Evidence that pinocytosis in lymphoid cells has a low capacity.

In contrast to adherent cells, human B and T lymphoblasts, marmoset monkey T lymphoblasts, and mouse T lymphoblasts do not form monolayers and have a poor ability to pinocytose. After a 10-min incubation of lymphoblasts at 37 degrees C, the level of internalized medium reached a plateau. During this time, lymphoblasts pinocytosed 3-4 femtoliters (1 fl = 10(-15) l) of medium per cell as calculated by the quantity of the entrapped pinocytic marker 5(6)-carboxyfluorescein. The levels of pinocytosed liquid did not increase during a subsequent 90-min incubation of cells at 37 degrees C. Adherent HeLa cells took up 27 fl of medium per cell per hour. Other types of adherent cells were reported by others to pinocytose 20 to 90 fl of medium per cell per hour. The process of pinocytosis in lymphoblasts appeared to be reversible since cells which were pre-loaded with carboxyfluorescein and then incubated at 37 degrees C in fresh medium lost the marker almost completely within 40 min. Similar results were obtained with horseradish peroxidase as the pinocytic marker. Further evidence that lymphoblasts have a low capacity for pinocytic internalization relative to adherent cells was obtained from the observation that Namalwa lymphoblasts were approximately 100 times more resistant to the cytotoxic action of the protein toxin gelonin than the adherent HeLa cells. Gelonin is a ribosome-inactivating toxin which is not capable of binding to cells, and its only mode for internalization appears to be pinocytosis. Ribosomes in cell lysates of the two lines were equally sensitive to gelonin. It is speculated that the poor pinocytic ability of lymphoid cells may reflect a fundamental difference between adherent and non-adherent cells and that this may impede the targeting of drugs into lymphoid cells.

Structure-function analysis of the interaction between Bax and the cytomegalovirus-encoded protein vMIA.

The viral mitochondrial inhibitor of apoptosis (vMIA) encoded by the human cytomegalovirus exerts cytopathic effects and neutralizes the proapoptotic endogenous Bcl-2 family member Bax by recruiting it to mitochondria, inducing its oligomerization and membrane insertion. Using a combination of computational modeling and mutational analyses, we addressed the structure-function relationship of the molecular interaction between the protein Bax and the viral antiapoptotic protein vMIA. We propose a model in which vMIA exhibits an overall fold similar to Bcl-X(L). In contrast to Bcl-X(L), however, this predicted conformation of vMIA does not bind to the BH3 domain of Bax and rather engages in electrostatic interactions that involve a stretch of amino acids between the BH3 and BH2 domains of Bax and an alpha-helical domain located within the previously defined Bax-binding domain of vMIA, between the putative BH1-like and BH2-like domains. According to this model, vMIA is likely to bind Bax preferentially in its membrane-inserted conformation. The capacity of vMIA to cause fragmentation of the mitochondrial network and disorganization of the actin cytoskeleton is independent of its Bax-binding function. We found that Delta131-147 vMIA mutant, which lacks both the Bax-binding function and cell-death suppression but has intact mitochondria-targeting capacity, is similar to vMIA in its ability to disrupt the mitochondrial network and to disorganize the actin cytoskeleton. vMIADelta131-147 is a dominant-negative inhibitor of the antiapoptotic function of wild-type vMIA. Our experiments with vMIADelta131-147 suggest that vMIA forms homo-oligomers, which may engage in cooperative and/or multivalent interactions with Bax, leading to its functional neutralization.

In vivo administration of lymphocyte-specific monoclonal antibodies in nonhuman primates. In vivo stability of disulfide-linked immunotoxin conjugates.

The stability in vivo and circulatory clearance of immunotoxins were assessed in rhesus monkeys. The immunotoxins studied were T cell-specific monoclonal anti-T11 antibodies conjugated by disulfide linkage to ribosome-inactivating toxins. Intact immunotoxin was detectable in the circulation of the monkeys following a single intravenous infusion. This was demonstrated by quantitative flow-cytometric analysis, gel-filtration, and sodium dodecyl sulfate-gel electrophoresis. This intact conjugate was shown to be functional in the plasma of the infused animals in an in vitro cytotoxicity assay. However, a number of factors contributed to bring the level of circulating immunotoxin to a less than optimal level. When conjugated to a ribosome-inactivating toxin, the antibody was cleared more rapidly than was the native antibody. Furthermore, following infusion, some breakdown of the conjugate occurred, resulting in the generation of detectable levels of circulating free antibody. The present data indicate the feasibility of using immunotoxins as therapeutic tools in man.

In vivo administration of lymphocyte-specific monoclonal antibodies in nonhuman primates. IV. Cytotoxic effect of an anti-T11-gelonin immunotoxin.

The cytotoxic effect of a lymphocyte-specific immunotoxin formed by disulfide conjugation of an anti-T11 monoclonal antibody with the ribosome-inactivating protein gelonin was assessed in vitro on peripheral blood T cells and in vivo on splenic and lymph node T cells of macaque monkeys. This immunotoxin was cytotoxic to proliferating peripheral blood T cells in vitro as measured by both direct and indirect assays. Two sequential intravenous infusions into macaque monkeys achieved plasma concentrations of immunotoxin far in excess of those shown to be cytotoxic for cultured T cells and coated all T cells in lymph nodes and spleen with intact immunotoxin for four days. However, the cytotoxic effect of the immunotoxin on T cells in vivo was considerably less than that predicted by the in vitro studies. Further experiments suggested that the state of activation of the targeted T cell population in vivo, or the appearance of anti-immunotoxin antibodies, which occurred in all infused monkeys, might attenuate immunotoxin-mediated cell killing in vivo. These studies illustrate the significant differences between the action of immunotoxin conjugates in vitro, and those seen when these conjugates are utilized as therapeutic agents in vivo.

An immunotoxin composed of a monoclonal antitransferrin receptor antibody linked by a disulfide bond to the ribosome-inactivating protein gelonin: potent in vitro and in vivo effects against human tumors.

An immunoconjugate was prepared containing a disulfide linker between a murine monoclonal antibody (5E9), which recognized the human transferrin receptor, and the ribosome-inactivating protein gelonin. This immunoconjugate was found to consist of two major species, 5E9-gelonin2 and 5E9-gelonin1, and a minor species of 5E9-gelonin3 and less than 10% of either free antibody or gelonin. 5E9-gelonin was extremely toxic in vitro to human tumor cell lines expressing the 5E9 antigen, including a Burkitt's lymphoma, an adult T-cell acute lymphocytic leukemia, an acute myelogenous leukemia, a promyelocytic leukemia, and a cervical carcinoma line. A 24-hour exposure to 10(-9) M immunoconjugate killed 90-99.9% of tumor cells, depending on the cell line. A 5E9-negative murine leukemia was not sensitive to this conjugate. Pharmacokinetic analysis of the disappearance of this immunoconjugate from the murine circulation revealed that it had a biphasic clearance, with an initial rapid phase with a half-life (t1/2) of 3 hours and a later, slower phase with a t1/2 of about 1 day. Analysis of blood samples by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed that a substantial degree of disulfide-linker breakdown occurred in vivo and that the 5E9-gelonin2 species was cleared more rapidly than the 5E9-gelonin1. With use of the same clonogenic assays used to measure in vitro toxicity, biologically active immunoconjugate could be detected in murine plasma for up to 24 hours after iv administration, but the concentration of immunoconjugate by this measure was considerably less than that predicted by SDS-gel electrophoresis. The ability to deliver immunoconjugate to tumor cells in vivo was studied with use of the Burkitt's lymphoma Namalwa as a xenograft in nude mice. It was possible to deliver substantial amounts of immunoconjugate to Namalwa cells in xenografted ascites with direct ip inoculation; lower but significant amounts of immunoconjugate could be delivered to this xenograft after systemic iv administration, provided the tumor burden was low. The 5E9-gelonin conjugate, when administered iv at the time of ip tumor inoculation, prolonged survival of nude mice bearing Namalwa or other human tumors as ascites xenografts and delayed or prevented the growth of subcutaneous nodules of Namalwa in an antigen-specific fashion after a single iv injection. Direct intratumoral administration also inhibited the growth of visible subcutaneous nodules of Namalwa. This immunoconjugate may be useful in the treatment of human cancer.

p53 status affects the rate of the onset but not the overall extent of doxorubicin-induced cell death in rat-1 fibroblasts constitutively expressing c-Myc.

To better understand the effects of p53 on the process of DNA damage-induced cell death, we examined the influence of p53 status on the rate of the onset and the overall extent of cell death induced by doxorubicin. We performed this study with Rat-1 fibroblasts, with Rat-1/myc cells which constitutively express c-Myc, and with Rat-1/myc/p53His175 cells derived from Rat-1/myc cells, which, in addition, express the full-length dominant-negative p53His175 mutant gene. The p53His175 mutant suppresses the transactivation function of endogenous p53 in these cells. In contrast to the parental Rat-1 cells, which exhibited only low levels of apoptosis within the first 24 h of treatment with 0.1 to 1 microM doxorubicin, similarly treated Rat-1/myc cells underwent massive and rapid apoptosis. Introduction of p53His175 into Rat-1/myc cells reversed this effect, indicating that Myc-accelerated doxorubicin-induced apoptosis requires functional p53. However, when the overall extent of cell death was measured using clonogenic assays, we found that greater than 90% of cells did not survive upon a 24-h pretreatment with doxorubicin at a concentration as low as 0.1 microM. Moreover, the effect of doxorubicin on all three cell lines was similar, irrespective of their p53 or c-Myc status. Taken together, our experiments indicate that: (a) constitutive expression of c-Myc accelerates the onset of doxorubicin-induced apoptosis in Rat-1 fibroblasts; (b) wild-type p53 function is necessary for this acceleration; and (c) neither overexpression of c-Myc nor the p53 status influences the overall extent of doxorubicin-induced cell death.

An immunotoxin prepared with blocked ricin: a natural plant toxin adapted for therapeutic use.

Ricin, the cytotoxic protein isolated from castor beans, is composed of two subunits, A-chain and B-chain. Ricin intoxicates cells by binding through its B-chain to galactose-terminated oligosaccharides found on the surface of all eukaryotic cells and then transferring its A-chain to the cytosol where it disrupts protein synthesis by inactivating ribosomes. In addition to binding, the B-chain plays an important, but not yet understood, role in the translocation of the A-chain through a cellular membrane to the cytosol. Blocking the two galactose-binding sites of native ricin by chemical modification with affinity ligands created an altered toxin, called blocked ricin, that has at least a 3500-fold lower binding affinity and is more than 1000-fold less cytotoxic than native ricin for Namalwa cells (a Burkitt's lymphoma line) but that has maintained the translocation function of the B-chain and the catalytic activity of the A-chain. Conjugation of blocked ricin to monoclonal antibodies that bind to cell surface antigens creates new cytotoxins that approach the potency of native ricin. These cytotoxins incorporate the three essential functions of natural toxins, i.e., binding to cells, transport through a membrane, and catalytic inactivation of an essential cellular process; but in addition they possess a defined cellular target specificity. Such potent immunotoxins may play an important therapeutic role in cancer treatment. Clinical trials with an anti-CD19-blocked ricin and an anti-CD33-blocked ricin conjugate against B-cell cancers and acute myeloblastic leukemia have begun.

Correlation between in vivo toxicity and preclinical in vitro parameters for the immunotoxin anti-B4-blocked ricin.

Anti-B4-blocked ricin (Anti-B4-bR) is an immunotoxin comprised of the anti-B4 monoclonal antibody and the protein toxin, "blocked ricin." In blocked ricin, the galactose-binding sites of the ricin B-chain which mediate nonspecific binding to cells are blocked by covalently linked affinity ligands prepared from N-linked oligosaccharides of fetuin. Blocked ricin consists of two species, one with two covalently attached ligands and one with three covalently attached ligands. In a Phase I dose escalation clinical trial, Anti-B4-bR was administered to patients with relapsed and refractory B-cell neoplasms by 7-day continuous infusion. Although several different lots of Anti-B4-bR had similar IC37 values as determined by in vitro cytotoxicity testing on cultured human cell lines, these lots differed in their in vivo toxicity when administered to patients. Thus, IC37 values alone were not sufficient to predict in vivo toxicity. We report that the degree of cell kill at concentrations of drug that saturate the B4 antigen and murine 50% lethal dose values provide additional parameters that may be predictive of in vivo cytotoxicity. Furthermore, we performed detailed cytotoxicity studies of the ricin species containing two and three covalently attached ligands, respectively. In vitro cytotoxicity testing using these samples revealed that Anti-B4-bR made with blocked ricin containing two covalently attached ligands is capable of depleting five logs of target cells in an in vitro cytotoxicity assay, while Anti-B4-bR comprised of blocked ricin with three ligands can deplete only one log of cells. Log cell kill at antigen saturating concentration, murine 50% lethal dose and biochemical analysis of the composition of blocked ricin are therefore important considerations for establishing the potential efficacy and safety of Anti-B4-bR.

Immunoconjugates containing novel maytansinoids: promising anticancer drugs.

The potential of immunoconjugates of cytotoxic drugs for the treatment of cancer has not yet been realized owing to the difficulty of delivering therapeutic concentrations of these drugs to the target cells. In an effort to overcome this problem we have synthesized maytansinoids that have 100- to 1000-fold higher cytotoxic potency than clinically used anticancer drugs. These maytansinoids are linked to antibodies via disulfide bonds, which ensures the release of fully active drug inside the cells. The conjugates show high antigen-specific cytotoxicity for cultured human cancer cells (50% inhibiting concentration, 10 to 40 pM), low systemic toxicity in mice, and good pharmacokinetic behavior.

Anti-B4-blocked ricin immunotoxin shows therapeutic efficacy in four different SCID mouse tumor models.

Anti-CD19 monoclonal antibody anti-B4 (IgG1) conjugated to the novel toxin-blocked ricin forms a potent immunotoxin, anti-B4-blocked ricin, that kills greater than 4.5 logs of CD19-positive cells in vitro after a 24-h exposure to a conjugate concentration of 5 x 10(-9) M (1.11 micrograms/ml). The efficacy of anti-B4-blocked ricin in vivo was assessed in survival models of SCID mice bearing either a human B-cell lymphoma (Namalwa), a human non-T and non-B acute lymphoblastic leukemia (Nalm-6), or a murine B-cell lymphoma transfected with the human CD19 gene (300B4). In one model, 5 x 10(7) tumor cells were injected i.p., and 1 h later the mice were treated with i.v. bolus injections of anti-B4-blocked ricin at 100 micrograms/kg/day for 5 days. Controls included similar treatment with anti-B4 antibody (72 micrograms/kg/day or 2 mg/kg/day for 5 days) alone or with the isotype-matched nonspecific immunotoxin, N901-blocked ricin (100 micrograms/kg/day). In a second model, 4 x 10(6) tumor cells were injected i.v., and 7 days later mice were treated i.v. as above. Anti-B4-blocked ricin showed efficacy by killing in vivo up to 3 logs of tumor cells, which was manifested in significant prolongation of the life of the treated animals. Only very limited or no effects were observed in animals treated with either anti-B4 antibody alone or N901-blocked ricin control conjugate. The concentration of anti-B4-blocked ricin in the blood of animals was 150 ng/ml after the first i.v. injection and about 800 ng/ml following the fifth injection of conjugate. This increase may be due to damage to the reticuloendothelial system by anti-B4-blocked ricin, since the rate of clearance of carbon from blood also decreased 5-fold after five injections as compared to the rate after only one injection. These studies indicate that anti-B4-blocked ricin has the potential to increase survival times of hosts with malignant disease.

Enhancement of the selectivity and antitumor efficacy of a CC-1065 analogue through immunoconjugate formation.

Bis-indolyl-(seco)-1,2,9a-tetrahydrocyclopropa[c]benz[e]indol-4-on e compounds are synthetic analogues of CC-1065 that are highly cytotoxic toward a broad spectrum of tumor cell lines. One of these compounds, called DC1, was conjugated to antibodies via novel cleavable disulfide linkers. Conjugates of DC1 with murine mAbs anti-B4 and N901 directed against tumor-associated antigens CD19 and CD56, respectively, proved to be extremely potent and antigen selective in killing target cells in culture. DC1 conjugates with humanized versions of anti-B4 and N901 antibodies were also constructed and demonstrated to be as cytotoxic and selective as the respective murine antibody conjugates. The anti-B4-DC1 conjugate showed antitumor efficacy in an aggressive metastatic human B-cell lymphoma survival model in SCID mice and completely cured animals hearing large tumors. Anti-B4-DC1 was considerably more effective in this tumor model than doxorubicin, cyclophosphamide, etoposide, or vincristine at their maximum tolerated doses.

Induction of apoptosis by tamoxifen-activation of a p53-estrogen receptor fusion protein expressed in E1A and T24 H-ras transformed p53-/- mouse embryo fibroblasts.

A fusion gene consisting of wild-type p53 linked to a modified ligand binding domain of the murine estrogen receptor has been constructed and should be a useful tool for studying controlled activation of wild-type p53 function in a variety of experimental cell systems. The protein product of this gene, p53ERTM, is expressed in cells constitutively but is not functional unless associated with tamoxifen or 4-hydroxytamoxifen. p53ERTM was introduced into p53-deficient mouse embryo fibroblasts (MEFs) expressing the E1A and T24 H-ras oncogenes. Activation of p53 in these transformed cells by the addition of tamoxifen or 4-hydroxytamoxifen resulted in apoptosis. In addition to engaging the apoptotic machinery, the tamoxifen-activated fusion protein exhibited other functions characteristic of wild-type p53, such as induction of WAF1 and MDM2 gene expression and activation of the p53-dependent spindle checkpoint in cells treated with nocodazole. Activation of p53ERTM expressed in p53-positive MEFs coexpressing E1A and ras had, at most, only a small cytotoxic effect. When three cell lines of transformed p53+/+ fibroblasts not expressing p53ERTM were tested for sensitivity to the DNA-damaging drug doxorubicin, the p53+/+ clones displayed either comparable sensitivity, or at most an increase in drug sensitivity of less than fourfold, as compared to several p53-/- cell lines. Our data show that restoration of wild-type p53 activity is sufficient to trigger apoptosis in p53-/- MEFs transformed with E1A and T24 H-ras and suggest that rare propagable clones of p53-normal MEFs expressing the E1A and T24 H-ras oncogenes have suffered compensatory alterations that compromise the ability to undergo p53-dependent apoptosis.

The adenine nucleotide translocator: a target of nitric oxide, peroxynitrite, and 4-hydroxynonenal.

Nitric oxide (NO), peroxynitrite, and 4-hydroxynonenal (HNE) may be involved in the pathological demise of cells via apoptosis. Apoptosis induced by these agents is inhibited by Bcl-2, suggesting the involvement of mitochondria in the death pathway. In vitro, NO, peroxynitrite and HNE can cause direct permeabilization of mitochondrial membranes, and this effect is inhibited by cyclosporin A, indicating involvement of the permeability transition pore complex (PTPC) in the permeabilization event. NO, peroxynitrite and HNE also permeabilize proteoliposomes containing the adenine nucleotide translocator (ANT), one of the key components of the PTPC, yet have no or little effects on protein-free control liposomes. ANT-dependent, NO-, peroxynitrite- or HNE-induced permeabilization is at least partially inhibited by recombinant Bcl-2 protein, as well as the antioxidants trolox and butylated hydroxytoluene. In vitro, none of the tested agents (NO, peroxynitrite, HNE, and tert-butylhydroperoxide) causes preferential carbonylation HNE adduction, or nitrotyrosylation of ANT. However, all these agents induced ANT to undergo thiol oxidation/derivatization. Peroxynitrite and HNE also caused significant lipid peroxidation, which was antagonized by butylated hydroxytoluene but not by recombinant Bcl-2. Transfection-enforced expression of vMIA, a viral apoptosis inhibitor specifically targeted to ANT, largely reduces the mitochondrial and nuclear signs of apoptosis induced by NO, peroxynitrite and HNE in intact cells. Taken together these data suggest that NO, peroxynitrite, and HNE may directly act on ANT to induce mitochondrial membrane permeabilization and apoptosis.

Anti-B4-blocked ricin: a phase I trial of 7-day continuous infusion in patients with B-cell neoplasms.

PURPOSE: This phase I trial was undertaken to determine the maximum-tolerated dose (MTD) and dose-limiting toxicities (DLTs) of the B-cell-restricted immunotoxin anti-B4-blocked ricin (anti-B4-bR) when it is administered by 7-day continuous infusion. PATIENTS AND METHODS: Thirty-four patients with relapsed and refractory B-cell neoplasms (26 non-Hodgkin's lymphoma [NHL], four chronic lymphocytic leukemia [CLL], four acute lymphoblastic leukemia [ALL]) received 7-day continuous infusion anti-B4-bR. Successive cohorts of at least three patients were treated at doses of 10 to 70 micrograms/kg/d for 7 days with the dose increased by 10 micrograms/kg/d for each cohort. The initial three cohorts of patients (10, 20, and 30 micrograms/kg/d x 7 days) also received a bolus infusion of 20 micrograms/kg before beginning the continuous infusion. RESULTS: The MTD was reached at 50 micrograms/kg/d x 7 days. The DLTs were National Cancer Institute Common Toxicity Criteria (NCI CTC) grade IV reversible increases in AST and ALT, and grade IV decreases in platelet counts. Adverse reactions included fevers, nausea, headaches, myalgias, hypoalbuminemia, dyspnea, edema, and capillary leak syndrome. Potentially therapeutic serum levels of anti-B4-bR could be sustained for 4 days in patients treated at the MTD. Two complete responses (CRs), three partial responses (PRs), and 11 transient responses (TRs) were observed. CONCLUSION: Anti-B4-bR can be administered safely by 7-day continuous infusion with tolerable, reversible toxicities to patients with relapsed B-cell neoplasms. Although occasional responses were seen, future trials will use anti-B4-bR in patients with lower tumor burdens to circumvent the obstacle of immunotoxin delivery to bulk disease.

A cytotoxicity assay utilizing a fluorescent dye that determines accurate surviving fractions of cells.

A cytotoxicity assay has been developed based on the measurement of the proliferative activity of surviving cells as quantified by a cell-incorporated fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The BCECF proliferative assay is fast (the results are obtained within 3-4 days depending on the cell line), accurate, not labor-intensive, does not require the use of radioisotopes or toxic compounds, and is amenable to automation. The BCECF proliferative assay was compared with two other indirect cytotoxicity tests, a trypan blue exclusion test and a BCECF viability test. Neither of these two latter assays reflected in any way the killing of cells by ricin. In contrast, using the BCECF proliferation assay, an optimal period of cell culturing after exposure to a toxin could be found so that the cytotoxicity values produced agreed with the surviving fractions of cells measured in a direct cytotoxicity assay. Under non-optimal conditions, the assay reflected the cell kill only qualitatively. Although it is common practice to conduct indirect cytotoxicity tests without validating them with a direct assay, our experiments demonstrate that the values obtained in such non-optimized indirect cytotoxicity tests may not parallel the cell kill and may, therefore, be meaningless.

Immobilization of protein molecules on liposomes. Anchorage by artificially bound unsaturated hydrocarbon tails.

A method for the immobilization of trypsin, a hydrophilic nonmembrane protein, on a liposomal surface has been developed. The technique consists of covalent coupling of linoleoyl residues to the protein globules and consequent binding of linoleoyl trypsin to liposomes by a detergent dilution method. The immobilized protein preserved its biological functions: specific esterolytic catalytic activity and ability to bind to a macromolecular trypsin protein inhibitor. Liposomes carrying immobilized trypsin were able to sequester glucose with the same efficiency as liposomes without trypsin.

Immunotoxins containing single-chain ribosome-inactivating proteins.

We have summarized what is currently known about the distribution, biological role, and the mechanism of action of the single chain ribosome-inactivating proteins and described the purification of one of them, gelonin, as an example. ITs have been made with several of these proteins and, depending upon the antibody used for conjugation, these immunoconjugates can show specific in vitro cytotoxicity which is similar to that shown by equivalent ITs prepared with ricin A chain. The most potent of these conjugates have shown antitumor efficacy in a variety of animal tumor models, including both syngeneic rodent tumors and xenografts in nude or immunosuppressed mice. An important point needs to be addressed, however, before concluding that ITs containing single chain toxins will be clinically useful. A major problem with this approach is that it is likely that both the antibody and the toxin components of these conjugates will be immunogenic. Both antitoxin and antixenogenic immunoglobulin responses have been shown to occur in animals after infusion of IT, although it has not yet been clearly demonstrated that such antibody responses adversely effect the pharmacokinetics or the efficacy of immunoconjugates. Thus, preliminary enthusiasm over the efficacy of these new reagents must be tempered with the knowledge that their use in the clinic may be limited by the host immune responses or other as yet undefined factors. The fact that there are many immunologically distinct single chain ribosome-inactivating proteins does suggest one way of evading the antitoxin response, by a sequential treatment with a panel of immunoconjugates, each containing a different single chain toxin.