Numerous studies in animal models have documented that ex vivo antigen-pulsed DCs are effective inducers of pathogen-specific immunity (59,63,66,67). However, the utility of ex vivo antigen-pulsed DCs for the prophylaxis or therapy of infection has not yet been extensively studied in humans. In the first reported DC clinical trial in HIV-infected patients, the safety and antigen-presenting properties of allogeneic or autologous DCs were investigated in seven HLA-A2 + , HIV-infected patients (46). Allogeneic DCs, obtained from the peripheral blood of HLA-identical, HIV-seronegative siblings using the density gradient procedure described earlier, were pulsed with recombinant HIV-1 MN gp160 or synthetic peptides corresponding to HLA-A*0201-restricted cytotoxic epitopes of envelope, Gag and Pol proteins. The antigen-pulsed cells were infused intravenously six to nine times at monthly intervals, and HIV-spe-cific immune responses were monitored.
One allogeneic DC recipient with a CD4+ T-cell count of 460/mm3 showed increases in envelope-specific CTLs and lymphocyte proliferative responses, as well as IFN-7 and IL-2 production. Two other allogeneic DC recipients with CD4+ T-cell counts of 434 and 560/mm3, respectively, also showed an increase in HIV envelope-specific lymphocyte proliferative responses. A recipient of autologous DC with a CD4+ T-cell count of 723/mm3 showed an increase in peptide-specific lymphocyte-proliferative responses after three infusions. There was a good correlation between the presence of specific virus sequences obtained by bulk plasma viral RNA sequencing and peptide-specific endogenous CTL responses measured by both direct and indirect CTL assays. Thus, these responses appeared to be recall responses. Three other allogeneic DC recipients with CD4+ T-cell counts <410/mm3 did not show increases in their HIV-specific immune responses.
No clinically significant adverse effects were noted in this study and CD4+ T-cell numbers and plasma HIV-1 RNA detected by reverse transcriptase polymerase chain reaction of all seven patients were stable during the study period. Thus, both allogeneic and autologous DC infusions were well tolerated, and in patients with normal or near normal CD4+ T-cell counts, administration of these antigen-pulsed cells enhanced the immune response to HIV. Future studies of HIV antigen-pulsed DC infusion in HIV-infected patients will be required to determine whether this approach is clinically beneficial.
In contrast to infectious disease, a number of groups are pursuing DC-based immunotherapy trials for cancer. In the first reported DC trial, our group assessed the effect of autologous DCs pulsed ex vivo with tumor-specific antigen in patients with malignant B-cell lymphoma who had failed conventional chemotherapy. Like other B-lymphocytes, the neoplastic cells in these patients express surface immunoglobulin receptors, and because B-cell lymphomas are monoclonal, all the cells of a given tumor express identical surface immunoglobulin. Moreover, this immunoglobulin is potentially immunogenic by virtue of its unique idiotypic determinants, which are formed by the combination of the variable regions of immunoglobulin heavy and light chains (127-129). To prepare idiotype proteins for this clinical study, patients underwent tumor biopsies, and the immunoglobulin (idiotype) produced by each tumor was "rescued" by somatic cell fusion techniques and purified from hybridoma supernatants (130). This protein, together with keyhole limpet hemocyanin, which served as a control antigen, was used to pulse autologous DCs obtained from the patients by leuka-pheresis, and the antigen-pulsed cells were administered to the patients by intravenous infusion. This procedure was repeated three times at monthly intervals with a booster immunization given 4-6 months later. Throughout the trial the patients were followed for the development of an immune response to the idiotype, and their tumor burden was monitored.
A report of the results obtained in our initial four patients has been published (108). All of these treated patients, as well as six not described in our published report, tolerated their infusions well, and none experienced clinically significant toxicity at any point during the study. In addition, most of the patients developed T-cell-mediated antiidiotype responses that were not observed prior to treatment initiation. The antiidiotype responses were specific for autologous tumor immunoglobulin compared with irrelevant, isotype-matched immunoglobulins. In addition to these proliferative responses, T-cells from one patient were expanded for several weeks in vitro in the presence of idiotype protein and shown to lyse autologous tumor hybridoma cells but not an isotype-matched, unrelated hybridoma. Most importantly, two of the patients experienced complete tumor regression, including one who entered the trial with bulky disease and remained in complete remission for more than 3 years. A third patient experienced a partial response, whereas three have had stable disease and three have experienced disease progression. Recently, a new cohort of patients has been vaccinated while in remission, and their follow-up is ongoing.
Two vaccine trials for melanoma have also been reported in which the immuno-genicity of DCs pulsed with a panel of melanoma-derived HLA-restricted peptides was investigated. Both trials utilized DC derived from monocytes by culture in GM-CSF and IL-4. Nestle et al. limited their clinical trial to patients expressing the HLA*A1 or A2 alleles and reported that 5 of 15 patients developed clinical responses including 2 who developed complete remissions (115). Induction of delayed-type hypersensitivity (as measured by skin testing) to the antigen was seen with this vaccination approach. Lotze et al. also reported the results of their clinical trial in melanoma with one complete response in their cohort of six patients (116).
Recently, we treated a cohort of advanced colorectal cancer patients with recombinant Flt3 ligand, a hematopoietic growth factor, and observed a 20-fold increase in circulating DC (119). Subsequently, these cells were isolated and loaded with a synthetic peptide derived from carcinoembryonic antigen (CEA) and mutated at a single amino acid position to make it a more potent T cell antigen. Following vaccination with these cells, more than half of the patients developed CD8 cytotoxic T cells that recognized tumor cells expressing endogenous CEA. Moreover, two of 12 patients experienced dramatic tumor regression and several other patients had stable disease. Finally, clinical response correlated with the expansion of CEA specific CD8 T cells, confirming the role of such cells in this treatment strategy. Based on these encouraging results, a number of investigators are now pursuing DC-based clinical trials in patients with a variety of malignancies.
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