Cdd2-11_Lundstrom.qxd 18/10/02 10:00 Page 19 FEATURE Breakthrough in cancer therapy: Encapsulation of drugs and viruses Kenneth Lundstrom & Teni Boulikas Regulon Inc, Switzerland & USA Cancer suffering and deaths still have devastating sociological dimensions despite multitudes of approaches and decades of research. However, a major breakthrough in cancer treatment is at hand. Novel liposome-based encapsulation technologies offer the potential for safe, efficient delivery of drugs, genes or even entire virus particles, specifically to tumors and metastases. Successful treatment of cancer has been one of the primary goals of medicine for the last two decades, but results have been fairly modest due to several factors. There are various forms of cancers that can affect most of the organs in the body, while several cancer types can co-occur and the disease can progress with variable aggressiveness. The spread of the cancerous tissue is also highly dependent on tissue origin and the time of diagnosis. Moreover, various cancer types can show completely different responses to treatment. Major obstacles for achieving high efficacy of cancer treatment include the limitations of surgical procedures, the inefficent and non-specific action of drugs and the strong side effects observed in patients after chemotherapy. These problems are caused by inefficient targeting of tumor tissue, particularly related to secondary tumors or metastases. Liposome formulations Liposome formulations and devise methods have been developed, which could be used for encapsulation of drugs or plasmid DNA. A variety of drug-loading methods are available. Lipophilic drug entrapment (passive loading) can be achieved by preparing a mixture of vesicle-forming lipids and the drug in a dried film, and hydrating the mixture to form liposomes; the drugs become trapped predominantly in the lipid bilayer of the vesicles. It is possible to capture hydrophilic drugs by passive loading, where the drug is dissolved in the aqueous medium used to hydrate a lipid film; however, encapsulation efficiencies are in the range of 5 to 20%. An alternative for capturing hydrophilic drugs involves reverse evaporation from an organic solvent. Amphipathic or ionizable hydrophilic drugs can also be loaded into liposomes against a transmembrane pH gradient, achieving even greater drugloading efficiencies. In general, two major problems are associated with drug encapsulation. First, encapsulation efficacy can be extremely low (<20%), severely limiting the use of this approach for large-scale production and clinical trials. Second, the gene delivery capacity and especially the potential of cell membrane penetration is limited for many of the liposomes developed. In an effort to improve the efficacy of gene SFV helper vector SFV expression vector Renewed hope The development of molecular biology technologies, and more recently, the birth of gene therapy has opened up new avenues, not only for fundamental research on cancer, but also for development of improved therapeutics. Although gene therapy is still in its infancy, massive work has been carried out on various viral and non-viral gene delivery systems and a variety of transfection reagents with potentially high delivery capacity have been developed. Unfortunately, transfection efficacy has been disappointingly low in most cases when administered in vivo. November 2002 In vitro transcriptions Co-transfections BHK cell Replication-deficient SFV particles Figure 1. Alphavirus expression system. Recombinant SFV particles are generated from DNA plasmids by in vitro RNA transcription followed by co-transfection of BHK cells. www.currentdrugdiscovery.com 19 Cdd2-11_Lundstrom.qxd 18/10/02 10:00 Page 20 FEATURE delivery, and furthermore to achieve cell/tissue-specific targeting, tumor-specific sequences have been introduced into the liposome structures as plasmid carriers, which has resulted in some success. such as cancer gene therapy, favor a highlevel but short-term expression pattern and in these cases, adenovirus or alphavirus vectors might be more appropriate. Viral vectors Alphaviruses are well characterized, single-stranded RNA viruses with an envelope structure. Three alphavirus species, Semliki Forest virus (SFV), Sindbis virus and Venezuelan equine encephalitis virus (VEE), have been developed as expression vectors and used for gene delivery both in vitro and in vivo. In particular, engineering of replication-deficient vectors has permitted high-level and short-term heterologous gene expression. This has been achieved by splitting the genome on two plasmids. The expression vector carries the nonstructural viral genes and a strong viral promoter, downstream of which the foreign gene of interest is inserted. The helper vector contains the structural genes, ie, the capsid and envelope protein genes. Packaging of recombinant particles takes place in mammalian host cells, for example baby hamster kidney (BHK) cells (Figure 1). Recently, a split helper system Viral vectors possess a natural ability to efficiently infect host cells. Another attractive feature is their capacity to promote high levels of transgene expression in infected cells or tissues. A variety of viral vectors have been engineered for gene delivery and recombinant purposes, including adenovirus, adeno-associated virus (AAV), alphaviruses, herpes simplex virus (HSV), lentivirus and retrovirus. As different vectors possess specific features and properties, their potential applications are therefore specifically defined. For instance, retroviruses, lentiviruses (a subfamily of retroviruses) and AAV all have the capacity to integrate into the host genome and HSV can establish a latent infection and persist for life in the host organism. For this reason, these vectors are useful when longterm expression of the therapeutic gene is required. However, many applications, Semliki Forest virus vectors A C B D Figure 2. Neuron-specific heterologous gene expression. A. Stereotactic injection of SFV-LacZ virus into rat brain resulted in high-level local expression of β-galactosidase. B. Larger magnification of A. C. SFV-GFP infection of rat hippocampal slice cultures demonstrated that >90% of the GFPpositive cells were of neuronal origin. D. Larger magnification of C. so, Stratum oriens; sp, Stratum pyramidale. 20 www.currentdrugdiscovery.com was developed, which has further reduced the possible recombination frequency to less than 4 x 10-17. SFV particles can also be generated in packaging cell lines harboring the viral structural genes. The titers of produced particles are reasonably high, approximately 109 to 1010 infectious particles per ml. For gene therapy applications, a further step of purification and concentration performed on a matrix affinity column is mandatory. Infection of neuronal cells Stereotactic injection of recombinant SFV carrying the LacZ gene into rat brain has shown that SFV can efficiently infect neuronal cells (Figure 2A to B). Similar observations were made for SFV-based green florescent protein (GFP) expression in hippocampal slice cultures (Figure 2C to D), SFV therefore has a great potential in the gene therapy of astrocytomas. Other SFV strains have revealed the potential of primary transduction of glial cells with implications in therapy of glioblastomas. SFV vectors are also able to infect a variety of cell types and may find applications in a variety of human malignancies. Generally, SFV vectors have shown high cytotoxicity to host cells and because of this, a very limited time of transgene expression. Obviously, for cancer therapy the capability to kill host cells should be considered as an advantage, but rapid cell death - within 2 to 3 days - will limit the full potential of the therapeutic gene. Therefore, it is beneficial to obtain vectors with lower cytotoxicity resulting in prolonged heterologous gene expression. For these purposes, novel, less cytotoxic SFV vectors have been developed. For instance, the SFV-PD vector has demonstrated substantially prolonged transgene expression in cell lines and hippocampal slice cultures. Due to their broad host range, SFV particles are potentially very interesting as gene delivery vectors, but their strong preference for neuronal expression can be a concern. Animal studies have indicated that although no spread of β-galactosidase is observed after stereotactic injection into rat striatum and amygdala, strong transgene expression is detectable in neurons at the injection site. In case of brain tumor therapy, this could cause a significant problem because of the non-selective November 2002 Cdd2-11_Lundstrom.qxd 18/10/02 10:00 Page 21 FEATURE expression in normal tissue as well as in tumors. Moreover, because alphavirus vectors are RNA-based there is no possibility of restricting expression by using tissue-specific promoters. Targeting of alphavirus-based expression has therefore relied almost completely on modifications of the viral envelope structure. It has previously been shown for Sindbis virus that the introduction of IgG binding domains of protein A into the E2 spike protein can alter the host range and result in antibodyspecific infection of host cells. In a similar way, the feasibility of the introduction of foreign sequences into the SFV envelope has been demonstrated. Although this type of targeting can contribute to improved efficacy of gene expression specifically in cancer tissue, modified methods will be required to maximize the therapeutic effect. One approach in this direction has been to develop technologies for liposomemediated drug or gene delivery. Encapsulation technology Many attempts have been made to encapsulate various drugs or small molecules into liposome structures. The advantages of this approach are numerous, as liposomes: • can slowly release drugs, encapsulated into the lumen of their lipid bilayer, into the blood stream of animals including humans • can evade the immune system and minimize allergic and other untoward reactions caused by drugs and proteins • change the pharmacokinetics of encapsulated drugs, often retaining the drug in circulation into body fluids for prolonged times • usually reduce substantially the toxicity of the drug • taken up by cells warrant entrance of chemicals and other molecules into otherwise inaccessible cells • can be targeted to tumors and preferentially accumulate in tumor tissues and their metastases by extravasation through their leaky neovasculature. Ideally, this would allow systemic drug delivery, extend the time of drug circulation and because of the targeting, less damage will be caused to the normal tissue and patients will suffer from fewer side effects. Two major obstacles have so far prevented a major breakthrough in tumor therapy November 2002 Figure 3. Systemic delivery of encapsulated plasmid DNA. A liposomally-encapsulated plasmid carrying the LacZ gene was systemically administered into SCID mice implanted with human tumors. X-gal staining revealed efficient targeting of β-galactosidase expression. using this approach. First, encapsulation efficacy has been disappointingly low, making the manufacturing process timeconsuming and expensive, and second, the generated liposomes have not been able to efficiently target tumors and/or penetrate into the cancerous tissue. Drug encapsulation Regulon has developed a new, patented method for liposome encapsulation that has demonstrated substantially improved efficacy (>90%). These liposomes are capable of both passive targeting of primary tumors and metastases, and have the ability to penetrate cell membranes. To demonstrate the efficacy of gene delivery, a mammalian expression vector carrying the LacZ gene was encapsulated and injected either intraperitoneally or intravenously into SCID mice implanted with human tumors. Whole animal carcass staining visualized the efficient targeting of β-galactosidase expression to tumors as well as their vasculature (Figure 3), while normal tissue showed hardly any β-galactosidase staining. Histological analysis also suggested that no damage related to the treatment occurred in non-cancerous tissues. Using a similar approach, cisplatin, commonly used in cancer therapy, was encapsulated into liposomes. The product called Lipoplatin has entered phase I and phase II trials as described below. Cisplatin is one of the most widely used and most effective cytotoxic agents in the treatment of epithelial malignancies such as lung, head and neck, ovarian, bladder and testicular cancer. However, its continued clinical use is impeded by its severe adverse reactions including renal toxicity, gastrointestinal toxicity, peripheral neuropathy, asthenia, and ototoxicity. Lipoplatin was developed in order to reduce the systemic toxicity of cisplatin while simultaneously improving the targeting of the drug to the primary tumor and its metastases and enhancing the time of circulation in body fluids and tissues. The therapy shows zero nephrotoxicity, ototoxicity, cardiotoxicity, hepatotoxicity, neuropathy, and no hair loss; the only adverse reactions are mild myelotoxicity and mild nausea. Phase II studies show that combination of Lipoplatin with radiation therapy, Gemzar or Taxol can considerably shrink lung, pancreatic and head and neck cancer as well as most other human malignancies, even in stage III or IV cancer patients, and as a second- or third-line treatment after failure of other chemotherapies. Viral encapsulation The efficient encapsulation of drugs, small molecules and DNA plasmids led to the www.currentdrugdiscovery.com 21 Cdd2-11_Lundstrom.qxd 18/10/02 10:00 Page 22 FEATURE Naked recombinant SFV particles Encapsulated SFV particles Figure 4. Systemic delivery of recombinant SFV particles. Intravenous or intraperitoneal administration of SFV-LacZ results in widespread infection, whereas encapsulated SFV-LacZ particles are targeted to tumors. idea of encapsulation of viral particles as a way of targeting the gene delivery and also protecting the normal tissue from damage as well as the generation of antibody responses against the viral vehicle (Figure 4). The feasibility of this approach was tested by administration of liposomeencapsulated recombinant SFV particles expressing β-galactosidase into SCID mice. X-gal staining revealed massive β-galactosidase expression in implanted tumors while only a minor signal was found around the injection site. Further administration of high concentrations of encapsulated SFV particles showed no toxicity in normal tissue. Based on the animal studies, it was possible to initiate a phase I/II trial on recombinantly expressed interleukin-12 (IL-12). The p40 and p35 subunits of IL-12 were introduced into the same SFV vector downstream of individual SFV 26S promoters, which resulted in secretion of high yields of enzymatically active recombinant IL-12 in various mammalian cell lines. Application of the SFV-PD vector resulted in elevated levels and prolonged duration of IL-12 expression. The encapsulated SFV-PD-IL12 has been named LipoVIL12. Lipoplatin trial A combined phase I and II study was conducted with Lipoplatin, which was administered as an 8-hour intravenous infusion. The treatment caused no renal toxicity (even of Grade 1), neuropathy, ototoxicity, hepatotoxicity, cardiotoxicity or allergic 22 www.currentdrugdiscovery.com reactions in any patient. The patients showed only mild nausea and vomiting, and Grade 1-3 bone marrow toxicity. The long circulation properties of Lipoplatin were confirmed by measured platinum levels in sera and urine excretion. Combination treatment (Lipoplatin + Gemzar) of patients with histologically confirmed advanced pancreatic, non-small lung-cell, head and neck and bladder cancers, showed significant clinical benefits in patients previously diagnosed as resistant to first- and second-line chemotherapy. patients indeed display higher levels of IL12 than sera from placebo-treated patients. ELISA tests on patients also revealed an increase in tumor necrosis factor-α (TNFα) and interferon-γ levels. The immunostimulation was clearly transient and the IL-12 activity returned to base levels after approximately 4 days. The encapsulation procedure substantially helped to overcome the major obstacles of systemic virus delivery. The liposome-coated SFV particles were targeted to tumors, stayed for an extended time in circulation and most importantly, were protected from clearance by immune cells and generation of antibodies against SFV particles. Because of the absence of immune responses, repeated administration of encapsulated SFV particles was feasible. Capturing the future Encapsulation technology has substantially improved the success rate in cancer therapy. The major obstacles characterized by inefficient drug or gene delivery and the severe side effects related to chemotherapy can be eliminated to a large extent. The encouraging results obtained from phase I and phase II trials indicate that the lipo- 200 97 LipoVIL12 trial Phase I trials were conducted with LipoVIL12 against advanced melanoma and renal cancer, refractory to other treatments. Treatment caused no toxicity to kidney, liver, bone marrow, neuronal, cardiovascular or any other organ. However, allergies including mild fever, chills, skin rush and tachycardia were observed due to the overexpression of the therapeutic cytokine IL-12. Previous studies on infection of cell lines with these SFV-IL-12 particles had demonstrated that infected cells produce extremely large amounts of SFVdriven human IL-12, representing the major synthesized protein (Figure 5). Therefore, intravenous infusion of LipoVIL12 was expected to sustain high levels of IL-12 protein that is also anticipated to be secreted into the bloodstream. Consistent with this, sera of treated 69 46 p40 p35 30 Figure 5. Metabolic labeling of BHK cells infected with SFV-PD-IL-12. BHK cells infected with SFV-PD vector expressing the p40 and p35 subunits of IL-12 were labeled with [35S]methionine at 18 hours postinfection and the expression verified by SDSPAGE and autoradiography. November 2002 Cdd2-11_Lundstrom.qxd 18/10/02 10:00 Page 23 FEATURE somes are applicable against various forms of cancer. Preliminary studies demonstrated that in addition to the cancer types mentioned above, efficacy was also obtained for breast, kidney, lung and prostate cancers. Another attractive feature of the liposomes is their capacity to target not only primary tumors but also metastases. This will substantially improve the therapeutic efficacy. The versatility of the encapsulation technology should also be acknowledged. Here, examples of a liposomally-coated drug, cisplatin, and a recombinant alphavirus expressing an immunostimulatory cytokine gene were presented. The application range is obviously almost unlimited as small molecules, oligonucleotides, DNA plasmids, peptides and proteins can be encapsulated. The technology should also be applicable to viruses other than alphaviruses such as AAV or lentivirus. Moreover, any foreign gene could in theory be introduced into the viral vectors, ranging from immunostimulatory genes to toxic genes as well as genes coding for enzymes necessary for normal cellular functions. Furthermore, various combination therapies including surgery, radiation or chemotherapy can be tested. With these possibilities in mind, it looks like a major breakthrough in cancer treatment is at hand, and it is likely that the technology could be used in the therapy of other diseases. Kenneth Lundstrom CSO Regulon Inc Chemin des Croisettes 22 CH-1066 Epalinges Switzerland Teni Boulikas CEO Regulon Inc 715 North Shoreline Boulevard Mountain View CA 94043 USA Email: [email protected] [email protected] FURTHER INFORMATION www.regulon.org www.gtmb.org FURTHER READING Lundstrom K (2002) Alphavirus-based vaccines. Current Opinion in Molecular Therapeutics 4(1):28-34. Martin F, Boulikas T (1998) The challenge of liposomes in gene therapy. Gene Therapy & Molecular Biology 1:173-214. Ohno K et al (1997) Cell-specific targeting of Sindbis virus vectors displaying IgG-binding domains of protein A. Nature Biotechnology 15:763-767. Polo JM et al (1999) Stable aplhavirus packaging cell lines for Sindbis virus- and Semliki Forest virus-derived vectors. Proceedings of National Academy of Science USA 96:4598-4603. Smerdou C, Liljeström P (1999) Two-helper RNA system for production of recombinant Semliki Forest virus particles. Journal of Virology 73:1499-1504. CALL FOR CONTRIBUTIONS In February, Current Drug Discovery will focus on Informatics, but will the new year have brought any new solutions to light? Are regulations curbing innovation? Are we any closer to an integrated solution? We want to hear about new technologies, new dynamics and new business insights YOU feel are important - from datamining to knowledge management to the latest buzz word proteomatics. If you have an interesting insight into this field and would like to write in this issue, contact: The Editor, Emma Jones, [email protected] November 2002 July 2001 www.currentdrugdiscovery.com Informatics Making it your world n eChemistry n Clinical data gathering n The evolution of analytics n Population-level DNA repositories From the publishers of www.currentdrugdiscovery.com 23