The role of ricin B chain in the intracellular trafficking of anti-CD5 immunotoxins

The mAb anti-CD5 was linked to purified ricin A chain (RTA) or intact ricin (Rc) containing B chain to determine the role of ricin B chain in the intracellular trafficking of anti-CD5 immunotoxins (IT). IT were radiolabeled with iodine-125 and then studied for their subcellular compartmentalization in an acute lymphoblastic leukemia T cell line, CEM. Ricin A chain IT was not as toxic to CEM cells as Rc-IT in protein synthesis inhibition assays. This difference was not attributed to differential binding or modulation of the CD5 determinant from the cell surface as measured by FACS analysis. However, we found a relationship between the toxicity of anti-CD5-Rc and anti-CD5-RTA and their ability to traffic to CEM lysosomes. Kinetic analysis of the transfer of radioimmunotoxin to the lysosomes showed that anti-CD5-Rc was trafficked significantly more slowly than anti-CD5-RTA, perhaps due to an extended period of time in the Golgi compartment. The possibility of a Golgi interaction was tested by adding monensin, a carboxylic ionophore that interrupts trafficking through the Golgi, to cells treated with anti-CD5-RTA. The addition of monensin caused anti-CD5-RTA to traffic in a manner identical to anti-CD5-Rc. We conclude that 1) B chain slows trafficking of anti-CD5-Rc to the lysosomes; 2) the rate-limiting step in the toxicity difference between anti-CD5-Rc and anti-CD5-RTA is the rate of transfer to the lysosomes; and 3) trafficking through the Golgi may be important for anti-CD5-IT toxicity.     

Evaluation of ricin A-chain immunotoxins directed against human T cells

We have synthesized four immunotoxins (ITs) by covalently coupling the A chain of ricin to murine monoclonal antibodies that recognize surface antigens on human T cells. Treatment of human peripheral blood lymphocytes with either 10.2-A, directed against the CD5 (Tp67) antigen, or 64.1-A, directed against the CD3 (Tp19) antigen, abolished protein synthesis in cells subsequently cultured with phytohemagglutinin (PHA). In contrast, two other ITs (9.6-A and 35.1-A), both directed against the CD2 (Tp50) antigen, had minimal effects on protein synthesis in PHA-stimulated cells. The binding of each IT to T cells was shown by immunofluorescence with fluorescein-conjugated goat anti-mouse immunoglobulin (FITC-GAMIg) and fluorescein-conjugated rabbit anti-ricin A-chain (FITC-RAR) antibodies. Activity of the ricin A chain in each IT was demonstrated by its ability to inhibit protein synthesis in a cell-free reticulocyte lysate assay. Ultrastructural immunoperoxidase analysis of IT internalization showed that ineffective and effective ITs were endocytosed at the same rate (50% of cells had labeled endosomes after 15 min). However, ineffective IT 35.1-A was more rapidly delivered to lysosomes (15-30 min) than effective ITs (10.2-A and 64.1-A) (greater than or equal to 30 min). The data support the hypothesis that there are several distinct pathways for internalization of ITs and that the ability of ricin A chain to reach and inactivate ribosomes may depend upon the specific membrane receptor involved in binding a given IT, its route of internalization, and the rate of entry of the IT into lysosomes.     

Whole ricin and recombinant ricin A chain idiotype-specific immunotoxins for therapy of the guinea pig L2C B cell leukemia

The therapeutic efficacy of whole ricin, or recombinant ricin A chain, coupled to a monoclonal antibody that reacts with the idiotype of the surface IgM expressed on guinea pig L2C lymphoblasts, was assessed. In vitro studies were done to characterize the immunotoxins (IT) and to demonstrate their specificity before use in vivo. The concentration of whole ricin IT (M6-Ricin) that inhibited protein synthesis by 50% (IC50) in L2C cells was 1.4 X 10(-9) M, in a 5-hr assay, in the presence of lactose to block non-antibody-directed toxicity. M6-Ricin did not inhibit protein synthesis in two control guinea pig cell lines that did not express the idiotype, nor did a whole ricin IT prepared with an isotype-matched monoclonal antibody of irrelevant specificity inhibit protein synthesis in L2C cells. Two recombinant ricin A chain IT, which differed from one another by a factor of 2 to 3 in the number of A chains conjugated per antibody molecule, were less effective in vitro than M6-Ricin (IC50 of greater than 5 X 10(-8) M). For in vivo experiments, the IT were given by the i.p. route 24 hr after the i.p. inoculation of 1 X 10(5) L2C cells. The highest doses of M6-Ricin and M6-Ricin A chain IT tested, 30 micrograms/kg and 3000 micrograms/kg, respectively, were within fourfold to fivefold of their maximum tolerated doses; no deaths or ill effects due to ricin toxicity were noted. These doses increased the median survival time of L2C-bearing guinea pigs to 31 to 34 days, compared with 15 days for untreated animals. This magnitude of increase in survival indicates that 99.999% (5 logs) of injected tumor cells were eliminated, thus accounting for the 12% long-term survival rate obtained. Median survival times for guinea pigs treated with 30 micrograms/kg of the A chain IT were 18 and 21 days for the two conjugates tested, and the median survival for guinea pigs treated with 3000 micrograms/kg of unconjugated antibody was 18 days. Our data demonstrate that recombinant A chain IT are active in vivo and that the B chain of ricin can potentiate IT activity in vivo. Although the potency differs by 100-fold, the therapeutic index of the intact ricin IT is similar to that of the ricin A chain IT.     

A flexible peptide spacer increases the efficacy of holoricin anti-T cell immunotoxins

Immunotoxins, constructed by chemically cross-linking an antibody and protein toxin, do not possess the high efficacy of the native toxin. Decreases in toxicity are due in part to the steric constraints imposed on the two macromolecules, which result in both decreased antibody binding and toxin function. In examining the structural features that influence efficacy in holotoxin-antibody conjugates, it was found that the incorporation of a 29-residue polypeptide, derived from the insulin B chain between the antibody and ricin moiety, resulted in an increase in both potency and efficacy. In a murine model system, potency of the peptide spacer conjugate was increased nearly 10-fold; however, when examined by the procedure used to purge bone marrow, the peptide spacer conjugate was not demonstrably more toxic to nontarget cells than the nonspacer conjugate. Thus, in addition to increases of efficacy and potency, this novel immunotoxin demonstrated increased specificity by approximately 10-fold. To test the general utility of peptide spacer inclusion, a T101-ricin conjugate was constructed with the peptide spacer. It yielded a protein synthesis inhibition rate of -0.6 log/h on MOLT-3 cells, greater than 10-fold more efficacious than a previously constructed nonspacer T101-ricin conjugate examined under similar conditions.     

Enhancing effects of monocytes on modulation of a lymphocyte membrane antigen

Redistribution, or modulation, of some cell surface antigens occurs in the presence of specific antibody. The phenomenon of antigenic modulation may therefore affect the use of antibodies as therapeutic agents. This study was undertaken to investigate modulation of the 65,000 dalton T65 antigen, present on normal and malignant T cells and some malignant B cells, which is recognized by the monoclonal antibody T101. To induce cell surface antigenic modulation, normal or leukemic lymphoid cells were cultured in the presence of monoclonal antibody T101 for 3-hr periods. Removal of monocytes from mononuclear cell preparations resulted in significantly lower degrees of T65 antigenic modulation. The degree of antigenic modulation could be increased by adding monocytes back to monocyte-depleted lymphocyte suspensions. Furthermore, maximal modulation occurred in the presence of monocytes at T101 concentrations that were 3 logs lower than in the absence of monocytes. The enhancing effect of monocytes was dependent on the Fc portion of the T101 antibody molecule, and presumably was mediated by cross-linking of antigen-antibody complexes on the surface membrane of the modulating cell by Fc receptors present on monocytes. Further experiments performed to examine the characteristics of this enhancement of antigenic modulation by monocytes indicated that autologous as well as allogeneic monocytes were effective, indicating that the enhancing phenomenon was not dependent upon recognition of major histocompatibility antigens. Viable monocytes were required, but pretreatment of monocytes with sodium azide to inhibit energy production, or indomethacin to inhibit prostaglandin synthesis had no effect on this phenomenon. Polymorphonuclear leukocytes did not mediate similar enhancement, although monocytic and myeloid cell lines U937, THP-1, and HL-60 did. Spent culture medium from modulated cultures and preparations containing IL 1 activity did not enhance modulation of the T65 surface antigen on lymphocytes, suggesting that direct contact between lymphocytes and monocytes is required to mediate the effect. The finding that leukemic cells from patients with CLL undergo modulation of the T65 antigen to a much lower degree in vitro than observed in vivo, and that this difference can be overcome by the addition of monocytes, suggests that monocytes or the reticuloendothelial system may augment antigenic modulation in vivo.     

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.     

Endocytosis of an antibody ricin A-chain conjugate (immuno-A-toxin) adsorbed on colloidal gold. Effects of ammonium chloride and monensin

An immunotoxin (IT) formed by a specific antibody coupled to the ricin A chain was adsorbed on colloidal gold particles (IT-Au). Binding and internalization of IT-Au in human lymphoblastic CEM cells were studied using electron microscopy. IT-Au showed specific cytotoxic activity toward the target cells. After 1 h at 4 degrees C, IT-Au were linked diffusely to the plasma membrane with 45% of the particles regrouped in clusters. Upon transfer to 37 degrees C, the particles carrying the ligand were regrouped more frequently and internalized into the cell by endocytosis through smooth microinvaginations or coated pits of the plasma membrane. After 15 min, IT-Au was observed in endocytic vacuoles, or receptosomes, in tubular structure near the Golgi apparatus and in lysosomes. Entry of IT-Au into lysosomes was rapid (around 50% of intracellular IT-Au particles after 30 min). NH4Cl or monensin, well-known potentiators of immunotoxin activity, when present in incubation medium, altered neither the processes nor the rate of IT-Au endocytosis. In the presence of either of these substances, IT-Au accumulated in the normal or often enlarged endocytic vacuoles, and entry into the lysosomes was slowed down (50% of particles after 2 h 15 min). We conclude that this intense slowing-down in the speed of IT-Au transportation into lysosomes and the functional modifications of these organelles help to explain the increased efficacy of immunotoxins in the presence of potentiators.     

Somatic cell mutants resistant to ricin, diphtheria toxin, and to immunotoxins

The human B-cell line Namalwa expresses the common acute lymphoblastic leukemia antigen (CALLA). Frame-shift mutants in Namalwa cell cultures were generated with ICR-191, and mutants were then selected for resistance to ricin or resistance to a conjugate of ricin with the anti-CALLA antibody J5 in the presence of lactose. Three mutants were found that were resistant to ricin and were in addition shown to be resistant to diphtheria toxin, to a J5-ricin conjugate, and to a conjugate between ricin B-chain and gelonin. The mutants, however, were sensitive to a J5-gelonin conjugate. These mutants expressed high levels of CALLA and/or receptors for ricin, and their cell-free translation systems appeared to be as sensitive to the inhibitory action of ricin A-chain and of gelonin as the translation system of wild-type Namalwa cells. The behavior of these mutants was consistent with the hypothesis that these cells possess an alteration of their surface that impedes the passage of ricin and diphtheria toxin across the plasma membrane. A fourth mutant was found to bind reduced quantities of ricin and was resistant to ricin but was sensitive to J5-ricin. The properties of this cell line provide evidence that the binding of antibody-ricin conjugates to cells via the ricin moiety may be prevented without impeding the cytotoxicity of the conjugates.     

Ricin A-chain containing immunotoxins directed against different epitopes on the CD2 molecule differ in their ability to kill normal and malignant T cells

Immunoconjugates consisting of mAb covalently coupled to plant or bacterial toxins or to cytotoxic drugs have provided novel experimental reagents for the treatment of malignancies and autoimmune diseases. In this report, we analyzed the efficacy of three ricin A chain-containing immunotoxins (IT-A) which recognize different epitopes on the CD2 molecule (E rosette receptor) on human T cells. Although all IT-A had similar binding avidities and A-chain activities, one (RFT11-A) was 100-1000-fold more effective in killing normal and malignant T cells than the others (35.1-A, 9.6-A). Immunoprecipitation experiments confirmed that all IT-A bound to the CD2 molecule. However, cross-blocking experiments, differential proteolysis with trypsin, and T cell co-activation experiments showed that the less effective IT-A, 35.1-A and 9.6-A, bound to an epitope far from the cell membrane (region I), whereas the more effective IT-A, RFT11-A bound to an epitope closer to the membrane (region II). Using cellular RIA and immunoelectron microscopy, it was shown that both RFT11-A and 35.1-A were rapidly internalized by T cells, but that their intracellular fates differed. The more toxic IT-A, RFT11-A, was retained for longer periods of time inside the cells and was more slowly degraded than the less effective IT-A, 35.1-A, which was rapidly transported to lysosomes, digested, and expelled. These results demonstrate that different IT-A targeting the same surface molecule can differ markedly in potency, and that the epitope recognized by an IT-A may have a significant impact on the ability of the IT-A to insert into cell membranes, translocate to the cytosol, and kill cells.     

Blockade of the galactose-binding sites of ricin by its linkage to antibody. Specific cytotoxic effects of the conjugates

A method is described for preparing specific cytotoxic agents by linking intact ricin to antibodies in a manner that produces obstruction of the galactose-binding sites on the B chain of the toxin and so diminishes the capacity of the conjugate to bind non-specifically to cells. The conjugates were synthesised by reacting iodoacetylated ricin with thiolated immunoglobulin and the components of conjugate with reduced galactose-binding capacity were separated by affinity chromatography on Sepharose (a beta-galactosyl matrix) and asialofetuin-Sepharose. Fluorescence-activated cell sorter (FACS) analyses revealed that the fraction of a monoclonal anti-Thy1.1-ricin conjugate that passed through a Sepharose column had markedly diminished capacity to bind non-specifically to Thy1.2-expressing CBA thymocytes and EL4 lymphoma cells. The fraction of conjugate that passed through an asialofetuin-Sepharose column displayed no detectable non-specific binding. Both fractions of conjugate were potent cytotoxic agents for Thy1.1-expressing AKR-A lymphoma cells in tissue culture. They reduced the [3H]leucine incorporation of the cells by 50% at a concentration of 2-5 pM. Comparable inhibition of EL4 cells was only achieved with 3000-7500-fold greater concentrations of conjugate. By contrast, the fraction of anti-Thy1.1-ricin that retained Sepharose-binding capacity showed marked non-specific binding and toxicity to EL4 cells. A conjugate with diminished galactose-binding capacity was also prepared from the W3/25 monoclonal antibody which recognises an antigen upon helper T-lymphocytes in the rat. It elicited powerful and specific toxic effects upon W3/25 antigen-expressing rat T-leukaemia cells. This finding is of particular importance because isolated ricin A-chain disulphide-linked to W3/25 antibody is not cytotoxic. The property of the B-chain in intact ricin conjugates that facilitates delivery of the A-chain to the cytosol thus appears to be independent of galactose recognition. It is concluded that the 'blocked' ricin conjugates combine the advantages of high potency, which is often lacking in antibody-A-chain conjugates, with high specificity, which previously was lacking in intact ricin conjugates.     

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.     

Interaction between ricin and Zajdela ascitic hepatoma cells

The binding to and toxicity of ricin on Zajdela hepatoma ascites cells were studied. The kinetic analysis of [125I]-ricin binding to hepatoma cells indicated that maximal specific binding was reached within 30 min. at 4 degrees C and 60 min. at 25 degrees C and that toxin binding to hepatoma cells was saturable. When the binding data were plotted according to the method of Scatchard, curvilinear graphs were obtained suggesting that hepatoma cells have both high and low affinity receptors for ricin. The number of high and low affinity receptors was identical at 4 and 25 degrees C, i.e., 8 x 10(5) and 1.2 x 10(7) sites per cell respectively. However, the capacity of hepatoma cells to bind ricin is stronger at 4 degrees C than at 25 degrees C. The toxic activity of ricin was totally abolished in the presence of lactose suggesting that ricin binding to cells occurs through binding sites containing galactosyl residues.     

Inhibition of the helper function of murine T cells with Fab'-anti-L3T4 ricin A chain immunotoxin

The ability of Fab'-anti-L3T4 A chain-containing immunotoxins to inhibit the helper function of keyhole limpet hemocyanin-specific T helper lymphocytes was evaluated. Keyhole limpet hemocyanin-specific T helper cells were prepared from the lymph nodes of primed mice and were enriched for T cells. Enriched populations of trinitrophenyl-specific B cells were prepared from spleens of normal mice. In the presence of antigen, the keyhole limpet hemocyanin-specific T helper cells were able to induce proliferation and differentiation of the trinitrophenyl-specific B cells. However, when the T helper cells were first treated with an immunotoxin composed of Fab' fragments of anti-L3T4 antibody coupled to ricin A chain (Fab'-anti-L3T4-A), they failed to induce proliferation and differentiation of the antigen-specific B cells. The concentrations of Fab'-anti-L3T4-A required to inhibit T cell help for the proliferation and differentiation of trinitrophenyl-specific B cells by 50% were 1 X 10(-9)M and 4 X 10(-10) M, respectively. Fab'-anti-L3T4 antibody alone did not inhibit T cell-induced B cell proliferation and differentiation by 50% at greater than 100-fold higher concentrations.     

Human chorionic gonadotropin-ricin A chain hybrid protein: a hormone analog for the study of signal transduction

We have prepared a disulfide cross-linked hybrid consisting of human chorionic gonadotropin and the toxic ricin A chain, utilizing N-succinimidyl-3-(2-pyridyldithio)-propionate. The purified hybrid exhibited cytotoxicity toward rat testis Leydig cells due to inhibition of protein synthesis within the cells, although it inhibited protein synthesis in a cell-free system of a rabbit reticulocyte lysate only after treatment of the hCG-A hybrid with dithiothreitol. On the other hand, pretreatment of the hCG-A hybrid with dithiothreitol completely abolished its cytotoxicity. The hCG-A hybrid stimulated the testosterone production by rat testis Leydig cells to a similar degree to hCG. The hCG-A hybrid could elicit full testosterone production in the presence of about 1/3-1/2 less accumulated cAMP compared with in the case of hCG stimulation. The steroidogenetic effect of the hCG-A hybrid was largely inhibited by anti-hCG antibody but not by anti-ricin A chain antibody. These results suggest that the hCG-A hybrid first binds to Leydig cells via an hCG receptor and then is internalized into the cells, and finally the inhibition of cellular protein synthesis occurs on the release of the ricin A chain from the hybrid. Furthermore, it was suggested that hCG may stimulate testosterone production via another second messenger system as well as via the cAMP system, since the hCG-A hybrid stimulated full testosterone production without maximal cAMP accumulation.     

Comparison of blocked and non-blocked ricin-antibody immunotoxins against human gastric carcinoma and colorectal adenocarcinoma cell lines

Summary To avoid non-specific binding of intact ricin-antibody conjugates, we prepared a new blocked thioether-linkedricin-antibody IT, in which the galactosebinding site of ricin had lost the ability to bind to galactosidic residues of Sepharose 6B gel. As carrier agent, the monoclonal antibody AR-3, which defines the CAR-3 tumour-associated antigenic determinant expressed selectively on different human carcinoma cell lines, was used. Purification of the new conjugate was performed in three sequential steps: (1) by HPLC gel filtration on TSK G3000SW to remove the unconjugated ricin: (2) by affinity chromatography on Affi-Gel Blue to separate the free antibody from the conjugate and (3) by affinity chromatography on Sepharose 6B to separate the galactose-binding IT from the non-binding moiety. The cytotoxicity of the blocked and non-blocked thioether-linked IT was compared with that of classical ricin-antibody IT conjugated via SPDP and that of ricin A chain IT. The comparison was made on two different target cell lines (KATO III human gastric carcinoma and HT-29 human colorectal carcinoma) versus two control cell lines (HL-60 promyelocytic pre-leukaemic and COLO38 melanoma). The results showed that the blocked thioether IT displayed a more selective toxicity to target cells than the non-blocked IT and was much more potent than the ricin A chain conjugate.This work was supported by Grants from the Italian National Research Council (C.N.R. PFTBMS n. 86.01476.57 and n. 86.01748.03), the Ministry of Public Education and the Italian Association for Cancer Research (AIRC) Abbreviations used: IT, immunotoxin; PBS, phosphate-buffered saline (100 mM sodium phosphate, 150 mM NaCl); SPDP, N-succinimidyl-3-(2-pyridyldithio)-propionate; PBS-EDTA, phosphatebuffered saline containing 1 mM EDTA; DMF, dimethylformamide; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SIA, N-hydroxuccinimidyl ester of iodoacetic acid; SATA, N-succinimidyl-S-acetylthiolacetate     

In vivo potentiation of ricin toxicity by monensin delivered through liposomes.

Monensin, a carboxylic ionophore, which is known to raise intravesicular pH, was intercalated in liposomes and its effect on the toxicity of ricin in mice was studied. The toxicity of ricin in vivo was found to be significantly enhanced by the administration of monensin intercalated in liposomes (liposomal monensin). The observed enhancement of the toxicity of ricin by monensin was highly dose-dependent and was maximal when ricin was injected within 60 min of monensin injection. The survival time was found to be reduced in the range of 8-20 h, depending on the dose of ricin used, by liposomal monensin. Stability of liposomes containing monensin as inferred from the release of entrapped calcein or FITC-dextran under both in vivo and in vitro conditions was comparable to that observed for liposomes without monensin. Liposomal monensin remains in circulation for 2 h and was cleared from the blood stream after 4 h. In contrast, 15 min was required for the clearance of monensin when administered in free form. Studies on the distribution of liposomal monensin and 125I-ricin in various tissues have revealed that monensin is mainly localized in the liver and spleen which are also the major sites for ricin accumulation. Our observation on the substantial enhancement of ricin toxicity in vivo by liposomal monensin strongly supports the potential usefulness of the latter as a potentiating agent in the enhancement of the toxicity of immunotoxin or hormonotoxin for selective elimination of cancer cells.     

Development of an antigen-specific CD8 suppressor effector clone in man

A long-term cultured IL-2-dependent keyhole limpet hemocyanin (KLH)-specific CD8 (T8) suppressor clone (5B9) was generated from a healthy donor hyperimmunized with KLH. The 5B9 clonal population suppressed in vitro anti-KLH antibody response but did not suppress anti-TT antibody response or PWM-driven IgG synthesis. Moreover, 5B9 cells could not suppress anti-TT antibody response even in the presence of free KLH. 5B9 cloned cells suppressed the anti-KLH antibody response of B cells cultured with CD4+4B4+ cells without requiring the presence of CD8+ cells. This KLH-specific CD8 suppressor clone is an effector type rather than an inducer type of suppressor T cell. The cloned cells expressed alpha- and beta-TCR proteins (defined by WT-31 antibody) on their cell surface. More importantly, the CD3:TCR complex was functionally important in the suppression induced by this clone, because after CD3 antigen modulation from its cell surface, the suppressor effector function was abolished.     

Toxicity of ligand and antibody-directed ricin A-chain conjugates recognizing the epidermal growth factor receptor

Approximately equal amounts of 125I-mAb 225 (a monoclonal antibody recognizing the human epidermal growth factor receptor) and 125I-labeled epidermal growth factor (125I-EGF) were bound by HeLa cells. However, these two EGF receptor binding moieties had different fates after binding. Sixty percent of cell-associated 125I-EGF was internalized. The majority of internalized 125I was released from the cell within 2 hr. In contrast, whereas only 30% of bound 125I-mAb 225 was internalized by HeLa cells, the internalized radioactivity remained cell-associated. EGF and mAb 225 were used to construct ricin A-chain (RTA) conjugates. The two chimeric molecules, EGF-RTA and mAb 225-RTA, were equally toxic to human HeLa cells. EGF-RTA was also toxic to murine 3T3 cells. In contrast, mAb 225-RTA was not toxic to 3T3 cells, consistent with the human EGF-receptor specificity of mAb 225. Neither conjugate was cytotoxic to EGF receptor-deficient 3T3-NR6 cells. Rapidity and potency of protein synthesis inhibition of HeLa cells were equivalent for the two chimeric conjugates, as was the degree to which colony-forming ability was reduced. However, ammonium chloride enhanced the toxicity of EGF-RTA but not mAb 225-RTA, suggesting that the two toxic chimeric toxins--like the unconjugated receptor-binding moieties--are processed differently by HeLa cells.     

Effects of Physical and Chemical Treatments on the Biological Activity of Ricin D

Effects of physical and chemical treatments on the cytoagglutinating activity, toxicity and inhibitory activity of cell-free protein synthesis of ricin D or its constituent polypeptide chains were investigated. The results indicated that the isolated polypeptide chains were much less stable than intact ricin D in acidic pH, heating as well as chemicals, and the Ala chain was more unstable than the lie chain.

Chemical modifications of ricin D with specific reagents revealed that the tryptophan and tyrosine residues as well as the carboxyl groups participated in the phenomena of cyto- agglutination and toxic action of ricin D, whereas arginine residues were considered not to be directly involved. Trinitrophenylation of free amino groups did not result in a loss of cytoagglutinating activity, whereas caused a loss of toxicity, suggesting that free amino groups in the lie chain were involved in the toxic action of ricin D.     

Mathematical modeling for antibody therapy in the setting of antigenic modulation

The in vivo efficacy of passive monoclonal antibody therapy is limited in certain systems by the process of antigenic modulation. We describe a compartmental model which addresses the kinetics of in vivo cell binding of murine monoclonal antibody T101, modulation of the T65 target antigen, serum levels of T101, and elimination of target cells. Observed data compare favorably to that predicted by the model. The model suggests that there is no rationale for administering T101 as a prolonged, continuous infusion for passive antibody therapy.