An ineffective monoclonal antibody-ricin A chain conjugate is converted to a tumouricidal agent in vivo by subsequent systemic administration of ricin B chain

Abstract

An immunotoxin comprising a tumour-specific monoclonal antibody (11/160) coupled to ricin A chain, although inactive in in vitro cytotoxicity assays against HSNtc sarcoma target cells, was found to be capable of significant tumouricidal activity in syngeneic rats if potentiated by ricin B chain. The 11/160-ricin A, when bound to tumour cells prior to their inoculation, led to a slight inhibition of tumour growth s.c. compared with untreated sarcoma cells or those coated with antibody alone. However, all tumours in these groups developed progressively (69/69), whereas in those rats receiving 15 micrograms or 150 micrograms ricin B chain i.v. 5 min after tumour cell inoculation, the 'take rate' was reduced to 75% and 30% respectively, and significantly longer latent periods were evident for those tumours which did develop. Ricin B chain similarly inhibited, in a dose-dependent manner, the lung colonisation potential of 11/160-ricin A coated HSNtc cells. No effects were obtained if the B chain treatment followed inoculation of untreated or antibody-coated cells, suggesting that systemically administered B chain is capable of gaining access to and activating antibody-ricin A chain conjugates bound to the surface of syngeneic sarcoma cells in lung or subcutaneous sites. Tumour inhibition was obtained in some instances with intervals of up to 24 h between inoculation of conjugate-coated tumour cells and B chain. Experiments are in progress to determine if such potentiation may be feasible in a therapeutic rather than a prophylactic setting using this syngeneic solid tumour system.

Cloning and expression of recombinant, functional ricin B chain.

“…To provide the A-chain-enhancing function of B chain, IT-As have been used in the presence of free B chain (9,10) or B-chain-containing ITs (IT-Bs) (11,12). Both approaches enhance the specific toxicity of IT-As in vitro and free B chains can also enhance specific killing by IT-As in vivo (13). With regard to IT-Bs, some lectin activity remains that could lead to nonspecific interactions in vivo.…”

Recombinant ricin toxin A chain cytotoxicity against carcinoembryonic antigen expressing tumour cells mediated by a bispecific monoclonal antibody and its potentiation by ricin toxin B chain

“…If, however, all the RTB had reassociated with r-RTA on an equimolar basis, the resultant 'reconstituted ricin' could have been present in a quantity more than adequate to kill 100% of the target cells. Further titration of RTB (Figure 2) or r-RTA (Figure 4) ed immunotoxins, because the need to avoid lectin activity is fundamental to their design, and in published studies of the effect of RTB or RTB conjugates on RTA immunotoxins the B chain lectin site has not been blocked (McIntosh et al, 1983;Eccles et al, 1987;Vitetta et al, 1983Vitetta et al, , 1984. The experiments shown in Figure 5, however, address this question.…”

“…RTB can be administered in free form, or conjugated to the same targeting antibody as the RTA immunotoxin or any antibody recognising the immunotoxin conjugate (McIntosh et al, 1983;Vitetta et al, 1983Vitetta et al, , 1984, and is thought to aid cellular incorporation of immunotoxin by facilitating translocation through the cell membrane (McIntosh & Thorpe, 1984). In cases where the targeted antigen is rapidly and efficiently internalised by the cell the lack of B chain may be advantageous but nonessential, but there are instances in which RTA immunotoxin is not readily incorported and the transport function of the B chain then becomes of major importance (McIntosh et al, 1983;Eccles et al, 1987 (Thorpe & Ross, 1982;Gregg et al, 1987;Cattel et al, 1988). Galactose blocking can be very effective in aiding selectivity of ricin-antibody conjugates in vitro, and a galactose-blocked ricin anti-iodiotype antibody conjugate has been tested in vivo against a guinea pig B cell leukaemia line (Gregg et al, 1987).…”