal antibody (Dako) and LMP2 antibody (gift of Dr Friedrich A. Grässer) were used in immunoperoxidase protocol. Images were photographed and processed as in Figure 3B. Original magnification, ×40.
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