Identification of the Angiogenic Endothelial Cell Growth Factor-1/Thymidine Phosphorylase as a Target for Immunotherapy of Cancer.

Characterization of the antigens recognized by tumor-reactive T cells isolated from patients successfully treated with allogeneic HLA-matched stem cell transplantation (SCT) can lead to the identification of clinically relevant target molecules. We isolated cytotoxic CD8⁺ T cell (CTL) clones from a patient successfully treated with donor lymphocyte infusion for relapsed multiple myeloma (MM) after allogeneic HLA-matched SCT who suffered only from mild graft-versus-host disease (GVHD). The CTL clones were isolated by direct cloning of T cells producing IFN-γ upon stimulation with irradiated bone marrow cells harvested from the patient before SCT. The tumor-reactivity of the CTL clones was demonstrated by the recognition of MM cells in the bone marrow using the CFSE-based cytotoxicity assay. In addition to tumor cells, the CTL clones also lysed the patient-derived EBV-transformed lymphoblastoid cell line (EBV-LCL) and PHA-blasts, whereas donor-derived EBV-LCL cells and PHA blasts were not recognized demonstrating that these CTL clones were directed against a minor histocompatibility antigen (mHag). Using cDNA expression cloning, the target molecule of a HLA-B7-restricted CTL clone was identified. The CTL clone recognized a mHag produced by a non-synonymous single nucleotide polymorphism in the angiogenic endothelial cell growth factor-1 (ECGF-1) gene also known as thymidine phosphorylase. Analysis of the expression of the ECGF-1 gene in a micro array study demonstrated high levels of expression in hematopoietic cells, in particular CD14⁺ monocytes and DC, but ECGF-1 expression could also be detected in lung, liver and heart. We confirmed the expression pattern of ECGF-1 using quantitative real-time RT-PCR. CD14⁺ cells showed the highest levels of expression, although expression in other hematopoietic cells, like CD4⁺, CD8⁺ and CD19⁺ cells could also be detected. In agreement with the microarray study, expression of ECGF-1 in lung, liver and heart was found. By immunohistochemical analysis, it has been demonstrated that the expression of ECGF-1 in these tissues was mainly due to the presence of macrophages, although weak expression in stroma cells could also be detected. Although the patient from whom the CTL clone was isolated had more than 1% circulating ECGF-1-specific CD8⁺ T cells as determined with tetramer staining, she only suffered from mild GVHD indicating that the low level of ECGF-1 expression in some normal tissues has no major detrimental side effects. ECGF-1 is not only expressed in hematological tumors like multiple myeloma, CML and AML, but is also expressed in various other tumors, like melanoma, breast carcinoma and renal cell carcinoma. In these solid tumors, ECGF-1 can be expressed by the tumor cells themselves or by tumor-infiltrating monocytes. ECGF-1 expression is positively correlated with microvessel density and seems to be an unfavorable prognostic factor. The ECGF-1-specific CTL clone recognized mHag-positive, HLA-B7-expressing CML as well as melanoma cells, demonstrating that the ECGF-1-specific T cells are not only reactive against hematological malignancies but also against solid tumors. Therefore, ECGF-1 is an interesting target for immunotherapy of both hematological and solid tumors.