Proteases As Activators for Cytotoxic Prodrugs in Antitumor Therapy ULRICH H

CANCER GENOMICS & PROTEOMICS 11 : 67-80 (2014)

Review

Proteases as Activators for Cytotoxic Prodrugs in Antitumor Therapy ULRICH H. WEIDLE, GEORG TIEFENTHALER and GUY GEORGES

Roche Pharma Research and Early Development (pRED), Roche Diagnostics GmbH, Penzberg, Germany

Abstract. Proteases are often overexpressed in tumor cells (membrane-type serine protease 1), as well as cathepsin B, a and/or the stromal compartment and can thus be exploited in cysteine protease (6), were thoroughly investigated (5). In tumor therapy to activate cytotoxic prodrugs as, for example, addition, the serine protease urokinase (uPA) and its specific in cytolytic fusion proteins, and for tumor imaging. Specifically, receptor (uPAR) control matrix degradation and many other we discuss cathepsin B-activated prodrug conjugates, antibody- biological processes by conversion of plasminogen into directed prodrug therapy, protease-activated peptide− plasmin and by receptor signaling based on the interaction of thapsigargin conjugates, protease-activated cytotoxic receptor uPAR with many extracellular protein ligands (7). The ligands and other cytotoxic proteins, protease-mediated deregulation of this system in several types of cancer has a activation of anthrax toxin, granzyme B as a therapeutic significant impact on prognosis and can serve as a possible principle in cytolytic fusion proteins, and tumor-imaging based target for therapeutic intervention (8-10). Other proteolytic on deregulated proteases. systems deregulated in cancer include kallikrein-related peptidases (11), a disintegrin and the metalloproteinase family The human degradome consists of roughly 600 proteases, of proteinases (ADAMs) (12), as well as several types of which are grouped into metallo-, serine, cysteine, threonine and cathepsins (6, 13). In the following, we summarize approaches aspartic proteases (1). They regulate physiological processes for tumor-specific activation of antitumoral agents capitalizing such as proliferation, migration, cell survival, apoptosis and on dysregulated and overexpressed proteases in several types protein turnover. In tumors, expression of proteases is of human cancer. We focus on MMP2, MMP9, MMP14, frequently dysregulated, thus, coopting these physiological matriptase, uPA and cathepsin B. processes, which are often based on complex interactions between tumors and their microenvironment (2). Matrix Cathepsin B-activated Drugs metalloproteinases (MMPs) are a family of proteins consisting of 26 members of secreted and transmembrane proteins (3) As outlined before, increased expression or altered localization which degrade the extracellular matrix and regulate the activity of proteases is typical in many types of cancer (2). Activation of other proteases, growth factors and receptors, thus of prodrugs through cleavage by cathepsin B is a common modulating signaling pathways often associated with pro- or principle in pre-clinical and clinical development of anticancer antitumoral outcome (4). The family consists of collagenases, drugs. Cathepsin B is a lysosomal cysteine protease which is stromelysins, gelatinases and membrane-type MMPs (5). In the overexperessed in numerous tumor types and can be context of tumor biology, MMP2 (gelatinase A), MMP9 associated with the external cell surface (14). Procathepsin B (gelatinase B), membrane-type1 protease MMP14, matriptase can be activated by several proteases. Activated cathepsin B can then convert pro-uPA to uPA, which finally activates plasminogen to plasmin, a serine protease, which can degrade several components of the extracellular matrix such as fibrin, Correspondence to: Ulrich H. Weidle, Roche Pharma Research and fibronectin, laminin and proteoglycans and which is able to Early Development (pRED), Roche Diagnostics GmbH, Im activate MMPs (15, 16). Nonnenwald 2, D 82372 Penzberg, Germany. E-mail: Several strategies for the construction of prodrug [email protected] therapeutics have been evaluated. A common motif here is the Key Words: Antibody−drug conjugates, antibody-directed enzyme combination of a therapeutic agent with an inactivating peptide prodrug therapy, Anthrax toxin, cytotoxic receptor ligand(s), substrate which is cleavable by proteases, thus leading to the granzyme B, probody, thapsigargin, review. liberation of the active therapeutic. In addition, more recently,

1109 -6535 /2014 67 CANCER GENOMICS & PROTEOMICS 11 : 67-80 (2014) such prodrug molecules were fused to a targeting moiety. ADCs consist of a targeting moiety, a linker, and a Alternatively, peptide-linked therapeutic agents can also be cytotoxic payload. The targeting module can be a ligand for a fused to a macromolecular carrier with or without a targeting receptor or an antibody-based moiety directed against a moiety (17). The targeting module can, for example, be a cancer-related antigen (26). After internalization of the ADC ligand for a receptor, or an antibody-based moiety. In the case antigen−receptor complexes, cytotoxic payloads such as of cathepsin B-activated prodrugs, conversion into the active calicheamycin, maytansine, duocarmycin, auristatin or drugs should occur in the lysosome, but due to possible irinotecan are released into the cytosol after cleavage of the association of cathepsin B with the cell surface in some types linker in the endosome (27). Chemically-unstable, enzyme- of cancer, extracellular activation of the substrate is also cleavable, but also stable, non-cleavable linkers have been possible. In addition, non-internalizable prodrug−receptor explored in this context (26). Chemically labile linkers are complexes will be activated exclusively at the cell surface. hydrazine-based, whereas stable, non-cleavable linkers make However, depending on the design of the substrate cleavage use of thioester chemistry (26). The activity of ADCs with site, prodrug activation in the serum by cathepsin B-like stable, non-cleavable linkers seems to depend on the activity is a critical issue. In the present review article, we degradation of the thioester-linked antibody component in the summarize selected approaches for the design of cathepsin B- lysosome, resulting in the release of the cytotoxic payload activated prodrugs, including cytotoxic antibody−drug (28). Molecules with enzyme-cleavable linkers, on the other conjugates (ADCs) which are at an advanced clinical stage. hand, rely on the hydrolysis of a valine-citrulline (vc) linker Linking cytotoxic agents to high-molecular weight carrier by cathepsin B. This results in the release of the cytotoxic molecules is a strategy to deliver drugs to tumors. The resulting payload, such as the tubulin binders monomethyl auristatin E modification of the pharmacokinetic properties of the cytotoxic (MMAE) and F (MMAF) (29, 30, 31). ADCs based on compound mediates tumor accumulation in pre-clinical models, cathepsin B cleavable linkers are among the most advanced so-called passive targeting (18). For example, FCE 28068 (also ADCs for the treatment of cancer. For example, brentuximab known as PK1) was constructed by linking doxorubicin to N-(2- vedotin, an IgG1 antibody directed against cluster of hydroxypropyl) methylacrylamide copolymers via a tetrapeptide differentiation-30 (CD30) which is linked to vc MMAE is spacer which is cleavable by cathepsins, allowing release of the approved for treatment of therapy-refractory Hodgkin’s active topoisomerase II inhibitor (19). This form of passive lymphoma and anaplastic large cell lymphoma (32, 33). tumor targeting is also known as the enhanced permeability and Similarly, CDX-011 (glembatumumab vedotin) is an ADC retention effect, resulting from enhanced permeability of the composed of a fully human IgG2 anti-glycoprotein non- tumor vasculature, facilitating extravasation of high-molecular metastatic melanoma protein b/osteoactivin (gpNMB) weight proteins such as drug conjugates from the blood vessels antibody conjugated to MMAE via a vc linker (34). CDX-011 (20). In a phase II study of FCE 28068 in chemotherapy- is presently in phase II clinical studies in melanoma and refractory patients with breast cancer, non-small cell lung breast cancer (35-36). In addition, a prostate-specific carcinoma, and colorectal cancer, 6/62 partial responses were membrane antigen (PSMA)-ADC composed of a fully- observed with limited side-effects thus supporting the concept humanized immunoglobulin G1 (IgG1) antibody and MMAE that polymer-based chemotherapeutics might have different and linked via a vc linker and is currently being evaluated in improved anticancer activities (21). In addition, FCE 28068 was patients with taxane-refractory metastatic prostate cancer in linked to galactosamine, resulting in drug PK2, which binds to phase I clinical trials (37, 38). ADCs with analogous design the hepatic asialoglycoprotein receptor, thus achieving liver- targeting nectin-4 and solute carrier family 4A4 (SLC4A4) specific doxorubicin delivery (22). Indeed, in a phase I study, are currently being evaluated in phase I clinical studies (27). liver-specific accumulation of PK2 after i.v. infusion was shown, Also undergoing phase I clinical studies are RG-7458, as well as targeting to primary hepatocellular tumors (22). targeting CD22 in patients with non-Hodgkin’s lymphoma, Similarly, the tubulin-binding compound paclitaxel−polyglumex and SGN75 and MDX 1203, two ADCs based on anti-CD70 (PPX) is a polymer−drug conjugate in which paclitaxel is linked antibodies. These are presently being evaluated in patients to a degradable polymer consisting of L-glutamic acid residues. with non-Hodgkin’s lymphoma and clear cell renal cancer The release of paclitaxel from the polymer backbone is (27). In summary, it seems that no general design for dependent on cathepsin B and other lysosomal proteases (23). optimized linkers can be deduced based on previous PPX was evaluated in a phase III trial versus docetaxel in experience. In addition, ADCs with non-cleavable linkers second-line treatment of patients with non-small cell lung cancer must be efficiently internalized to exert their cytotoxic (24, 25). Although PPX and docetaxel led to similar survival function. On the other hand, ADCs with cleavable linkers rates, different toxicity profiles were noted. Compared to may mediate cytotoxicity by extracellular cleavage at the docetaxel, PPX-treated patients had less frequent febrile tumor cell surface in addition to cytotoxic pathways. neutropenia and alopecia, however, increased neurotoxicity was Therefore, toxicity directed against antigen-negative tumor noted for PPX in comparison to docetaxel. cells is expected only for ADCs with cleavable linkers (26).

68 Weidle et al : Proteases as Mediators of Cytotoxicity in Tumor Therapy (Review)

Antibody-directed Enzyme Prodrug molecule (47). For second-generation approaches, humanized Therapy (ADEPT) antibodies and CPG2 with removed B- and T-cell epitopes are under consideration (48). ADEPT is based on pre-targeting antibody−enzyme complexes or corresponding fusion proteins to tumors, Protease-cleavable Peptide−Thapsigargin followed by administration of an inactive prodrug which is (TG) Conjugates converted to an active drug on the tumor cell surface by these enzymes (39-41). After administration of the antibody-enzyme The peptide−TG drug platform is based on coupling a conjugates, the unbound conjugate is allowed to be cleared chemically modified amino acid containing TG with protease- from the circulation. This can be accelerated by using cleavable peptides (49). TG is a sesquiterpene lactone isolated mannosylated conjugates (42), or a second antibody (43). For from the plant Thapsia garganica (50). TG is a lipohilic effective treatment, the prodrug has to be an optimal and molecule and an inhibitor of the ubiquitously expressed specific substrate for the enzyme and must not be able to sarcoplasmic/endoplasmic reticulum (ER) calcium ATPase penetrate cells. The inhibitory concentration 50 (IC 50 ) of the pump (SERCA) (51, 52). The SERCA pump is essential for liberated drug should be below 10 nM, and the ratio of the the function of the ER. SERCA inhibition leads to a 3-5 fold 2+ IC 50 s of the prodrug and drug should exceed 1,000 (40). The increase in intracellular free Ca accompanied by the released drug must be able to penetrate tumor cells and its depletion of the ER Ca 2+ , pool resulting in cell death plasma half-life should be short enough to prevent leakage into independent of proliferation status (53, 54). Because the healthy tissues, but long enough to induce a bystander effect toxicity of TG is not cell type-specific, systemic administration on neighbouring tumor cells. Activation of prodrug can be of TG would be associated with significant toxicity. Therefore, mediated by proteases, glycosidases, alkaline phosphatase, prodrug molecules were designed with a carrier peptide that penicillin amidase, β- lactamase or cysteine deaminase (41). does not allow TG to enter the cell until the TG analog is In the following, we focus on prodrug activation by liberated by proteolysis. A PSMA-targeted prodrug was proteases. Most thoroughly explored, also in terms of evaluated (55). PSMA is a membrane-bound protease with its translational applications in clinical studies, is catalytic domain exposed to the extracellular space. It exhibits carboxypeptidase G2 (CPG2). CPG2 is a bacterial, Zn 2+ - dual exopeptidase activity as both a pteroyl poly γ glutamyl dependent, homodimer forming exopeptidase, which can exopeptidase (folate hydrolase) and N-acetylated α- linked acid cleave a deactivating glutamate moiety from benzoic acid dipeptidase (56, 57). PSMA is expressed in tumor-associated mustard (42, 43). Initial pre-clinical and clinical studies were endothelium, thus potentially enabling TG prodrug activation performed with chemically produced antibody−enzyme in the tumor microenvironment (53). The TG-based prodrug conjugates. Subsequently, in order to overcome the variability G202 (Figure 1A), consisting of a PSMA-specific peptide of random chemical conjugation which results in product conjugated to an analog of TG, induced tumor regression in heterogeneity, recombinantly produced antibody several xenograft models at doses which were minimally toxic moiety−enzyme fusion proteins were explored (42, 43). Such to the host (55). Critical issues are, however, the expression of a fusion protein consisting of a single-chain variable domain PSMA in the proximal tubules of the kidney and in the brain fragment (scFv) directed against carcinoembryonic antigen (58). Ongoing clinical studies will show whether a therapeutic (CEA), and CPG2 produced in Pichia pastoris for activation window combining efficacy with an acceptable toxicity profile of a methotrexate-based prodrug was investigated in more can be defined. detail. This fusion protein, MFECP, bore branched mannose A new emerging strategy for tumor therapy is ablation of residues due to post-translational N-glycosylation (44). supporting stromal cells in the tumor microenvironment by However, despite rapid clearance via mannose receptors, targeting cancer-associated fibroblasts (59). Fibroblast activation tumor-to-plasma ratios of 1,400:1 in the LS174T colon protein (FAP), a post-prolyl endopeptidase predominantly xenograft model and 339:1 in the SW122 colon xenograft expressed in cancer-associated fibroblasts with a limited pattern model were recorded (45). Since the fusion protein localized of expression in normal tissues has emerged as an interesting specifically to CEA-expressing xenografts in the mouse, the target (60). A FAP-activated peptidyl-TG prodrug killed FAP- prodrug was administered 6 h after administration of the positive human cancer cells at low nanomolar concentrations, fusion protein. Cycles could be repeated without obvious efficiently reduced growth of MCF-7 and LNCaP tumor toxicity (45). Consequently, this concept was explored in xenografts, concurrent with ablation of tumor stroma (61). clinical studies in heavily pre-treated patients with CEA- However, activation of the prodrug in plasma was observed expressing tumors. Treatment resulted in stable disease eight indicating a possible limitation of this approach (61). Ongoing weeks after therapy in 11 out of 28 patients (46). Three cycles efforts are based on activation of TG-based prodrugs by of treatment could be applied after administration of prostate-specific antigen and human kallikrein-2, proteolytic cyclosporine to suppress immune responses against the fusion enzymes secreted by the prostate with expression orders of

69 CANCER GENOMICS & PROTEOMICS 11 : 67-80 (2014) magnitude greater than in other tissues (50, 62). Inactivation of domain (Figure 1B and C). In order to achieve tumor-specific the enzymatic activity of prostate-specific antigen and human activation of CD95, CD95L is only unmasked after binding of kallikrein-2 in the circulation by serum proteins is an the fusion protein to FAP-positive tumor cells and subsequent encouraging aspect of this approach (63, 64). cleavage of the linker by tumor-associated proteases. The cleaved scFv-CD95L remains membrane-bound and thereby Protease-based Activation of Cytotoxic Receptor mimics membrane CD95L. It was shown that activation of Ligands and Other Cytotoxic Proteins CD95L in vitro is dependent on CD95 binding and protease- dependent processing. In vivo efficacy was demonstrated by Use of tumor necrosis factor (TNF) as an antitumoral agent is intra-tumoral injection of FAP-transfected HT1080 cells hampered by its severe systemic toxicity, allowing its clinical together with the prodrug one day after tumor inoculation with application only in the limb perfusion setting (65). Similarly, 1.5×10 6 cells. This resulted in 80% tumor growth inhibition CD95−CD95L interaction can be exploited for tumor therapy which was not observed when non-transfected HT1080 cells due to its pro-apoptotic effect after delivery of CD95L or an were co-injected. A critical issue of the approaches, as agonistic antibody to tumor cells. However, this approach is outlined, is the potential activation of the fusion proteins by also limited because of the induction of apoptosis in normal serum proteases. Therefore, efficient targeting of the tumor liver cells, resulting in acute liver failure and thus the necessity with these effector fusion moieties is mandatory because for local activiation of CD95L in tumor tissue (66). In order to expression of tumor-associated proteases is not strictly tumor- tackle these issues, an antibody-based targeting moiety was specific. fused to CD95L or TNF via a linker which can be cleaved by Mellitin is a 26 amino acid pore-forming protein derived tumor-associated proteases such as uPA, tissue type PA, MMP2 from honey bee venom (72). Formation of pores in the cell or MMP9, to effect specific tumor targeting. In the following membrane induces cell lysis. In order to target the cytotoxic examples, the antibody moiety is directed against FAP (67). activity of mellitin to MMP2-overexpressing tumor cells, a A TNF-selectokine, a homotrimeric fusion protein biologically-inactive fusion protein consisting of mellitin and consisting of a scFv antibody fragment directed against FAP avidin connected by an MMP2 cleavage site was designed as a targeting module, a trimerization domain derived from (73). This fusion protein mediated strong cytotoxic activity tenascin C, TNF, and a linker consisting of protease cleavage after activation by MMP2 in MMP2-overexpressing tumor sites followed by the extracellular domain (ECD) of TNF cells, while only little cytotoxicity was seen in cells with low receptor-1 (TNFR1) was evaluated (68). This cell-bound MMP2 activity. In vivo tumor shrinkage was also shown. selectokine mimics membrane-bound TNF. The unique feature Fusogenic membrane glycoproteins were identified in of this TNF selectokine is the presence of the ECD of TNFR1 several viruses inducing cell-cell fusion and syncytia as an intra-molecular inhibitor, thus creating a prodrug. The formation, which ultimately results in cytotoxicity and cell prodrug exerts minimal TNF activity, but can be activated death (74). The Gibbon Ape Leukemia Virus envelope several thousand-fold after cleavage by tissue-type glycoprotein (GALV) was assessed for its potential for plasminogen activator (tPA). Activated prodrug was shown to targeted killing of glioma cells. Thus, GALV was linked to the bear TNFR-dependent cytotoxic activity in targeted and extracellular domain of CD40 ligand as a binding blocking adjacent FAP-negative cells. Making use of the same species moiety, via an MMP or factor Xa protease-cleavable linker, or crossreactive anti-FAP moiety, analogous murine TNF a non-cleavable linker. Transfection of constructs with constructs were evaluated in which the natural processing site cleavable linkers selectively induced cytotoxicity in glioma for TNF α converting enzyme was mutated in order to avoid cells in contrast to non-cleavable constructs (75). Targeting in potential cleavage of the prodrug (69). Such cell surface- this case is necessary due to the ubiquitous expression of the targeted fusion proteins induced strong cytotoxicity in human GALV receptor, pituitary-specific positive transcription factor- HT1080 fibrosarcoma cells expressing MMPs and transfected 1 (PIT-1). Adenoviral vectors expressing fusion proteins, as with FAP. Antigen-positive and -negative cells were affected outlined above, were shown to exhibit significant antitumoral in a juxtatropic mode. These findings were extrapolated to potential for gene therapy of glioma gene therapy (76). human TNF prodrugs bearing either a uPA-selective or a dual uPA−MMP-2-specific linker (70). The same basic principle Protease-mediated Activation of Anthrax was applied to the design of CD95L-based prodrugs (71). Toxin for Tumor Therapy Again, FAP-transfected HT1080 cells were used for the in vitro and in vivo evaluation of the corresponding prodrug Bacillus anthracis , a bacterium that forms highly resistant molecules. These prodrugs are composed of an scFv-directed spores, can cause lethal infections after inhalation or ingestion. against FAP, followed by the ECD of CD95L, a protease- The causative agent for lethal infections is anthrax toxin which sensitive linker which can be cleaved by MMP-2 and related consists of three components which individually are non-toxic: proteases and the ECD of CD95 as carboxyterminal inhibitory protective antigen, lethal factor (LF) and edema factor (EF)

70 Weidle et al : Proteases as Mediators of Cytotoxicity in Tumor Therapy (Review)

Figure 1. Display of G202 (A) and cluster of differentiation 95 ligand (CD95L)-based (B) prodrugs. A: Schematic presentation of G202. The toxic entity thapsigargin (orange) is fused via an aliphatic linker (pink) to a cleavable prostate specific membrane antigen (PSMA)-specific peptide (grey). Protease cleavage sites are indicated by arrows. B: CD95L-based prodrug. The proteins are displayed as ribbons coloured according to domain function. The CD95L prodrug is composed of the CD95 receptor (magenta, pdbcode 3TJE) as the protecting moiety, followed by a trimerization coiled-coil domain of tenascin C (shown in green, helix modeling is based on pbdcode 2F3M), a cleavable linker (grey), a modeled scFv directed against fibroblast activation protein (FAP) (sc FAP) heavy and light variable domains displayed in dark and light blue and their corresponding linker shown in pink, a polyaspartate flag (red) and finally the CD95L domain as an effector (orange, pdbcode 4MSV). The lower left shows the cyclic configuration of the CD95L-based prodrug, bringing the ligand and the corresponding receptor together. The protease cleavage site is indicated by an arrow. The trimeric prodrug is shown in the middle and lower right. The head-to-tail configuration is not shown. The models were generated based on available structural data of domains or entities and were assembled and minimized using DiscoveryStudio40 (128, 129).

(77, 78). The unique processing and assembly of the processed by proteases such as furin, and the remaining components of the anthrax toxin was exploited in the design of receptor-bound moiety of protective antigen forms a ring- anti-tumoral agents and for the delivery of other toxins into shaped heptamer which transports EF, LF, or both of them into tumor cells. The action of anthrax toxin starts by binding of the cell to exert their cytotoxic function. The resulting protective antigen to two broadly-expressed receptors: tumor complexes are referred to as lethal toxin or edema toxin. These endothelial marker-8 and capillary morphogenesis protein-2 complexes are internalized by receptor-mediated endocytosis (79). After binding to its receptors, protective antigen is via a clathrin-dependent process, and after formation of a

71 CANCER GENOMICS & PROTEOMICS 11 : 67-80 (2014) channel in the membrane of the endosome, LF and EF are Granzyme B as Effector Moiety for transported into the cytosol. LF is a zinc-dependent Cytolytic Fusion Proteins metalloprotease that cleaves mitogen-activated protein kinase kinases (MAPKK). EF is a calmodulin-dependent adenylate Receptor−ligand or antibody-based moieties directed against cyclase which leads to intracellular elevation of adenosine- tumor antigens genetically fused to granzyme B as a cytotoxic monophosphate or cyclic adenosine monophosphate levels, principle are presently pre-clinically evaluated as anti-tumor thus interfering with the balance of intracellular signaling (79, agents (88-92). Granzyme B is member of a family of serine 80). Anti-angiogenic effects were observed for both LF and proteases with five different human granzymes (A, B, H, K and EF due to their effects on MAPKK inhibition and cAMP M). They are stored in the granules of natural killer cells and levels in endothelial cells. LF interferes with MAPK signaling cytotoxic T-lymphocytes. Granzyme B is an inducer of granule- in Ha-ras transformed NIH-3T3 cells, inhibits their growth in mediated apoptosis. Granzyme B cytolytic fusion proteins as soft-agar, reverts their transformed phenotype and inhibits described above are internalized and induce apoptosis after their growth in athymic mice, partly through inhibition of release from the endosome. Once in the cytosol, granzyme B angiogenesis (81). Human melanoma cells were shown to be can directly activate different apoptotic pathways and at equisitely sensitive to LF in combination with a small different levels, ensuring induction of apoptosis even when molecule MAPK inhibitor, inducing apoptosis, whereas some apoptotic pathways are blocked. Important granzyme B normal melanocytes were not killed but arrested in the G1- substrates include caspase-3 and other initiator and effector phase of the cell cycle (82). Due to the broad expression of caspases, the inhibitor of caspase-activated DNase, and BH3- the receptors, tumor-specific targeting of anthrax toxin is interacting domain death agonist (BID). In addition, granzyme essential to generate a therapeutic window. Activation of B can cleave further death-substrates such as poly(ADP modified anthrax toxins on the surface of tumor cells is a ribose)polymerase, DNA-dependent protein kinase, straightforward strategy. MMPs such as MMP2, MMP9, components of the cytoskeleton and the nuclear mitotic MMP14 and matriptase, as well as uPA are proteases which apparatus as well as proteins involved in stress response and are overexpressed on the surface of tumor cells or the cellular homeostasis. Numerous variations of granzyme B surrounding stromal cells. Replacement of the furin cleavage fusion proteins were studied. Transforming growth factor- α and site in protective antigen by those specific for MMPs or uPA vascular endothelial growth factor (VEGF) (Figure 2A) were leads to tumor-specific activation of protective antigen. the targeting domains in the context of ligand−granzyme B Making use of MMP-activated LF, antitumor activity was fusions, while CD64, GP240, Lewis Y antigen, human observed in tumor xenograft models harbouring the V600E epidermal growth factor receptor 2 (HER2) and human activating rapidly accelerated fibrosarcoma B (BRAF) luteinizing hormone receptor were evaluated as targets for mutation, but also in xenografts with non-mutated BRAF (83). antibody-based moieties of granzyme B-derived cytolytic In addition, anthrax toxin inhibits tumor angiogenesis by fusion proteins (88). Despite encouraging in vitro and in vivo interference with endothelial cell proliferation, migration and efficacy data in various tumor-derived cell lines and xenograft tube formation for which MAPK activation plays a crucial models, several problems preventing optimal efficacy of such role. Delivery of other toxin effector moieties such as the granzyme B-based cytolytic fusion proteins have emerged: (i) ADP-ribosylation domain of pseudomonas exotoxin into granzyme B inhibitor PI-9 is expressed in several tumor types tumor cells making use of the properties of the anthrax toxin and their corresponding cell lines, limiting efficacy; and (ii) complex is another potential application of this system. Thus, granzyme B possesses a strong positive surface charge which fusion of passenger proteins with amino acids 1-254 of LF causes non-specific binding to negatively charged cell surfaces. allows their translocation into, and eventually killing of cells This leads to adsorption and off-target effects, as well as expressing PA receptors with toxic effector molecules such as trapping of the internalized antigen−fusion proteins in the ADP-ribosylating moiety of pseudomonas exotoxin, the A endosomes, resulting in insufficient delivery of granzyme B subunit of diphtheria toxin, and the A subunit of Shiga toxin into the cytosol. Consequently, highly variable efficacy of the (84, 85, 86). Similarly, when amino acids 1-254 of LF were corresponding cytolytic fusion proteins was observed. One combined with mutated protective antigen proteins, the recent achievement is the generation of granzyme B mutants resulting fusion protein (FP59) mediated selective killing of insensitive to inhibition by PI-9 by mutating positions MMP-overexpressing tumor cells such as fibrosarcoma, breast interacting with the variable reactive center loop of PI-9, which and melanoma (83). Analogous observations were made with is its primary site of interaction with proteases. Importantly, uPA activated protective antigen which resulted in selective one of these variants, granzyme B R201K, exhibits the same killing of uPAR-expressing tumor cells (87). A critical issue enzymatic activity in the absence or presence of PI-9 (93). is that neither the MMP nor uPA-activation systems are strictly Mutation of sequences responsible for interaction with tumor-specific, thus side-effects after administration of these heparan-sulfate-containing molecules resulted in diminished agents are an issue. cell binding but had a negative impact on cytotoxicity (94).

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Figure 2. Display of a targeted granzyme B-based fusion protein and two different versions of a probody. A: Granzyme B-vascular endothelial growth factor 121 (VEGF121) fusion protein. The model is based on the structures of granzyme B (orange, pdbcode 3PW9) and VEGF121 (blue, pbdcode 1FQ4). The models were generated based on available structural data of domains or entities and were assembled and minimized using DiscoveryStudio40 (128, 129). B: Schematic presentation of a probody. The principle design is derived from a probody directed against vascular cellular adhesion molecule 1 (VCAM1) (117). The peptide with lid function (magenta) has affinity to the antibody paratope. Dark and light orange variable domains were taken from a non-related Fab complex (pdbcode 4RNRX), the peptide is linked via a flexible glycine-serine linker (pink) and a cleavable sequence (grey) to the heavy chain variable domain (VH domain). The constant part of the antibody is shown in grey. Protease cleavage sites are shown in grey. C: Schematic presentation of a bi-specific probody. Targeting to antigen 1 is mediated by the V domains shown in dark and light blue. The molecule is based on the knob-into-hole differentiation of the two fragment crystallization region (Fcs), highlighted in dark and light grey. V-domains of an antibody directed against antigen 2 are fused to the C-termini of the Fc portions by connectors with and without a protease cleavage site. VH and VL domains are disulfide bridged. Binding functionality is reconstituted after cleavage of one of the two linkers. The cleavable element is shown in grey and G4S in pink. The schematic presentation is based on the description of a bi-specific prodbody directed against human epidermal growth factor receptor 3 (HER3) and cellular proto-oncogene of mesenchymal epithelial transition factor (c MET) (120). The protease cleavage site is indicated by an arrow and the disulfide bridge connecting VL and VH domains is shown in green.

Residues 96-103 and 221-226 confer a positive surface charge penetrating peptides or membrane transfer sequences into the on mature granzyme B, causing interactions with corresponding cytolytic fusion proteins; and (iii) insertion of glycosaminoglycans. Substitution of such cationic residues was synthetic, multifunctional linkers, as outlined below (98). These shown to have no impact on the enzymatic and pro-apoptotic linkers are composed of a membrane transfer peptide flanked activity of granzyme B but reduced binding to antigen-negative by an endosomal cleavable peptide and a cytosolic cleavable cells (95). Improved efficacy of granzyme B-based cytolytic peptide (98). Potential immunogenicity after introduction of fusion proteins was achieved by co-administration with amino acid substitutions has not yet been properly investigated. chloroquine, which accumulates in acidic compartments and An interesting example for the improvement of efficacy is a lysosomes and causes osmotic rupture of these vesicles (95- fusion protein between a scFv directed against HER2, a 97). Further approaches for improved endosomal release are fusogenic pH-sensitive peptide, and granzyme B (99). Tumor the incorporation of (i) protein transduction domains derived cells resistant to lapatinib or Herceptin and expressing MDR1 from pseudomonas exotoxin or diphtheria toxin; (ii) cell- showed no crossresistance to the granzyme B-based fusion

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protein, and IC 50 s between 20 and 90 nM were noted for stability was reduced, resulting in complete cleavage of the various HER2-positive breast cancer cell lines. Significant adapter after 1 h, whereas the adapter-free fusion protein was tumor growth suppression was noted in vivo in mice bearing stable with no evidence for cleavage after 24 h. Consequently, BT474 breast tumors after i.v. administration (99). a fusion protein bearing a modified adapter with deleted endosomal cleavable peptide was evaluated. Thus, while Protease-cleavable Adapters for Improved cytotoxicity of this molecule was still 10-fold better than the Cytolytic Fusion Proteins adapter-free fusion protein, almost no cleavage was observed after 24 h in the presence of serum. Cytolytic fusion proteins are composed of a targeting module Finally, the findings as outlined above indicate that an and a protein-based cytotoxic moiety. The targeting module adapter cannot be transferred between fusion proteins by can be a ligand for a cell surface receptor or an antibody-based default to improve cytotoxicity and stability but has to be moiety, the therapeutic principle as a rule is an enzyme with optimized specifically for each fusion protein under cytotoxic properties. The concept of cleavable adapters was consideration. raised (98, 100) to improve the characteristics of cytolytic fusion proteins. The prototype adapter is composed of a Activation of Antibodies with Impaired cytosolic cleavable peptide, a cell-penetrating peptide (CPP) Functionality by Proteases which is a membrane transfer peptide, and an endosomal cleavable peptide (98). For example, a cytosolic cleavable Antibody-based therapies often exploit targets which play a peptide was designed by incorporating cleavage sites for crucial role in physiological processes and therefore can be caspases 1, 3 and 7 and a cytosolic protease from yeast into a associated with serious toxicities (108). For example, blockage corresponding peptide sequence (101). After cleavage, the of delta-like ligand 4, a notch ligand, leads to inhibition of compound is trapped within the cell, converting a formerly angiogenesis in pre-clinical models. However, liver pathology, cell-permeable compound into a cell-impermeable drug. CPPs gut and T-cell toxicity, and induction of vascular neoplasms can penetrate membranes by an energy-independent process were observed in mice and rats (109, 110, 111). Similarly, the or by an endocytosis-related process (102, 103). CPPs are of anti-angiogenic agent bevacizumab, a VEGF-neutralizing diverse origin, 10-16 amino acids long and can be grouped monoclonal antibody, can cause side-effects such as into peptides composed of basic amino acids such as poly- hypertension, proteinuria and gastrointestinal symptoms, and lysine or poly-arginine, α- helical, and β- sheet peptides (102, in rare cases, gastrointestinal perforation and arterial 103). Examples are TAT from HIV, penetratin from thromboembolic complications (112). Skin toxicity as dose- Drosophila homeotic transcription factor antennapedia, or the limiting toxicity was observed after treatment of cancer patients preS2-domain of hepatitis B virus surface protein (104). The with antibodies or antibody conjugates directed against EGFR function of membrane transfer peptides and CPPs is to (113, 114). An increased risk of serious infection and a dose- improve cellular uptake and deliver the cytotoxic molecule dependent risk for malignancies was observed in patients with directly into the cytosol. Endosomal cleavable peptides on the rheumatoid arthritis after anti-TNF antibody therapy (115). other hand are based on dedicated translocation sequences HER2-directed therapy with trastuzumab caused an incidence which are activated after protease cleavage and usually are of 0.2-3.8% of class III congestive heart failure in the adjuvant derived from diphtheria toxin or pseudomonas exotoxin (105). trastuzumab trials of patients with HER2-positive breast cancer. One exemplary adapter was designed to improve the uptake This was probably due to the expression of HER2 on and transport of the catalytic cytotoxic moiety into the cytosol cardiomyocytes in addition to tumor tissues (116). This list of and subsequently trap it in the cell. This trapping might be serious target-related side-effects could be extended to responsible for the reduced cytotoxicity which was associated numerous other examples. Therefore, the probody concept, with long circulation times and damage of target-negative cells activation of an inactive preform of an antibody by tumor- (98). Saporin linked to EGF with or without adapter was associated proteases was raised (117) (Figure 2B). Accordingly, evaluated, and reduced non-specific cytotoxicity was noted by a probody with masked antibody binding site was designed by inserting a cleavable adapter, as outlined (101). Similarly, tethering a peptide to the N-terminus of an antibody-derived treatment of human EGFR -transfected murine tumor cells with binding domain via a flexible linker containing a protease- adapter-containing and adapter-free fusion protein showed cleavage site in the context of a scFv-Fc format (117). In a enhanced efficacy for the adapter-containing fusion protein proof-of-concept experiment, such a probody directed against (106). Surprisingly, however, in vitro cytotoxicity was not vascular cell adhesion molecule-1, a marker of atherosclerotic affected by the insertion of an adapter (101). Another example plaques, was evaluated (117). In vitro activation of the probody is a fusion protein between a humanized scFv directed against with MMP1 resulted in a 200-fold increase of binding affinity CD64 and human ribonuclease angiogenin (107). Insertion of and restored binding to tissue sections from atherosclerotic an adapter improved cytotoxicity 20-fold, however, serum mice ex vivo demonstrating MMP-dependent probody

74 Weidle et al : Proteases as Mediators of Cytotoxicity in Tumor Therapy (Review) accumulation in aortic plaques. Analysis of frozen aorta tissue in active matriptase on the cell surface (121, 122). Using a sections from mice injected with such probodies revealed large recombinant human antibody against an epitope covering the plaques that stained strongly for the presence of the antibody to active site of matriptase, it was shown that tumor epithelium vascular cell adhesion molecule-1 and probody. Regarding can be visualized selectively (123, 124). Live cell fluorescence possible oncological applications of the probody technology, imaging showed that the antibody localized only to the surface antibodies against matriptase were successfully used for of matriptase-positive tumor cells, and immunofluorescence imaging of colon cancer xenografts, pointing to matriptase as a experiments with tissue microarray sections from primary and possible activator of probodies (118). Tumor-specific activation metastatic colon cancer indicated active matriptase in 68% of of therapeutic antibodies potentially results in an improved primary and metastatic colon cancer specimens (122). Near therapeutic index (119). For example, the EGFR-targeting infrared and single-photon emission computed tomographic antibody cetuximab was converted into a probody by fusing a imaging visualized matriptase in human colon cancer tumors masking peptide which blocks binding to EGFR to the N- in a patient-derived xenograft model (122). The antibody used terminus of the L-chain. The masking peptide (21 amino acids) in these studies was crossreactive with the murine matriptase was fused to a cleavable linker (26 amino acids), sensitive to ortholog epithin, and radiolabeled antibody was taken-up by uPA, matriptase, and legumain. This probody possessed in vitro tumor xenografts. Specificity of the interaction was activity equivalent to cetuximab, was activated ex vivo by demonstrated by inhibition of uptake of the antibody after xenograft tumors, and suppressed tumor growth in vivo in injection of ecotin, a macromolecular inhibitor of matriptase, mouse xenograft models with efficacy equivalent to cetuximab. before injection of the radiolabeled antibody. Accordingly, However, due to the lack of crossreactivity of cetuximab with active site probes may become important tools for the rodent EGFR, final conclusions are limited. This probody was elucidation of the role of proteases in the pathogenesis of shown to be activated by human tumor samples in vitro . The different types of cancer. probody was nearly inert in the circulation and possessed markedly improved safety with respect to cutaneous toxicity Concluding Remarks and increased plasma half-life in non-human primates. Therefore, this probdody could be dosed safely at much higher Personalized medicine is one of the key issues of cancer levels than cetuximab (119). Finally, proof-of-concept therapy (125-127) meaning that patients are treated based on experiments have shown that bi-specific antibodies with molecular characteristics as revealed by diagnostic tests restricted binding functionality can be activated by proteolytic and/or high throughput sequencing. Overexpressed plasma processing (120). The principal design of such a bi-specific membrane-associated receptors, oncogenic driver mutations probody is shown in Figure 2C. A bispecific antibody which and other deregulated cancer promoting pathways are targets binds HER3 in a conventional, bivalent IgG-like manner and for small molecule- or antibody-derived therapeutics, which carries in addition a cellular proto-oncogene of resulting in inhibition of migration, induction of apoptosis, mesenchymal epithelial transition factor (C-MET) binding inhibition of angiogenesis, stimulation of an antitumoral moiety composed of a variable heavy chain (VH)- and a immune response, or synthetic lethality. Knowledge about, variable light chain (VL)-domain which can be connected by and specification of, the expression pattern of proteases in a disulfide bond was designed (120). The H- and L- chain tumors, and quantification of the ratio of inactive domains are linked to the C-termini of the constant heavy chain proenzymes versus enzymatically active ones in individual 3 domains, respectively. One of the connectors contains a tumors could advise treatment with prodrugs activated by the protease cleavage site for MMP2, MMP9 or uPA. After proteases characteristic of the individual tumors. Finally, cleavage of the protease-sensitive connector, steric hindrance these findings will also have an impact on optimal tumor is resolved and antigen access and affinity in vitro is fully imaging. A further future application is the administration of restored. cytolytic fusion proteins with proteases such as granzyme B as an effector moiety. Proteases and Imaging References Tumor-associated expression of enzymatically active matriptase has been exploited for tumor imaging (122). 1 López-Otín C and Matrisian LM: Emerging roles of proteases in Matriptase is a type II transmembrane protease of the serine tumour suppression. Nat Rev Cancer 7: 800-808, 2007. 2 Coussens LM, Fingleton B and Matrisian LM: Matrix protease family. It is found on the surface of epithelial cells metalloproteinase inhibitors and cancer: Trials and tribulations. and its activity is tightly regulated by its cognate inhibitor, Science 295 : 2387-2392, 2002. hepatocyte growth factor activator inhibitor-1. In normal 3 Overall CM and López-Otín C: Strategies for MMP inhibition in tissues, the ratio of matriptase to its inhibitor is low. This ratio cancer: innovations for the post-trial era. Nat Rev Cancer 2: 657- increases during progression of some types of cancer, resulting 672, 2002.

75 CANCER GENOMICS & PROTEOMICS 11 : 67-80 (2014)

4 Hadler-Olsen E, Winberg JO and Uhlin-Hansen L: Matrix 22 Seymour LW, Ferry DR, Anderson D, Hesslewood S, Julyan PJ, metalloproteinases in cancer: Their value as diagnostic and Poyner R, Doran J, Young AM, Burtles S and Kerr DJ: Hepatic prognostic markers and therapeutic targets. Tumour Biol 34 : drug targeting: Phase I evaluation of polymer-bound doxorubicin. 2041-2051, 2013. J Clin Oncol 20 : 1668-1676, 2002. 5 Vartak DG and Gemeinhart RA: Matrix metalloproteases: 23 Shaffer SA, Baker-Lee C, Kennedy J, Lai MS, de Vries P, Buhler Underutilized targets for drug delivery. J Drug Target 15 : 1-20, K and Singer JW: In vitro and in vivo metabolism of paclitaxel 2007. poliglumex: I dentification of metabolites and active proteases. 6 Mohamed MM and Sloane BF: Cysteine cathepsins: Cancer Chemother Pharmacol 59 : 537-548, 2007. Multifunctional enzymes in cancer. Nat Rev Cancer 6: 764- 24 Chipman SD, Oldham FB, Pezzoni G and Singer JW: Biological 775, 2006. and clinical characterization of paclitaxel poliglumex (PPX, CT- 7 D’Alessio S and Blasi F: The urokinase receptor as an entertainer 2103), a macromolecular polymer-drug conjugate. Int J of signal transduction. Front Biosci (Landmark Ed) 14 : 4575- Nanomedicine 1: 375-383, 2006. 4587, 2009. 25 Paz-Ares L, Ross H, O’Brien M, Riviere A, Gatzemeier U, Von 8 Schmitt M, Harbeck N, Thomssen C, Wilhelm O, Magdolen V, Pawel J, Kaukel E, Freitag L, Digel W, Bischoff H, García- Reuning U, Ulm K, Höfler H, Jänicke F and Graeff H: Clinical Campelo R, Iannotti N, Reiterer P, Bover I, Prendiville J, impact of the plasminogen activation system in tumor invasion Eisenfeld AJ, Oldham FB, Bandstra B, Singer JW and Bonomi and metastasis: Prognostic relevance and target for therapy. P: Phase III trial comparing paclitaxel poliglumex vs. docetaxel Thromb Haemost 78 : 285-296, 1997. in the second-line treatment of non-small-cell lung cancer. Br J 9 Min HY, Doyle LV, Vitt CR, Zandonella CL, Stratton-Thomas Cancer 98 : 1608-1613, 2008. JR, Shuman MA and Rosenberg S: Urokinase receptor 26 Ducry L and Stump B: Antibody-drug conjugates: Linking antagonists inhibit angiogenesis and primary tumor growth in cytotoxic payloads to monoclonal antibodies. Bioconjug Chem syngeneic mice. Cancer Res 56 : 2428-2433, 1996. 21 : 5-13, 2010. 10 Blasi F and Carmeliet P: uPAR: A versatile signalling 27 Sassoon I and Blanc V: Antibody-drug conjugate (ADC) clinical orchestrator. Nat Rev Mol Cell Biol 3: 932-943, 2002. pipeline: A review. Methods Mol Biol 1045 : 1-27, 2013. 11 Oikonomopoulou K, Diamandis EP and Hollenberg MD: 28 Erickson HK, Park PU, Widdison WC, Kovtun YV, Garrett LM, Kallikrein-related peptidases: proteolysis and signaling in cancer, Hoffman K, Lutz RJ, Goldmacher VS and Blättler WA: the new frontier. Biol Chem 391 : 299-310, 2010. Antibody-maytansinoid conjugates are activated in targeted 12 Murphy G: The ADAMs: Signalling scissors in the tumour cancer cells by lysosomal degradation and linker-dependent microenvironment. Nat Rev Cancer 8: 929-941, 2008. intracellular processing. Cancer Res 66 : 4426-4433, 2006. 13 Benes P, Vetvicka V and Fusek M: Cathepsin D−many functions of 29 Widdison WC, Wilhelm SD, Cavanagh EE, Whiteman KR, Leece one aspartic protease. Crit Rev Oncol Hematol 68 : 12-28, 2008. BA, Kovtun Y, Goldmacher VS, Xie H, Steeves RM, Lutz RJ, 14 Koblinski JE, Ahram M and Sloane BF: Unraveling the role of Zhao R, Wang L, Blättler WA and Chari RV: Semisynthetic proteases in cancer. Clin Chim Acta 291 : 113-135, 2000. maytansine analogues for the targeted treatment of cancer. J Med 15 Danø K, Andreasen PA, Grøndahl-Hansen J, Kristensen P, Chem 49 : 4392-4408, 2006. Nielsen LS and Skriver L: Plasminogen activators, tissue 30 Carter PJ and Senter PD: Antibody−drug conjugates for cancer degradation, and cancer. Adv Cancer Res 44 : 139-266, 1985. therapy. Cancer J 14 : 154-169, 2008. 16 Baramova EN, Bajou K, Remacle A, L’Hoir C, Krell HW, Weidle 31 Beck A, Haeuw JF, Wurch T, Goetsch L, Bailly C and Corvaïa UH, Noel A and Foidart JM: Involvement of PA/plasmin system N: The next generation of antibody−drug conjugates comes of in the processing of pro-MMP-9 and in the second step of pro- age. Discov Med 10 : 329-339, 2010. MMP-2 activation. FEBS Lett 405 : 157-162, 1997. 32 Senter PD and Sievers EL: The discovery and development of 17 Choi KY, Swierczewska M, Lee S and Chen X: Protease- brentuximab vedotin for use in relapsed Hodgkin lymphoma and activated drug development. Theranostics 2: 156-178, 2012. systemic anaplastic large cell lymphoma. Nat Biotechnol 30 : 18 Seymour LW: Passive tumor targeting of soluble macromolecules 631-637, 2012. and drug conjugates. Crit Rev Ther Drug Carrier Syst 9: 135-187, 33 Minich SS: Brentuximab vedotin: A new age in the treatment of 1992. Hodgkin lymphoma and anaplastic large cell lymphoma. Ann 19 Duncan R, Kopecek J, Rejmanová P and Lloyd JB: Targeting of Pharmacother 46 : 377-383, 2012. N-(2-hydroxypropyl)methacrylamide copolymers to liver by 34 Naumovski L and Junutula JR: Glembatumumab vedotin, a incorporation of galactose residues. Biochim Biophys Acta 755 : conjugate of an anti-glycoprotein non-metastatic melanoma 518-521, 1983. protein B mAb and monomethyl auristatin E for the treatment of 20 Seymour LW, Ulbrich K, Steyger PS, Brereton M, Subr V, melanoma and breast cancer. Curr Opin Mol Ther 12 : 248-257, Strohalm J and Duncan R: Tumour tropism and anti-cancer 2010. efficacy of polymer-based doxorubicin prodrugs in the treatment 35 Keir CH and Vahdat LT: The use of an antibody−drug conjugate, of subcutaneous murine B16F10 melanoma. Br J Cancer 70 : 636- glembatumumab vedotin (CDX-011), for the treatment of breast 641, 1994. cancer. Expert Opin Biol Ther 12 : 259-263, 2012. 21 Seymour LW, Ferry DR, Kerr DJ, Rea D, Whitlock M, Poyner 36 Hoashi T, Sato S, Yamaguchi Y, Passeron T, Tamaki K and R, Boivin C, Hesslewood S, Twelves C, Blackie R, Schatzlein A, Hearing VJ: Glycoprotein nonmetastatic melanoma protein b, a Jodrell D, Bissett D, Calvert H, Lind M, Robbins A, Burtles S, melanocytic cell marker, is a melanosome-specific and Duncan R and Cassidy J: Phase II studies of polymer- proteolytically released protein. FASEB J 24 : 1616-1629, 2010. doxorubicin (PK1, FCE28068) in the treatment of breast, lung 37 Wang X, Ma D, Olson WC and Heston WD: In vitro and in vivo and colorectal cancer. Int J Oncol 34 : 1629-1636, 2009. responses of advanced prostate tumors to PSMA ADC, an

76 Weidle et al : Proteases as Mediators of Cytotoxicity in Tumor Therapy (Review)

auristatin-conjugated antibody to prostate-specific membrane 54 Christensen SB, Andersen A, Kromann H, Treiman M, Tombal antigen. Mol Cancer Ther 10 : 1728-1739, 2011. B, Denmeade S and Isaacs JT: Thapsigargin analogues for 38 Akhtar NH, Pail O, Saran A, Tyrell L and Tagawa ST: Prostate- targeting programmed death of androgen-independent prostate specific membrane antigen-based therapeutics. Adv Urol 973820, cancer cells. Bioorg Med Chem 7: 1273-1280, 1999. 2012. 55 Denmeade SR, Mhaka AM, Rosen DM, Brennen WN, Dalrymple 39 Andrady C, Sharma SK and Chester KA: Antibody-enzyme fusion S, Dach I, Olesen C, Gurel B, Demarzo AM, Wilding G, proteins for cancer therapy. Immunotherapy 3: 193-211, 2011. Carducci MA, Dionne CA, Møller JV, Nissen P, Christensen SB 40 Tietze LF and Schmuck K: Prodrugs for targeted tumor therapies: and Isaacs JT: Engineering a prostate-specific membrane antigen- Recent developments in ADEPT, GDEPT and PMT. Curr Pharm activated tumor endothelial cell prodrug for cancer therapy. Sci Des 17 : 3527-3547, 2011. Transl Med 4: 140ra86, 2012. 41 Melton RG and Sherwood RF: Antibody−enzyme conjugates for 56 Pinto JT, Suffoletto BP, Berzin TM, Qiao CH, Lin S, Tong WP, cancer therapy. J Natl Cancer Inst 88: 153-165, 1996. May F, Mukherjee B and Heston WD: Prostate-specific 42 Bagshawe KD: Antibody-directed enzyme prodrug therapy membrane antigen: a novel folate hydrolase in human prostatic (ADEPT) for cancer. Expert Rev Anticancer Ther 6: 1421-1431, carcinoma cells. Clin Cancer Res 2: 1445-1451, 1996. 2006. 57 Carter RE, Feldman AR and Coyle JT: Prostate-specific 43 Springer CJ, Antoniw P, Bagshawe KD, Searle F, Bisset GM and membrane antigen is a hydrolase with substrate and Jarman M: Novel prodrugs which are activated to cytotoxic pharmacologic characteristics of a neuropeptidase. Proc Natl alkylating agents by carboxypeptidase G2. J Med Chem 33 : 677- Acad Sci USA 93 : 749-753, 1996. 681, 1990. 58 Chang SS, O’Keefe DS, Bacich DJ, Reuter VE, Heston WD and 44 Medzihradszky KF, Spencer DI, Sharma SK, Bhatia J, Pedley Gaudin PB: Prostate-specific membrane antigen is produced in RB, Read DA, Begent RH and Chester KA: Glycoforms obtained tumor-associated neovasculature. Clin Cancer Res 5: 2674-2681, by expression in Pichia pastoris improve cancer targeting 1999. potential of a recombinant antibody−enzyme fusion protein. 59 Brennen WN, Isaacs JT and Denmeade SR: Rationale behind Glycobiology 14 : 27-37, 2004. targeting fibroblast activation protein-expressing carcinoma- 45 Sharma SK, Pedley RB, Bhatia J, Boxer GM, El-Emir E, Qureshi associated fibroblasts as a novel chemotherapeutic strategy. Mol U, Tolner B, Lowe H, Michael NP, Minton N, Begent RH and Cancer Ther 11 : 257-266, 2012. Chester KA: Sustained tumor regression of human colorectal 60 Wolf BB, Quan C, Tran T, Wiesmann C and Sutherlin D: On the cancer xenografts using a multifunctional mannosylated fusion edge of validation-cancer protease fibroblast activation protein. protein in antibody-directed enzyme prodrug therapy. Clin Mini Rev Med Chem 8: 719-727, 2008. Cancer Res 11 : 814-825, 2005. 61 Brennen WN, Rosen DM, Wang H, Isaacs JT and Denmeade SR: 46 Mayer A, Francis RJ, Sharma SK, Tolner B, Springer CJ, Martin Targeting carcinoma-associated fibroblasts within the tumor J, Boxer GM, Bell J, Green AJ, Hartley JA, Cruickshank C, Wren stroma with a fibroblast activation protein-activated prodrug. J J, Chester KA and Begent RH: A phase I study of single Natl Cancer Inst 104 : 1320-1334, 2012. administration of antibody-directed enzyme prodrug therapy with 62 Olsson AY, Bjartell A, Lilja H and Lundwall A: Expression of the recombinant anti-carcinoembryonic antigen antibody-enzyme prostate-specific antigen (PSA) and human glandular kallikrein fusion protein MFECP1 and a bis- iodo phenol mustard prodrug. 2 (hK2) in ileum and other extraprostatic tissues. Int J Cancer Clin Cancer Res 12 : 6509-6516, 2006. 113 : 290-297, 2005. 47 Bagshawe KD and Sharma SK: Cyclosporine delays host 63 Otto A, Bär J and Birkenmeier G: Prostate-specific antigen forms immune response to antibody−enzyme conjugate in ADEPT. complexes with human alpha 2-macroglobulin and binds to the Transplant Proc 28 : 3156-3158, 1996. alpha 2-macroglobulin receptor/LDL receptor-related protein. J 48 Chester KA, Baker M and Mayer A: Overcoming the Urol 159 : 297-303, 1998. immunologic response to foreign enzymes in cancer therapy. 64 Mikolajczyk SD, Millar LS, Kumar A and Saedi MS: Human Expert Rev Clin Immunol 1: 549-559, 2005. glandular kallikrein, hK2, shows arginine-restricted specificity 49 Denmeade SR and Isaacs JT: Engineering enzymatically activated and forms complexes with plasma protease inhibitors. Prostate ‘molecular grenades’ for cancer. Oncotarget 3: 666-667, 2012. 34 : 44-50, 1998. 50 Christensen SB, Skytte DM, Denmeade SR, Dionne C, Møller 65 Eggermont AM and ten Hagen TL: Isolated limb perfusion for JV, Nissen P and Isaacs JT: A Trojan horse in drug development: extremity soft-tissue sarcomas, in-transit metastases, and other targeting of thapsigargins towards prostate cancer cells. unresectable tumors: credits, debits, and future perspectives. Curr Anticancer Agents Med Chem 9: 276-294, 2009. Oncol Rep 3: 359-367, 2001. 51 Thastrup O, Cullen PJ, Drøbak BK, Hanley MR and Dawson AP: 66 Wajant H, Gerspach J and Pfizenmaier K: Tumor therapeutics by Thapsigargin, a tumor promoter, discharges intracellular Ca 2+ design: Targeting and activation of death receptors. Cytokine stores by specific inhibition of the endoplasmic reticulum Ca 2+ - Growth Factor Rev 16 : 55-76, 2005. ATPase. Proc Natl Acad Sci USA 87 : 2466-2470, 1990. 67 Kelly T: Fibroblast activation protein-alpha and dipeptidyl 52 Breckenridge DG, Germain M, Mathai JP, Nguyen M and Shore peptidase IV (CD26): cell-surface proteases that activate cell GC: Regulation of apoptosis by endoplasmic reticulum pathways. signaling and are potential targets for cancer therapy. Drug Resist Oncogene 22 : 8608-8618, 2003. Updat 8: 51-58, 2005. 53 Denmeade SR, Jakobsen CM, Janssen S, Khan SR, Garrett ES, 68 Wüest T, Gerlach E, Banerjee D, Gerspach J, Moosmayer D and Lilja H, Christensen SB and Isaacs JT: Prostate-specific antigen- Pfizenmaier K: TNF-Selectokine: A novel prodrug generated for activated thapsigargin prodrug as targeted therapy for prostate tumor targeting and site-specific activation of tumor necrosis cancer. J Natl Cancer Inst 95 : 990-1000, 2003. factor. Oncogene 21 : 4257-4265, 2002.

77 CANCER GENOMICS & PROTEOMICS 11 : 67-80 (2014)

69 Gerspach J, Müller D, Münkel S, Selchow O, Nemeth J, Noack 84 Arora N, Klimpel KR, Singh Y and Leppla SH: Fusions of M, Petrul H, Menrad A, Wajant H and Pfizenmaier K: anthrax toxin lethal factor to the ADP-ribosylation domain of Restoration of membrane TNF-like activity by cell surface Pseudomonas exotoxin A are potent cytotoxins which are targeting and matrix metalloproteinase-mediated processing of a translocated to the cytosol of mammalian cells. J Biol Chem TNF prodrug. Cell Death Differ 13 : 273-284, 2006. 267 : 15542-15548, 1992. 70 Gerspach J, Németh J, Münkel S, Wajant H and Pfizenmaier K: 85 Milne JC, Blanke SR, Hanna PC and Collier RJ: Protective Target-selective activation of a TNF prodrug by urokinase-type antigen-binding domain of anthrax lethal factor mediates plasminogen activator (uPA) mediated proteolytic processing at translocation of a heterologous protein fused to its amino- or the cell surface. Cancer Immunol Immunother 55 : 1590-1600, carboxy-terminus. Mol Microbiol 15 : 661-666, 1995. 2006. 86 Arora N and Leppla SH: Fusions of anthrax toxin lethal 71 Watermann I, Gerspach J, Lehne M, Seufert J, Schneider B, factor with shiga toxin and diphtheria toxin enzymatic Pfizenmaier K and Wajant H: Activation of CD95L fusion protein domains are toxic to mammalian cells. Infect Immun 62 : prodrugs by tumor-associated proteases. Cell Death Differ 14 : 4955-4961, 1994. 765-774, 2007. 87 Liu S, Bugge TH and Leppla SH: Targeting of tumor cells by 72 Hristova K, Dempsey CE and White SH: Structure, location, and cell surface urokinase plasminogen activator-dependent anthrax lipid perturbations of melittin at the membrane interface. Biophys toxin. J Biol Chem 276 : 17976-17984, 2001. J 80: 801-811, 2001. 88 Weidle UH, Georges G and Brinkmann U: Fully human 73 Holle L, Song W, Holle E, Wei Y, Wagner T and Yu X: A matrix targeted cytotoxic fusion proteins: New anticancer agents on the metalloproteinase 2 cleavable melittin/avidin conjugate horizon. Cancer Genomics Proteomics 9: 119-133, 2012. specifically targets tumor cells in vitro and in vivo . Int J Oncol 89 Hehmann-Titt G, Schiffer S, Berges N, Melmer G and Barth S: 22 : 93-98, 2003. Improving the therapeutic potential of human granzyme B for 74 Bateman A, Bullough F, Murphy S, Emiliusen L, Lavillette D, targeted cancer therapy. Antibodies 2: 19-49, 2013. Cosset FL, Cattaneo R, Russell SJ and Vile RG: Fusogenic 90 Oberoi P, Jabulowsky RA and Wels WS: Selective induction of membrane glycoproteins as a novel class of genes for the local cancer cell death by targeted granzyme B. Antibodies 2: 130- and immune-mediated control of tumor growth. Cancer Res 60 : 151, 2013. 1492-1497, 2000. 91 Kurschus FC and Jenne DE: Delivery and therapeutic potential 75 Johnson KJ, Peng KW, Allen C, Russell SJ and Galanis E: of human granzyme B. Immunol Rev 235 : 159-171, 2010. Targeting the cytotoxicity of fusogenic membrane glycoproteins 92 Rosenblum MG and Barth S: Development of novel, highly in gliomas through protease-substrate interaction. Gene Ther 10 : cytotoxic fusion constructs containing granzyme B: Unique 725-732, 2003. mechanisms and functions. Curr Pharm Des 15 : 2676-2692, 76 Allen C, McDonald C, Giannini C, Peng KW, Rosales G, Russell 2009. SJ and Galanis E: Adenoviral vectors expressing fusogenic 93 Losasso V, Schiffer S, Barth S and Carloni P: Design of human membrane glycoproteins activated via matrix metalloproteinase granzyme B variants resistant to serpin B9. Proteins 80 : 2514- cleavable linkers have significant antitumor potential in the gene 2522, 2012. therapy of gliomas. J Gene Med 6: 1216-1227, 2004. 94 Bird CH, Sun J, Ung K, Karambalis D, Whisstock JC, Trapani 77 Liu S, Schubert RL, Bugge TH and Leppla SH: Anthrax toxin: JA and Bird PI: Cationic sites on granzyme B contribute to structures, functions and tumour targeting. Expert Opin Biol Ther cytotoxicity by promoting its uptake into target cells. Mol Cell 3: 843-853, 2003. Biol 25 : 7854-7867, 2005. 78 Abrami L, Liu S, Cosson P, Leppla SH and van der Goot FG: 95 Jabulowsky RA, Oberoi P, Bähr-Mahmud H, Dälken B and Anthrax toxin triggers endocytosis of its receptor via a lipid raft- Wels WS: Surface charge-modification prevents sequestration mediated clathrin-dependent process. J Cell Biol 160 : 321-328, and enhances tumor-cell specificity of a recombinant granzyme 2003. B-TGF α fusion protein. Bioconjug Chem 23 : 1567-1576, 2012. 79 Cryan LM and Rogers MS. Targeting the anthrax receptors, 96 Kurschus FC, Fellows E, Stegmann E and Jenne DE: Granzyme TEM-8 and CMG-2, for anti-angiogenic therapy. Front Biosci B delivery via perforin is restricted by size, but not by heparan (Landmark Ed) 16 : 1574-1588, 2011. sulfate-dependent endocytosis. Proc Ntl Acad Sci USA 105 : 80 Shapira A and Benhar I: Toxin-based therapeutic approaches. 13799-13804, 2008 Toxins 2: 2519-2583, 2010. 97 Kurschus FC, Kleinschmidt M, Fellows E, Dornmair K, 81 Duesbery NS, Webb CP, Leppla SH, Gordon VM, Klimpel KR, Rudolph R, Lilie H and Jenne DE: Killing of target cells by Copeland TD, Ahn NG, Oskarsson MK, Fukasawa K, Paull redirected granzyme B in the absence of perforin. FEBS Lett KD and Vande Woude GF. Proteolytic inactivation of MAP- 562 : 87-92, 2004. kinase-kinase by anthrax lethal factor. Science 280 : 734-737, 98 Keller J, Heisler I, Tauber R and Fuchs H: Development of a 1998. novel molecular adapter for the optimization of immunotoxins. 82 Koo HM, VanBrocklin M, McWilliams MJ, Leppla SH, J Control Release 74 : 259-261, 2001. Duesbery NS and Vande Woude GF. Apoptosis and 99 Cao Y, Mohamedali KA, Marks JW, Cheung LH, Hittelman melanogenesis in human melanoma cells induced by anthrax WN and Rosenblum MG: Construction and characterization of lethal factor inactivation of mitogen-activated protein kinase novel, completely human serine protease therapeutics targeting kinase. Proc Natl Acad Sci USA 99 : 3052-3057, 2002. HER2/NEU. Mol Cancer Ther 12 : 979-991, 2013. 83 Liu S, Netzel-Arnett S, Birkedal-Hansen H and Leppla SH: 100 Hetzel C, Bachran C, Tur MK, Fuchs H and Stöcker M: Tumor cell-selective cytotoxicity of matrix metalloproteinase- Improved immunotoxins with novel functional elements. Curr activated anthrax toxin. Cancer Res 60 : 6061-6067, 2000. Pharm Des 15 : 2700-2711, 2009.

78 Weidle et al : Proteases as Mediators of Cytotoxicity in Tumor Therapy (Review)

101 Heisler I, Keller J, Tauber R, Sutherland M and Fuchs H: A 118 LeBeau AM, Lee M, Murphy ST, Hann BC, Warren RS, Delos cleavable adapter to reduce nonspecific cytotoxicity of Santos R, Kurhanewicz J, Hanash SM, VanBrocklin HF and recombinant immunotoxins. Int J Cancer 103 : 277-282, 2003. Craik CS: Imaging a functional tumorigenic biomarker in the 102 Säälik P, Elmquist A, Hansen M, Padari K, Saar K, Viht K, transformed epithelium. Proc Natl Acad Sci USA 110 : 93-98, Langel U and Pooga M: Protein cargo delivery properties of cell- 2013. penetrating peptides. A comparative study. Bioconjug Chem 15 : 119 Desnoyers LR, Vasiljeva O, Richardson JH, Yang A, Menendez 1246-1253, 2004. EEM, Liang TW, Wong C, Bessette PH, Kamath K, Moore SJ, 103 Jones AT: Gateways and tools for drug delivery: endocytic Sagert JG, Hofstetter DR, Han F, Gee J, Flandez J, Markham pathways and the cellular dynamics of cell penetrating peptides. K, Nguyen M, Krimm M, Wong KR, Liu S, Daugherty, West Int J Pharm 354 : 34-38, 2008. JW and Lowman HB: Tumor-specific activation of EGFR- 104 Fuchs H, Bachran C, Heisler I and Sutherland M: A closer look targeting probody enhances therapeutic index. Sci Transl Med at protein transduction domains as a tool in drug delivery. Curr 5: 207ra144, 2013. Nanosci 1: 117-124, 2005. 120 Metz S, Panke C, Haas AK, Schanzer J, Lau W, Croasdale R, 105 Hudson TH and Neville DM Jr: Enhancement of immunotoxin Hoffmann E, Schneider B, Auer J, Gassner C, Bossenmaier B, action: manipulation of the cellular routing of proteins. Cancer Umana P, Sustmann C and Brinkmann U: Bispecific antibody Treat Res 37 : 371-389, 1988. derivatives with restricted binding functionalities that are 106 Fuchs H, Bachran C, Li T, Heisler I, Dürkop H and Sutherland activated by proteolytic processing. Protein Eng Des Sel 25 : 571- M: A cleavable molecular adapter reduces side effects and 580, 2012. concomitantly enhances efficacy in tumor treatment by targeted 121 Parr C, Sanders AJ and Jiang WG: Hepatocyte growth factor toxins in mice. J Control Release 117 : 342-350, 2007. activation inhibitors − therapeutic potential in cancer. Anticancer 107 Hetzel C, Bachran C, Fischer R, Fuchs H, Barth S and Stöcker Agents Med Chem 10 : 47-57, 2010. M: Small cleavable adapters enhance the specific cytotoxicity of 122 Saleem M, Adhami VM, Zhong W, Longley BJ, Lin CY, Dickson a humanized immunotoxin directed against CD64-positive cells. RB, Reagan-Shaw S, Jarrard DF and Mukhtar H: A novel J Immunother 31 : 370-376, 2008. biomarker for staging human prostate adenocarcinoma: 108 Hansel TT, Kropshofer H, Singer T, Mitchell JA and George AJ: overexpression of matriptase with concomitant loss of its The safety and side effects of monoclonal antibodies. Nat Rev inhibitor, hepatocyte growth factor activator inhibitor-1. Cancer Drug Discov 9: 325-338, 2010. Epidemiol Biomarkers Prev 15 : 217-227, 2006. 109 Yan M, Callahan CA, Beyer JC, Allamneni KP, Zhang G, 123 Sun J, Pons J and Craik CS: Potent and selective inhibition of Ridgway JB, Niessen K and Plowman GD: Chronic DLL4 membrane-type serine protease 1 by human single-chain blockade induces vascular neoplasms. Nature 463 : E6-7, 2010. antibodies. Biochemistry 42 : 892-900, 2003. 110 Wu Y, Cain-Hom C, Choy L, Hagenbeek TJ, de Leon GP, Chen 124 Farady CJ, Egea PF, Schneider EL, Darragh MR and Craik CS: Y, Finkle D, Venook R, Wu X, Ridgway J, Schahin-Reed D, Dow Structure of a Fab-protease complex reveals a highly specific GJ, Shelton A, Stawicki S, Watts RJ, Zhang J, Choy R, Howard non-canonical mechanism of inhibition. J Mol Biol 380 : 351- P, Kadyk L, Yan M, Zha J, Callahan CA, Hymowitz SG and 360, 2008. Siebel CW: Therapeutic antibody targeting of individual Notch 125 Hayden, C.E: News. Personalized cancer therapy gets closer. receptors. Nature 464 : 1052-1057, 2010. Genetic testing allows doctors to select best treatment. Nature 111 Li JL, Jubb AM and Harris AL: Targeting DLL4 in tumors shows 458 : 131-132, 2009. preclinical activity but potentially significant toxicity. Future 126 Simon R and Roychowdhury S: Implementing personalized Oncol 6: 1099-1103, 2010. cancer genomics in clinical trials. Nat Rev Drug Discov 12 : 358- 112 Kamba T and McDonald DM: Mechanisms of adverse effects of 369, 2013. anti-VEGF therapy for cancer. Br J Cancer 96 : 1788-1795, 2007. 127 Martini M, Vecchione L, Siena S, Tejpar S and Bardelli A: 113 Segaert S and Van Cutsem E: Clinical signs, pathophysiology and Targeted therapies: how personal should we go? Nat Rev Clin management of skin toxicity during therapy with epidermal growth Oncol 9: 87-97, 2011. factor receptor inhibitors. Ann Oncol 16 : 1425-1433, 2005. 128 Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, 114 Tijink BM, Buter J, de Bree R, Giaccone G, Lang MS, Staab A, Weissig H, Shindyalov IN and Bourne PE: The Protein Data Leemans CR and van Dongen GA: A phase I dose escalation Bank (www.pdb.org). Nucleic Acids Res 28 : 235-242, 2000. study with anti-CD44v6 bivatuzumab mertansine in patients with 129 Accelrys Software Incorp., Discovery Studio Modeling incurable squamous cell carcinoma of the head and neck or Environment, Release 3.1, San Diego: Accelrys Software Inc., esophagus. Clin Cancer Res 12 : 6064-6072, 2006. 2007. 115 Bongartz T, Sutton AJ, Sweeting MJ, Buchan I, Matteson EL and Montori V: Anti-TNF antibody therapy in rheumatoid arthritis and the risk of serious infections and malignancies: systematic review and meta-analysis of rare harmful effects in randomized controlled trials. JAMA 295 : 2275-2285, 2006. 116 Perez EA: Cardiac toxicity of ErbB2-targeted therapies: What do we know? Clin Breast Cancer 8(Suppl 3) : S114-120, 2008. 117 Erster O, Thomas JM, Hamzah J, Jabaiah AM, Getz JA, Schoep TD, Hall SS, Ruoslahti E and Daugherty PS: Site-specific Received February 21, 2014 targeting of antibody activity in vivo mediated by disease- Revised March 6, 2014 associated proteases. J Control Release 161 : 804-812, 2012. Accepted March 7, 2014