Study of the plasma clearance of antibody – ricin‐A‐chain immunotoxins

Abstract

In recent years, antibody -ricin-A-chain immunotoxins have been investigated as anti-neoplastic agents. To achieve in vivo therapy it is necessary that the immunotoxin remains in circulation at a sufficiently high level for a sufficiently long time to allow binding to tumor cells to occur. Therefore, examination of the pharmacology of immunotoxins may elucidate the reasons for the poor in vivo tumoricidal effect of immunotoxin described before. In this study the plasma clearance of antibody -ricin-A-chain immunotoxins, after intravenous injection in animals of different species, has been examined. Sensitive and reproducible techniques were developed to monitor the level of immunotoxin and its constituents in the blood. It is shown that immunotoxins are rapidly eliminated from the bloodstream. Neither the properties of the antibody moiety nor the nature of the linkage binding rich A-chain to antibody account for the disappearance of immunotoxin from the plasma. On the other hand, we found that the rapid clearance of immunotoxin is due to the mannose residues on the rich A-chain moiety which are specifically recognized by liver cells. When immunotoxin is administrated together with yeast mannan, which enhances the level of active immunotoxin in circulation by inhibition of liver uptake, the anti-cancer efficacy of immunotoxin in vivo is drastically improved.Progress has been made in rendering monoclonal antibodies highly cytotoxic by coupling them to the A-chain subunit of the phytotoxin ricin (RTA). Such hybrid molecules, termed immunotoxins (ITS), are now currently prepared according to standardized methods and their biological properties have been studied in detail [l -41. Although these conjugates showed specific cytotoxicity, their efficiency varies greatly from one IT to another. Obviously, the most attractive use of the IT is its in vivo application as a therapeutic agent for cancer. However, the results of initial trials in which ITS have been used therapeutically in animals showed, with one exception [3], that, even when the tumor load was small, the anti-cancer effects were relatively unimpressive [lo, 111. To improve the cancerocidal action of these conjugates, a detailed examination of potential agonists of the action of IT in vivo is needed. Site-specific toxin delivery requires the fulfillment of two sequential steps. First, IT should localize on the desired target site and subsequently bind to the relevant cell surface antigen; secondly, the toxin subunit should be translocated into the cytoplasm to inhibit protein synthesis and thus induce cell death.It has been shown from in vitro studies that to achieve high efficiency of this latter step it is necessary to administer most ITS together with IT enhancers. It has recently been demonstrated that IT enhancers that promote RTA delivery into the cytosol could also be effective in vivo (unpublished work). In order to achieve efficient binding, the IT must remain in the systemic circulation, in its active form, a sufficiently long time and at a sufficie...

The Aspergillus toxin restrictocin is a suitable cytotoxic agent for generation of immunoconjugates with monoclonal antibodies directed against human carcinoma cells

“…A second possible advantage of restrictocin over ricin A chain or other glycosylated toxins is its absence of glycosylation. The mannose-rich oligosaccharide side chains of ricin A chain apparently lower the potency of ricin A chain conjugates in two ways: in vitro by creating an energy barrier which blocked the passage through the lipophilic core of the cell membrane [26] and in viva by deterring fast clearance from the blood stream [27]. In agreement with these and other results [34], we found a longer clearance of restrictocin compared to that of the intact ricin.…”

Antibody-Based Immunotoxins for the Treatment of Cancer

“…The development of MAbs enabled the coupling of ricin to a specific targeting agent [84]. Because the ricin toxin A chain (RTA) by itself has an unwanted affinity to hepatic cells [87][88][89], several strategies have been developed to prevent this binding. Deglycosylated A chain (dgA) is one strategy that has been taken, since the glycosylated side residues of RTA are responsible for the non-specific binding to hepatic cells [87,88,90].…”

Toxin selection and modification: Utilization of the A chain of ricin

“…Ricin A chain and its conjugates with antibodies have a short plasma halflife in animals [22] or in humans [31] due to the presence of carbohydrate structures with terminal mannose residues on the asparagines in position 10 and 236. Mannose receptors on Kupffer cells in the liver capture A chains and ITs, thus eliminating them rapidly from the circulation.…”

“…2. The presence of terminal mannose residues on ricin A chain, which is a glycoprotein [21], is another inconvenience, since conjugates containing it are then captured in vivo by liver receptors recognizing these sugars [22][23][24][25]. However, chemical deglycosylation is an easy way to obtain, in large quantities, an A chain derivative with long plasma half-life, while preserving all other biological properties of the native protein [26,27].…”