Overexpression of the cytokine transforming growth factor-beta 2 (TGF-beta2) is a hallmark of various malignant tumors including pancre¬atic carcinoma, malignant glioma, metastasizing melanoma, and metastatic colorectal carcinoma. This is due to the pivotal role of TGF-beta2 as it regulates key mechanisms of tumor develop¬ment, namely immunosuppression, metastasis, angiogenesis, and proliferation. The antisense technology is an innovative technique offering a targeted approach for the treatment of differ¬ent highly aggressive tumors and other diseases. Antisense oligonucleotides are being developed to inhibit the production of disease-causing pro¬teins at the molecular level. The immunothera-peutic approach with the phosphorothioate oli-godeoxynucleotide AP 12009 for the treatment of malignant tumors is based on the specific inhibition of TGF-beta2. After providing pre¬clinical proof of concept, the safety and efficacy of AP 12009 were assessed in clinical phase I/II open-label dose-escalation studies in recurrent or refractory high-grade glioma patients. Me¬dian survival time after recurrence exceeded the current literature data for chemotherapy. Cur¬rently, phase I/II study in advanced pancreatic carcinoma, metastatic melanoma, and metastatic colorectal carcinoma and a phase IIb study in recurrent or refractory high-grade glioma are ongoing. The preclinical as well as the clinical results implicate targeted TGF-beta2 suppression as a promising therapeutic approach for malig¬nant tumor therapy.
16.1 Introduction
Pancreatic cancer is the fourth leading cause of cancer-related deaths in men and women in virtu-ally all industrialized countries (Jemal et al. 2005, 2006). Its incidence cuts across all racial and so-cio-economic barriers. Outcome is nearly always fatal with a 1-year survival rate of about 20% and a 5-year survival rate of less than 5% (Cardenes et al. 2006; Jemal et al. 2005, 2006). The majority of patients exhibit a very aggressive adenocarci-noma (85%). These tumor types are less aggres¬sive and are often curable. Pancreatic tumors are difficult to detect at early stages and, due to their nonspecific symptoms, extremely hard to diagnose. Therefore, most patients present with locally advanced or metastatic disease resulting in high mortality and very short life expectancy. This is at least partially due to the observed resis¬tance of pancreatic cancer to chemotherapy and radiation therapy. Currently, complete surgical resection remains the only therapeutic option with a potential for cure. However, only a low proportion of patients (only 15%-20%) are suit¬able candidates for surgical resection (Cardenes et al. 2006; Siech et al. 2001). The median sur¬vival of these patients who undergo successful resection is approximately 12-19 months with a 5-year survival rate of 15%-20%. Risk factors for pancreatic cancer include advanced age, obesity, diabetes, and chronic pancreatitis (Li et al. 2004; Lowenfels and Maisonneuve 2006). However, cigarette smoking is considered the most signifi¬cant and avoidable risk factor, causing more than 25% of the pancreatic cancer cases (Li et al. 2004;
Lowenfels and Maisonneuve 2006). Because of the lethality of this disease and the failure of standard treatment to date, future efforts will be focused on the advances that are being made in the understanding and delineation of the genetic and molecular cell biology of cancer cells.
During the last three decades, the interest in new therapeutics such as those based on anti-sense technology has strongly increased. Today, the improved technology and ability of chemi¬cal synthesis of antisense oligodeoxynucleotides (ODNs) has become a routine process and offers researchers the possibility to target almost any single gene (Schlingensiepen et al. 1993). This technology has become a powerful research tool in molecular biology, biochemistry, and microbi¬ology, and has tremendous potential in the fields of functional genomics, drug discovery, and clin¬ical therapy, especially oncology. A major cause of cancer lies in defective gene regulation. Such mutations can result either in an overproduction or in abnormal production of proteins promoting dysfunctional growth and tumor development. Antisense drugs are able to block the blueprint (messenger ribonucleic acid, mRNA) of a can¬cer gene and specifically inhibit its conversion into the pathogenic cancer protein. Preventing the formation of such pathogenic factors means combating cancer disease directly at its roots.
Transforming growth factor beta (TGF-beta) is a multifunctional cytokine that has been iden-tified as a key factor in tumor development. TGF-beta is a vital factor controlling several signaling cascades with oncogenic potential, including immunosuppression, epithelial to mesenchymal transition (EMT), metastasis and invasion, an-giogenesis, and proliferation. Overexpression of the TGF-beta isoform TGF-beta2 is a hallmark of various malignant tumors, e.g., pancreatic carcinoma, malignant brain tumors, malignant melanoma, and metastatic colorectal carcinoma. Thus, targeting this key factor to suppress sev¬eral cancer mechanisms simultaneously right at their origin offers a very promising therapeutic approach.
The phosphorothioate oligodeoxynucleotide (S-ODN) AP 12009 is used for the treatment of malignant tumors based on the specific inhibi¬tion of TGF-beta2. This chapter describes this an-tisense technology in general and gives an over-view of oligodeoxynucleotide modifications and their delivery to cells. Furthermore, the status of preclinical and clinical trials with AP 12009 for the treatment of pancreatic carcinoma, malig¬nant glioma, malignant melanoma, and meta-static colorectal carcinoma is presented.
16.2 Targeted Therapies
Until recently the traditional therapy for patients with advanced pancreatic cancer was palliative 5-fluorouracil (5-FU)-based chemotherapy (Au-erbach et al. 1997; Cardenes et al. 2006). Novel approved chemotherapeutic agents such as gem-citabine (Gemzar, Eli Lilly, Indianapolis) and ox-aliplatin (Eloxatin, Sanofi-Aventis, Paris), as well as new therapeutic approaches including tyrosine kinase inhibitors, e.g., erlotinib (Tarceva, Genen-tech, South San Francisco; Osi Pharmaceuticals, Melville, NY) plus chemotherapy, have demon¬strated a survival benefit and improved quality of life in patients with advanced disease (Moore et al. 2005). However, the best combinational therapy still results in median survival of less than 1 year. Furthermore, the high risk of severe side effects and possible resistance to chemother-apeutic agents has evoked considerable interest in molecular pathways of tumors and new treat-ment strategies such as targeted therapies.
Conventional chemotherapeutic treatments aim at rapidly dividing cells. However, even highly proliferative healthy cells such as blood cells, cells in the hair follicles, and cells lining the gastrointestinal tract are attacked. Similarly, con-ventional radiation therapy affects some healthy cells surrounding the radiated tumor during treatment. Newer radiation therapy techniques can reduce but not fully eliminate this damage. This treatment-related damage of healthy tissue induces chemotherapy's and radiotherapy's well-known side effects. Targeted therapy acts by interfering with specific molecules needed for carcinogenesis and tumor growth. Monoclonal antibodies are one example for targeted therapy. Targeted cancer therapies can be more effective and may offer the advantage of reduced treat-ment-related side effects and improved outcomes due to their action restricted only to the target. Recent phase II and III trials with molecular targeted therapies in advanced pancreatic cancer include approaches using monoclonal antibodies [e.g., cetuximab, Erbitux (ImClone Systems, New York; an anti-EGFR), trastuzumab, Herceptin (Genentech, South San Francisco; anti-HER-2), bevacizumab, Avastin (F. Hoffmann-La Roche, Basel; anti-VEGF)], small molecules [e.g., gefitinib, Iressa (Astra Zeneca, London; EGFR inhibitor), erlotinib, Tarceva (EGFR inhibitor)], protein inhibitors (e.g., marimastat or BAY 12-9566, both matrix metalloproteinase inhibi-tors), and antisense therapeutics (e.g., GTI-2501, complementary to the subunit R1 of ribonucleo-tide reductase) (Cardenes et al. 2006; Lee et al.
2006).
Herein we report on the antisense therapy using the S-ODN AP 12009 for the treatment of pancreatic carcinoma and other solid tumors overexpressing TGF-beta2.
16.2.1 The Antisense Mechanism
In 1978 Zamecnik and Stephenson published their exciting results on the successful blockade of the replication of the Rous sarcoma virus by adding a synthetic oligodeoxynucleotide directed against a specific sequence of the viral genome (Zamecnik and Stephenson 1978). Only two de¬cades later, in 1998, the first antisense compound named Vitravene (Novartis Ophthalmics Eu¬rope, Basel; fomivirsen sodium), an S-ODN, was approved by the Food and Drug Administration (FDA) for the treatment of cytomegalovirus-in-duced retinitis in patients with acquired immu-nodeficiency syndrome (AIDS) (Roehr 1998). Especially in the field of oncology, a number of antisense compounds have been developed that are currently in clinical trials for the treatment of different types of tumors (Coppelli and Grandis 2005; Dean and Bennett 2003; Lahn et al. 2005; Schlingensiepen et al. 2006).
In contrast to gene therapy, which aims at replacing, removing, or introducing genes to correct a genetic defect or a mutation, antisense drugs neither alter human genes nor have any ef-fect on genetic information. Antisense molecules are a mirror image of the genetic blueprint or sequence that contains the necessary informa¬tion for the production of the targeted protein. By binding to the blueprint (mRNA), antisense-molecules render the contained information il¬legible, thereby inhibiting the protein produc¬tion (Fig. 16.1).
Different antisense mechanisms are under discussion concerning how the translation of the targeted protein is inhibited; these are (1) sterical blockade of the ribosome (Schlingensiepen et al. 1997), which physically prevents the progres¬sion of splicing or translation, and (2) RNase H-induced mRNA cleavage (Akhtar and Agrawal 1997). RNase H is an endoribonuclease that spe-cifically hydrolyzes the phosphodiester bonds of
the target RNA.
16.2.1.1 Chemical Modifications
of Antisense Oligodeoxynucleotides
Native ODNs contain phosphodiester linkages in their nucleotide backbone making them highly soluble in aqueous solutions but also very sus-ceptible to degradation by exo- and endonucle-ases within minutes (Shaw et al. 1991; Wick-strom 1986). Established modifications of the ODN chemistry aim at an optimal combination of long half-life due to nuclease resistance, suffi¬cient cellular uptake, good hybridization charac¬teristics, specific binding affinity, and reduction of nonspecific interactions, which could cause toxicities (for review see Mahato 2005). The first chemically synthesized modified ODNs were methylphosphonates (Me-ODNs) with a neutral methyl group replacing the negative charge-bear¬ing oxygen of the phosphodiester bond (Miller et al. 1981). Although Me-ODNs demonstrate an excellent nuclease resistance in biological sys¬tems (Tidd and Warenius 1989), their lipophilic nature leads to solubility problems in comparison with other analogs (Brysch and Schlingensiepen 1994; Mahato 2005). Furthermore, this type of oligonucleotides exhibits insufficient duplex for-mation presumably caused by steric hindrance by the methyl group, resulting in poor antisense activity that cannot activate RNase H activity (Crooke 1999; Miller et al. 1981).
In S-ODNs, one of the nonbridging oxygens of the phosphate backbone is substituted by a sulfur atom (Eckstein 1983). S-ODNs show analogous characteristics to unmodified ODNs such as their net charge and aqueous solubil¬ity, but exhibit a significantly higher stability in vitro and in vivo (Shaw et al. 1991). Further¬more, S-ODNs show excellent antisense activity. Pharmacokinetic experiments in rats, mice, and monkeys have shown that S-ODNs are cleared from plasma biphasically (Agrawal et al. 1995). As observed in preclinical models as well as in humans, the pharmacokinetics of S-ODNs are largely independent of the sequence; thus, differ-ent S-ODNs have shown a similar pharmacoki-netic profile (Geary et al. 2001). Immediately af¬ter administration, they are rapidly distributed into different tissues and organs. Major sites of accumulation are liver and kidney followed by spleen, bone marrow, and lymph nodes (Agrawal et al. 1995, 1991; Mahato 2005). Excretion from the human body occurs primarily via the urine, with up to 30% being excreted within 24 h and 70% within 10 days after a single intravenous ad¬ministration (Agrawal et al. 1991, 1995). After intravenous administration, S-ODNs are not de¬tectable in the brain since they are not able to pass the blood-brain barrier (BBB) (Agrawal
et al. 1991).
Further chemical modifications in ODNs in-clude 2'-0-methyl and 2'-0-methoxy-ethyl oli-gonucleotides showing increased nuclease resis¬tance and oligonucleotide:RNA binding affinities (Agrawal et al. 1997). Other chemical modifica¬tions of ODNs such as N3'-P5' phosphoroami-dates and morpholino oligonucleotides enhance stability, target affinity, and bioavailability (Kur-reck 2003). Another class of oligonucleotide-based compounds consisting of small interfer¬ing RNAs has recently become widely used for gene knockdown in vitro and in vivo (Coppelli and Grandis 2005). So far, none of these com¬pounds is in advanced clinical trials. The key fac¬tors are cellular uptake, the therapeutic activity of the individual antisense compounds, and the sequences themselves, rather than the chemical modifications alone.
Apart from selecting the optimal gene area, it is crucial to avoid interaction with proteins via certain base sequences, which may result in non-specific effects. Antisense compounds may con-tain special motifs such as G-quartets or CG-rich sequences (CpG motif). Four consecutive gua-nosines exhibit a nonspecific antiproliferative action and inhibit enzymatic activities in several cell types (Burgess et al. 1995; Yaswen et al. 1993). CpG motifs may activate defense mechanisms in humans, leading to a natural and acquired im¬mune response (Krieg 2002).
The future therapeutic success of antisense compounds will depend, as is the case with any targeted therapy, on the careful selection of op-timal targets, dosing, schedules, and clinical trial design. The ideal drug candidate should drive tumor progression and should not have redun-dant pathways, as is case with PKC, for example.
16.2.2 Delivery of Oligodeoxynucleotides into Cells
Antisense ODNs must be internalized into tar¬get cells in sufficient amounts to exert their in¬hibiting effects by targeted downregulation of RNA encoding disease-inducing genes. Owing to their anionic nature and their size, phosphodi-ester and phosphorothioate ODNs (S-ODNs) are unable to cross the lipophilic cell membrane by passive diffusion. It is well accepted that cellular uptake of S-ODNs is energy-, temperature-, and time-dependent (Levin 1999). The mechanism of cellular uptake can vary depending on the chemical structure and the concentration of the oligonucleotide. Whereas at low concentrations S-ODN uptake is predominantly achieved via a receptor-like mechanism, at higher concentra¬tions adsorptive endocytosis, pinocytosis and ca-veolar potocytosis are described (Lysik and Wu-
Pong 2003; Mahato et al. 2005; Stein et al. 1993;
Zamecnik et al. 1994).
In vitro uptake of free antisense ODNs into cultured cell lines is in some cases inefficient. Depending on the cell type, in vitro uptake of
ODNs is generally enhanced using different vec-tors. A variety of viral and nonviral possibilities of oligonucleotide delivery has been developed for basic and clinical research. Viral vectors in¬clude retroviral, adenoviral, and adeno-associ-ated viral vectors, which introduce their DNA into the cells with high efficiency. A major ob¬stacle of viral vectors in vivo but not in vitro is the host's immune response including both the adaptive response (Yang and Wilson 1995) and the innate immune system (Plank et al. 1996; Sung et al. 2001). Despite the observed limita¬tions on the usage of viral vectors, especially re¬garding safety, they are still the most used gene transfer vehicles (Gardlik et al. 2005).
Nonviral methods make use of cationic lipid complexes, liposomes (see below), polymers, and other reagents. Furthermore, ODNs may be in-ternalized mechanically, i.e., by generating tran-sient permeabilization of the plasma membrane to allow penetration of naked ODNs into cells by diffusion. However, these methods are not useful in vivo and their relevance for gene func¬tion analysis remains questionable. Therefore, plasmid or liposomal complexes are the most commonly used nonviral vectors and repre¬sent attractive tools in gene therapy due to their relatively simple production, low toxicity, and low host immunogenicity (Gardlik et al. 2005). Shen and colleagues demonstrated that the use of a cationic liposome elicits enhanced efficacy of ODNs for the inhibition of TGF-beta2 expres¬sion in the human promonocytic leukemia cell line U937 (Shen et al. 1999). All of these cationic delivery systems internalize ODNs via an endo-cytotic mechanism. In contrast to the in vitro situation, many reports have shown that in vivo uptake of S-ODNs does not depend on cationic carrier liposomes (Braasch and Corey 2002; Tari and Lopez-Berestein 2001).
In vitro, most of the ODNs designated for clinical studies were delivered in the presence of carrier liposomes in order to facilitate the ODN uptake. In contrast, during the selection process toward the development of the herein described antisense S-ODN AP 12009, inhibition of TGF-beta2 expression without carriers was crucial. Im¬portantly, in preclinical experiments performed with and without the carrier protein Lipofectin
(Invitrogen, Carlsbad, CA; a transfection reagent) AP 12009 showed similar effects (see below).
16.3 The Target: Transforming Growth Factor-Beta 2
TGF-beta is a multifunctional cytokine playing various roles in cell functions, including mor-phogenesis, cell proliferation, and migration, and is a key regulator of the immune system. Three isoforms of TGF-beta are described in mam¬mals: TGF-beta1, TGF-beta2, and TGF-beta3. A unique gene on different chromosomes encodes each isoform. All three human isoforms show a different temporal and spatial expression. Major activities of TGF-betas include inhibition of cell proliferation by blocking the cell cycle in late G1 phase, immunosuppressive effects, and enhanc-ing the formation of extracellular matrix. The transcriptional regulation of TGF-beta1 is dif-ferent from that of TGF-beta2 and TGF-beta3 as the latter are mostly under hormonal and devel-opmental control (Roberts 1998).
TGF-beta is synthesized as homomeric pro-proteins in vivo, which need to be activated to bind to the signaling receptors (Murphy-Ullrich and Poczatek 2000; Wakefield and Roberts 2002). The so-called latency-associated protein (LAP) is generated by removal of the N-terminus of the mature TGF-beta by a furin-like peptidase. The LAP is noncovalently associated with a homodi-mer of mature TGF-beta (Li et al. 2006). TGF-beta is secreted as a complex, which consists of the inactive, mature TGF-beta, the LAP, and the latent TGF-beta binding protein (LTBP) (Annes
et al. 2003; Oeklue and Hesketh 2000). Extracel-lular activation of this complex is a critical step in the regulation of TGF-beta function, includ¬ing plasmin-dependent and plasmin-indepen-dent pathways (Derynck and Zhang 2003; Piek et al. 1999; Wakefield and Roberts 2002; Yingling
et al. 2004).
Although TGF-beta1 and TGF-beta2 share various similar receptor binding and signaling properties, some crucial differences have been described. In general, the TGF-beta ligand binds to receptors on the cell surface forming a bi-di-meric receptor complex consisting of two pairs of subunits known as receptor type I (TBR-I, usually ALK5) and type II (TBR-II). A mem¬brane-anchored proteoglycan, known as type III receptor (TBR-III or betaglycan), aids this pro-cess by capturing TGF-beta for presentation to the signaling receptors I and II. Importantly, the type III receptor is particularly important for TGF-beta2, which cannot bind TBR-II indepen-dently and thus depends on the presence of TBR-III to signal—a unique feature that distinguishes TGF-beta2 from TGF-beta1 and TGF-beta3
(Blobe et al. 2001).
The biological activities of TGF-betas are mod-ulated by binding proteins with alpha-2-macro-globulin (A2M) as the major binding protein for TGF-beta1 and TGF-beta2 in plasma (Daniel-pour and Sporn 1990; O'Connor-McCourt and Wakefield 1987). A2M is a homotetrameric gly-coprotein that inhibits various proteinases and serves as a regulator and major carrier of various cytokines (Crookston et al. 1994). It is one of the most abundant proteins in human plasma with a concentration of 2-4 mg/ml. Both isoforms, TGF-beta1 and TGF-beta2, bind reversibly and covalently to native A2M and A2M-methylamine. It has been shown that A2M significantly inhib¬its the receptor binding and biological activity of TGF-betas (O'Connor-McCourt and Wakefield 1987). However, TGF-beta2 is more affected due to a distinctive interaction pattern with A2M compared to TGF-beta1 and other cytokines (Crookston et al. 1994). First, TGF-beta2 reveals substantially higher affinity to A2M and therefore an increased complex formation (Danielpour and Sporn 1990; Liu et al. 2001). Second, it is the only growth factor that binds with equiva¬lent affinity to both A2M and A2M-methylamine (Crookston et al. 1994). In experiments using na¬tive A2M as well as the activated form A2M-me-thylamine, TGF-beta2 shows the highest affinity to both proteins compared to other cytokines including TGF-beta1, nerve growth factor-beta (NGF-beta), platelet-derived growth factor-BB (PDGF-BB), tumor necrosis factor (TNF-alpha), and basic fibroblast growth factor (Crookston et
al. 1994).
The significance of TGF-beta has become in-creasingly evident since it obviously elicits two opposed mechanisms depending on the respec-tive environment (Akhurst and Derynck 2001; Wakefield and Roberts 2002). In normal cells of epithelial origin as well as in early well-dif-ferentiated tumor cells of epithelial origin, the TGF-beta pathway restricts cell growth, differen-tiation, and cell death. However, during progres-sion of cells toward fully malignant tumor cells, these cells undergo changes resulting in reduced expression of TGF-beta receptors, increased ex-pression of TGF-beta ligands, and resistance to growth inhibition by TGF-beta (Moustakas et al. 2002; Wakefield and Roberts 2002).
The crucial role of TGF-beta2 in pancreatic cancer progression and aggressiveness was dem-onstrated in an animal model consisting of human pancreatic cancer cells grown either ectopically in subcutaneous tissue or orthotopically in the pancreas (Choudhury et al. 2004). In this model, TGF-beta2 expression clearly correlated with tu¬mor aggressiveness and metastatic behavior. The far more aggressive orthotopic tumors not only demonstrated a larger size, shorter latent period, higher metastasis, and more extensive invasion of the stomach, but also a higher expression of TGF-beta2 compared to the less aggressive sub¬cutaneous tumors. In another study on human pancreatic tissue samples, immunohistochemi-cal analysis has shown that all three mammalian isoforms of TGF-beta (TGF-beta1, -beta2, and -beta3) were overexpressed (Friess et al. 1993). However, only the TGF-beta2 isoform was sig¬nificantly correlated with advanced tumor stage and a more aggressive phenotype. Pancreatic cancer patients bearing TGF-beta2 producing tumors showed the shortest postoperative sur¬vival period in contrast to patients with tumors producing TGF-beta1, TGF-beta3, or none of the TGF-beta isoforms (Friess et al. 1993).
16.3.1 Targeted Therapy with
the TGF-Beta2 Inhibitor AP 12009
16.3.1.1 Preclinical Experiments
In vitro experiments were performed to evalu¬ate the specificity and efficacy of the TGF-beta2 specific phosphorothioate ODN AP 12009 by employing human tumor cell cultures as well as peripheral blood mononuclear cells (PBMC) from healthy donors and from patients (Schlin-gensiepen et al. 2006).
The efficacy of AP 12009 in reducing TGF-beta2 secretion of human pancreatic carcinoma cells was determined by measuring the TGF-beta2 concentration in culture supernatants us¬ing an enzyme-linked immunosorbent assay (ELISA). Treatment with AP 12009 complexed with the liposomal carrier Lipofectin significantly inhibited TGF-beta2 production compared to Lipofectin alone in all human pancreatic cancer cell lines tested. Importantly, comparable data were obtained in experiments without Lipofectin indicating that AP 12009 alone is able to inhibit TGF-beta induced tumor-promoting effects.
Furthermore, AP 12009 was shown to revert the strong immunosuppressive effects exerted by TGF-beta2. TGF-beta has multiple immunosup-pressive properties including inhibition of T cell proliferation and inhibition of T cell differen¬tiation into cytotoxic T lymphocytes (CTLs) and
helper T cells (Gorelik and Flavell 2001). TGF-
beta inhibits these immune cell functions includ-ing cell-dependent cytotoxicity (Weller and Fon-tana 1995). Treatment with AP 12009 enhances the cytotoxic antitumor response of human lymphokine activated killer (LAK) cells directed against pancreatic carcinoma cells.
The invasion of neoplastic cells into healthy tissue is a pathologic hallmark of highly aggres-sive tumors such as pancreatic carcinoma, malig-nant melanoma, or malignant glioma.
The key mechanism for infiltration of tumor cells into healthy tissue leading to metastasis is tumor cell motility. TGF-beta, produced by tumor cells, acts directly on the tumor cells by in-ducing EMT (Janji et al. 1999), and by increasing motility, invasiveness, and metastasis (Dumont and Arteaga 2000; Oft et al. 1998). AP 12009 in-hibits the migration of cancer cells in vitro. The motility of pancreatic cancer cells was measured employing an in vitro spheroid migration model (Nygaard et al. 1998). Tumor cells spontane¬ously form round shaped clusters (spheroids) when cultured in medium on agar-coated plates, which prevents their adherence to the plastic surface. The spheroids can be transferred into culture medium without agar where the tumor cells start migrating off the spheroids. AP 12009 inhibits migration of the pancreatic tumor cells with the spheroids remaining compact after 24 h. In contrast, untreated and recombinant human
(rh-) TGF-beta2 treated cells migrate and, as a consequence, the spheroids dissolve.
Similar results as described for pancreatic can-cer cells were obtained for other cancer cells in-cluding human malignant glioma and malignant melanoma cell cultures (Jachimczak et al. 1993, 1996; Schlingensiepen et al. 2006). Importantly, all experiments were performed in the presence as well as in the absence of a liposome carrier and showed comparable efficiency to naked and Lipofectin-complexed AP 12009 in various cell lines test.
16.3.1.2 Toxicological Studies
In the current clinical trials of AP 12009 are be¬ing developed for the treatment of TGF-beta2-overproducing tumors such as malignant gli-oma, pancreatic carcinoma, metastatic colorectal carcinoma, and metastatic melanoma. Whereas AP 12009 is administered systemically by intra¬venous infusion in the indications for pancreatic carcinoma, metastatic colorectal carcinoma, and melanoma, in the case of high-grade glioma the same substance is applied locally by convection-enhanced delivery (CED) directly into the brain tumor tissue.
Local toxicity studies applying AP 12009 by the intrathecal and intracerebral routes were performed in rabbits and monkeys in order to match the intended human mode of applica¬tion in malignant glioma as close as possible. AP 12009 showed excellent local tolerability in rabbits and monkeys when administered by intra-thecal bolus injection. Neither clinical signs of toxicity nor substance-related histomorphologi-cal changes were observed. The application of AP 12009 via continuous intracerebral infusion focally resulted in a mild to moderate lympho-cytic leptomeningo-encephalitis. Changes are considered a reversible immunological reaction to AP 12009. Local tolerance tests of AP 12009 in rabbits after intravenous, intraarterial, intramus-cular, paravenous, and subcutaneous application led neither to macroscopic nor to microscopic changes.
Acute toxicology studies in mice and rats as well as subchronic toxicity studies in rats and in cynomolgus monkeys were performed employ¬ing intravenous infusion. Liver and kidney were identified as target organs. The observed changes match the common toxic effects reported for S-ODNs (Henry et al. 1997; Levin et al. 1998). Detailed methods and results were reported by Schlingensiepen et al. (2005).
The pharmacological effects of AP 12009 on the cardiovascular system, complement ac-tivation, and hematological parameters corre-sponded well to the effects reported for other phosphorothioate ODNs as a class of compounds
(Mahato 2005).
AP 12009 showed neither mutagenic effect in the Salmonella typhimurium strains nor indica¬tions of mutagenic properties in cultured human peripheral lymphocytes with respect to chro-mosomal or chromatid damage. Furthermore, AP 12009 showed no mutagenic properties in the mouse bone marrow micronucleus study us¬ing intravenous administration.
16.3.1.3 Clinical Studies: Systemic Application
In pancreatic carcinoma cells, all three mam-malian isoforms of TGF-beta (TGF-beta1, TGF-beta2, and TGF-beta3) are expressed. However, only excessive expression of TGF-beta2 is signifi¬cantly associated with pancreatic cancer progres¬sion (Friess et al. 1993).
Spurred by the clinical data in recurrent or refractory high-grade glioma patients (see Sect. 3.1.4) and the impressive antitumor ac¬tivity in a wide variety of preclinical assays (Schlingensiepen et al. 2006), the clinical stud¬ies for other solid tumors were initiated. A mul-ticenter dose-escalation phase I/II trial with AP 12009 in adult patients suffering from ad-vanced pancreatic carcinoma (AJCC stage IVA or IVB) as well as metastatic melanoma (AJCC/ UICC stage III or IV) and advanced metastatic colorectal carcinoma (AJCC/UICC stage III or IV), is currently ongoing. The primary endpoint is the assessment of the maximum tolerated dose (MTD) as well as the dose-limiting toxicities. Secondary objectives include safety and tolerabil-ity of AP 12009 and its potential antitumor activ¬ity. Adult patients (18-75 years) with advanced tumors who are not or no longer amenable to established therapies are eligible for this dose¬escalation study. Karnofsky performance status (KPS) should be at least 80%. Patients receive the study drug intravenously via an implanted port system at weekly intervals. Up to ten treatment cycles are to be applied per patient.
The majority of patients already treated re-ceived more than the minimum number of two cycles. One of them received ten full cycles. So far, AP 12009 revealed a good safety profile. The MTD has not yet been reached. Further dose es-calations are ongoing.
16.3.1.4 Clinical Studies: Local Application in High-Grade Glioma Patients
The TGF-beta2 isoform is specifically overex-pressed in malignant gliomas (Frankel et al. 1999; Maxwell et al. 1992). The increased levels of TGF-beta2 are associated with disease stage and causative for the immunodeficient state of patients (Bodmer et al. 1989; Kjellman et al.
2000; Maxwell et al. 1992).
In three phase I/II dose-escalation stud¬ies (G001, G002, and G003) a total of 24 adult patients with recurrent or refractory malig¬nant glioma, i.e., anaplastic astrocytoma (AA,
WHO grade III) or glioblastoma (GBM, WHO
grade IV), and evidence of tumor progression were treated with AP 12009 (Schlingensiepen et al. 2006). In these studies, the drug was ad-ministered intratumorally using CED over a 4- or 7-day period. The CED application allows AP 12009 to bypass the BBB. The BBB serves as a natural defense system by blocking the entry of foreign substances, including bacteria and toxins but also many therapeutic agents (Bobo et al. 1994). While conventional diffusion is characterized by a steep drop in drug concentra-tion close to the catheter tip, CED creates a ho-mogeneous drug concentration extending over several centimeters in diameter (Lieberman et al. 1995). To facilitate multiple cycles of AP 12009, the investigational drug was infused through an implanted port system connected to the intratu-moral catheter. AP 12009 proved to be well toler¬ated and revealed a good safety profile. Since two complete remissions in two different dose groups were observed (see below), further dose escala¬tion was not necessary. MTD was not reached.
Although the clinical phase I/II trials were pri-marily designed to assess safety, survival times as well as tumor response data were obtained. Data on antitumor activity from 24 patients included several patients with stabilization of disease and two patients with complete tumor remission, both of them long-lasting without recurrence. One of these two patients, diagnosed with AA, was treated with only one course of AP 12009. At baseline four tumor lesions had been detected, which were spread over both hemispheres. Only one lesion had been infused with one cycle of AP 12009, but all lesions had disappeared several months after start of treatment despite an ini¬tial and temporary increase in tumor volume at the beginning of the treatment. The patient died from a myocardial infarction without any signs of tumor, 25 months after start of AP 12009 treat¬ment. The second patient, also diagnosed with AA, received a total of 12 cycles of AP 12009 over the course of the three phase I/II studies (G001,
G002, and G003; Fig. 16.2).
Prior to AP 12009 treatment, he had been treated with surgery, radiation, and chemo-therapy [temozolomide (TMZ) after the first re-lapse], followed by a second incomplete surgery. After an initial stabilization following the second cycle, the enhancing lesion continued to increase until 10 months after baseline G001 (Fig. 16.2b), inducing a significant edema. The central read¬ing of the magnetic resonance image (MRI) 20 months after the start of AP 12009 treatment (in G001) was evaluated as partial response (PR, 83% tumor reduction, Fig. 16.2c); there was com-plete response after 22 months. The patient is known to still be alive today; the MRI in August 2006 (Fig. 16.2d) showed no recurrence. Survival of this patient after the first recurrence is now 307 weeks (71 months); it has been 286 weeks (66 months) since treatment with AP 12009 be¬gan (status 01 August 2007).
As of 01 August 2007 the median overall sur-vival after recurrence for AA patients treated with AP 12009 was 146.6 weeks (range 32.0¬306.6 weeks), and for GBM patients treated with AP 12009 44.0 weeks (range 18.9-87.9 weeks). The most recent and accurate survival data after start of therapy that clearly distinguish between recurrent AA and GBM are available for the current gold standard treatment TMZ. The re-
ported median overall survival for TMZ alone is 42.0 weeks (9.7 months) for recurrent AA (The-odosopoulos et al. 2001), and 31.8 (7.3 months) (Yung 2000; Yung et al. 2000) or 32.0 weeks (7.4 months) (Theodosopoulos et al. 2001) for recurrent GBM. These results were reported for patients with high-grade glioma who received TMZ as first treatment after recurrence. In the adjuvant treatment of newly diagnosed glioma, the combination of TMZ with radiotherapy has improved median overall survival from 12.1 to 14.6 months (Stupp et al. 2005).
The phase IIb clinical trial of AP 12009-G004 is an international, open-label, active-controlled dose-finding study in high-grade glioma pa¬tients. The main trial objective is the compari¬son of two different doses of AP 12009 (10 uM or 80 uM) against standard chemotherapy. In all, 145 patients with either recurrent or re¬fractory AA (WHO grade III) or GBM (WHO
grade IV) are receiving either one of the two doses of AP 12009 or standard chemotherapy [TMZ or procarbazine/CCNU (lomustine)/ vincristine = PCV, if TMZ was already given].
AP 12009 is applied intratumorally by CED dur-ing a 6-month active treatment period at weekly intervals. The primary efficacy endpoint is tumor response after radiological evaluation. The main secondary efficacy endpoints are overall survival and 12-month survival. As in the previous stud-ies, preliminary data show long-lasting responses both in recurrent or refractory AA and GBM
patients (Bogdahn et al. 2006; Hau et al. 2006).
Especially in recurrent or refractory AA patients, very promising efficacy data have been docu-mented compared to current standard treatment
with TMZ or PCV.
16.4 Summary
Despite tremendous advances in cancer research and the development of new therapies, patients with malignant tumors such as advanced pancre-atic carcinoma, metastatic melanoma, metastatic colorectal carcinoma, and malignant glioma still face a poor prognosis. The severe morbidity and mortality of these malignant tumor types makes the identification of factors associated with their incidence an important area of both preclinical and clinical research. Antisense technology is a new and innovative method offering a causal approach for the treatment of various highly ag-gressive diseases. Antisense compounds inhibit the production of disease-causing proteins at the molecular level and combat tumor development directly at its roots. Preclinical experiments us¬ing the TGF-beta2 specific phosphorothioate
ODN AP 12009 revealed the potential of this
compound to reverse TGF-beta2 induced immu-nosuppression as well as inhibition of tumor cell proliferation and tumor cell migration. Initial clinical studies have demonstrated AP 12009 to be well tolerated and safe. Furthermore, the first evidence of efficacy of AP 12009 antisense ther¬apy in recurrent or refractory high-grade glioma has been provided.
These data confirm that the blockade of TGF-beta2, a key factor in tumorigenesis, in tumor tissue by AP 12009 represents a novel and promising therapeutic approach for malignant tumors such as advanced pancreatic carcinoma and malignant glioma. This approach aims at a reduction of tumor-promoting effects and, most importantly, an enhancement of the antitumor immune response.
16.1 Introduction
Pancreatic cancer is the fourth leading cause of cancer-related deaths in men and women in virtu-ally all industrialized countries (Jemal et al. 2005, 2006). Its incidence cuts across all racial and so-cio-economic barriers. Outcome is nearly always fatal with a 1-year survival rate of about 20% and a 5-year survival rate of less than 5% (Cardenes et al. 2006; Jemal et al. 2005, 2006). The majority of patients exhibit a very aggressive adenocarci-noma (85%). These tumor types are less aggres¬sive and are often curable. Pancreatic tumors are difficult to detect at early stages and, due to their nonspecific symptoms, extremely hard to diagnose. Therefore, most patients present with locally advanced or metastatic disease resulting in high mortality and very short life expectancy. This is at least partially due to the observed resis¬tance of pancreatic cancer to chemotherapy and radiation therapy. Currently, complete surgical resection remains the only therapeutic option with a potential for cure. However, only a low proportion of patients (only 15%-20%) are suit¬able candidates for surgical resection (Cardenes et al. 2006; Siech et al. 2001). The median sur¬vival of these patients who undergo successful resection is approximately 12-19 months with a 5-year survival rate of 15%-20%. Risk factors for pancreatic cancer include advanced age, obesity, diabetes, and chronic pancreatitis (Li et al. 2004; Lowenfels and Maisonneuve 2006). However, cigarette smoking is considered the most signifi¬cant and avoidable risk factor, causing more than 25% of the pancreatic cancer cases (Li et al. 2004;
Lowenfels and Maisonneuve 2006). Because of the lethality of this disease and the failure of standard treatment to date, future efforts will be focused on the advances that are being made in the understanding and delineation of the genetic and molecular cell biology of cancer cells.
During the last three decades, the interest in new therapeutics such as those based on anti-sense technology has strongly increased. Today, the improved technology and ability of chemi¬cal synthesis of antisense oligodeoxynucleotides (ODNs) has become a routine process and offers researchers the possibility to target almost any single gene (Schlingensiepen et al. 1993). This technology has become a powerful research tool in molecular biology, biochemistry, and microbi¬ology, and has tremendous potential in the fields of functional genomics, drug discovery, and clin¬ical therapy, especially oncology. A major cause of cancer lies in defective gene regulation. Such mutations can result either in an overproduction or in abnormal production of proteins promoting dysfunctional growth and tumor development. Antisense drugs are able to block the blueprint (messenger ribonucleic acid, mRNA) of a can¬cer gene and specifically inhibit its conversion into the pathogenic cancer protein. Preventing the formation of such pathogenic factors means combating cancer disease directly at its roots.
Transforming growth factor beta (TGF-beta) is a multifunctional cytokine that has been iden-tified as a key factor in tumor development. TGF-beta is a vital factor controlling several signaling cascades with oncogenic potential, including immunosuppression, epithelial to mesenchymal transition (EMT), metastasis and invasion, an-giogenesis, and proliferation. Overexpression of the TGF-beta isoform TGF-beta2 is a hallmark of various malignant tumors, e.g., pancreatic carcinoma, malignant brain tumors, malignant melanoma, and metastatic colorectal carcinoma. Thus, targeting this key factor to suppress sev¬eral cancer mechanisms simultaneously right at their origin offers a very promising therapeutic approach.
The phosphorothioate oligodeoxynucleotide (S-ODN) AP 12009 is used for the treatment of malignant tumors based on the specific inhibi¬tion of TGF-beta2. This chapter describes this an-tisense technology in general and gives an over-view of oligodeoxynucleotide modifications and their delivery to cells. Furthermore, the status of preclinical and clinical trials with AP 12009 for the treatment of pancreatic carcinoma, malig¬nant glioma, malignant melanoma, and meta-static colorectal carcinoma is presented.
16.2 Targeted Therapies
Until recently the traditional therapy for patients with advanced pancreatic cancer was palliative 5-fluorouracil (5-FU)-based chemotherapy (Au-erbach et al. 1997; Cardenes et al. 2006). Novel approved chemotherapeutic agents such as gem-citabine (Gemzar, Eli Lilly, Indianapolis) and ox-aliplatin (Eloxatin, Sanofi-Aventis, Paris), as well as new therapeutic approaches including tyrosine kinase inhibitors, e.g., erlotinib (Tarceva, Genen-tech, South San Francisco; Osi Pharmaceuticals, Melville, NY) plus chemotherapy, have demon¬strated a survival benefit and improved quality of life in patients with advanced disease (Moore et al. 2005). However, the best combinational therapy still results in median survival of less than 1 year. Furthermore, the high risk of severe side effects and possible resistance to chemother-apeutic agents has evoked considerable interest in molecular pathways of tumors and new treat-ment strategies such as targeted therapies.
Conventional chemotherapeutic treatments aim at rapidly dividing cells. However, even highly proliferative healthy cells such as blood cells, cells in the hair follicles, and cells lining the gastrointestinal tract are attacked. Similarly, con-ventional radiation therapy affects some healthy cells surrounding the radiated tumor during treatment. Newer radiation therapy techniques can reduce but not fully eliminate this damage. This treatment-related damage of healthy tissue induces chemotherapy's and radiotherapy's well-known side effects. Targeted therapy acts by interfering with specific molecules needed for carcinogenesis and tumor growth. Monoclonal antibodies are one example for targeted therapy. Targeted cancer therapies can be more effective and may offer the advantage of reduced treat-ment-related side effects and improved outcomes due to their action restricted only to the target. Recent phase II and III trials with molecular targeted therapies in advanced pancreatic cancer include approaches using monoclonal antibodies [e.g., cetuximab, Erbitux (ImClone Systems, New York; an anti-EGFR), trastuzumab, Herceptin (Genentech, South San Francisco; anti-HER-2), bevacizumab, Avastin (F. Hoffmann-La Roche, Basel; anti-VEGF)], small molecules [e.g., gefitinib, Iressa (Astra Zeneca, London; EGFR inhibitor), erlotinib, Tarceva (EGFR inhibitor)], protein inhibitors (e.g., marimastat or BAY 12-9566, both matrix metalloproteinase inhibi-tors), and antisense therapeutics (e.g., GTI-2501, complementary to the subunit R1 of ribonucleo-tide reductase) (Cardenes et al. 2006; Lee et al.
2006).
Herein we report on the antisense therapy using the S-ODN AP 12009 for the treatment of pancreatic carcinoma and other solid tumors overexpressing TGF-beta2.
16.2.1 The Antisense Mechanism
In 1978 Zamecnik and Stephenson published their exciting results on the successful blockade of the replication of the Rous sarcoma virus by adding a synthetic oligodeoxynucleotide directed against a specific sequence of the viral genome (Zamecnik and Stephenson 1978). Only two de¬cades later, in 1998, the first antisense compound named Vitravene (Novartis Ophthalmics Eu¬rope, Basel; fomivirsen sodium), an S-ODN, was approved by the Food and Drug Administration (FDA) for the treatment of cytomegalovirus-in-duced retinitis in patients with acquired immu-nodeficiency syndrome (AIDS) (Roehr 1998). Especially in the field of oncology, a number of antisense compounds have been developed that are currently in clinical trials for the treatment of different types of tumors (Coppelli and Grandis 2005; Dean and Bennett 2003; Lahn et al. 2005; Schlingensiepen et al. 2006).
In contrast to gene therapy, which aims at replacing, removing, or introducing genes to correct a genetic defect or a mutation, antisense drugs neither alter human genes nor have any ef-fect on genetic information. Antisense molecules are a mirror image of the genetic blueprint or sequence that contains the necessary informa¬tion for the production of the targeted protein. By binding to the blueprint (mRNA), antisense-molecules render the contained information il¬legible, thereby inhibiting the protein produc¬tion (Fig. 16.1).
Different antisense mechanisms are under discussion concerning how the translation of the targeted protein is inhibited; these are (1) sterical blockade of the ribosome (Schlingensiepen et al. 1997), which physically prevents the progres¬sion of splicing or translation, and (2) RNase H-induced mRNA cleavage (Akhtar and Agrawal 1997). RNase H is an endoribonuclease that spe-cifically hydrolyzes the phosphodiester bonds of
the target RNA.
16.2.1.1 Chemical Modifications
of Antisense Oligodeoxynucleotides
Native ODNs contain phosphodiester linkages in their nucleotide backbone making them highly soluble in aqueous solutions but also very sus-ceptible to degradation by exo- and endonucle-ases within minutes (Shaw et al. 1991; Wick-strom 1986). Established modifications of the ODN chemistry aim at an optimal combination of long half-life due to nuclease resistance, suffi¬cient cellular uptake, good hybridization charac¬teristics, specific binding affinity, and reduction of nonspecific interactions, which could cause toxicities (for review see Mahato 2005). The first chemically synthesized modified ODNs were methylphosphonates (Me-ODNs) with a neutral methyl group replacing the negative charge-bear¬ing oxygen of the phosphodiester bond (Miller et al. 1981). Although Me-ODNs demonstrate an excellent nuclease resistance in biological sys¬tems (Tidd and Warenius 1989), their lipophilic nature leads to solubility problems in comparison with other analogs (Brysch and Schlingensiepen 1994; Mahato 2005). Furthermore, this type of oligonucleotides exhibits insufficient duplex for-mation presumably caused by steric hindrance by the methyl group, resulting in poor antisense activity that cannot activate RNase H activity (Crooke 1999; Miller et al. 1981).
In S-ODNs, one of the nonbridging oxygens of the phosphate backbone is substituted by a sulfur atom (Eckstein 1983). S-ODNs show analogous characteristics to unmodified ODNs such as their net charge and aqueous solubil¬ity, but exhibit a significantly higher stability in vitro and in vivo (Shaw et al. 1991). Further¬more, S-ODNs show excellent antisense activity. Pharmacokinetic experiments in rats, mice, and monkeys have shown that S-ODNs are cleared from plasma biphasically (Agrawal et al. 1995). As observed in preclinical models as well as in humans, the pharmacokinetics of S-ODNs are largely independent of the sequence; thus, differ-ent S-ODNs have shown a similar pharmacoki-netic profile (Geary et al. 2001). Immediately af¬ter administration, they are rapidly distributed into different tissues and organs. Major sites of accumulation are liver and kidney followed by spleen, bone marrow, and lymph nodes (Agrawal et al. 1995, 1991; Mahato 2005). Excretion from the human body occurs primarily via the urine, with up to 30% being excreted within 24 h and 70% within 10 days after a single intravenous ad¬ministration (Agrawal et al. 1991, 1995). After intravenous administration, S-ODNs are not de¬tectable in the brain since they are not able to pass the blood-brain barrier (BBB) (Agrawal
et al. 1991).
Further chemical modifications in ODNs in-clude 2'-0-methyl and 2'-0-methoxy-ethyl oli-gonucleotides showing increased nuclease resis¬tance and oligonucleotide:RNA binding affinities (Agrawal et al. 1997). Other chemical modifica¬tions of ODNs such as N3'-P5' phosphoroami-dates and morpholino oligonucleotides enhance stability, target affinity, and bioavailability (Kur-reck 2003). Another class of oligonucleotide-based compounds consisting of small interfer¬ing RNAs has recently become widely used for gene knockdown in vitro and in vivo (Coppelli and Grandis 2005). So far, none of these com¬pounds is in advanced clinical trials. The key fac¬tors are cellular uptake, the therapeutic activity of the individual antisense compounds, and the sequences themselves, rather than the chemical modifications alone.
Apart from selecting the optimal gene area, it is crucial to avoid interaction with proteins via certain base sequences, which may result in non-specific effects. Antisense compounds may con-tain special motifs such as G-quartets or CG-rich sequences (CpG motif). Four consecutive gua-nosines exhibit a nonspecific antiproliferative action and inhibit enzymatic activities in several cell types (Burgess et al. 1995; Yaswen et al. 1993). CpG motifs may activate defense mechanisms in humans, leading to a natural and acquired im¬mune response (Krieg 2002).
The future therapeutic success of antisense compounds will depend, as is the case with any targeted therapy, on the careful selection of op-timal targets, dosing, schedules, and clinical trial design. The ideal drug candidate should drive tumor progression and should not have redun-dant pathways, as is case with PKC, for example.
16.2.2 Delivery of Oligodeoxynucleotides into Cells
Antisense ODNs must be internalized into tar¬get cells in sufficient amounts to exert their in¬hibiting effects by targeted downregulation of RNA encoding disease-inducing genes. Owing to their anionic nature and their size, phosphodi-ester and phosphorothioate ODNs (S-ODNs) are unable to cross the lipophilic cell membrane by passive diffusion. It is well accepted that cellular uptake of S-ODNs is energy-, temperature-, and time-dependent (Levin 1999). The mechanism of cellular uptake can vary depending on the chemical structure and the concentration of the oligonucleotide. Whereas at low concentrations S-ODN uptake is predominantly achieved via a receptor-like mechanism, at higher concentra¬tions adsorptive endocytosis, pinocytosis and ca-veolar potocytosis are described (Lysik and Wu-
Pong 2003; Mahato et al. 2005; Stein et al. 1993;
Zamecnik et al. 1994).
In vitro uptake of free antisense ODNs into cultured cell lines is in some cases inefficient. Depending on the cell type, in vitro uptake of
ODNs is generally enhanced using different vec-tors. A variety of viral and nonviral possibilities of oligonucleotide delivery has been developed for basic and clinical research. Viral vectors in¬clude retroviral, adenoviral, and adeno-associ-ated viral vectors, which introduce their DNA into the cells with high efficiency. A major ob¬stacle of viral vectors in vivo but not in vitro is the host's immune response including both the adaptive response (Yang and Wilson 1995) and the innate immune system (Plank et al. 1996; Sung et al. 2001). Despite the observed limita¬tions on the usage of viral vectors, especially re¬garding safety, they are still the most used gene transfer vehicles (Gardlik et al. 2005).
Nonviral methods make use of cationic lipid complexes, liposomes (see below), polymers, and other reagents. Furthermore, ODNs may be in-ternalized mechanically, i.e., by generating tran-sient permeabilization of the plasma membrane to allow penetration of naked ODNs into cells by diffusion. However, these methods are not useful in vivo and their relevance for gene func¬tion analysis remains questionable. Therefore, plasmid or liposomal complexes are the most commonly used nonviral vectors and repre¬sent attractive tools in gene therapy due to their relatively simple production, low toxicity, and low host immunogenicity (Gardlik et al. 2005). Shen and colleagues demonstrated that the use of a cationic liposome elicits enhanced efficacy of ODNs for the inhibition of TGF-beta2 expres¬sion in the human promonocytic leukemia cell line U937 (Shen et al. 1999). All of these cationic delivery systems internalize ODNs via an endo-cytotic mechanism. In contrast to the in vitro situation, many reports have shown that in vivo uptake of S-ODNs does not depend on cationic carrier liposomes (Braasch and Corey 2002; Tari and Lopez-Berestein 2001).
In vitro, most of the ODNs designated for clinical studies were delivered in the presence of carrier liposomes in order to facilitate the ODN uptake. In contrast, during the selection process toward the development of the herein described antisense S-ODN AP 12009, inhibition of TGF-beta2 expression without carriers was crucial. Im¬portantly, in preclinical experiments performed with and without the carrier protein Lipofectin
(Invitrogen, Carlsbad, CA; a transfection reagent) AP 12009 showed similar effects (see below).
16.3 The Target: Transforming Growth Factor-Beta 2
TGF-beta is a multifunctional cytokine playing various roles in cell functions, including mor-phogenesis, cell proliferation, and migration, and is a key regulator of the immune system. Three isoforms of TGF-beta are described in mam¬mals: TGF-beta1, TGF-beta2, and TGF-beta3. A unique gene on different chromosomes encodes each isoform. All three human isoforms show a different temporal and spatial expression. Major activities of TGF-betas include inhibition of cell proliferation by blocking the cell cycle in late G1 phase, immunosuppressive effects, and enhanc-ing the formation of extracellular matrix. The transcriptional regulation of TGF-beta1 is dif-ferent from that of TGF-beta2 and TGF-beta3 as the latter are mostly under hormonal and devel-opmental control (Roberts 1998).
TGF-beta is synthesized as homomeric pro-proteins in vivo, which need to be activated to bind to the signaling receptors (Murphy-Ullrich and Poczatek 2000; Wakefield and Roberts 2002). The so-called latency-associated protein (LAP) is generated by removal of the N-terminus of the mature TGF-beta by a furin-like peptidase. The LAP is noncovalently associated with a homodi-mer of mature TGF-beta (Li et al. 2006). TGF-beta is secreted as a complex, which consists of the inactive, mature TGF-beta, the LAP, and the latent TGF-beta binding protein (LTBP) (Annes
et al. 2003; Oeklue and Hesketh 2000). Extracel-lular activation of this complex is a critical step in the regulation of TGF-beta function, includ¬ing plasmin-dependent and plasmin-indepen-dent pathways (Derynck and Zhang 2003; Piek et al. 1999; Wakefield and Roberts 2002; Yingling
et al. 2004).
Although TGF-beta1 and TGF-beta2 share various similar receptor binding and signaling properties, some crucial differences have been described. In general, the TGF-beta ligand binds to receptors on the cell surface forming a bi-di-meric receptor complex consisting of two pairs of subunits known as receptor type I (TBR-I, usually ALK5) and type II (TBR-II). A mem¬brane-anchored proteoglycan, known as type III receptor (TBR-III or betaglycan), aids this pro-cess by capturing TGF-beta for presentation to the signaling receptors I and II. Importantly, the type III receptor is particularly important for TGF-beta2, which cannot bind TBR-II indepen-dently and thus depends on the presence of TBR-III to signal—a unique feature that distinguishes TGF-beta2 from TGF-beta1 and TGF-beta3
(Blobe et al. 2001).
The biological activities of TGF-betas are mod-ulated by binding proteins with alpha-2-macro-globulin (A2M) as the major binding protein for TGF-beta1 and TGF-beta2 in plasma (Daniel-pour and Sporn 1990; O'Connor-McCourt and Wakefield 1987). A2M is a homotetrameric gly-coprotein that inhibits various proteinases and serves as a regulator and major carrier of various cytokines (Crookston et al. 1994). It is one of the most abundant proteins in human plasma with a concentration of 2-4 mg/ml. Both isoforms, TGF-beta1 and TGF-beta2, bind reversibly and covalently to native A2M and A2M-methylamine. It has been shown that A2M significantly inhib¬its the receptor binding and biological activity of TGF-betas (O'Connor-McCourt and Wakefield 1987). However, TGF-beta2 is more affected due to a distinctive interaction pattern with A2M compared to TGF-beta1 and other cytokines (Crookston et al. 1994). First, TGF-beta2 reveals substantially higher affinity to A2M and therefore an increased complex formation (Danielpour and Sporn 1990; Liu et al. 2001). Second, it is the only growth factor that binds with equiva¬lent affinity to both A2M and A2M-methylamine (Crookston et al. 1994). In experiments using na¬tive A2M as well as the activated form A2M-me-thylamine, TGF-beta2 shows the highest affinity to both proteins compared to other cytokines including TGF-beta1, nerve growth factor-beta (NGF-beta), platelet-derived growth factor-BB (PDGF-BB), tumor necrosis factor (TNF-alpha), and basic fibroblast growth factor (Crookston et
al. 1994).
The significance of TGF-beta has become in-creasingly evident since it obviously elicits two opposed mechanisms depending on the respec-tive environment (Akhurst and Derynck 2001; Wakefield and Roberts 2002). In normal cells of epithelial origin as well as in early well-dif-ferentiated tumor cells of epithelial origin, the TGF-beta pathway restricts cell growth, differen-tiation, and cell death. However, during progres-sion of cells toward fully malignant tumor cells, these cells undergo changes resulting in reduced expression of TGF-beta receptors, increased ex-pression of TGF-beta ligands, and resistance to growth inhibition by TGF-beta (Moustakas et al. 2002; Wakefield and Roberts 2002).
The crucial role of TGF-beta2 in pancreatic cancer progression and aggressiveness was dem-onstrated in an animal model consisting of human pancreatic cancer cells grown either ectopically in subcutaneous tissue or orthotopically in the pancreas (Choudhury et al. 2004). In this model, TGF-beta2 expression clearly correlated with tu¬mor aggressiveness and metastatic behavior. The far more aggressive orthotopic tumors not only demonstrated a larger size, shorter latent period, higher metastasis, and more extensive invasion of the stomach, but also a higher expression of TGF-beta2 compared to the less aggressive sub¬cutaneous tumors. In another study on human pancreatic tissue samples, immunohistochemi-cal analysis has shown that all three mammalian isoforms of TGF-beta (TGF-beta1, -beta2, and -beta3) were overexpressed (Friess et al. 1993). However, only the TGF-beta2 isoform was sig¬nificantly correlated with advanced tumor stage and a more aggressive phenotype. Pancreatic cancer patients bearing TGF-beta2 producing tumors showed the shortest postoperative sur¬vival period in contrast to patients with tumors producing TGF-beta1, TGF-beta3, or none of the TGF-beta isoforms (Friess et al. 1993).
16.3.1 Targeted Therapy with
the TGF-Beta2 Inhibitor AP 12009
16.3.1.1 Preclinical Experiments
In vitro experiments were performed to evalu¬ate the specificity and efficacy of the TGF-beta2 specific phosphorothioate ODN AP 12009 by employing human tumor cell cultures as well as peripheral blood mononuclear cells (PBMC) from healthy donors and from patients (Schlin-gensiepen et al. 2006).
The efficacy of AP 12009 in reducing TGF-beta2 secretion of human pancreatic carcinoma cells was determined by measuring the TGF-beta2 concentration in culture supernatants us¬ing an enzyme-linked immunosorbent assay (ELISA). Treatment with AP 12009 complexed with the liposomal carrier Lipofectin significantly inhibited TGF-beta2 production compared to Lipofectin alone in all human pancreatic cancer cell lines tested. Importantly, comparable data were obtained in experiments without Lipofectin indicating that AP 12009 alone is able to inhibit TGF-beta induced tumor-promoting effects.
Furthermore, AP 12009 was shown to revert the strong immunosuppressive effects exerted by TGF-beta2. TGF-beta has multiple immunosup-pressive properties including inhibition of T cell proliferation and inhibition of T cell differen¬tiation into cytotoxic T lymphocytes (CTLs) and
helper T cells (Gorelik and Flavell 2001). TGF-
beta inhibits these immune cell functions includ-ing cell-dependent cytotoxicity (Weller and Fon-tana 1995). Treatment with AP 12009 enhances the cytotoxic antitumor response of human lymphokine activated killer (LAK) cells directed against pancreatic carcinoma cells.
The invasion of neoplastic cells into healthy tissue is a pathologic hallmark of highly aggres-sive tumors such as pancreatic carcinoma, malig-nant melanoma, or malignant glioma.
The key mechanism for infiltration of tumor cells into healthy tissue leading to metastasis is tumor cell motility. TGF-beta, produced by tumor cells, acts directly on the tumor cells by in-ducing EMT (Janji et al. 1999), and by increasing motility, invasiveness, and metastasis (Dumont and Arteaga 2000; Oft et al. 1998). AP 12009 in-hibits the migration of cancer cells in vitro. The motility of pancreatic cancer cells was measured employing an in vitro spheroid migration model (Nygaard et al. 1998). Tumor cells spontane¬ously form round shaped clusters (spheroids) when cultured in medium on agar-coated plates, which prevents their adherence to the plastic surface. The spheroids can be transferred into culture medium without agar where the tumor cells start migrating off the spheroids. AP 12009 inhibits migration of the pancreatic tumor cells with the spheroids remaining compact after 24 h. In contrast, untreated and recombinant human
(rh-) TGF-beta2 treated cells migrate and, as a consequence, the spheroids dissolve.
Similar results as described for pancreatic can-cer cells were obtained for other cancer cells in-cluding human malignant glioma and malignant melanoma cell cultures (Jachimczak et al. 1993, 1996; Schlingensiepen et al. 2006). Importantly, all experiments were performed in the presence as well as in the absence of a liposome carrier and showed comparable efficiency to naked and Lipofectin-complexed AP 12009 in various cell lines test.
16.3.1.2 Toxicological Studies
In the current clinical trials of AP 12009 are be¬ing developed for the treatment of TGF-beta2-overproducing tumors such as malignant gli-oma, pancreatic carcinoma, metastatic colorectal carcinoma, and metastatic melanoma. Whereas AP 12009 is administered systemically by intra¬venous infusion in the indications for pancreatic carcinoma, metastatic colorectal carcinoma, and melanoma, in the case of high-grade glioma the same substance is applied locally by convection-enhanced delivery (CED) directly into the brain tumor tissue.
Local toxicity studies applying AP 12009 by the intrathecal and intracerebral routes were performed in rabbits and monkeys in order to match the intended human mode of applica¬tion in malignant glioma as close as possible. AP 12009 showed excellent local tolerability in rabbits and monkeys when administered by intra-thecal bolus injection. Neither clinical signs of toxicity nor substance-related histomorphologi-cal changes were observed. The application of AP 12009 via continuous intracerebral infusion focally resulted in a mild to moderate lympho-cytic leptomeningo-encephalitis. Changes are considered a reversible immunological reaction to AP 12009. Local tolerance tests of AP 12009 in rabbits after intravenous, intraarterial, intramus-cular, paravenous, and subcutaneous application led neither to macroscopic nor to microscopic changes.
Acute toxicology studies in mice and rats as well as subchronic toxicity studies in rats and in cynomolgus monkeys were performed employ¬ing intravenous infusion. Liver and kidney were identified as target organs. The observed changes match the common toxic effects reported for S-ODNs (Henry et al. 1997; Levin et al. 1998). Detailed methods and results were reported by Schlingensiepen et al. (2005).
The pharmacological effects of AP 12009 on the cardiovascular system, complement ac-tivation, and hematological parameters corre-sponded well to the effects reported for other phosphorothioate ODNs as a class of compounds
(Mahato 2005).
AP 12009 showed neither mutagenic effect in the Salmonella typhimurium strains nor indica¬tions of mutagenic properties in cultured human peripheral lymphocytes with respect to chro-mosomal or chromatid damage. Furthermore, AP 12009 showed no mutagenic properties in the mouse bone marrow micronucleus study us¬ing intravenous administration.
16.3.1.3 Clinical Studies: Systemic Application
In pancreatic carcinoma cells, all three mam-malian isoforms of TGF-beta (TGF-beta1, TGF-beta2, and TGF-beta3) are expressed. However, only excessive expression of TGF-beta2 is signifi¬cantly associated with pancreatic cancer progres¬sion (Friess et al. 1993).
Spurred by the clinical data in recurrent or refractory high-grade glioma patients (see Sect. 3.1.4) and the impressive antitumor ac¬tivity in a wide variety of preclinical assays (Schlingensiepen et al. 2006), the clinical stud¬ies for other solid tumors were initiated. A mul-ticenter dose-escalation phase I/II trial with AP 12009 in adult patients suffering from ad-vanced pancreatic carcinoma (AJCC stage IVA or IVB) as well as metastatic melanoma (AJCC/ UICC stage III or IV) and advanced metastatic colorectal carcinoma (AJCC/UICC stage III or IV), is currently ongoing. The primary endpoint is the assessment of the maximum tolerated dose (MTD) as well as the dose-limiting toxicities. Secondary objectives include safety and tolerabil-ity of AP 12009 and its potential antitumor activ¬ity. Adult patients (18-75 years) with advanced tumors who are not or no longer amenable to established therapies are eligible for this dose¬escalation study. Karnofsky performance status (KPS) should be at least 80%. Patients receive the study drug intravenously via an implanted port system at weekly intervals. Up to ten treatment cycles are to be applied per patient.
The majority of patients already treated re-ceived more than the minimum number of two cycles. One of them received ten full cycles. So far, AP 12009 revealed a good safety profile. The MTD has not yet been reached. Further dose es-calations are ongoing.
16.3.1.4 Clinical Studies: Local Application in High-Grade Glioma Patients
The TGF-beta2 isoform is specifically overex-pressed in malignant gliomas (Frankel et al. 1999; Maxwell et al. 1992). The increased levels of TGF-beta2 are associated with disease stage and causative for the immunodeficient state of patients (Bodmer et al. 1989; Kjellman et al.
2000; Maxwell et al. 1992).
In three phase I/II dose-escalation stud¬ies (G001, G002, and G003) a total of 24 adult patients with recurrent or refractory malig¬nant glioma, i.e., anaplastic astrocytoma (AA,
WHO grade III) or glioblastoma (GBM, WHO
grade IV), and evidence of tumor progression were treated with AP 12009 (Schlingensiepen et al. 2006). In these studies, the drug was ad-ministered intratumorally using CED over a 4- or 7-day period. The CED application allows AP 12009 to bypass the BBB. The BBB serves as a natural defense system by blocking the entry of foreign substances, including bacteria and toxins but also many therapeutic agents (Bobo et al. 1994). While conventional diffusion is characterized by a steep drop in drug concentra-tion close to the catheter tip, CED creates a ho-mogeneous drug concentration extending over several centimeters in diameter (Lieberman et al. 1995). To facilitate multiple cycles of AP 12009, the investigational drug was infused through an implanted port system connected to the intratu-moral catheter. AP 12009 proved to be well toler¬ated and revealed a good safety profile. Since two complete remissions in two different dose groups were observed (see below), further dose escala¬tion was not necessary. MTD was not reached.
Although the clinical phase I/II trials were pri-marily designed to assess safety, survival times as well as tumor response data were obtained. Data on antitumor activity from 24 patients included several patients with stabilization of disease and two patients with complete tumor remission, both of them long-lasting without recurrence. One of these two patients, diagnosed with AA, was treated with only one course of AP 12009. At baseline four tumor lesions had been detected, which were spread over both hemispheres. Only one lesion had been infused with one cycle of AP 12009, but all lesions had disappeared several months after start of treatment despite an ini¬tial and temporary increase in tumor volume at the beginning of the treatment. The patient died from a myocardial infarction without any signs of tumor, 25 months after start of AP 12009 treat¬ment. The second patient, also diagnosed with AA, received a total of 12 cycles of AP 12009 over the course of the three phase I/II studies (G001,
G002, and G003; Fig. 16.2).
Prior to AP 12009 treatment, he had been treated with surgery, radiation, and chemo-therapy [temozolomide (TMZ) after the first re-lapse], followed by a second incomplete surgery. After an initial stabilization following the second cycle, the enhancing lesion continued to increase until 10 months after baseline G001 (Fig. 16.2b), inducing a significant edema. The central read¬ing of the magnetic resonance image (MRI) 20 months after the start of AP 12009 treatment (in G001) was evaluated as partial response (PR, 83% tumor reduction, Fig. 16.2c); there was com-plete response after 22 months. The patient is known to still be alive today; the MRI in August 2006 (Fig. 16.2d) showed no recurrence. Survival of this patient after the first recurrence is now 307 weeks (71 months); it has been 286 weeks (66 months) since treatment with AP 12009 be¬gan (status 01 August 2007).
As of 01 August 2007 the median overall sur-vival after recurrence for AA patients treated with AP 12009 was 146.6 weeks (range 32.0¬306.6 weeks), and for GBM patients treated with AP 12009 44.0 weeks (range 18.9-87.9 weeks). The most recent and accurate survival data after start of therapy that clearly distinguish between recurrent AA and GBM are available for the current gold standard treatment TMZ. The re-
ported median overall survival for TMZ alone is 42.0 weeks (9.7 months) for recurrent AA (The-odosopoulos et al. 2001), and 31.8 (7.3 months) (Yung 2000; Yung et al. 2000) or 32.0 weeks (7.4 months) (Theodosopoulos et al. 2001) for recurrent GBM. These results were reported for patients with high-grade glioma who received TMZ as first treatment after recurrence. In the adjuvant treatment of newly diagnosed glioma, the combination of TMZ with radiotherapy has improved median overall survival from 12.1 to 14.6 months (Stupp et al. 2005).
The phase IIb clinical trial of AP 12009-G004 is an international, open-label, active-controlled dose-finding study in high-grade glioma pa¬tients. The main trial objective is the compari¬son of two different doses of AP 12009 (10 uM or 80 uM) against standard chemotherapy. In all, 145 patients with either recurrent or re¬fractory AA (WHO grade III) or GBM (WHO
grade IV) are receiving either one of the two doses of AP 12009 or standard chemotherapy [TMZ or procarbazine/CCNU (lomustine)/ vincristine = PCV, if TMZ was already given].
AP 12009 is applied intratumorally by CED dur-ing a 6-month active treatment period at weekly intervals. The primary efficacy endpoint is tumor response after radiological evaluation. The main secondary efficacy endpoints are overall survival and 12-month survival. As in the previous stud-ies, preliminary data show long-lasting responses both in recurrent or refractory AA and GBM
patients (Bogdahn et al. 2006; Hau et al. 2006).
Especially in recurrent or refractory AA patients, very promising efficacy data have been docu-mented compared to current standard treatment
with TMZ or PCV.
16.4 Summary
Despite tremendous advances in cancer research and the development of new therapies, patients with malignant tumors such as advanced pancre-atic carcinoma, metastatic melanoma, metastatic colorectal carcinoma, and malignant glioma still face a poor prognosis. The severe morbidity and mortality of these malignant tumor types makes the identification of factors associated with their incidence an important area of both preclinical and clinical research. Antisense technology is a new and innovative method offering a causal approach for the treatment of various highly ag-gressive diseases. Antisense compounds inhibit the production of disease-causing proteins at the molecular level and combat tumor development directly at its roots. Preclinical experiments us¬ing the TGF-beta2 specific phosphorothioate
ODN AP 12009 revealed the potential of this
compound to reverse TGF-beta2 induced immu-nosuppression as well as inhibition of tumor cell proliferation and tumor cell migration. Initial clinical studies have demonstrated AP 12009 to be well tolerated and safe. Furthermore, the first evidence of efficacy of AP 12009 antisense ther¬apy in recurrent or refractory high-grade glioma has been provided.
These data confirm that the blockade of TGF-beta2, a key factor in tumorigenesis, in tumor tissue by AP 12009 represents a novel and promising therapeutic approach for malignant tumors such as advanced pancreatic carcinoma and malignant glioma. This approach aims at a reduction of tumor-promoting effects and, most importantly, an enhancement of the antitumor immune response.
