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Notes: Summary 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 μ g or 150 μ g 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.

Notes: Summary The object of our current investigations is to explore the potential of antibodies for localisation and treatment of disseminated disease, using as a model rat monoclonal antibodies (mAbs) raised against syngeneic tumourspecific antigens. As part of this study, antibodies of differing isotypes with specificity for either HSN or MC24 sarcoma were labelled with125iodine and injected intravenously into normal rats or those bearing paired tumours in contralateral flanks. The blood clearance rates of the radiolabelled antibodies were found to be influenced by immunoglobulin subclass (IgG2b 〉 IgG2a 〉 IgG1) and to be increased non-specifically by the presence of growing tumours. The tumour and normal tissue distributions of the antibodies tested were also found to vary according to their apparent degree of interaction with host Fc-receptor-bearing cells, to the extent that tumour specificity in vitro was not necessarily reflected in selectivity of localisation in vivo. Three IgG2b monoclonal antibodies showed preferential uptake in the spleens of syngeneic rats and non-specific accumulation in tumours. This effect was not observed with antibodies of IgG2a or IgG1 subclass, and was abolished by the use of IgG2b F(ab′)2 preparations. In spite of the use of immunoglobulin fragments, varying the assay time and testing tumours of different sizes, specific tumour localisation was low with all seven monoclonal antibodies tested. The maximum uptake achieved was less than 1% of the injected dose of antibody per gram of tumour. Much higher levels of antibody localisation have been reported for human tumour xenografts growing in nude mice, but these are rarely achieved in other systems. We propose that the use of autologous monoclonal antibodies recognising tumour-associated antigens of relatively low epitope density in syngeneic hosts provides a valid alternative model in which to investigate the factors limiting more effective, specific immunolocalisation of malignant disease.

Notes: Summary In the preceding paper it was suggested that the tumour localisation of125I-labelled syngeneic rat monoclonal antibodies (mAbs) may be limited in immunocompetent hosts by the presence of competing endogenous serum antibodies. In syngeneic congenitally athymic (nu/nu) and cyclosporin-A-treated rats (both of which fail to mount immune responses to tumour antigens) increased uptake of mAbs in tumour tissue was obtained compared with that in immunocompetent animals. However, in the case of IgG2b and IgG1 mAbs, this appeared to be due primarily to enhanced “non-specific” localisation mediated by Fc binding, since it was abolished by the use of F(ab′)2 fragments with two out of three mAbs tested. Normal tissue distribution was also influenced by host immune status: in nu/nu rats the uptake of IgG2b mAbs in the spleen was up to fivefold higher than that previously found in normal animals and the levels in liver were also increased. This effect was not seen in cyclosporin-A-treated hosts, suggesting that the reticuloendothelial system of congenitally athymic animals contains cells with enhanced IgG2b-FcR activity. This hypothesis was strengthened by the observation that splenic uptake was reduced by either the use of F(ab′)2 fragments, or prior “blockade” of Fc receptors by “cold competition” with excess unlabelled IgG2b mAbs. This blockade could not be effected by mAbs of any other isotype or by IgG2b F(ab′)2 fragments. The former manoeuvre resulted in higher tumour specificity ratios but usually at the expense of reduced levels of tumour associated radiolabelled mAb. The latter was found to increase “absolute” tumour localisation by up to 35%. In an attempt to characterise further and compare the Fc receptor activity of intratumour and intrasplenic host cells. The distribution of IgG2b mAbs was assayed in 3-week, 8-week and 12-week-old rats. We were able operationally to distinguish the activity of these two categories of cells, suggesting that they represent either different lineages or differentially activated subpopulations: the splenic IgG2b binding was fully expressed in weanling nu/nu rats whereas the FcR activity of cells infiltrating MC24 sarcoma was limited in 3-week-old compared with 8–12-week-old hosts. A further difference was apparent in the subclass “preference” of FcR binding: in immunodeprived rats both IgG1 and IgG2b mAbs were able to bind to tumour-infiltrating host cells, but uptake of IgG1 mAbs in the spleen was always low and not reduced further by the use of F(ab′)2 fragments. These results demonstrate the extent to which the interactions between specific isotypes of rat mAbs and different subpopulations of host FcR-positive cells in both normal and malignant tissues may influence biodistribution patterns, particularly in athymic rodents. These interactions may occur to a greater or lesser extent across species barriers, and account should be taken of this in all studies designed to assay rodent mAbs for their potential usefulness in the diagnosis and treatment of human tumours.