Rat monoclonal antibodies to the external domain of the product of the C‐erbB‐2 proto‐oncogene

Abstract

With the breast carcinoma cell line BT 474 used as a source of antigen, four rat monoclonal antibodies (MAbs) (3 IgG2a and I IgA) have been prepared against the external domain of the product of the c-erbB-2 proto-oncogene. All 4 antibodies stain frozen sections of tissues that over-express the product of the c-erbB-2 proto-oncogene, and competitive binding assays showed that the antibodies recognized 2 non-overlapping epitopes. Representative antibodies from the two groups (ICR12 and 13) were shown to specifically immunoprecipitate a 190 kDa protein from 35S-methionine-labelled breast carcinoma cells where the c-erbB-2 is amplified (BT 474 and MDA-MB 361). Two of the antibodies (ICR12 and ICR17) bind to the denatured antigen in Western blots and ICR12 stains formolsaline-fixed sections of breast carcinoma tissue that over-expresses the product of the c-erbB-2 proto-oncogene. These antibodies should be useful not only for immunocytochemical diagnoses but also for radio-immunoscintigraphic and therapeutic applications.

The human EGF receptor as a target for cancer therapy: six new rat mAbs against the receptor on the breast carcinoma MDA-MB 468

“…MAb ALN/1 1/53 is directed against a membrane antigen on the rat sarcoma HSN (Dean et al, 1984), ICR12 (Styles et al, 1990) and ICR55 are directed against distinct epitopes on the extracellular domain of the c-erbB-2 p185. Antibodies ICRIO and ICR16, prepared against the receptor on HN5 cells (Modjtahedi et al, 1992), were used as positive controls.…”

“…The proteins of HN5, SKOV3 or A431 cells were radiolabelled by incubation for 16-20 h with 35S-methionine (200 pCi/80 ml-1 flask containing 5 ml methionine-free DMEM), and cell lysates were prepared using Triton-X 100 as described previously (Styles et al, 1990 Effect of antibodies to the EGFR on growth of tumour cells in culture About 5 x 103 cells in 100 j4 of DMEM containing 2% FCS were seeded into each well of a 96-well plate. After incubation for 4 h at 37°C, 100 ftl aliquots of dilutions of each mAb (starting at 50 gLg ml) were added in triplicate to the wells and the cultures were incubated at 37°C.…”

“…Since our intention is to test the effectiveness of antibodies in the clinic we report here the preparation of a second series of antibodies using as immunogen the EGFR over-expressing breast carcinoma (Filmus et al, 1985) searching particularly for IgG2b antibodies with growth inhibitory properties. (Styles et al, 1990).…”

Signaling pathways required for matrix metalloproteinase‐9 induction by betacellulin in head‐and‐neck squamous carcinoma cells

“…The rat MAb ICR-62 directed against the extracellular domain of human EGFR has been described previously, 17 as has the rat MAb against the extracellular domain of c-erbB-2 (ICR-12). 18 Mouse anti-c-erbB-3 (Ab-5, clone H3.105.5) and antic-erbB-4 (Ab-3, clone H4.72.8) neutralizing MAbs were obtained from Neomarkers (Fremont, CA). Antisera utilized for immunoprecipitation include mouse MAbs F4 (Sigma, Poole, UK) and E12020 (Transduction Laboratories, Exeter, UK) directed against the intracellular domains of EGFR and rabbit antibodies to the cytoplasmic domains of c-erbB-2 (sc-284), B-3 (sc-285) and B-4 (sc-283) (Santa Cruz Biotechnology, Santa Cruz, CA).…”

“…18 The mouse anti-c-erbB-3 (clone H3.105.5) and anti-c-erbB-4 (clone H4.72.8) neutralizing MAbs block HRG-␤1-induced phosphorylation of c-erbB-3 and c-erbB-4, respectively. 24 SIHN-006 cells were pretreated with ICR-12 MAb (800 nM), H3.105.5 MAb (10 g/ml) or H4.72.8 MAb (10 g/ml) for 30 min before a 10 min treatment with BTC (10 nM); and tyrosine phosphorylation of either c-erbB-2, c-erbB-3 or c-erbB-4 was analyzed (Fig.…”