Diphtheria toxin-based targeted toxins that target glioblastoma multiforme

Abstract

Targeted toxins (TTs) are compounds that bind cell surface receptors overexpressed on cancer cells. After the molecule enters the cell, the toxin component migrates to the ribosomes where it blocks protein synthesis killing the cell. Most of these proteins are recombinant and contain a carrier ligand or antibody attached to a modified bacterial toxin such as diphtheria toxin (DT). With respect to cancer, these fusion proteins are active against brain tumor cells that are resistant to chemotherapy and radiation therapy. The toxicity profile for TTs is acceptable and they have been safe in animal studies and demonstrated a therapeutic response in early clinical trials. Those barriers to the successful treatment of brain tumors include poor tumor penetration, immune recognition of DT and the heterogeneity of cancer.     

Immunotoxins

Immunotoxins are molecules which contain a protein toxin connected to a targeting antibody. The goal of therapy is for the molecule to bind selectively to cancer cells, or to cells mediating autoimmune disease, internalise and then for the toxin to kill the cell. Several immunotoxins meeting this definition are in preclinical and clinical development, but none are approved yet for use in general practice. One close relative of immunotoxins is the growth factor fusion toxin, wherein the targeting antibody is replaced with a growth factor that selectively binds and this ligand is fused in a recombinant fashion to a protein toxin. One such molecule, containing human interleukin-2 (IL-2) fused to truncated diphtheria toxin (DT), has recently been approved under the name Ontak, and others are under development. A newer class of immunotoxins, termed recombinant immunotoxins, contain the variable or antigen binding domains of an antibody fused in recombinant fashion to a toxin. Recombinant immunotoxins, like growth factor fusion toxins, can be produced efficiently from bacteria and have a defined structure with respect to the linkage between the toxin and the ligand. However, they can, like conventional immunotoxins, be directed to antigens other than growth factor receptors, including receptor subunits. Several recombinant immunotoxins are under clinical testing and major responses have been reported, particularly in haematological malignancies. Some of these molecules may enter clinical practice in the future as targeted therapy, which is a modality distinct from those of chemotherapy, surgery and radiation therapy.     

Diphtheria fusion protein therapy of chemoresistant malignancies

Patients with widespread cancer respond initially to combination chemotherapy, immunotherapy, and/or radiotherapy, but most relapse with chemoresistant disease. Novel methods of killing resistant neoplastic stem cells are needed. One such approach is therapy with targeted toxins composed of tumor cell selective ligands covalently linked to group I peptide toxins (group II and III peptide toxins act on the cell surface). The targeted toxin is delivered to the cell by a tumor selective ligand. Once bound, the ligand-receptor complex is internalized. The catalytic domain escapes to the cytosol. The toxin then enzymatically modifies a critical cell function (protein synthesis, p21 Rho activity, protein kinase signaling, cyclic AMP signaling or others). The irreversibly damaged cells fails to divide and, eventually, undergoes lysis or programmed cell death. Targeted peptide toxins used to date in the treatment of chemotherapy refractory cancers include ricin toxin, Pseudomonas exotoxin, pokeweed antiviral protein, saporin, gelonin and diphtheria toxin. In this review, we have focused on the applications of genetically engineered diphtheria toxin for cancer therapy.     

Immunotoxins for targeted cancer therapy

Immunotoxins constitute a new modality for the treatment of cancer, since they target cells displaying specific surface-receptors or antigens. Immunotoxins contain a ligand such as a growth factor, monoclonal antibody, or fragment of an antibody which is connected to a protein toxin. After the ligand subunit binds to the surface of the target cell, the molecule internalizes and the toxin kills the cell. Bacterial toxins which have been targeted to cancer cells include Pseudomonas exotoxin and diphtheria toxin, which are well suited to forming recombinant single-chain or double-chain fusion toxins. Plant toxins include ricin, abrin, pokeweed antiviral protein, saporin and gelonin, and have generally been connected to ligands by disulfide-bond chemistry. Immunotoxins have been produced to target hematologic malignancies and solid tumors via a wide variety of growth factor receptors and antigens. Challenges facing the clinical application of immunotoxins are discussed.     

Immunotoxins

Immunotoxins are molecules which contain a protein toxin connected to a targeting antibody. The goal of therapy is for the molecule to bind selectively to cancer cells, or to cells mediating autoimmune disease, internalise and then for the toxin to kill the cell. Several immunotoxins meeting this definition are in preclinical and clinical development, but none are approved yet for use in general practice. One close relative of immunotoxins is the growth factor fusion toxin, wherein the targeting antibody is replaced with a growth factor that selectively binds and this ligand is fused in a recombinant fashion to a protein toxin. One such molecule, containing human interleukin-2 (IL-2) fused to truncated diphtheria toxin (DT), has recently been approved under the name Ontak, and others are under development. A newer class of immunotoxins, termed recombinant immunotoxins, contain the variable or antigen binding domains of an antibody fused in recombinant fashion to a toxin. Recombinant immunotoxins, like growth factor fusion toxins, can be produced efficiently from bacteria and have a defined structure with respect to the linkage between the toxin and the ligand. However, they can, like conventional immunotoxins, be directed to antigens other than growth factor receptors, including receptor subunits. Several recombinant immunotoxins are under clinical testing and major responses have been reported, particularly in haematological malignancies. Some of these molecules may enter clinical practice in the future as targeted therapy, which is a modality distinct from those of chemotherapy, surgery and radiation therapy.     

Recombinant toxins in haematologic malignancies and solid tumours

Recombinant toxins constitute a new modality for the treatment of cancer, since they target cells displaying specific surface-receptors or antigens. They are fusion proteins, which contain toxin and ligand regions, and are produced in Escherichia coli. The ligand may be a growth factor or a fragment of an antibody, and the toxin is usually one of the two bacterial toxins: Pseudomonas exotoxin and diphtheria toxin. Compared to the earlier generation chemical conjugates of ligands and toxins, recombinant toxins have many advantages, including homogeneity with respect to the connection between the ligand and toxin, ease and yield of production and small size. A variety of chemotherapy-resistant haematologic and solid tumours have been targeted with recombinant toxins, and clinical trials with many of them have recently demonstrated their effectiveness. Moreover, their unwanted toxic effects are different from those of most chemotherapeutic agents, supporting the expectation that they can be combined with existing modalities to improve the clinical resources available to treat cancer in humans.     

Recombinant antibody fragments and immunotoxin fusions for cancer therapy

Recombinant immunotoxins consist of Fv regions of cancer specific antibodies fused to truncated bacterial toxins. Many recombinant immunotoxins contain a truncated version of Pseudomonas Exotoxin as a toxic moiety. This toxin is modified in such a manner that by itself it does not bind to normal human cells, but it retains all other functions of cytotoxicity. The recombinant antibody fragments target the modified toxin to cancer cells which are killed, either by direct inhibition of protein synthesis, or by concomitant induction of apoptosis. Cells that are not recognized by the antibody fragment, because they do not carry the cancer antigen, are spared. Many factors influence the in vivo anti-tumor activity of recombinant immunotoxins. Among them are considerations of which types of cancer and at what stages may be the best targets for immunotoxin therapy and tumor specificity of the antigen that is targeted by the recombinant antibody. Also the affinity of immunotoxins and their ability to enter and penetrate into tissues and tumors, which in turn is dependent on the size of the protein. And, because one very important factor is the stability of immunotoxins, a great deal of protein-engineering is required to stabilize the recombinant antibody moiety of immunotoxins. Excellent activity and specificity can be observed for many recombinant immunotoxins in in vitro assays using cultured cancer cells as well as in animal tumor models. Ongoing clinical trials provide examples where the promising preclinical data correlate with successful results in experimental cancer therapy.     

Targeted drug delivery via the folate receptor

The folate receptor is a highly selective tumor marker overexpressed in greater than 90% of ovarian carcinomas. Two general strategies have been developed for the targeted delivery of drugs to folate receptor-positive tumor cells: by coupling to a monoclonal antibody against the receptor and by coupling to a high affinity ligand, folic acid. First, antibodies against the folate receptor, including their fragments and derivatives, have been evaluated for tumor imaging and immunotherapy clinically and have shown significant targeting efficacy in ovarian cancer patients. Folic acid, a high affinity ligand of the folate receptor, retains its receptor binding properties when derivatized via its gamma-carboxyl. Folate conjugation, therefore, presents an alternative method of targeting the folate receptor. This second strategy has been successfully applied in vitro for the receptor-specific delivery of protein toxins, anti-T-cell receptor antibodies, interleukin-2, chemotherapy agents, gamma-emitting radiopharmaceuticals, magnetic resonance imaging contrast agents, liposomal drug carriers, and gene transfer vectors. Low molecular weight radiopharmaceuticals based on folate conjugates showed much more favorable pharmacokinetic properties than radiolabeled antibodies and greater tumor selectivity in folate receptor-positive animal tumor models. The small size, convenient availability, simple conjugation chemistry, and presumed lack of immunogenicity of folic acid make it an ideal ligand for targeted delivery to tumors.     

Effective tumor targeting: strategies for the delivery of Armed Antibodies

It has long been realized that the presence of tumor-associated antigens offers an excellent opportunity for targeted cancer therapy and hence an improved clinical benefit for cancer patients. Advances in the field of antibody engineering as well as the characterization of toxins, such as diphtheria toxin and Pseudomonas exotoxin A, have enabled the routine construction of recombinant immunotoxins, which we have termed Armed Antibodies. The selective toxicity and mechanism of action of these molecules could potentially provide an excellent clinical alternative to conventional anticancer agents which have many unacceptable side effects. Although considerable clinical success has been achieved using immunotoxin therapy, particularly for B-cell malignancies, the treatment of solid tumors remains highly challenging. To successfully treat solid tumors that are not amenable to local therapy, immunotoxins must be designed to permit repeat systemic administration. This review outlines some of the strategies currently being employed in the design of the Viventia Biotech Inc Armed Antibodies to minimize the development of immunogenicity and to remove the potential for toxicity in non-target tissues.     

Immunotoxins for targeted cancer therapy

Immunotoxins are proteins that contain a toxin along with an antibody or growth factor that binds specifically to target cells. Nearly all protein toxins work by enzymatically inhibiting protein synthesis. For the immunotoxin to work, it must bind to and be internalized by the target cells, and the enzymatic fragment of the toxin must translocate to the cytosol. Once in the cytosol, 1 molecule is capable of killing a cell, making immunotoxins some of the most potent killing agents. Various plant and bacterial toxins have been genetically fused or chemically conjugated to ligands that bind to cancer cells. Among the most active clinically are those that bind to hematologic tumors. At present, only 1 agent, which contains human interleukin-2 and truncated diphtheria toxin, is approved for use in cutaneous T-cell lymphoma. Another, containing an anti-CD22 Fv and truncated Pseudomonas exotoxin, has induced complete remissions in a high proportion of cases of hairy-cell leukemia. Refinement of existing immunotoxins and development of new immunotoxins are underway to improve the treatment of cancer.     

Antibody engineering for targeted therapy of cancer: recombinant Fv-immunotoxins

Recombinant Fv-immunotoxins are a new class of biologic anticancer agents composed of a recombinant antibody fragment linked to a very potent bacterial toxin. These potent molecules are designed to specifically bind and kill cancer cells that express a specific target antigen on their cell surface. Recombinant Fv-immunotoxins are an excellent example for the concept of rational drug design. They combine the progress in understanding cancer biology, -the recent knowledge on the mechanisms of malignant transformation and the special properties of cancer cells, -with the enormous developments in recombinant DNA technology and antibody engineering. Recombinant Fv immunotoxins were developed for solid tumors and hematological malignancies and have been characterized intensively for their biological activity in vitro and in vivo in animal models. The excellent in vitro and in vivo activities of recombinant Fv-immunotoxins have lead to their pre-clinical development and to the initiation of clinical trial protocols. Recent trials have demonstrated potent clinical efficacy in patients with malignant diseases that are refractory to traditional modalities of cancer treatment. It is thus suggested that this strategy can be developed into a separate modality of cancer treatment with the basic rationale of specifically targeting cancer cells on the basis of their unique surface markers combined with potent effective biological toxic agents that directly kill the cancer cell. Efforts are now being made to improve the current molecules and to develop new agents with better clinical efficacy. In this review, we will describe the rationale in designing Fv-immunotoxins and will review current progress made in using these agents for cancer treatment.     

Pseudomonas Exotoxin A Based Toxins Targeting Epidermal Growth Factor Receptor for the Treatment of Prostate Cancer

The epidermal growth factor receptor (EGFR) was found to be a valuable target on prostate cancer (PCa) cells. However, EGFR inhibitors mostly failed in clinical studies with patients suffering from PCa. We therefore tested the targeted toxins EGF-PE40 and EGF-PE24mut consisting of the natural ligand EGF as binding domain and PE40, the natural toxin domain of Pseudomonas Exotoxin A, or PE24mut, the de-immunized variant thereof, as toxin domains. Both targeted toxins were expressed in the periplasm of E.coli and evoked an inhibition of protein biosynthesis in EGFR-expressing PCa cells. Concentration- and time-dependent killing of PCa cells was found with IC₅₀ values after 48 and 72 h in the low nanomolar or picomolar range based on the induction of apoptosis. EGF-PE24mut was found to be about 11- to 120-fold less toxic than EGF-PE40. Both targeted toxins were more than 600 to 140,000-fold more cytotoxic than the EGFR inhibitor erlotinib. Due to their high and specific cytotoxicity, the EGF-based targeted toxins EGF-PE40 and EGF-PE24mut represent promising candidates for the future treatment of PCa.     

Novel approaches for targeted cancer therapy

The clinical use of chemotherapeutic agents against malignant tumors is successful in many cases but suffers from major drawbacks. One drawback is lack of selectivity, which leads to severe side effects and limited efficacy; and another is the emergence/selection of drug-resistance. To limit non-specific toxicity and to improve the efficiency of cancer therapy, "tumor markers", which are proteins generally overexpressed on the surface of tumor cells, can be selectively targeted. Growth factor receptors are one of the most extensively studied tumor markers. The implication of growth factor receptors in the pathogenesis and evolution of cancer has clearly been established and therefore, provides a rationale for therapeutic intervention. The targeting of cytotoxic substances to tumor markers with "magic bullets" is an old idea that raised high expectations but also disappointment. Over the past decade, newly gained understanding of mechanisms for targeted therapy have brought new hopes. Pharmacological agents that selectively target and block the action of growth factors and their receptors have been attempted, such as monoclonal antibodies (mAbs) (whole molecule or fragments), bispecific antibodies, mAbs conjugated to drugs, toxins or radioisotopes, small peptidic and peptidomimetic molecules in free form or conjugated to drugs, anti-sense oligonucleotides, immunoliposomes-encapsulated drugs, and small molecule inhibitors. This review will focus on current developments of selective targeting and bypassing drug resistance in the management of growth factor receptor-overexpressing tumors.     

Trastuzumab,a humanized anti-HER2 monoclonal antibody,for the treatment of breast cancer

The HER2/neu gene encodes a 185 kDa transmembrane receptor (HER2) that belongs to the epidermal growth factor receptor family and has intrinsic tyrosine kinase activity. HER2 is overexpressed in 25-30% of breast cancers and is suggested to have a direct role in the pathogenesis and clinical aggressiveness of HER2 overexpressing tumors. A murine monoclonal antibody, 4D5, directed against the extracellular domain of HER2, is a potent inhibitor of growth of human breast cancer cells overexpressing HER2 in vitro and in xenograft models. To facilitate clinical investigation, 4D5 was humanized by inserting the complementary determining regions of 4D5 into the framework of a consensus human IgG1. The resulting recombinant humanized anti-HER2 MAb, trastuzumab, was found to inhibit the growth of human cancer cells and tumor xenografts overexpressing HER2. Data from phase II trials in women with breast cancer whose tumors overexpress HER2 have shown that trastuzumab has a favorable toxicity profile, is active as a single agent and induces long-lasting objective tumor responses. In combination studies, there was no evidence that trastuzumab enhanced the toxicity of cisplatin and the pharmacokinetic parameters of trastuzumab were unaltered by coadministration of cisplatin. Furthermore, clinical response rates were higher than those reported with either agent alone in a similar patient population. Results of a multicenter, phase III clinical trial of chemotherapy (doxorubicin- or paclitaxel-based) plus trastuzumab as compared to chemotherapy alone in patients with advanced breast cancers overexpressing HER2 showed a significant enhancement in the effects of chemotherapy on time to disease progression, response rates and survival with coadministration of trastuzumab, without increases in overall severe adverse events. Myocardial dysfunction syndrome, similar to that observed with anthracyclines, was reported more commonly with chemotherapy plus trastuzumab. Positive results from clinical studies led to the approval of trastuzumab in the U.S in October 1998 for the treatment of metastatic breast cancer in patients with tumors overexpressing HER2. Since then, the MAb has also been marketed in Switzerland and Canada.     

Bacteria and bacterial toxins as therapeutic agents for solid tumors

Patients with advanced solid tumors frequently relapse and succumb to their metastatic disease after developing resistance to conventional treatment modalities such as chemotherapy and radiotherapy. In these patients, novel strategies of targeting widespread tumors are urgently needed. The increasing knowledge of the underlying pathogenetic mechanisms has led to the identification of numerous molecules that are overexpressed in various tumors and accumulate at the cell surface. The use of genetically modified bacteria and their toxins targeting these surface molecules has emerged as a promising new treatment strategy in refractory cancers. This review focuses on bacterial toxins such as Diphtheria toxin (DT), Pseudomonas exotoxin A (PE) and Clostridium perfringens enterotoxin (CPE). In addition, the use of anaerobic bacteria such as Clostridium, Salmonella and Bifidobacterium spp. as drug-delivery systems targeting hypoxic tumor areas will be discussed as a new therapeutic modality of advanced solid tumors.     

Immunotoxins: The Role of the Toxin ?

Immunotoxins are antibody-toxin bifunctional molecules that rely on intracellular toxin action to kill target cells. Target specificity is determined via the binding attributes of the chosen antibody. Mostly, but not exclusively, immunotoxins are purpose-built to kill cancer cells as part of novel treatment approaches. Other applications for immunotoxins include immune regulation and the treatment of viral or parasitic diseases. Here we discuss the utility of protein toxins, of both bacterial and plant origin, joined to antibodies for targeting cancer cells. Finally, while clinical goals are focused on the development of novel cancer treatments, much has been learned about toxin action and intracellular pathways. Thus toxins are considered both medicines for treating human disease and probes of cellular function.     

Immunoconjugates: Applications in Targeted Drug Delivery for Cancer Therapy

Monoclonal antibodies can be produced against virtually any molecule, and unlike polyclonal anti-sera, they are highly specific. There has been great improvement in the monoclonal antibody production technique since its inception in 1975. The idea behind using monoclonals to direct cancer treatments is based on the fact that surfaces of tumor contain a wide variety of proteins, some of which are specific to the tumor type. Monoclonal antibodies that bind to such tumor-specific antigens could be used, either alone or as conjugates of drugs and toxins (immunoconjugates), to selectively seek out and destroy these tumor cells. Targeted drug delivery therapy of tumor using monoclonals or their conjugates has been reported by many investigators, and the early results are quite promising. However, many obstacles still have to be overcome before immunoconjugates become a valuable agent in the treatment of human diseases including cancer.     

Receptor-targeted gene delivery viafolate-conjugated polyethylenimine

A novel synthetic gene transfer vector was evaluated for tumor cell-specific targeted gene delivery. The folate receptor is a tumor marker overexpressed in more than 90% of ovarian carcinomas and large percentages of other human tumors. Folic acid is a high affinity ligand for the folate receptor that retains its binding affinity upon derivatization via its gamma carboxyl. Folate conjugation, therefore, presents a potential strategy for tumor-selective targeted gene delivery. In the current study, we investigated a series of folate conjugates of the cationic polymer polyethylenimine (PEI) for potential use in gene delivery. A plasmid containing a luciferase reporter gene (pCMV-Luc) and the folate receptor expressing human oral cancer KB cells were used to monitor gene transfer efficiency in vitro. Transfection activity of polyplexes containing unmodified polyethylenimine was highly dependent on the positive to negative charge (or the N/P) ratio. Folate directly attached to PEI did not significantly alter the transfection activity of its DNA complexes compared to unmodified PEI. Modification of PEI by polyethyleneglycol (PEG) led to a partial inhibition of gene delivery compared to unmodified PEI. Attaching folates to the distal termini of PEG-modified PEI greatly enhanced the transfection activity of the corresponding DNA complexes over the polyplexes containing PEG-modified PEI. The enhancements were observed at all N/P ratios tested and could be blocked partially by co-incubation with 200 μM free folic acid, which suggested the involvement of folate receptor in gene transfer. Targeted vectors based on the folate-PEG-PEI conjugate are potentially useful as simple tumor-specific vehicles of therapeutic genes.     

Therapeutic potential of anticancer immunotoxins

Immunotoxins are chimeric proteins consisting of a tumor-specific ligand (antibody, growth factor or peptide) linked to a modified toxin. These molecules bind to cell surface receptors and are subsequently internalized by endocytosis, resulting in cell death. Advances in protein engineering and phage display have enabled the selection of high-affinity targeting moieties. Denileukin diftitox is the only FDA-approved immunotoxin, although others such as BL22 are currently in different phases of development. This review elaborates the key findings of the important clinical studies relating to various chimeric toxins.     

Small molecule toxins targeting tumor receptors

Targeting toxic therapeutics to tumors through receptors over expressed on the surface of cancer cells can reduce systemic toxicity and increase the effectiveness of the targeted compounds. Small molecule targeted therapeutics have a number of advantages over toxic immunoconjugates including better tumor penetration, lack of neutralizing host immune response and superior flexibility in selection of drug components with optimal specificity, potency and stability in circulation. Three major components of the targeted drug, the toxic warhead, tumor-specific ligand and the linker can influence the properties of each other and thus have to be optimized for each system. All receptor-targeted drugs are delivered inside the cells through endocytosis and undergo processing liberating the toxins in endosomes and lysosomes. Common delivery route defines a number of general requirements for each drug component. The review addresses currently known possible receptor targets and their ligands along with toxins that have been used and that have a potential to be successfully applied in tumor targeting. Linkers that are stable in circulation, but efficiently cleaved in lysosomes constitute an essential component of receptor-targeted drugs and are evaluated in greater detail.