Targeting cell-impermeable prodrug activation to tumor microenvironment eradicates multiple drug-resistant neoplasms

The tumor microenvironment is notably enriched with a broad spectrum of proteases. The proteolytic specificities of peptide substrates provide modular chemical tools for the rational design of cell-impermeable prodrugs that are specifically activated by proteases extracellularly in the tumor microenvironment. Targeting cell-impermeable prodrug activation to tumor microenvironment will significantly reduce drug toxicity to normal tissues. The activated prodrug attacks both tumor and stroma cells through a "bystander effect" without selectively deleting target-producing cells, therefore further minimizing resistance and toxicity. Here, we showed that legumain, the only asparaginyl endopeptidase of the mammalian genome, is highly expressed by neoplastic, stromal, and endothelial cells in solid tumors. Legumain is present extracellularly in the tumor microenvironment, associated with matrix as well as cell surfaces and functional locally in the reduced pH of the tumor microenvironment. A novel legumain-activated, cell-impermeable doxorubicin prodrug LEG-3 was designed to be activated exclusively in the tumor microenvironment. Upon administration, there is a profound increase of the end-product doxorubicin in nuclei of cells in tumors but little in other tissues. This tumor microenvironment-activated prodrug completely arrested growth of a variety of neoplasms, including multidrug-resistant tumor in vivo and significantly extended survival without evidence of myelosuppression or cardiac toxicity. The tumor microenvironment-activated prodrug design can be extended to other proteases and chemotherapeutic compounds and provides new potentials for the rational development of more effective functionally targeted cancer therapeutics.     

Lymphoid procoagulant response to bacterial endotoxin in the rat.

A number of species respond to bacterial endotoxin (lipopolysaccharide [LPS]) wherein cells of the monocyte-macrophage lineage are rapidly induced either directly or via T-cell collaboration to initiate the extrinsic coagulation protease pathway. This results in fibrin formation and deposition as well as consumption of plasma coagulation proteins. It has been claimed that this cellular response, basic to the Shwartzman reaction, is lacking in rats and may account for the more limited severity of the Shwartzman reaction in this species. We examined the in vitro lymphoid procoagulant response in Fischer 344, Brown Norway, and Lewis rats. When peripheral blood mononuclear cells (PBM) were stimulated in vitro with LPS, a procoagulant activity (PCA) response was observed when assayed by acceleration of clotting of recalcified human or rat platelet-poor plasma. The response was rapid, with a maximum achieved at 4 h. PCA was not physically dissociated from viable PBM by 5 mM EDTA, which is consistent with the presence of an intrinsic plasma membrane initiator molecule rather than calcium-bound gamma-carboxylated glutamic acid-containing proteases. The induction of monocyte PCA was prevented by incubation of cells with cycloheximide or actinomycin D, implicating a new biosynthetic requirement. Cultivation of PBM with warfarin did not diminish the function of the effector PCA, nor did vitamin K augment the function of the endotoxin-induced PCA, indicating that the functional activity was not attributable to gamma-carboxylated glutamic acid-containing proteins. No inhibition of the cellular PCA molecule was produced by serine protease inhibitors. The LPS-induced PCA appeared to involve a tissue factor-like molecule since both factors X and VII were required in mediating PCA. Isolation of monocytes and T lymphocytes from LPS-stimulated PBM demonstrated that PCA was present in the monocyte-rich fraction. When isolated rat T lymphocytes and monocytes were separately exposed to LPS, PCA was not induced. In contrast, when the cells were combined, LPS induced PCA, indicating that the PCA response involved cellular collaboration between cells present in T lymphocyte and monocyte populations.     

Drug-Injecting Street Youth: A Comparison of HIV-Risk Injection Behaviors Between Needle Exchange Users and Nonusers

Injection drug use, the second most common risk factor associated with HIV infection in the United States after sexual transmission, is especially prevalent among street youth, many of whom routinely share their drug injection equipment. To prevent sharing behavior among street youth, a needle exchange program for youth was established in Hollywood, California. This study compared the drug use and needle-sharing behaviors of youth who were users of the needle exchange with those of youth who were not. Results indicate that while demographic characteristics and drug injection frequency were similar in the two groups, needle exchange users were significantly less likely to share needles, share other injection equipment, use other drugs to help them come down, use unsterile needles when high or craving drugs, and report difficult access to sterile needles. These findings suggest that needle exchange programs may reduce needle-sharing behavior among street youth.     

Hybridoma antibodies to human von Willebrand factor

Twenty-seven stable subclones of seven independent cellular hybrids producing murine monoclonal antibodies to human von Willebrand factor (vWF) have been established. The specificity of the hybridoma antibodies for vWF has been substantiated by a variety of methods including binding to highly purified vWF, absence of binding to plasma or cryoprecipitate from severe von Willebrand's disease, binding to different size multimers in normal plasma, and binding to low molecular weight multimers in type IIA von Willebrand's disease plasma. Monoclonality of the hybridoma derived antibodies has been sought by repeated cloning at limiting dilutions. All seven specificities of hybridoma antibodies cross-react to a variable degree with porcine vWF and all but one with bovine vWF, indicating that they bind to structural loci that are relatively though not identically conserved between species. Hybridoma antibodies bind native as well as denatured vWF, suggesting that the epitopes may be determined to a considerable extent by primary structure rather than entirely by tertiary or quaternary conformation. The hybridoma antibodies define precise epitopes in the vWF molecule, extend the horizon of direct analysis of vWF to the intramolecular level, and may be useful in assigning functional loci to parts of the molecule.     

Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody

Gram-negative bacteremia poses a major health problem, causing one-half of cases of lethal septic shock acquired during hospitalization. Bacterial lipopolysaccharide (LPS) and the inflammatory cytokines, tumor necrosis factor (TNF) and interleukin-1 (IL-1), have been shown to be essential mediators of septic shock. Among the effects of these mediators is a coagulopathy that may be triggered by induced expression of tissue factor (TF) on macrophages and endothelial cells. We now report that 500 micrograms/kg of either immunoglobulin G (IgG) or Fab fragments of a monoclonal antibody against TF administered to baboons as a pretreatment attenuates the coagulopathy and protects against LD100 Escherichia coli. This study provides direct evidence of an essential effector role for TF in septic shock.     

Discriminating Neoantigenic Differences Between Fibrinogen and Fibrin Derivatives

Discrimination between the physiological cleavage fragments of fibrinogen and fibrin offers an approach to differentiation between fibrinogenolytic processes and fibrinolysis after coagulation. By use of the cleavage-associated neoantigen of fibrinogen (fg-D(neo)) as a molecular marker, characteristic differences between the D regions of fibrinogen derivatives and fibrin derivatives can be demonstrated. The expression of fg-D(neo) by X, Y, D:E complex, and D-fragments of fibrinogen or fibrin is shown to be quantitative and unitary. Characteristic differences between fg-D(neo) sites present on fibrinogen cleavage fragments, as contrasted to fibrin cleavage fragments, are indicated by different competitive inhibition slopes, and appear to reflect differential binding affinity of selected anti-fg-D(neo) antibodies for the specific molecular site. There is a linear relationship between the slope of quantitative competitive inhibition and the relative molar ratio of fibrinogen and fibrin derivatives. Identical immunochemical expressions are observed in vitro and in vivo, and support the thesis that cleavage in vivo is produced by plasmin. The differential immunochemical features of fg-D(neo) expression may be the result of stable conformational and/or subtle structural differences between the D region of fibrinogen and fibrin cleavage fragments and suggest that precise changes in the D region are associated with the fibrin transition. These molecular features not only provide additional insight into the molecular immunology and structure of fibrinogen, but also appear to offer a new molecular approach to discrimination between fibrinogenolytic mechanisms as contrasted to fibrinolysis secondary to coagulation.