Search

The overall goal of gene therapy is to cure or stabilize a disease process that results from the production of a mutant protein (for example, the chloride channel protein important in cystic fibrosis) or overproduction of a normal protein (such as the products of certain oncogenes). We can achieve this goal by replacing the defective gene or by reducing the overexpression of the target gene using an antisense strategy, thus reducing the production of the diseasepromoting protein (1,2). For either method, it is critical to transfer DNA into target cells in a concentration high enough to be effective in modifying the disease. DNA must be delivered to the desired cell population in an intact state, whereby it can be efficiently transcribed and ultimately translated. The method of gene transfer must be highly efficient and nontoxic, and the delivery system must be relatively easy to prepare and administer (3). There is a great deal of optimism surrounding the development of gene therapy as an effective strategy for management of many different human diseases. The active agent used to procure gene therapy is likely to consist of oligonucleotides, ribozymes, or a DNA sequence that can be transcribed into a message capable of eliciting a therapeutic response. Unlike conventional small-molecule therapeutics however, gene therapy requires the use of a carrier system to deliver the active agent directly into the target cell population. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide formed in the lung tissue in ischemia/reperfusion or released from activated leukocytes causes oxidative injury of the vascular endothelial cells (1-3). H2O2-degrading enzyme, catalase, has been extensively explored in order to protect cells and tissues against H2O2-mediated injury (4). Catalase, however, has short lifetime in the bloodstream and provides only marginal protective effect after intravascular administration in animals (5). Catalase modification (conjugation with polyethylene glycol (6) or encapsulation in liposomes [7]), prolongs catalase lifetime in the circulation and facilitate its cellular uptake. These modifications, however, do not provide it with an affinity to the endothelial cells. In order to provide catalase with such an affinity, catalase could be chemically conjugated with a carrier antibodies recognizing the surface endothelial antigens (8). A monoclonal antibody (MAb) against such an antigen, angiotensin-converting enzyme (anti-ACE MAb 9B9, produced by Dr. Sergei M. Danilov [9]), accumulates in the pulmonary endothelium after systemic injection (10). Therefore, MAb 9B9 may serve as an affinity carrier for targeting of catalase to the pulmonary endothelial cells, for specific augmentation of their antioxidative defense. [ABSTRACT FROM AUTHOR]

Malaria is a serious public health problem that affects about 300-500 million people and claims 1.5-2.7 million deaths every year. One-third of all humans live in zones where they risk catching it (1). The situation is aggravated because the malarial parasites are rapidly developing resistance to the existing antimalarial drugs, like chloroquine (2), when given in classical pharmaceutical forms. Studies on the molecular basis of chloroquine resistance suggest that enhanced active efflux of the drug from the cells infected with resistant parasite strain prevents drug accumulation to toxic levels within the cytosol of the infected erythrocytes (3-5). It has been shown that erythrocytes infected with chloroquine-resistant parasite accumulate less chloroquine than those with sensitive parasites (6,7). Furthermore, inhibiting the chloroquine efflux by Ca2+-channel blockers render the resistant cells fully sensitive to chloroquine (8), indicating that the antimalarial activity of the chloroquine is directly related to its concentration within the parasite food vacuole (9). The mechanism by which this concentration effect is achieved is unclear, but it is believed to involve binding to a putative chloroquine receptor (10). [ABSTRACT FROM AUTHOR]

Recombinant toxins are cytotoxic proteins that are encoded by DNA sequences that can be expressed in prokaryotic or eukaryotic cells. The proteins contain both a ligand, for binding to cells, and a toxin, for killing the cells. In recombinant toxins the connection between the toxin and ligand is encoded by the DNA and not produced by chemical conjugation. Since recombinant toxins must bind specifically to cell surface molecules to kill cells, the number of possible recombinant toxin molecules that can bind to a cell is in the hundreds or thousands; hence, the toxins must be very potent. [ABSTRACT FROM AUTHOR]

Targeting pharmaceuticals to the infarcted myocardium has two primary objectives: the diagnostic imaging of the infarcted myocardium and the delivery of therapeutic agents to compromised myocardial areas. Various diagnostic and therapeutic agents (such as radiolabeled compounds, thrombolytic enzymes, proteolytic drugs, and antioxidants) have been proposed for visualization or treatment of the infarcted myocardium. However, none of these reagents has the specificity for targeting the compromised myocardium. Therefore, the availability of a target-specific delivery system should increase the efficacy of diagnosis and therapy. Furthermore, the existence of such a targeted delivery system may pave the way for the use of new pharmaceuticals that by themselves can be harmful to normal tissues. [ABSTRACT FROM AUTHOR]

Research into the treatment of cancer has often been driven by the idea that a common biochemical pathway might exist in all tumors, providing an ideal target for therapy. However, it is now clear that a great variety of genetic changes contributes to the development of individual cancers, and that no two cancers are identical. Nevertheless, a common feature in solid tumors is the disorganized way in which the blood vessels develop. Networks of tumor capillaries, linked to the blood supply by arterioles and venules, are necessary for tumors to grow but, in contrast to capillaries supplying normal tissue, are haphazard in both their structure and their dynamics. It may be possible to use this common feature as a basis for selective therapy. In this chapter we provide a short review of the vasculature of solid tumors, then describe methods by which drugs might be used to inhibit tumor blood flow. Finally, we review some of the methods available for measurement of tumor blood flow. [ABSTRACT FROM AUTHOR]

Specific targeting of radioisotopes or toxic drugs to tumors for cancer detection and treatment is an enticing but elusive goal. It has proved difficult to achieve adequate concentration ratios between tumor and normal tissues to improve on standard diagnostic and therapeutic methods. [ABSTRACT FROM AUTHOR]

This chapter will describe methods that may be used to deliver agents to HIV-infected cells. These materials may be used for therapeutic or experimental purposes. There are several general approaches to delivering compounds to human immunodeficiency cells (HIV)-infected cells. All cells may be exposed to materials that only have an effect or become activated in HIV-infected cells. Examples include drugs that are specific for HIV-encoded enzymes, such as reverse transcriptase or protease, or genes that are expressed under the control of the HIV-LTR. Lack of specificity is a major limitation to this approach; for example, reverse transcriptase inhibitors also inhibit cellular DNA polymerases and cellular transcription factors clan initiate low-level-transcription off the HIV-LTR, even in the absence of tat. The alternative approach, which is the subject of this chapter, is to target the materials specifically to the infected cells. We have used monoclonal antibodies (MAbs) to deliver toxins to HIV-infected cells, but others have used this approach to deliver antiviral agents, liposomes, and even genes. [ABSTRACT FROM AUTHOR]

The huge molecular radius of immunoglobulins would seem to be a major drawback for the targeting of solid tumors, because of slow extravasation into tumor interstitium and along plasma half-life. The permeability of normal continuous capillary endothelia to intravascular solutes of different molecular sizes has been determined in animals, mainly for macromolecules, and different sources give data consistent with the graph in Fig. 1 (1-3). The position of whole antibodies (IgG, mol wt 150 kDa and effective molecular radius 5.5 nm) is well to the right of albumen (66 kDa and 3.5 nm), and they are therefore very slowly extravasated in normal tissues. A F(ab')2 fragment (100 kDa, 5.06 nm) should not extravasate much faster than the intact molecule on this basis and a monomeric Fab' fragment (50 kDa, 3.48 nm) still has quite a high molecular radius (4), so a much smaller molecule would be necessary to equilibrate very quickly with extracellular fluid (ECF). [ABSTRACT FROM AUTHOR]

Selective cytotoxicity is an important goal of specific drug targeting. Toward this end, toxins isolated primarily from higher plants and bacteria have been coupled to monoclonal antibodies (MAbs) and evaluated for their clinical efficacy in cancer, AIDS, and immunological diseases (1,2). Immune responses against murine monoclonal antibodies MAbs (3,4) and antitoxin antibodies have been detected in both animals and humans treated with immunotoxins (ITs) (5-7) and present a major obstacle to the successful application of this technology. Although development of humanized antibodies have alleviated some of these effects (8, and references therein), the toxins themselves remain a problem. Consequently, the identification of human proteins to be used as components of immunoconjugates is highly desirable [ABSTRACT FROM AUTHOR]

Folic acid (Mr = 441, Fig. 1) is a vitamin essential for de novo nucleotide synthesis and one-carbon metabolism. The ability to acquire folate, therefore, is important to the viability of proliferating cells. [ABSTRACT FROM AUTHOR]

Liposomes are useful drug delivery vehicles since they may protect encapsulated drugs from enzymatic degradation and rapid clearance in vivo, or alter biodistribution, potentially leading to reduced toxicities (1,2). A major limitation to the development of many specialized applications is the problem of directing liposomes to tissues where they would not normally accumulate. Consequently, a great deal of effort has been made over the years to develop liposomes that have targeting vectors attached to the bilayer surface. These vectors have included ligands such as oligosaccharides (3,4), peptides (5,6), proteins (7,8) and vitamins (9). Most studies have focused on antibody conjugates since procedures for producing highly specific monoclonal antibodies (MAbs) are well established. In principle it should be possible to deliver liposomes to any cell type as long as the cells are accessible to the carrier. In practice it is usually not this simple since access to tissue, competition, and rapid clearance are formidable obstacles. It has also been shown that antibodies become immunogenic when coupled to liposomes (10,11), although in similar experiments with ovalbumin we have demonstrated that immunogenicity can be suppressed by formulating the liposomes with the cytotoxic drug doxorubi-cin (12). Such issues as these suggest that the development of antibody-targeted liposomes for in vivo applications will present difficult challenges. [ABSTRACT FROM AUTHOR]

The technological development and application of bispecific antibodies for biological research have advanced steadily since the idea of creating hybrid reagents with dual specificity was first promulgated by Nisonoff and Rivers (1). It was realized that appropriately designed bispecific antibodies could provide a unique means for selectively delivering biologically active agents onto the surface of target cells so that they could ultimately be internalized (2-7). Hybrid constructs developed in my laboratory used a specific antibody to reversibly bind the effector molecule within its combining site, whereas the second antibody or ligand component accurately targeted the complex to selected sites on the cell membrane Fig. 1). Those target receptor sites, along with the attached hybrid antibody complex, are subsequently taken inside the cell via receptor-mediated endocytosis. Cytotoxic drugs and toxins were chosen for delivery via the bispecific reagent because the entry of these potent molecules into target cells is signaled by an easily measured intracellular activity (2-7). [ABSTRACT FROM AUTHOR]

Immunotoxins based on human and humanized ribonuclease may have potential for cancer therapy while exhibiting less toxic side effects and stimulating less of an immune response in humans than immunotoxins based on plant and bacterial toxins (1). Both recombinant RNase fusion proteins (2-4 see alsoChapter 6, this volume) and chemical RNase conjugates have been made and characterized. The cytotoxic potential of targeted ribonuclease was first demonstrated with bovine RNase conjugated to transferrin or an antibody directed against the human transferrin receptor (5). Antibody RNase conjugates have also been shown to have potent anti-tumor activity against human glioma cells in athymic mice (6) and to enhance the activity of vincristine in mdr1 multidrug-resistant colon cancer cells in vitro and in vivo (7). Recently, RNase chemically conjugated to an antibody against CD22 was found to specifically kill Daudi lymphoma cells in cell culture at picomolar concentrations (IC50, 10-50 pM) and to exhibit potent antitumor activity in SCID mice with disseminated Daudi lymphoma (unpublished data). Methods for linking RNase to specific cell binding ligands are described. [ABSTRACT FROM AUTHOR]

Immunotoxins (ITs) are chimeric proteins consisting of an antibody linked to a toxin. The antibody confers specificity (ability to recognize and react with the target), whereas the toxin confers cytotoxicity (ability to kill the target) (1-3). ITs have been used in both mice and humans to eliminate tumor cells, autoimmune cells, and virus-infected cells (4-6. [ABSTRACT FROM AUTHOR]