Cytoreductive effects of anti‐transferrin receptor immunotoxins in a multicellular tumor spheroid model

Abstract

We have evaluated the sensitivity to immunotoxins (IT) of monolayer and of 200-250 microns multicellular tumor spheroid (MTS) cultures obtained with human breast (MCF7) and glioblastoma (U118) tumor cells and with rat glioblastoma (9L) cells. Monolayer MCF7 and U118 cells were highly sensitive to antitransferrin receptor (anti-TfnR) ricin A chain (RTA)-IT (Tfn-RTA and MAb OKT9-RTA) treatment in the presence of the intracellular RTA-IT enhancing agent human serum albumin-monensin (HSA-Mo) conjugate. A 790- to 2000-fold higher concentration of anti-TfnR IT was instead required to reduce by 50% the volume of individually treated MCF7 spheroids, as evaluated by applying the Gompertz growth model. Monolayer 9L cells showed 230- to 5700-fold lower sensitivity to Tfn-RTA IT than MCF7 and U118 monolayers, yet 9L spheroid cells were almost as sensitive to anti-TfnR IT as monolayer 9L cultures. Binding studies performed with [125I]-Tfn and FITC-labelled anti-TfnR MAb revealed that 9L monolayers and MTS expressed 4.1-fold and 8.8-fold lower amounts of TfnR than MCF7 monolayers and MTS, respectively. However, Tfn bound to TfnR sites of 9L and of MCF7 cells with comparable affinity. Experiments carried out with the diphtheria toxin mutant CRM107 linked to Tfn confirmed the pattern observed with RTA-IT. Monolayers and spheroids showed no considerable differences in sensitivity to ricin toxin. Collectively, these results indicated that the efficacy of IT against 3-D tumors is heavily influenced by the number of target Ag expressed by the tumor cells, as well as by the affinity of IT/toxin-cell interaction.

An anti-transferrin receptor-avidin fusion protein exhibits both strong proapoptotic activity and the ability to deliver various molecules into cancer cells

“…Low or no expression of the TfR is unlikely to explain the result seen with 9L, which has been successfully treated with an anti-TfR immunotoxin (38). Instead, these findings are consistent with previous studies showing that hematopoietic cells are generally more sensitive to the antiproliferative effects of anti-TfR monoclonal Abs than other cell types (43).…”

“…Anti-rat TfR IgG3-Av contains the variable regions of the mouse monoclonal Ab OX26, which binds specifically to the rat TfR without blocking Tf binding (6). Chemical conjugation of OX26 with recombinant ricin A chain yielded an immunotoxin exhibiting strong inhibition of protein synthesis in Y3-Ag1.2.3, NBT II, and 9L cell lines (38,39). These findings suggested that anti-rat TfR IgG3-Av could be used to deliver molecules into rat cancer cells through receptor-mediated endocytosis, and we have now demonstrated that anti-rat TfR IgG3-Av can deliver different biotinylated molecules into Y3-Ag1.2.3 cells.…”

Coupled cellular trafficking and diffusional limitations in delivery of immunotoxins to multicell tumor spheroids

“…This distribution is likely to depend on the cell line and receptor type. A 50% reduction in the average number of transferrin binding sites in MTS as compared to monolayers was reported for 9L cells (Chignola et al, 1994), similar to the reduction we calculate for our system. No such reduction was found in MCF7 cells (Chignola et al, 1994), however, indicating that the effect of geometry on receptor distribution is not universal to all cell lines.…”

“…Chignola et al (1994) reported that antitransferrin receptor-ricin A conjugates were only slightly less toxic to 9L MTS than to 9L monolayers, but were much less toxic to MCF7 MTS than MCF7 monolayers. These Figure 9.…”

“…MTS size was monitored using the calibrated eyepiece micrometer of an inverted microscope. The longest diameter, Di 1 , and the perpendicular diameter, Di 2 , were measured, and an average radius, R avg , and volume, V MTS , were calculated using the following equations (Chignola et al, 1994):…”