798. Epstein Barr Virus-Specific Cytotoxic T Lymphocytes (EBV-CTL) Expressing an Anti-CD30 Chimeric T-Cell Receptor (cTcR) for the Treatment of Hodgkin's Disease (HD)

Abstract

HD is a suitable disease for immunotherapy, and in patients with EBV-related HD, adoptive transfer of EBV-CTLs has produced disease responses. For non-EBV-related HD, the CD30 molecule, which is present on the malignant cells of almost all patients with HD, can be targeted using T-lymphocytes genetically modified to express an anti-CD30 cTcR {Cancer Res,1998;58:1116}. Transgenic T cells expressing a cTcR have been used in clinical trials for HIV patients, but they produced only minimal effects mostly because artificial receptors lack co-stimulation signals required for the full activation of resting T-cells. We have shown that EBV-CTL can fulfill this need, since the co-stimulatory signals delivered by EBV-infected B cells after native receptor engagement ensure full functionality when the CTL subsequently bind to tumor cells through their cTcR {Blood,2002;99:2009}. We first evaluated whether EBV-CTL can be redirected to kill CD30+ HD cell lines and whether they retain their specificity and antigen repertoire. EBV-CTLs were prepared from 8 EBV+ healthy donors using weekly stimulation with irradiated autologous EBV-transformed lymphoblastoid cell lines (LCL) in the presence of IL-2 (50U/mL). CTL were transduced after the 3rd stimulation and were further expanded with 3-4 weekly LCL/IL-2 stimulations. The expansion rate of the transduced CTL was similar to that of control EBV-CTL. Transduced CTL retained killing of their autologous LCL targets through their native receptor (64.4|[plusmn]|16.4% at 20:1 E:T ratio), and became able to lyse CD30+ malignant lymphoma targets through their cTcR (e.g. HDLM=45.4|[plusmn]|16.4% and Karpas-299=42.5|[plusmn]|16.4%). Killing of CD30+ tumor cells was significantly inhibited when target cells where preincubated with an anti-CD30 blocking antibody (16.5|[plusmn]|12%). Of potential concern, however, is that CD30 is also expressed by activated normal T-lymphocytes: expression was undetectable on resting T cells, but increased to 3-32% on day 4-7 after stimulation with autologous LCL. Fortunately, expression dwindles to 3-6% by two weeks as an EBV-specific line emerges, suggesting that CD30 is expressed only in the early phases of T-cell activation. As anticipated from these data, therefore, expression of a CD30 cTcR did not impair the antigenic repertoire of the EBV-CTL, which retained the same pattern of immunodominant MHC class I epitopes (detected by tetramer binding) as control cells. We also performed co-culture experiments to evaluate whether infusion of CTL-CD30 cTcR could cross-compromise the primary reactivation of other virus-specific CTL. Autologous EBV-CTLs engineered to express the CD30-cTCR were added to cultures of PBMC stimulated to reactivate cytomegalovirus- or adenovirus-specific CTL. In 4/4 donors, the percentage of CMV pp65+ T cells did not change, while generation of adenovirus-specific T cells (Hexon-tetramer+) was significantly reduced in only 1/3 donor. These data support the feasibility of using EBV-CTL bearing a cTcR for CD30 to treat EBV negative HD.