One milligram in 0.5 ml of PBS was used in each injection. and identified by MS. We isolated 2,114 mAbs with unique sequences and identified 21 distinct Ags highly expressed on several carcinomas. Of those 2,114 mAbs 356 bound specifically to one of the 21 Ags. After preparing complete IgG1 Abs the assay for Ab-dependent cell-mediated cytotoxicity (ADCC) and the assay in cancer-bearing athymic mice were performed to examine antitumor activity. The mAbs converted to IgG1 revealed effective ADCC as well as antitumor activity assay for Ab-dependent cell-mediated cytotoxicity (ADCC) using 22 clones against 10 Ags (EGFR, ALCAM, ICAM-1, EpCAM, HGFR, TfR, ITGA3, EMMPRIN, PTP-LAR, and CD44) using the cell lines listed in Table 4. As can be seen, those FLAG tag Peptide mAbs gave a positive reading that ranged between 5% and 95% for cell killing. The FLAG tag Peptide details using anti-EGFR Abs and anti-EpCAM Ab are in Fig. 1 and assay using three mAbs against two of the Ags (EGFR and EpCAM) in cancer-bearing athymic mice. As can be seen in Fig. 1the anti-EGFR Abs showed a strong antitumor activity against tumor cell line A431. When we compared our mAbs (048-006 and 059-152) against EGFR with cetuximab it appeared that they had a very similar level of antitumor activity. The anti-EpCAM Ab also prevented the growth of HT29 (Fig. 1mAb 048-006 was very effective in inhibiting the binding of EGF to the EGFR, whereas mAb 059-152 only partially prevented the binding reaction. Rabbit Polyclonal to FZD4 The phosphorylation assay (Fig. 1and (13). In brief, the phages (2 1013 cfu) were mixed with cells (0.2C1 108) in 1.6 ml of solution A (1% BSA, MEM, and 0.1% NaN3), and AgCAb complexes on the cell surface were formed. The cell and phage suspension was overlaid on the organic solution in an Eppendorf tube. After the tube was centrifuged, water and organic layers were discarded. The collected cells were suspended in solution A. This process was repeated three times. Finally, the cells were suspended in PBS and frozen in liquid nitrogen. The frozen cells were thawed FLAG tag Peptide and mixed with DH12S. The phages were prepared. This screening round was performed repeatedly three times. After three rounds of screenings, DH12S infected with recovered phages was spread on plates. Approximately 200 colonies were picked up. Thirty-three cancer cell lines listed in Table 1 were used as Ags. Immunostaining of Fresh Tumors. Tumor tissues and the neighboring normal tissues resected by operation were used for immunostaining. They were fixed with 4% paraformaldehyde in 0.1 M cacodylic buffer (pH 7.4) by microwave irradiation as described previously (14). FLAG tag Peptide Identification of Ag. Membrane protein analysis was performed according to Zhao (15). Proteins present on the cell surface were biotinylated according to the manufacturer’s instruction by using the EZ-Link Sulfo-NH-LC Biotinylation kit (Pierce). After the cells were homogenized with a Dounce homogenizer, the proteinCmembrane complexes were banded between 0.25 M and 1.25 M sucrose layers by centrifugation. The complexes were dissolved in a detergent mixture: 50 mM Hepes (pH 7.6), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 1% octyl glucoside. scFv-CL fused with cp3 was converted to scFv-CL fused with protein A domains (scFv-CL-PP) (16). scFv-PP form was covalently bound to beads that were CNBr-activated Sepharose 4B (GE Health Care Bioscience). Ab-bound beads were used for immunoprecipitation as described by David (17). MS analysis was performed according to Geuijen (8). Preparation of IgG1. ScFv was converted to IgG1 and prepared by using a high-level expression vector (18). Using IgG1 mAbs we examined ADCC, effects on binding of EGF to EGFR, effects on phosphorylation of EGFR, and antitumor activity in athymic nude mice. ADCC. The enzymatic activity of lactic dehydrogenase released from the target cells was measured for estimation of ADCC (19). Various cell lines were used as targets for the mAbs. Cells were derived from the following cancers: NCI-H1373, lung adenocarcinoma; CCF-RC1, renal clear cell carcinoma; A431, vulva epidermoid carcinoma; ACHN, renal adenocarcinoma; HT-29, colorectal adenocarcinoma; SKOv3, ovarian adenocarcinoma; CW-2, colorectal adenocarcinoma; EBC-1, lung squamous cell carcinoma; NCI-H441, lung papillary adenocarcinoma; HepG2, hepatocellular carcinoma; MKN45, gastric adenocarcinoma; MIAPaca-2, pancreatic carcinoma; PC14, ling carcinoma. Effector cells were prepared from blood of healthy volunteers and used in a ratio of 100:1 (106 to 104 in 200 l) (20). Effects of Anti-EGFR mAbs on the Function of EGFR. Binding of EGF to EGFR on the cell surface was estimated according to Yang (21). Phosphorylation of EGFR induced by EGF was measured according to Matar (22). Antitumor Activity em in Vivo /em . Two different methods were adopted. In the first method each mouse was injected with 5 106 cells, and when the tumor grew to 0.2 cm3 mAb therapy was initiated. Treatments consisted of twice-weekly i.p. injections of mAb for.