Improving T-cell therapy for relapsed EBV-negative Hodgkin lymphoma by targeting upregulated MAGE-A4

Abstract

Purpose: Patients with Hodgkin lymphoma (HL) relapsing after hematopoietic stem cell transplant have limited options for long-term cure. We have shown that infused cytotoxic T cells (CTL) targeting Epstein Barr virus (EBV)-derived proteins induced complete remissions in EBV(+) HL patients. A limitation of this approach is that up to 70% of relapsed HL tumors are EBV-negative. For these patients, an alternative is to target the cancer/testis antigen MAGE-A4 present in EBV antigen-negative HL tumors. Furthermore, epigenetic modification by clinically available demethylating agents can enhance MAGE-A4 expression in previously MAGE-negative tumors.

Experimental design: We explored the feasibility of combining adoptive T cell therapy with epigenetic modification of tumor antigen expression. We further characterized MAGE-A4-specific T-cell phenotype and function, and examined the effects of the epigenetic modifying drug decitabine on these T cells.

Results: Cytotoxic T cells were generated specifically recognizing MAGE-A4 expressed by autologous HL targets and tumor cell lines. Decitabine-previously shown to increase tumor antigen expression in HL-did not compromise MAGE-A4-specific T-cell phenotype and function. In patients treated with decitabine, expanded MAGE-A4-specific T cells had a broader antitumor T cell repertoire, consistent with increased antigen stimulation in vivo.

Conclusions: Adoptive transfer of MAGE-A4-specific T cells, combined with epigenetic modifying drugs to increase expression of the protein, may improve treatment of relapsed HL.

FIGURE 1. Generation of MAGE-A4 specific T cells from healthy donors

A). Expansion of MAGE specific T cells after 2 stimulations with MAGE pulsed antigen presenting cells. Fold change was calculated from weekly cell counts using the trypan blue exclusion method. B). Phenotype of CD3+ cells after at least 2 stimulations was determined using flow cytometry of surface receptors listed on the x-axis. The median is shown in black bars. C). Shows the specificity of MAGE-specific CTL lines generated from 15 donors as measured by IFNγ release (IFNγELIspot) in response to stimulation with MAGE-A4 pepmix. Results are expressed as SFC/1×10⁵ input cells. Control was IFNγ release in response to stimulation with irrelevant pepmix. The black bars show the median interferon gamma secretion. CTL lines were able to kill MAGE-loaded autologous PHA blasts. D). and HLA A2 matched MAGE positive HL cell line L1236. E). Specific lysis was evaluated by standard Cr⁵¹ release assay - E:T ratio of 40:1, 20:1, 10:1 and 5:1. F). Further epitope mapping of the NPARYEFLWGPRALAETSYV 20mer commonly seen in A2+ donors show that 3 donors each recognize a unique and distinct 9mer epitopes as shown using IFNγ ELISPOT assay. Results are expressed as SFC/1×105 input cells +/− SD.

FIGURE 2. Generation of MAGE-A4 T cells from patients with Hodgkin’s lymphoma

A). Phenotype analysis of cells obtained using flow cytometry are shown and are similar to the phenotype of CTL derived from healthy donors. Black bars show the median percentage for each marker. B). Specificity of the CTL was evaluated by IFNΓ ELIspot assay. CTL responses to MAGE-A4 pepmix versus irrelevant pepmix are shown. Data from nine HL patients is shown. IFNγ positive Spot Forming Cells (SFC)/1×10⁵ input cells are shown for each CTL line. The black bars show the median interferon gamma secretion for each group. C). Cytolytic activity of evaluable T cell lines generated from Hodgkin’s lymphoma patients is evaluated using autologous PHA blasts pulsed with the MAGE-A4 peptide mix as targets in a chromium release assay.

FIGURE 3. MAGE specific T cell phenotype and function are unaffected by Decitabine

A). Phenotype of the MAGE-specific T cells pre and post treatment with 5′ aza-2′ deoxycytidine is shown. B). T cell secretion of interferon-gamma in response to MAGE-A4 peptides is shown pre and post decitabine treatment. Black bars indicate the IFNγ response in CTL lines incubated with decitabine (gray bars) versus without decitabine (black bars).

FIGURE 4. Increased frequency of MAGE-A4 specific T cells in vivo in patients receiving a regimen containing decitabine

T cells obtained from the peripheral blood of HL patients taken before and after treatment with an HDAC inhibitor with or without decitabine were expanded in vitro, and MAGE specific responses were evaluated in an IFNγ ELISpot assay. Results are shown for patients who received decitabine containing regimens (A and B) versus those who did not (C and D).