Cancer Biology & Therapy 13:3, 123–129; February 1, 2012; G 2012 Landes Bioscience
`
`REVIEW
`
`Fibroblast activation protein
`A potential therapeutic target in cancer
`
`Rui Liu,1 Hui Li,1 Liang Liu,1 Jinpu Yu1 and Xiubao Ren1,2,*
`
`1Key Laboratory of Cancer Prevention and Therapy; Tianjin Medical University Cancer Institute and Hospital; Tianjin, China; 2Department of Biotherapy; Tianjin Medical University
`Cancer Institute and Hospital; Tianjin, China
`
`Keywords: fibroblast activation protein, seprase, serine protease, tumor, stroma, inhibitor, therapy
`
`The concept of targeting antigens selectively expressed on the
`surface of tumor capillary endothelial cells or in tumor stroma
`has emerged as a promising strategy for cancer therapeutics.
`Identification of stromal targets for anticancer therapy and
`development of selective inhibitors of these targets are of
`great clinical
`interest. Fibroblast activation protein (FAP), a
`member of the serine protease family, selectively expressed in
`the stromal
`fibroblasts associated with epithelial cancers,
`whereas with low or undetectable expression in the resting
`fibroblasts of normal adult tissues. The proteolytic activity of
`FAP has been shown to support tumor growth and prolifera-
`tion, making it a potential
`target
`for novel anticancer
`therapies, such as those by immune-based approaches.
`
`Introduction
`
`The clinical benefits of conventional cancer therapies to induce
`potent immunity in cancer patients are hampered by the genetic
`instability of tumor cells, leading to their escape from immune
`surveillance through generating drug-resistant variants. The growth
`of solid neoplasms beyond a diameter of 1–2 mm requires a
`supporting tumor stroma to ensure the supply of nutrients for
`tumor cells to survive and continuously grow.1 Thus, immuniza-
`tion against epitopes expressed by the tumor stroma can also
`potentially suppress tumor growth.
`is an
`FAP,
`formerly known as F19 cell surface antigen,
`inducible cell surface glycoprotein originally identified in 1986 in
`cultured fibroblasts using the monoclonal antibody (mAb) F19.2
`In 1994, the so-called F19 cell surface antigen was renamed the
`fibroblast activation protein (FAP).3
`In 1990, FAP was independently identified by a different group
`as a gelatinase expressed by the aggressive melanoma cell line LOX
`and was given the name “seprase” for surface expressed protease.4
`Subsequent cloning of FAP and seprase revealed that they are
`the same cell-surface serine protease.5 Expression of FAP is highly
`restricted to cancer-associated fibroblasts. It is not expressed
`in resting fibroblasts in normal tissue, but can be induced to
`express in nontransformed, activated stromal
`fibroblasts. The
`
`*Correspondence to: Xiubao Ren; Email: rwziyi@yahoo.com
`Submitted: 08/30/11; Revised: 11/04/11; Accepted: 11/07/11
`http://dx.doi.org/10.4161/cbt.13.3.18696
`
`cancer-specific distribution of FAP makes it an emerging novel
`therapeutic target in cancer.
`
`The Structure and Enzymatic Activity of FAP
`
`The structure of FAP. FAP is a type II transmembrane glyco-
`protein consisting of 760 amino acids. It belongs to the family
`of post-proline dipeptidyl aminopeptidase, known as dipeptidyl
`peptidase-IV activity and/or structure homologs (DASH), which
`includes
`the well-studied dipeptidyl peptidase 4 (DPP4 or
`CD26), as well as DPP2, DPP8 and DPP9. Human FAP gene
`is located on chromosome 2q23, and shares similar genomic
`organization and 89% amino-acid-sequence identity, including a
`perfectly conserved catalytic triad, with the mouse FAP gene,
`which is also located on chromosome 2.6 The FAP monomer has
`five potential N-glycosylation sites, 13 cysteine residues, three
`segments corresponding to the highly conserved catalytic domains
`of serine proteases, a hydrophobic transmembrane segment and a
`short cytoplasmic tail of six amino acids.7 Homodimerization to
`form a 170-kDa dimer is necessary for the enzymatic activity of
`FAP.8 FAP harbors 48% identical amino acid sequence with the
`T cell activation antigen CD26 (DPP4). The sequences of FAP
`and CD26 are most closely related in the putative extracellular
`domain, especially near the C-terminus.5 The FAP catalytic triad
`is composed of residues Ser624, Asp702, and His734, and is located at
`the interface of the β-propeller and a/β hydrolase domain. The
`eight bladed β-propeller domain is situated on the top of the
`catalytic triad and may serve as a gate to selectively filter protein
`access to the catalytic triad.9
`Study on the transcriptional regulation of FAP gene shows that
`EGR1 regulates FAP expression through binding to its promoter.
`Downregulation of EGR1 results in a significant reduction of
`endogenous FAP mRNA expression.10 Further
`studies
`are
`required to determine other
`transcription factors
`that may
`regulate FAP promoter.
`Enzymatic activity of FAP. In vitro studies have shown that
`FAP has both dipeptidyl peptidase activity that removes P2-Pro1
`dipeptides from the N-terminus of the substrate and endopepti-
`dase activity against substrates containing a Gly2-Pro1 motif,
`including a collagenolytic activity capable of degrading gelatin and
`type I collagen.11 Both functions utilize a common active site
`Ser624. Recently, it was reported that a2-antiplasmin is an impor-
`tant physiological substrate of FAP endopeptidase activity.12
`Moreover, neuropeptide Y, B-type natriuretic peptide, substance
`
`www.landesbioscience.com
`
`Cancer Biology & Therapy
`
`123
`
`© 2012 Landes Bioscience.
`Do not distribute.
`
`Petitioner GE Healthcare – Ex. 1022, p. 123
`
`

`

`P and peptide YY are the most efficiently hydrolysed substrates of
`FAP dipeptidyl peptidase activity.13
`Based on its structure, mutations of FAP with compromised
`protease activities have been engineered, including FAPS624A,
`which lacks dipeptidyl peptidase and endopeptidase activity, as
`well as FAPA657S, which retains its dipeptidyl peptidase activity
`but lacks endopeptidase activity.14,15 These mutant forms of FAP
`provide important tools for defining the in vivo relevance of the
`distinct activities and physiologic substrates of FAP.
`
`Tissue Distribution of FAP
`
`FAP is transiently expressed in some fetal mesenchymal tissues.
`The presence of FAP protein is normally restricted to endometrial
`cells. For the endometrium, FAP expression decreases during the
`midsecretory phase. No expression was found in samples of
`atrophic endometrium.15 FAP is also expressed during diseases
`associated with activated stroma,
`including wound healing,
`rheumatoid arthritis, osteoarthritis, cirrhosis and pulmonary
`fibrosis.16-20 Thus, the function of FAP in tissue remodeling
`becomes an intriguing topic. FAP is expressed selectively by
`cancer-associated fibroblasts (CAFs) and pericytes rather than
`tumor cells in more than 90% of human epithelial malignancies,
`including colorectal, ovarian, breast, bladder and lung. Benign
`and premalignant epithelial tumors, including fibroadenomas and
`phylloides tumors of the breast and colorectal adenomas, generally
`lack FAP+ stromal cells.21 As to bone and soft tissue sarcomas,
`expression of FAP is clearly independent of
`the malignant
`potential of the tumor, and is rather related to the histogenesis of
`the tumor cells.22 Based on the highly regulated expression and
`restricted distribution of FAP, it has been identified as a marker
`fibroblasts. Ossama Abbas et al.23
`of reactive tumor stromal
`reported the differentiation of morpheaform/infiltrative basal cell
`carcinoma from desmoplastic trichoepithelioma using FAP as a
`marker. They also observed a gradient in the pattern of FAP
`staining with prominent expression noted in fibroblasts directly
`surrounding the tumor cells, a more diffusive pattern in the distal
`part of the peritumoral stroma, and minimal or absent expression
`in adjacent normal tissue. FAP also serves as a surface protein
`marker that can define MSCs from bone marrow (BM) cells,
`raising the possibility that MSCs may be among very few, if any,
`cell types in the adult human body that express FAP.24
`A soluble form of FAP lacking the transmembrane domain has
`recently been discovered in human serum as antiplasmin-cleaving
`enzyme (APCE).25 APCE cleaves the Pro12-Asn13 bond of
`Met-a2AP to a more active form, Asn-a2AP, and thus suppresses
`fibrinolysis.26
`
`FAP in Tumorigenesis and Cancer Progression
`
`FAP as a tumor promoter. FAP is thought to promote tumor cell
`growth and proliferation. The role of FAP in breast cancer has
`been investigated using human breast cancer cell
`lines that
`naturally express FAP (MDA-MB-435 and MDA-MB-436).27
`Suppression of FAP expression using anti-sense oligonucleotides
`rendered these cells
`sensitivity to serum starvation, whereas
`
`control transfectants with high levels of FAP expression grew well
`in the absence of serum. Therefore, breast cancer cells with high
`FAP levels are less dependent on exogenous serum factors for
`growth and have gained independence on normal growth
`regulatory controls. Independence on normal growth regulation
`is a key characteristic of malignantly transformed cells that
`distinguishes them from normal cells. It has been reported that
`injection with pFAP-transfected CT26 cells resulted in a larger
`average tumor volume than tumors formed from the control cells.
`This group also constructed a DNA vaccine directed against
`FAP. This vaccine significantly suppressed primary tumor and
`pulmonary metastases primarily through CD8+T cell mediated
`killing in tumor-bearing mice.28 Moreover, HEK293 cells trans-
`fected to constitutively express murine FAP, when xenografted
`into SCID mice, were 2–4 times more likely to develop tumors
`and showed a 10- to 40-fold enhancement of tumor growth
`compared with control transfectants. They also demonstrated
`inhibition of FAP enzymatic activity with anti-FAP antibodies was
`associated with growth attenuation of HT-29 xenografts.29 This
`indicates that FAP has a potent effect on tumor cell growth. Chen
`et al.30 reported that FAP increased the invasion, proliferation and
`migration of HO-8910PM ovarian cancer cells. In addition, in
`patients with pancreatic adenocarcinoma, higher FAP expression
`is associated with worse clinical outcome.31 Taken together, a role
`of FAP in mediating tumor growth is becoming clearer.
`FAP as a tumor suppressor. In contrast, other studies suggest
`that FAP has tumor suppressive activity and show that this activity
`is independent of its enzymatic activity. Elevated expression of
`FAP in cancer causes dramatic promotion or suppression of tumor
`growth, depending on the model system investigated.32 It was
`observed that expression of FAP, or a catalytic mutant of FAP,
`decreased the tumorigenicity of mouse melanoma cells in animals
`and restored contact inhibition and growth factor dependence.33
`A second independent observation from studies of studies of
`somatic cell hybrids between normal
`fibroblasts and HeLa
`carcinoma cells
`identified FAP as a potential
`inhibitor of
`tumorigenesis. FAP was one of eight genes differentially expressed
`in nontumorigenic hybrid lines; consistent with the hypothesis
`that FAP is involved in suppressing the tumorigenic phenotype.34
`In addition, the degree of FAP expression in breast cancer stromal
`cells was associated with a longer survival of patients.35
`In summary, there is an obvious discrepancy between FAP
`function in tumor promotion and tumor suppression. Some
`researchers propose that the factor that determines this must
`reside in the signaling molecules that are available for interaction
`with FAP on the cells. Thus, FAP executes its biological functions
`in a cell-context dependent manner through a combination of its
`protease activity and its ability to form complexes with other cell-
`surface molecules. However, the role of FAP in tumor growth and
`invasion, and the exact molecular mechanisms
`the enzyme
`utilizes, still remains largely unknown.
`
`The Role of FAP in the Tumor Microenvironment
`
`Given the obvious discrepancy on FAP function in tumor
`promotion and tumor suppression, increasing attentions are being
`
`124
`
`Cancer Biology & Therapy
`
`Volume 13 Issue 3
`
`© 2012 Landes Bioscience.
`Do not distribute.
`
`Petitioner GE Healthcare – Ex. 1022, p. 124
`
`

`

`the “tumor microenvironment (TME).”
`paid to the role of
`Tumors are composed of heterogeneous populations of cells,
`including infiltrating inflammatory and immune cells, endothelial
`cells and mesenchymal-derived smooth muscle cells, pericytes,
`and CAFs.36 Stromal cells communicate with each other as well as
`with cancer cells and immune cells directly through cell contacts
`and indirectly through paracrine signaling, protease secretion, and
`modulation of the ECM. This complex communication network
`is pivotal to a supportive microenvironment for tumorigenesis,
`angiogenesis, and metastasis. Fibroblasts are one of the most
`crucial components of the tumor microenvironment, and can
`promote the growth and invasion of cancer cells through the
`synthesis, deposition and remodeling of the extracellular matrix
`(ECM), involved in angiogenesis and deregulation of antitumor
`immune responses. FAP is emerging as an important factor in the
`pro-oncogenic function of these stromal cells, although further
`studies are required to define the mechanisms involved.
`Modification of the ECM. Neoplastic cells must attach to
`adhesion proteins of the ECM, proteolytically degrade the ECM,
`and migrate to distant sites in order to invade surrounding tissues
`and ultimately metastasize. The remodeling of ECM and tumor
`invasion involves profound changes in the secretion of ECM
`proteins, such as collagens, fibronectin, tenascin, and laminin, as
`well as ECM protein degradation mediated by specific protease
`and protease inhibitors, such as urokinase plasminogen activator
`(uPA) and MMPs. FAP functions as an active serine protease
`capable of degrading type I collagen, and dipeptidyl-peptidase
`activity, thus suggesting that FAP could possibly act directly as
`an ECM-degrading or indirectly as regulatory protease involved
`in the activation/modification of other ECM-proteases/protease
`inhibitors. Lee et al.37 reported that FAP remodels the ECM
`through modulating protein levels, as well as through increasing
`levels of
`fibronectin and collagen fiber organization. FAP-
`dependent architectural/compositional alterations of the ECM
`promote tumor invasion along characteristic parallel fiber orienta-
`tions, as demonstrated by enhanced directionality and velocity of
`pancreatic cancer cells on FAP+ matrices. This phenotype can be
`reversed by inhibition of FAP enzymatic activity during matrix
`production resulting in the disorganization of the ECM and
`impeded tumor invasion. Wang et al.38 discovered that over-
`expression of FAP in the human hepatic stellate cell line LX-2
`caused increased migration through ECM proteins and an
`induction of MMP-2 and adhesion proteins. In invadopodia,
`FAP associates with a3β1 integrin, DPPIV, MMP-2, membrane-
`type 1 MMP and uPA.39-42 The resulting complexes appear to
`enhance cell invasion by co-operative roles in ECM-degradation
`and adhesion. The exact natures of the homodimer and hetero-
`dimer complexes of FAP are poorly understood. As to astroglial
`tumors, FAP is highly expressed on the surface of glioma cells
`and contributes to diffuse glioma invasion through extracellular
`matrix components. In contrast, siRNA knockdown of FAP in a
`glioma cell line showed decreased invasion through brain extra-
`cellular matrix proteoglycan brevican and denatured collagen.43
`Additionally, William et al.12 reported that following collagen I
`cleavage by MMP-1, FAP digests collagen I into smaller peptides,
`suggesting that FAP synergizes with other proteases to cleave
`
`partially degraded or denatured collagen I and III as ECM is
`excavated. Furthermore, in tumor mouse models FAP depletion
`increases accumulation of collagen that is not directly cleaved.44
`Waster et al.45 also reported that UV radiation stimulated FAP-
`driven migration and invasion in fibroblasts, melanocytes and
`primary melanoma cells. Another evidence of FAP in cancer
`metastasis is that a significant correlation between FAP RNA
`expression and incidence of LN metastases was
`found in
`medullary thyroid carcinoma.46 Interestingly, a recent
`study
`reported cancer cells expressing wild type FAP or FAPS624A
`degrade ECM more extensively, accumulate higher levels of
`matrix metalloproteinase-9 (MMP-9) in conditioned medium, are
`more invasive in type I collagen gels, and have altered signaling
`compared with control transfectants that do not express FAP and
`form slow growing tumors. Thus,
`they conclude that
`the
`proteolytic activity of FAP participates in matrix degradation,
`but other functions of the protein stimulate increased tumor
`growth.47 Overall, these data reveal a vital role of FAP in ECM
`remolding.
`Involvement in angiogenesis. For tumors to grow and integrate
`into the surrounding tissue, they must gain a blood supply for
`sustained growth. The first evidence for a pro-angiogenic function
`for FAP shows that tumors derived from FAP expressing human
`breast cancer cells have a 3-fold higher microvessel density as
`compared with tumors from cells not expressing FAP. It is
`thought that FAP promotes growth of breast tumors at least in
`part by driving angiogenesis.48 This notion is also supported by
`studies showing that FAP mRNA is upregulated by endothelial
`cells undergoing reorganization and capillary morphogenesis.49 In
`contrast, it is reported that FAP depletion decreases blood vessel
`density of tumor xenografts in mice.50 In addition, depletion of
`FAP+ cells causes rapid hypoxic necrosis of both cancer and
`stromal cells in immunogenic tumors through a process involving
`interferon-c (IFN-c) and tumor necrosis factor a (TNF-a), which
`have previously been shown to be involved in the suppression of
`angiogenesis.51 In corneal stroma, FAP could express where the
`new vessels reached, which indicates the close correlation of FAP
`and angiogenesis.52 These findings suggest that FAP can alter the
`tumor microenvironment at least partially by driving angiogenesis.
`
`Clinical Applications of FAP
`
`The enzymatic activity of FAP provides a potentially important
`new therapeutic target
`in a variety of human malignancies.
`Therefore, it is important to develop selective FAP inhibitors in
`preclinical investigation. Henry et al.53 reported that stromal FAP
`is more prominent in early-stage colorectal cancer and smaller
`colorectal tumor xenografts. Furthermore, increased FAP is an
`adverse prognostic indicator in patients with advanced metastatic
`disease. This study also suggests that the effects of FAP inhibition
`should be investigated in early-stage tumors, which harbor higher
`levels of FAP.
`Inhibition of FAP protease activity. Jonathan et al.11 reported
`that abrogation of FAP enzymatic activity attenuates tumor
`growth, indicating that the enzymatic activity of this protein plays
`an important role in the promotion of
`tumor growth, and
`
`www.landesbioscience.com
`
`Cancer Biology & Therapy
`
`125
`
`© 2012 Landes Bioscience.
`Do not distribute.
`
`Petitioner GE Healthcare – Ex. 1022, p. 125
`
`

`

`provides an attractive target for therapeutics designed to alter
`FAP-induced tumor growth through targeting its enzymatic
`function. The data provide a strategy for exploiting small
`molecule
`inhibitors of
`the protease
`activity of FAP and
`investigating their anticancer activity in preclinical models.
`Administration of Val-boro-Pro (PT-100; Talabostat) attenuates
`tumor growth in a variety of tumor models in mice.54 In addition,
`PT-100 promotes
`the growth of primitive hematopoietic
`progenitor cells by increasing granulocyte-colony stimulating
`factor (G-CSF), interleukin-6 (IL-6) and IL-11 production by
`bone marrow stromal cells, making it a therapeutic candidate for
`the treatment of neutropenia and anemia.55 However,
`this
`compound also inhibits multiple intracellular and extracellular
`dipeptidyl peptidases (e.g., FAP, CD26/DPPIV, DPP7), so that
`its effect cannot be directly attributed to FAP inhibition. In
`clinical trials, Phase II trial of Val-boro-Pro demonstrates minimal
`clinical activity in patients with previously treated metastatic
`colorectal cancer.56 In addition, Phase II trial of talabostat and
`docetaxel
`in advanced non-small cell
`lung cancer shows no
`evidence that talabostat enhanced the clinical activity of docetaxel
`in patients with NSCLC.57 PT630 is more specific than
`talabostat, effectively inhibiting FAP and DPPIV but not the
`intracellular family members. Santos et al.44 found PT630 has
`potent anticancer effects in several mouse models. However, all of
`these methods have limitations and may be associated with a
`greater risk of side effects than inhibition of FAP, often arising
`from the fact that the DPPIV and multiple intracellular dipeptidyl
`peptidases have normal functions outside of the tumor. With
`regard to presented challenges, it is important to develop selective
`FAP inhibitors for the use of target validation. In addition,
`research with catalytic mutants suggests that at least a portion of
`the biological functions of FAP reside in non-proteolytic domains
`of FAP. Thus, inhibiting the protease activity may have profound
`effects on some tumors but little effect or even growth-promoting
`effects on others. The role of FAP in a particular tumor type must
`be understood in future studies.
`Targeting FAP. The combinatorial approach targeting both the
`oncogenic pathways intrinsic to neoplastic cells and the pathways
`that mediate the pro-tumorigenic effects of the non-transformed
`stromal component is becoming a widely accepted strategy for
`targeted anticancer therapy.
`Several groups have developed FAP-specific mAb for imaging
`tumors. Although a humanized anti-FAP antibody (mAb F19;
`sibrotuzumab) is well tolerated,58 it shows no beneficial effect in a
`phase II trial for metastatic colorectal cancer,59 and the antibodies
`did not inhibit FAP enzymatic activity. It was recently reported
`that a monoclonal anti-FAP antibody conjugated to maytansi-
`noid, FAP5-DM1,
`induced long-lasting inhibition of
`tumor
`growth and complete regression in stroma-rich xenograft models
`of lung, pancreatic, and head and neck cancers in immune-
`deficient mice, with no evidence of toxicity.60 However, further
`studies will be required to determine the clinical application.
`Recent alternative approaches that utilize or localize FAP
`enzyme activity have shown potential. Potent cytotoxic agents,
`preferably ones that do not cause hemolysis, could be coupled to
`an FAP-specific peptide to generate an inactive prodrug that is
`
`selectively activated by FAP-expressing cells within the tumor
`stroma. Intratumoral
`injection of an FAP-activated protoxin
`produces significant lysis and growth inhibition of human breast
`and prostate cancer xenografts with minimal toxicity to the host
`animal.61 A novel FAP-triggered photodynamic molecular beacon
`(FAP-PPB) comprised of a disease-specific linker, a photosensi-
`tizer (PS), and a fluorescence and singlet oxygen (O2) quencher
`(Q)
`is a potential
`tool
`for epithelial cancer detection and
`treatment. In vitro and in vivo experiments have validated the
`FAP-specific activation of FAP-PPB in cancer cells and mouse
`xenografts.62 In addition, Huang et al.63 conjugated Doxorubicin
`(Dox) with a FAP-specific dipeptide to develop a FAP-targeting
`prodrug of Dox (FTPD). They demonstrated that FAP-cleaved
`FTPD exhibited significantly higher cytotoxicity against 4T1 cells
`in vitro than the uncatalyzed prodrug. Additionally, FTPD
`produced similar anticancer efficacy in 4T1 tumor-bearing mice
`to free Dox without obvious cardiotoxic effect. These findings
`suggest that such FAP-based prodrug strategy is promising to
`achieve targeted delivery of anticancer agents.
`CAFs are key modulators of the immune TME and that their
`elimination in vivo has profound effects on immune polarization
`in the TME.64 Importantly, with highly restricted of FAP to
`CAFs, the development FAP-based immunotherapy could be
`optimized for anticancer therapy in a clinical setting. Sequential
`application of
`scFv-IL-872 and dimeric IgG1-TNF fusion
`proteins
`significantly enhanced antitumor
`activity in mice
`when compared either to a single-agent treatment or sequential
`application of non-targeted cytokines, indicating that the tumor-
`restricted sequential application of
`IL-872 and TNF is a
`therapy.65 A group recently
`promising approach for cancer
`developed a new tumor vaccine, FAPtau-MT, which was
`produced by conjugating 1-methyl-tryptophan (1-MT), a specific
`inhibitor of Indolamine2, 3-dioxygenase (IDO) to FAP. The
`vaccine breaks tumor immune tolerance as a local IDO inhibitor.
`Most importantly, administration of the FAPtau-MT vaccine did
`not lead to pregnancy failiure in mice carrying allogeneic fetuses.66
`A bispecific fusion protein, AntiFAP-mGITRL (murine gluco-
`corticoid-induced tumor necrosis factor-related receptor), is able
`to costimulate CD8+ and CD4+ effector T cells resulting in
`increased proliferation,
`IFN-gamma
`and IL-2 production.
`In suppression assays, membrane-bound antiFAP-mGITRL is
`100-fold more effective in overcoming Treg-mediated suppression
`than unbound fusion protein. These studies suggest that targeted
`tumor
`therapy with antiFAP-mGITRL fusion protein could
`induce tumor
`rejection while minimizing autoimmune side
`effects.67 Future studies need to address the mechanism how a
`FAP-directed immune response affects tumor growth, whether by
`directly affecting the tumor-associated fibroblast or via collateral
`damage inducing a local inflammatory response.
`FAP can serve as a novel target for active vaccination against
`cancer, especially if combined with chemotherapy. Tumor tissue
`of FAP-vaccinated mice revealed markedly decreased collagen type
`I expression and up to 70% greater uptake of chemotherapeutic
`drugs. Most importantly, pcDNA3.1/V5-His-TOPO-Fap-vacci-
`nated mice treated with chemotherapy show a 3-fold prolonged
`survival and marked suppression of tumor growth, with 50% of
`
`126
`
`Cancer Biology & Therapy
`
`Volume 13 Issue 3
`
`© 2012 Landes Bioscience.
`Do not distribute.
`
`Petitioner GE Healthcare – Ex. 1022, p. 126
`
`

`

`the animals completely rejecting a tumor cell challenge.68 This
`strategy opens a new venue for the combination of immunothera-
`pies and chemotherapies.
`Yet there are challenges to be met in the future preclinical
`studies required to develop FAP inhibitors to their clinical
`application. Although these studies provide the proof of principle,
`the development of additional animal models that recapitulate
`the properties of human tumors will be important in future
`studies. This includes endogenous tumor models in immune-
`competent mice, even though the endogenous tumor models
`currently available in mice may not typically exhibit the dramatic
`desmoplastic response seen in many epithelial-derived tumors
`in patients.69
`Importantly, however, besides the involvement of FAP in
`tumorigenesis, it also plays a role in biological function such as
`embryonic development and tissue remodeling. Thus, a deliberate
`effort must be made to use to determine whether or not the use of
`FAP inhibitors is likely to have beneficial or deleterious effects on
`normal tissue. Further clinical trials of FAP inhibitors will be
`required to design to define the potential risks in cancer patients.
`
`Conclusion
`
`It is increasingly recognized that the stromal compartment plays a
`crucial role in tumorigenesis and invasion. FAP is a product
`overexpressed by CAFs, the predominant component of cancer
`stoma in most types of cancer. In comparison to tumor cells, FAP
`may represent a more viable therapeutic target
`for cancer
`immunotherapy. Since its discovery in 1986, a great amount of
`research has been performed on the localization and expression of
`this protease. However, the role of FAP in tumor growth and
`invasion, as well as the exact molecular mechanisms the enzyme
`utilizes remain unknown. FAP does seem to have an important
`role in malignant cell invasion and metastasis through partici-
`pating in angiogenesis, deregulation of antitumor
`immune
`responses and synthesis, deposition and remodeling of ECM.
`The availability of a potent and selective, in vitro and in vivo
`applicable FAP inhibitor opens new perspectives for further
`studies of the physiological function of FAP. Future studies on the
`contribution of FAP to tumor growth and invasion will constitute
`an essential step toward stroma-targeted anticancer therapy.
`
`5.
`
`References
`that do not heal.
`1. Dvorak HF. Tumors: wounds
`Similarities between tumor
`stroma generation and
`wound healing. N Engl J Med 1986; 315:1650-9;
`PMID:3537791
`2. Rettig WJ, Chesa PG, Beresford HR, Feickert HJ,
`Jennings MT, Cohen J, et al. Differential expression of
`cell surface antigens and glial fibrillary acidic protein in
`human astrocytoma subsets. Cancer Res 1986; 46:
`6406-12; PMID:2877731
`3. Rettig WJ, Su SL, Fortunato SR, Scanlan MJ, Raj BK,
`Garin-Chesa P, et al. Fibroblast activation protein:
`purification,
`epitope mapping
`and induction by
`growth factors. Int J Cancer 1994; 58:385-92; PMID:
`7519584; http://dx.doi.org/10.1002/ijc.2910580314
`4. Aoyama A, Chen WT. A 170-kDa membrane-bound
`protease is associated with the expression of invasiveness
`by human malignant melanoma cells. Proc Natl Acad
`Sci U S A 1990; 87:8296-300; PMID:2172980; http://
`dx.doi.org/10.1073/pnas.87.21.8296
`Scanlan MJ, Raj BK, Calvo B, Garin-Chesa P, Sanz-
`Moncasi MP, Healey JH, et al. Molecular cloning of
`fibroblast activation protein alpha, a member of the
`serine protease family selectively expressed in stromal
`fibroblasts of epithelial cancers. Proc Natl Acad Sci
`U S A 1994; 91:5657-61; PMID:7911242; http://dx.
`doi.org/10.1073/pnas.91.12.5657
`6. Niedermeyer J, Enenkel B, Park JE, Lenter M, Rettig
`WJ, Damm K, et al. Mouse fibroblast-activation
`protein–conserved Fap gene organization and bio-
`chemical function as a serine protease. Eur J Biochem
`1998; 254:650-4; PMID:9688278; http://dx.doi.org/
`10.1046/j.1432-1327.1998.2540650.x
`7. Chen WT, Kelly T. Seprase complexes in cellular
`invasiveness. Cancer Metastasis Rev 2003; 22:
`259-69; PMID:12785000; http://dx.doi.org/10.1023/
`A:1023055600919
`8. Goldstein LA, Ghersi G, Piñeiro-Sánchez ML,
`Salamone M, Yeh Y, Flessate D, et al. Molecular
`cloning of seprase: a serine integral membrane protease
`from human melanoma. Biochim Biophys Acta 1997;
`1361:11-9; PMID:9247085
`9. Aertgeerts K, Levin I, Shi L, Snell GP, Jennings A,
`Prasad GS, et al. Structural and kinetic analysis of the
`substrate specificity of human fibroblast activation
`protein alpha. J Biol Chem 2005; 280:19441-4; PMID:
`15809306; http://dx.doi.org/10.1074/jbc.C500092200
`
`10. Zhang J, Valianou M, Cheng JD. Identification and
`characterization of the promoter of fibroblast activation
`protein. [Elite Ed]. Front Biosci (Elite Ed) 2010; 2:
`1154-63; PMID:20515787; http://dx.doi.org/10.2741/
`E175
`11. Cheng JD, Valianou M, Canutescu AA, Jaffe EK, Lee
`HO, Wang H, et al. Abrogation of fibroblast activation
`protein enzymatic activity attenuates tumor growth.
`Mol Cancer Ther 2005; 4:351-60; PMID:15767544
`12. Christiansen VJ, Jackson KW, Lee KN, McKee PA.
`Effect of
`fibroblast activation protein and alpha2-
`antiplasmin cleaving enzyme on collagen types I, III,
`and IV. Arch Biochem Biophys 2007; 457:177-86;
`PMID:17174263; http://dx.doi.org/10.1016/j.abb.2006.
`11.006
`13. Keane FM, Nadvi NA, Yao TW, Gorrell MD.
`Neuropeptide Y, B-type natriuretic peptide, substance
`P and peptide YY are novel substrates of fibroblast
`activation protein-a. FEBS J 2011; 278:1316-32;
`PMID:21314817;
`http://dx.doi.org/10.1111/j.1742-
`4658.2011.08051.x
`14. Meadows SA, Edosada CY, Mayeda M, Tran T, Quan
`C, Raab H, et al. Ala657 and conserved active site
`residues promote fibroblast activation protein endo-
`peptidase activity via distinct mechanisms of transition
`state stabilization. Biochemistry 2007; 46:4598-605;
`PMID:17381073; http://dx.doi.org/10.1021/bi062227y
`15. Dolznig H, Schweifer N, Puri C, Kraut N, Rettig WJ,
`Kerjaschki D, et al. Characterization of cancer stroma
`markers:
`in silico analysis of an mRNA expression
`database for fibroblast activation protein and endosialin.
`Cancer Immun 2005; 5:10; PMID:16076089
`16. Mathew S, Scanlan MJ, Mohan Raj BK, Murty VV,
`Garin-Chesa P, Old LJ, et al. The gene for fibroblast
`activation protein alpha (FAP), a putative cell surface-
`bound serine protease expressed in cancer stroma and
`wound healing, maps to chromosome band 2q23.
`Genomics 1995; 25:335-7; PMID:7774951; http://dx.
`doi.org/10.1016/0888-7543(95)80157-H
`17. Bauer S, Jendro MC, Wadle A, Kleber S, Stenner F,
`Dinser R, et al. Fibroblast activation protein is
`expressed by rheumatoid myofibroblast-like synovio-
`cytes. Arthritis Res Ther 2006; 8:R171; PMID:
`17105646; http://dx.doi.org/10.1186/ar2080
`
`Jones D,
`JM, Kevorkian L, Young DA,
`18. Milner
`Wait R, Donell ST, et al. Fibroblast acti