An antibody delivery system for regulated expression of therapeutic levels of monoclonal antibodies in vivo.

Monoclonal antibody (mAb) delivery by gene transfer in vivo may be an attractive alternative to current mAb therapies for applications that require long-term therapy. This article describes a transfer system that allows inducible high-level expression of unmodified mAbs in vivo. A recombinant adeno-associated viral (rAAV) vector is used that comprises an expression cassette consisting of a dimerizer-regulated promoter that drives expression of the antibody heavy and light chains linked by a 2A self-processing peptide and a furin cleavage site. Following intravenous injection of the rAAV vector, serum mAb levels >1 mg/ml were attained by administration of the inducer, rapamycin. Antibody expression could be rapidly shut off by discontinuing treatment with rapamycin. By optimizing the furin cleavage sequence, this system generated native antibody in vivo, decreasing the likelihood of a host immune response to foreign sequences. In summary, this optimized mAb expression system allows regulated high-level expression of native full-length mAbs in vivo and may offer a new opportunity for delivery of therapeutic mAbs in the clinic.     

Controlled-release and local delivery of therapeutic antibodies

Human and humanised antibodies are now poised to become a major new class of protein-based therapeutic agents. A significant fraction of new drugs in clinical testing (approximately 20% in 2002) are antibody classes. Monoclonal antibodies (mAbs) with high affinities against newly discovered disease targets, both cellularly and extracellularly, are now clinically proven to elicit high bioactivities against numerous diseases, including tumours, infections, asthma, inflammation, arthritis and osteoporosis. Clinical humanised antibody delivery is typically intravenous, with large multiple doses (grams) required for systemic volumes of distribution. Due to the relatively high costs of both this drug type, and its common mode of administration, alternatives are sought where doses might be reduced and the bioavailability and efficacy enhanced. Local, controlled-release methods that deliver antibodies locally to site of disease, offer new possibilities with these potential advantages. However, protein drugs frequently exhibit formulation challenges when packaged in delivery vehicles, and as globular proteins, antibodies are no exception. Several examples of mAb controlled-release and local delivery strategies against several disease targets are reviewed. Importantly, several antibody delivery methods work in tandem with existing clinically-accepted therapeutics, sometimes exhibiting potentiating or synergistic effects in animal models with small molecule, systemically administered drugs.     

Controlled-release and local delivery of therapeutic antibodies

Introduction: As HIV continues to spread worldwide, new therapies which have the potential to treat and cure infected patients need to be developed. The results observed with the “Berlin patient” who received a bone marrow transplant with HIV-resistant hematopoietic stem cells highlight the potential of HIV gene therapy to be used as an alternative treatment. With the discovery of TRIM5α, an HIV inhibitor and species-specific restriction factor, a new molecule can be evaluated as an HIV gene therapeutic. Nonhuman primate TRIM5α orthologs restrict HIV infection, whereas human TRIM5α does not. However, upon genetic modification, variations to human TRIM5α have been made which are capable of potent HIV restriction.

Areas covered: This review seeks to cover the discovery and biology of various HIV-restrictive nonhuman primate TRIM5α orthologs, modifications made to human TRIM5α to enable HIV restriction, and the use of these molecules in an HIV gene therapy setting.

Expert opinion: Engineered human TRIM5α molecules, demonstrated to be strong inhibitors of HIV infection, have the potential of being used as new HIV therapeutics in human gene therapy clinical trials. By combining TRIM5α with other highly potent anti-HIV molecules, the generation of an HIV-resistant immune system and potential cure for infected patients may be accomplished.     

In vitro and in vivo evaluation of an oral system for time and/or site-specific drug delivery.

Aim of this work was the evaluation of an oral system (Chronotopic) designed to achieve time and/or site-specific release. The system consists in a drug-containing core, coated by a hydrophilic swellable polymer which is responsible for a lag phase in the onset of release. In addition, through the application of an outer gastroresistant film, the variability in gastric emptying time can be overcome and a colon-specific release can be sought relying on the relative reproducibility of small intestinal transit time. For this study, cores containing antipyrine as the model drug were prepared by tableting and both the retarding and enteric coatings were applied in fluid bed. The release tests were carried out in a USP 24 paddle apparatus. The in vivo testing, performed on healthy volunteers, envisaged the HPLC determination of antipyrine salivary concentration and a gamma-scintigraphic investigation. The in vitro release curves presented a lag phase preceding drug release and the in vivo pharmacokinetic data showed a lag time prior to the detection of model drug in saliva. Both in vitro and in vivo lag times correlate well with the applied amount of the hydrophilic retarding polymer. The gamma-scintigraphic study pointed out that the break-up of the units occurred in the colon. The obtained results showed the capability of the system in delaying drug release for a programmable period of time and the possibility of exploiting such delay to attain colon-targeted delivery according to a time-dependent approach.     

Noninvasive gene transfer to the lung for systemic delivery of therapeutic proteins

This study evaluates the use of vectors based on adeno-associated viruses (AAVs) to noninvasively deliver genes to airway epithelial cells as a means for achieving systemic administration of therapeutic proteins. We intranasally delivered AAV vectors to mice in which the same AAV2 genome encoding a cellular marker was packaged in capsids from AAV1, 2, or 5 (AAV2/1, AAV2/2, or AAV2/5, respectively). Gene expression levels achieved in both airways and alveoli were higher with AAV2/5 than with AAV2/1 and were undetectable with AAV2/2. The same set of vectors encoding a secreted therapeutic protein, erythropoietin (Epo), under the control of a lung-specific promoter (CC10) was intranasally delivered to mice, resulting in polycythemia with the highest levels of serum Epo obtained with AAV2/5 vectors. After a single intranasal administration of this vector, secretion of Epo was documented for 150 days. Similarly, intranasal administration of an AAV2/5-CC10-factor IX vector resulted in secretion of functional recombinant protein in the bloodstream of hemophiliac, factor IX-deficient mice. In addition, we demonstrate successful readministration of AAV2/5 to the lung 5 months after the first delivery of the same vector. In conclusion, we show that intranasal administration of AAV vectors results in efficient gene transfer to the lung only when the vector contains the AAV5 capsid and that this noninvasive route of administration results in sustained secretion of therapeutic proteins in the bloodstream.     

In vivo myocardial gene transfer: optimization and evaluation of intracoronary gene delivery in vivo

Clinical reports suggest that intracoronary delivery of adenoviruses encoding angiogenic growth factors, or their transactivators, has a therapeutic benefit. However there has not been a systematic assessment of the transfection efficiency of this technique in vivo. In rabbits we investigated the efficiency of myocardial gene transfer following intracoronary infusion of 1 x 10(-10) -1 x 10(12) p.f.u. of adenovirus in combination with interventions to enhance transfection. In five standard short axis sections, we were barely able to detect reporter gene expression following unmodified intracoronary infusion. Efficiency was not enhanced by the exclusion of blood and the increase of intracoronary dwell time through occlusive engagement of the left coronary ostium enabled by oxygenated perfluorocarbon emulsion as viral diluent. Of the interventions and pretreatments designed to increase vascular permeability, VEGF, calcium-free viral diluent and adenosine, only the latter tended to increase efficiency. However an intervention designed to increase the myocardial transcapillary gradient, by increasing venular pressure with pulmonary artery occlusion and arteriolar pressure with occlusion of the aorta above the coronary ostia, increased transfection efficiency by two orders of magnitude. Unfortunately the clinical utility of this technique may be limited by accompanying cardiac dilation and marked elevations in intracardiac pressure.     

In vivo gene transfer to pancreatic beta cells by systemic delivery of adenoviral vectors

Type 1 diabetes results from autoimmune destruction of pancreatic beta cells. This process might be reversed by genetically engineering the endocrine pancreas in vivo to express factors that induce beta cell replication and neogenesis and counteract the immune response. However, the pancreas is difficult to manipulate and pancreatitis is a serious concern, which has made effective gene transfer to this organ elusive. Thus, new approaches for gene delivery to the pancreas in vivo are required. Here we show that pancreatic beta cells were efficiently transduced to express beta-galactosidase after systemic injection of adenovirus into mice with clamped hepatic circulation. Seven days after vector administration about 70% of pancreatic islets showed beta-galactosidase expression, with an average of about 20% of the cells within positive islets being transduced. In addition, scattered acinar cells expressing beta-galactosidase were also observed. Thus, this approach may be used to transfer genes of interest to mouse islets and beta cells, both for the study of islet biology and gene therapy of diabetes and other pancreatic disorders.     

In vivo antigen delivery by a Salmonella typhimurium type III secretion system for therapeutic cancer vaccines

Bacterial vectors may offer many advantages over other antigen delivery systems for cancer vaccines. We engineered a Salmonella typhimurium vaccine strain to deliver the NY-ESO-1 tumor antigen (S. typhimurium-NY-ESO-1) through a type III protein secretion system. The S. typhimurium-NY-ESO-1 construct elicited NY-ESO-1-specific CD8+ and CD4+ T cells from peripheral blood lymphocytes of cancer patients in vitro. Oral administration of S. typhimurium-NY-ESO-1 to mice resulted in the regression of established NY-ESO-1-expressing tumors. Intratumoral inoculation of S. typhimurium-NY-ESO-1 to NY-ESO-1-negative tumors resulted in delivery of antigen in vivo and led to tumor regression in the presence of preexisting NY-ESO-1-specific CD8+ T cells. Specific T cell responses against at least 2 unrelated tumor antigens not contained in the vaccine were observed, demonstrating epitope spreading. We propose that antigen delivery through the S. typhimurium type III secretion system is a promising novel strategy for cancer vaccine development.     

In vitro and in vivo evaluation of a matrix-in-cylinder system for sustained drug delivery.

A matrix-in-cylinder system for sustained drug delivery, consisting of a hot-melt extruded ethylcellulose (EC) pipe surrounding a drug containing HPMC-Gelucire 44/14 core, was evaluated in vitro and in vivo. In an aqueous medium, the HPMC-Gelucire core forms a gel plug, which releases the drug-through the open ends of the EC pipe--by means of erosion. The influence of hydrodynamic and mechanical stress and the effect of different 'physiologically relevant' dissolution media on the in vitro drug release were investigated. From these in vitro dissolution tests, it was concluded that the EC pipe has a protective effect on the drug containing HPMC-Gelucire core. It largely protects the core against hydrodynamics and mechanical stress. Furthermore, drug release from the matrix-in-cylinder system was only slightly affected by the composition of the dissolution medium. A randomised crossover in vivo study in dogs revealed that the matrix-in-cylinder system containing propranolol hydrochloride has an ideal sustained release profile with constant plasma levels maintained over 24 h. Moreover, administration of the matrix-in-cylinder system resulted in a 4-fold increase in propranolol bioavailability when compared with a commercial sustained release formulation (Inderal).     

In vivo cell type-specific gene delivery with retroviral vectors that display single chain antibodies

Cell type-specific gene delivery will be essential for in vivo gene therapy. Our laboratory has previously developed retroviral vector particles, derived from spleen necrosis virus, SNV, which display the antigen-binding site of an antibody on the viral surface. Such particles infected only human cells in vitro, which expressed a receptor recognized by the antibody. To test cell type-specific gene delivery in vivo, a mouse model system has been developed. Antibiotic resistant human target and non-target cells were injected into the peritoneum of SCID mice. Subsequently, a vector solution containing 106 infectious particles, which display scAs against the human her2neu cell surface protein, was injected. Cells were recovered from the peritoneum, subjected to antibiotic selection, and tested for the expression of a lacZ gene transduced by the retroviral vector. We found that human target cells, which express her2neu, were infected in vivo. However, neither human cells that do not express her2neu, nor normal mouse cells were infected by such viral particles. These data give proof of principle that retroviral vector-mediated, cell type-specific gene delivery can be obtained in vivo.     

In vivo evaluation of riboflavin-containing microballoons for floating controlled drug delivery system in healthy human volunteers.

Hollow microspheres (microballoons) floatable in JPXIII no. 1 solution were developed as a floating controlled drug delivery system. Microballoons (MB) were prepared by the emulsion solvent diffusion method utilizing enteric acrylic polymers dissolved in a mixture of dichloromethane and ethanol. To assess the usefulness of the intragastric floating properties of MB in sustained pharmacological action, nonfloating microspheres (NF) possessing riboflavin release profiles equivalent to those of MB were prepared. Riboflavin powder, riboflavin-containing MB, and riboflavin-containing NF were administrated orally to each of three healthy volunteers. Riboflavin pharmacokinetics was investigated via analysis of urinary excretion of riboflavin. As a result, although urinary excretion of riboflavin following administration of MB was not sustained in the fasted state, urinary excretion of MB was significantly sustained in comparison to riboflavin powder and NF in the fed condition. Additionally, the excretion half-life time (t(1/2)) of MB was prolonged significantly by feeding. Furthermore, MB provided significantly high total urinary excretion (%) of riboflavin compared to NF in the fasted and the fed conditions. In view of the finding regarding similar riboflavin release profiles of MB and NF in JP XIII no. 1 solution (pH 1.2) and no. 2 solution (pH 6.8), the intragastric floating properties of MB appeared to be beneficial with respect to sustained pharmacological action.     

In vivo evaluation of an adenoviral vector encoding canine factor VIII: high-level, sustained expression in hemophiliac mice.

Hemophilia A is the most common severe hereditary coagulation disorder and is caused by a deficiency in blood clotting factor VIII (FVIII). Canine hemophilia A represents an excellent large animal model that closely mimicks the human disease. In previous studies, treatment of hemophiliac dogs with an adenoviral vector encoding human FVIII resulted in complete correction of the coagulation defect and high-level FVIII expression [Connelly et al. (1996). Blood 88, 3846]. However, FVIII expression was short term, limited by a strong antibody response directed against the human protein. Human FVIII is highly immunogenic in dogs, whereas the canine protein is significantly less immunogenic. Therefore, sustained phenotypic correction of canine hemophilia A may require the expression of the canine protein. In this work, we have isolated the canine FVIII cDNA and generated an adenoviral vector encoding canine FVIII. We demonstrate expression of canine FVIII in hemophiliac mice at levels 10-fold higher than those of the human protein expressed from an analogous vector. Canine FVIII expression was sustained above human therapeutic levels (50 mU/ml) for at least 1 year in hemophiliac mice.     

Nonviral in vivo delivery of therapeutic small interfering RNAs

Since its discovery in the late 1990s, RNA interference (RNAi) has gained much attention as a powerful strategy for silencing activity. Instrumental for this naturally occurring targeting mechanism is the intracellular presence of a target gene-specific small interfering RNA (siRNA). Therefore, the in vivo delivery of highly specific siRNA molecules represents one major goal in the further development of RNAi-based approaches for clinical applications. For the non-viral delivery of siRNAs, except for local or topical administration, various routes of application and delivery vehicles/strategies have been explored so far, including the systemic injection of pure, unmodified or chemically modified siRNAs, physical methods such as hydrodynamic injection or electropulsation, encapsulation of siRNAs in liposomes, lipoplexes or cationic lipids, formation of nanoplexes through complexation of siRNAs in cationic or other carriers, or chemical coupling of siRNAs to specific carrier molecules. Therefore, approaches to establish the clinical application of RNAi may rely on a combination of biosciences and nanotechnology; in particular, for the identification of optimal siRNAs against optimal target molecules, and the development of sophisticated delivery systems.     

Possible therapeutic applications of single-chain antibodies in systemic autoimmune diseases

B cells participate in the induction and maintenance of systemic autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, via production of pathogenic autoantibodies, contributing to the formation of immune complexes. Immune complex deposition in the kidney and joints causes inflammation and organ destruction, and chemokine production enhances T cell activation and tissue damage. The development of the disorder depends on several factors, for example, genetic susceptibility, environmental factors or immune dysregulation. Traditional therapies, which aimed at the alleviation of symptoms, are giving way to biological therapies with the potential of disrupting disease progression. This article focuses on antibody therapies, especially on the applications of single-chain antibodies, as new biological agents for the treatment of systemic autoimmune disorders.     

In vivo three-dimensional spectroscopic photoacoustic imaging for monitoring nanoparticle delivery

In vivo monitoring of nanoparticle delivery is essential to better understand cellular and molecular interactions of nanoparticles with tissue and to better plan nanoparticle-mediated therapies. We developed a three-dimensional ultrasound and photoacoustic (PA) imaging system and a spectroscopic PA imaging algorithm to identify and quantify the presence of nanoparticles and other tissue constituents. Using the developed system and approach, three-dimensional in vivo imaging of a mouse with tumor was performed before and after intravenous injection of gold nanorods. The developed spectroscopic PA imaging algorithm estimated distribution of nanoparticle as well as oxygen saturation of blood. Moreover, silver staining of excised tumor tissue confirmed nanoparticle deposition, and showed good correlation with spectroscopic PA images. The results of our study suggest that three-dimensional ultrasound-guided spectroscopic PA imaging can monitor nanoparticle delivery in vivo.     

Evaluation of the duration of human factor VIII expression in nonhuman primates after systemic delivery of an adenoviral vector

An E1/E2a/E3-deficient adenoviral vector encoding an epitope-tagged (flagged) human factor VIII (FVIII) cDNA was delivered systemically to four cynomolgus monkeys. Analysis of liver biopsy samples revealed the presence of vector DNA at all points in the study (day 7, 28, and 56), with vector copy number declining approximately 10-fold between day 7 and day 56. Immunoprecipitation/Western analyses detected human flagged FVIII in the plasma of all monkeys and expression persisted for 14-28 days. Peak plasma FVIII levels ranged from 50 to 100 ng/ml. Bethesda assays revealed no inhibitor in two animals, the development of a low-level transient inhibitor in one animal, and an inhibitor titer that continued to increase for the duration of the study in one animal. Other treatment-related changes included modest increases in liver enzymes, an increase in interleukin-6 (IL-6) levels, and a transient decrease in platelets in all four animals. These data indicate that early generation adenoviral vectors do not support the long-term expression of FVIII in nonhuman primates.     

Erbium:YAG fractional laser ablation for the percutaneous delivery of intact functional therapeutic antibodies

The physicochemical properties and stability requirements of therapeutic proteins necessitate their parenteral administration even for local therapy; however, unnecessary systemic exposure increases the risk of avoidable side-effects. The objective of this study was to use fractional laser ablation to enable the delivery of intact, functional therapeutic antibodies into the skin in vitro and in vivo. The laser-assisted delivery of Antithymocyte globulin (ATG) and Basiliximab - FDA-approved therapeutics for the induction of immunosuppression - was investigated. In vitro delivery experiments were performed using dermatomed porcine ear and human abdominal skins; an in vitro/in vivo correlation was shown using C57 BL/10 SCSnJ mice. Antibody transport was quantified by using ELISA methods developed in-house. Results showed that increasing the pore number from 300 to 450 and 900, increased total antibody delivery (sum of amounts permeated and deposited); e.g., for ATG, from 1.18 ± 0.10 to 3.98 ± 0.64 and 4.97 ± 0.83 μg/cm², respectively — corresponding to 19.7-, 66.3- and 82.8-fold increases over the control (untreated skin). Increasing laser fluence from 22.65 to 45.3 and 135.9 J/cm² increased total ATG delivery from 1.70 ± 0.65 to 4.97 ± 0.83 and 8.70 ± 1.55 μg/cm², respectively. The Basiliximab results confirmed the findings with ATG. Western blot demonstrated antibody identity and integrity post-delivery; human lymphocyte cytotoxicity assays showed that ATG retained biological activity post-delivery. Immunohistochemical staining was used to visualize ATG distribution in the epidermis. Total ATG delivery across porcine ear and human abdominal skin was statistically equivalent and an excellent in vitro/in vivo correlation was observed in the murine model. Based on published data, the ATG concentrations achieved in the laser-porated human skins were in the therapeutic range for providing local immunosuppression. These results challenge the perceived limitations of transdermal delivery with respect to biopharmaceuticals and suggest that controlled laser microporation provides a less invasive, more patient-friendly “needle-less” alternative to parenteral administration for the local delivery of therapeutic antibodies.