Detection of Spontaneous CD4+ T-Cell Responses in Melanoma Patients against a Tyrosinase-Related Protein-2–Derived Epitope Identified in HLA-DRB1*0301 Transgenic Mice

Abstract

Purpose: The frequently expressed differentiation antigen tyrosinase-related protein-2 (TRP-2) has repeatedly been described as a target of spontaneous cytotoxic T-cell responses in melanoma patients, suggesting that it might be an ideal candidate antigen for T cell^based immunotherapy. As a prerequisite for immunization, T-cell epitopes have to be identified.Whereas a number of HLA class I^presented TRP-2^derived epitopes are known, information about HLA class IIp resented antigenic ligands recognized by CD4 + T helper (Th) cells is limited. Experimental Design: The search forTRP-2^derived Th epitopes was carried out by competitive in vitro peptide binding studies with predicted HLA-DRB1*0301ligands in combination with peptide and protein immunizations of HLA-DRB1*0301 transgenic mice. In vivo selected candidate epitopes were subsequently verified for their immunogenicity in humanT-cell cultures.Results: This strategy led to the characterization of TRP-2 60-74 as an HLA-DRB1*0301^restricted Th epitope. Importantly, TRP-2 60-74^r eactive human CD4 + Th cell lines, specifically recognizing target cells loaded with recombinant TRP-2 protein, could be established by repeated peptide stimulation of peripheral blood lymphocytes from several HLA-DRB1*03 + melanoma patients. Even short-term peptide stimulation of patients' peripheral blood lymphocytes showed the presence of TRP-2 60-74^r eactive T cells, suggesting that these T cells were already activated in vivo. Conclusion: Peptide TRP-2 60-74 might be a useful tool for the improvement of immunotherapy and immune monitoring of melanoma patients.

Phage idiotype vaccination: first phase I/II clinical trial in patients with multiple myeloma

“…We demonstrate a fast method for generating tumor-specific vaccinations with a maximal production success rate. Endotoxins were efficiently removed from phage preparations by an optimized purification protocol [7,9,10] and the resulting phage vaccine compositions could be readily employed in the vaccination studies. Previous methods based on generating a hybridoma cell line or PCR amplification of the Id from small numbers of tumor cells are costly and have failure rates as high as 15% with long production times of 3 to 13 months to vaccine completion [11,12].…”

“…Contaminating endotoxins were removed by two-phase Triton X-114 separation as described elsewhere [8,9], resulting in an endotoxin concentration of < 1 unit/ml [7]. Chemically linked Id-phage was generated as described [7] by coupling purified paraproteins to bacteriophages (50 mg/ml in PBS) with 0.1% (v/v) glutaraldehyde/water and an Id protein/phage ratio of 1:10 (w/w).…”

Identification of T-cell epitopes for cancer immunotherapy

“…The degeneracy of HLA class II binding motifs allows the search and prediction of promiscuous pan-class II binding peptides, which are obviously more widely applicable for vaccine development. HLA class II peptide binding predictions have successfully led to the identification of promiscuous T-helper epitopes in among others: NY-ESO-1, 174 TRP-2, 175 hTERT, 176 and Melan-A/MART-1. 177 Identification of HLA class I and class II ligands by MS/MS As discussed above, starting from pre-existing CTL with unknown or known specificity, the combination of microscale liquid chromatography coupled to MS/MS with functional immunoassays has been used for the identification or confirmation of tumor-specific CTL epitopes, respectively.…”

Screening of Human Tumor Antigens for CD4+ T Cell Epitopes by Combination of HLA-Transgenic Mice, Recombinant Adenovirus and Antigen Peptide Libraries

“…Thus, after extensive T cell culture only a subgroup of predicted peptides can be verified as natural epitopes. Though different CD4 + T cell epitopes from tumor antigens such as CEA [22], p53 [23] or TRP-2 [24] have been defined by in silico prediction, we set out to overcome the drawbacks of this strategy by setting up a comprehensive screening approach based on the immunization of HLA-transgenic mice with recombinant adenovirus encoding human melanoma antigens and subsequent scanning of the T cell responses in vitro with the help of combinatorial antigen-specific peptide libraries. This approach allowed us to directly concentrate our efforts on naturally processed tumor antigen-specific epitopes presented by a given MHC class II restriction element.…”