Suppression of metastasis formation by a recombinant single chain antibody-toxin targeted to full-length and oncogenic variant EGF receptors

Abstract

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.

Sequence-specific Peptide Aptamers, Interacting with the Intracellular Domain of the Epidermal Growth Factor Receptor, Interfere with Stat3 Activation and Inhibit the Growth of Tumor Cells

“…Prior to growth factor stimulation the cells were kept for 18 h in serum-free medium. Renca EGFR and Renca-ErB2 cells (36) were kept in RPMI medium supplemented with 10% FCS, 0.25 mg/ml Zeocin, and 0.48 mg/ml G418.…”

“…Expression of the aptamer was induced with 0.5 mM isopropyl-1-thio-␤-D-galactopyranoside for 4.5 h at room temperature. Purification of the aptamer under denaturing conditions was done as published before (36). Briefly the bacteria were lysed in 8 M urea, purified via a Ni 2ϩ -Sepharose column (Amersham Pharmacia), and eluted with 250 mM Imidazole in 4 M urea.…”

3PO, a novel nonviral gene delivery system using engineered Ad5 penton proteins

“…Directing nonviral vectors to cellular receptors by the incorporation of specific ligands, such as transferrin, asialoglycoprotein, or epithelial growth factor (EGF), has provided an efficient means of crossing the plasma membrane by receptor-mediated internalization. [1][2][3][4][5][6][7][8][9][10][11][12][13] The cellular endosome encapsulating the vector following endocytosis, however, has proven to be a major barrier to nuclear delivery and gene expression by nonviral vectors. 14 Without endosome penetration, lysosomal enzymes eventually degrade the DNA, resulting in low transduction.…”

Peptide mimotopes recognized by antibodies cetuximab and matuzumab induce a functionally equivalent anti-EGFR immune response

“…To investigate this further, we analyzed the ability of the phage clones to interfere with cell binding and cytotoxic activity of the EGFR-specific antibody-toxin scFv(225)-ETA that carries the binding domain of cetuximab fused to truncated Pseudomonas exotoxin A (ETA) (Wels et al, 1995;Azemar et al, 2000). EGFR-expressing Renca-lacZ/EGFR renal carcinoma cells (Schmidt et al, 1999) were treated with scFv(225)-ETA after preincubation of antibody-toxin with a 10-fold molar excess (based on pIII fusion peptides present) of phage before cell killing was determined in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assays. Although control phage did not inhibit cell binding of scFv(225)-ETA resulting in more than 60% cell killing at the chosen conditions, cetuximab and all tested phage showing KTL-or YPLG-containing peptides efficiently Figure 1 Selection of cetuximab-and matuzumab-binding peptide-displaying phage by delayed infectivity panning.…”