Toward The Rational Design of Cell Fate Modifiers: Notch Signaling as a Target for Novel Biopharmaceuticals

A. Zlobin, M. Jang and L. Miele*

Cardinal Bernardin Cancer Center, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA

*Address correspondence to this author at the Cardinal Bernardin Cancer Center, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA; Tel: 708-327-3362; Fax: (708)-327-3238; Email: lmiele@luc.edu

Abstract: Recent advances in our understanding of highly conserved mechanisms that control cell fate determination are paving the way towards rationally designed biologics that modulate specific cell fate decisions. Cell fate decisions leading to proliferation, differentiation or apoptosis are crucial elements in the pathogenesis of countless human diseases. Biopharmaceuticals designed to regulate such processes in specific cell types in vivo or ex vivo have vast potential applications in oncology, stem cell technology, immunomodulation and neuropathology. One of the most conserved mechanisms controlling cell fate determination is based upon Notch-ligand interactions and subsequent signaling events. Recent studies have shown that this mechanism regulates cell differentiation, proliferation and apoptosis in a wide variety of cell maturation processes and in neoplastic cells. These observations identify the Notch signaling network as a promising drug target for numerous indications. In this review, we describe: 1) potential drug targets in the Notch signaling network; 2) the Notch agonists and antagonists developed so far, including recombinant proteins, antibody-based agents, synthetic peptides, antisense oligonucleotides and gene therapy approaches, as well as possible strategies to design novel Notch-targeting biopharmaceuticals; 3) the possible clinical applications of such biopharmaceuticals and 4) a model strategy for the selection and developement of a Notch-targeting biopharmaceutical.

INTRODUCTION

     Notch genes encode evolutionarily conserved transmembrane receptors that regulate cell fate determination [1-7]. In recent years, Notch signaling has been identified as a crucial mechanism controlling numerous cell fate decisions during development and postnatal life in organisms from Drosophila to humans [1; 5-7]. Strong experimental evidence indicates that Notch signaling regulates all three branches of the cell fate decision tree: differentiation, cell cycle progression and apoptotic cell death. Notch activation promotes proliferation and inhibits differentiation of bone marrow stem cells and it prevents differentiation of neural and glial precursors. Recent data suggest a possible connection of Notch-1 with the pathogenesis of Alzheimer’s disease (see below). Notch signaling plays multiple, crucial roles in T cell development, and most likely in hematopoiesis and the immune response. Transformed cells of virtually all embryonic lineages and various human malignancies overexpress Notch receptors and ligands. Chronic Notch activation is likely to be a survival advantage for neoplastic cells, in light of the recently discovered anti-apoptotic activity of Notch signaling.

     In summary, numerous observations indicate that the Notch signaling network, including Notch receptors, ligands, mediators, modulators and target genes, is one of the most attractive targets for biopharmaceutical development identified in recent years. Promising areas of clinical development for Notch-targeting biologics include cancer treatment, stem cell technology, immunomodulation and the treatment of neurodegenerative disorders. In this review, we shall describe in separate sections: 1) the drug targets, including Notch receptors, ligands, mediators and modulators, and the current knowledge about their biological roles; 2) the agents, including Notch agonists and antagonists that have been recently developed, and possible alternative strategies for the development of novel candidates; 3) the potential clinical applications of Notch-targeting biopharmaceuticals and 4) a model strategy for the selection and development of a putative Notch-targeting cell fate modifier. For a comprehensive discussion of Notch biology, the reader is referred to excellent recent reviews [1-7].

DRUG TARGETS

a)    Notch Receptors: Biological Functions and Expression Patterns

     Notch signaling is thought to mediate primarily interactions between contiguous cells during cell-cell contact. This is because Notch receptors are activated by ligands that are predominantly cell membrane-associated. However, a soluble form of the Drosophila Notch ligand Delta has been recently identified, suggesting that Notch may also mediate interactions between non-contiguous cells [8]. In general terms, Notch receptors regulate cell fate determination in three different situations [1; 7]: 1) lateral specification/inhibition, in which initially identical cells with multiple possible differentiation fates regulate each other’s fates; 2) inductive signaling in which one cell type regulates another cell type’s differentiation choices and 3) cell-autonomous effects, in which a cell regulates it own fate through Notch signaling. The latter may be due to expression of ligand and receptor by the same cell, in a fashion similar to autocrine production of growth factors, or to ligand-independent activation of Notch. A classical paradigm of lateral specification/ inhibition is Drosophila neurogenesis [9-11]. In this model, putative stochastic oscillations in the levels of expression of Notch and its ligand Delta in neuroectodermal cells identify cells that will commit to the neuronal phenotype. Recently, such oscillations in the levels of Notch and Delta have been shown to be controlled by Wingless signaling [12; 13]. Neuronal precursor cells express higher levels of Delta and lower levels of Notch than surrounding cells. Each neuronal precursor, through Delta, activates Notch in the cells surrounding it, and causes them to further upregulate Notch expression. The activation of Notch in the cells surrounding neuronal precursors prevents them from differentiating towards the neuronal lineage. Subsequently, these cells switch to an epidermal differentiation program. Thus, notch deletions or loss of function mutations result in excessive numbers of neuroectodermal cells differentiating towards a neuronal fate. This is defined a “neurogenic” phenotype [9-11]. The C. elegans notch homologues LIN-12 and GLP-1 play analogous lateral specification roles during the development of uterine and vulvar precursors and of germ cells and pharyngeal epithelium respectively [5; 14-16]. Examples of inductive signaling can be found in mammalian odontogenesis [17; 18] and hematopoiesis [for review, see 6]. In these cases, Notch signaling is used to mediate communications between different cell types (ameloblasts and dental mesenchyme, bone marrow stroma and hematopoietic precursors). Cell-autonomous effects of Notch have been observed in Drosophila [19-21]. The anti-apoptotic effect of Notch-1 in murine erythroleukemia (MEL) cells [22] may fall within this category.

     The effects of Notch signaling on cell fate decisions in vertebrates have been extensively studied in tissue culture, ex vivo systems and transgenic animals. Notch signaling appears to be a highly conserved mechanism, which is used in cell fate determination control in many different tissues. In general, Notch activation modulates the response of a cell to a differentiation stimulus, rather than directly specifying a cell fate. Notch and its ligands have been shown to be essential for Xenopus neurogenesis [23-25] and mesoderm segmentation [26]. In mice, Notch-1 is necessary for embryonic development and targeted disruption of the NOTCH-1 gene results in disorganized somitogenesis and embryonic lethality [27; 28]. Similarly, targeted disruption of Notch ligand genes JAGGED-1 or -2 and the NOTCH-2 gene result in severe developmental defects or embryonic lethality [29-31]. Expression of constitutively active forms of Notch receptors inhibits terminal differentiation in vitro in murine models of myogenesis and granulocytopoiesis [32-35]. In chick retina explants, expression of constitutively active Notch-1 inhibits differentiation of retinal progenitors to ganglion cells, while NOTCH-1 antisense oligonucleotides increase differentiation towards a neuronal phenotype [36]. Similarly, ligand-induced activation of Notch-1 inhibits oligodendrocyte maturation in vitro [37]. Overall, these data suggest that in many contexts Notch activation inhibits or delays differentiation toward a specific fate until the cell is able to respond to signals which specify an alternate fate. However, in some experimental models, such as CD4 versus CD8 and a/b versus g/d T-cell receptor (TCR) lineage decisions in thymocytes [38; 39] and in vitro adipocyte differentiation [40], Notch signaling appears to be required for correct processing of differentiation stimuli.

     Notch receptors and ligands are widely expressed in postnatal animals. Notch-1 and -2 mRNA can be detected in various organs of humans and mice, particularly thymus, spleen, lung, heart, testis, ovary and central nervous system, with partially overlapping organ distributions [41; 42]. The same is true of Notch ligands Jagged-1 and Delta-1 in human organs [43]. Notch-1 and-2 are expressed in CD34⁺ human bone marrow stem cells and other hematopoietic precursors and are thought to participate in the control of cell fate decisions during hematopoiesis [6; 34; 44-46]. Bone marrow stromal cells express the Notch ligand Jagged-1 [46; 47], as do thymic stromal cells [48]. There is strong evidence that Notch-1 participates in the regulation of T cell development [38; 39; 49-52]. Notch-3 is expressed in the developing central and peripheral nervous system [53] and in the thymus [48] and developing pancreas [54]. Mutations of the NOTCH-3 gene in humans are associated with CADASIL, a neurological disorder characterized by multiple subcortical strokes and dementia [55]. Notch-4 is thought to be expressed primarily in endothelial cells during development and adult life [56].

     Recent data indicate that, in addition to its well-characterized effects on cell differentiation, Notch signaling affects cell cycle progression and apoptotic cell death. In HL-60 promyelocytic leukemia cells and CD34⁺ bone marrow stem cells, constitutively active Notch-1 and Notch-1 stimulation by Notch ligand Jagged-2 accelerated progression through G₁ [57]. Transfected constitutively active Notch-1 inhibits apoptosis induced by glucocorticoids [50] and by TCR engagement [51] in T cell hybridomas. Moreover, spontaneously expressed Notch-1 inhibits pharmacologically induced apoptosis in MEL cells [22]

b)    Structure and Processing of Notch Receptors and Ligands

     Notch receptors have an evolutionarily conserved structural organization. Vertebrate NOTCH genes are strongly related to each other and to Drosophila notch [58; 59]. Humans and mice have 4 NOTCH genes, denominated NOTCH-1 through 4. Similarly, multiple Notch ligands have been identified in vertebrates [60-66]. These are homologous to the Drosophila ligands Delta and Serrate and the C. elegans ligand Lag-2, and thus, are commonly identified as DSL from the initials of Delta, Serrate and Lag-2 [3; 67]. Mammalian Delta homologues are denominated “Delta-like” and mammalian Serrate homologues are denominated “Jagged”. [60; 62]. Notch proteins are synthesized as single polypeptide precursors, which are proteolytically processed to a heterodimeric, mature form [see Fig. (1)]. During receptor maturation, Notch pre-proteins are cleaved into an extracellular subunit (N^EC) containing multiple EGF-like repeats and a transmembrane subunit including the intracellular region (N^TM) [68], a single pass transmembrane domain and a short extracellular tail. This cleavage is catalyzed by a furin-like convertase [69]. It is still unclear whether the N^EC subunit is further processed by an ADAM family protease [70-72]. Such proteases have been shown to facilitate Notch signaling [70-72]. However, more recently the Drosophila ADAM family protease, Kuzbanian, has been shown to process the Notch ligand Delta [8]. Therefore, the effect of ADAM proteases on Notch signaling may be indirect. The number of EGF repeats in the Notch pre-proteins varies from 36 repeats in Drosophila Notch and mammalian Notch-1 and-2 to the 13 and 10 repeats of C. elegans Lin-12 and Glp-1 respectively. In Drosophila Notch, EGF repeats 11 and 12 are responsible for binding both ligands Delta and Serrate [73]. These repeats are highly conserved in mammalian Notch receptors, particularly Notch-1 and -2, and are thought to be the main ligand -binding site of these receptors. The N^TM subunit contains 6 ankyrin-like repeats, a polyglutamine region (OPA) and a proline/glutamic acid/serine/threonine rich (PEST) sequence [1]. A sequence denominated RAM23, immediately distal to the transmembrane region and proximal to the ankyrin repeats is thought to be a high affinity binding site for transcription factors of the CSL group (see below) [74]. The N^TM subunit is further cleaved to release an intracellular fragment (N^IC) during Notch signaling in a presenilin-dependent step (see below). Notch ligands also contain multiple EGF-like domains and an additional, N-terminal cysteine-rich domain (the DSL domain) that appears to be responsible for Notch binding [67], as well as short cytoplasmic tails.

c)     Notch Signaling Components

     Notch signaling involves several putative pathways and is still incompletely understood [Fig.(2)]. [1; 7; 33; 35; 75-77]. In mammals, most of the available data have been obtained studying Notch-1 or Notch-2, which appear to be functionally interchangeable in most systems. It is still unclear whether individual Notch receptors have “private” or preferred signaling pathways. However, recent observations indicate that Notch-3 may in some instances antagonize Notch-1 [54; 78]. This suggests the possibility of different signaling pathways for different Notch receptors, or different effects on the same molecular targets. We shall summarize the most clearly identified mediators and modulators of Notch signaling, since many of these may be drug targets in their own rights. For clarity, we shall describe separately: i) the “classical” signaling pathway mediated by transcription factors of the CSL family; ii) CSL-independent pathways and iii) modulators of Notch signaling.

Panel b: Mediators and modulators of Notch activities Top: Notch-binding proteins that are putative mediators of Notch signaling. In addition to CSL transcriptional regulators and Deltex, direct binding of several other key signaling molecules to Notch has been described. These include: the chromatin-remodeling factor EMB-5 in C. elegans, the p50 subunit of NF-kB in human transformed T cell lines, the c-Abl accessory protein Disabled (Dab) in neurons and the orphan nuclear receptor Nur77 in mouse T-cell hybridomas.

Bottom: Notch-binding proteins that are thought to modulate Notch signaling. The secretory protein Fringe in Drosophila and its mammalian homologues Lunatic, Radical and Manic Fringe modulate Notch-ligand interactions extracellularly. The C. elegans proteins Sel-1, Sel-9 and Sel-10 have been shown to modulate Notch protein turnover/intracellular trafficking. In Drosophila, Numb downregulates Notch signaling by binding to the intracellular region of Notch. Numb affects cell fate by distributing asymmetrically in daughter cells during mitosis, and thus, causing different cells to have different levels of Notch signaling. Disheveled is a downstream effector of Wingless, the Drosophila counterpart of Wnt oncogenes. It binds to the C-terminal portion of N^IC, and is thought to mediate cross-talk between Notch and Wingless signaling. Notchless is a WD40 domain containing protein that downregulates Notch signaling by binding Notch and Suppressor of Deltex [Su(D)] is a putative ubiquitin-ligase. Su(D) downregulates Notch activity, possibly by catalyzing C-terminal ubiquitination (UQ) of Notch, which may lead to Notch degradation.

i.     CSL-Mediated Signaling

     It is well established that the transcriptional regulator Suppressor of Hairless [Su(H)] is the primary mediator of Drosophila Notch [79]. Drosophila Su(H) is the prototype of a family that includes C. elegans Lag-1 and mammalian CBF-1(also known as RBP-Jk). This is often referred to as the CSL family, from the initials CBF-1, Su(H), Lag-1 [3]. Both in Drosophila and mammalian cells, Notch-ligand interactions appear to induce release and nuclear access of the intracellular portion of Notch (N^IC), which is accompanied by activation of CSL-dependent transcription [80; 81]. Release of N^IC requires a proteolytic cleavage within or near the membrane. Recently, Notch cleavage and CSL-mediated signaling have been shown to require presenilin-1 [82-85]. Presenilins 1 and 2 are multiple-pass transmembrane proteins which are mutated in most cases of familial, early onset Alzheimer’s disease [86-89]. Presenilin homologues had been previously found to facilitate Notch processing and intracellular trafficking in C. elegans [90; 91]. A physical interaction between presenilin-1 and Notch, occurring prior to cleavage of Notch pre-protein has been observed [92]. Whether presenilin-1 directly cleaves Notch in response to ligand binding remains to be established.

     The cleaved N^IC is thought to enter the nucleus and interact with transcription factors of the CSL family. A high affinity CSL interaction site has been mapped to the RAM23 region [74], and a lower affinity binding site is present in the ankyrin region [93]. The mammalian member of the CSL family, CBF-1/RBP-Jk, is a ubiquitous transcriptional regulator that functions as a repressor in the absence of Notch [77; 94]. This repressor activity is thought to be mediated by complexes including CBF-1, a nuclear co-repressor and histone deacetylase HDAC-1 [95]. Nuclear co-repressors that form complexes with CBF-1 include SMRT (silencing mediator of retinoid and thyroid hormone receptors) [95], nCoR (nuclear co-repressor) [95] and a CBF-1-specific co-repressor denominated CIR [96]. Notch binding leads to dissociation of co-repressors and HDAC-1 from CBF-1. CBF-1 is then thought to become a transcriptional activator, which upregulates the expression of Notch target genes. Thus, according to this model a general mechanism of Notch activation would involve binding of N^IC subunits to CSL/co-repressor/HDAC-1 complexes, release of co-repressors/HDAC-1 and conversion of CSL molecules to transcriptional activators. CSL factors would then induce the expression of a set of genes that mediate Notch functions.

     Several of these target genes whose expression is upregulated by CSL in response to Notch activation have been identified. Many of them encode downstream transcriptional regulators. A large group of Notch target genes in Drosophila is collectively identified as the “Enhancer of Split” [97-99]. Mammalian homologues have been identified for many of these genes. The Enhancer of Split group includes several bHLH transcription factors that are thought to mediate Notch effects by inhibiting the activity and/or the expression of differentiation-inducing, cell type-specific bHLH factors [100; 101]. Mammalian homologues of these bHLH genes are denominated HES (Hairy/Enhancer of Split) [75; 102-105]. The Enhancer of Split group also includes the Drosophila groucho gene and its mammalian homologue, Enhancer of Split-2. These genes encode bHLH transcriptional co-repressors that bind other bHLH proteins and may affect chromatin structure [102; 106-110]. Additional targets of Notch-mediated transcriptional regulation are: 1) The transcriptional repressor Mastermind  in Drosophila [111] and 2) the p100/NF-kB2 gene in mammalian cells [77; 112; 113].

     In summary, Notch activation results in CSL-dependent expression of a set of effector genes, including the “Enhancer of Split” group and others. These Notch-effector genes encode transcriptional regulators, which in turn modulate cell fate by affecting the function of tissue-specific bHLH transcription factors (e.g., Enhancer of Split) or through other molecular targets (e.g.NF-kB).

ii.    CSL-Independent Signaling

     Not all the effects of Notch signaling appear to be mediated by CSL-dependent signaling [33]. In mammalian cells, a CBF-1-independent pathway has been described, which involves the intracellular Notch-binding protein Deltex and possibly c-Jun N-terminal kinase (JNK, a member of the MAP kinase family) [76; 114]. Deltex is an evolutionarily conserved cytoplasmic, zinc-finger protein which binds to the ankyrin repeats of Notch and does not seem to enter the nucleus after Notch activation [115]. Through Deltex-mediated effects on JNK, Notch signaling may cross-talk with Ras signaling [115]. Additionally, Guan et al. [116] have shown that a constitutively active form of human Notch-1 construct interacts with p50-containing NF-kB complexes. When this construct was co-transfected with p50 and p65 NF-kB subunits, its effects on NF-kB- dependent reporter gene expression depended upon the stoichiometry of the system. Since the construct used did not contain the main CBF-1 binding RAM23 region, this effect is most likely CBF-1 independent. The protein kinase c-Abl has been proposed to participate in Notch-1 signaling in neurons [117]. This effect is suggested to be mediated by the Abl-accessory protein Disabled, which binds N^IC [117]. Direct interaction of constitutively active Notch-1 constructs with the orphan nuclear receptor nur77, which results in inhibition of nur77-dependent transcription, has been recently demonstrated [51]. In C. elegans, the transcriptional regulator EMB-5 interacts with the intracellular domain of Lin-12 and is necessary for signaling [118]. In summary, several different proteins in addition to CSL family transcriptional regulators have been found to physically associate with Notch receptors in various models and are putative mediators of Notch signaling. The relative physiologic roles of these putative Notch mediators in different cell types remain to be established.

iii.   Modulation of Notch Signaling

     Another group of proteins modulates the activity or processing of Notch receptors. In Drosophila, several proteins can bind the intracellular domain of Notch and downregulate Notch signaling. These include the membrane protein Numb [119; 120], the protein Disheveled which mediates cross talk between Notch and Wingless (the Drosophila homologue of wnt oncogenes) [121; 122] and the WD40 motif-containing protein Notchless [123]. The ubiquitin ligase Suppressor of Deltex is involved in downregulating Notch signaling [124; 125], suggesting that ubiquitination may lead to Notch degradation. In C. elegans, accessory proteins Sel-1, Sel-9 and Sel-10 negatively regulate Lin-12/Notch signaling by affecting the processing of Notch proteins or their trafficking to the plasma membrane [126-128]. Finally, secretory proteins that modulate Notch-ligand interactions in vivo have been identified. These include Drosophila Fringe [129; 130] and its mammalian homologues Lunatic Fringe, Radical Fringe and Manic Fringe [131-135].

     It should be emphasized that the biological effects of Notch signaling have been shown time and again to be context dependent [7]. The relative roles of the various Notch mediators, targets and modulators in different cell types are still poorly understood. As more information is gathered on the Notch signaling network, individual components of it will be identified as promising drug targets in specific tissues or indications. The level of Notch expression in a given cell is also likely to be an important factor. Prominent gene dosage effects have been demonstrated in Drosophila [136]. This suggests that the amount of Notch proteins can influence which signaling pathway(s) are triggered. The intracellular concentrations, physical state (monomers, multimers, supramolecular complexes) subcellular localization, time course of expression and post-translational modifications of Notch receptors, mediators, targets and modulators are likely to affect the ultimate biological effects of Notch activation or inhibition in a specific cell type. As is the case for most key mediators of cell fate, cross-talk with other signaling pathways adds a further dimension to the range of possible in vivo effects of Notch activation or inhibition. Signals delivered by cytokines [45], growth factors [137], steroid hormones [50] and Wingless/Wnt proteins [121] have been shown to interact with the Notch signaling network.

NOTCH-TARGETING BIOPHARMA-CEUTICALS

a)    Notch Antagonists

     Several approaches have been used to downregulate Notch signaling and could be exploited for biopharmaceutical purposes.

i)     Recombinant Proteins and Antibodies

     We have previously described a recombinant Notch antagonist consisting of a soluble “decoy” ligand binding region [40]. This antagonist consists of EGF repeats 11 and 12 of human Notch-1 (rh11-12), expressed in E. coli in soluble, disulfide bonded form using the expression vector pLD101 [138; 139]. EGF repeats 11 and 12 have been shown to be both necessary and sufficient for Delta and Serrate binding in Drosophila Notch [140]. These repeats are highly conserved in mammalian Notch-1 and -2. In 3T3 L1 cells, which require Notch-1 signaling to differentiate into adipocytes, rh11-12 significantly inhibited differentiation at 10^-7 M and abolished it at 10^-6 M. This effect corresponded to that of abrogating Notch-1 expression with an antisense construct [40]. This approach should be effective for Notch-2 as well, since EGF repeats 11 and 12 are highly conserved in Notch-2. The ligand-binding regions of Notch-3 and -4 are less characterized. However, once specific EGF repeats necessary for ligand binding in Notch-3 and -4 are identified, soluble decoy antagonists could easily be designed for these receptors as well. Potential advantages of such antagonists include ease of manufacturing and relatively small molecular mass (approximately 10 kDa for a 2-EGF repeats protein) which is likely to allow extravascular biodistribution. On the other hand, small molecular mass may also lead to rapid elimination via the kidney, and therefore, short biological half life. One can envision the design of chimeric proteins including Notch decoys fused to other subunits to modulate pharmacokinetics and biodistribution. For example a fusion with an immunoglobulin Fc fragment, as was successfully used in the case of p75 TNF receptor [141-144], may significantly affect the pharmacokinetics of a Notch decoy by increasing its molecular mass, as well as its biodistribution, if needed, by binding to Fc receptors.

     In the same paper where the effects of rh11-12 were first described [40], we provided proof of concept for an antibody-based strategy to design Notch antagonists. A rabbit polyclonal antibody raised against rh11-12 had Notch antagonist activity in 3T3 L1 cells, effectively blocking differentiation in that model. This suggests that monoclonal antibodies (mAbs) to ligand binding regions of Notch receptors could be used as Notch antagonists. Indeed, biologically active Notch-1 mAbs have been obtained using the same strategy [145] and will be described in detail elsewhere. Should it prove advantageous to block specific Notch ligands rather than receptors (for example, to increase the tissue specificity of a pharmacological effect), mAbs directed to unique or poorly conserved epitopes in the DSL domain could be developed. Alternatively, mAbs could be developed against non-conserved EGF repeats, using steric hindrance as a mechanism for blocking Notch-ligand interactions.

     Using mAb-based antagonists to inhibit Notch signaling has several potential advantages. Mab-based therapeutics have a long history of safe clinical use and are currently enjoying a resurgence, thanks to engineered, chimeric and humanized molecules and to a careful choice of epitopes. Recently, several safe and effective non-conjugated mAb therapeutics have been approved by the FDA and have entered mainstream clinical use. Especially the antineoplastic products Rituximab (Rituxan) [146-148] and Trastuzumab (Herceptin) [149; 150] have considerable promise in CD20-positive B cell lymphomas and in Her-2/Neu-expressing epithelial cancers respectively. Antibody engineering has considerably reduced the problem of mAb immunogenicity leading to loss of activity upon chronic treatment. Sink effects due to Fc receptor binding can also be alleviated by mutagenizing residues needed for receptor binding or deleting parts of the Fc region. The main disadvantage of mAb therapeutics remains poor tissue penetration into extravascular compartments. This may or may not be a problem for clinical use, depending on the specific indication and mode of administration (see below). Additionally, engineered forms of mAbs including recombinant F(ab)’ fragments, and single-chain Fv fragments can be used to improve tissue penetration. Such agents are currently included by the FDA in the same broad category as intact immunoglobulins from a regulatory standpoint [151]. Possible uses of mAb-based Notch-antagonists may include hematologic malignancies, the local treatment of unresectable Notch-expressing tumors and immune modulation (see below).

     An alternative strategy to Notch antagonist design could involve Fringe proteins. These are secretory, extracellular proteins that modulate Notch-ligand interactions (see above) [129-134]. In principle, such proteins could be used in recombinant form as Notch antagonists. The pharmacological effects of recombinant Numb are likely to be more complex than a simple inhibition of Notch signaling. For example, Drosophila Fringe specifically inhibits Notch-Serrate interactions and thus favors Notch-Delta interactions. Once the specific functions of the several mammalian Fringe proteins are clarified, one can envision using Fringe-derived biopharmaceuticals to modulate specific Notch-ligand interactions. Theoretically, this could provide a degree of pharmacological specificity similar to mAb directed to specific Notch ligands.

ii)    Antisense Drugs and Gene Therapy Vectors

     Antisense drugs and constructs have been used successfully to downregulate Notch signaling. Austin et al. [36] used antisense oligonucleotides directed to 3 different regions of the Notch-1 mRNA to show that reduced Notch-1 expression accelerates the differentiation of ganglion cell precursors in chick retina. The effect was shown to be sequence-specific. Similarly, Garces et al. [40] transfected an antisense construct encompassing the ankyrin repeat region of Notch-1 into 3T3 L1 cells. This construct, expressed under a cytomegalovirus (CMV) promoter, completely abolished the expression of Notch-1 protein in the cells.

     We [22; 145] used antisense phosphorothioate oligonucleotides to 3 different regions of Notch-1 cDNA to induce apoptosis in MEL cells. Similar results were obtained by using a 1.1 kb antisense cDNA construct expressed under a CMV promoter [22; 145]. The latter construct was directed to the 5’ end of the Notch-1 mRNA, a region with less sequence similarity to Notch-2 than the ankyrin repeats. A 50-60% downregulation of Notch-1 protein levels was observed in logarithmically growing cells with this construct. In combination with a differentiation-inducing agent, which itself downregulated Notch expression, the antisense construct led to accelerated disappearance of Notch protein which in turn resulted in massive apoptosis.

     In summary, antisense strategies can be used to downregulate Notch signaling, either in the form of synthetic antisense modified oligonucleotides or of gene transfer vectors. Antisense gene therapy is a potentially attractive approach to biopharmaceutical modulation of Notch signaling. Antisense drugs or constructs could be designed to target any component of the Notch signaling pathway even in the absence of protein structural data. For example, one may want to specifically downregulate one of the Notch mediators, such as Deltex or HES-1. Using “conventional” pharmaceuticals, this would require a cell-permeable drug that specifically interacts with and inhibits the function of the target protein. Despite the progress of combinatorial chemistry, identifying such a specific agent may be quite difficult in the absence of a high-throughput assay to measure the function of the target protein or of structural information on the putative active site(s) of the target protein. In the case of Notch signaling (see above) many of the mediators of Notch action have multiple biochemical activities which are still incompletely understood and at present, would be difficult to assay in an automated system.

     Despite a still incomplete understanding of their mechanism(s) of action and persistent technical hurdles, antisense nucleic acid therapeutics are attracting increasing scientific and clinical interest. In preclinical models, there are numerous examples of successful therapeutic use of antisense agents to downregulate specific genes. Clinical trials of antisense gene therapy agents for numerous indications are underway and an antiviral antisense drug (Fomivirsen) has been licensed by the FDA [152; 153]. Agents that have been used to induce antisense effects in vitro and in vivo include modified synthetic oligonucleotides [154-165] and several classes of viral vectors based on adenovirus, adeno-associated virus and retroviruses [166-181]. Antisense ribozymes that cleave the target mRNA utilizing catalytic RNA mechanisms are also in preclinical and clinical development [167; 174; 179; 182-188]. A thorough discussion of the merits and pitfalls of antisense drugs and gene therapy vectors is beyond the scope of this article, and the reader is referred to excellent recent reviews on the subject [189-197]. However, the available data indicate that antisense agents targeting Notch receptors and/or Notch signaling pathway components are a promising strategy for drug development. In this setting, a possible advantage of using antisense strategy may be the ability to downregulating the amount of intracellular Notch, rather than targeting only Notch molecules exposed at the cell surface, Specific indications (see below) will dictate the most suitable type of agent and delivery system.

b)    Notch Agonists

i.     Recombinant Proteins and Peptides

     Since Notch receptors are exposed at the cell surface, recombinant proteins and synthetic peptides derived from natural Notch ligands are potentially useful Notch agonists. A soluble, recombinant forms of human Jagged-1 and a synthetic peptide derived from it have Notch agonist activity in vitro [46]. This peptide corresponds to Jagged-1 residues 188-204, i.e., the sequence CDDYYYGFGCNKFCRPR. This sequence is part of the DSL region and is highly conserved between human Jagged-1 and Jagged-2. The three-dimensional structure of Jagged proteins has not been resolved yet. However, the high Cys content of the DSL region, together with secondary structure prediction analysis (Chou-Fasman and Garnier-Robson) suggest that the bioactive peptide derives from a region with high probability to form turns, stabilized by disulfide bonds. The high Cys content of the peptide is a potential pitfall, as oxidation in solution may lead to random disulfide formation, aggregation and loss of biological activity upon storage. This may account, at least in part, for the relatively high peptide concentrations (10^-5 M) needed to obtain significant biological activity in vitro. Thus, it may be desirable to attempt the design of optimized peptides in which Cys residues are replaced by other amino acids, or conformationally constrained peptides that can only form one intramolecular disulfide bond which stabilizes an active conformation. Rational design [198; 199] and/or combinatorial peptide libraries [200] may help in this task. These studies, in turn, may lead to the design of pharmacologically useful peptide mimetic Notch agonists [201].

     The large-scale production of intact recombinant soluble Notch ligands would most likely require eukaryotic cell substrates, due to the very large number of disulfide bonds associated with multiple EGF repeats. However, shorter recombinant proteins derived from Notch ligands (e.g., soluble DSL domains) could be produced in bacterial hosts in disulfide-bonded form. We have previously shown that native, disulfide-bonded proteins can be produced in E. coli using a modification of a common expression system [138; 139; 202]. We have used the same expression system to produce a biologically active form of human Notch-1 EGF repeats 11-12 [40, see above].

     An important caveat in the design of Notch agonists is the possibility of mixed agonist/antagonist effects. In Drosophila, soluble forms of Delta and Serrate have been shown to inhibit Notch activation [203]. On the other hand, Delta is processed to a soluble form that activates Notch by the ADAM protease Kuzbanian [8]. This apparent contradiction may be explained by differences in the structures of the soluble ligands tested so far, or by different responsiveness of the various cell systems used to such ligands [7]. A possible mechanism for such differential responsiveness to Notch agonists is supported by experimental evidence. The effect of exogenous Delta on Drosophila developing neurons depends upon the level of endogenous Delta [21]. This suggests that interactions between Notch and ligand expressed in the same cell modulate responsiveness to exogenous ligand [21]. This is relevant to Notch agonist design, since co-expression of Notch and Notch ligands (Notch-1, Notch-2, Jagged-1 and Delta-1) has been detected in cervical cancer specimens [43]. It should be pointed out that Delta can mediate cell-cell homotypic adhesion [204], which suggests Delta-Delta interaction. Depending on the relative affinities of ligand-ligand and ligand-receptor interactions, one may speculate that at low doses, a soluble ligand may sequester other ligand molecules in homotypic complexes, thus effectively reducing the concentration of free ligand molecules available for Notch binding and producing an antagonist effect. At higher doses, once endogenous Notch ligands have been saturated, one would observe a pure agonist effect since all additional exogenous ligand would be free to interact with Notch. This model predicts that the predominant effect (antagonist or agonist) of an exogenous Notch ligand would be dictated by the relative concentrations of endogenous and exogenous ligand. Thus would result in different dose-response relationships in different cells/tissues. One possible way of avoiding such complex dose-response relationships is to design Notch agonists that are incapable of interacting with endogenous ligands. Short recombinant proteins derived from DSL regions of specific ligands and synthetic peptide/peptide mimetic drugs may offer more promise than intact Notch ligands in this respect, provided that acceptable potency can be attained. However, the relative clinical usefulness of recombinant Notch ligands, recombinant protein fragments from Notch ligands and synthetic peptide/peptide mimetic drugs will be dictated also by other pharmacological considerations, such as pharmacokinetics, volume of distribution, access to extravascular compartments etc.

ii.    Gene Therapy

     In principle, a gene therapy approach to activating Notch signaling can be envisioned. Forms of Notch receptors lacking all or most extracellular subunits behave as constitutively active receptors, and have been widely used in cell culture and transgenic animals to activate Notch signaling. However, such forms of Notch have transforming activity in conjunction with some viral oncogenes [205] and cause T-cell lymphomas when introduced into mouse hematopoietic progenitor cells [206]. Thus, the potential use of constitutively active forms of Notch in gene therapy is plagued by obvious safety concerns, even if inducible vectors are used. Alternatively, it may be possible to transduce specific downstream Notch mediators (e.g., Deltex), or to attain inducible activation of CSL transcription factors to produce a partial Notch-like effect. This approach is used in nature by Epstein-Barr virus. The EBNA2 viral protein mimics Notch signaling by converting CBF-1 from a transcriptional repressor into a transcriptional activator [94; 207; 208]. Thus, at least in theory, targeted delivery of an EBNA2-derived protein capable of interacting with CBF-1 in an inducible expression vector may attain Notch activation-like effects in target cells. Whether this strategy presents the same safety pitfalls as using constitutively active Notch is unknown.

     Potential indirect ways of upregulating Notch signaling may rely on inhibition of Suppressor of Deltex-mediated receptor ubiquitination/ degradation, or on downregulation of negative modulators of Notch activity such as Notchless or Numb. This could be obtained using antisense approaches or, in theory, cell-permeable synthetic drugs that inactivate these targets.

POTENTIAL APPLICATIONS OF NOTCH-TARGETING BIOPHARMACEUTICALS

a)    Cancer

     Considerable evidence suggests that agents affecting Notch signaling may have clinical applications in the treatment of human malignancies [for review, see 209]. Notch expression and/or signaling are commonly altered in transformed cells and in spontaneous human tumors. Two distinct phenomena have been described: 1) increased expression of apparently intact Notch receptors and ligands and 2) mutations that produce constitutively active Notch receptors.

     Strong overexpression of Notch-1 and -2 has been observed in cervical carcinomas and pre-neoplastic lesions of the cervical epithelium, as well as other epithelial malignancies [210; 211] and neurological malignancies (M. Gunel, personal communication) In the cervix, overexpression of Notch-1 is associated with dramatic changes in the subcellular distribution of Notch-1 protein during the progression from pre-neoplastic CIN 3 lesion to microinvasive carcinoma. Pre-neoplastic lesions show mainly cytoplasmic Notch immunoreactivity with antibodies that recognize N^IC, while strong nuclear immunoreactivity was observed in carcinomas [211]. This phenomenon was present in 100% of the cervical cancer specimens studied so far [210; 211]. Moreover, Notch ligands Jagged-1 and Delta-1 were also increased in cervical carcinomas, concomitantly with Notch-1 and -2 overexpression [43]. Similarly, overexpression of Notch-1 has been described in colon adenocarcinomas and lung squamous carcinomas [210]. These data suggest that Notch signaling is chronically upregulated during the progression of cervical cancer and quite possibly, of several other epithelial cancers.

     Consistent with clinical observations, strong expression of Notch-1 (the most studied member of the family) can be readily detected in transformed cell lines of many different lineages, from cervical and endometrial carcinomas to T-cell acute lymphoblastic leukemia (T-ALL), acute promyelocytic leukemia, erythroleukemia, glioblastoma multiforme, neuroblastoma and medulloblastoma to pleural mesothelioma. This suggests that increased expression of Notch receptors, and possibly ligands, is a common molecular consequence of transformation, regardless of cell type. Interestingly, the human NOTCH-1, -2 and -3 genes have been mapped to chromosomal regions associated with hematopoietic malignancies of lymphoid, myeloid and erythroid lineages [212].

     Constitutively active forms of Notch have transforming activity in vitro and in vivo, and are associated with certain spontaneous human malignancies. Notch-1 deletions of all or most extracellular domain N^EC, resulting from a 9:7 chromosomal translocation, are associated with approximately 10% of the cases of T-ALL [41; 213]. A similar form of Notch-1 induced T-cell lymphomas when transduced into bone marrow precursors that were infused into syngeneic mice [206]. Similarly, mutations resulting in activated Notch-1 cooperate with c-myc in the pathogenesis of thymomas in MMTVD/c-myc transgenic mice [214]. In addition, constitutively active, truncated forms of Notch-4 cause breast cancer in mice [215-218] and are found in various transformed cell lines [219]. Constitutively active Notch-1 and Notch-2, when co-transduced with adenovirus oncogene E1A, transform primary rat kidney cells in vitro [205], with Notch-1 slightly more potent than Notch-2. As we have seen, EBV immortalizing protein EBNA2, which is necessary for EBV-induced transformation, mimics Notch-1 and -2 signaling by binding and activating CBF-1 [207; 208] .

     In summary, numerous observations suggest that chronically active Notch signaling is associated with the transformed phenotype in many cell types. Overexpression of apparently full-length Notch receptor(s) and ligand(s) is observed in several common epithelial malignancies and tumor cell lines. In other cases, activated Notch signaling results from deletions that produce constitutively active Notch receptors.

     Until recently, it was unknown whether overexpressed Notch receptors were functionally active in transformed cells or simply represented markers of loss of differentiation. Recently, three different groups including us have independently discovered that Notch-1 inhibits apoptotic cell death in various transformed cell lines. [22; 50; 51]. This suggests that increased Notch signaling is advantageous for transformed cells, similar to what has been observed with other anti-apoptotic genes such as Bcl-2 [220-223], and may explain the widespread occurrence of Notch overexpression in transformed cells in vivo and in vitro. This newly discovered anti-apoptotic function of Notch in transformed cells makes it a potential target for anti-neoplastic agents, and suggests that Notch antagonists may be potential drug candidates in this setting. We [22; 145] found that both Notch-1 antisense phosphorothioate oligonucleotides and enforced expression of Notch-1 antisense mRNA induced apoptosis in MEL cells. This effect was potentiated when antisense Notch-1 treatment was used in conjunction with a hexamethylene-bisacetamide (HMBA), a differentiation-inducing antineoplastic agent which is the prototype of the hybrid polar drug class [224-229]. HMBA itself downregulated Notch expression. In cells expressing antisense Notch-1, Notch-1 expression was abolished early during HMBA treatment. This in turn caused MEL cells to abort the differentiation program and undergo massive apoptosis. Even in the absence of HMBA, Notch-1 antisense expressing cells were significantly more likely than vector-transfected controls to undergo spontaneous apoptosis when cell density increased, possibly due to cell stress/growth factor deprivation. These results point to a possible therapeutic strategy including the use of Notch antagonists or antisense gene therapy in combination with cytotoxic or differentiation-inducing antineoplastic agents [145]. The mechanism of action of Notch antagonists in this setting, and the most effective combinations of antineoplastic agents and Notch antagonists, are currently under investigation in our laboratory.

     As for possible safety concerns related to in vivo use of Notch antagonists in an oncologic setting, reversible thymocyte depletion is likely to result from Notch-1 inhibition, due to the anti-apoptotic effect of Notch-1 in the thymus [50; 51] and/or its role in early thymocyte maturation [52]. Importantly, myelosuppression is not likely to occur. Mice carrying an inducible Cre/lox mediated inactivation of the Notch-1 gene did develop thymocyte depletion but did not develop myelosuppression even though gene inactivation in the bone marrow approached 100% [52]. B cell maturation was likewise not affected. A possible interpretation of these results is that Notch-1-deficient bone marrow precursors are not capable of developing into thymocytes. This is supported by recent data indicating that constitutively active Notch-1 expressed in mouse bone marrow progenitors causes the development of immature T cells in the bone marrow and blocks B cell development [230]. Other lineages may not be affected due to partial functional redundancy between Notch-1 and Notch-2. The latter is also expressed in the bone marrow [6; 45] and is very abundant in the spleen [42]. This suggests that the systemic use of reversible antagonists of Notch-1 would not result in severe myelosuppression. Other potential toxicity target organs are the brain and the testis (see above), but both these organs are relatively inaccessible to parenterally administered biologics unless special techniques are used. Due to our incomplete understanding of the physiological roles of the various mammalian Notch receptors and their possible functional redundancy, it is difficult to predict the possible side effects of Notch-2, -3 and -4 inhibition. It is likely that combined suppression of both Notch-1 and -2 would result in more severe effects on the hematopoietic and immune systems than inhibition of either receptor individually [6]. In terms of virus-mediated gene therapy, inducible antisense/ribozyme retroviral vectors are an attractive possibility for cancer treatment. Due to the property of retroviruses to integrate only into mitotically active cells, one could envision using such a vector expressing antisense Notch at a time when a large fraction of neoplastic cells are mitotically active (e.g., after a cycle of chemo/radiotherapy or bulk resection). A second cycle of treatment with a differentiation-inducing or cytotoxic antineoplastic drug coupled with induction of antisense expression would then be used to trigger apoptosis into neoplastic cells. This approach would avoid retroviral integration into mitotically quiescent pools of stem cell compartments, and limit the potential toxicity of Notch antisense to virally transduced, mitotically active cells. This therapeutic strategy is currently under investigation in our lab, concomitantly with a screening of common human malignancies for Notch expression. Such an approach may be considered for repeated intralesional or local treatment of unresectable malignancies (e.g., ovarian carcinomas, melanomas, gliomas, late-stage epithelial malignancies). Similar treatment schemas can be envisioned using modified antisense oligonucleotides in place of retroviral vectors.

b)    Stem Cell Technology

     “Stem cells” and multipotent progenitor cells are a highly promising area of experimental therapeutics. Three major areas of application are envisioned for such cell therapies. Reconstitutive treatments, in which stem cells are used to replenish a depleted pool in vivo; gene therapy applications in which therapeutic genes are transduced into appropriate progenitor cells which are then reintroduced into the patient and immunotherapy, e.g., for ex vivo production of dendritic cells to be used in tumor vaccine applications. Autologous hematopoietic precursor cells isolated from peripheral blood or from bone marrow are attracting considerable interest for bone marrow reconstitution after myeloablative chemo- and radio-therapy in oncologic indications and in severe autoimmune disorders, as well as for immunotherapeutic applications [231-246]. Cord blood-derived hematopoietic precursors are being actively investigated for similar indications [247-251]. Neural stem cells and oligodendrocyte progenitor cells hold great promise in the treatment of neurological lesions, neurode-generative disorders and demyelinating disorders [252-259]. Similar considerations apply to retinal stem cells [260] keratinocyte stem cells [261], limbal stem cells for corneal reconstruction [262; 263] and liver stem cells [264].

     A major obstacle to the widespread clinical development of stem cell therapeutics is the difficulty of obtaining large-scale in vitro expansion of highly undifferentiated cell populations without loss of multipotency. Several ingenious culture systems have been devised, which use combinations of cytokines, growth factors, feeder cells, special culture surfaces or other conditions to expand stem cell populations ex vivo and manipulate their differentiation potential [231; 244; 245; 265-281]. However, the field of ex vivo expansion and manipulation of stem cells is still in its infancy, especially in terms of industrial scale systems capable of delivering lot-to-lot consistent cell populations. Ideally, such systems should not rely on feeder cells, in order to avoid the need to validate the removal of such cells and their byproducts from the therapeutic product. From a biological standpoint, in order to obtain sustained amplification without loss of multipotency, one needs to create conditions that favor proliferation and inhibit differentiation and/or death in the stem cell population. Activation of Notch signaling has precisely these effects in neuronal precursors, glial precursors, and bone marrow hematopoietic precursors. Inhibition of neuronal differentiation was the first Notch function to be discovered in Drosophila (see above) and it is conserved in vertebrates. [23-25; 36; 53]. Indeed, Delta-Notch interactions have been shown to inhibit the differentiation of mammalian neural stem cells [282]. Moreover, activation of Notch-1 inhibits mammalian oligodendrocyte maturation in vitro [37]. The Notch ligand Jagged-1 is expressed by bone marrow stromal cell lines that support long term maintenance of hematopoietic progenitor cells [46]. Co-culture with stromal cells expressing Jagged-1 inhibited G-CSF induced differentiation [46]. This effect could be mimicked by soluble, recombinant forms of Jagged-1 and by the Jagged-1 derived peptide described above [46]. Similarly, Notch-2 has been shown to inhibit the differentiation of myeloid precursors in response to GM-CSF [45]. Expression of constitutively active Notch-1 and exposure to Jagged-2 expressing 3T3 cells inhibited the differentiation of human cord blood CD34⁺ hematopoietic precursors and accelerated their progression through G₁ [57]. Finally, Jagged-1 mediated Notch activation has been shown to maintain human CD34⁺ hematopoietic precursors in an immature state [283].

     Taken together, these data strongly suggest that biopharmaceuticals acting as Notch agonists may be used to enhance the ex vivo expansion of both neural and hematopoietic precursors for therapeutic applications. Whether other stem cell types may be similarly affected by Notch agonists in unclear at this time. However, the expression of Notch-1 and -2 in basal cells of some mucosal epithelia [210, L. Shelly and L.M., unpublished], and of Notch-1, -2 and -3 in developing teeth [18] suggests that this may be the case.

c)     Immunomodulation

     Considerable evidence points to multiple roles of Notch signaling in the immune system. [for review, see 284]. However, our knowledge in this field is still incomplete, and thus the possible therapeutic uses of Notch-targeting biopharma-ceuticals in the setting of immunomodulation remain speculative. It is well established that Notch-1 plays an important role in T-cell maturation. The expression of Notch-1 is highest in very immature, CD4-, CD8-, double negative thymocytes, declines in CD4+, CD8+ double positive cells and rises again in mature, CD4+ or CD8+ single positive thymocytes [49]. Several groups have shown that Notch-1 is necessary for CD4 versus CD8 [38] and ab versus gd T-cell receptor (TCR) [39] cell fate decisions. Moreover, Notch-1 has been shown to inhibit apoptosis induced by glucocorticoids [50] and by TCR stimulation [51]. This suggests that Notch-1 plays a role in the process of selection that eliminates potentially autoreactive T-cells via TCR stimulation-induced apoptosis and ineffective T cells, possibly via glucocorticoid-induced apoptosis [285]. Finally, Notch-1 appears to be necessary for the development of very immature thymocyte precursors [52]. Notch-3 may also be involved in regulating thymocyte development [48]. Interestingly, mature, single-positive T-cells that exit the thymus express Notch-1 [49], suggesting that Notch-1 may have a role not only in the development of T-cells but also in their mature function. The expression of both Notch-1 and -2 in the spleen [41; 42] and peripheral lymphoid organs (L. Shelly and L.M., unpublished) supports this hypothesis. However, the possible roles of Notch-1 in the context of the immune response have not been investigated in detail. By analogy, with what has been observed in other systems, it is tempting to speculate that Notch-1 may inhibit the “differentiation” program that is triggered after activation of T-cells by a cognate interaction. A similar role may be played by Notch-2 in B-cells. Indeed, recent experimental evidence suggests that Delta-1 Notch-1 signaling may be involved in the establishment of peripheral tolerance [286; 287]. In this case, Notch agonists may be used for immunosuppressive or tolerogenic purposes (e.g., in the setting of transplantation/graft versus host diseases) while Notch antagonists may amplify an immune response (e.g., in combination with a tumor vaccine). Further investigations will be needed to test these hypotheses. An additional area that deserves further investigation is the possible role of Notch signaling in the maturation of professional antigen-presenting cells (e.g., dendritic cells) from bone marrow precursors (see above). It may be possible to modulate the in vitro maturation of such cells by using Notch-targeting biologics. This in turn would have applications in the area of immunotherapy with ex-vivo generated, antigen-loaded and/or genetically manipulated dendritic cells.

d)    Neurodegenerative Disorders

     Notch signaling has been known for many years to play a pivotal role in the development of the central nervous system [5; 7; for reviews see 9; 11]. More recently, Notch signaling has been implicated in neuropathology. NOTCH-3 gene mutations are associated with CADASIL, a syndrome characterized by multiple subcortical strokes and leukoencephalopathy with progressive dementia [55]. Interest in a possible connection between Notch signaling and Alzheimer’s disease has been steadily increasing ever since the C. elegans presenilin-like molecule Sel-12 was found to facilitate Notch signaling, presumably by regulating Notch processing [90; 91]. The recent demonstrations of functional interactions between presenilins and Notch receptors has attracted further attention upon a possible connection between Notch signaling and Alzheimer’s disease. It is generally thought that mutant, dysfunctional presenilins occurring in patients affected with familial, early onset Alzheimer’s disease directly or indirectly increase neuronal death [87-89; 288-290] Presenilins have been proposed to regulate neuronal cell death in mammals by affecting the function of the endoplasmic reticulum [288]. As we have seen, presenilin-1 appears to be necessary for Notch processing and signaling, although the detailed mechanism(s) remain somewhat unclear [82-85; 92]. Moreover, Presenilin-1 facilitates Notch signaling in primary neurons [291]. Presenilin-1 mutants derived from familial Alzheimer disease cases appear to be unable to facilitate C. elegans Notch signaling [292]. Mice carrying a presenilin-1 gene “knockout” show phenotypes similar to Notch-1 “knockout” mice and have greatly reduced expression of Notch-1 and Delta-like-1 mRNA [50; 293]. Expression of Notch-1, -2 and -3 has been observed in mature neurons of various species [294-296]. This suggests that Notch signaling has a biological function in mature neurons. Interestingly, in patients with sporadic (non-familial) Alzheimer’s disease, significantly increased Notch-1 protein expression was observed in the hippocampus [296], suggesting that altered Notch expression may be associated with this disorder. Whether the overexpressed Notch protein in these patients is correctly processed and functional remains to be established, Notch overexpression in this context may represent accumulation of incorrectly processed or targeted protein. In light of the recent findings that Notch can prevent cell death in some systems and the interaction between Notch and presenilins, it is tempting to speculate that Notch expression may protect mature neurons from apoptotic cell death. Should that be the case, one might envision a possible use for Notch agonists in reducing neuronal loss associated with Alzheimer’s disease. Due to the drug delivery problems posed by the blood-brain barrier, it is difficult to imagine using recombinant proteins or even peptides in this clinical setting. Nevertheless, once the precise structural requirements of Notch-agonist interactions are elucidated, it should be possible to design appropriate peptide mimetics or identify by combinatorial screening synthetic drugs that pass the blood-brain barrier and activate Notch signaling.

     The inhibitory effect of Notch-1 on oligodendrocyte maturation [37] suggests another potential area of interest: that of demyelinating disorders, such as multiple sclerosis. One can envision Notch agonists to be used ex vivo to expand oligodendrocyte precursors as a prelude to cell/gene therapy of demyelinating disorders (see above, stem cell technology).

A MODEL STRATEGY FOR THE SELECTION AND DEVELOPMENT OF A NOTCH-TARGETING BIOPHARMA-CEUTICAL

     Based on the information discussed above, it is possible to envision a model strategy for the selection, design, evaluation and development of a Notch-targeting cell fate modifier. The following steps may be typical of such a strategy:

a)    Determining the Pattern of Expression and Possible Function(s) of Notch Signaling Components in the Target Tissue or Cells

     The context-dependent nature of Notch effects implies that the effects of inhibiting or activating signaling by a particular Notch receptor may vary depending on the target tissue or cell (see above). Recent evidence indicates that different Notch receptors may have different and even antagonistic functions in some tissues [45; 54; 78]. Thus, the rational design of a Notch-targeting agent should begin with an exploration of expression patterns of Notch receptors and ligands, and if necessary downstream mediators, in tissues relevant to the planned indication either as targets for a therapeutic effect or as potential targets for toxic effects. It should be kept in mind that such expression patterns may be affected by disease conditions (e.g., increased expression in pre-neoplastic lesions or in cancer cells). DNA array technology could be used to rapidly screen various tissues or pathological specimens for expression of all known members of the Notch signaling network. Information obtained in such studies would facilitate the selection of a molecular target (Notch receptor, ligand or mediator). If tissue-specific downstream mediators are found, these would be potential candidates for the development of tissue-specific agents. A clear understanding of the effects of Notch signaling in the target tissue(s) would greatly facilitate not only the choice of agents (agonists, antagonists, modulators of Notch signaling targeting specific members of the group) but also the design of therapeutic strategies. For example, an immunomodulatory agent may be most effective if administered before, simultaneously or after a tumor vaccine, depending on the specific role of Notch signaling in the relevant immunological process(es).

b)    Determining Whether the Target is a Wild-Type or Mutant Molecule

     Constitutively active forms of Notch receptors have been identified in various transformed cells (see above). These mutant Notch receptors usually result from chromosomal translocations or deletions leading to loss of all or most extracellular domain N^EC.  Similarly, mutations in the N^EC domain of Notch-3 are associated with CADASIL (see above). Obviously, intracellular, constitutively active Notch receptors would have to be targeted using biopharmaceuticals that act intracellularly, such as antisense or ribozyme agents or cell-permeable, small molecular inhibitors. Similar considerations apply to Notch receptors that are exposed at the cell surface but carry extracellular mutations. In the case of mutant Notch targets, it may be possible to design agents that specifically target mutant molecules, such as antisense molecules that specifically target a translocation-derived chimeric mRNA or mAbs directed against a mutant epitope within N^EC.

c)     Selecting the Optimal Drug Delivery Strategy for the Intended Indication

     Drug delivery considerations will be critical in the choice of an appropriate Notch-targeting agent. Local or regional delivery strategies may be used for viral gene therapy agents, non-viral gene therapy delivery systems or in some cases for mAb agents. Examples of such strategies may include intralesional treatment of a malignant glioma or a skin cancer, or intraperitoneal treatment of a locally disseminated ovarian cancer. Under such conditions, poor extravascular biodistribution may not be a major disadvantage and indeed may be desirable to prevent potential toxic effects resulting from systemic distribution of the agent. On the other hand, if systemic administration is necessary, small recombinant proteins, peptides or antisense oligonucleotides may be preferable to viral gene therapy vectors. Despite their generally low rate of distribution to extravascular compartments, mAbs may also be used systemically, as proven by the successful use of mAbs in the treatment of solid tumors (see above). Special drug delivery problems may arise if the CNS is the target organ (e.g., in neurodegenerative disorders), especially if the blood-brain barrier is intact. In  such cases, one can envision the design of synthetic, lipid-soluble drugs based on data obtained in vitro with biologics. Such drugs would preferably target components of the Notch signaling network that are specficically altered in the CNS or are expressed preferentially in the CNS.  Ex vivo applications such as stem cell technology require different considerations. In such cases, it would be important to determine whether a soluble or immobilized agent is most effective. Also, removal of the Notch-targeting agent from the cells prior to administration will likely be necessary. This would, ofcourse, favor extracellularly active recombinant proteins, peptides or antibodies over cell-permeable agents.

d)    Establishing the Safety Profile of the Candidate Agent(s)

     Once one or more candidate agents have been selected, a crucial step in determining their potential for clinical development and their optimal use(s) will be a thorough assessment of their in vivo safety. Such an assessment is not necessarily straightforward with bio-pharmaceuticals, due to the species-specifc nature of many such agents and the frequent absence of truly relevant animal models. Unless evolutionarily conserved molecular regions are targeted, “parallel” models, i.e., agents that specifically target the mouse or rat homologue of the intended human target are likely to offer the most realistic assessment of potential toxicities associated with candidate agents. In the case of agents targeting mutant forms of Notch, animal safety studies would be of limited usefulness beyond generic toxicity. Even with the best possible animal safety data, well designed phase 1 clinical trials will be necessary to identify unexpected adverse effects, the safest dose ranges and administration regimens. For life-threatening indications, such as inoperable malignancies, even signficant potential toxicities would not necessarily preclude clinical development of a promising candidate agent. In such cases, intralesional or regional administration routes may be preferable.

e)     Evaluating the Efficacy of the Candidate Agent(s), Alone or in Combination with other Treatments

     A thorough pre-clinical evaluation process based on a solid understanding of the biochemistry and biology of the intended molecular targets greatly increases the likelihood of succss in the clinical development of biopharmaceuticals. At the same time, promising indications for such agents are more easily identified once the role played by individual molecular targets in specific disease entities is elucidated. Decision processes, similar to the one briefly sketched above, should allow knowledge-based design, selection, pre-clinical and early clinical evaluation of cell fate modifiers targeting the Notch signaling network. Agents selected through such strategies would likely be promising candidates for advanced clinical development in phase 2 and phase 3 efficacy trials.

CONCLUSIONS AND FUTURE DIRECTIONS

     Recent advances in the molecular biology of cell fate determination have identified several novel drug targets with vast potential clinical applications. Among these targets, the network consisting of Notch receptors, ligands, intracellular mediators and modulators holds great promise for the development of novel biopharmaceuticals. Eventhough, our knowledge of Notch signaling and biology is still incomplete, a number of important potential applications for biopharmaceuticals that stimulate or inhibit Notch signaling have become apparent. Some such agents have been already generated, and these results in turn suggest strategies to develop new ones. Recombinant proteins, mAbs, synthetic peptides, antisense oligonucleotides and gene therapy vectors targeting Notch signaling have been developed and are potential candidates for drug development.

     The road towards the development of safe and effective cell fate modifiers that target Notch signaling is open. Choosing the most promising agents for clinical development in specific indications, improving the available Notch-targeting biopharmaceuticals and designing new ones are the next challenges. Progress in these areas will proceed hand in hand with advances in our understanding of the physiological roles of each Notch receptor and ligand and of the Notch signaling alterations associated with specific human diseases.

Acknowledgements

     The authors are grateful to Prof. Barbara Osborne (University of Massachusetts) for helpful discussions and comments on this manuscript. We also express our gratitude to the Illinois Department of Public Health and the National Institutes of Health (RO1CA84065-01) for supporting our work.

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