Sustained complete responses in patients with lymphoma receiving autologous cytotoxic T lymphocytes targeting Epstein-Barr virus latent membrane proteins

Abstract

Purpose: Tumor cells from approximately 40% of patients with Hodgkin or non-Hodgkin lymphoma express the type II latency Epstein-Barr virus (EBV) antigens latent membrane protein 1 (LMP1) and LMP2, which represent attractive targets for immunotherapy. Because T cells specific for these antigens are present with low frequency and may be rendered anergic by the tumors that express them, we expanded LMP-cytotoxic T lymphocytes (CTLs) from patients with lymphoma using autologous dendritic cells and EBV-transformed B-lymphoblastoid cell lines transduced with an adenoviral vector expressing either LMP2 alone (n = 17) or both LMP2 and ΔLMP1 (n = 33).

Patients and methods: These genetically modified antigen-presenting cells expanded CTLs that were enriched for specificity against type II latency LMP antigens. When infused into 50 patients with EBV-associated lymphoma, the expanded CTLs did not produce infusional toxicities.

Results: Twenty-eight of 29 high-risk or multiple-relapse patients receiving LMP-CTLs as adjuvant therapy remained in remission at a median of 3.1 years after CTL infusion. None subsequently died as a result of lymphoma, but nine succumbed to complications associated with extensive prior chemoradiotherapy, including myocardial infarction and secondary malignancies. Of 21 patients with relapsed or resistant disease at the time of CTL infusion, 13 had clinical responses, including 11 complete responses. T cells specific for LMP as well as nonviral tumor-associated antigens (epitope spreading) could be detected in the peripheral blood within 2 months after CTL infusion, but this evidence for epitope spreading was seen only in patients achieving clinical responses.

Conclusion: Autologous T cells directed to the LMP2 or LMP1 and LMP2 antigens can induce durable complete responses without significant toxicity. Their earlier use in the disease course may reduce delayed treatment-related mortality.

Trial registration: ClinicalTrials.gov NCT00671164.

Fig 1.

Characteristics of latent membrane protein (LMP) –specific cytotoxic T lymphocyte (CTL) lines derived from patients with Epstein-Barr virus (EBV) –positive lymphoma. Bars indicate median values. (A) Phenotype of LMP-specific CTL lines at time of freezing, showing predominance of CD3+ and CD8+ T cells. Recognition of LMP1 and LMP2 in interferon gamma (IFN-γ) enzyme-linked immunospot (ELISPOT) assay by CTLs, generated with APCs transduced with (B) Ad5f35LMP2 or (C) Ad5f35ΔLMP1-1-LMP2 vector. Spot counts in response to target antigen stimulation by IFN-γ ELISPOT assay of LMP-CTL lines are shown as solid circles. (D) LMP2-specific CTL lines demonstrate cytotoxicity against autologous lymphoblastoid cell lines (LCLs) and LMP2 pepmix–pulsed phytohemagglutinin (PHA) blasts but not against unpulsed or LMP1-pulsed PHA blasts, at effector cell to target cell (E:T) ratio of 20:1. (E) LMP1/2-specific CTL lines demonstrate similar cytotoxicity against autologous LCL and LMP2 pepmix–pulsed PHA blasts but less against LMP1-pulsed PHA blasts. Unpulsed PHA blasts were not killed. (F) LMP1- and LMP2-specific activity in CTL line generated from patient with relapsed Hodgkin lymphoma. Results represent patients whose CTLs recognized both LMP1 and LMP2. LMP-specific CTL line from this patient showed killing of autologous (auto) LCL and PHA blasts only if pulsed with LMP1 or LMP2 pepmix. There was no killing of PHA blasts alone. Allo, allogeneic; SFC, spot-forming cell.

Fig 2.

Outcomes in 29 patients in first or later remission who received latent membrane protein (LMP) –cytotoxic T lymphocyte (CTL) as adjuvant therapy. (A) Remissions were sustained in all but one patient. (B) Two-year event-free survival for 29 patients treated with either LMP2- (n = 9) or LMP1/2-specific CTLs (n = 20; P = .22). (C) Cumulative incidence of death resulting from lymphoma or nonlymphoma causes (eg, cardiac disease, secondary malignancy, infection).

Fig 3.

Outcomes in patients who received latent membrane protein (LMP) –cytotoxic T lymphocytes (CTLs) as treatment for relapsed or refractory disease. (A) Distribution of clinical responses among 21 patients. (B) Increased proportion of responding versus nonresponding patients had CTLs with specific activity against LMP1 by interferon gamma enzyme-linked immunospot assay. (C) This relationship was not apparent when analysis was based on CTLs with LMP2-specific activity. (D) Two-year event-free survival for patients treated in LMP2-CTL protocol (n = 8) versus LMP1/2-CTL protocol (n = 13; P = .626). (E) Cumulative risk of death resulting from lymphoma or other causes. CR, complete response; NR, no response; PR, partial response; SFC, spot-forming cell.

Fig 4.

Frequency of latent membrane protein (LMP) –specific and tumor antigen–specific T cells in responding versus nonresponding patients. Immune reconstitution was evaluated in individual patients who received LMP–cytotoxic T lymphocytes. (A-D) Peripheral blood T cells were incubated with LMP1 or LMP2 pepmixes. Number of interferon gamma (IFN-γ) spot-forming cells (SFCs) per 2 × 10⁵ mononuclear cells was measured in enzyme-linked immunospot (ELISPOT) assays. Gold lines represent individual LMP-specific T cells; blue lines represent mean of LMP-specific T cells over time. Note greater frequencies of reactivity for responding patients. (A) LMP1 responders. (B) LMP2 responders. (C) LMP1 nonresponders. (D) LMP2 nonresponders. (E) Evidence for epitope spreading in 12 patients with non-Hodgkin or Hodgkin lymphoma. Peripheral blood T cells were incubated with lymphoma antigen pepmixes (MAGE A4, survivin, PRAME); number of IFN-γ SFCs per 2 × 10⁵ mononuclear cells was measured in ELISPOT assays.

Fig 4.

Frequency of latent membrane protein (LMP) –specific and tumor antigen–specific T cells in responding versus nonresponding patients. Immune reconstitution was evaluated in individual patients who received LMP–cytotoxic T lymphocytes. (A-D) Peripheral blood T cells were incubated with LMP1 or LMP2 pepmixes. Number of interferon gamma (IFN-γ) spot-forming cells (SFCs) per 2 × 10⁵ mononuclear cells was measured in enzyme-linked immunospot (ELISPOT) assays. Gold lines represent individual LMP-specific T cells; blue lines represent mean of LMP-specific T cells over time. Note greater frequencies of reactivity for responding patients. (A) LMP1 responders. (B) LMP2 responders. (C) LMP1 nonresponders. (D) LMP2 nonresponders. (E) Evidence for epitope spreading in 12 patients with non-Hodgkin or Hodgkin lymphoma. Peripheral blood T cells were incubated with lymphoma antigen pepmixes (MAGE A4, survivin, PRAME); number of IFN-γ SFCs per 2 × 10⁵ mononuclear cells was measured in ELISPOT assays.