Jean-Charles Cerottini, Pedro Romero, Daniel E. Speiser
Ludwig Institute for Cancer Research, Lausanne, Switzerland

Melan-A/MART-1: Immunogenicity and Vaccine Strategies

Melan-A/MART-1 is a representative member of the category of differentiation tumor antigens. It is expressed in normal melanocytes and in the majority of primary and metastatic melanomas. The Melan-A/MART-1 gene encodes a type III protein of 118 amino acids that is localized mainly in the endoplasmic reticulum and the transgolgi network¹. Its function remains unknown. An immunodominant HLA-A2 restricted epitope has been mapped to residues 26 – 35, which are located within the putative transmembrane region. We have designed single amino acid substituted peptide analogs with enhanced antigenicity and immunogenicity² as well as doubly substituted non-natural amino acid peptide analogs that are stable to serum proteases³.

Detailed studies of the cytolytic T lymphocyte (CTL) response to the Melan-A/MART-1 antigen with a variety of assays, including the use of fluorescent HLA-A2/peptide analog multimers, have revealed the positive selection of a broad repertoire of Melan-A/MART-1 specific CD8 T cells in the human thymus and the maintenance of a high frequency of these T cells in the periphery of the majority of healthy HLA-A2 individuals, which remain in a naïve state despite the expression of antigen in skin melanocytes, a phenomenon akin to immunological ignorance⁴. In contrast, Melan-A/MART-1 specific T cells undergo in vivo proliferation and differentiation in two thirds of HLA-A2 metastatic melanoma patients. High avidity, antigen-experienced specific CTL migrate and accumulate at the tumor sites. However, we have observed that, while these cells are functionally competent when sampled from the peripheral blood mononuclear lymphocyte pool, those freshly assayed from the tumor sites display reduced IFNg production in response to antigen. In addition, the Melan-A/MART-1 specific T cells residing in metastatic lymph nodes, but not those residing in blood or subcutaneous or visceral metastases, exhibit virtually no perforin message and protein. Of interest, once released from the tumor mass and transferred to in vitro culture in the presence of tumor cells and cytokines, they rapidly recover normal functional activity before undergoing proliferation⁵.

We have carried out phase I clinical trials of vaccination with natural or substituted peptide analogs together with immunological adjuvants in patients with metastatic melanoma. Our strategy, which is to rapidly test various parameters such as dose, route of administration, type of molecular adjuvant, with the aim of selecting strongly immunogenic vaccines, is based on quantitative and qualitative monitoring of Melan-A/MART-1 antigen-specific CD8 T-cell responses. A unique advantage offered by the Melan-A/MART-1 antigenic system is the existence of a baseline frequency of specific T cells that is measurable directly ex vivo with fluorescent multimers in the majority of the patient population. Thus, there is no need to use any in vitro expansion step prior to measuring the response. We standardized laboratory immune monitoring to reach standards for medical diagnostic procedures (high reproducibility, low intra-individual variations, thresholds to distinguish responder patients from non-responders). We analyzed four parameters for antigen specific CD8 T cells in peripheral blood: frequency of IFNg Elispot forming cells, frequency of multimer labeled CD8 T cells and the proportion of these T cells expressing the cell surface CD45RA and CD28 molecules^6,7. The shifts of post-vaccine values relative to pre-vaccine values for each parameter were converted into a combined score that weights the magnitude of the increase of the specific T-cell activity in responder patients. Significant activation of antigen-specific CD8 T cells was detected in PBMC from 12/17 patients vaccinated with peptides emulsified in mineral oil-based adjuvant. T-cell responses correlated significantly with inflammatory skin reactions at vaccine injection sites, demonstrating that immune monitoring data were relevant for in vivo ongoing immunity. Repeated vaccination resulted in enhanced T-cell activity in > 90 % of the responder patients, indicating that vaccine induced activity was sustained. Although all patients were vaccinated with a mixture of Melan-A and influenza peptides, responses to the two antigens occurred independently. Thus, non-responsiveness was antigen-specific rather than patient specific. Interestingly, vaccine responsiveness was significantly more frequent in patients with antigen-specific CD8 T cells that were pre-activated before vaccination. Therefore, endogenous stimulation by tumor derived antigen contributed to T-cell activation observed after immunotherapy. This is in line with T-cell activation being a process requiring several triggering steps, and indicates that vaccination with peptide in conjunction with mineral oil-based adjuvant is capable to promote some, but not all, of these steps.

An essential parameter that remains to be assessed in the context of vaccine-induced Melan-A/MART-1 specific T-cell responses is that of the avidity of responding T cells. Thus far the estimation of TCR avidity has relied on indirect functional assays. The availability of mutant multimers offers the possibility to develop direct assays of TCR avidity. We have developed new multimeric MHC class I/peptide reagents allowing to visualize low, intermediate and high avidity T cells⁸.

In conclusion, ex vivo monitoring of the response elicited by Melan-A/MART-1 peptide-based vaccines provides precise quantitative data on T-cell expansion and qualitative information on T-cell differentiation in vivo. This approach thus allows to us rapidly test new vaccine formulations and to determine whether they can recruit naïve T cells, and/or promote differentiation to fully active effector T cells. Results from such phase I/II studies form the basis to select optimized vaccines for subsequent clinical efficacy testing in large-scale phase III trials.

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