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`Molecular
`Cancer
`Therapeutics
`
`Review
`
`Rationale Behind Targeting Fibroblast Activation
`Protein–Expressing Carcinoma-Associated
`Fibroblasts as a Novel Chemotherapeutic Strategy
`
`W. Nathaniel Brennen1, John T. Isaacs2, and Samuel R. Denmeade1,2
`
`Abstract
`
`The tumor microenvironment has emerged as a novel chemotherapeutic strategy in the treatment of cancer.
`This is most clearly exemplified by the antiangiogenesis class of compounds. Therapeutic strategies that target
`fibroblasts within the tumor stroma offer another treatment option. However, despite promising data obtained
`in preclinical models, such strategies have not been widely used in the clinical setting, largely due to a lack of
`effective treatments that specifically target this population of cells. The identification of fibroblast activation
`protein a (FAP) as a target selectively expressed on fibroblasts within the tumor stroma or on carcinoma-
`associated fibroblasts led to intensive efforts to exploit this novel cellular target for clinical benefit. FAP is a
`membrane-bound serine protease of the prolyl oligopeptidase family with unique post-prolyl endopeptidase
`activity. Until recently, the majority of FAP-based therapeutic approaches focused on the development of
`small-molecule inhibitors of enzymatic activity. Evidence suggests, however, that FAP’s pathophysiological
`role in carcinogenesis may be highly contextual, depending on both the exact nature of the tumor microen-
`vironment present and the cancer type in question to determine its tumor-promoting or tumor-suppressing
`phenotype. As an alternative strategy, we are taking advantage of FAP’s restricted expression and unique
`substrate preferences to develop a FAP-activated prodrug to target the activation of a cytotoxic compound
`within the tumor stroma. Of note, this strategy would be effective independently of FAP’s role in tumor
`progression because its therapeutic benefit would rely on FAP’s localization and activity within the tumor
`microenvironment rather than strictly on inhibition of its function. Mol Cancer Ther; 11(2); 257–66. Ó2012 AACR.
`
`Introduction
`
`There is an increasing awareness of the necessity to
`understand a tumor within the context of its surround-
`ings, i.e., the tumor microenvironment. Investigations that
`take into consideration the complex network of interac-
`tions and regulatory signals that exist between the stroma
`and tumor itself have become essential for the full eluci-
`dation of both oncogenesis and tumor progression. The
`stroma associated with a tumor commonly contributes a
`significant portion of the mass of many malignancies, and
`it can account for >90% of the tumor mass in carcinomas
`characterized by a desmoplastic reaction, such as breast,
`colon, and pancreatic carcinomas (1). It is well documen-
`ted that the tumor is dependent on the reactive stroma for
`survival and growth signals, as well as the nutritional
`
`Authors' Affiliations: 1Department of Pharmacology and Molecular
`Sciences, and 2Sidney Kimmel Comprehensive Cancer Center at Johns
`Hopkins, Johns Hopkins University, Baltimore, Maryland
`
`Corresponding Author: Samuel R. Denmeade, Department of Oncology,
`Johns Hopkins University School of Medicine, Cancer Research Building I,
`Rm. 1M43, 1650 Orleans St., Baltimore MD 21231. Phone: 410-955-8875;
`Fax: 410-614-8397; E-mail: denmesa@jhmi.edu
`
`doi: 10.1158/1535-7163.MCT-11-0340
`Ó2012 American Association for Cancer Research.
`
`support required for maintenance of the primary mass.
`Additionally, the ability of the stroma to not only con-
`tribute to but also potentially drive the progression of
`cancerous cells into a highly aggressive and metastatic
`phenotype has only recently begun to be truly appreciated
`(2, 3), even though the first observations linking nonma-
`lignant cells of the tumor microenvironment to tumori-
`genesis were made more than a century ago.
`The stroma has been shown to undergo morphological
`alterations; recruit reactive fibroblasts, macrophages, and
`lymphocytes; increase secretion of growth factors and
`proteases; induce angiogenesis; and produce an altered
`extracellular matrix (ECM) when associated with a trans-
`formed epithelium (4). The tumor and its microenviron-
`ment exist in a dynamic and interconnected network of
`reciprocal interactions that can influence such varied
`processes as proliferation, migration, invasion, survival,
`and angiogenesis, to name a few. These effects are medi-
`ated through both paracrine and autocrine stimulation by
`a variety of growth factors and cytokines,
`including
`transforming growth factor b (TGF-b), basic fibroblast
`growth factor (bFGF), VEGF, platelet-derived growth
`factor (PDGF), and interleukins [IL (4)]. These growth
`factors can be liberated from the ECM through the action
`of proteases, such as the matrix metalloproteinases
`(MMP), in addition to being secreted from cancer cells
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`Brennen et al.
`
`and activated fibroblasts. The presence of these growth
`factors, together with the remodeling of the ECM and
`induction of neovascularization, leads to a tumor micro-
`environment that is conducive to the growth, progression,
`and eventual metastasis of the tumor and has been termed
`a "reactive" stroma. The induction of a desmoplastic or
`reactive stroma is associated with a poor prognosis in
`multiple carcinomas, including breast, pancreatic, and
`colorectal cancers (5–7).
`Fibroblasts in particular have been shown to consis-
`tently undergo several changes in both morphology
`and expression profiles when present in the tumor
`microenvironment (8). Indeed, the presence of activated
`fibroblasts that have acquired a myofibroblast-like phe-
`
`notype within the tumor microenvironment serves as a
`primary indicator of reactive stroma formation (4).
`Evidence suggests that these activated fibroblasts, also
`known as carcinoma-associated fibroblasts (CAF), are
`central to regulating the dynamic and reciprocal inter-
`actions that occur among the malignant epithelial cells,
`the ECM, and the numerous noncancerous cells that are
`frequently found within this tumor milieu, including
`endothelial, adipose, inflammatory, and immune cells
`(Fig. 1; ref. 9).
`CAFs have been implicated in nearly all stages of onco-
`genesis, from initiation through progression to metastasis,
`and have been shown to enhance epithelial cell growth,
`tumorigenicity, angiogenesis, and the metastatic potential
`
`Figure 1. CAFs can promote tumorigenesis directly through multiple mechanisms, including increased angiogenesis, proliferation, invasion, and inhibition of
`tumor cell death. These effects are mediated through the expression and secretion of numerous growth factors, cytokines, proteases, and extracellular matrix
`proteins, such as SDF-1, FGF2, VEGF, TGF-b, HGF, tenascin-c, LOX, and the MMPs. CAFs can additionally influence tumorigenesis indirectly
`through effects on a multitude of other cell types, including adipocytes and inflammatory and immune cells. Furthermore, paracrine signals (examples listed
`around the perimeter of the web) derived from these accessory cells feed back to promote tumor growth. Ac, acetyl; AFC, 7-amino-4-(trifluoromethyl)
`coumarin; bFGF, basic fibroblast growth factor; CCL2, chemokine (C-C motif) ligand 2; Col, collagen; DPP-II (IV, 6, 7, 8, 9, 10), dipeptidyl peptidase-II (IV, 6, 7, 8,
`9, 10); FN, fibronectin; GM-CSF, granulocyte macrophage colony-stimulating factor; HGF, hepatocyte growth factor; IGF2, insulin-like growth factor 2; LOX,
`lysyl oxidase; SDF-1, stromal cell-derived factor 1; SFRP-1, secreted frizzled-related protein 1; SPARC, secreted protein, acidic and rich in cysteine; TNC,
`tenascin-c.
`
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`of transformed cells compared with their normal fibroblast
`counterparts (2, 9). Knockout of the TGF-b type II receptor
`(TGFbR2) using the fibroblast-specific protein 1 promoter
`(Tgfbr2fspKO) resulted in a loss of TGF-b responsiveness in
`stromal fibroblasts and led to the development of prostatic
`intraepithelial neoplasia, a precursor lesion of prostate
`cancer, in mice (10). CAFs grown with initiated but
`nontumorigenic human prostatic epithelium in male
`athymic mice resulted in tumors 500 times larger than
`controls grown with normal fibroblasts (11). Compara-
`ble studies involving the coimplantation of CAFs with a
`variety of neoplastic cells, including breast, ovary, and
`pancreas, into immunodeficient mice showed similar
`increases in tumorigenicity (12–14). Bone marrow–
`derived mesenchymal stem cells, which are known to
`localize to malignant tissues where they have the ability
`to differentiate into CAFs or myofibroblast-like cells,
`have been shown to enhance the metastatic spread of
`breast cancer cells up to 7-fold (15). These results clearly
`suggest a role for CAFs in tumor initiation, progression,
`and malignancy.
`
`Fibroblast Activation Protein and the Post-Prolyl
`Peptidase Family
`
`A key characteristic of CAFs is the expression of fibro-
`blast activation protein a [FAP (16, 17)], which was orig-
`inally identified as an inducible antigen expressed in
`reactive stroma (16, 18). Subsequently, it was indepen-
`dently identified as a gelatinase expressed by aggressive
`melanoma cell lines and given the name seprase [for
`surface expressed protease (19)]. Subsequent cloning
`revealed that FAP and seprase are the same cell-surface
`serine protease (17).
`FAP is a type II integral membrane serine protease of the
`prolyl oligopeptidase family (also known as the S9 fam-
`ily), and it is further classified into the dipeptidyl pepti-
`dase (DPP) subfamily (S9B), of which dipeptidyl pepti-
`dase IV (DPPIV/CD26) is the prototypical member.
`Enzymes in this class are distinguished by their ability
`to cleave the Pro-Xaa peptide bond (where Xaa represents
`
`FAP Is a Novel Therapeutic Target
`
`any amino acid), and they have been shown to play a role
`in cancer by modifying bioactive signaling peptides
`through this enzymatic activity (20). FAP, like all enzy-
`matically active members of the subfamily, is a dipepti-
`dase characterized by its ability to cleave after a proline
`residue (Table 1; ref. 21). The crystal structure of FAP has
`confirmed that the enzyme exists as a homodimer and that
`dimerization is necessary for enzymatic function (22).
`There is also evidence that FAP can additionally form
`heterodimers with DPPIV that are localized to invadopo-
`dia of migrating fibroblasts (23, 24). Normal, healthy adult
`tissues have no detectable FAP expression outside areas of
`tissue remodeling or wound healing; however, FAP-pos-
`itive cells are observed during embryogenesis in areas of
`chronic inflammation, arthritis, and fibrosis, as well as in
`soft tissue and bone sarcomas (23, 25). Additionally,
`expression of FAP has been detected on mesenchymal
`stem cells derived from human bone marrow (26, 27).
`A soluble form of FAP has been found in both bovine
`serum (28) and human plasma (29). Currently, the func-
`tional significance of this soluble form of FAP, as well as
`the role of the full-length membrane-bound form, is poor-
`ly understood. Even the mechanism leading to FAP’s
`presence in the plasma is not known. Whether FAP’s
`presence in the plasma is the result of shedding from the
`membrane surface or the biosynthesis of an alternatively
`spliced isoform is not clear at this point. Despite our poor
`understanding of how FAP enters the circulation, its
`presence there raises the possibility of using serum levels
`of FAP as a biomarker for cancer prognosis. Sequencing
`has shown that this extracellular, soluble form of FAP
`found in human plasma is highly homologous to anti-
`plasmin-cleaving enzyme (APCE), which has been shown
`to cleave a
`2-antiplasmin into a form that cross-links to
`fibrin more efficiently, resulting in greater plasmin inhi-
`bition (29). The suggested cleavage site within a
`2-anti-
`plasmin is not conserved evolutionarily, which implies
`that this is probably not the primary function for which
`FAP originally diverged from DPPIV during a duplication
`event (30). Neuropeptide Y (NPY), B-type natriuretic
`peptide (BNP), substance P, and peptide YY (PYY) were
`
`Table 1. Characteristics of known post-prolyl peptidases
`
`Prolyl peptidase
`
`Enzymatic activity
`
`Cellular localization
`
`References
`
`DPPIV
`FAP
`DPP6
`DPP8
`DPP9
`DPP10
`AAP
`POP
`DPPII (DPP7)
`PCP
`
`Dipeptidase
`Dipeptidase/endopeptidase
`Inactive
`Dipeptidase
`Dipeptidase
`Inactive
`Acylpeptide hydrolase
`Prolyl oligopeptidase
`Dipeptidase
`Prolyl carboxypeptidase
`
`þ
`
`þ
`
`channel)
`
`channel)
`
`Membrane
`Membrane
`Membrane (Kv
`Cytoplasm
`Cytoplasm
`Membrane (Kv
`Cytoplasm
`Cytoplasm
`Intracellular vesicles
`Lysosome
`
`(25, 36–38)
`(25, 36, 38)
`(25, 37)
`(25, 37, 38)
`(25, 37, 38)
`(25, 37)
`(38, 39)
`(38)
`(37, 38)
`(38)
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`FAP and DPPIV are the only 2 enzymatically active
`members of the family that are synthesized as integral
`membrane proteins with extracellular domains, there are
`distinct differences in their enzymatic properties (Table 1;
`refs. 25, 40). Unique to FAP among the DPPIV family is its
`collagen type I-restricted gelatinase activity (41, 42),
`which classifies it as both an endopeptidase and an
`exopeptidase.
`
`FAP Expression in the Tumor Microenvironment
`
`In contrast to DPPIV, FAP is not expressed in normal,
`healthy adult tissues outside of granulation tissue during
`times of wound repair (40). However, studies showed that
`in the disease state, FAP expression was detected on the
`surface of fibroblasts in the stroma surrounding >90% of
`the epithelial cancers examined,
`including malignant
`breast, colorectal, skin, and pancreatic cancers, as well as
`in some soft tissue and bone sarcomas (16, 18). In a small
`study, FAP expression was also detected in the stroma of
`all 7 human prostate cancer specimens examined (43).
`FAP expression has also been observed on the surface of
`fibroblasts or pericytes in areas of tumor angiogenesis
`(23, 35, 44).
`To date, FAP expression has been most extensively
`characterized in breast tissue. In 14 samples analyzed,
`strong (12/14) to moderate (2/14) expression of FAP was
`observed in the stroma of human breast carcinomas but
`not in malignant epithelial cells or adjacent normal tissue
`(16). Furthermore, minimal or no expression was
`observed in samples obtained from fibrocystic disease
`(10/10) or fibroadenomas (2/2) in the same study. In
`another study, Ariga and colleagues (45) analyzed tissue
`samples from 112 Japanese women diagnosed with inva-
`sive ductal carcinoma of the breast, and they confirmed
`that FAP expression is exclusively localized to the stroma
`adjacent to FAP-negative tumor cells but is not present in
`the stroma of normal tissues. The semiquantitation of FAP
`levels in these samples showed strong expression in 61 of
`112 patients and low levels of expression in the remaining
`51 samples. Longer overall and disease-free survival rates
`were associated with increased FAP expression in that
`study, and a multivariate analysis showed FAP expres-
`sion levels to be an independent prognostic factor (45).
`In contrast, in a study examining FAP expression in
`patients with colon cancer, elevated levels were associated
`with aggressive disease as well as an increased risk of
`recurrence and metastasis (46). This observation led to
`multiple phase I and II trials to evaluate FAP as a ther-
`apeutic target in the treatment of colorectal cancer (47–49).
`Additionally, FAP expression has been associated with an
`overall poorer prognosis in multiple other cancer types,
`including pancreatic (50), hepatocellular (51), colon (52),
`ovarian (53), and gastrointestinal carcinomas (54). The
`mechanisms underlying these seemingly contradictory
`observations regarding FAP’s role in tumorigenesis are
`still unknown, but they may be related to differences in the
`tumor microenvironment among different tumor types,
`
`Brennen et al.
`
`recently identified as N-terminal dipeptide substrates
`for FAP in vitro, and further investigation into the phys-
`iological relevance of these substrates should prove
`interesting (31).
`FAP appears to be conserved among chordates, with
`especially high homology in many mammals, including
`primates, rodents, dogs, and ungulates; however, homo-
`logs have also been found in zebrafish and 2 amphibian
`species of the Xenopus genus. Both the FAP and DPPIV
`genes are located on the 2q23 locus. This proximity,
`coupled with their high degree of homology (48% overall
`amino acid sequence identity), suggests a common ances-
`try, and it is believed that FAP evolved from DPPIV via a
`gene duplication event (30).
`The FAP homolog found in the mouse genome [herein
`termed murine FAP (mFAP)] is expressed on the surface
`of reactive stromal fibroblasts, and it shares an 89%
`sequence identity, including the catalytic triad, with the
`human enzyme (32). FAP expression is observed during
`mouse embryogenesis in primitive mesenchymal cells in
`areas undergoing active tissue remodeling (33); however,
`/
`FAP
`mice are viable and manifest no apparent devel-
`opmental defects (34). This lack of phenotype is likely the
`result of compensation by other proteases. It is also pos-
`sible, however, that defects in these FAP-null mice may
`only manifest under the appropriate stressed or patho-
`genic conditions. Like its human counterpart, mFAP
`expression is not observed in normal adult murine tissues
`outside areas of tissue remodeling, such as wound healing
`(34). Of
`interest, FAP-null mice have displayed a
`decreased tumorigenicity, at least in the context of endog-
`enous K-rasG12D-driven lung cancer and syngeneic CT26
`colon tumors (35).
`In addition to FAP and DPPIV, the prolyl oligopepti-
`dase family includes DPP6, DPP8, DPP9, DPP10, prolyl
`oligopeptidase [POP, also known as prolyl endopeptidase
`(PEP)], and acylaminoacyl peptidase [AAP, also known as
`acylpeptide hydrolase (APH); Table 1; refs. 25, 36–39].
`Prolyl carboxypeptidase (PCP) and DPPII (also known as
`DPP7) of the S28 family are structurally related proteases
`with similar enzymatic activity that are localized to lyso-
`somes and intracellular vesicles, respectively (Table 1;
`refs. 25, 36–39). The substrate preferences for many of
`these post-prolyl peptidases are not entirely known, but
`similar to DPPIV, most have dipeptidase activity (Table 1).
`AAP is enzymatically distinct in that it cleaves intracel-
`lular N-acylated amino acids from the NH2-terminus of
`peptides, resulting in a single free N-acetyl amino acid as
`part of the protein catabolism pathway (Table 1; ref. 39).
`POP is a cytoplasmic protease whose oligopeptidase
`activity allows it to cleave after internal proline residues
`in short (<30 aa) peptide sequences (Table 1). This is in
`contrast to most members of the family, which are limited
`to exopeptidase activity. DPP6 and DPP10 are inactive
`due to an amino acid substitution in the catalytic triad, but
`they were recently found to be critical components of
`þ
`voltage-gated potassium (Kv
`) channels (Table 1; ref. 37).
`Despite FAP’s high homology to DPPIV and the fact that
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`FAP Is a Novel Therapeutic Target
`
`(2- to 4-fold) and growth (10- to 40-fold) in mFAP-
`transfected HEK293 human embryonic kidney cells
`grown as xenografts compared with mock-transfected
`controls. Administration of polyclonal rabbit antisera
`that was shown to inhibit FAP enzymatic activity sig-
`nificantly attenuated the growth of HT-29 human colo-
`rectal xenografts (32). In another study, Huang and
`colleagues (59) generated FAP-expressing human breast
`cancer cells (MDA-MB-231) that formed tumors with
`increased growth rates and a 3-fold higher microvessel
`density compared with vector controls when implanted
`into the mammary fat pads of murine hosts. Of interest,
`both FAP-positive cells and vector controls grew at the
`same rate in vitro, suggesting that FAP’s effect on tumor
`growth is mediated through the tumor microenviron-
`ment in vivo. Combined with data showing an upregu-
`lation of FAP transcription in endothelial cells under-
`going capillary morphogenesis and reorganization (61),
`this suggests that this tumor-promoting effect may be
`due in part to making the tumor microenvironment more
`conducive to angiogenesis. Most convincingly, using both
`syngeneic colon and endogenous K-rasG12D–driven lung
`models of murine cancer in which they recapitulated the
`physiologic stromal-restricted expression of FAP, Santos
`and colleagues (35) showed that both pharmacologic inhi-
`bition and genetic deletion of FAP resulted in decreased
`tumor proliferation and altered stromatogenesis.
`More recently, Kraman and colleagues (27) implicated
`FAP-expressing cells in immunosuppression, and selec-
`tive elimination of this population of cells using trans-
`genic mice expressing the diphtheria toxin receptor under
`the control of the FAP promoter restored host immuno-
`logical control of tumor growth. A significant proportion
`of the FAP-expressing cells identified in this study, which
`are likely responsible for this immunomodulatory capa-
`bility, share known markers (CD45/CD34þ/Sca-1þ)
`associated with multipotent MSCs. MSCs are known to
`be immune-privileged due to a lack of antigenic stimula-
`tory molecules, including major histocompatability com-
`plex class II antigens and costimulatory molecules, in
`addition to promoting an immunosuppressive and anti-
`inflammatory local environment (62). Circulating bone
`marrow–derived MSCs have been shown to express FAP
`by multiple groups, including our own (S. Chen and J.T.
`Isaacs, unpublished data) and are known to traffic to
`tumor sites at frequencies comparable to those observed
`in previous studies (26, 27). Of importance, FAP activity
`itself was not shown to mediate this immunosuppres-
`sive activity, because the LL2 carcinoma cells themselves
`were shown to express FAP. This indicates that inhibition
`of FAP activity alone by pharmacological agents will not
`restore host immunological defenses.
`In contrast, other studies showed that expression of FAP
`decreased tumorigenicity in mouse models of melanoma
`(63), and it was associated with longer survival in patients
`with invasive ductal carcinoma of the breast (45). These
`conflicting observations suggest
`that
`the physiologic
`response to FAP may depend not only on the in vivo
`
`including variations in the ECM, as well as the immune
`and inflammatory cell infiltrates present.
`It is a well-known phenomenon that fibroblasts and
`other stromal cells of murine origin constitute the stroma
`surrounding tumorigenic human cell line xenografts in
`immunodeficient mice (32). Both murine fibroblasts in the
`tumor microenvironment and mouse embryonic fibro-
`blasts grown in vitro (33) were found to express mFAP
`transcripts. Similar to human FAP expression patterns,
`mFAP has not been detected in normal adult mouse
`tissues. Using a polyclonal antibody produced within
`their laboratory, Cheng and colleagues (32) showed abun-
`dant mFAP expression in the stroma surrounding human
`HT-29 xenografts. Data from our laboratory, obtained with
`the same antibody, support these observations and show
`that murine stromal cells invade human tumor xenografts
`to various degrees depending on the xenograft being used
`and that a subset of these invading cells expresses mFAP
`(W.N. Brennen and S.R. Denmeade, unpublished data).
`
`Role of FAP in the Biology of Cancer
`
`Currently, not a lot is known about the regulation of
`FAP expression, and further investigations are necessary
`to fully elucidate the mechanisms underlying FAP’s
`dichotomous role in tumorigenesis. Zhang and colleagues
`(55) characterized the minimal FAP promoter and showed
`that early growth response 1 (EGR1) is an important
`regulator of FAP transcription. Of note, the EGR1 tran-
`scription factor itself has also been shown to have con-
`tradictory roles in tumorigenesis depending on the tumor
`type. Furthermore, treatment with TGF-b, 12-O-tetrade-
`conyl-phorbol-13-acetate (TPA), and retinoids is known to
`induce the upregulation of FAP expression on fibroblasts
`in vitro, whereas stress induced by serum starvation has
`no effect (56). Of interest, retinoids have been shown to
`have both chemopreventive and chemotherapeutic ben-
`efits in multiple cancer types (57). TGF-b is known to act as
`either a tumor promoter or suppressor, depending on the
`tumor type and stage of the disease. TGF-b is a potent
`inducer of the reactive phenotype in fibroblasts, and its
`regulation of FAP may underlie the context-dependent
`promotion or suppression of tumor growth that has been
`observed clinically.
`Although the physiologic substrates of FAP have yet to
`be fully determined, investigators are beginning to eluci-
`date a role for FAP in cancer biology. It has been proposed
`that FAP plays a role in matrix digestion and invasion
`through its gelatinase activity (58). The cleavage product
`generated from NPY in the presence of FAP has been
`shown to be proangiogenic, which may explain the cor-
`relation observed between FAP expression and increased
`microvessel density in tumors (31, 35, 59).
`Using a variety of in vivo models, researchers have
`directly implicated FAP in tumor promotion by show-
`ing increases in tumor incidence, growth, and micro-
`vessel density (32, 35, 59, 60). Cheng and colleagues
`(32) reported an increase in both tumor incidence
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`and it stimulates the production of immunomodulatory
`cytokines and chemokines through a mechanism that is
`not completely understood (65, 66). In phase II clinical
`trials, talabostat has been tested alone or in combination as
`therapy for metastatic colorectal cancer (49), NSCLC (67),
`stage IV melanoma (68), and chronic lymphocytic leuke-
`mia (66). Although one complete response (in NSCLC)
`and a few partial responses were reported in these studies,
`no clinical benefit could be attributed to talabostat or
`combination therapy over the single-agent, standard-of-
`care arms in the studies. However, no dose-limiting toxi-
`cities were observed in these trials, and the most com-
`monly reported adverse event linked to talabostat was
`peripheral edema, likely resulting from stimulation of IL-
`6 or other immunomodulatory effects (66). On the basis of
`these trial results, investigators initiated 2 phase III trials
`in which talabostat was administered to patients with late-
`stage NSCLC in combination with either docetaxel or
`pemetrexed. However, these trials were halted at the
`interim evaluation. As reported by Kennedy (69) in the
`Wall Street Journal, these trials were terminated early
`because neither the primary nor the secondary goals were
`being met, and the patient group in the docetaxel-com-
`bination study appeared to have a lower survival rate than
`the group in the placebo arm. Additional therapies involv-
`ing immunoliposomes that target FAP to deliver TNF are
`also in preclinical development (70).
`FAP inhibition may not inhibit the growth of all tumor
`types and may even promote tumor growth, based on
`current conflicting data regarding FAP’s role within the
`tumor microenvironment. Furthermore, FAP activity may
`not be as critical as the role of the FAP-expressing cell
`itself; consequently, merely inhibiting its enzymatic activ-
`ity alone may not be as beneficial as eliminating the cell
`type in question altogether. A more viable strategy, which
`would circumvent this uncertainty regarding FAP’s role
`in tumorigenesis, would be to take advantage of the
`protein’s restricted tumor expression and unique enzy-
`matic activity to selectively target FAP-activated pro-
`drugs designed to deliver very potent cytotoxic agents to
`the tumor microenvironment (Fig. 2). This strategy entails
`the systemic administration of an inactive prodrug com-
`posed of a cytotoxin coupled to a peptide carrier contain-
`ing a FAP cleavage site. This peptide carrier inactivates the
`prodrug by preventing it from crossing the cell membrane
`and consequently from reaching its intracellular target.
`The prodrug circulates throughout the body in this non-
`toxic, inactive form until it is proteolytically activated by
`FAP, which is present on CAFs localized to the tumor
`microenvironment. The cytotoxin itself has no inherent
`specificity; therefore, once it becomes activated, it non-
`specifically targets any cell in close proximity to the region
`of activation, including fibroblasts, tumor cells, and endo-
`thelial cells. This propensity to kill neighboring cells that
`do not express the target once the prodrug has been
`activated is a phenomenon known as the bystander effect,
`and it can lead to a greater antitumor effect. This strategy
`should allow increased delivery of the drug specifically to
`
`Brennen et al.
`
`tumor microenvironment but also on the exact context of
`the expression within different microenvironments.
`
`Potential for Therapeutic Exploitation of FAP by a
`Novel Prodrug
`
`The unique enzymatic activity and highly restricted
`expression of FAP in the reactive stroma associated with
`>90% of epithelial cancers examined thus far make it a
`very attractive candidate for tumor-specific therapies. A
`number of potential therapeutic strategies can be envi-
`sioned, including inhibition of enzymatic function by a
`small molecule or antibody, and immunotherapies that
`deliver radioisotopes, drugs, or toxins to the tumor stro-
`ma. These approaches are currently being developed by
`several pharmaceutical companies, and promising prog-
`ress along these lines is being made. Significantly, target-
`ing of FAP-expressing cells in tumor-bearing mice using
`an oral DNA vaccine resulted in a considerable increase in
`intratumoral drug uptake, likely due to a concomitant
`decrease in the amount of type I collagen present (64).
`Two phase I studies have been done to evaluate the
`biodistribution of a 131I-labeled mFAP monoclonal anti-
`body (mAb F19) in presurgical patients with hepatic
`metastases from colorectal cancer and in soft-tissue sar-
`coma. These studies showed selective accumulation of
`the F19 mAb in tumors, with minimal localization to any
`other normal tissue, indicating selective FAP expression
`within the tumor microenvironment (47). In another
`study, Scott and colleagues (47) used a humanized version
`of F19 mAb sibrotuzumab, which is under development
`by Boehringer Ingelheim Pharma KG, and tested its safety
`and distribution in 26 patients with colorectal and non–
`small cell lung cancer (NSCLC). They observed no objec-
`tive tumor response, but they did see selective uptake in
`tumors 24 to 48 hours after infusion, with no significant
`normal organ uptake (47). In a phase II study, Hofheinz
`and colleagues (48) treated 25 patients with colorectal
`cancer with unconjugated sibrotuzumab. Although the
`therapy was found to be safe and well tolerated, no
`responses were observed and the trial was stopped. Stud-
`ies to evaluate the effects of radioisotope- or toxin-labeled
`antibodies are currently under development.
`Another FAP-targeted therapeutic strategy would be to
`inhibit its enzymatic function. A number of studies have
`characterized the dipeptide substrate requirements for
`FAP, leading to the development of small-molecule inhi-
`bitors that selectively inhibit FAP over other prolyl pep-
`tidases by companies such as Boehringer Ingelheim (65),
`Point Therapeutics (35), and Genentech (21). For example,
`Genentech developed a boronic acid–based inhibitor (Ac-
`Gly-prolineboronic acid) with a Ki of 23 nm and a rea-
`sonable (9-fold)
`to significant
`(5,400-fold) selectivity
`against all other members of the prolyl peptidase family
`(21). Point Therapeutics also developed a boronic acid–
`based inhibitor, Val-prolineboronic acid (PT-100, or tala-
`bostat), that is being evaluated in a variety of cancer types
`(66). Talabostat selectively inhibits both FAP and DPPIV,
`
`262
`
`Mol Cancer Ther; 11(2) February 2012
`
`Molecular Cancer Therapeutics
`
`Petitioner GE Healthcare – Ex. 1016, p. 262
`
`

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`FAP Is a Novel Therapeutic Target
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