Mechanisms involved in serum‐dependent inactivation of the immunotoxin enhancers monensin and carrier‐protein—monensin

Abstract

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...

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“…We confirmed the results of Candiani et al 30 and Franceschi et al 40 who showed that monensin is unsuitable for use in vivo because it forms complexes with serum components thereby losing its ability to enhance IT. Human serum also abrogated the enhancing activity of nigericin.…”

“…30,40 We confirmed that enhancement by both monensin (Figure 2) and nigericin (data not shown) was completely blocked when the assay was performed in 50% human serum. We found that the activity of CD22-rec ricin A was also impaired by human serum.…”

“…We assume that CD22-rec ricin A is inhibited by complexation of the IT with serum components as described for monensin. 30,40 Inhibition of activity might also be induced by covalent binding of ␣ 2 -macroglobulin to rec ricin A as has been described for deglycosylated ricin A chain and its ITs by Ghetie et al 45 who found a substantial amount of complexes formed after 24 h. However, we could not detect these high molecular complexes with the immunoblotting assay used. Besides, the kinetics of CD22-rec ricin A activity are too fast 6 to allow these complexes to form.…”

“…Serum has been described to inhibit the IT enhancing activity of monensin. 30,40 Therefore the effect of the enhancers in combination with serum was assessed.…”

Escape mechanisms of human leukemic cells to long‐term immunotoxin treatment in an in vitro experimental model

“…The addition of Mo greatly increases the kinetics of anti CD5-RTA IT cytotoxicity in vitro . However, in vivo administration of Mo is severely restricted by the Mo-inactivating properties of serum proteins (Candiani et al, 1992;Jansen et al, 1992;Franceschi et al, 1994).The slow rates of cell killing observed with anti-CD5-RTA IT are an obstacle to anti-CD5 IT applications in vivo. Moreover, 2 further limitations may prevent the successful outcome of anti-CD5 IT treatments in the clinics: (1) if CD5 modulation occurs before a sufficient number of IT molecules have been internalized and routed to the cytosol; and (2) if the rate of target-cell proliferation is faster than the rate of IT-mediated cell killing, the net effect of the IT treatment might be too small to be of appreciable therapeutic value.…”