Distribution of endocytosed molecules to intracellular acidic environments correlates with immunotoxin activity.

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).

Carrier protein-monensin conjugates: enhancement of immunotoxin cytotoxicity and potential in tumor treatment.

We have investigated the potentiation of transferrin [Tfn]-toxin [Tfn-ricin toxin A chain (RTA) and Tfn-So6 saporin toxin] and monoclonal antibody-RTA conjugates by monensin (Mo) and by a human serum albumin (HSA)-monensin conjugate in vitro. The in vivo survival and in vitro and in vivo toxicity of HSA-Mo were also studied; monensin was chemically linked to HSA carrier protein via a disulfide bridge. HSA-Mo was 2-13-fold less toxic than Mo for cells in vitro. HSA-Mo was active in the same concentration range as Mo in potentiating mAb-RTA and Tfn-toxin conjugates reactive with Tfn receptors expressed by different cell lines in monolayer cell cultures. Multicell tumor spheroid cultures were used to investigate the target cell killing effect of cytotoxic conjugates and HSA-Mo in three-dimensional structures mimicking the properties of nonvascularized micrometastases. Spheroids 300-400 microns were as sensitive to Tfn-RTA and HSA-Mo in combination as monolayer cells. After 24 h incubation at 37 degrees C in human serum about 2% HSA-Mo molecules remained available for immunotoxin potentiation and about 10% after 24 h incubation in human cerebrospinal fluid. BALB/c mice tolerated injections of 2 mg/kg HSA-Mo i.v. and of 16 mg/kg i.p. The HSA-Mo half-life in the serum of BALB/c mice was 0.5 h. Following i.v. injection about 0.5% of the initial HSA-Mo persisted in the circulation at 24 h.

Sensitivity of human glioma cells to cytotoxic heteroconjugates.

Several anti-human glioma cytotoxic conjugates were studied in vitro. Monoclonal antibodies (MAbs) to the GE2 glioma-associated antigen (anti-GE 2) and MAbs to HLA-DR antigens (D1/12) or human diferric transferrin (Tfn) were linked to the potent cytotoxin ricin (anti-GE 2-ricin) or to its A subunit (anti-GE 2-RTA, D1/12-RTA, Tfn-RTA). Anti-GE 2-RTA had low cytotoxic activity in both the absence and the presence of lysosomotropic substances inhibiting intracellular degradation. Anti-GE 2-ricin was about 1,000 times more toxic than RTA alone, but showed only 14-fold target specificity. D1/12-RTA was about 20 times more toxic than RTA and its cytotoxic effect increased about 6- to 7-fold when cell-surface HLA-DR antigen expression was enhanced by IFN-gamma treatment. Human diferric Tfn linked to RTA demonstrated the highest cytotoxic activity, being about 5,000 times more toxic than RTA alone for glioma cells and about 6,000 times more toxic for Jurkat cells in the presence of the carboxylic ionofore monensin. Ricin toxin was only about 5 times more toxic for Jurkat and glioma cells than Tfn-RTA-monensin. Tfn-RTA was over 100,000 times more potent than the chemotherapeutic agent BCNU in reducing glioma cell survival in vitro. Addition of 80% human pooled cerebrospinal fluid (CSF) reduced Tfn-RTA toxicity about 10-fold. Kinetics of Tfn-RTA cytotoxicity at non-saturating concentrations indicated that over 80% of target cells could be killed within 8-10 hr in the absence and within 10-12 hr in the presence of human pooled CSF.

A C terminus cysteine of diphtheria toxin B chain involved in immunotoxin cell penetration and cytotoxicity.

The role of diphtheria toxin (DT) B-chain subdomains in DT cytotoxicity and immunotoxin mechanism of action has been investigated. OKT3 (mAb to the CD3 surface Ag of human T lymphocytes) was conjugated to DT or the DT mutant CRM 1001, which has a cys----tyr substitution at position 471 of the B chain. OKT3-CRM 1001 immunotoxin was about 1400-fold less cytotoxic for CD3 Jurkat cells than OKT3-DT and had a 12-fold slower kinetics of protein synthesis inactivation, CRM 1001 killed DT-sensitive Vero cells at a 5000-fold higher concentration than DT. Its cell surface-binding activity was comparable to DT. Based on kinetics of cell inactivation, toxicity determination at low extracellular pH and Triton X-114 distribution, it was concluded that CRM 1001 is defective in at least one crucial step of toxin penetration and is unable to cross cell membranes as efficiently as DT. The substituted cysteine appears to be important for DT translocating functions. Data on the function of DT B-chain subdomains are relevant for the study of whole toxin conjugates and their mechanism of action.