Immunotoxins to the murine transferrin receptor: intracavitary therapy of mice bearing syngeneic peritoneal tumors.

Two rat monoclonal antibodies to the murine transferrin receptor (TFR) were each conjugated to recombinant ricin toxin A chain (rRTA). The monoclonal antibodies, R17 217 and YE1/9.9, bound to partially overlapping antigenic determinants on the murine TFR that were distinct from the transferrin binding site. Immunotoxins R17 217-rRTA and YE1/9.9-rRTA were potent cytotoxins for mouse cell lines in vitro. Animal toxicity studies showed that they were 10 to 20 times more toxic to mice than were control immunotoxins, including the anti-human-TFR immunotoxin 454A12-rRTA. The mouse toxicity of i.p. injected R17 217-rRTA could be partially blocked by the i.p. or i.v. administration of unconjugated R17 217 monoclonal antibody. I.p. but not i.v. administration of antimouse-TFR immunotoxins prolonged the survival of mice given implants of syngeneic peritoneal P388D1 lymphoid tumors. The anti-mouse-TFR immunotoxins killed at least 99.9% of the P388D1 tumor cells in vivo. In addition, i.p. treatment with immunotoxin R17 217-rRTA was able to prolong the survival of mice given an implant of a murine ovarian teratocarcinoma. The results show that anti-TFR immunotoxins can be efficacious in homologous species when the tumor is confined to a specific body cavity and immunotoxin can be delivered into that cavity.

Recombinant ricin A chain conjugated to monoclonal antibodies: improved tumor cell inhibition in the presence of lysosomotropic compounds.

Recombinant ricin A chain was chemically linked to monoclonal antibodies directed toward human breast cancer cells, a human T-cell differentiation antigen, and mouse transferrin receptor. Three types of immunotoxins were prepared; in two of them the antibody was linked to recombinant ricin A chain by a disulfide bond and in the third, a nonreducible thioether bond was used. Immunotoxins containing a nonreducible linkage may have some advantage over conjugates containing a reducible linkage because of improved stability in vivo. Conjugation of recombinant ricin A chain through either the endogenous thiol group or through a derivatized amino group produced immunotoxins with comparable cytotoxicity. The thioether conjugate was 1000-fold less cytotoxic to target tumor cells than the respective disulfide-linked immunotoxin. However, addition of monensin, a monocarboxylic ionophore, greatly enhanced the cytotoxicity of the thioether-linked immunotoxin. Monensin increased the immunotoxin activity better than other lysosomotropic reagents that were tested. The increase in activity of recombinant ricin A chain-containing immunotoxins mediated by monensin argues against a role for contaminating ricin B chain in potentiation.

Evaluation of monoclonal antibodies for the development of breast cancer immunotoxins.

Eighty-five antibodies recognizing breast cancer-selective antigens were conjugated to ricin toxin A-chain using a disulfide linkage. The cytotoxicities of the resulting immunotoxins were determined on breast cancer cells and normal human fibroblasts. Twenty-four antibodies formed immunotoxins that were toxic to at least one breast cancer cell line at concentrations of 10 nM or less but were nontoxic to human fibroblast lines used as negative controls. Some of the breast tumor-selective immunotoxins were as toxic as a conjugate between monoclonal anti-transferrin receptor and ricin toxin A-chain (50% inhibition of cellular protein synthesis at approximately 0.1 nM). Another set of four immunotoxins were indiscriminately toxic to human breast tumor cell lines, two human fibroblast cell lines, and a human lymphoblastoid line. Several of the antibodies the toxin conjugates of which specifically killed breast cancer cell lines may be useful in cancer therapy, since they show a wide range of binding to individual breast tumors and cell lines and a limited range of binding to normal tissue types.

Antibody-Pseudomonas exotoxin A conjugates cytotoxic to human breast cancer cells in vitro.

Breast tumor selective antibodies (MAB) conjugated to Pseudomonas exotoxin A (PE) formed immunotoxins with potent cytotoxicities for human breast tumor cell lines. The most effective of the MAB-PE conjugates were about 500-fold more toxic to the breast tumor target cell lines than to the nontarget human fibroblast cell line. Specificity of cytotoxicity by MAB-PE conjugates was demonstrated by protection of target cells in the presence of excess unconjugated homologous antibody. A MAB-PE conjugate inhibited protein synthesis more rapidly than the corresponding MAB-ricin toxin A chain (RTA) conjugate. Generally, there was a direct correlation between the cytotoxicity of RTA and PE when conjugated to the same MAB: MABs that made highly cytotoxic RTA conjugates made effective PE conjugates and MABs that made poorly cytotoxic RTA conjugates made PE conjugates of low cytotoxicity; however, one MAB-PE immunotoxin was cytotoxic to the human breast tumor cell lines, whereas the corresponding MAB-RTA immunotoxin was noncytotoxic at the highest dose tested. In contrast to MAB-RTA conjugates, which require a cleavable (disulfide) linkage for maximal in vitro cytotoxicity, MAB-PE conjugates were about equally cytotoxic when linked by either a cleavable or noncleavable (thioether) bond.

Inhibition of human tumor growth by intraperitoneal immunotoxins in nude mice.

Intracavitary administration of immunotoxins may play a role in the control of malignant effusions. Selection of immunotoxins for this form of therapy is based on their prior evaluation in preclinical studies. Monoclonal antibodies (mAb) 454A12 (antitransferrin receptor), and 260F9 are directed against antigens which are present on tumor cells in pleural and peritoneal effusions of patients with adenocarcinoma of the breast and ovary. In the present study, immunotoxins derived by conjugating these mAb to recombinant ricin A (rRA) were shown to be cytotoxic to human ovarian adenocarcinoma HEY cells in vitro and in vivo. In the in vitro assay 454A12-rRA and 260F9-rRA were 1000-fold and 10-fold, respectively, more cytotoxic than free rRa against HEY cells, and both immunotoxins were potentiated approximately 1000-fold by monensin. For in vivo studies HEY cells were injected i.p. into nude mice at a challenge dose (3 x 10(5) cells) which produced carcinomatosis with ascites, leading to death 30 days following injection. Administration of 454A12-rRA i.p. following the challenge dose resulted in a complete cure, whereas administration of 260 F9-rRA with monensin significantly prolonged survival. The greater cytotoxicity of 454A12-rRA than 260F9-rRA against HEY cells could be accounted for by the greater number of binding sites and higher internalization rate for 454A12-rRA and mAb 454A12 than 260F9-rRA and mAb 260F9, respectively. These results suggest a potential role for 454A12-rRA and 260F9-rRA plus monensin in the intracavitary therapy of malignant effusions associated with carcinoma of breast and ovary. In the case of 260F9-rRA, this represents the first preliminary indication of the suitability of this immunotoxin for intracavitary therapy of malignancies.

Selective elimination of breast cancer cells from human bone marrow using an antibody-Pseudomonas exotoxin A conjugate.

A pancarcinoma monoclonal antibody (NR-LU-10), homogeneously reactive with human breast cancer cells, was conjugated to Pseudomonas exotoxin A. The immunotoxin was evaluated for its potential for purging breast cancer cells from human bone marrow. The immunotoxin NR-LU-10 antibody did not react with normal bone marrow preparations yet readily detected 1% contamination of bone marrow by MCF-7 breast cancer cells added to normal bone marrow without significantly inhibiting the colony-forming ability of bone marrow progenitor cells. NR-LU-10-Pseudomonas exotoxin A has potential for purging bone marrow of breast cancer cells without impairing the growth of bone marrow progenitor cells.

Distribution and physical properties of BCA200, a Mr 200,000 glycoprotein selectively associated with human breast cancer.

Of 122 mouse monoclonal antibodies selective for human breast cancer, 13 immunoprecipitated an acidic glycoprotein from SK-Br-3 and ZR-75-30 human breast cancer cells. The antigen (BCA200) migrates with an apparent molecular weight of 200,000 on reducing and 180,000 on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting a single polypeptide chain with a folded domain stabilized by a disulfide bond. Cross-blocking and sandwich immunoassays detected at least three distinct antigenic determinants on BCA200. Scatchard experiments measured 1,000,000 to 5,000,000 antigen copies per SK-Br-3 cell. The tissue distribution of BCA200 was studied using two monoclonals to different epitopes. Neither antibody stained any cells in human blood. When frozen sections of 20 normal human tissues were immunoperoxidase stained, the only positive structures were mucinous glands of colon, transitional epithelium of bladder, sweat glands of skin, and acinar epithelium of breast. Antibody 454C11 stained 16 of 21 breast tumor frozen sections and 9 of 12 breast cancer cell lines, while antibody 520C9 stained 5 of 20 breast tumors and 4 of 10 breast cancer lines. Cross-reaction was observed with lung, prostatic, pancreatic, endometrial, and ovarian cancer, but not with lymphoma, melanoma, colon, stomach, bladder, or esophageal cancer. When conjugated to ricin A chain, 10 of 13 antibodies produced immunotoxins selectively cytotoxic to SK-Br-3 breast cancer cells.

Antitumor activity of an immunotoxin in a nude mouse model of human ovarian cancer.

An immunotoxin composed of an antibody to the human transferrin receptor (454A12) and ricin A chain (RTA) was shown to inhibit the growth of NIH:OVCAR-3 tumors in a nude mouse model of human ovarian cancer. Inhibition of tumor growth by 454A12-RTA was related to the dose administered. The antitumor activity of the immunotoxin was blocked by coinjection of excess antibody with immunotoxin. An immunotoxin made using 454A12 and recombinant ricin A chain (rRTA) had an activity similar to that made with native RTA. The administration of 10 micrograms or greater of the immunotoxin 454A12-RTA/rRTA had significant antitumor activity. The injection of 30 micrograms of an irrelevant immunotoxin, MOPC21-RTA, or 30 to 500 micrograms of the 454A12 antibody had no antitumor activity.

Characterization of translational inhibitors from Phytolacca americana. Amino-terminal sequence determination and antibody-inhibitor conjugates.

Two translational inhibitors (pokeweed antiviral protein and pokeweed antiviral protein II) isolated from the leaves of the pokeweed plant, Phytolacca americana, were characterized as to their behavior during reverse-phase HPLC and their amino-terminal sequences. Alignment of the sequences demonstrated that a substantial degree of homology was present (10 of 29 identical residues). Pokeweed antiviral protein was shown by reverse-phase chromatography to be composed of at least two components, pokeweed antiviral proteina and pokeweed antiviral proteinb, which comigrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis, shared identical N-terminal amino-acid sequences through residue 31, and had similar specific activities in a cell-free translation inhibition assay. Pokeweed antiviral protein II was covalently coupled to a monoclonal antibody that recognizes the transferrin receptor (anti-transferrin receptor). The disulfide-linked conjugate inhibited protein synthesis in the human breast tumor cell line MCF-7, whereas anti-transferrin receptor, pokeweed antiviral protein II, or an immunotoxin composed of an irrelevant antiserum and pokeweed antiviral protein II, were nontoxic. The inhibitory dose 50% of anti-transferrin receptor-pokeweed antiviral protein II for MCF-7 cells was 0.7 nM, whereas the corresponding ricin A chain conjugate (anti-transferrin receptor-ricin A chain) was more potent with a inhibitory dose 50% of 0.1 nM. Pokeweed antiviral protein II can be added to the growing list of translation inhibitors that are effective as components of immunotoxins in vitro. Additional studies will be needed to determine whether pokeweed antiviral protein II immunotoxins provide advantageous properties for in vivo applications.

Dosimetry of rhenium-186-labeled monoclonal antibodies: methods, prediction from technetium-99m-labeled antibodies and results of phase I trials.

Rhenium-186 is a beta-emitting radionuclide that has been studied for applications in radioimmunotherapy. Its 137 keV gamma photon is ideal for imaging the biodistribution of the immunoconjugates and for obtaining gamma camera data for estimation of dosimetry. Methods used for determining radiation absorbed dose are described. We have estimated absorbed dose to normal organs and tumors following administration of two different 186Re-labeled immunoconjugates, intact NR-LU-10 antibody and the F(ab')2 fragment of NR-CO-02. Tumor dose estimates in 46 patients varied over a wide range, 0.4-18.6 rads/mCi, but were similar in both studies. Accuracy of activity estimates in superficial tumors was confirmed by biopsy. Prediction of 186Re dosimetry from a prior 99mTc imaging study using a tracer dose of antibody was attempted in the NR-CO-02 (Fab')2 study. Although 99mTc was an accurate predictor of tumor localization and the mean predicted and observed radiation absorbed doses to normal organs compared favorably, 186Re dosimetry could not be reliably predicted in individual patients. The methods described nevertheless provide adequate estimates of 186Re dosimetry to tumor and normal organs.

Rhenium-186-labeled chimeric antibody NR-LU-13: pharmacokinetics, biodistribution and immunogenicity relative to murine analog NR-LU-10.

A mouse-human chimeric monoclonal antibody (NR-LU-13), with the same pancarcinoma antigen recognition site as a previously studied murine monoclonal antibody (NR-LU-10), was radiolabeled with 186Re using a bifunctional chelate. Nine patients (ages 31-81 yr) with metastatic adenocarcinoma received 186Re NR-LU-13. A single intravenous dose of 42 mg NR-LU-13 labeled with 25 mCi/m2 (two patients) or 60 mCi/m2 (seven patients) was administered. Mean serum disappearance half-time values for the chimeric 186Re NR-LU-10). Fifty percent of the radiolabel was excreted in the urine by 6 days. Tumor localization was demonstrated by gamma camera imaging in seven of nine patients. The percent injected dose per gram in a single tumor biopsy specimen was 0.003% at 72 hr postinjection. Absorbed dose to bone marrow was 1.5 +/- 0.7 rads/mCi and resulted in reversible myelosuppression in five of six evaluable patients who received 60 mCi/m2: median WBC nadir = 2500/microliters; median platelet nadir = 85,500/microliters. Low grade fever, nausea, slight elevations of liver function tests and mild allergic reactions were seen in some patients. The chimeric antibody elicited low levels of anti-NR-LU-13 antibody in six of eight evaluable patients (75%), in contrast to NR-LU-10 which elicited higher levels of human anti-mouse antibody in all patients. This pilot study demonstrates the ability of the chimeric antibody to target tumors with reduced (but not absent) immunogenicity and delayed clearance relative to the murine antibody.

Clinical experience with rhenium-186-labeled monoclonal antibodies for radioimmunotherapy: results of phase I trials.

Rhenium is a radionuclide with physical and chemical properties suitable for radioimmunotherapy. Two Phase I trials were carried out using 186Re-labeled murine monoclonal antibodies. Patients with refractory metastatic epithelial carcinoma received single doses of either 186Re-labeled intact NR-LU-10, a pancarcinoma antibody, 25-120 mCi/m2 (n = 15) or 186Re-labeled F(ab')2 fragment of NR-CO-02, an anti-CEA variant antibody, 25-200 mCi/m2 (n = 31). Prior to radioimmunotherapy, tumor localization of antibody was confirmed by 99mTc-labeled NR-LU-10 Fab or 99mTc-labeled NR-CO-02 F(ab')2 imaging. Dose-limiting myelosuppression was observed at 120 mCi/m2 following 186Re-NR-LU-10 intact antibody and at 150 mCi/m2 following NR-CO-02 F(ab')2 fragment in heavily pretreated patients. In patients with minimal prior therapy, a maximum tolerated dose for NR-CO-02 F(ab')2 was not reached by 200 mCi/m2. Non-marrow toxicity was minimal. Human anti-mouse antibody developed in all patients receiving intact NR-LU-10, and in 86% patients receiving F(ab')2 NR-CO-02. One patient treated with 186Re NR-CO-02 achieved a partial response. We conclude that 186Re-labeled antibody can be safely administered with significant toxicity limited to marrow.

Clinical experience with Tc-99m nofetumomab merpentan (Verluma) radioimmunoscintigraphy.

Tc-99m nofetumomab merpentan (Verluma), consisting of a Fab fragment of the pancarcinoma murine antibody NR-LU-10, has been previously evaluated as a diagnostic imaging agent in staging patients with lung cancer. The authors have taken advantage of the pancarcinoma reactivity of this antibody to select patients with a variety of carcinomas for radioimmunotherapy trials. These have included gastrointestinal, breast, ovary, pancreas, kidney, cervix, and bladder carcinoma. This article documents the range of tumor types and locations that can be identified by gamma camera imaging with this radioimmunoconjugate. Tumor was positively identified in 92% of 107 patients studied. In 15 patients, the images led to suspicion of previously unknown disease. The authors conclude that this radioimmunoconjugate is useful in assessing patients with advanced disease. Additional studies may be warranted to explore further the potential benefit of this diagnostic imaging agent in evaluating the extent of disease in patients with a variety of carcinomas.

Response of primary human mammary tumor cell cultures to a monoclonal antibody-recombinant ricin A chain immunotoxin.

Malignant epithelial tumor cells were isolated and cultured from ten human mammary specimens of cancerous origin. The 260F9 monoclonal antibody (MAB) bound to frozen sections of all of the human breast tumors tested and to primary cultured cells from the tumors. Cultured cells from all ten breast tumors were sensitive to the clonal inhibitory effects of immunotoxin 260F9 MAB-recombinant ricin A chain. At an immunotoxin concentration of 200 ng/ml (about 1 nM), inhibition of colony formation was greater than 99% for all ten tumors.

Influence of iron on yields of extracellular products in Pseudomonas aeruginosa cultures.

The effect of the iron content of the medium on the yields of extracellular products by seven distinct strains of Pseudomonas aeruginosa was examined. All strains showed at least an 85% decrease in toxin A yields when grown in medium containing 5.0 mug of iron per ml (high iron) as compared to 0.05 mug/ml (low iron), whereas bacterial growth increased approximately twofold. During the course of examining extracellular products produced by P. aeruginosa, we found many strains that produced an extracellular factor which agglutinated erythrocytes. This hemagglutinin was nondialyzable, heat stable, and resistant to Pronase and trypsin. The effect of iron on extracellular yields of hemagglutinin was strain dependent; four of seven strains showed decreases in hemagglutinin yields in high-iron medium. Similarly, the effect of increasing the iron concentration of the growth medium on yields of total extracellular proteases or on elastase was strain dependent. The amount of total extracellular protein was decreased by at least 31% in the high-iron medium for all strains of P. aeruginosa examined. Detailed studies on one strain (WR-9) showed that, in the presence of increasing amounts of iron in the medium, the extracellular yields of toxin A, protease, and hemagglutinin were decreased in a similar manner. In addition, the kinetics of release of these extracellular products were similar at a given iron concentration. Thus it appears that the yields of other extracellular products of P. aeruginosa besides toxin A are influenced by the concentration of iron in the growth medium.

Expression of soluble and fully functional ricin A chain in Escherichia coli is temperature-sensitive.

Linkage of ricin A chain (RA) to a cell surface binding antibody or other ligand can result in a potent cytotoxic agent. We expressed the primary sequence for RA in Escherichia coli to facilitate production and to obtain protein free of naturally occurring contaminants, i.e. ricin B chain. Differences in the level of expression and in the characteristics of the expressed protein were noted when several different host/vector systems were tested. Recombinant RA (rRA) was expressed directly under control of the phage lambda major leftward promoter (PL) and the E. coli trp promoter. It was also expressed fused to E. coli alkaline phosphatase sequences, both in the same reading frame for secretion and out-of-reading frame for expression in a cistron-like arrangement. Expression in the PL promoter system, which is temperature-regulated, was achieved at 37 degrees C as well as at 42 degrees C. The protein expressed at these different temperatures had grossly different properties. Whereas rRA expressed at 37 degrees C was soluble and fully active, that produced at 42 degrees C was aggregated, insoluble, and reduced in activity. Soluble rRA could be converted to the insoluble form by incubation at 42 degrees C in vivo, but not in vitro. Hence, this difference in properties does not simply reflect an inherent thermal instability of the protein. Conditions present in vivo, including the possible association with other proteins, are apparently required for this effect on rRA.

Production of exoenzyme S during Pseudomonas aeruginosa infections of burned mice.

Antisera which distinguished between Pseudomonas aeruginosa exoenzyme S and toxin A neutralized the adenosine diphosphate ribosyl transferase activity of the homologous, but not the heterologous, enzyme. Skin extracts and sera from burned mice infected with the exoenzyme S-producing strain P. aeruginosa 388 contained adenosine diphosphate ribosyl transferase activity that was not found in skin extracts or sera from uninfected mice. On the basis of immunological reactivity and enzymatic properties, the adenosine diphosphate ribosyl transferase activity present in skin extracts and sera from P. aeruginosa 388-infected mice was identified as exoenzyme S. Active elongation factor 2 levels in tissues from strain 388-infected mice were normal at 24 h postinfection, indicating that strain 388 does not produce detectable amounts of toxin A in vivo. An unexpected finding in this investigation was the presence of exoenzyme S-inactivating activity in the sera from some nonimmunized animals.

Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A.

Pseudomonas aeruginosa exoenzyme S is an adenosine diphosphate ribosyltransferase distinct from Pseudomonas toxin A. Exoenzyme S catalyzes the transfer of radioactivity from all portions of radiolabeled NAD+ except nicotinamide. Digestion of the radiolabeled product(s) formed in the presence of [adenine-14C]NAD+ and exoenzyme S with snake venom phosphodiesterase yields only AMP, suggesting that ADP-ribose is present as monomers and not as poly(ADP-ribose). Exoenzyme S does not catalyze the transfer of ADP-ribose from NAD+ to elongation factor 2, as do toxin A and diphtheria toxin, but to one or more other proteins present in crude extracts of wheat germ or rabbit reticulocytes and in partially purified preparations of elongation factor I. The ADP-ribosyltransferase activity of exoenzyme S is distinct from toxin A by several tests: it is not neutralized by toxin A antibody, it is destroyed rather than potentiated by pretreatment with urea, and it is more heat stable. These latter observations and the substrate specificity suggest that exoenzyme S is different from any previously described prokaryotic ADP-ribosyltransferase.

Incidence of exotoxin production by Pseudomonas species.

Pseudomonas aeruginosa exotoxin A has been shown to catalyze the transfer of the adenosine 5'-diphosphate (ADP)-ribose moiety of nicotinamide adenine dinucleotide onto elongation factor 2, resulting in the inhibition of mammalian protein synthesis. The enzymatic activity (ADP-ribosyl [ADPR]-transferase) is thought to account for the toxicity of exotoxin A. The distribution of the expression of exotoxin A within Pseudomonas species was examined. Laboratory strains as well as clinical isolates of Pseudomonas aeruginosa were tested. The production of exotoxin A was determined by assaying for ADPR-transferase activity in dialyzed frozen (-20 degrees C) and thawed cell-free supernatants from 22-h cultures or in 10-fold-concentrated supernatants. In addition, toxin production was detected immunologically using a modified Elek test. Exotoxin A production was detected in approximately 90% of the 111 isolates of P. aeruginosa. In contrast, none of the other species of Pseudomonas examined produced exotoxin A detectable by either ADPR-transferase activity or immunological reactivity.