Current Pharmaceutical Design - Volume 15, Issue 23, 2009

Immunotoxins are anticancer agents with at least two functional components, one of which binds to a tumor-specific, internalizing cell-surface antigen and one that confers cellular toxicity. The two components may be joined covalently in vitro by chemical conjugation or they may be expressed as a fusion protein generated by recombinant DNA technology. The binding moiety is usually a monoclonal antibody derivative or a cytokine, ultimately of mammalian origin, whereas the cytotoxic moiety is an enzyme that may originate from any source, including plants and bacteria as well as mammals. After selective binding to a tumor-specific receptor, immunotoxins are internalized and translocated into the cytosol. The cytotoxic component then induces cell death through its catalytic activity. Beyond the first recombinant immunotoxins to undergo clinical studies were based on Pseudomonas exotoxin A, a format pioneered by Pastan and colleagues. One of the major challenges with this format was its immunogenicity - treated patients produced neutralizing antibodies against the toxin, reducing the effectiveness of multiple treatment cycles. This special issue of Current Pharmaceutical Design considers recent developments in immunotoxin technology that aim to overcome this difficulty by incorporating modified or novel cytotoxic components into immunotoxins. The new generation of recombinant immunotoxins includes molecules that incorporate cytotoxic human enzymes, or non-human enzymes with reduced immunogenicity. In the first review, RJ Kreitman [1] summarizes results from recent clinical studies with immunotoxins based on Pseudomonas exotoxin A. These studies involved immunotoxins targeting CD25 (LMB-2) and CD22 (BL22 and HA22) in patients with hematologic malignancies, and showed promising results against a range of leukemias and lymphomas. The review considers recent strategies to reduce the immunogenicity of the bacterial toxin, e.g. by removing immunodominant epitopes or by suppressing the patient's immune system nonspecifically. Several research groups have investigated the possibility of replacing immunogenic bacterial toxins with human enzymes, to generate fully human immunotoxins. Rybak et al. [2] were the first to consider this approach, using human ribonucleases (RNases) as the cytotoxic component. Their review discusses the most recent developments in the field of immunoRNase fusion proteins, focusing on the results of pre-clinical studies featuring immunoRNases targeting CD22 and CD30.

Recombinant immunotoxins are proteins composed of fragments of monoclonal antibodies fused to truncated protein toxins. No agents of this class are approved yet for medical use, although a related molecule, denileukin diftitox, composed of interleukin-2 fused to truncated diphtheria toxin, is approved for relapsed/refractory cutaneous T-cell lymphoma. Recombinant immunotoxins which have been tested in patients with chemotherapy-pretreated hematologic malignancies include LMB-2 (anti-CD25), BL22 (CAT-3888, anti-CD22) and HA22 (CAT-8015, anti-CD22), each containing an Fv fragment fused to truncated Pseudomonas exotoxin. Major responses were observed with LMB-2 in adult T-cell leukemia, chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma, Hodgkin's disease, and hairy cell leukemia (HCL). BL22 resulted in a high complete remission rate in patients with HCL, particularly those without excessive tumor burden. HA22, an improved version of BL22 with higher affinity to CD22, is now undergoing phase I testing in HCL, CLL, non-Hodgkin's lymphoma, and pediatric acute lymphoblastic leukemia.

Ribonucleases degrade RNA, now considered an important drug target. The parent member of this protein superfamily is bovine pancreatic RNase A that functions as a digestive enzyme. Other physiological roles and activities have been ascribed to more recently discovered members of this superfamily. Angiogenin was isolated by following angiogenic activity from cell culture media conditioned by colon cancer cells. ONCONASE kills tumor cells in vitro and in vivo and has advanced to a phase IIIb confirmatory clinical trial for the treatment of unresectable malignant mesothelioma. All three of these RNA degrading enzymes have been used to generate immunoRNases; chemical conjugates and ligand- RNase fusion proteins, for cancer therapy. The properties of each of these RNases are described along with the increasingly sophisticated construction of recombinant immunoRNases. The advantages of using RNase as an antibody payload is compared to using plant or bacterial toxins in the construction of immunotoxins, a related strategy for specifically killing malignant cells.

Recombinant fusion proteins are an expanding, important class of novel therapeutic agents. The designs of these constructs typically involve a cell-targeting motif genetically fused to a highly toxic class of enzymes capable of ruthlessly attacking critical cellular machinery once delivered successfully to the cytoplasm of the target cell. Initial development of this class of constructs typically contained recombinant growth factors or single-chain antibodies as the celltargeting motif fused to highly cytotoxic plant or bacterial toxins. This review describes second-generation molecules composed of cell-targeting molecules fused to highly cytotoxic human enzymes capable of generating intense apoptotic response once delivered to the cytoplasm. The human serine protease granzyme B has been shown to be extremely effective as a cytotoxic molecule when incorporated into numerous cell-targeting constructs. The biological activity of GrBcontaining constructs rivals that of plant or bacterial toxins and appears to represent a new generation and class of completely human proteins with unique biological activities.

Certain characteristics of tumor cells make it possible to develop rational strategies for targeting tumors without harming normal cells. These include the presence of cell surface molecules that characterize the current state of the tumor (e.g. CD30 on Hodgkin lymphoma cells) and the genetic and epigenetic changes that activate oncogenes and inactivate tumor suppressor genes (e.g. the inactivation of tumor suppressor gene DAPK2 in Hodgkin lymphoma cells, which blocks apoptosis). We have developed a novel tumor-targeting fusion protein by combining a selective ligand (CD30L) with a constitutively active version of DAPK2 (DAPK2'-CD30L), thus increasing tumor specificity and reducing systemic toxicity. We showed that this immunokinase fusion protein induces apoptosis specifically in CD30+/DAPK2- tumor cells in vitro and significantly prolonged overall survival in a disseminated Hodgkin lymphoma xenograft SCID mouse model. Therapeutic strategies based on the cell-specific restoration of a defective, tumor-suppressing kinase demonstrate the feasibility of targeted therapy using recombinant immunokinases

Immunotoxins (ITs) are protein-based drugs combining a target-specific binding domain (usually derived from an antibody) and a cytotoxic domain to kill target cells. They are among the most promising new therapeutic tools to fight cancer, and several clinical trials have been completed with encouraging results. Although the targeted elimination of malignant cells is an elegant concept, there are numerous practical challenges that limit the clinical use of ITs, including inefficient cellular uptake, low cytotoxicity and off-target effects. Here we present some of the strategies that have been developed to improve the efficacy of ITs, particularly those involving the incorporation of functional peptide sequences into recombinant ITs to improve target binding, modify plasma half life and distribution, boost tumor penetration, enhance cellular uptake and increase cytotoxic efficiency.

Immunotoxins are powerful tools to specifically eliminate deviated cells. Due to the side effects of the original immunotoxins, they were only considered for the treatment of cancer as in these cases, the potential favourable effect outweighed the unwanted toxic side effects. Over time, many improvements in the construction of immunotoxins have been implemented that circumvent, or at least strongly diminish, the side effects. In consequence this opens the way to employ these immunotoxins for the treatment of non-life threatening diseases. One such category of disease could be the many chronic inflammatory disorders in which an uncontrolled interaction between inflammatory cells leads to chronicity. In several of these chronic conditions, activated macrophages, which are characterised by an increased expression of CD64, are known to play a key role. In this review we discus the data presently available on elimination of activated macrophages through CD64 immunotoxins in several animal models for chronic disease. A chemically linked complete antibody with the plant toxin Ricin-A, proved very effective and provided proof of concept. Subsequently, the development towards genetically engineered, fully human, multivalent single chain based immunotoxins that have diminished immunogenicity, is discussed. The data show that the specific elimination of activated macrophages through CD64 is indeed beneficial for the course of disease. As opposed to other methods used to inactivate or eliminate macrophages, with the CD64 based immunotoxins only the activated population is killed. This may open the way to apply these immunotoxins as therapeutics in chronic inflammatory disease.

Targeted delivery of cytotoxic agents limits the severe toxic side-effects of anti-cancer drugs on healthy tissues. Annexin A5 is a well explored probe to target phosphatidylserine (PS)-expressing cells in vivo. Our novel understanding of the cellular and molecular mechanism of annexin A5 as a cell-entry agent and the finding that PS is expressed on living tumour as well as endothelial cells in the tumour vasculature, will allow the development of lead compounds for anticancer therapy.

Controlled drug delivery to eye is one of the most challenging fields of pharmaceutical research. Low drugcontact time and poor ocular bioavailability due to drainage of solution, tear turnover and its dilution or lacrimation are the problems associated with conventional systems. In addition, anatomical barriers and physiological conditions of eye are also important parameters which control designing of drug delivery systems. Nanosized carriers like micro/nanosuspensions, liposome, niosome, dendrimer, nanoparticles, ocular inserts, implants, hydrogels and prodrug approaches have been developed for this purpose. These novel systems offer manifold advantages over conventional systems as they increase the efficiency of drug delivery by improving the release profile and also reduce drug toxicity. Conventional delivery systems get diluted with tear, washed away through the lacrimal gland and usually require administering at regular time intervals whereas nanocarriers release drug at constant rate for a prolonged period of time and thus enhance its absorption and site specific delivery. This review presents an overview of the various aspects of the ocular drug delivery, with special emphasis on nanocarrier based strategies, including structure of eye, its barriers, delivery routes and the challenges/ limitations associated with development of novel nanocarriers. The recent progresses in therapy of ocular disease like gene therapy have also been included so that future options should also be considered from the delivery point of view. Recent progress in the delivery of proteins and peptides via ocular route has also been incorporated for reader benefit.

The aetiology of obesity is multi-factorial. Recent research has identified a novel association between endotoxaemia (circulating lipopolysaccharide in the systemic circulation) and low-grade inflammation in the adipose organ, which may contribute to obesity. The mechanisms for the low-grade elevation of circulating lipopolysaccharide in obesity are poorly understood. Vitamin D has been increasingly recognised for its pleiotropic actions beyond maintenance of musculoskeletal health. The parathyroid-vitamin D axis is altered in obesity. Circulating vitamin D levels are lower in obese individuals. The regulatory role of vitamin D in the immune system and colonic mucosa may explain the under-appreciated contribution of vitamin D deficiency in the obese to the pathogenesis of endotoxaemia and adipose inflammation. We propose a hypothetical model linking metabolic endotoxaemia with vitamin D deficiency in obesity. A therapeutic approach involving the use of probiotics and vitamin D metabolites in the obese is described.