Vivian B. Dissette

A Phase I/II Trial Testing Immunization of Hepatocellular Carcinoma Patients with Dendritic Cells Pulsed with Four α-Fetoprotein Peptides

α-Fetoprotein (AFP) is a self protein expressed by fetal liver at high levels, but is transcriptionally repressed at birth. AFP is up-regulated in hepatocellular carcinomas, and patients with active disease could have plasma levels as high as 1 mg/mL. We previously identified four immunodominant HLA-A*0201-restricted peptides [hAFP137-145 (PLFQVPEPV), hAFP158-166 (FMNKFIYEI), hAFP325-334 (GLSPNLNRFL), and hAFP542-550 (GVALQTMKQ)] derived from human AFP that could stimulate specific T cell responses in healthy donor peripheral blood lymphocytes in vitro. We conducted a phase I/II clinical trial in which HLA-A*0201 patients with AFP-positive hepatocellular carcinoma were immunized with three biweekly intradermal vaccinations of the four AFP peptides pulsed onto autologous dendritic cells (DC). DCs were prepared from adherent peripheral blood mononuclear cells cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 7 days. Sixteen subjects were enrolled and 10 were treated. Peripheral blood lymphocytes were isolated from these patients before, during, and after AFP peptide/DC immunization and were tested ex vivo with MHC tetramer and IFNγ ELISPOT analysis. Six of 10 subjects expanded statistically significant levels of AFP-specific T cells postvaccine to at least one peptide by MHC tetramer. Also, 6 of 10 subjects increased IFNγ producing AFP-specific T cell responses to at least one of the peptides postvaccination, by ELISPOT. We conclude that the human T cell repertoire is capable of responding to the AFP self antigen after the administration of AFP peptide-pulsed DC even in an environment of high circulating levels of this oncofetal antigen.

A herpes simplex virus transcript abundant in latently infected neurons is dispensable for for establishment of the latent state

We have previously reported that a novel herpes simplex virus RNA transcript partially overlapping the gene encoding ICPO and expressed from the opposite DNA strand is abundant in sensory neurons of mice harboring a latent infection [J.G. Stevens, E.K. Wagner, G.B. Devi-Rao, M.L. Cook, and L.T. Feldman, Science 235, 1056-1059 (1987)]. This finding suggested that this transcript might be involved in establishment, maintenance, or reactivation of latent virus. To determine the function of this latency-associated transcript (LAT), we have examined the latency characteristics of a deletion mutant which is unable to express the LAT gene. Although no viral transcripts could be found in the lumbosacral ganglia of mice surviving rear footpad infection with this deletion virus, a latent infection had been established since infectious virus could be induced and detected after explanation and cocultivation of ganglia with permissive cells in culture. These results indicate that HSV-1 LAT expression is not an absolute requirement for establishment of the latent state.

T Cell Responses to HLA-A*0201-Restricted Peptides Derived from Human α Fetoprotein

α fetoprotein (AFP)-derived peptide epitopes can be recognized by human T cells in the context of MHC class I. We determined the identity of AFP-derived peptides, presented in the context of HLA-A*0201, that could be recognized by the human (h) T cell repertoire. We screened 74 peptides and identified 3 new AFP epitopes, hAFP137–145, hAFP158–166, and hAFP325–334, in addition to the previously reported hAFP542–550. Each possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent class I binding assay. The peptides were stable for 2–4 h in an off-kinetics assay. Each peptide induced peptide-specific T cells in vitro from several normal HLA-A*0201 donors. Importantly, these hAFP peptide-specific T cells also were capable of recognizing HLA-A*0201+/AFP+ tumor cells in both cytotoxicity assays and IFN-γ enzyme-linked immunospot assays. The immunogenicity of each peptide was tested in vivo with HLA-A*0201/Kb-transgenic mice. After immunization with each peptide emulsified in CFA, draining lymph node cells produced IFN-γ on recognition of cells stably transfected with hAFP. Furthermore, AFP peptide-specific T cells could be identified in the spleens of mice immunized with dendritic cells transduced with an AFP-expressing adenovirus (AdVhAFP). Three of four AFP peptides could be identified by mass spectrometric analysis of surface peptides from an HLA-A*0201 human hepatocellular carcinoma (HCC) cell line. Thus, compelling immunological and physiochemical evidence is presented that at least four hAFP-derived epitopes are naturally processed and presented in the context of class I, are immunogenic, and represent potential targets for hepatocellular carcinoma immunotherapy.

Role of dendritic cell phenotype, determinant spreading, and negative costimulatory blockade in dendritic cell-based melanoma immunotherapy.

MART-127–35-peptide-pulsed immature dendritic cells (DCs) resulted in immunologic and clinical activity in a prior phase 1 trial. A phase 2 cohort expansion was initiated to further characterize the phenotype and cytokine milieu of the DC vaccines and their immunologic activity in vitro and to further examine a possible link between clinical activity and determinant spreading. In an open-label phase 2 trial, 107 autologous ex vivo generated DCs pulsed with the HLA-A*0201 immunodominant peptide MART-127–35 were administered to 10 subjects with stage II–IV melanoma. The experimental vaccines were administered intradermally in a biweekly schedule for a total of three injections, and blood for immunologic assays was obtained before each administration and at three time points after. DC vaccine preparations had wide intra- and interpatient variability in terms of cell surface markers and preferential cytokine milieu, but they did not correlate with the levels of antigen-specific T cells after vaccination. Of four patients with measurable disease, one had stable disease for 6 months and another has a continued complete response for over 2 years, which is confounded by receiving a closely sequenced CTLA4 blocking antibody. The DC vaccines induced determinant spreading in this subject, and CTLA4 blockade reactivated T cells with prior antigen exposure. The DC phenotype and cytokine profile do not correlate with the ability to induce antigen-specific T cells, while determinant spreading after DC immunization may be a marker of an efficient antitumor response. Sequential CTLA4 blockade may enhance the immune activity of DC-based immunotherapy.

Adenovirus MART-1-engineered autologous dendritic cell vaccine for metastatic melanoma.

We performed a phase 1/2 trial testing the safety, toxicity, and immune response of a vaccine consisting of autologous dendritic cells (DCs) transduced with a replication-defective adenovirus (AdV) encoding the full-length melanoma antigen MART-1/Melan-A (MART-1). This vaccine was designed to activate MART-1–specific CD8+ and CD4+ T cells. Metastatic melanoma patients received 3 injections of 106 or 107 DCs, delivered intradermally. Cell surface phenotype and cytokine production of the DCs used for the vaccines were tested, and indicated intermediate maturity. CD8+ T-cell responses to MART-127-35 were assessed by both major histocompatibility complex class I tetramer and interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISPOT) before, during, and after each vaccine and CD4+ T-cell responses to MART-151-73 were followed by IFN-γ ELISPOT. We also measured antigen response breadth. Determinant spreading from the immunizing antigen MART-1 to other melanoma antigens [gp100, tyrosinase, human melanoma antigen-A3 (MAGE-A3)] was assessed by IFN-γ ELISPOT. Twenty-three patients were enrolled and 14 patients received all 3 scheduled DC vaccines. Significant CD8+ and/or CD4+ MART-1–specific T-cell responses were observed in 6/11 and 2/4 patients evaluated, respectively, indicating that the E1-deleted adenovirus encoding the cDNA for MART-1/Melan-A (AdVMART1)/DC vaccine activated both helper and killer T cells in vivo. Responses in CD8+ and CD4+ T cells to additional antigens were noted in 2 patients. The AdVMART1-transduced DC vaccine was safe and immunogenic in patients with metastatic melanoma.

Intratumoral Immune Cell Infiltrates, FoxP3, and Indoleamine 2,3-Dioxygenase in Patients with Melanoma Undergoing CTLA4 Blockade

Purpose: CTL-associated antigen 4 (CTLA4)-blocking monoclonal antibodies induce long-term regression of metastatic melanoma in some patients, but the exact mechanism is unknown. In this study, biopsies of selected accessible tumor lesions from patients treated with tremelimumab were examined to further elucidate the mechanism of its antitumor activity. Experimental Design: Fifteen tumor biopsies from 7 patients who had been treated with tremelimumab (CP-675,206) were collected. Samples were analyzed for melanoma markers, immune cell subset markers, the presence of the T regulatory-specific transcription factor FoxP3 and the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO). Results: Clinically responding lesions had diffuse intratumoral infiltrates of CD8+ T cells that were markedly increased in cases where comparison with a baseline biopsy was available. Nonregressing lesions had sparse, patchy CD8+ intratumoral infiltrates. Patients with regressing lesions had an increased frequency of CD8+ cells with or without a concomitant increase in CD4+ cells. Two of 3 responding patients with paired samples showed a slight increase in the number of FoxP3+ cells in the postdosing biopsies. In patients with regressing lesions who had paired samples, the intensity of IDO staining in macrophages and/or melanoma cells showed no clear pattern of change postdosing. Conclusions: Administration of tremelimumab was associated with massive intratumoral infiltrates of CD8+ CTLs in patients with regressing tumors but had varying effects on intratumoral infiltrates of CD4+ and FoxP3+ cells or intratumoral expression of IDO.

Generation of T-cell immunity to a murine melanoma using MART-1-engineered dendritic cells

The murine melanoma B16 expresses the murine counterpart of the human MART-1/Melan-A (MART-1) antigen, sharing a 68.6% amino acid sequence identity. In this study, mice were vaccinated with bone marrow-derived murine dendritic cells genetically modified with a replication-incompetent adenoviral vector to express the human MART-1 gene (AdVMART1). This treatment generated a protective response to a lethal tumor challenge of unmodified murine B16 melanoma cells. The response was mediated by major histocompatibility complex class I-restricted cytotoxic T lymphocytes specific for MART-1 antigen, which produced high levels of interferon-gamma when reexposed to MART-1 in vitro and lysed targets in a calcium-dependent mechanism suggestive of perforin/granzyme B lysis. MART-1 was presented by the dendritic cells used for vaccination and not by epitopes cross-presented by host antigen-presenting cells. In conclusion, dendritic cells genetically modified to express the human MART-1 antigen generate potent murine MART-1-specific protective responses to B16 melanoma.

Immunosensitization of Tumor Cells to Dendritic Cell-Activated Immune Responses with the Proteasome Inhibitor Bortezomib (PS-341, Velcade)

Proteasome inhibition results in proapoptotic changes in cancer cells, which may make them more sensitive to immune effector cells. We established a murine model to test whether the proteasome inhibitor bortezomib could sensitize established B16 melanoma tumors to dendritic cell (DC)-activated immune effector cells. Day 3-established s.c. B16 tumors had significantly decreased tumor outgrowth when treated with a combination of bortezomib and DC, regardless of whether the DC were loaded or not with a tumor Ag. In vivo Ab-depletion studies demonstrated that the effector cells were NK and CD8+ cells, but not CD4+ cells. NF-κB nuclear transcription factor assay and gene-expression profiling of B16 treated with bortezomib was consistent with inhibition of NF-κB target genes leading to a proapoptotic phenotype. In vitro lytic assays demonstrated that TNF-α, but not perforin, Fas-ligand, or TRAIL, was responsible for bortezomib-sensitized B16 cytotoxicity. In conclusion, the proteasome inhibitor bortezomib can pharmacologically sensitize tumor cells to the lytic effects of DC-activated immune effector cells.

Human dendritic cell maturation by adenovirus transduction enhances tumor antigen-specific T-cell responses.

Abstract: Dendritic cells (DCs) have been shown to require a degree of maturation to stimulate antigen-specific, type 1 cytotoxic T lymphocytes in numerous murine models. Limited data in humans suggest that immature DCs (DC) can induce tolerance, yet a variety of nonmatured DC used clinically have induced antigen-specific type 1 T cells in vivo to various tumor-associated antigens. Use of adenovirus to engineer DCs is an efficient method for delivery of entire genes to DC, but the data on the biologic effects of viral transduction are contradictory. The authors demonstrate that DCs transduced with adenovirus (AdV) clearly become more mature by the phenotypic criterion of upregulation of CD83 and downregulation of CD14. Transduced DCs also decrease production of IL-10, and a subset of transduced DCs produce increased levels of IL-12 p70. This level of maturation is superior to that achieved by treatment of these cells with tumor necrosis factor-&agr; or interferon-&agr; but less pronounced than with CD40L trimer or CD40L + interferon-γ. Maturation by AdV transduction alone leads to efficient stimulation of antigen-specific T cells from both healthy donors and patients with advanced cancer using two defined human tumor-associated antigens, MART-1 and AFP. Given the pivotal role of DCs in immune activation, it is important to understand the direct biologic effects of AdV on DCs, as well as the impact these biologic changes have on the stimulation of antigen-specific T cells. This study has important implications for the design of DC-based clinical trials.

Long-term expression of a reporter gene from latent herpes simplex virus in the rat hippocampus

A problem in utilizing herpes simplex virus (HSV) as a vector for expression of foreign genes in CNS neurons has been the inability to facilitate long-term expression of the engineered genes. Previously, we showed that the murine moloney leukemia virus LTR would drive beta-galactosidase (beta-gal) transcription for extended periods from the latent viral genome in sensory, but not motor neurons. In this communication we further evaluate the utility of the LTR promoter for use in long-term expression vectors. Following stereotactic injection of 8117/43 (an ICP4 minus, non-replicating virus with the LTR driving the beta-gal gene, or KD6 (an ICP4 minus non-replicating virus not expressing beta-gal) into the hippocampus of rats, polymerase chain reaction (PCR) analysis of viral DNA after 2 months indicated that latent infections were established. Assaying by both x-gal staining and reverse transcriptase PCR we demonstrate that (1) beta-gal can be detected for at least 6 months in hippocampal neurons, and (2) although the number of beta-gal transcripts in these cells drops considerably by 2 weeks, they can be detected during the period studied. These studies indicate that the LTR promoter is active and affords long-term expression in the CNS, albeit at comparatively low levels compared to those observed at acute times.