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Review Advances in targeted therapeutic agents Nicholas C Nicolaides†, Philip M Sass & Luigi Grasso Morphotek, Inc., Exton, PA, USA 1. Overview of the targeted therapeutic landscape 2. Classes of TTAs 3. Nucleic acid TTAs 4. Target identification and Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Eisai Inc on 02/18/11 For personal use only. selection 5. TTAs in development 6. Expert opinion Importance of the field: The number of disease-associated protein targets has significantly increased over the past decade due to advances in molecular and cellular biology technologies, human genetic mapping efforts and information gathered from the human genome project. The identification of gene products that appear to be involved in supporting the underlying cause of disease has offered the biopharmaceutical industry an opportunity to develop compounds that can specifically target these molecules to improve therapeutic responses and lower the risk of unwanted side effects that are commonly seen in traditional small chemical-based medicines. Areas covered in this review: An overview of targeted drug therapies is presented in this review. We include a review of the various classes of targeted therapeutic agents, the types of disease-associated molecules being targeted by these agents and the challenges currently being encountered for the successful development of these various platforms for the treatment of disease. What the reader will gain: An understanding of the current targeted therapy landscape, the discovery and selection of disease-specific gene products that are being targeted, and an overview of targeted therapies in preclinical and clinical studies. A description of the various targeted therapeutic platforms, target selection criteria and examples of each are discussed in order to provide the reader with the current status of the field and emerging areas of targeted therapy discovery and development. Take home message: Novel medications are in demand for the treatment of serious medical conditions including cancer, autoimmune, infectious and metabolic diseases. Targeted therapies offer a way to develop very specific treatments for serious medical conditions while concomitantly resulting in little to no off-target toxicity. Targeted therapies provide an opportunity to develop personalized medicines with superior treatment modalities for the patient and a better quality of life. Keywords: antibodies, antibody fragments, antisense oligodeoxynucleotides, cell surface receptors, fusion proteins, genetic pathways, genetics, genomics, immunotoxins, protein design, protein--drug conjugates, proteomics, RNAi, targeted therapies Expert Opin. Drug Discov. (2010) 5(11):1123-1140 1. Overview of the targeted therapeutic landscape Targeted therapy is generally considered as the use of a compound that can specifically bind a disease-associated gene product and results in the suppression or activation of its biological activity thereby altering the disease state. In this broad definition, targeted therapeutic agents (TTAs) may include small chemical entities (SCEs), peptides, recombinant proteins, antibodies, antibody fragments and nucleic acid-based agents. Targeted SCEs are mostly comprised of compounds that can block enzymes, ion channels, cell surface proteins and transmembrane receptors. Peptides and recombinant proteins include molecules that can target soluble proteins, cell surface proteins/receptors and non-protein antigens. Antibodies and 10.1517/17460441.2010.521496 © 2010 Informa UK, Ltd. ISSN 1746-0441 All rights reserved: reproduction in whole or in part not permitted 1123 Advances in targeted therapeutic agents Article highlights. . . . . Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Eisai Inc on 02/18/11 For personal use only. . . . Targeted therapies offer opportunity to generate person-specific medicine; an overview of this area of drug development is presented. Targeted therapeutic agents include mAbs, antibody fragments, bifunctional antibodies and recombinant protein receptors; an overview of these various agents is provided. Protein immunotoxins have improved the therapeutic activity of recombinant protein therapies. Protein--chemotoxin conjugates provide another alternative to improve the therapeutic benefit of target-specific proteins. Pathogenic targeting proteins offer a means of discovering disease-specific targets and serve as possible targeting agents; examples of this class are provided. Nucleic acid targeting molecules represent one of the most promising target-specific agents for therapy. Disease-specific targets are the key for maximizing the potential of targeted therapeutic agents; methods for identifying and validating targets are discussed. This box summarizes key points contained in the article. antibody fragments are molecules that can specifically bind to distinct regions (epitopes) on a protein or non-protein-based molecule (antigen) and perturb its activity. Antibody-based therapeutics contain immunoglobulin-type structures with a protein backbone comprising of a variable antigen-binding domain that provides target specificity and, in the case of full length antibodies, a constant region (Fc) that can engage with immune cells and/or immunomodulatory factors to elicit immune-effector activity as a means to kill target cells. Nucleic acid-based TTAs encompass short DNA or RNA strands consisting of complementary nucleic acids that can form double-stranded complexes with specific sequences encoded by an mRNA within the cell that in turn results in degradation of steady-state message and suppression of the encoded protein. Nucleic acid agents include antisense deoxyribonucleic acids and inhibitory RNA (RNAi), miRNA and siRNA. Experimental and clinical studies have demonstrated the ability of several SCEs to be capable of targeting a biological molecule based on its chemical structure; however, off-target effects of the parent compound or metabolites have typically resulted in side effects due to binding of homologous family member proteins or non-related proteins, respectively, whose function is critical for normal organ function and not involved in the disease. Examples of these include the broad spectrum tyrosine kinase inhibitors sorafinib and sunitinib as well as the bcr-abl kinase inhibitor imatinib, which have all demonstrated clinical activity and suppression of a focused target kinase but have subsequently been shown to suppress the biological activity of a larger class of kinase proteins [1,2]. Because of the inability of SCEs and/or their metabolites to strictly bind one target, here we focus on TTAs that elicit their 1124 pharmacological activity via the ability to provide a more stringent binding to a single target molecule while completely avoiding metabolite-mediated side effects because of their degradation into ubiquitous biological subunits (i.e., amino acids or nucleic acids). This class of targeting agent includes mAbs, antibody fragments, recombinant fusion proteins, peptides and nucleic acid fragments. Despite the complexity of manufacturing, antibody and non-antibody protein-based therapeutics remain as some of the most successful drugs in the marketplace. A representative sampling of therapeutic proteins, soluble receptor and recombinant fusion proteins is shown in Tables 1 and 2, respectively, which covers the diverse array of disease indications for which this class of therapeutic proteins are approved. The development and application of proteinbased TTAs have significantly advanced since insulin, the first recombinant protein, was successfully manufactured [3]. Therapeutic proteins continue to represent a high potential for treating disease as numerous novel platform technologies have been developed that are able to improve their expression titers, purification, stability and overall manufacturing in addition to their pharmacokinetic and pharmacodynamic profiles. Furthermore, the use of protein-based TTAs to deliver small chemical compounds via targeting of cell surface proteins that are exclusively expressed by diseaseassociated cells have expanded their pharmaceutical potential by combining traditional SCE drug development expertise with a target-specific delivery system [4]. The development of conjugate-based targeting agents has especially created a great deal of interest in oncology by the use of antibodies, antibody fragments and fusion proteins to deliver toxic payloads to malignant tissues. This approach offers the opportunity of delivering cytotoxic compounds to tumors while avoiding toxic side effects due to suppression of targets involved in pathways important for normal tissue function [5]. The development of compounds that can specifically inhibit the translation of mRNA encoding for a diseaseassociated protein provides one of the most powerful applications of targeted therapy. Because these sequence-specific compounds are capable of blocking the synthesis of specific proteins whose function is needed to promote and/or maintain the disease state, mRNA targeting compounds offer the potential for better efficacy and less toxicity in treating disease [6]. In light of these benefits, many years of research have focused on refining the development and clinical application of antisense oligodeoxynucleotide (AS)-based therapies and the more recent RNAi platforms, siRNA and miRNA. Despite the many advances achieved in enhancing the utility of this platform, several major hurdles still remain for these agents to be able to specifically deliver intact nucleic-acid compounds to disease tissues and elicit robust, long-term suppression of a disease-specific gene message for clinical benefit. A review of these platforms and efforts to improve their therapeutic use are provided in the sections below. Expert Opin. Drug Discov. (2010) 5(11) Nicolaides, Sass & Grasso Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Eisai Inc on 02/18/11 For personal use only. Table 1. Approved recombinant proteins. Protein class Molecule (indication) Trade name (company) Recombinant erythropoeitin Erythropoietin (anemia) Recombinant insulin Insulin (diabetes) Recombinant HGH HGH (growth disorders) Recombinant ILs IL-2* IL-11z GM-CSF (neutrophilsz) G-CSF (neutrophilsz) Epogen (Amgen) Procrit/eprex (J&J) Aranesp (Amgen) Epogin (Roche) Espo (Kirin) Dynepo (Shire) Novolog (Novo Nordisk) Humalog (Eli Lilly) Apidra (Sanofi-Aventis) Humulin (Eli Lilly) Lantus (Sanofi-Aventis) Levemir (Novo Nordisk) Genotropin (Pfizer) Nutropin (Roche) Norditropin (Novo Nordisk) Humatrope (Eli Lilly) Saizen (MerckSerono) Serostim (MerckSerono) Omnitrope (Sandoz) Valtropin (BioPartners) Proleukin (Prometheus) Neumega (Pfizer) Leukine (Bayer) Neupogen (Amgen) GRAN (Kirin) Neutrogin (Roche) Avonex (BiogenIDEC) Rebif (MerckSerono) Betaferon (BayerShering) IntronA (MerckShering Plough) Roferon (Roche) Pegasys (Roche) Actimmune (Intermune) NovoSeven (Novo Nordisk) ReFacto (Pfizer Wyeth) Recombinate (Baxter) Advate (Baxter) Kogenate (Bayer) Helixate (CSL Behring) BeneFIX (PfizerWyeth) Recothrom (Zymogenetics) Activase (Roche) Actilyse (Boehringer Ingelheim) Abbokinase (Abbot Labs) Rapilysin/retavase (EKR Therapeutics) TNKase (Roche) Metalyse (Boehringer Ingelheim) Streptase (ZLB Behring) Recombinant CSFs Recombinant INFs Recombinant blood clotting factors Recombinant plasminogen activators INF-b1b§ INF-a2b{ INF-a2a# INF-g 1b** Factor VIIazz Factor VIII§§ Factor IX{{ Thrombinzz Alteplase## Urokinase*** Reteplase## Tenecteplase## Streptokinase## *Metastatic melanoma and renal cell carcinoma. z To enhance production of these cells after chemotherapy. § Multiple sclerosis. { Melanoma. # Hepatitises B and C. **Chronic granulomatous disease and severe, malignant osteopetrosis. zz To aid in hemostasis. §§ Control of hemorrhagic episodes in hemophilia A. {{ Control of hemorrhagic episodes in hemophilia B. ## Thrombolytic agent for treatment of myocardial infarction/stroke. ***Pulmonary embolism. HGH: Human growth hormone. Expert Opin. Drug Discov. (2010) 5(11) 1125 Advances in targeted therapeutic agents Table 2. Approved fusion and soluble receptor recombinant proteins. Generic name Molecule (indication) Trade name (company) Etanercept Abatacept Soluble TNF-a receptor (treatment of inflammatory disease) CTLA4--IgG fusion protein (treatment rheumatoid arthritis) Alefacept Rilonacept LFA3--IgG1 Fc fusion (treatment of severe chronic plaque psoriasis) Soluble IL-1 receptor--IgG1 Fc fusion (treatment of cryopyrin-associated periodic syndrome) Enbrel (Amgen) Orencia (Bristol-Myers Squibb) Amevive (Astellas) Arcalyst (Regeneron) 2. Classes of TTAs mAbs, antibody fragments, bifunctional antibodies and recombinant protein receptors Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Eisai Inc on 02/18/11 For personal use only. 2.1 A number of recombinant proteins have been developed over the years that can specifically suppress or replace the biological activity/deficiency of an endogenous protein responsible for promoting and/or maintaining human disease. A representation of the various classes of these proteins for the broad variety of disease indications they are used for is provided in Tables 1 and 2. For the purposes of this review, we focus on TTAs that can specifically bind and antagonize a soluble factor, a cell surface protein or an mRNA target within a disease-associated cell as well as provide an overview of the types of TTAs that can specifically deliver cytotoxic compounds via disease-specific cell surface proteins. This last class of TTAs includes mAbs, antibody fragments, peptides, recombinant fusion proteins, protein--SCE cytotoxin conjugates and nucleic acid-based therapies. mAbs Over the past decade, the use of therapeutic mAbs has demonstrated significant success in specifically targeting and suppressing the activity of proteins modulating diseaseassociated pathways [7]. mAbs have been shown to elicit their pharmacological activity by a number of mechanisms. They can bind and sequester soluble factors, thereby suppressing the ability of these factors to interact with cell surface receptors or proteins that in turn activate biological pathways within the cell. They can bind to pathogenic agents and remove them from the patients’ system via clearance mechanism. mAbs can also bind to cell surface proteins of disease cells and recruit immunological molecules such as complement or immune cells (macrophages, neutrophils or NK cells) to specifically kill the mAb-bound cell via complementdependent cytotoxicity (CDC) or antibody dependent cytotoxicity, respectively. These mechanisms of action are referred to as immune-effector function. There have been 30 mAbs approved for treating a wide array of diseases including cancer, hematological and bone disorders as well as inflammatory and infectious diseases. Table 3 provides an overview of these mAbs and their approved indications. In addition to these compounds, > 300 antibodies are in various stages of development for a wide range of potential indications. 2.1.1 1126 A description of a subset of these development-stage antibodies being pursued across the different disease indications is included in Table 4. While canonical full length IgG-type mAbs have been the predominate antibody isotypes to achieve clinical and regulatory approval, the pursuit of antibody therapies using IgM isotype and antibody fragment-based TTAs has increased as these platforms have matured and offer additional therapeutic benefits. For example, the ability to deliver robust target binding, specificity, unique epitope recognition, enhanced tissue penetration and/or high efficiency immuneeffector activity in addition to the improved technologies that support their scaleable manufacturing have all made IgM and antibody fragment-based TTAs a reasonable alternative to IgG-based therapies. While it is generally understood that IgMs have the ability to effectively kill antigen expressing cells via the highly potent CDC mechanism, their broad pursuit as pharmaceutical products has been limited due to their inherent low-binding affinities as well as the technical difficulties in scaleable cGMP manufacturing due to their large molecular mass (> 750,000 kDa) [8,9]. Recent studies and new technologies have enabled the advancement of IgM development in light of the recognition that an IgM’s low affinity-binding is offset by its high avidity, which is enabled by the presence of 10 (pentameric IgM) or 12 (hexameric IgM) antigen-binding domains, thereby providing good targeting and disease tissue retention. In addition, new manufacturing technologies have yielded cell-based systems producing high titers of IgM under scaleable GMP manufacturing conditions in quantities comparable to IgGs as well as new analytical methods that have advanced the characterization of these large biomolecular structures for regulatory documentation requirements [10]. Due to these major advancements and breakthroughs in technology and with future success in clinical trials, the pursuit of IgMs as bona fide TTAs, particularly for oncology indications, may become more accepted by the biopharmaceutical industry [11]. Antibody fragments Antibody fragment-based TTAs are molecules that are engineered to contain the antigen-binding site of an antibody within a small scaffold thereby enabling the molecule to retain target specificity as well as high-binding affinity. These platforms, called fabs (fragment antigen-binding region containing an antibody V region without the Fc domain), 2.1.2 Expert Opin. Drug Discov. (2010) 5(11) Nicolaides, Sass & Grasso Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Eisai Inc on 02/18/11 For personal use only. Table 3. Approved monoclonal antibodies. Target First approved indication Generic name (manufacturer) Anthrax toxin a4 Integrin CD11a CD20 Anthrax inhalation MS Psoriasis B-cell lymphoma CD3 CD3 and EPCAM CD33 CD52 Complement Ca5 EGFR Chronic lymphocytic leukemia Organ transplant rejection Ovarian cancer ascites Acute myeloid leukemia B-cell leukemia Paroxysmal nocturnal hemoglobinuria Colorectal cancer GPIIb/IIIa HER2 IgE IL-12 and IL-23 IL-1b IL-2Ra Squamous head and neck cancer Ischemia Breast cancer Atopic asthma Psoriasis Cryopyrin-associated periodic syndrome Organ transplant rejection RANK ligand RSV-F protein TNF-a Osteoporosis RSV infections Rheumatoid arthritis VEGF Crohn’s disease Colorectal cancer Macular degeneration Raxibacumab (Human Genome Science) Natalizumab (BiogenIDEC) Efalizumab (Genetech) Rituximab (Roche/BiogenIDEC) Ibritumomab (Celldex) Tositumomab (GSK) Ofatumumab (GSK/Genmab) Muromonab-CD3 (J&J) Catumaxomab (Trion) Gemtuzumab (Pfizer) Alemtuzumab (Genzyme) Eculizumab (Alexion) Cetuximab (Eli Lilly) Panitumumab (Amgen) Nimotuzumab (Biocon) Abciximab (J&J/Eli Lilly) Trastuzumab (Roche) Omalizumab (Roche) Ustekinumab (J&J) Canakinumab (Novartis) Daclizumab (Roche/Sanofi-Aventis) Basiliximab (Novartis) Denosumab (Amgen) Palivizumab (AstraZeneca) Infliximab (J&J) Adalimumab (Abott/Eisai) Golimumab (J&J) Certoluzimab (UCB) Bevacizumab (Roche) Ranibizumab (Roche) RANK: Receptor activator of NF-kB; RSV: Respiratory syncytial virus. scFVs (a single chain fab), domain antibodies, diabodies or non-immunoglobulin protein domains that can be structurally altered to have antigen-specific binding, offer flexibility in their use to develop small antigen-specific therapeuticbinding proteins or as conjugates linked to other scaffolds to improve bio-distribution and/or pharmacology [12,13]. Several types of antibody fragment technologies exist. These include single domain antibodies derived from human variable heavy chain or variable light chain domains (i.e., scFVs, dAbs) as well as naturally occurring single domain antibodies that are derived from shark or camelidae immune systems. An advanced camelidae single domain antibody platform called Nanobody technology has been applied broadly to develop a number of therapeutic antibodies to a wide array of disease antigens based on the need of obtaining good target penetration, which was not optimal using canonical tetrameric mammalian-derived IgG type antibodies. These single chain antibodies lack a light chain that is normally found within most mammalian-derived antibodies yet retain the ability to get high affinity and target specificity through a specific spatial arrangement of the antigen-binding domain regions on a single heavy chain backbone [14]. Preclinical experiments have demonstrated the ability of nanobodies to bind to therapeutic targets in regions not as accessible using IgG/ IgM type mAbs offering the ability to develop a broader library of targeted agents depending on the need for maximal target suppression. Finally, a more distinct antibody-like fragment platform that utilizes the ability of protein-binding domains contained within host proteins such as fibronectin (FN), T-cell receptors, ankyrins, and ‘A-domain’ (Figure 1), has been manipulated to develop libraries of peptides with altered amino-acid residues within their protein-binding domains to generate target-specific fragments for therapeutic use [15]. One such platform called adnectins has been developed that takes advantage of the inherent promiscuous protein-binding properties that FN-binding domains contain. Experimental data have shown that adnectins are capable of being engineered to specifically target binding to a broad number of antigens by altering amino-acid residues within or flanking the FN-binding domain. This feature enables these molecules to act in a manner similar to an antibody fragment, concomitant with a low molecular mass. This combination enables the use of these compounds to target disease-associated proteins by tailoring the FN-binding domain to bind to certain epitopes on a target antigen by in vitro engineering, which is not possible using other antibody-based platforms that require Expert Opin. Drug Discov. (2010) 5(11) 1127 Advances in targeted therapeutic agents Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Eisai Inc on 02/18/11 For personal use only. Table 4. Therapeutic mAbs under development. Generic name Antibody target Lead indication Adecatumumab Amatuximab Belimumab Bertilimumab Blinatumomab Briakinumab Canakinumab Catumaxomab CDX-011 ADC Cixutumumab Conatumumab CT-011 EB10 Elotuzumab Farletuzumab Figitumumab Galiximab Ipilimumab J591 (111In) Lexatumumab Lucatumumab Mapatumumab MEDI-547 MNRP1685A MORAb-004 MORAb-028 Ofatumumab Olaratumab Pritumumab Robatumumab Siltuximab Telimomab aritox Trastuzumab TRC105 Tremelimumab EPCAM Mesothelin LymphostatB Eotaxin 1 CD19/CD3 IL-12 and IL-23 IL1-b Epcam/CD3 GPNMB IGFR1 Trail R2 PD1 receptor FLT3 CS1 glycoprotein Folate receptor a IGFR1 CD80 CTLA-4 PSMA Trail R2 CD40 Trail R1 EphA2 Neuropilin-1 Endosialin GD2 CD20 Anti-PDGFR a Vimentin IGFR1 IL-6 CD5 HER2 CD105 CTLA4 Prostate cancer Mesothelioma Lupus Allergic disorders Lymphoma Psoriasis COPD Ovarian cancer Melanoma Islet cell carcinoma Breast cancer Follicular lymphoma AML Multiple myeloma Ovarian cancer NSCLC Hodgkin’s lymphoma Melanoma Prostate cancer Pediatric Hodgkin’s lymphoma Non-Hodgkin’s Multiple cancers Multiple cancers Multiple cancers Melanoma Follicular lymphoma Ovarian cancer Brain cancer Ovarian cancer Castleman disease Graft rejection Breast cancer Prostate cancer Bladder cancer AML: Acute myeloid leukemia; COPD: Chronic obstructive pulmonary disease. immune recognition and precursor backbones derived from the host’s immune response [16]. Figure 1 reviews and provides a schematic diagram of the various antibody and antibody fragment technologies. Bifunctional antibodies In addition to the ability to generate smaller targetspecific binding proteins that can offer different advantages over canonical IgG and IgM type mAbs, another use of antibody fragment-based TTAs is the ability to engineer them as a bifunctional antibody capable of binding to a disease-associated target and a receptor on immune cells to facilitate immune-mediated cell killing. Research has found that antitumor responses and overall survival are highly correlated with the degree of inflammatory cells infiltrated into tumors [17]. Hosts containing tumors that are infiltrated with high numbers of T cells have been shown to have a longer overall survival and greater antitumor effects than those lacking T-cell infiltration [18]. To enhance T-cell recruitment 2.1.3 1128 to tumors, an antigen-binding domain from one of the various platforms described above that can recognize a diseaseassociated protein is assembled with an antibody domain that can bind to a T-cell receptor to form a bifunctional antibody that can bind target cells and recruit T cells via direct T-cell receptor binding. Bifunctional antibodies can contain or lack an Fc domain depending on whether macrophage recruitment is desired. Several bifunctional antibodies have been developed using this platform. The most advanced is catumaxomab, an Fc containing bifunctional antibody that recognizes the EpCam cell surface protein on tumor cells and the CD3 receptor on T cells. This antibody has shown the ability to suppress the growth of ascites in ovarian cancer patients and is currently approved in Europe for treating ascites in patients with ovarian cancer [19]. Another bifunctional mAb is blinatumomab, an antibody being developed for the treatment of leukemia and lymphoma. This TTA is a nonFc containing bifunctional mAb that binds CD19 antigen on malignant cells and CD3 receptor on T cells. Clinical studies have shown it to have robust therapeutic activity in cancers that do not respond to standard treatment. This class of TTA adds yet an additional opportunity to leverage targetspecific binding molecules and the use of the host’s immune system to fight against diseased tissues. Antibody fragment-based TTAs represent one of the most contemporary fields of research and development that can complement traditional IgG and IgM mAb platforms to improve on therapeutic efficacy of targeting antigens of new and approved therapeutic antibodies. While antibody fragments have certain limitations such as short serum halflife and the inability to deliver immune-effector activity by the natural mechanism, they have unique attributes when compared to standard full length mAbs. Because of their small molecular size, these antibodies offer some advantages over standard antibodies including: i) the ability to penetrate tissue and have access to epitopes that may not be accessible by larger mAbs due to steric hindrance; ii) the ability to obtain better tissue penetration than the larger IgG/IgM-based mAbs; iii) a shorter serum half-life than full length antibodies that depending on the preferential means of treatment maybe more advantageous and iv) competitive cost of goods due to manufacturing using less expensive prokaryotic-based fermentation systems which are still more cost-effective despite the vast improvement observed in mammalian cell-based biological manufacturing. While these features offer significant advantages, the ultimate fate of these agents as valid therapeutics will depend on their ability to deliver clinically meaningful outcomes in pivotal clinical trials. Recombinant protein receptors Recombinant protein receptors that target soluble factors or cell surface proteins have been shown in clinical studies to be effective in treating a variety of diseases. This class of molecules consists of a fusion between the ligand-binding domain of a receptor and the constant region (Fc) of the 2.1.4 Expert Opin. Drug Discov. (2010) 5(11) VH /V L Nicolaides, Sass & Grasso Mo CL no y od iab me r D CH1–3 IgG IgM H Vh A- do bo o an ma in dy Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Eisai Inc on 02/18/11 For personal use only. N Camelid Ig Non-Ig domain Figure 1. Top left: IgG is a hetero-tetrameric protein formed by two light chains (light green and red) and two heavy chains (dark green and red). CL region; CH1-3, heavy chain constant domains. The VL and the VH regions pair up to form the antigen-binding domain. In a ‘diabody’ (top middle), a VL/VH domain specific for one ligand (green) is assembled with a VL/ VH domain specific for another ligand (orange). Top right: a pentameric IgM is composed of five IgG-like structures (monomer) linked together by disulfide bonds. A J-chain (not shown here) may be present to stabilize this multimer. Bottom left: A camelid antibody is a homodimeric protein. Unlike an IgG, the camelid constant region (red) contains only two domains, while the variable region (VhH, green) is represented by a single antigen-binding domain. If separated from the rest of the molecule, a VhH domain forms a ‘nanobody’. Bottom right: An ‘A-domain’ contains ~ 35 amino acids including 6 conserved cysteine residues forming disulfide bonds (S-S). They occur naturally in some human receptors, including lowdensity lipoprotein-related proteins and are known to form various tandem repeat combinations binding > 100 different natural ligands. A-domain shuffling technology allows deriving non-immunoglobulin domain molecules with new ligand-binding specificities. CL: Light chain constant; VH: Heavy chain variable; VL: Light chain variable. immunoglobulin IgG1 protein which provides protein stability, improved systemic delivery and an anchor for purification during manufacturing [20]. One of the most commercially successful recombinant protein receptors to date is etanercept (Table 2), a fusion protein containing a ligand-binding domain from the TNF-a receptor2 (TNFR) soluble receptor fused to the Fc immunoglobulin region [21]. Etanercept exerts its therapeutic action by sequestering the soluble TNF-a inflammatory-associated cytokine, which in turn suppresses membrane-associated TNFR activity in inflammatory cells and the inflammatory cascade underlying a number of diseases, including rheumatoid arthritis (RA) and inflammatory bowel disease. Etanercept has been shown in numerous clinical trials to be efficacious and has the ability to suppress the aberrant immune cascade associated with inflammatory disease and in turn improve patient health [22]. Other recombinant fusion receptor-based TTAs include abatacept, alefacept, aflibercept and rilonacept (Table 2). Abatacept is approved for use in the treatment of RA in patients nonresponsive to disease-modifying antirheumatic drugs [23]. It is a fusion protein consisting of the high affinity binding site for the B7 co-stimulatory receptor expressed by antigen presenting cells (APCs) and the Fc immunoglobulin domain. In host immune response, APCs interact with T cells via antigenic presentation of processed proteins and interaction of the B7 and CD28 co-receptors expressed by the APC and T cells, respectively. Abatacept’s mechanism of action is to block the ability of the CD28 protein to bind the B7 receptor on APCs, which in turn suppresses immune cascades that underlie several inflammatory disorders including RA. Alefacept is a fusion protein consisting of the lymphocyte function-associated antigen 3 fused to the Fc immunoglobulin domain. It has been approved for treatment of patients with moderate to severe chronic plaque psoriasis and functions by suppressing the activation of T cells via binding to the CD2 T-cell surface protein thereby blocking the ability of T cells to interact with co-stimulatory receptors on APCs [24]. Aflibercept and rilonacept are fusion proteins containing cytokine receptor-binding domains for the VEGF and IL-1 cytokines, respectively, fused to the Fc domain. Aflibercept is being tested in Phase III clinical studies for the treatment of neovascular diseases while rilonacept is approved for treating a subset of rare genetic autoimmune disorders [25,26]. Receptor fusion proteins have been proven to be very effective in specifically blocking the biological activity of growth stimulatory/activation molecules. While a subset of this class of TTAs have been mentioned here, there are several others in development that are Expert Opin. Drug Discov. (2010) 5(11) 1129 Advances in targeted therapeutic agents specific for blocking ligands associated with a variety of diseases (Table 5). Protein immunotoxins Another recombinant protein-based platform incorporates the antigen-binding domain of an antibody or a cell surface ligand fused to a cytotoxic protein (toxin) that when internalized via a cell surface receptor/protein is able to specifically kill the host cell. An example of immunotoxin is denileukin diftitox, a recombinant protein consisting of the IL-2 cytokine fused to the diphtheria toxin (DT) fragments A and B. On binding to the IL-2 receptor on T cells, the compound is internalized and kills host cells by DT’s ability to block protein translation via binding to the elongation factor eEF-2. The drug is approved for treatment of cutaneous T-cell lymphoma and represents a class of toxin fusion proteins that have demonstrated robust therapeutic activity in treating cancer [27]. A number of immunotoxins are currently being pursued in clinical trials to treat cancer, inflammatory and infectious diseases (Table 6). Preclinical and clinical results have shown promise in the ability of these TTAs to be active in effectively eradicating disease-promoting cells whereby non-toxin containing fragments were less effective. An example of an immunotoxin fusion protein is the SS1-P molecule that contains a mouse IgG1 derived antigen-binding fragment that is specific for the mesothelin tumor-associated antigen fused to the Pseudomonas exotoxin PE38. Preclinical and clinical trials have shown the ability of SS1-P to specifically bind and kill tumor cells expressing mesothelin [28]. While early stage clinical studies have shown patient benefit, the broad use of this agent has been limited by the highly immunogenic nature of PE38 toxin in mammalian hosts thereby restricting repeated administration. Efforts are being pursued to generate less- or non-immunogenic PE38 toxin as well as other pathogens that can be potentially used for chronically treated diseases [29]. Other attempts at developing immunotoxins include the use of antibody fragments that recognize GP120 expression of HIV infected cells fused to either ricin or PE38 toxins, and the BL22 antibody-immunotoxin that targets CD22 and contains the PE38 toxin [30,31]. Clinical studies of patients with relapsed/refractory hairy cell leukemia have shown that single agent BL22 is able to elicit a 47% complete response and a 25% partial response. A second generation BL22 is currently being developed with the goal of creating an anti-CD22-immunotoxin fusion that has a higher antigen-binding affinity to improve on efficacy and target cell specificity. Immunotoxins are able to leverage the use of a target-specific binding domain and a highly potent toxin to selectively kill disease-associated cells while avoiding toxicity in cells not expressing the antigen. In light of the positive preclinical and early-stage clinical results, interest in pursuing immunotoxin therapeutic agents is increasing; however, hurdles still exist for the efficient development of this class. Immunotoxins are highly toxic once internalized into a cell; hence, developing molecules to cell surface antigens that are Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Eisai Inc on 02/18/11 For personal use only. 2.2 1130 expressed primarily on disease tissue is critical to avoid toxicities of normal cells. Another developmental challenge for this class is the ability to develop an immunotoxin that can deliver highly toxic effects to target cells but is not recognized by the host’s immune system after long-term repeat dosing to avoid drug neutralization. The refinement of these areas will undoubtedly enable the expansion of this class of targeted agent across various disease indications. Protein--drug conjugates Antibody and non-antibody protein-based TTAs have demonstrated pharmacological activity across a wide spectrum of diseases; however, in most part, their effects in cancer and in a subset of other disease types have been less than ideal for delivering long-term clinical benefits to patients. One area being actively pursued to improve TTA pharmacological activity is the use of proteins (antibody and non-antibody) conjugated to chemotoxins or radionuclides (Table 7). These efforts aim to deliver highly cytotoxic chemical-based agents or radioactive isotopes (radionuclides) to disease cells via disease-associated cell surface antigens [32,33]. Technologies that enable robust linkage of targeting proteins to cytotoxins such as radionuclides, chemotherapeutic SCEs and nucleic acid-based targeting agents have led to the establishment of a variety of novel therapeutic approaches. The protein carriers themselves have varied from full length antibodies to recombinant proteins and small polypeptides while different linkage chemistries coupling cytotoxin to the protein carrier have been implemented depending on where in the tissue it is most desirable to have the cytotoxin liberated from the carrier, if at all. The success of antibody conjugates using radionuclides, such as 90Y-labeled-ibritumomab tiuxetan and 131 I-labeled tositumomab, in treating refractory lymphoma as well as the success of the chemotoxin-antibody conjugate gemtuzumab ozogamicin support the concept of using a targeting agent coupled to a cytotoxin to treat cancers and other diseases in cases where nontoxin-conjugated biologicals have little therapeutic activity [34-36]. In the case of the two radionuclide conjugated mAbs mentioned above, both antibodies target the CD20 antigen, which is expressed on B-cell lymphomas, and both were approved for use in rituximab-refractory lymphoma. Rituximab is a chimeric mouse-human IgG1 mAb directed to CD20 and approved for treatment of B-cell lymphomas. Clinical studies using these molecules showed a statistical improvement in patients treated with these conjugates as compared to rituximab or chemotherapy alone. While both are approved, their use in clinical practice is limited due to the complexity of handling radioisotope-labeled antibody before and after patient treatment. These limitations have fostered the generation of alternate molecules, including nonradioactive cytotoxins such as calichaemycin, the cytotoxic molecule contained in the gemtuzumab ozogamicin antibody, as well as others that can be effectively conjugated to an antibody, protein or peptide targeting agent without affecting the pharmacokinetic or 2.3 Expert Opin. Drug Discov. (2010) 5(11) Nicolaides, Sass & Grasso Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Eisai Inc on 02/18/11 For personal use only. Table 5. Recombinant receptor fusion proteins under development. Compound name Ligand-binding domain Disease indication Target Ref. ActRIIA-IgG1 ActRIIB Aflibercept ALK1-Fc APG101 Atacicept Belatacept Briobacept FP1039 sEGFR501.F YSPSL Type II activin receptor IIA Type II activin receptor IIB VEGFR Activin like receptor CD95 receptor TACI receptor CTLA-4 BAFF receptor FGFR EGFR PSGL-1 Anemia Neuro-muscular disease Angiogenesis cancer Angiogenesis cancer Glioblastoma multiforme Lymphoma Graft rejection Rheumatoid arthritis Cancer Cancer Delayed graft function Activin Myostatin VEGF GDF CD95 ligand BLyS CD80 and CD86 BAFF ligand FGF1, FGF2, FGF4 EGF P-selectin [91] [92] [25] [93] [94] [95] [96] [97] [98] [99] [100] Table 6. Immunotoxins under development. Immunotoxin name Target Disease indication Toxin Ref. Anti-B4-blocked ricin IL13PE H22ETA scFv35-ETA Affitoxin VB6-845 FR betaPE 26292(Fv)-PE38 SS1P BL22 CD19-ETA scFv(MUC1)-ETA CD19 IL13Ra2 CD64 Acetylcholine receptor g HER2 EpCAM Folate receptor b CD123/IL3Ra Mesothelin CD22 CD19 MUC1 Lymphocytic leukemia Pancreatic cancer Arthritis Rhabdo-myosarcoma HER2+ cancers Various cancers Inflammation Acute myeloid leukemia Lung cancer Hairy cell leukemia Lymphocytic leukemia Breast cancer Ricin PE PE PE PE Bouganin PE PE PE PE PE PE [101] [102] [103] [104] [105] [106] [107,108] [109] [28] [31] [110] [111] PE: Pseudomonas exotoxin. Table 7. Protein--chemotoxin/radionuclide conjugates. Compound name Target Disease indication Chemotoxin/radionuclide Ref. Trastuzumab-DM1 Lorvotuzumab mertansine PTX:C225 BT062 J591-111In MAb 81C6 SGN-35 SGN-75 131 I-TM601 HER2 CD56 EGFR CD138 PSMA Tenascin C CD30 CD70 Annexin A2 Breast cancer Myeloma Various cancers Myeloma Prostate cancer Glioblastoma Hodgkin’s lymphoma Non-Hodgkin’s lymphoma Glioma Maytansinoid Maytansinoid Paclitaxel Maytansinoid Various isotopes 111 Iodide Monomethyl auristatin E Monomethyl auristatin F 131 Iodide [39-41] [112] [113] [114] [115] [116] [43] [117] [53] pharmacodynamic properties of the carrier protein (i.e., diminished ability to maximally access its target expressed by the disease tissue). In addition to the cytotoxic agents used in the commercially approved protein conjugates described above, two chemotoxins that are being broadly pursued by biopharmaceutical companies developing protein conjugate TTAs are the maytansine and auristatin microtubule disrupting agents [37,38]. These molecules have both shown broad cytotoxic activity on mammalian cells; however, when conjugated to a protein carrier these compounds lose their cytotoxic activities which is regained on liberation of intact cytotoxin. The increased use of these agents has been swayed by the refinement of linker chemistries that provide greater stability of the cytotoxin--protein conjugate in serum and effective intracellular cytotoxin--antibody dissociation by cleavage of Expert Opin. Drug Discov. (2010) 5(11) 1131 Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Eisai Inc on 02/18/11 For personal use only. Advances in targeted therapeutic agents the linker in the highly reducing environment of the cell or by intracellular enzymes, depending on the type of linker used. While several antibody conjugates using these cytotoxins are in various stages of clinical development (a few examples are listed in Table 7), two of the most advanced therapeutic conjugates include trastuzumab-DM1 (T-DM1) and brentuximab vedotin (SGN35). T-DM1 is an antibody conjugate comprised of the anti-HER2 antibody, trastuzumab, linked to the maytansine derivative DM1 via a modified disulfide linker that is resistant to reduction in serum [39]. The conjugate has been tested in a single arm Phase II clinical trial in female patients with HER2-positive advanced breast cancer whose disease has progressed after trastuzumab treatment, and an average of six other chemotherapeutic regimens including anthracycline, taxane and capecitabine. Results of the study showed that approximately a third of patients treated with T-DM1 had tumor regressions with limited toxicities [40]. Based on the positive outcome of this Phase II study, the drug has recently been filed for marketing approval with the US FDA while additional Phase II and larger Phase III clinical studies are being pursued [41]. SGN35 is an antibody conjugate consisting of a mAb that targets the cell surface antigen CD30, which is overexpressed by Hodgkin’s lymphoma cells and a subset of other hematologic cancers, linked to an auristatin microtubule inhibitor. The linkage of this conjugate occurs via a cathepsin protease sensitive substrate [42]. In the low pH intracellular environment, cathepsin is a highly active protease that can cleave the conjugate linker and produce a liberated active cytotoxic agent. This cathepsinsensitive linker is highly stable in the extracellular serum environment because of the presence of protease inhibitors and high pH which significantly reduces cathepsin’s proteolytic activity. In a SGN35 Phase I open-labeled study in patients with relapsed or refractory Hodgkin’s lymphoma, 54% of patients achieved an objective response, 39% of whom exhibited a complete response. Clinical studies using the non-conjugated anti-CD30 antibody had no objective responders, again demonstrating the ability of protein--cytotoxin conjugates to increase therapeutic benefit [43]. As the use of antibody--cytotoxin conjugates increases in development and begins to deliver clinically successful results, it is likely that more first generation compounds of this class will be pursued. As discussed above, antibody--cytotoxin conjugates offer a great deal of promise in enhancing the therapeutic activity of antibody or antibody-fragment-based TTAs. In light of the improved therapeutic potential, a number of development candidates are currently being pursued as first or second generation therapeutics [44]. Despite their therapeutic potential, a number of challenges still remain for their clinical and commercial success. The effectiveness of cytotoxin conjugates depends in part on the inherent features of the mAb used as the targeting component of the conjugate. Less desired properties of the mAb include: i) insufficient tumor penetration; ii) relatively long serum half-life, which may 1132 lead to liberation of the cytotoxin in serum and higher side effects; iii) limited ability of targeting epitopes within an antigen that can support maximal conjugate internalization and iv) technical challenges of universal, large scale conjugation of cytotoxins for GMP manufacturing at a reasonable cost of goods. Smaller targeting proteins such as antibody fragment platforms offer the ability to circumvent some of these potential challenges in developing protein drug conjugates. Studies using antibody fragments have demonstrated the ability of these agents to have better tumor penetration [45], preferred binding specificities [46] and lower serum half-lives that may avoid prolonged circulation and unwanted conjugate degradation in serum leading to toxic side effects via organ accumulation. Furthermore, smaller protein conjugates offer the ability to use alternative manufacturing approaches to maximize success in cGMP manufacturing by maintaining high quality production at a reasonable cost of goods in contrast to mammalian cell fermentation that is required for manufacturing fulllength mAbs. These factors need to be considered when deciding to develop a protein--cytotoxin conjugate that will probably be influenced by the nature of the disease, type of antigen being targeted and market dynamics. Pathogenic targeting proteins Antibody and antibody fragment conjugate-based TTAs offer many benefits for developing disease-specific therapies; however, a major drawback of using an antibody-based targeting agent is the limited frequency by which a cell surface target is expressed across a heterogeneous disease. In cancer, many cell surface targets have been identified that appear to be tumor-specific but the frequency of expression is quite variable from one tumor type to another thereby limiting the breadth of which an approved antibody/protein--conjugate TTA can be used across different cancer indications. The identification of broadly expressed molecular targets that are present on a diseased cell and not on normal tissues and can be selectively targeted by a protein conjugate is limited. Nevertheless, several disease-specific antigens have been identified as a result of epigenetic mechanisms, alternative splicing, gene rearrangement and overexpression that have been targeted and been able to deliver robust clinical activity, supporting the notion that this class of strictly expressed target exist and is important for maximizing the therapeutic potential of TTAs [47,48]. While efforts are currently being pursued to identify more disease-specific targets using a variety of genomic and proteomic discovery approaches (discussed below), disease-specific targets have been identified by naturally occurring pathogenic agents that are able to bind to these targets expressed on a broad class of normal and diseased cell types. Research has shown that some of these naturally occurring agents are able to identify differentially expressed and conformation-specific proteins that are not easily recognized by nucleic acid or proteomic analysis nor may be easily targeted using traditional protein/antibody approaches [49]. 2.4 Expert Opin. Drug Discov. (2010) 5(11) Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Eisai Inc on 02/18/11 For personal use only. Nicolaides, Sass & Grasso The identification of targets and binding agents using natural product screens from pathogenic organisms may provide additional opportunities for effective targeting of molecules. One source of natural proteins capable of binding to specific mammalian cell surface proteins comes from the venom of insects and reptiles. An example of this class is chlorotoxin, one of several dozen proteins found in the venom of the Israeli scorpion, Leiurus quinquestriatus. Screening of venom subunits identified proteins that were able to suppress the growth of stimulated epithelial cells [50]. Subsequent analysis revealed the ability of a 4 kDa peptide, later identified as chlorotoxin, to have preferential binding to tumor versus normal cells. Recently, molecular studies have identified that chlorotoxin binds and internalizes into a wide range of tumor types via annexin A2, a ubiquitously expressed intracellular protein in normal tissues that is found expressed on the extracellular membrane of transformed cells thereby giving it disease specificity [51]. Preclinical studies have shown the ability of chlorotoxin to deliver radionuclides, complex dyes, as well as nanoparticles preferentially to tumor cells in vitro and in vivo [52]. Synthetic chlorotoxin has been tested in clinical trials and showed the ability to deliver conjugated 131I radionuclide to tumors via local and systemic delivery while no detectable uptake was observed in normal tissues. The platform is currently being expanded to identify delivery of other potential compounds to tumors for diagnosis and therapy while the radionuclide conjugate strategy continues to be advanced [53]. Other natural compounds have also been shown to internalize on binding to cell-specific surface proteins. These proteins include vacuolating toxin A, which enters human cells via sphingomyelin; hepatitis C viral coat protein, which enters cells via claudin-1; and crotamine, a toxin from rattlesnake venom that enters cells via heparan sulfate proteoglycans [54-56]. Natural proteins with broad diseased cell-binding properties may enable shorter development and target discovery timelines and offer the ability to treat a wider class of disease indications due to the selected nature of a pathogenic molecule to maximize its ability to penetrate its infected host for propagation. 3. Nucleic acid TTAs Suppression of gene expression via knock down of mRNA steady-state levels or suppression of translation offers some of the most powerful opportunities to expand targeted therapy. Some examples of nucleic acid-based TTAs being developed across a differing spectrum of disease indications are listed in Table 8. Early attempts to regulate disease pathways by decreasing mRNA levels used antisense deoxynucleotide (AS) that can bind to complementary RNA transcripts and either block translation or decrease message via RNase H mediated degradation [57]. While gene-specific suppression has been proven in cell-based assays and in in vivo animal models, recapitulation of AS effects in human clinical trials has been unsuccessful [58]. Over the past 2 decades, enhanced efforts have been used to create improved AS that are more stable in serum, have enhanced cellular uptake and improved intracellular mRNA targeting [59]. In addition, the discovery of naturally occurring small RNAis has led to the development of the novel siRNA and miRNA platform technologies for targeted therapy. The two most advanced nucleic acidbased TTAs to date are Vitravene, an AS to treat CMV retinitis, and mipomersen, an AS compound that targets apolipoprotein B (apoB) to reduce low-density lipoprotein cholesterol (LDL-C) levels in patients with high cholesterol [60,61]. The use of mipomersen as a single agent in a Phase II clinical study showed its ability to suppress apoB and lower LDL-C in all patients treated [61]. In addition to this program, which is currently in Phase III randomized controlled clinical studies, over a dozen other antisense programs are ongoing that target mRNAs encoding for proteins associated with cancer, cardiovascular, metabolic, neurodegenerative, inflammatory and infectious disease [62]. Many development hurdles still exist in fully maximizing the therapeutic potential of nucleic acid-based TTAs. These include further development of nucleic acid chemistries and carrier strategies that can enhance the preferential accumulation of nucleic acid fragments in disease cells versus normal, as well as maintain sufficient steady-state serum and intracellular concentrations for maximizing binding and suppression of perpetually transcribed mRNA. Strategies to improve stability by using phosphorothioated oligodeoxynucleotides in which a non-bridging oxygen atom in the phosphate backbone is replaced by sulfur have proven to be effective in increasing serum and intracellular stability. Additional technologies are being attempted to further improve serum stability, cellular uptake, target selectivity, reduced toxicity, while maintaining support of RNase H activity for DNA--RNA degradation [63]. Similar modification strategies are being pursued with RNAi-based therapies to enhance stability and pharmacokinetic profiles [64]. While improvements to the nucleic acid moiety of these agents are needed, delivery to target cells also needs to be improved to help offset some of the development hurdles listed above. In vivo studies using RNAibased therapies have shown off-target RNA suppression as a result of inducing host immune responses which subsequently alters gene expression in target cells as well as intracellular offtarget gene expression, again suggesting the need for better nucleic acid TTA delivery [65,66]. Early attempts to address nucleic acid delivery have used the lipid encapsulation methods as well as direct linkage to carrier proteins such as folate, transferrin, somatostatin and antibodies to shuttle nucleic acid fragments across the bilipid cell membrane through pathways that allow nucleic acid fragments to remain intact [67-70]. These studies demonstrated the ability to improve internalization in vitro; however, their therapeutic activity in vivo has been equivocal. Several reasons may explain these results, including uptake by receptor pathways expressed ubiquitously among disease and normal cell types. Other reasons may include conjugate instability in serum and intracellular Expert Opin. Drug Discov. (2010) 5(11) 1133 Advances in targeted therapeutic agents Table 8. Nucleic acid-based TTAs. Compound name Target Disease indication Nucleic acid platform Ref. Vitravene Mipomersen ISIS113715 OGX-011 AIR645 ALN-RSV01 QPI-1002 CMV Apolipoprotein B Protein tyrosine phosphatase-1B Clusterin IL-4Ra RSV nucleocapsid protein p53 CMV-induced retinitis Cholesterol reduction Type 2 diabetes Cancer Asthma RSV infection Acute kidney injury PT antisense PT antisense PT antisense PT antisense PT antisense siRNA siRNA [60] [61] [118] [119] [120] [121] [122] oligonucleotide oligonucleotide oligonucleotide oligonucleotide oligonucleotide Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Eisai Inc on 02/18/11 For personal use only. CMV: Cytomegalovirus; PT: Phosphorothioated; RSV: Respiratory syncytial virus; TTA: Targeted therapeutic agent. trafficking of the conjugate that may result in sequestering the nucleic acid in a compartment of the cell that prohibits its ability to access and bind target mRNA [71,72]. Whatever the reason for the observed discordance between in vitro and in vivo studies, improving delivery and intracellular activity of nucleic acid-based TTA will undoubtedly enhance the overall therapeutic potential of these agents. 4. Target identification and selection Although TTAs with their ability to deliver molecules to cells expressing disease-causing pathways promise the potential for improving the treatment of a wide variety of diseases, important factors need to be considered in fully realizing their pharmacological potential. As discussed above, a major hurdle for developing successful TTAs is the generation of targeting molecules that can deliver robust pharmacological activity. The other is developing TTAs to bona fide diseaseassociated targets that are exclusively expressed in the disease versus normal tissues. Targets may include those that are widely expressed in normal and disease tissues but in the disease state are more accessible to TTAs or are present in an altered conformation or contain a different post-translational form that may enable a TTA to specifically bind to the disease-associated conformation that is absent in normal cells [73]. An important factor to consider when searching for disease-specific targets is selecting the method to be used to identify and validate them. A number of approaches have been used to identify disease-specific targets including differential RNA and protein analysis, immunological approaches and genetic polymorphisms. Here, we discuss these platforms and types of targets identified that may be suitable for TTA-based strategies. Methods for target identification Several platforms and technologies have been used in an attempt to identify targets suitable for disease-specific therapies. These include genomic-based technologies such as microarrays, serial analysis of gene expression and RNA subtraction analysis that can identify differential expression patterns of mRNA in normal versus diseased tissues [74]. In general, a good correlation exists between steady-state 4.1 1134 mRNA and protein levels; however, post-transcriptional mechanisms and post-translational modification of proteins have been shown to result in differential levels in diseased versus normal tissues [75]. In light of this caveat, proteinbased differential analysis has proven to be an effective method for identifying disease-associated targets. In particular, proteomic technologies such as 3D electrophoresis and quantitative mass spectrometry have been helpful in identifying proteins whose overexpression is observed in the disease state [76]. While target discovery via these platforms have yielded a number of candidates that are being pursued for targeted therapy, these techniques omit the identification of proteins whose tertiary structures are modified to induce disease-associated signaling [77]. These types of targets can be best identified using immune-based systems that can generate immunological reagents such as antibodies to identify aberrantly folded proteins whose altered structure may be associated with disease state but are not obvious using denaturing or differential expression platforms. Immunological-based platforms include the use of patient B cells to identify those producing antibodies to antigens restricted to the disease tissue, whole disease cell immunization of rodent hosts and SEREX, a platform that utilizes the serological analysis of recombinant complementary DNA to identify humoral and cellular immune responses in patients with disease [78-82]. These methods have led to the discovery of many disease-specific antigens that are being targeted in advanced clinical trials such as folate receptor a, mesothelin, NY-ESO-1 and a number of other antigens whose diseaseassociated epitope may not have been found using traditional large scale RNA/protein differential analysis methods. Finally, in cancer, the identification of chromosomal regions that are amplified or deleted as well as genetic loci that accumulate point mutations or translocations offers the ability to identify gene pathways that are involved in initiating, expanding or maintaining the disease state. The use of altered genomic information along with other platforms that measure altered steady-state mRNA levels may help identify gene products involved in supporting a disease phenotype. An example of this would be the case whereby a disease-associated gene product whose expression is only moderately enhanced in the disease state versus normal is not obvious as a candidate Expert Opin. Drug Discov. (2010) 5(11) Nicolaides, Sass & Grasso in contrast to gene products exhibiting dramatic increases in expression in the disease versus the normal cell. However, combining low overexpression with incidence of genetic mutation may help flag a particular gene product as a target candidate for therapy. These types of combined metaanalytical approaches may be beneficial in increasing the list of ‘bona fide’ disease-specific targets which may often be overlooked because of highly expressed gene products that are easily detected using large scale gene expression technologies. to deliver robust clinical benefit to patients. As discussed here, traditional nucleic acid or proteomic analysis and pursuit of candidates based on the level of expression may not always yield the best therapeutic target. Strategies that incorporate expression profiling, evidence of disease involvement via genetic or genomic locus modification(s) in addition to biological validation will help in identifying targets whose antagonism via TTAs may result in significant clinical benefit. 5. Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Eisai Inc on 02/18/11 For personal use only. 4.2 TTAs in development Selection of targets and criteria Because of the plethora of disease-associated targets identified using the garden of discovery technologies, it is crucial to have available algorithms that can prioritize those that have a higher probability of supporting the disease state. An ideal target is one in which its involvement in a disease is supported by strong scientific rationale. The most definitive of targets are those that are found mutated or containing a functional polymorphism that is highly correlative with and can recapitulate the manifestations of a disease pathway in experimental systems. Targets of this class have been identified via genetic mapping studies of families affected with a particular disease and, in the case of cancer, by comparing genetic information of malignant versus nonmalignant tissue [83,84]. Traditional genetic mapping of affected kindred has led to the discovery of several disease-associated targets that encode for intracellular and extracellular proteins. In cancer, several germline genetic mutations have been identified in genes that predispose individuals to cancer, including BRCA1, MLH1, MSH2 and the RB1 genes. Unfortunately, most of these targets encode for inactivating mutations in proteins that are intracellular and not accessible via protein-based TTAs. A unique feature of cancer is the ability to isolate cells from the transformed tissue and compare nucleic acid and protein products in the tumor material from the normal tissue cells using differential expression analysis and genomic technologies as described above. This approach has led to the identification of a number of gene targets that are mutated via point mutation or translocation that in turn supports the disease state [85,86]. Targets such as BCR-ABL, KIT, RAS and PTEN have been identified by these methods. Again, most of these targets encode for intracellular proteins which are not easily amenable to non-SCE or protein-based TTAs, but could be inhibited by nucleic acid-based TTA strategies. Genetic mapping of other disease types has also led to the discovery of a handful of extracellular targets which are mutated in the disease state and are amenable to therapy via a broader array of TTAs. These include targets such as ACVR1, a mutated cell surface receptor that causes constitutive signaling in the rare genetic disease fibrodysplasia ossificans progressive, a disorder that leads to ossification of muscle [87]. A combination of diverse target identification methods coupled with validation strategies will aid in identifying ideal disease-associated targets that can be manipulated by TTAs Here, we present the use of TTA platforms in developing novel therapies for treating a broad spectrum of diseases. Monoclonal IgG1-type antibodies and recombinant fusion proteins containing the receptor of a ligand have been the most successful agents of this class to date. A number of preclinical and clinical stage compounds are currently being pursued using a variety of next generation targeting platforms as discussed above. Tables 4 -- 8 provide a sampling overview of TTAs from these various platforms being developed to treat diseases across various therapeutic areas. The successful clinical development of one or more of these newer TTAs will undoubtedly support their broader pursuit by the biopharmaceutical sector as companies attempt to deliver novel medicines to treat diseases with unmet medical needs. In addition, with the new legislative challenges being placed on healthcare, the need for showing potential patient benefit of drugs is increasing as international regulatory agencies try to limit the high costs of drugs in various healthcare reform initiatives. The implementation of TTAs will enable biopharmaceutical engineers in developing diagnostic tools that can be used to predetermine whether a targeted pathway is intact in patients selected to receive a particular TTA-based medicine. Meeting these criteria will support the ability of novel TTAs to receive approval and reimbursement that will help pharmaceutical companies recoup a portion of their R&D investment, and in turn deliver patient benefit and provide those companies resources to support the development of additional innovative drugs. 6. Expert opinion Targeted therapy offers the medical community and patients an opportunity to vastly improve treatment and potentially cure disease. This era of targeted biopharmaceuticals provides an exciting time to leverage the combined information of the human genome along with the number of novel drug discovery technologies to create innovative medicines that can specifically bind to a disease target and suppress its associated pathway to relieve patients from morbidity and decrease disease-associated mortalities. While the concept of targeted therapy has been around for > 2 decades, the time to realize the potential benefits of this class of therapy has never been nearer. mAbs and other recombinant therapeutic proteins Expert Opin. Drug Discov. (2010) 5(11) 1135 Expert Opin. Drug Discov. Downloaded from informahealthcare.com by Eisai Inc on 02/18/11 For personal use only. Advances in targeted therapeutic agents have paved the way for demonstrating how TTAs can improve patient health. These TTAs have been developed with the notion that a patient prescreen can identify those that may benefit from targeted therapy. Two examples of successful TTAs that use a patient prescreen to determine therapeutic treatment are the HER2 screen of patients with advanced breast cancer for treatment with Herceptin and the identification of leukemia patients containing the Philadelphia chromosome for treatment with Gleevec [88]. With the advent of new antibody fragment technologies, nucleic acid-based TTAs along with the current mAb and recombinant fusion protein therapies ongoing, the future of personalized medicine is at the cusp of delivering promising treatments in helping patients combat disease without the risk of experiencing unwanted side effects that commonly occur with chemical-based drugs [89]. As drug developers gain more experience and insight into how these new platforms perform as monotherapies or in combination therapy from analysis of large randomized controlled clinical trials, they will be able to further refine TTAs to make them even more amenable for robust treatment. This experience will be similar to the one that led to the refinement of therapeutic mAbs as they evolved from a platform with unproven therapeutic potential in the 1970s to one of the most successful drug-based platforms in modern medicine [90]. As in the case of mAb therapies, further optimization of next generation TTA platforms by engineering features to improve target-specific delivery, optimal drug stability and robust manufacturing along with selecting bona fide disease-associated targets via applying stringent criteria (i.e., expression restricted to disease tissues, accessible by TTA in disease state, target required to support disease cell growth/maintenance, etc.) should support further developmental success of new compounds that have clinically relevant effects on the targeted disease. If these two conditions are met, we believe that there will be a continuum of regulatory successes utilizing TTAs. Moreover, the pursuit of TTAs will be further endorsed by the growing focus of international regulatory agencies whose priority is to support affordable drug pricing of therapies that can show patient benefit as well as exhibit good safety and efficacy results in pivotal clinical trials. 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Science 1999;285:1733-7 Affiliation Nicholas C Nicolaides†, Philip M Sass & Luigi Grasso † Author for correspondence Morphotek, Inc., 210 Welsh Pool Road, Exton, PA 19341, USA Tel: +1 610 423 6100; E-mail: [email protected] 1140 Expert Opin. Drug Discov. (2010) 5(11)