Uptake of native and deglycosylated ricin A-chain immunotoxins by mouse liver parenchymal and non-parenchymal cells in vitro and in vivo

The therapeutic activity of ricin A-chain immunotoxins is undermined by their rapid clearance from the bloodstream of animals by the liver. This uptake has generally been attributed to recognition of the mannose-terminating oligosaccharides present on ricin A-chain by receptors present on the non-parenchymal (Kupffer and sinusoidal) cells of the liver. However, we demonstrate here that, in the mouse, the liver uptake of a ricin A-chain immunotoxin occurs in both parenchymal and non-parenchymal cells in equal amounts. This is in contrast to the situation in the rat, where uptake of the immunotoxin is predominantly by the non-parenchymal cells. Recognition of sugar residues on the A-chain portion of the immunotoxin plays an important role in the liver uptake by both cell types in both species. However it is not the only mechanism since, firstly, an immunotoxin containing ricin A-chain which had been effectively deglycosylated with metaperiodate and cyanoborohydride was still trapped to a significant extent by hepatic non-parenchymal cells after it was injected into mice. Secondly, deglycosylation, while eliminating uptake of the free A-chain by parenchymal and non-parenchymal cells in vitro, only reduced the uptake of an immunotoxin by either cell type by about half. Thirdly, the addition of excess D-mannose or L-fucose inhibited the uptake of free A-chain by mouse liver cell cultures by more than 80% but only inhibited the uptake of the native A-chain immunotoxin by about half and had little effect on the uptake of the deglycosylated ricin A-chain immunotoxin. Recognition of the antibody portion of the immunotoxin by liver cells seems improbable, since antibody alone or an antibody-bovine serum albumin conjugate were not taken up in appreciable amounts by the cultures. Possibly attachment of the A-chain to the antibody exposes sites on the A-chain that are recognised by liver cells in vitro and in vivo.     

Trichokirin, a ribosome-inactivating protein from the seeds of Trichosanthes kirilowii Maximowicz. Purification, partial characterization and use for preparation of immunotoxins

A protein, here named trichokirin, was extracted from the seeds of Trichosanthes kirilowii and purified by ion-exchange and gel-filtration chromatography. Trichokirin is a basic glycoprotein of apparent relative molecular mass of 27,000 with a strong ribosome-inactivating activity. Alignment of the trichokirin, trichosanthin and momordin N-terminal sequences shows a substantial degree of homology. Trichokirin was conjugated to a monoclonal antibody directed against the Thy 1.2 antigen with the cleavable dimethyl 3,3'-dithiobispropionimidate cross-linking reagent. This immunotoxin selectively killed leukemia cells expressing the Thy 1.2 antigen. The addition of ammonium chloride, which increases the cytotoxicity of ricin A-chain immunotoxins, blocks that of the trichokirin immunotoxin, suggesting that they enter cells by different mechanisms. In vivo studies showed that the pharmacokinetic properties of the trichokirin immunotoxin could be more advantageous than those of the ricin A-chain immunotoxins for in vivo applications.     

Carbohydrate-specifically polyethylene glycol-modified ricin A-chain with improved therapeutic potential

Ricin A-chain, which exhibits excellent cytotoxicity to tumor cells, has been widely used as an immunotoxin source. However, it has the fatal shortcoming of poor pharmacokinetics due to the tremendous liver uptake via carbohydrate-mediated recognition. Modification of proteins with polyethylene glycol, PEGylation, has the advantages of shielding the specific sites and prolonging the biological half-life. In this study, the carbohydrate-specific PEGylation of ricin A-chain was considered to be a novel approach to overcome this limitation. The carbohydrate group of ricin A-chain was oxidized by sodium m-periodate and further specifically conjugated with hydrazide-derivatized PEG. For a comparative study, the PEGylated ricin A-chain at amino groups was prepared using the hydroxysuccinimide ester-derivatized PEG. The carbohydrate-specifically PEGylated ricin A-chain showed a markedly lower liver uptake and systemic clearance compared with the amine-directly PEGylated ricin A-chain as well as the unmodified ricin A-chain. Furthermore, carbohydrate-specifically PEGylated ricin A-chain showed a significantly higher in vitro ribosome-inactivating activity than the amine-directly PEGylated ricin A-chain. These findings clearly demonstrate that the carbohydrate-specificity as well as PEGylation plays an important role in improving the in vivo pharmacokinetic properties and in vitro bioactivity. Therefore, these results suggest that the therapeutic use of immunotoxins constructed using this carbohydrate-specifically PEGylated ricin A-chain has potential as a cancer therapy.     

Comparison of the cytotoxic potency of T101 Fab, F(ab')2 and whole IgG immunotoxins

The in vitro killing of the human CEM cell line was studied by using ricin A-chain immunotoxins constructed with either the whole IgG or the Fab and F(ab')2 fragments of the same T101 (anti-CD5) antibody. In the presence of ammonium chloride as an activator, the "whole" immunotoxin as well as the "fragment" immunotoxins did not show any significant difference in the cell killing efficacy. In contrast, without the activator, the efficacy of the T101 immunotoxin was greatly improved when fragments were used. Indeed, at a saturating dose, a cytoreduction of three orders of magnitude was obtained with the fragment immunotoxins vs less than one order of magnitude for the whole immunotoxin, as assessed in a clonogenic assay. This enhancing effect was related to better cell killing kinetics, because with a similar amount of A-chain molecules bound per cell, T101 fragment immunotoxins achieved a twofold faster protein synthesis inactivation rate than the corresponding whole IgG immunotoxin. No significant difference in activity was shown between monovalent (Fab) and divalent (F(ab')2) forms of fragment immunotoxins. The observation that T101 fragment immunotoxins were more potent than intact immunotoxins was extended to another fragment immunotoxin constructed with an antibody (F111.98) directed against a different epitope of the CD5 Ag. In another model (anti-CD22 1G11 antibody on Raji cells), the fragment immunotoxin did not show any superiority over the IgG immunotoxin which was by itself very potent, strongly suggesting an Ag-dependent phenomenon.     

In vivo cytotoxic efficacy of immunotoxins prepared from anti-CD5 antibody linked to ricin A-chain

Summary The antitumoral efficacy of various anti-CD5 immunotoxins, prepared with whole monoclonal antibody (mAb), F(ab)₂ or Fab fragment linked to native ricin A-chain (RTA) or partially deglycosylated ricin A-chain (dRTA), was examined in vivo in ascitic nude mice bearing a large burden of Ichikawa human tumour cells. We first demonstrated that after systemic administration of IgG-RTA or F(ab)₂-dRTA, the cytotoxic activity of immunotoxin molecules specifically bound to tumour cells was preserved. Secondly we showed, by using different immunotoxins with various targeting capacities, that their cytotoxic effect in vivo was related to the number of immunotoxin molecules bound per cell. However, even when antigen saturation was achieved after i.p. injection, the cytotoxic effect did not exceed 53% of the tumour burden. By contrast, when the immunotoxin was administered i.p. or i.v. with the enhancer monensin conjugated to human serum albumin and injected i.p., 90% of the tumour cells were killed. This potentiating effect was demonstrated even when the tumour localisation was as low as 5% of the saturation level. Such an effect could be completely prevented by addition of unconjugated monoclonal antibody, demonstrating the specificity of the immunotoxin-induced cytotoxicity in the presence of the enhancer. However this enhancement was demonstrated whatever the route of immunotoxin administration, i.p. or i.v., but was only observed when the enhancer was injected i.p. and not i.v.. These results emphasize the importance of optimizing the therapeutic course to improve the antitumoral efficacy of immunotoxins.     

Purification of immunotoxins containing ricin A-chain and abrin A-chain using Blue Sepharose CL-6B

We describe a method for separating antibody from immunotoxins by affinity chromatography on Cibacron blue F3GA coupled to Sepharose (Blue Sepharose). The antibody did not bind to the gel. The immunotoxins were bound by their ricin A-chain or abrin A-chain moiety and could be recovered in high yield and purity using mild elution conditions. The method is suitable for the large-scale purification of immunotoxins.     

Brefeldin-A enhancement of ricin A-chain immunotoxins and blockade of intact ricin, modeccin, and abrin

After binding, the protein toxins ricin, abrin, and modeccin are endocytosed and processed through the cell's vesicular system in a poorly understood fashion, prior to translocation to the cytosol. The role of the Golgi apparatus in toxin processing was studied using brefeldin-A (BFA), a fungal metabolite which blocks Golgi function. At concentrations that inhibit secretion of interleukin-2 (IL-2), BFA blocks ricin, modeccin, and abrin intoxication of a lymphocyte derived cell line (Jurkat). Paradoxically, BFA enhances the toxicity of two ricin A-chain immunotoxins targeted against distinct cell surface determinants. BFA concentrations which are optimal for immunotoxin enhancement are below those needed to affect ricin intoxication or IL-2 secretion. BFA blockade of ricin does not involve effects on ricin endocytosis, toxin translocation to the cytosol, or the enzymatic activity of toxin A-chain. In contrast, BFA has no effect on immunotoxin processing but does enhance the immunotoxin translocation step. It is concluded that: 1) intact Golgi function is required for holotoxin processing. 2) Intact Golgi function is not required for holotoxin translocation. 3) Golgi function is tightly linked to immunotoxin translocation. 4) BFA has effects on vesicular routing in addition to the block of Golgi function in secretion which has been reported.     

Comparison of the cellular internalization of antibodies used either as immunotoxins or in ADEPT

The internalization into tumor cells of two antibodies (C242 and 454A12), which make potent immunotoxins when linked to ricin A-chain, and an antibody (A5B7), which does not make a potent immunotoxin but has proven useful in ADEPT, was evaluated. The 454A12 antibody was rapidly taken into the cells, 50% of the antibody being internalized after 2 h. The C242 antibody was internalized more slowly, approx 50% being taken up by the cells in 24 h. With A5B7, less than 10% of the antibody was internalized after 24 h. Internalization of the C242 antibody was accompanied by the appearance of antibody degradation products in the cell medium after 2 h, and this degradation could be inhibited by addition of a metabolic inhibitor that prevented cell internalization. In contrast, minimal degradation of the A5B7 antibody could be detected up to 24 h after binding to the cells. In conclusion, both 454A12 and C242 antibodies, which make potent immunotoxins, were internalized into tumor cels. The A5B7 antibody, which does not make a potent immunotoxin, was not internalized, and this property may be one reason why A5B7 has proved useful for delivery of enzymes in ADEPT.     

Retinoic acid disrupts the Golgi apparatus and increases the cytosolic routing of specific protein toxins

All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.     

Rationale for clinical use of immunotoxins in cancer and autoimmune disease

Immunotoxins constructed by conjugating monoclonal antibodies to plant and bacterial toxin molecules are being evaluated clinically for the treatment of cancer and as immunosuppressive agents in treating autoimmune diseases. Immunoconjugates constructed with ricin A-chain and in certain indications, whole ricin have been most extensively investigated. The experience with these immunotoxins has highlighted issues to be dealt with in order to improve therapeutic efficacy. Immunotoxins containing ricin A-chain conjugated to monoclonal antibody reacting with the CD5 molecule on T lymphocytes has proved most efficacious in treating acute graft versus host disease (aGvHD) in patients receiving bone marrow transplants as part of a regimen of high dose chemotherapy in leukaemias and lymphomas. This involved immunotoxin used after the onset of a GvHD or prophylactically to reduce the development of the condition. Immunotoxin treatment of leukaemias and lymphomas is also showing promise with clinical responses being observed. In comparison, treatment of solid cancers such as colorectal cancer and malignant melanoma has not yet proved effective. Factors to be resolved in order to improve treatment include better pharmacokinetic properties of immunotoxins, improved tumour penetration and the use of antibody cocktails to accommodate antigenic heterogeneity of tumours.     

Selective cytotoxic effect of an immunotoxin on human erythroid tumor cells]

The possibility of efficient directed elimination of human erythroblastoid cells by the conjugate of IgM-monoclonal antibody HAE9 directed against the erythroblast antigen and the A-chain of a plant toxin ricin has been demonstrated. The conjugate contained 2 molecules of A-chain per one antibody molecule. The efficiencies of the cytotoxic effect of native ricin and the conjugate were compared according to the number of binding sites on the surface of K562 cells as well as to the internalization rate of these molecules. As was shown, that the number of binding sites for the antibody approaches 2.7.10(4) molecules/cell, K a being equal to 1.7.10(8) M-1 while for ricin these indices constitute 2.4.10(5) and 4.6.10(8) M-1. Almost 100% of antibodies and 36% of ricin are internalized within 10 min at 37 degrees C. At a concentration 10(-11) of native ricin and 10(-10) of immunotoxin the 50% inhibition of growth of K562 cells carrying the erythroblast antigen on their surface is observed.     

Ricin A-Chain and Ricin A-Chain Immunotoxins Rapidly Damage Human Endothelial Cells: Implications for Vascular Leak Syndrome

The results of Phase I/II clinical trials indicate that ricin A-chain-containing immunotoxins cause vascular leak syndrome, characterized by hypoalbuminemia with resultant weight gain and edema. Vascular leak syndrome may be a dose-limiting factor during treatment with ricin A-chain-containing immunotoxins. In this report, we determined the effect of ricin A-chain and ricin A-chain-containing immunotoxins on human umbilical vein endothelial cells with the aim of developing an in vitro model to study vascular leak syndrome. The major findings of our study are: (1) Human umbilical vein endothelial cells undergo rapid and dramatic changes in morphology after treatment with ricin A-chain and ricin A-chain-containing immunotoxins. These changes include rounding of the cells and, eventually, the formation of gaps between them. (2) The permeability of human umbilical vein endothelial cell monolayers to passage of molecules increases after exposure to ricin A-chain or ricin A-chain-containing immunotoxins and this is consistent with the morphologic changes. (3) Human umbilical vein endothelial cells bind ¹²⁵ I-rRTA in a dose-dependent manner but binding is not specific. (4) Human umbilical vein endothelial cells are moderately more sensitive to ricin A-chain-induced inhibition of protein synthesis and proliferation than simian virus-transformed mouse endothelial cells. (5) The morphologic changes are observed 1 h after exposure to the toxins, whereas inhibition of protein synthesis is not detectable until 4 h after a similar exposure. The in vitro model represents a first step in dissecting the complex events which occur in cancer patients who develop vascular leak syndrome after treatment with ricin A-chain-containing immunotoxins.     

Potentiation of anti-carcinoembryonic antigen immunotoxin cytotoxicity by monoclonal antibodies reacting with co-expressed carcinoembryonic antigen epitopes

The initial step in ricin A-chain (RTA)-immunotoxin-mediated cell cytotoxicity involves binding to the target cell Ag through the antibody moiety. One of the factors influencing this is the affinity of the antibody component for the target cell Ag. Multiple epitopes on carcinoembryonic Ag have been mapped providing a range of mAb of known specificity. These have been used to show that the cytotoxicity of an immunotoxin containing RTA conjugated to an anti-carcinoembryonic Ag mAb (228-RTA) is potentiated by mAb recognizing different epitopes. The potentiating antibodies also increased the level of target cell binding of antibody 228. Cross-linking of cell bound antibody was not involved because monovalent fragments of a potentiating antibody were effective. The potentiating antibodies modified the binding affinity of 228 antibody increasing the t1/2 of antibody at the tumor cell surface. This increased the dwell time of cell bound antibody and using conjugates of 228 linked to albumin-tetramethylrhodamine it was shown to enhance conjugate endocytosis. These investigations indicate that enhanced antibody affinity leads to increased endocytosis of bound immunoconjugate and potentiates cytotoxicity.     

Hepatotoxicity of immunotoxins made with saporin, a ribosome-inactivating protein from Saponaria officinalis

Immunotoxins were prepared by conjugating saporin, a ribosome-inactivating protein from Saponaria officinalis, to a monoclonal antibody against the Thy1.1 antigen, or to its F(ab')2 fragment. The immunotoxins were eight- to 16-fold more toxic to mice than free saporin. Injection of the immunotoxins induced necrosis of the liver and spleen, whereas free saporin caused necrosis of the epithelium of the kidney tubules. The cytoplasm of the hepatic parenchymal cells was affected by the immunotoxins, lesions being apparent in the rough endoplasmic reticulum and, later, in the mitochondria. These changes were associated with a reduced capacity to synthesise proteins both in the intact liver and by isolated liver microsomes. Studies of the in vivo distribution showed that 90% of the free saporin was removed from the bloodstream, mainly by the kidneys, within 10 min of injection. By contrast, the immunotoxins persisted in the blood for several hours and the only organ in which they consistently accumulated was the liver. The hepatotoxic effect of the immunotoxins was not due to their binding to liver cells via the antigen-binding sites or the Fc-piece of the antibody moiety, nor was it due to hepatic recognition of carbohydrate in the immunotoxin. It is concluded that free saporin, although capable of entering liver cells, is filtered so rapidly by the kidney that liver damage does not occur to a significant extent. Filtered saporin, however, is reabsorbed by renal tubules, whose epithelial cells are damaged. The antibody-saporin conjugate is too large to filter at the glomerulus and so has greater opportunity to penetrate into and to damage the hepatic parenchymal cell.     

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

Summary 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.     

Cytotoxicity of a conjugate of ricin A-chain and monoclonal anti-allotypic antibody

A conjugate was developed between ricin A-chain and monoclonal antiallotypic antibody specific for L-chains of the rat Ig. Incubation of spleen cells with the conjugate (10(-7) M) resulted in a strong decrease of 1a-positive B-lymphocyte growth. At the same time spleen cells that lacked this antigen were not affected by immunotoxin (IT). Both the isolated antibodies and ricin A-chain did not inhibit cell growth in the same concentrations. IT injection to 10-day-old heterozygous rats (1a/1b) resulted in a partial suppression of Ig production with 1a-allotype. The conjugate was 4.5 times more effective than the isolated antibodies used to form it. The in vivo and in vitro differences in IT cytotoxicity were probably connected with insufficient efficacy of conjugate targeting in vivo.     

In vitro synergism between hybrid immunotoxins and chemotherapeutic drugs: relevance to immunotherapy of prostate carcinoma

Summary Cultured prostate carcinoma cells incubated in the presence of a novel hybrid immunotoxin and ricin A chain exhibited synergy with the chemotherapeutic drugs vinblastine, methotrexate, and bleomycin. No cooperative effect was noted with adriamycin. Under conditions where individual components of immunotoxin or chemotherapeutic drug mixtures were nontoxic or minimally toxic the immunotoxin-drug mixture exhibited marked impact on ¹⁴C amino acid incorporation into prostate carcinoma cells. Analysis of drug-treated cells by flow cytometry indicated that cells exposed to vinblastine and bleomycin bound hybrid immunotoxin antibody to a greater extent than cells not exposed to these drugs. Adriamycin did not exhibit synergistic cytotoxicity with hybrid immunotoxin. Also, adriamycin did not enhance antibody binding as evaluated by flow cytometry. The fact that hybrid monoclonal antibody-ricin A chain (HIT-RAC) conjugates inhibited uptake of ¹⁴C amino acids 3 to 10-fold within 48 h of incubation with target cells and that this inhibition was further increased 2 to 3-fold in conjunction with three out of four chemotherapeutic drugs tested may be attributed to the unique cytotoxicity imposed by the hybrid immunotoxins. The RAC moiety is not chemically coupled to antibody but instead occupies one of the antigen-combining sites of the molecule. In this manner, RAC is closely juxtaposed to the cell membrane of the target cell and is anchored in this position via binding of the remaining antigen-combining site to p40 prostate restricted antigen.     

The removal of carbohydrates from ricin with endoglycosidases H, F and D and alpha-mannosidase

Recently, several investigators have explored the possibility of targeting ricin to designated cell types in animals by its linkage to specific antibodies. There is evidence, however, that the mannose-containing oligosaccharide chains on ricin are recognised by reticuloendothelial cells in the liver and spleen and so cause the immunotoxins to be removed rapidly from the blood stream. In the present study we analysed the carbohydrate composition of ricin and examined enzymic methods for removing the carbohydrate. The carbohydrate analysis ricin A-chain revealed the presence of one residue of xylose and one of fucose in addition to mannose and N-acetylglucosamine which had been detected previously. The B-chain contained only mannose and N-acetylglycosamine. Ricin A-chain is heterogeneous containing two components of molecular weight 30 000 and 32 000. Strong evidence was found that the heavier form of the A-chain contains an extra carbohydrate unit which is heterogeneous with respect to concanavalin A binding and sensitivity to endoglycosidase H. The lower molecular weight form of A-chain did not bind concanavalin A and was insusceptible to endoglycosidases. Only one of the two high mannose oligosaccharide units on the isolated B-chain could be removed by endoglycosidases H or F, whereas both were removable after denaturation of the polypeptide by SDS. Both the isolated A- and B-chains were sensitive to alpha-mannosidase. Intact ricin was resistant to endoglycosidase treatment and was only slightly sensitive to alpha-mannosidase. The addition of SDS allowed endoglycosidase H to remove both of the B-chain oligosaccharides from intact ricin and increased the toxin's sensitivity to alpha-mannosidase. In conclusion, extensive enzymic deglycosylation of ricin may only be possible if the A- and B-chains are first separated, treated with enzymes and then recombined to form the toxin.     

Comparison of multiple anti-CEA immunotoxins active against human adenocarcinoma cells

Summary Anti-carcinoembryonic antigen (CEA) immunotoxins constructed with multiple anti-CEA antibodies (goat and baboon polyclonal, and three murine monoclonal antibodies) by covalently linking them to the A chain of ricin via a disulfide bond all function as potent and specific toxins for CEA-bearing cells, suggesting that the CEA molecule is capable of directing productive internalization of ricin A chain. The high potency of anti-CEA immunotoxins apparently makes addition of ricin B chain unnecessary for high toxic efficiency, as in some other systems, because presence of the B chain reduces target cell specificity. Several characteristics of the immunotoxins which might account for their cytotoxic potency were studied. Equilibrium association constants of the goat, baboon, and murine monoclonal C-19 antibodies with fluid-phase CEA were determined by using Langmuir plots and were found to be 8.79, 6.61, and 8.13×10⁹ M ^–1, respectively, indicating the high and similar affinities of the three antibodies toward CEA. Radioimmunoassay binding studies of the three immunotoxins with ¹²⁵I-CEA showed that the antibody portions of the molecules retained the ability to form complexes with CEA after conjugation to ricin A chain. The maximum number of anti-CEA antibody molecules bound per cell, as demonstrated by ¹¹¹In-labeled C-19 binding assays with CEA-bearing cell lines, varied from 2.65×10⁵ per cell for HT29 to 2.01×10⁶ for LoVo, with an intermediate value of 1.17×10⁶ per cell for WiDr. Cytotoxicity of the immunotoxins was assessed by inhibition of protein synthesis and expressed as a median inhibitory dose (ID₅₀). Comparison of the ID₅₀'s of each immunotoxin on the three cell lines has shown that the immunotoxin made of the monoclonal C-19 antibody is in general 6 to 7 times more cytotoxic than the goat and baboon antibody immunotoxins. The affinity of CEA-antibody binding is probably an important, but not a sole factor in determining the immunotoxin potency. The fact that the antibodies with very similar affinity toward fluid phase CEA make immunotoxins of different potency might indicate that interactions with membrane-bound CEA are more complex and/or the efficiency of internalization of various immunotoxins is different. An important factor in immunotoxin action appears to be the CEA content in target adenocarcinoma cells.Supported in part by the NIH BRSG grant SO7RRO5712, the American Cancer Society, Mass. Div. Research Grant 1543-C-1, and by the Aid for Cancer Research (Boston) award to L. V. L., and by RO1 CA 29160 and RO1 CA 39748 grants to T. W. G.     

Inhibition of hematopoietic progenitor colony growth by a monoclonal antibody against the transferrin receptor: comparison of unconjugated antibody with an immunotoxin containing recombinant ricin A chain

We studied an immunotoxin consisting of recombinant ricin A chain (rRA) conjugated to 454A12 MoAb, a monoclonal antibody which recognizes an epitope on the human transferrin receptor, and compared the ability of 454A12 MoAb-rRA immunotoxin to inhibit the growth of erythroid burst-forming units (BFU-e) and myeloid colony-forming units (CFU-c) with unconjugated 454A12 MoAb. A significant reduction in BFU-e colony growth was observed at 0.001 microgram/ml of 454A12 MoAb-rRA versus 0.1 microgram/ml of unconjugated 454A12 MoAb (p = 0.005). Comparison of the effects of 454A12 MoAb-rRA and 454A12 MoAb on myeloid colony development gave markedly different results. Unconjugated antibody had no effect on CFU-c colony growth; in contrast, 0.01 microgram/ml of 454A12 MoAb-rRA reduced the number of colonies from 139 per 1 X 10(5) to 75 per 1 X 10(5) cells plated (p = 0.0005). No myeloid progenitor colonies developed at 0.1 microgram/ml of immunotoxin. These observations suggest that 454A12 MoAb-rRA inhibits growth by a potent, ricin A chain-mediated toxic effect on any proliferating cells expressing transferrin receptors, whereas the 454A12 MoAb exerts a selective inhibitory effect primarily on erythroid progenitors by perturbing the transferrin cycle. While growth factor receptors expressed on hematopoietic cells represent promising targets for immunotoxin therapy, our data indicate that an immunotoxin could inhibit cellular proliferation by a different mechanism than the corresponding unconjugated MoAb. Depending on the antibody used, these differences may be important in trials using immunotoxins for in vivo treatment or in vitro purging of malignant hematopoietic cells.