Cytotoxic strategies which are directed to tumorassociated antigens might be most bene®cial for cancer patients with minimal tumor load such as in an adjuvant setting after initial therapy. We have recently described a highly potent single chain antibody-toxin, scFv(14E1)-ETA, which consists of the variable domains of the antibody 14E1 genetically fused to a truncated form of Pseudomonas exotoxin A. ScFv(14E1)-ETA speci®cally recognizes the human epidermal growth factor receptor (EGFR) and the oncogenically activated receptor variant EGFRvIII, which have been implicated in the development of various human malignancies. Here we have investigated the antimetastatic activity of bacterially expressed scFv(14E1)-ETA and its disul®de-stabilized derivative ds-scFv(14E1)-ETA in a novel model for disseminated disease which is based on murine renal carcinoma cells subsequently transfected with the E. coli b-galactosidase gene, and human full-length or variant EGFR cDNAs. Intravenous injection of these RencalacZ/EGFR and Renca-lacZ/EGFRvIII cells in syngenic Balb/c mice led to the formation of pulmonary metastases which were readily detectable upon excision of the lungs and X-gal staining. Systemic treatment of mice with scFv(14E1)-ETA resulted in the complete suppression of Renca-lacZ/EGFRvIII metastasis formation and drastically reduced the number of pulmonary Renca-lacZ/EGFR tumor nodules. The ds-scFv(14E1)-ETA derivative where the antibody variable regions are connected by an arti®cial disul®de bond displayed improved thermal stability at physiological temperature but due to reduced cytotoxic activity was less potent than the original scFv(14E1)-ETA in metastasis suppression.
Cellular transformation does not necessarily require the expression of proteins with neoantigenic properties, and for this reason, immunosurveillance does not register all tumor cells. They frequently express potentially immunogenic components, but are able to escape elimination by immune mechanisms. One explanation for this escape is poor antigen presentation by the tumor cells, resulting in little or no measurable antitumor immunity in immunocompetent hosts. T cells remain naive or even become anergic to the tumor cells. Reasons for the deficient antigen presentation by the tumor cells include the reduced or absent expression of major histocompatibility complex (MHC) molecules and the absence of tumor antigens in the groove of class I or class II MHC molecules as a consequence of defective protein processing. Other reasons are the absence or inadequate levels of expression of adhesion molecules, the absence or inadequate levels of costimulatory molecules or the expression of lymphocyte suppressive cytokines like transforming growth factor (TGF-β) or interleukin 10 (IL-10) by tumor cells (1-5).
The ErbB2 receptor tyrosine kinase is often overexpressed in human malignancies and causally involved in transformation. High levels of ErbB2 in tumor cells correlate with an unfavorable prognosis. This makes the ErbB2 receptor an interesting target for tumor therapy, and several strategies have been designed to direct drugs to ErbB2-expressing cells. We established a novel cellular model that allows preclinical evaluation of ErbB2-directed drugs in immunocompetent animals. Renal carcinoma (Renca) cells are an established tumor cell line that originated in Balb/c mice. Upon intravenous transplantation, these cells form pulmonary metastases in Balb/c mice. The transforming genetic lesions in these cells are not fully characterized, but do not seem to involve alterations in ErbB2 gene expression. We transfected Renca cells with the gene encoding the human ErbB2 receptor to provide a target structure for specific drugs and with the bacterial lacZ gene to provide a sensitive means of detection of the tumor cells in the transplanted animals. These genetically modified cells form lung metastasis and can be easily visualized on the surface of lung tissue by staining with an X-gal solution. This allows a quantitative analysis of the number of ErbB2-expressing pulmonary metastasis. We previously used these Renca cells to evaluate the efficacy of an ErbB2-specific tumor toxin on pulmonary metastases in an adjuvant and a palliative treatment setting. In both cases, we achieved a dramatic reduction of disseminated lung lesions. Here we show that even at an advanced stage of metastasis formation, the ErbB2-specific toxin is able to efficiently reduce the number of pulmonary tumors.
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