Abstract
The potential of mouse monoclonal antibodies for recognising different antigens associated with human small cell lung cancer (SCLC) to form active immunotoxins was assessed by an indirect in vitro screening assay. The screening agent used was a conjugate made by linking ricin A chain to a sheep anti-mouse IgG Fab' fragment via a disulphide bond. The monoclonal antibodies SWA11 and SWA20 both mediated the toxic effects of ricin A chain against the HC12 classic SCLC cell line in dose-dependent fashion. The SWA11 antibody was the more effective; in combination with the screening agent at a concentration of 1 x 10(-7) M, it inhibited the incorporation of [3H] leucine into HC12 cells by 94% compared with only 44% inhibition in the case of SWA20. An immunotoxin made by the direct chemical conjugation of ricin A chain to SWA11 exhibited selective toxic effects upon HC12 cells in tissue culture inhibiting the incorporation of [3H] leucine by 50% at a concentration (IC50) of 6.2 x 10(-10) M and by 98% at 1 x 10(-7) M. SWA11-ricin A chain had an IC50 of 4.4 x 10(-10) M against the NCI-H69 classic SCLC cell line but showed no cytotoxic activity against the human lung adenocarcinoma cell line NCI-H23 at a concentration of 1 x 10(-8) M.
Highlights
The ability of the two anti-small cell lung cancer (SCLC) monoclonal antibodies, SWA1I and SWA20, to mediate ricin A chain toxicity against the human classic SCLC cell line HC12 was tested by an indirect screening assay in vitro
Both monoclonal antibodies bound to a high proportion of HC12 cells by indirect immunofluorescence analysis, SWA1I giving the greater intensity of fluorescence staining on cells treated with the antibodies at identical concentration
We have shown that an indirect screening procedure accurately predicted the cytotoxic potency of ITs made by linking ricin A chain to the SWAl 1 and SWA20 monoclonal antibodies against the human classic SCLC cell line HC12 in tissue culture
Summary
Castor bean cake derived from the seeds of Ricinus communis of Sri Lankan origin was a gift of Croda Premier Oils, Hull, Humberside, England. The anti-SCLC mouse monoclonal antibodies, SWAl 1 and SWA20, were purified from hybridoma supernatants as described by Smith et al (1989). The control mouse monoclonal antibody raised against vesicular stomatitis virus, 2AL-1, was purified from ascitic fluid (Forrester et al, 1984). All three antibodies are of the IgG2a subclass. The human classic SCLC cell line HC12 (Duchesne et al, 1987) was the gift of Dr G. The human classic SCLC cell line NCI-H69 and the human lung adenocarcinoma cell line NCIH23 (Carney et al, 1985) were kindly provided by Dr V.
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