Expression of Lewis carbohydrate antigens in metastatic lesions from human prostatic carcinoma.

BACKGROUND: Monoclonal antibodies B1 and B3 react with Lewis(y) and related carbohydrate antigens, which are abundant in many solid tumors. These antibodies, when conjugated to a toxin, have been used to target a variety of cancers. Treatment options for advanced prostate cancer are very limited, and there is a need to develop new therapies. In this study, we have asked whether antibodies B1 and B3 react with metastatic lesions from human prostatic carcinoma. METHODS: Indirect streptavidin-biotin peroxidase immunohistochemistry was performed on formalin-fixed specimens from prostate cancer metastases. A total of 6 lymph node metastatic samples from patients who did not receive endocrine treatment and specimens of 14 distant metastases from patients who failed hormonal therapy were obtained. RESULTS: Of the samples, 6 lymph node and 11 distant metastases stained for B1. In the case of B3 staining, 6 lymph node and 10 distant metastatic lesions were positive. In about half of these metastatic samples, more than 40% of cells were immunoreactive with either antibody. Two metastatic samples stained neither for B1 nor for B3 antibody. In general, B1 staining intensity was stronger in samples in which more than 40% of cells were positive. CONCLUSIONS: Our results suggest that B1 and B3 immunoconjugates could be applied to target a substantial percentage of prostate cancer metastases.

Identification of epitopes on a mutant form of Pseudomonas exotoxin using serum from humans treated with Pseudomonas exotoxin containing immunotoxins.

PE38 is a 38-kDa derivative of the 66-kDa Pseudomonas exotoxin (PE) in which the cell binding domain of PE (domain Ia, amino acids 1-252) and a portion of domain Ib (amino acids 365-380) are deleted. The immunotoxins LMB-1 and LMB-7 contain PE38 and kill cancer cells by exploiting the cytotoxic action of PE38. The major human B cell epitopes of PE38 were mapped by measuring the reactivity of 45 serum samples from patients treated with the PE38-containing immunotoxins LMB-1 or LMB-7 to two panels of overlapping synthetic peptides representing the sequence of PE38. One panel of peptides is ten amino acids long and overlap by seven amino acids, and the second panel of peptides is twenty amino acids long and overlap by ten. Five major epitopes were identified: amino acids 274-283, 470-492, 531-540, 555-564, and the C-terminal amino acids 596-609. Two minor epitopes were identified as well: amino acids 501-510 and 582-589. These epitopes are predominantly located on the surface of the protein. The amino acids believed to be critical for binding are highly solvent-accessible residues. The results of the human antibody response to peptides are compared to the pattern of reactivity previously identified with serum samples obtained from monkeys administered LMB-1 and LMB-7. The epitopes between monkey and human are almost identical, demonstrating similarity in the response of antibody repertoires between the two species and providing further support that these are the immunodominant epitopes. This information is critical for genetically engineering less immunogenic immunotoxins and provides a foundation for the development of a vaccine against pseudomonal infections which plague immunocompromised individuals and individuals with cystic fibrosis.

Child abuse: attitudes and perceptions of health profession students--a pilot study.

Child abuse and neglect is a serious social problem with global dimensions. The purpose of this study was to assess attitudes and perceptions of students of medicine, dentistry and public health at three Boston institutions about child abuse and neglect issues that they may encounter in their future professional lives. Among others, we investigated how participants rank the public health importance of child abuse and neglect in comparison with violent acts against other social groups, their willingness to report it and their potential interest to supplement their knowledge with additional course work. Two hundred and fourteen students participated in the study by completing our questionnaire. The results of the statistical analyses indicate that child abuse is considered the most serious problem, followed by domestic violence, child neglect and abuse of the handicapped and the elderly. We also documented that a significant educational need exists in this group, regarding diagnostic methods and interventions aimed to reduce the incidence and the impact of violence or neglect against children.

Treatment of advanced solid tumors with immunotoxin LMB-1: an antibody linked to Pseudomonas exotoxin.

Immunotoxin LMB-1 is composed of monoclonal antibody B3 chemically linked to PE38, a genetically engineered form of Pseudomonas exotoxin. B3 recognizes a carbohydrate antigen (Le(Y)) present on many human solid tumors. LMB-1 has excellent antitumor activity in nude mice bearing Le(Y)-positive tumors. We conducted a phase I study of 38 patients with solid tumors who failed conventional therapy and whose tumors expressed the Le(Y) antigen. Objective antitumor activity was observed in 5 patients, 18 had stable disease, 15 progressed. A complete remission was observed in a patient with metastatic breast cancer to supraclavicular nodes. A greater than 75% tumor reduction and resolution of all clinical symptoms lasting for more than six months was observed in a colon cancer patient with extensive retroperitoneal and cervical metastasis. Three patients (two colon, one breast cancer) had minor responses. The maximum tolerated dose of LMB-1 is 75 microgram/kg given intravenously three times every other day. The major toxicity is vascular leak syndrome manifested by hypoalbuminemia, fluid retention, hypotension and, in one case, pulmonary edema. Although immunotoxins have been evaluated in clinical studies for more than two decades, this is the first report of antitumor activity in epithelial tumors.

Recombinant immunotoxins.

Pseudomonas exotoxin has been genetically modified so that it targets cancer cells. This was accomplished by deleting its cell binding domain and replacing it with Fv fragments of antibodies that react with breast, colon, and other cancers. Several recombinant immunotoxins are now in clinical trials.

Efficacy of compartmental administration of immunotoxin LMB-1 (B3-LysPE38) in a rat model of carcinomatous meningitis.

LMB-1 (B3-LysPE38) is an immunotoxin composed of the tumor-reactive monoclonal antibody B3 and a genetically engineered form of Pseudomonas exotoxin. Monoclonal antibody B3 reacts with a carbohydrate epitope that is found on a number of solid tumors (e.g., breast, ovarian, and lung carcinomas) that frequently invade the intrathecal space, causing neoplastic meningitis. The Pseudomonas exotoxin has been engineered to remove the binding domain to eliminate nonspecific binding. A model of human neoplastic meningitis using rats bearing the human epidermoid carcinoma A431 was used for therapeutic studies of immunotoxin LMB-1. Therapy was initiated 3 days after injection of the tumor cells, which was one third of the median survival time of untreated rats. A single intrathecal injection of 40 microgram increased median survival from 9 days with saline injection to 16 days (78%, P < 0.001), and a single dose of 200 microgram increased median survival to 25 days (188%, P < 0. 001). Three doses of 40 or 200 microgram given on days 3, 6, and 8 significantly increased the median survival of 9.5 days associated with saline injection to 40.5 days (326% increase) and 33.0 days (247% increase), respectively, with two long-term survivors (191-day survival) in each treatment group. LMB-1 had no therapeutic effect on the treatment of two B3 antigen-negative neoplastic meningitis models. Treatment of the antigen-positive A431 neoplastic meningitis with B3 alone or a nonspecific monoclonal, MOPC, coupled to the engineered Pseudomonas exotoxin produced no survival effects. Nontumor-bearing athymic rats showed no toxicity with a single dose of either 40 microgram or 200 microgram, or 3 doses of 40 microgram. However, when they were given three doses of 200 microgram, these rats showed weight loss and loss of neurological function, and two of eight animals died. These studies indicate that, in the range of the most therapeutically effective dosage, the immunotoxin LMB-1 is tolerated in the intrathecal space and should be considered for human intrathecal trials.

Immunotoxins containing Pseudomonas exotoxin that target LeY damage human endothelial cells in an antibody-specific mode: relevance to vascular leak syndrome.

Vascular leak syndrome (VLS) was originally found to be a major dose-limiting toxicity in humans with cancer treated with several immunotoxins (ITs) containing ricin A chain or blocked ricin. Recently, VLS has also been observed in patients treated with an IT containing the murine monoclonal antibody (MAb) B3 coupled to LysPE38, a recombinant truncated form of Pseudomonas exotoxin (PE) A. Antibody B3 (IgG1k) recognizes LewisY and related carbohydrate epitopes present on many human solid tumors, and B3-LysPE38 showed excellent antitumor activity in nude mice bearing tumors that express the B3 antigen. In the clinical trial, the development of VLS has prevented the administration of the amount of IT necessary to achieve blood levels required for good therapeutic responses. We have now investigated the effects of several PE-based ITs on different human endothelial cell lines to elucidate the mechanism of VLS induced by ITs containing PE. To assess the cytotoxic effect of IT on endothelial cells, various ITs were incubated with cells for 2 or 20 h, and the incorporation of [3H]leucine into protein was measured. The endothelial cells studied were human umbilical vein endothelial cells, human lung-derived microvascular endothelial cells (HUVECs), human adult dermal microvascular endothelial cells, human pulmonary artery endothelial cells, and human aortic endothelial cells. We found that both B3-LysPE38 (LMB-1), a chemical conjugate of MAb B3 with PE38, as well as B3(Fv)-PE38 (LMB-7), a recombinant single chain immunotoxin, inhibited protein synthesis, with 50% inhibitory concentrations between 600 and 1000 ng/ml for 20-h incubation in HUVECs, human lung-derived microvascular endothelial cells, and human adult dermal microvascular endothelial cells but not on human pulmonary artery endothelial cells. The cytotoxic effect was specific since PE38 itself or PE coupled to several other antibodies did not inhibit protein synthesis in these cells even at 10,000 ng/ml. Further evidence that the cytotoxicity of B3-containing ITs is due to specific B3 binding to endothelial cells comes from the fact that the cytotoxicity can be blocked by excess free MAb B3. HUVECs undergo overt morphological changes after treatment with B3-LysPE38 or B3(Fv)PE38. Gaps between the cells are formed after a 20-h exposure but not after 2 h. These studies suggest that VLS in patients is due to capillary damage caused by prolonged exposure to high concentrations of LMB-1.

Administration of disulfide-stabilized Fv-immunotoxins B1(dsFv)-PE38 and B3(dsFv)-PE38 by continuous infusion increases their efficacy in curing large tumor xenografts in nude mice.

B1 (dsFv)-PE38 and B3(dsFv)-PE38 are recombinant immunotoxins in which the Fv fragments of MAbs B1 and B3, respectively, are stabilized by an engineered interchain disulfide bond and are fused at their C-termini to a modified Pseudomonas exotoxin from which the cell-binding domain has been deleted (PE38). Both immunotoxins have been shown to be specifically cytotoxic toward human cancer cell lines which express Le gamma-related carbohydrates on their surface, and when given i.v., eradicated 30- to 50-mm3 s.c. A431 tumors growing in nude mice. A major advantage of dsFv-immunotoxins is their stability at 37 degrees C compared with the relatively unstable single-chain Fvs. This allows them to be given continuously by osmotic pumps placed in the peritoneal cavity. In an attempt to increase the therapeutic index of the immunotoxins, we have now delivered them continuously for 6 days through mini-osmotic pumps placed in the peritoneal cavity of tumor-bearing nude mice. Using this mode of administration, we were able to maintain a constant level of immunotoxin in the serum which was non-toxic to the mice, but caused complete regressions of large 150- to 200-mm3 tumors which lasted for over a month at 1/11 of the LD50 with B1(dsFv)-PE38 and 1/6 of the LD50 with B3(dsFv)-PE38. Complete regression of tumors of similar size could also be achieved by i.v. bolus injections of these immunotoxins at 1/7 of the LD50 with B1(dsFv)-PE38) and 1/3 of the LD50 with B3(dsFv)-PE38. These results suggest that in patients it may be advantageous to administer dsFv-immunotoxins by continuous infusion, since a larger therapeutic index is achieved.

Intrathecal administration of single-chain immunotoxin, LMB-7 [B3(Fv)-PE38], produces cures of carcinomatous meningitis in a rat model.

LMB-7 [B3(Fv)-PE38] is a single-chain immunotoxin constructed from the murine monoclonal antibody B3 and a truncated from of Pseudomonas exotoxin PE38. Antibody B3 recognizes a carbohydrate epitope found on solid tumors that frequently invade the intrathecal space and cause neoplastic meningitis. We tested the therapeutic value of intrathecally administered LMB-7 by using a model of human neoplastic meningitis in athymic rats. This model is representative of a clinical situation in that antibody B3 cross-reacts with a number of normal tissues that can be used to monitor potential systemic toxicity. Treatment was begun 3 days after A431 tumor implantation. Without treatment, the animals median survival was 10 days. Intrathecal administration of 10 micrograms of LMB-7 in 40 microliters on days 3, 5, and 7 produced 4 of 10 and 8 of 10 long-term survivors (> 170 days) in two experiments. Of the long-term survivors, 2 of 4 and 7 of 8 survivors had no microscopic evidence of tumor and were considered histologic cures. Lack of significant toxicity in the effective dose range and specificity make LMB-7 an excellent candidate for intrathecal treatment of neoplastic meningitis in humans.

Transforming growth factor-beta1 circulates in normal human plasma and is unchanged in advanced metastatic breast cancer.

A method has been developed to determine true plasma transforming growth factor beta (TGF-beta) levels by using the platelet alpha granule-specific marker, platelet factor 4, to correct for the TGF-beta contributed by platelets degranulated ex vivo. TGF-beta levels were measured on acid-ethanol extracts of human plasma using isoform-specific sandwich enzyme-linked immunosorbent assays. Normal human subjects had 4.1 +/- 2.0 ng/ml TGF-beta1 (range, 2.0-12.0; n = 42), <0.2 ng/ml TGF-beta2, and <0.1 ng/ml TGF-beta3 in their plasma. There were no significant changes with age or with hormonal status, but any given individual showed fluctuations of up to 3-fold in measured plasma TGF-beta levels due to unknown factors. Of 28 patients with advanced metastatic breast cancer, 2 had greatly elevated TGF-beta1 levels, while the rest were in the normal range. The presence of physiologically significant levels of TGF-beta1 in the plasmas of normal human subjects may indicate previously unsuspected endocrine roles for this peptide, while TGF-beta2 and TGF-beta3 appear to act only in a local autocrine/paracrine fashion.

Comparative biodistribution of indium- and yttrium-labeled B3 monoclonal antibody conjugated to either 2-(p-SCN-Bz)-6-methyl-DTPA (1B4M-DTPA) or 2-(p-SCN-Bz)-1,4,7,10-tetraazacyclododecane tetraacetic acid (2B-DOTA).

The biodistribution of indium-111/yttrium-88-labeled B3 monoclonal antibody, a murine IgG1k, was evaluated in non-tumor-bearing mice. B3 was conjugated to either 2-(p-SCN-Bz)-6-methyl-DTPA (1B4M) or 2-(p-SCN-Bz)-1,4,7,10 tetraazacyclododecane tetra-acetic acid (2B-DOTA) and labeled with 111In at 1.4-2.4 mCi/mg and 88Y at 0.1-0.3 mCi/mg. Non-tumor-bearing nude mice were co-injected i.v. with 5-10 microCi/4-10 micrograms of 111In/88Y-labeled B3 conjugates and sacrificed at 6 h and daily up to 168 h post-injection. Mice injected with 111In/88Y-(1B4M)-B3 showed a similar biodistribution of the two radiolabels in all tissues except the bones, where significantly higher accretion of 88Y than 111In was observed, with 2.8% +/- 0.2% vs 1.3% +/- 0.16% ID/g in the femur at 168 h, respectively (P < 0.0001). In contrast, mice receiving the 111In/88Y-(DOTA)-B3 conjugate showed significantly higher accumulation of 111In than 88Y in most tissues, including the bones, with 2.0% +/- 0.1% vs 1.2% +/- 0.09% ID/g in the femur at 168 h, respectively (P < 0.0001). Whereas the ratios of the areas underneath the curve (%ID x h/g) in the blood, liver, kidney and bone were 0.96, 1.12, 1.13, and 0.74 for 111In/88Y-(1B4M)-B3 and 0.84, 1.23, 1.56, and 1.31 for 111In/88Y-(DOTA)-B3, respectively, ratios approximately 1 were observed between 111In-(1B4M)-B3 and 88Y-(DOTA)-B3. In summary, while neither 1B4M nor DOTA was equally stable for 111In and 88Y, the fate of 88Y-(DOTA)-B3 could be closely traced by that of 111In-(1B4M)-B3.

Antitumor activity and pharmacokinetics in mice of a recombinant immunotoxin containing a disulfide-stabilized Fv fragment.

Disulfide-stabilized Fvs (dsFv) are recombinant Fv fragments of antibodies in which the inherently unstable VH-VL heterodimer is stabilized by a disulfide bond engineered between structurally conserved framework positions of VH and VL. We have recently described a recombinant immunotoxin, B3(dsFv)-PE38KDEL, that is composed of such a dsFv connected to a truncated form of Pseudomonas exotoxin (PE38KDEL). This disulfide-stabilized immunotoxin is indistinguishable in activity and specificity from its single-chain immunotoxin counterpart (Brinkmann et al., Proc. Natl. Acad. Sci. USA, 90: 7538-7542, 1993). We have now constructed and evaluated the stability, pharmacokinetics, and antitumor effect of a very similar disulfide-stabilized immunotoxin B3(dsFv)-PE38. This immunotoxin is specifically cytotoxic to human cancer cell lines such as A431 that express the B3 antigen on their surface. In addition, the dsFv-immunotoxin is more stable at 37 degrees C in human serum than the corresponding single-chain immunotoxin B3(Fv)-PE38. The survival of the disulfide-stabilized immunotoxin in the circulation of mice was determined by a bioassay on cultured A431 cells after administering the immunotoxin i.v. The half-life in blood was 23 min. To determine the therapeutic effects of the disulfide-stabilized immunotoxin, it was given i.v. to immunodeficient mice bearing s.c. human epidermoid carcinomas. The dsFv-immunotoxin caused complete regression of tumors with no toxic effect on mice. The antitumor effect was similar to that reported for the single-chain Fv-immunotoxin. Our data show that dsFv-immunotoxins retain full in vitro as well as in vivo activity when compared to scFv-immunotoxins. Because dsFv-immunotoxins have full activity, are more stable, and can be produced with significantly improved yields compared to scFv-immunotoxins, the dsFv-immunotoxins may be more useful for therapeutic applications than scFv-immunotoxins.

Evaluation of the serum stability and in vivo biodistribution of CHX-DTPA and other ligands for yttrium labeling of monoclonal antibodies.

UNLABELLED: Serum stability and in vivo biodistribution of both A and B isomers of the 2-(p-isothiocyanatobenzyl) (p-SCN-Bz)-cyclohexyldiethylenetriaminepentaacetic acid ligand (CHX-DTPA), a recently developed backbone-substituted derivative of DTPA, were evaluated and compared to those of 2-(p-SCN-Bz)-6-methyl-DTPA (1B4M-DTPA) and 2-(p-SCN-Bz)-1,4,7,10-tetraazacyclododecane tetra-acetic acid (2B-DOTA). METHODS: Stability of 88Y-labeled ligands (0.1 microM) was evaluated in serum for up to 17 days. For biodistribution, ligands were conjugated to monoclonal antibody (Mab) B3, a murine IgG1k, and labeled with 88Y at 0.1-0.3 mCi/mg. Nontumor-bearing nude mice were injected intravenously with 1-2 microCi/4-10 micrograms of 88Y-labeled B3-conjugates and killed at 6 hr and daily up to 168 hr postinjection. Indium-111-(1B4M)-B3 was co-injected in all mice as internal control. RESULTS: Serum stability of 88Y-DOTA failed to show any significant release of activity, whereas pseudo-first-order dissociation rate constants of 3.97 x 10(-3), 2.54 x 10(-3) and 1.46 x 10(-2) (day-1) were calculated for 88Y-1B4M, 88Y-CHX-A and 88Y-CHX-B, respectively. Accordingly, cortical bone uptake of 88Y was significantly higher for all DTPA-derivative chelates than for DOTA. CONCLUSIONS: While none of the DTPA-derivative chelates could challenge DOTA in its ability to hold the radioytrium, significant differences were observed in the kinetic inertness of the A and B isomers of CHX, indicating that the CHX-B ligand is not as suitable for 90Y-labeling of Mabs.

Evaluation of a new DTPA-derivative chelator: comparative biodistribution and imaging studies of 111In-labeled B3 monoclonal antibody in athymic mice bearing human epidermoid carcinoma xenografts.

Biodistribution and imaging characteristics of monoclonal antibody (MAb) B3 conjugated to either the 2-(p-isothiocyanatobenzyl)-cyclohexyl-DTPA (CHX-B) or 2-(p-isothiocyanatobenzyl)-6-methyl-DTPA (1B4M) and labeled with 111In, were evaluated in nude mice bearing A431 human epidermoid carcinoma xenografts. MAb B3, is a murine IgG1k reacting with a carbohydrate antigen abundantly expressed by most carcinomas. Both 111In-(CHX-B)-B3 and 111In-(1B4M)-B3 showed good tumor targeting with peak values observed at 72 h with 27.6 +/- 7.6 and 25.4 +/- 1.7% ID/g, respectively (P > 0.05). High tumor-to-organ ratios were also observed and, confirmed by the imaging results. In particular, tumor-to-liver ratios increased from 5.0 +/- 0.9 at 24 h to 9.2 +/- 2.0 at 168 h for 111In-(CHX-B)-B3 and from 4.5 +/- 0.6 to 8.9 +/- 3.5 for 111In-(1B4M)-B3. This was mainly the result of low liver accumulation of both 111In-(CHX-B)-B3 and 111In-(1B4M)-B3, with only 2.48 +/- 0.46 and 2.5 +/- 0.9% ID/g at 168 h, respectively (P > 0.05). Our findings indicate that either CHX-B or 1B4M can be successfully used for 111In-labeling of MAbs and that 111In-B3 may represent a promising radioimmunoimaging agent.

Cytotoxicity and antitumor effects of growth factor-toxin fusion proteins on human glioblastoma multiforme cells.

The prognosis of glioblastoma multiforme remains poor despite advances in treatment by surgery, irradiation, and chemotherapy. Many malignant gliomas overexpress growth factor receptors. The possibility of targeting these receptors with selective cytotoxic molecules constructed by fusing deoxyribonucleic acid (DNA)-encoding mutant forms of Pseudomonas exotoxin A (PE) with complementary DNA-encoding growth factors was investigated. Several recombinant toxins have been produced, including those in which transforming growth factor (TGF)-alpha, insulin-like growth factor (IGF)-I, and acidic fibroblast growth factor (FGF) were fused to mutant forms of PE lacking the native cell-binding domain. These recombinant proteins are cytotoxic to cells that express specific cell-surface receptors. The cytotoxic activity of TGF-alpha, IGF-I, and acidic FGF chimeric toxins was tested in vitro against human glioblastoma cell lines. Each recombinant toxin exhibited potent and specific killing of cells. The TGF-alpha-PE40 construct was cytotoxic to seven of the eight cell lines and was active at concentrations as low as 0.5 ng/ml (1.1 x 10(-11) M). The acidic FGF-PE40 toxin was also active on seven of the eight cell lines but was 50-fold less active than the TGF-alpha-PE40. The IGF-I-PE40 construct was active on only two cell lines. To determine the possible therapeutic effect in animals, TGF-alpha-PE40 was administered to nude mice bearing subcutaneous human glioblastoma xenografts. The animals were treated for 7 days via a continuous infusion pump placed in the peritoneal cavity. A constant serum level of TGF-alpha-PE40 was achieved that was nontoxic to the mice yet caused a reduction in tumor volume and retarded growth beyond the treatment period. The overexpression of the epidermal growth factor receptor in glioblastomas multiforme and the potency and specificity of the TGF-alpha-PE40 construct designed to target this receptor suggests that TGF-alpha-PE40 has the potential to be an effective antitumor agent for the adjuvant therapy of these carcinomas.

Polyethylene glycol-modified chimeric toxin composed of transforming growth factor alpha and Pseudomonas exotoxin.

Modification of proteins with monomethoxy-polyethylene glycol (mPEG) has been shown to prolong circulation time and to reduce immunogenicity. To make a mPEG-modified recombinant toxin that retained cytotoxic activity but had a longer residence time in circulation, we have constructed an altered form of TGF alpha-PE40, a recombinant toxin composed of human transforming growth factor alpha (TGF alpha) fused to a fragment of Pseudomonas exotoxin (PE38) devoid of its cell-binding domain. In the newly designed protein, termed TGF alpha R29-L2-CH2-PE38QQ delta (TCP), there are no lysine residues in the TGF alpha and PE38 portions. Human IgG4 constant region CH2 and a tetradecapeptide linker; L2, are inserted between TGF alpha and PE38. Together, L2 and CH2 contain 13 lysine residues as potential modification sites for mPEG. mPEG conjugates of TCP (PEG-TCP) were generated and the products were resolved by ion exchange chromatography. Two PEG-TCP species termed B4 and B6 retained 15 and 4% of cytotoxicity, respectively, and 26% of their receptor binding activity compared with the unmodified TCP. Both B4 and B6 had prolonged circulation times in the blood and reduced toxicity in animals. The mean residence times of B4 and B6 were 37 and 68 min, respectively, compared to 7 min for TCP. When administered i.v. to tumor bearing mice, both B4 and B6 produced marked antitumor effects whereas the unmodified TCP had none. Also, the immunogenicity of PEG-TCP was 5-10 times less than that of TCP. We suggest that the prolonged circulating time and reduced toxicity of PEG-TCP compensate for a diminished cytotoxic activity and enlarge significantly the therapeutic window of this chimeric toxin.

Preclinical evaluation of 111In-labeled B3 monoclonal antibody: biodistribution and imaging studies in nude mice bearing human epidermoid carcinoma xenografts.

Biodistribution and imaging characteristics of monoclonal antibody B3 were evaluated in nude mice bearing A431 human epidermoid carcinoma xenografts. B3 is a murine IgG1k, recently isolated, reacting with a carbohydrate epitope abundantly and uniformly expressed by most carcinomas. B3 was conjugated to a new backbone-substituted derivative of diethylenetriaminepentaacetic acid, 2-(p-isothiocyanato benzyl)-cyclohexyl-diethylenetriaminepentaacetic acid, and labeled with 111In. Tumor-bearing mice were given i.v. injections of approximately 5 microCi of either 111In-B3 or 111In-MOPC-21, an isotype-matched control, and sacrificed in groups of five at 6 h and daily up to 168 h. Imaging was performed at 24, 72, and 144 h. Significant differences were observed in tumor uptake at all time points with peak values at 48 h (25 +/- 5.2% versus 6.3 +/- 0.4% of the injected dose/g tissue) (mean +/- SD) for 111In-B3 and 111In-MOPC-21, respectively (P < 0.001). All tumor to organ ratios increased with time for 111In-B3. In particular, tumor:liver ratios rose from 3.2 +/- 0.6 at 24 h to 6.3 +/- 1.2 at 168 h. Imaging results showed selective and progressive accumulation of 111In-B3 at the tumor site, whereas 111In-MOPC-21 did not show specific localization. In summary, 111In-labeled B3 demonstrated good and specific tumor targeting, which warrants its future clinical evaluation.