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.

a-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 as1mg/mL.We previously identified four immunodominant HLA-A*0201-restricted peptides [hAFP [137][138][139][140][141][142][143][144][145] (PLFQVPEPV), hAFP [158][159][160][161][162][163][164][165][166] (FMNKFIYEI), hAFP [325][326][327][328][329][330][331][332][333][334] (GLSPNLNRFL), and hAFP 542-550 (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*0201patients 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 afterAFP peptide/DC immunization and were tested ex vivo with MHC tetramer and IFNg ELISPOTanalysis. Six of 10 subjects expanded statistically significant levels of AFP-specificTcells postvaccine to at least one peptide by MHC tetramer. Also, 6 of10 subjects increased IFNg producing AFP-specificTcell responses to at least one of the peptides postvaccination, by ELISPOT. We conclude that the humanTcell repertoire is capable of responding to the AFP self antigen after the administration of AFP peptidepulsed DC even in an environment of high circulating levels of this oncofetal antigen.We originally reported that the self antigen a-fetoprotein (AFP) could be recognized by both murine and human T cells and serve as a tumor rejection antigen in a murine tumor model (1, 2). AFP is produced by 50% to 80% of hepatocellular carcinomas (HCC), and its measurement in serum has played an important role in diagnosis and monitoring responses to treatment for the last several decades (3). AFP is expressed by the fetal liver with serum levels of 1 mg/mL, but is transcriptionally repressed shortly after birth (4 -6). Our ability to generate potent AFP-specific T cell immunity to murine AFP in mice and to human AFP in in vitro human T cell cultures clearly indicates that, despite being exposed to high plasma levels of this protein during embryonic development, some AFP-specific T cells are not deleted during ontogeny.Using a combination of strategies (HLA-A*0201/K b transgenic mice, human T cell cultures, and mass spectrometric analysis), we identified four immunodominant AFP-derived peptides that are naturally processed and presented in the context of HLA-A*0201 (1, 7 -9). At least three of these peptides could be isolated from the surface of an HLA-A*0201/AFP-positive human HCC cell line, HepG2. These peptides can stimulate T ...

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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 + Tcells 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