Synthesis and Biological Evaluation of Chlorinated Analogs of Leukotoxin Diol.

This study documents that chlorinated analogs of leukotoxin diol 1, in which the vic-diol has been replaced with vic-chlorides (2), induce caspase 3 activity and apoptosis on HepG2 cells in a dose-dependent manner in analogy to the parent diol. This suggests that chlorides may substitute for hydroxyls in certain lipids as bioisosteres in defined biological settings.

The IL-4 and IL-13 pseudomonas exotoxins: new hope for brain tumor therapy.

Targeting cell surface receptors with cytotoxins or immunotoxins provides a unique opportunity for brain tumor therapy. The authors have discovered that receptors for two cytokines, interleukin (IL)-4 and IL-13, are overexpressed on tumor biopsy samples and on cell lines derived from a variety of human tumors, including brain tumors. These investigators have demonstrated that the structure of these cytokine receptors on tumor cells is different from that found on normal immune cells. In human solid tumor cells, IL-4 binds to two chains (IL-4Ra and IL-13Ra1), whereas IL- 13 binds to three chains in many solid tumor cells, including glioma cells (to IL-4Ra, IL-13Ra1, and IL-13Ra2). To target IL-4Rs and IL-13Rs, the authors generated two recombinant fusion cytotoxins composed of IL-4 or IL-13 and a mutated form of pseudomonas exotoxin (PE), which for simplicity are called IL4-PE and IL13-PE in this paper. These chimeric cytotoxins are highly toxic in vitro to human tumor cell lines and primary cell cultures, including glioma cells, and in vivo to animal models of human tumors, including gliomas. In contrast, normal cells, including immune, endothelial, and brain cells, are spared from their cytotoxic effects. Based on numerous preclinical studies, IL13-PE (also known as IL13-PE38QQR or cintredekin besudotox) has been tested in four Phase I/II clinical trials. The agent IL13-PE was administered intracranially by using convection-enhanced delivery (CED). The drug was delivered through catheters placed either directly into the tumor bed or in the peritumoral region after resection of the lesion. The CED of IL13-PE was fairly well tolerated, with a reasonable benefit/risk profile for treatment of patients with glioma. Based on Phase I/II clinical trials, the Phase III Randomized Evaluation of CED of IL13-PE Compared to Gliadel Wafer with Survival Endpoint Trial (also known as the PRECISE Trial) in patients with initial recurrence of glioblastoma multiforme has recently been completed. Patients are being monitored for safety of the agents, duration of overall survival, and quality of life.

Depletion of CCR5-expressing cells with bispecific antibodies and chemokine toxins: a new strategy in the treatment of chronic inflammatory diseases and HIV.

The chemokine receptor CCR5 is expressed on the majority of T cells and monocytes in the inflammatory infiltrate of diseases such as rheumatoid arthritis, renal diseases, and multiple sclerosis. In contrast, little expression of CCR5 is found on peripheral blood leukocytes. A specific depletion of CCR5(+) cells could therefore be a useful strategy to reduce the cellular infiltrate in chronic inflammations. Moreover, CCR5 is the major coreceptor for M-tropic HIV-1 strains. Depletion of CCR5(+) leukocytes may help to eliminate cells latently infected with HIV-1. We designed two constructs that specifically destroy chemokine receptor-positive cells. The first construct, a bispecific Ab, binds simultaneously to CCR5 and CD3. Thereby it redirects CD3(+) T cells against CCR5(+) target cells. The Ab specifically depletes CCR5(+) T cells and monocytes, but is inactive against cells that do not express CCR5. Furthermore, ex vivo the bispecific Ab eliminated >95% of CCR5(+) monocytes and T cells from the synovial fluid of patients with arthritis. Also, we designed a fusion protein of the chemokine RANTES and a truncated version of Pseudomonas. exotoxin A. The fusion protein binds to CCR5 and down-modulates the receptor from the cell surface. The chemokine toxin completely destroyed CCR5(+) Chinese hamster ovary cells at a concentration of 10 nM, whereas no cytotoxic effect was detectable against CCR5(-) Chinese hamster ovary cells. Both constructs efficiently deplete CCR5-positive cells, appear as useful agents in the treatment of chronic inflammatory diseases, and may help to eradicate HIV-1 by increasing the turnover of latently infected cells.

Development of streptococcal pyrogenic exotoxin C vaccine toxoids that are protective in the rabbit model of toxic shock syndrome.

Streptococcal pyrogenic exotoxin C (SPE C) is a superantigen produced by many strains of Streptococcus pyogenes that (along with streptococcal pyrogenic exotoxin A) is highly associated with streptococcal toxic shock syndrome (STSS) and other invasive streptococcal diseases. Based on the three-dimensional structure of SPE C, solvent-exposed residues predicted to be important for binding to the TCR or the MHC class II molecule, or important for dimerization, were generated. Based on decreased mitogenic activity of various single-site mutants, the double-site mutant Y15A/N38D and the triple-site mutant Y15A/H35A/N38D were constructed and analyzed for superantigenicity, toxicity (lethality), immunogenicity, and the ability to protect against wild-type SPE C-induced STSS. The Y15A/N38D and Y15A/H35A/N38D mutants were nonmitogenic for rabbit splenocytes and human PBMCs and nonlethal in two rabbit models of STSS, yet both mutants were highly immunogenic. Animals vaccinated with the Y15A/N38D or Y15A/H35A/N38D toxoids were protected from challenge with wild-type SPE C. Collectively, these data indicate that the Y15A/N38D and Y15A/H35A/N38D mutants may be useful as toxoid vaccine candidates.

Development of a recombinant interleukin-4-Pseudomonas exotoxin for therapy of glioblastoma.

About 12,000 Americans are diagnosed with malignant astrocytoma each year. Despite surgery, radiotherapy, and chemotherapy, the prognosis of these patients remains poor. Targeted toxins based on the identification of novel antigens or receptors provide a promising new approach to treating cancer. We have identified one such cell surface protein in the form of interleukin (IL)-4 receptors (IL-4R) on human malignant astrocytoma. Normal brain tissues from frontal cortex and temporal lobe cortex do not express IL-4R. To target IL-4R, we generated a chimeric fusion protein composed of IL-4 and Pseudomonas exotoxin (IL4-PE). This toxin is highly cytotoxic to IL-4R-bearing human brain cancer cells. Preclinical toxicologic experiments were performed in mice, rats, and guinea pigs to determine an maximum tolerated dose. Intrathecal administration in cynomolgus monkeys produced high cerebrospinal fluid levels without any central nervous system or other abnormalities. When IL4-PE was injected into the right frontal cortex of rats, localized necrosis was observed at 1,000 but not < or =100 microg/ml doses. Intravenous administration of this biologic to monkeys produced reversible grade 3 or grade 4 elevations of hepatic enzymes in a dose-dependent manner. These results indicate that localized administration can produce nontoxic levels of IL4-PE that may have significant activity against astrocytoma. In vivo experiments with nude mice have demonstrated that IL4-PE has significant antitumor activity against human glioblastoma tumor model. Intratumor administration of IL4-PE has been initiated for the treatment of malignant astrocytoma in a phase I clinical trial.

Synthesis and characterization of a polyvalent Escherichia coli O-polysaccharide-toxin A conjugate vaccine.

A 12-valent Escherichia coli O-polysaccharide (O-PS)-toxin A conjugate vaccine was formulated. Nonpyrogenic, low-molecular-weight O-PS was derived from lipopolysaccharides (LPS) of the following serotypes: O1,O2,O4,O6,O7,O8,O12, O15,O16,O18,O25, and O75. Individual O-PS were covalently coupled to Pseudomonas aeruginosa toxin A using adipic acid dihydrazide as a spacer molecule and carbodiimide as a coupling agent. On a weight basis, the final multivalent vaccine was composed of 43% O-PS and 57% toxin A. The vaccine was nontoxic nad nonpyrogenic in anti-LPS immunoglobulin G (IgG) antibody titers. When passively transferred to mice, immune rabbit IgG conferred statistically significant (p < 0.05) protection against a challenge with 9 of the 12 vaccine serotypes. For two serotypes, although the mortality rate declined by > or 50% in the passively immunized versus the control group, the difference did not reach statistical significance. The degree of protection provided by passively transferred IgG was influenced by both the anti-LPS antibody levels in the IgG preparation and the virulence of the challenge strain. Active immunization of mice with either conjugate vaccine or killed E. coli whole cells did not confer protection. This was most probably due to the fact that these antigens induced a meagre anti-LPS IgG antibody response.

[Synthesized leukotoxin: its biological activity and establishment of radioimmunoassay].

Leukotoxin, 9: 10-epoxy-12-octadecenoic acid, was reported to exist in human burned skin and neutrophils, and to have toxic effects in experimental animals and antifungal effects against rice blast disease. Leukotoxin was regarded as a toxic and/or defensive substance in living beings. The author synthesized leukotoxin from linoleic acid with peracetic acid and purified practically by thin layer chromatography. The leukotoxin synthesized was injected into guinea pigs intravenously and caused a systemic convulsion of the animal body and cardiac arrest. The leukotoxin synthesized, on the other hand, was conjugated with bovine serum albumin (BSA) by means of the mixed anhydride technique and immunized in rabbits. After immunization of leukotoxin conjugated with BSA over 4 months, the author succeeded in producing anti-leukotoxin antiserum for the first time. According to a titration test of antiserum, sixty-folds diluted antiserum was found to bind approximately 50% of methylated leukotoxin labeled with carbon 14. And unlabeled leukotoxin was detected at least 5 ng in this radioimmunoassay by use of polyethylene glycol precipitation. This antiserum had a strong specificity to leukotoxin and no cross-reactivity to the other analogs tested. The role of leukotoxin in living creatures had not been clarified yet. Therefore both the leukotoxin synthesized by this simple procedure and the anti-leukotoxin antibodies would aid the study of the mechanism of its biological activities and its histochemical investigations.