No abstract

Ospedale Ci-ile Atfaggiore B.go Trento. 37100 1 erona. Itats.Summarv The cvtoreductive effects of anti-transferrin receptor (anti-TfnR)

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.

We have investigated the internalization to low pH intracellular compartments of transferrin (Tfn), diphtheria toxin (DT) and of anti-cell surface antibodies (MAb) by a cytofluorometric assay based on low pH quenching of fluorescein (FITC) emission. FITC-labelled Tfn, anti-CD3, anti-CD5 and anti-Thy 1.2 MAb internalization resulted in a progressively lower FITC quenching effect. Following internalization, a distinction could be made between molecules that enter low pH compartments without undergoing intracellular degradation (e.g., Tfn, anti-CD3 MAb) and molecules that are internalized through low pH organelles and are then degraded within the cell (e.g., DT). A strict correlation was observed between quenching of internalized FITC-protein fluorescent emission and the cytotoxic activity of DT-based immunotoxins (IT).

The immunotoxin-enhancing properties of monensin and of human-serum-albumin-monensin conjugates are severely impaired in the presence of human serum. In this study we have therefore investigated the interaction between serum proteins and monensin leading to the inactivation of monensin function as immunotoxin potentiator. We found that the binding of monensin-specific mAb to thioether-cross-linked or disulfide-cross-linked protein-monensin conjugates is negatively affected by serum, as indicated by immunoenzymic (ELISA) and radioimmunobinding analysis. Size-exclusion chromatography of serum samples indicated that the greatest blocking effect is due to protein components of 40-90 kDa eluting as a broad peak (peak 4). Analysis of the proteins contained within peak 4 by ion-exchange chromatography followed by microsequencing revealed that the major components of peak no. 4 were transferrin, human serum albumin and immunoglobulin fragments. Investigations on the nature of the interactions between serum proteins and monensin leading to monensin inactivation were conducted by affinity chromatography of serum on immobilized human-serum-albumin-monensin conjugates, size-exclusion chromatography, SDSPAGE analysis of serum-treated human-serum-albumin-monensin conjugates, and evaluation of the stability of immobilized human-serum-albumin-bound '251-monensin following treatment with serum. Addition of esterase inhibitors (e.g. EDTA, 4-nitrophenyl phosphate) or prior treatment of the serum at 56°C partially reversed the serum effects observed. We conclude that serum proteins block the immunotoxin-enhancing effect of monensin and of human-serum-albumin-monensin conjugates by multiple mechanisms involving hydrophobic and covalent interactions and enzyme-mediated cleavage of protein-bound monensin.Hybrid molecules formed by mAbAigands chemically or genetically linked to toxins (immunotoxins) have been proposed as a new class of anti-tumor pharmacological reagents [l]. In particular, immunotoxins made with mAb linked to ricin A chain (RTA) have been extensively studied in vitro and in animal models [2-111, and their anti-tumor potential clinically evaluated [ 12-171. Although RTA-immunotoxins show high target-cell selectivity and low in vivo toxicity [12,13,[15][16][17], their cytotoxic potency is variable, depending on the internalization rate [18, 191, the intracellular distribution (11, 20-231, the target-cell type [24, 251 and other parameters. As a result, clinical trials conducted with RTA-immunotoxins have met with limited success [12-171. To increase the anti-target cell potency of RTA-immunotoxins, lysosomotropic amines (e.g. chloroquine, methylamine, ammonium chloride) or ionophores (e.g. monensin,Correspondence to M. Colombatti, Istituto di Immunologia e Malattie Infettive, Universiti di Verona, c/o Policlinico Borgo Roma, 1-37134 Verona, ItalyAbbreviations. HSA, human serum albumin; RIB, radioimmunobinding ; Tfn, transferrin; Nph-P, 4-nitrophenyl phosphate; RTA, rich toxin A chain; SPDP, N-succinimidyl-3-(2-pyridyl...