Haya Lorberboum-Galski

TAT-based drug delivery system – new directions in protein delivery for new hopes?

There has been great progress in the use of TAT-based drug delivery systems for the delivery of different macromolecules into cells in vitro and in vivo, thus circumventing the bioavailability barrier that is a problem for so many drugs. There are many advantages to using this system, such as the ability to deliver these cargoes into all types of cells in culture and into all organs in vivo. This system can even deliver cargoes into the brain across the blood–brain barrier. In addition, the ability to target specific intracellular sub-localizations such as the nuclei, the mitochondria and lysosomes further expands the possibilities of this drug delivery system to the development of sub-cellular organelle-targeted therapy. The therapeutic applications seem almost unlimited, and the use of the TAT-based delivery system has extended from proteins to a large variety of cargoes such as oligonucleotides, imaging agents, low molecular mass drugs, nanoparticles, micelles and liposomes. In this review the most recent advances in the use of the TAT-based drug delivery system will be described, mainly discussing TAT-mediated protein delivery and the use of the TAT system for enzyme replacement therapy.

Tryptase activates peripheral blood mononuclear cells causing the synthesis and release of TNF-α, IL-6 and IL-1β: possible relevance to multiple sclerosis

Abstract Presence of mast cells and an increase in the concentration of their products has been reported in multiple sclerosis (MS) plaques. The most abundant secretory mediator of the human mast cell is the tetrameric protease tryptase. We demonstrate that tryptase can activate peripheral mononuclear cells (PBMCs), isolated from healthy donors as well as MS patients for the release of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β. Cytokine secretion was significantly higher in secondary progressive (SP) MS patients and healthy control (HC) individuals than in relapsing–remitting (RR) patients. Our findings suggest that tryptase is, most probably, an important mediator of inflammation in MS.

Adenocarcinoma Cells Are Targeted by the New GnRH-PE66 Chimeric Toxin through Specific Gonadotropin-releasing Hormone Binding Sites

Luteinizing hormone-releasing hormone, also termed gonadotropin-releasing hormone (GnRH), accounts for the hypothalamic-pituitary gonadal control of human reproduction. The involvement of GnRH has been demonstrated in several carcinomas of hormone-responsive tissues. Exploiting this common feature, we constructed a Pseudomonas exotoxin (PE)-based chimeric toxin (GnRH-PE66) aimed at targeting those cancer cells bearing GnRH binding sites. We report here the strong growth inhibition and killing of a surprisingly wide variety of cancers, confined to the adenocarcinoma type. These cancer cells arising from hormone-responsive tissues, as well as non-responsive ones, express specific GnRH binding sites as indicated by the marked killing of ovarian, breast, endometrial, cervical, colon, lung, hepatic, and renal adenocarcinoma. This cytotoxicity is specific as it could be blocked upon addition of excess GnRH. The specificity of GnRH-PE66 chimeric toxin was also confirmed by GnRH binding assays, and its ability to prevent the formation of colon cancer xenografts in nude mice is presented. Although the functional role of specific GnRH binding sites in human carcinomas remains obscure, GnRH-PE66 displays considerable targeting potential and its use as a therapeutic agent for cancer should be considered.

INTERLEUKIN 2-BAX : A NOVEL PROTOTYPE OF HUMAN CHIMERIC PROTEINS FOR TARGETED THERAPY

During the past few years many chimeric proteins have been developed to target and kill cells expressing specific surface molecules. Generally, these molecules carry a bacterial or plant toxin that destroys the unwanted cells. The major obstacle in the clinical application of such chimeras is their immunogenicity and non‐specific toxicity. We have developed a new generation of chimeric proteins, taking advantage of apoptosis‐inducing proteins, such as the human Bax protein, as novel killing components. The first prototype chimeric protein, IL2‐Bax, directed toward IL2R‐expressing cells, was constructed, expressed in Escherichia coli and partially purified. IL2‐Bax increased the population of apoptotic cells in a variety of target T cell lines, as well as in human fresh PHA‐activated lymphocytes, in a dose‐dependent manner and had no effect on cells lacking IL2R expression. The IL2‐Bax chimera represents an innovative approach for constructing chimeric proteins comprising a molecule that binds a specific cell type and an apoptosis‐inducing protein. Such new chimeric proteins could be used for targeted treatment of human diseases.

TAT-mediated Delivery of LAD Restores Pyruvate Dehydrogenase Complex Activity in the Mitochondria of Patients with LAD Deficiency

Modern medicine offers no cure for mitochondrial disorders such as lipoamide dehydrogenase (LAD) deficiency. LAD is the E3 subunit shared by alpha-ketoacid dehydrogenase complexes in the mitochondrial matrix, and these complexes are crucial for the metabolism of carbohydrates and amino acids. We propose a novel concept for restoring the activity of an immense multicomponent enzymatic complex by replacing one mutated component, the LAD subunit. Our approach entails the fusing of LAD with the transactivator of transcription (TAT) peptide, which is capable of rapidly crossing biological membranes, thereby allowing TAT-LAD to be delivered into cells and their mitochondria where it can replace the mutated endogenous enzyme. Our results show that TAT-LAD is indeed delivered into the cells and their mitochondria, where it is processed, restoring LAD activity to normal values and, most importantly, increasing the activity of pyruvate dehydrogenase complex. We report here, for the first time, that TAT-mediated replacement of one mutated component restores the activity of an essential mitochondrial multicomponent enzymatic complex in cells of patients with enzyme deficiencies. We believe that this approach involving TAT-mediated enzyme replacement therapy (ERT) can be applied to the treatment of LAD deficiency as well as to other mitochondrial and metabolic disorders.

A Novel Approach for Enzyme Replacement Therapy THE USE OF PHENYLALANINE HYDROXYLASE-BASED FUSION PROTEINS FOR THE TREATMENT OF PHENYLKETONURIA

Metabolic diseases arise from mutations in key enzymes of major metabolic pathways. One promising approach for the treatment of such diseases is based on the administration of a wild-type enzyme to substitute the activity of the impaired enzyme by the use of enzyme replacement therapy, yet it is important to deliver this enzyme to the specific deficient tissue. We suggest a new concept for the treatment of metabolic diseases using fusion proteins. We examined the feasibility of this concept in the well characterized metabolic disease, phenylketonuria (PKU), which results from a mutation in the liver enzyme phenylalanine hydroxylase (PAH). PAH is a key enzyme in the metabolic pathway of phenylalanine. Deficiency in PAH leads to high and persistent levels of this amino acid in the plasma of PKU patients, causing permanent neurological damage. Currently a low protein diet is still considered the only effective treatment for most PKU patients. To restore PAH activity in the liver of PKU patients, we constructed PAH-based fusion proteins with delivery moieties based on the HIV-transactivator of transcription peptide, and fragments of human hepatocyte growth factor aiming to specifically target PAH to the liver. We show that these new fusion proteins can be delivered into a variety of human liver cell lines and retain PAH activity after being internalized. We also show that plasma phenylalanine levels were dramatically lowered in mice treated with PAH-based fusion proteins after intravenous administration. We therefore suggest an alternative concept for the treatment of PKU using targeted fusion proteins, which may also be applied to the treatment of other metabolic diseases.

Detection of minimal residual disease state in chronic myelogenous leukemia patients using fluorescence in situ hybridization.

Detection of minimal residual disease and relapse remain major problems in chronic myelogenous leukemia (CML) patients following bone marrow transplantation (BMT). In order to disclose the 9;22 Philadelphia translocation, we used a fluorescence in situ hybridization (FISH) technique. BCR and ABL gene fragments were used as probes for the detection of the BCR/ABL fusion product in peripheral blood and bone marrow cells from 11 CML patients in which 5 were post-BMT. The sensitivity and specificity of this approach were compared to conventional cytogenetic and polymerase chain reaction (PCR) methods. FISH demonstrated a high degree of sensitivity (1%) for the detection of the BCR/ABL translocation in these patients. A linear correlation was found between FISH detection of the BCR/ABL fusion product and routine chromosomal analysis (r = 0.995; p < 0.001). Detection of the BCR/ABL signal by FISH was observed in all patients showing a positive PCR signal. A significant reduction in BCR/ABL signal was observed post-transplant (p < 0.001). However, the BCR/ABL translocation was detected in four of five transplanted patients immediately (0.75-2.5 months) following transplant and was found in patients with a low expression of the translocation.

Human toxin-based recombinant immunotoxins/chimeric proteins as a drug delivery system for targeted treatment of human diseases

Introduction: The development of specific immunosuppressive reagents remains the major goal in the treatment of human diseases. One such approach is the use of recombinant immunotoxins/chimeric proteins, composed of targeting and killing moieties, fused at the cDNA level. Most of these ‘magic bullets’ use bacterial or plant toxins to induce cell death. These toxins are extremely potent, but they also cause severe toxicity and systemic side effects that limit the maximal doses given to patients. Moreover, being of non-human origin, they are highly immunogenic, and the resulting neutralizing antibody production impairs their efficacy. Areas covered: This review describes recombinant immunotoxins/chimeric proteins composed of the classical delivering, cell-targeting molecules, fused to highly cytotoxic human proteins capable of generating an intense apoptotic response within the target cell. This review focuses on the new ‘Human Killing Moieties’ of these targeted proteins and describes recent progress in the development of these promising molecules. Expert opinion: Human toxin-based immunotoxins/chimeric proteins for the targeted delivery of drugs are still in their early stages of development. However, they are certain to advance in the very near future to become an extra weapon in the everlasting war against human diseases, mainly cancer.

Targeted cancer therapy with gonadotropin-releasing hormone chimeric proteins.

Tumor-associated antigens (TAAs) have been identified mainly to determine cancer prognosis. In the past few years, TAAs have been used in the development of treatment modalities such as tumor vaccination. This review describes an additional application of TAAs: as a target for specific antitumor treatment. Since TAAs are overexpressed on the tumor cell surface, they can be targeted to deliver drugs directly to cancer cells. One such delivery system exploits chimeric proteins. Chimeric proteins are a class of targeted molecules designed to recognize and specifically destroy cells that overexpress specific receptors. These molecules, designed and constructed by gene fusion techniques, comprise both cell-targeting and cell-killing moieties. The authors’ laboratory has developed a number of chimeric proteins using gonadotropin-releasing hormone (GnRH) as the targeting moiety. These chimeras recognize a GnRH binding site that is expressed on adenocarcinoma cells. GnRH was fused to a large number of killing moieties, including bacterial and human proapoptotic proteins. All GnRH-based chimeric proteins selectively killed adenocarcinoma cells both in vitro and in vivo. Utilizing chimeric proteins for targeted therapy represents a new and exciting therapeutic modality for the treatment of cancer in humans.

Apoptosis-Inducing Human-Origin Fcε-Bak Chimeric Proteins for Targeted Elimination of Mast Cells and Basophils: A New Approach for Allergy Treatment

During the past few years, many chimeric proteins have been developed to specifically target and kill cells expressing specific surface molecules. Generally these molecules carry a bacterial or plant toxin to destroy the unwanted cells. The major obstacle regarding these molecules in their clinical application is the immunogenicity and nonspecific toxicity associated with bacterial or plant toxins. We lately reported a new approach for construction of chimeric proteins: we successfully replaced bacterial or plant toxins with human apoptosis-inducing proteins. The resulting chimeras were shown to specifically induce apoptosis in the target cells. Taking advantage of the human apoptosis inducing proteins Bak and Bax as novel killing components, we have now constructed new chimeric proteins targeted against the human FcεRI, expressed mainly on mast cells and basophils. These cells are the main effectors of the allergic response. Treatment of the target cells with the new chimeric proteins, termed Fcε-Bak/Bax, had a dramatic effect on cell survival, causing apoptosis. The effect was specific to cells expressing the FcεRI of both human and, very unexpectedly, also of mouse origin. Moreover, interaction of the chimeric proteins with the mast cells did not cause degranulation. Fcε-Bak/Bax are new chimeric proteins of human origin and, as such, are expected to be both less immunogenic and less toxic and, thus, may be specific and efficient reagents for the treatment of allergic diseases.