Abstract

Identification and targeting of antigens involved in malignant transformation, survival advantage of tumor cells or their metastatic spread might represent a promising approach in the development of immunotherapeutic strategies to treat malignant diseases. Using oligonucleotide microarray analysis we found that matrix metalloproteinase- (MMP-)7 is extensively upregulated in a broad variety of malignant tissues. MMPs are a family of endopeptidases that degrade extracellular matrix (ECM). MMP-7 can promote cancer invasion by proteolytic cleavage of ECM and basement membrane proteins. In addition, MMP-7 has been shown to accelerate the proliferation of umbilical vein endothelial cells in vitro and promote angiogenesis. Thus, targeting MMP-7 may affect both tumor cells and tumor vessels. In order to analyze the feasibility to induce MMP-7-specific CTLs, monocyte derived dendritic cells (DCs) were generated from a healthy donor, electroporated with full length MMP-7-in-vitro-transcript (IVT) that coded for the entire MMP-7-protein and used as antigen presenting cells (APCs) for the in vitro induction of antigen-specific CTLs. These in vitro generated CTLs demonstrated an antigen-specific and HLA class I-restricted cytolytic activity against tumor cells endogenously expressing MMP-7. The specificity and HLA-restriction was confirmed in cold target inhibition assays and by using blocking antibodies. In the next set of experiments, we wanted identify HLA class I-presented MMP-7-peptides that are recognized by our CTL lines. To accomplish this, peptides bound to HLA class I molecules were isolated from surgically removed RCC specimens that overexpressed MMP-7. By applying mass spectrometry-based peptide sequencing of the HLA ligands, a HLA-A3-binding peptide derived from the novel antigen MMP-7 was identified. To analyze the immunogenicity of the new MMP-7 derived peptide with respect to its ability to elicit primary T cell responses, we induced MMP-7-specific CTLs in vitro. DCs derived from a HLA-A3+ healthy donor were pulsed with the HLA-A3-binding antigenic peptide and utilized as APCs. The induced CTL lines demonstrated antigen-specific killing: T cells only recognized autologous DCs loaded with the cognate MMP-7-A3 peptide or HLA matched tumor cells expressing MMP-7, whereas they did not lyse target cells pulsed with an irrelevant HLA-A3-binding peptide. The objective of our study is the development of a DC-based adoptive immunotherapy for patients with malignant diseases. Therefore, we sought to analyze, whether our previous findings could be applied in patients with malignant diseases in an autologous setting. To achieve this, we generated DCs from an HLA-A3+ ALL patient in complete remission after chemotherapy and used them as APCs for CTL induction after electroporation with MMP-7-IVT. The autologous ALL blasts, frozen at the time of diagnosis, were used as targets in Cr-release assays. These cells were efficiently lysed by the CTLs specific for MMP-7, confirming the expression analysis by RT-PCR. Importantly, the normal autologous purified DCs, CD3+ T and CD19+ B cells were not recognized. In conclusion, our study describes the identification of a novel broadly expressed T-cell epitope derived from the MMP-7-protein that represents an interesting candidate to be applied in immunotherapies of human malignancies capable of targeting neo-vascularization and malignant cells in developing tumors.

Author notes

Disclosure: No relevant conflicts of interest to declare.