Blocked and non-blocked ricin immunotoxins against the CD4 antigen exhibit higher cytotoxic potency than a ricin A chain immunotoxin potentiated with ricin B chain or with a ricin B chain immunotoxin

Abstract

An immunotoxin consisting of ricin A chain linked to the monoclonal antibody M-T151, recognising the CD4 antigen, was weakly toxic to the human T-lymphoblastoid cell line CEM in tissue culture. The incorporation of [3H]leucine by CEM cells was inhibited by 50% at an M-T151--ricin-A-chain concentration (IC50) of 4.6 nM compared with an IC50 of 1.0 pM for ricin. In contrast, immunotoxins made by linking intact ricin to M-T151 in such a way that the galactose-binding sites of the B chain subunit were either blocked sterically by the antibody component or were left unblocked, were both powerfully cytotoxic with IC50 values of 20-30 pM. The addition of ricin B chain to CEM cells treated with M-T151--ricin-A-chain enhanced cytotoxicity by only eight-fold indicating that isolated B chain potentiated the action of the A chain less effectively than it did as an integral component of an intact ricin immunotoxin. Ricin B chain linked to goat anti-(mouse immunoglobulin) also potentiated weakly. Lactose completely inhibited the ability of isolated ricin B chain to potentiate the cytotoxicity of M-T151--ricin-A-chain and partially (3- to 4-fold) inhibited the cytotoxicity of the blocked and non-blocked ricin immunotoxins. Thus, in this system, the galactose-binding sites of the B chain contributed to cell killing regardless of whether isolated B chain was associated with the A chain immunotoxin or was present in blocked or non-blocked form as part of an intact ricin immunotoxin. The findings suggest that the blocked ricin immunotoxin may become unblocked after binding to the target antigen to re-expose the cryptic galactose-binding sites. However, the unblocking cannot be complete because the maximal inhibition of [3H]leucine incorporation by the blocked immunotoxin was only 80% compared with greater than 99% inhibition by the non-blocked immunotoxin.

Antitumour activity of a sterically blocked ricin immunotoxin on a human colorectal adenocarcinoma grafted subcutaneously in nude mice

“…Nevertheless, as previously suggested [ 18,29,33], the saccharide-binding ability of the ricin B chain should be not totally abolished by the steric hindrance of the antibody molecule on the linked ricin moiety.…”

“…This procedure has been successfully applied by our group [3,4] and others [13,19,20,29,33]. We prepared a ricin-antibody conjugate, lacking the ability to bind the galactosidic residues of Sepharose 6B, a so-called blocked immunotoxin.…”

Systemic immunotoxin therapy of cancer: advances and prospects

“…In every case, intoxication requires that the enzymic portion of the toxin is transferred to the cytosol from an initial binding site on the cell surface and depends on the co-ordinated action of several parts of the toxin molecule with distinct functions (Figure 2 (Ogata et al, 1990). A second feature of PE important to toxicity is the presence of an amino acid sequence at the C-terminus of the catalytic fragment which resembles the consensus sequence KDEL responsible for signalling the retention of soluble proteins within the endoplasmic reticulum (Chaudhary et al, 1990b (Wawrzynczak et al, 1988 (Wawrzynczak et al, 1991d (Hertler & Frankel, 1989). …”

Blocked ricin-conjugated T cell immunotoxins: Effect of anti-CD6-blocked ricin on normal T cell function

“…It has been shown, however, that in most cases IT containing ricin A chain are less cytotoxic than native ricin. It is believed that the B chain is necessary for optimal internalization or transmembrane migration [13,27,40,43,45]. In this re-gard, we have used blocked ricin as the cytotoxic effector molecule, which consists of an intact ricin molecule in which the galactose-binding sites of the B chain have been blocked with covalently attached affinity ligands [21].…”

“…The natural mechanism of action of this two-chain toxin involves the binding of the toxin to the cell surface via galactose-binding sites on the ricin B chain, followed by internalization of the toxin, and eventually by inactivation of ribosomes by the ricin A chain [9,12]. Attempts to circumvent non-specific cell attachment of ricin via ricin B chain in IT have included treatment of cells in the presence of high concentrations of lactose [37], decreasing galactose binding by steric hindrance achieved by cross-linking ricin to the antibody [38,43] or, more frequently, using IT composed of mAb attached to purified ricin A chain [1,3,25]. It has been shown, however, that in most cases IT containing ricin A chain are less cytotoxic than native ricin.…”