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PubMed2006

Peptide substrate profiling defines fibroblast activation protein as an endopeptidase of strict Gly(2)-Pro(1)-cleaving specificity.

Fibroblast activation protein (FAP) is a serine protease of undefined endopeptidase specificity implicated in tumorigenesis. To characterize FAP's P(4)-P(2)(') specificity, we synthesized intramolecularly quenched fluorescent substrate sets based on the FAP cleavage site in alpha(2)-antiplasmin (TSGP-NQ). FAP required substrates with Pro at P(1) and Gly or d-amino acids at P(2) and preferred small, uncharged amino acids at P(3), but tolerated most amino acids at P(4), P(1)(') and P(2)('). These substrate preferences allowed design of peptidyl-chloromethyl ketones that inhibited FAP, but not the related protease, dipeptidyl peptidase-4. Thus, FAP is a narrow specificity endopeptidase and this can be exploited for inhibitor design.

Amino Acid Chloromethyl Ketones↗
PubMed2005

Antiplasmin-cleaving enzyme is a soluble form of fibroblast activation protein.

Circulating antiplasmin-cleaving enzyme (APCE) has a role in fibrinolysis and appears structurally similar to fibroblast activation protein (FAP), a cell-surface proteinase that promotes invasiveness of certain epithelial cancers. To explore this potential relationship, we performed comparative structure/function analyses of the 2 enzymes. APCE from human plasma and recombinant FAP (rFAP) exhibited identical pH optima of 7.5, extinction coefficients (in(280 nm)(1%)) of 20.2 and 20.5, common sequences of tryptic peptides, and cross-reactivity with FAP antibody. APCE and rFAP are homodimers with monomeric subunits of 97 and 93 kDa. Only homodimers appear to have enzymatic activity, with essentially identical kinetics toward Met-alpha2-antiplasmin (Met-alpha2AP) and peptide substrates. APCE and rFAP cleave both Pro3-Leu4 and Pro12-Asn13 bonds of Met-alpha2AP, but relative kcat/Km values for Pro12-Asn13 are about 16-fold higher than for Pro3-Leu4. APCE and rFAP demonstrate higher kcat/Km values toward a peptide modeled on P4-P4' sequence surrounding the Pro12-Asn13 primary cleavage site than for Z-Gly-Pro-AMC and Ala-Pro-AFC substrates. These data support APCE as a soluble derivative of FAP and Met-alpha2AP as its physiologic substrate. Conversion of Met-alpha2AP by membrane or soluble FAP to the more easily fibrin-incorporable form, Asn-alpha2AP, may increase plasmin inhibition within fibrin surrounding certain neoplasms and have an impact on growth and therapeutic susceptibility.

Amino Acid Sequence↗
PubMed2006

Effect of fibroblast activation protein and alpha2-antiplasmin cleaving enzyme on collagen types I, III, and IV.

The circulating enzyme, alpha2-antiplasmin cleaving enzyme (APCE), has very similar sequence homology and proteolytic specificity as fibroblast activation protein (FAP), a membrane-bound proteinase. FAP is expressed on activated fibroblasts associated with rapid tissue growth as in embryogenesis, wound healing, and epithelial-derived malignancies, but not in normal tissues. Its presence on stroma suggests that FAP functions to remodel extracellular matrix (ECM) during neoplastic growth. Precise biologic substrates have not been defined for FAP, although like APCE, it cleaves alpha2-antiplasmin to a derivative more easily cross-linked to fibrin. While FAP has been shown to cleave gelatin, evidence for cleavage of native collagen, the major ECM component, remains indistinct. We examined the potential proteolytic effects of FAP or APCE alone and in concert with selected matrix metalloproteinases (MMPs) on collagens I, III, and IV. SDS-PAGE analyses demonstrated that neither FAP nor APCE cleaves collagen I. Following collagen I cleavage by MMP-1, however, FAP or APCE digested collagen I into smaller peptides. These peptides were analogous to, yet different from, those produced by MMP-9 following MMP-1 cleavage. Amino-terminal sequencing and mass spectrometry analyses of digestion mixtures identified several peptide fragments within the sequences of the two collagen chains. The proteolytic synergy of APCE in the cleavage of collagen I and III was not observed with collagen IV. We conclude that FAP works in synchrony with other proteinases to cleave partially degraded or denatured collagen I and III as ECM is excavated, and that derivative peptides might function to regulate malignant cell growth and motility.

Amino Acid Sequence↗
PubMed2002

Intrahepatic expression of the hepatic stellate cell marker fibroblast activation protein correlates with the degree of fibrosis in hepatitis C virus infection.

BACKGROUND: Activated hepatic stellate cells (HSCs), recognised by their alpha smooth muscle actin immunoreactivity, are primarily responsible for liver fibrosis. However, the presence of alpha smooth muscle actin positive HSCs is not always associated with the development of liver fibrosis. Recently, other markers of human HSCs including the gelatinase fibroblast activation protein (FAP) and glial fibrillary acidic protein have been identified. AIMS: We examined the relationship between the expression of these HSC markers and the severity of liver injury in patients with chronic hepatitis C virus infection. METHODS: Liver tissue from 27 patients was examined using immunohistochemistry. Linear correlation analysis was used to compare staining scores with the stage and grade of liver injury. RESULTS-CONCLUSIONS: FAP expression, seen at the tissue-remodelling interface, was strongly and significantly correlated with the severity of liver fibrosis. A weaker correlation was seen between glial fibrillary acidic protein expression and fibrosis stage. This contrasted with the absence of a relationship between alpha smooth muscle actin and the fibrotic score. A correlation was also observed between FAP expression and necroinflammatory score. In summary, FAP expression identifies a HSC subpopulation at the tissue-remodelling interface that is related to the severity of liver fibrosis.

Actins↗
PubMed1999

Fibroblast activation protein: a cell surface dipeptidyl peptidase and gelatinase expressed by stellate cells at the tissue remodelling interface in human cirrhosis.

Fibroblast activation protein (FAP) is a cell surface-bound protease of the prolyl oligopeptidase gene family expressed at sites of tissue remodelling. This study aimed to delineate the expression of FAP in cirrhotic human liver and examine its biochemical activities. Seventeen cirrhotic and 8 normal liver samples were examined by immunohistochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR). Hepatic stellate cells (HSC) were isolated and immunostained. Recombinant FAP and immunopurified, natural FAP were analyzed for protease activities and similarities to dipeptidyl peptidase IV (DPPIV), a structurally related enzyme. FAP-specific messenger RNA and immunoreactivity were detected in cirrhotic, but not normal, livers. FAP immunoreactivity was most intense on perisinusoidal cells of the periseptal regions within regenerative nodules (15 of 15 cases); this pattern coincides with the tissue remodelling interface. In addition, human FAP was expressed by cells within the fibrous septa (10 of 15 cases). Cell morphology, location, and colocalization with glial fibrillary acidic protein (GFAP) indicated that FAP is present on HSC in vivo. Similarly, isolated HSC expressed FAP in vitro. Both natural FAP from cirrhotic liver and recombinant FAP were shown to have gelatinase and dipeptidyl peptidase activities. FAP is a cell-bound, dual-specificity dipeptidyl peptidase and gelatinase expressed by activated HSC at the tissue remodelling interface in human cirrhosis. FAP may contribute to the HSC-induced extracellular matrix (ECM) changes of cirrhosis.

Actins↗
PubMed2001

Generation of human high-affinity antibodies specific for the fibroblast activation protein by guided selection.

Four completely human antibody derivatives [single-chain-antibody fragments (scFvs)] with specificity for the general tumor stroma marker fibroblast activation protein (FAP) were isolated by guided selection. Highly diverse IgG, IgM and IgD isotypes comprising heavy-chain variable domain libraries were generated using cDNAs derived from diverse lymphoid organs of a multitude of donors. Three of the human scFvs were converted into bivalent minibodies and expressed in eukaryotic cells for further functional characterization. Binding-competition studies and analysis by fluorescence-activated cell sorting showed high-affinity binding (10--20 nM) for two clones and recognition of the same epitope as the murine guiding antibody. The minibodies were successfully used for immunohistology of a variety of human carcinoma biopsies, revealing specific staining of stromal fibroblasts. Therefore, they should be suitable for in vivo diagnostic and tumor-targeting studies and, because of their completely human origin, be superior to murine or humanized antibody derivatives.

Amino Acid Sequence↗
PubMed2003

Expression and targeting of human fibroblast activation protein in a human skin/severe combined immunodeficient mouse breast cancer xenograft model.

Antigens and receptors that are highly expressed on tumor stromal cells, such as fibroblast activation protein (FAP), are attractive targets for antibody-based therapies because the supporting stroma and vessel network is essential for a solid neoplasm to grow beyond a size of 1-2 mm. The in vivo characterization of antibodies targeting human stromal or vessel antigens is hindered by the lack of an appropriate mouse model system because xenografts in standard mouse models express stromal and vessels elements of murine origin. This limitation may be overcome by the development of a human skin/mouse chimeric model, which is established by transplanting human foreskin on to the lateral flank of severe combined immunodeficient mice. The subsequent inoculation of breast carcinoma MCF-7 cells within the dermis of the transplanted human skin resulted in the production of xenografts expressing stromal and vessel elements of human origin. Widespread expression of human FAP-positive reactive stromal fibroblasts within xenografts was seen up to 2 months posttransplantation and postinjection of cells. Human blood vessel antigen expression also persisted at 2 months posttransplantation and postinjection of cells with murine vessels coexisting with the human vascular supply. The model was subsequently used to evaluate the biodistribution properties of an iodine-131-labeled humanized anti-FAP monoclonal antibody (BIBH-7). The results showed high specific targeting of the stromal compartment of the xenograft, indicating that the model provides a useful and novel approach for the in vivo assessment of the immunotherapeutic potential of molecules targeting human stroma and angiogenic systems.

Animals↗
PubMed2005

Fibroblast activation protein increases apoptosis, cell adhesion, and migration by the LX-2 human stellate cell line.

Injury and repair in chronic liver disease involve cell adhesion, migration, apoptosis, proliferation, and a wound healing response. In liver, fibroblast activation protein (FAP) has both collagenase and dipeptidyl peptidase IV (DPIV) activities and is expressed only by activated hepatic stellate cells (HSC) and myofibroblasts, which produce and degrade extracellular matrix (ECM). FAP was colocalized with collagen fibers, fibronectin, and collagen type I in human liver. FAP function was examined in vitro by expressing green fluorescent protein FAP fusion protein in cell lines cultured on collagen-I, fibronectin, and Matrigel. Glutamates at 203 and 204 as well as serine624 of FAP were essential for peptidase activity. Human embryonic kidney 293T cells overexpressing FAP showed reduced adhesion and migration. FAP overexpression in the human HSC line LX-2 caused increased cell adhesion and migration on ECM proteins as well as invasion across transwells in the absence or presence of transforming growth factor beta-1. FAP overexpression enhanced staurosporine streptomyces-stimulated apoptosis in both cell lines. Interestingly, the enzyme activity of FAP was not required for these functions. Overexpressing FAP increased the expression of matrix metalloproteinase-2 and CD44 and reduced integrin-beta1 expression in 293T cells, suggesting potential pathways of FAP-mediated impairment of cell adhesion and migration in this epithelial cell line. In conclusion, these findings further support a pro-fibrogenic role for FAP by indicating that, in addition to its enzymatic functions, FAP has important nonenzymatic functions that in chronic liver injury may facilitate tissue remodeling through FAP-mediated enhancement of HSC cell adhesion, migration, and apoptosis.

Antigens, Neoplasm↗
PubMed1997

Mouse fibroblast activation protein: molecular cloning, alternative splicing and expression in the reactive stroma of epithelial cancers.

The growth of solid neoplasms requires the recruitment of a supporting stroma. In most epithelial cancers, this stromal compartment comprises newly formed blood vessels and abundant, reactive stromal fibroblasts. Tumor stromal fibroblasts are not transformed but differ from resting fibrocytes in normal adult tissues by an altered pattern of gene expression. In human cancers, this includes induction of the cell-surface-bound fibroblast-activation protein (FAP), a member of the serine protease family encoded by the FAP gene on chromosome 2. In this study, we have cloned a complementary DNA for Fap, the murine homologue of FAP. The predicted murine FAP protein, mFAP, shares 89% amino-acid-sequence identity with human FAP, including a perfectly conserved catalytic triad. Cultured mouse embryo fibroblasts and mouse embryonic tissues were found to express Fap transcripts. In addition, the host-derived, fibroblast-rich stroma of human epithelial-cancer xenografts grown in immunodeficient mice also expresses Fap. Sequencing of reverse-transcription-PCR products indicates that 3 distinct Fap splice variants can be detected in tissues. Our findings suggest a close similarity in structure and tissue expression of FAP in different species. By extending the analysis of FAP to the mouse, new in vivo test systems become available for genetic and therapeutic manipulations and for the study of FAP regulation and function in embryonic development and in epithelial cancers.

Adenocarcinoma↗
PubMed2001

Construction of a bispecific single chain antibody for recruitment of cytotoxic T cells to the tumour stroma associated antigen fibroblast activation protein.

Bispecific antibodies directed against tumour associated antigens and the T cell receptor component CD3 for recruitment and tumour targeted activation of T cells represent a novel class of highly specific immunotherapeutics for cancer. We here describe the construction, eukaryotic expression and in vitro functional activity of a new T cell activating bispecific reagent, termed TTS for T cell targeting to the tumour stroma, comprised of a CD3 specific single chain antibody derivative (scFv) fused C-terminally to a 'fibroblast activation protein' (FAP) specific scFv that targets cytotoxic effector cells to FAP. FAP is highly expressed in the vascularised tumoural stroma of most lung, breast and colon carcinomas. It thus represents a selectively tumour associated, yet common marker of many solid tumours and is a potentially ideal candidate marker for efficient targeting of immune effector cells.

Animals↗
PubMed2006

Fibroblast activation protein alpha is expressed by chondrocytes following a pro-inflammatory stimulus and is elevated in osteoarthritis.

Arthritis is characterised by the proteolytic degradation of articular cartilage leading to a loss of joint function. Articular cartilage is composed of an extracellular matrix of proteoglycans and collagens. We have previously shown that serine proteinases are involved in the activation cascades leading to cartilage collagen degradation. The aim of this study was to use an active-site probe, biotinylated fluorophosphonate, to identify active serine proteinases present on the chondrocyte membrane after stimulation with the pro-inflammatory cytokines IL-1 and oncostatin M (OSM), agents that promote cartilage resorption. Fibroblast activation protein alpha (FAPalpha), a type II integral membrane serine proteinase, was identified on chondrocyte membranes stimulated with IL-1 and OSM. Real-time PCR analysis shows that FAPalpha gene expression is up-regulated by this cytokine combination in both isolated chondrocytes and cartilage explant cultures and is significantly higher in cartilage from OA patients compared to phenotypically normal articular cartilage. Immunohistochemistry analysis shows FAPalpha expression on chondrocytes in the superficial zone of OA cartilage tissues. This is the first report demonstrating the expression of active FAPalpha on the chondrocyte membrane and elevated levels in cartilage from OA patients. Its cell surface location and expression profile suggest that it may have an important pathological role in the cartilage turnover prevalent in arthritic diseases.

Animals↗
PubMed2006

Laser capture microdissection of epithelial cancers guided by antibodies against fibroblast activation protein and endosialin.

Transcriptional profiling of cancer biopsies is used extensively to identify expression signatures for specific cancer types, diagnostic and prognostic subgroups, and novel molecular targets for therapy. To broaden these applications, several challenges remain. For example, the integrity of RNA extracted even from small tissue samples has to be insured and monitored. Moreover, total tumor RNA may hide the marked histologic heterogeneity of human cancers. A principle approach to this heterogeneity has been provided by laser capture microdissection performed on antibody-stained tissue sections (immuno-LCM; iLCM). In this study, we have established a procedure to assess the quality of RNA obtained from tissue sections, coupled with immunostaining using antibodies to different tumor stromal markers, and subsequent iLCM to selectively capture the cancer stroma compartments. The procedure was applied to 53 frozen specimens of human epithelial cancers. Sections were stained for histopathological evaluation, and RNA was isolated from adjacent serial sections. RNA quality was assessed by the Agilent-Bioanalyzer (Agilent, Palo Alto, CA) and by multiplex RT-PCR. Two thirds of the specimens were found to yield good to excellent RNA quality. For microdissection of the tumor stroma with reactive fibroblasts and tumor blood vessels, a rapid incubation protocol with antibodies against fibroblast activation protein (FAP) and against endosialin was developed to ensure RNA integrity for subsequent iLCM. Using these procedures, RNA from distinct tumor compartments can be isolated, analyzed, amplified, and used for transcription profiling.

Antibodies, Neoplasm↗
PubMed2006

Fibroblast activation protein (FAP) is upregulated in myelomatous bone and supports myeloma cell survival.

The microenvironment plays a critical role in facilitating cancer progression and metastasis. We previously demonstrated the ability of osteoclasts to support primary myeloma plasma cell (MM PC) growth. Our study on the role of the bone marrow (BM) microenvironment in myeloma, using global gene expression profiling, has identified fibroblast activation protein (FAP) as one of 28 genes significantly overexpressed in cocultured osteoclasts. Because FAP has been previously implicated in tumorigenesis and shown to be selectively expressed by the reactive stroma of epithelial tumours, we focused our study on the role of this serine protease in myeloma. Using quantitative polymerase chain reaction amplification, we demonstrated upregulation of FAP by cocultured osteoclasts and mesenchymal stem cells, and in whole myelomatous human bone in SCID-hu mice. Immunohistochemical analysis of myelomatous bone sections revealed FAP expression by osteoclasts, osteogenic cells, fibrotic stroma and certain adipocytes and vascular endothelial cells. FAP was not expressed in PCs by all these methods. Inhibition of FAP expression with the use of small-interference RNA reduced MM PC survival in cocultures. Our results indicate that FAP is critical for the interaction of MM cells with the BM microenvironment--a potential therapeutic target in myeloma.

Adipocytes↗
PubMed2005

Induction of CD4(+) and CD8(+) T-cell responses to the human stromal antigen, fibroblast activation protein: implication for cancer immunotherapy.

PURPOSE: The propensity of tumor cells to escape immune elimination could limit, if not defeat, the long-term benefits of effective immunotherapeutic protocols. Immunologic targeting of tumor stroma could significantly reduce the ability of tumors to evade immune elimination. Murine studies have shown that inducing immunity against angiogenesis-associated products engenders potent antitumor immunity without significant pathology. It is, however, not known whether T cells corresponding to stromal products are present in humans. In this study, we describe a method to screen for human stromal products that have not triggered significant tolerance and could therefore serve as candidate antigens for cancer immunotherapy. EXPERIMENTAL DESIGN: To identify candidates for human stromal antigens, we used an in vitro-screening method to determine whether dendritic cells transfected with mRNA encoding products, which are overexpressed in the tumor stroma, are capable of stimulating cytotoxic CD8(+) (CTL) responses from human peripheral blood mononuclear cells. RESULTS: CTL responses could be consistently generated against fibroblast activation protein (FAP) but not against matrix metalloproteinase-9 (MMP-9) or MMP-14. To enhance the immunogenicity of the mRNA-translated FAP product, a lysosomal targeting signal derived from lysosome-associated membrane protein-1 (LAMP-1) was fused to the COOH terminus of FAP to redirect the translated product into the class II presentation pathway. Dendritic cells transfected with mRNA encoding the FAP-LAMP fusion product stimulated enhanced CD4(+) and CD8(+) T-cell responses. CONCLUSION: This study identifies FAP, a protease preferentially expressed in tumor-associated fibroblasts, as a candidate human stromal antigen to target in the setting of cancer immunotherapy, and shows that differential expression of stromal products is not a sufficient criteria to indicate its immunogenicity in a vaccination setting.

Animals↗
PubMed2005

Characterization of cancer stroma markers: in silico analysis of an mRNA expression database for fibroblast activation protein and endosialin.

Standardized, high-throughput RNA detection with microarray chips allows for the construction of genome-wide databases for tissue specimens suitable for in silico electronic Northern blot (eNorthern) analysis of marker genes. We used the BioExpress database, which contains transcriptional profiles of normal and cancer samples, to examine two putative markers of cancer stroma: fibroblast activation protein-alpha (FAP-alpha) and endosialin. Analyses for FAP-alpha showed that normal tissues generally lack RNA signals, with the exception of endometrium. Typing of tumors revealed prominent FAP-alpha signals in cancer types marked by desmoplasia, and localization of FAP-alpha in reactive cancer stroma was confirmed by immunohistochemistry. A subset of sarcomas displayed prominent FAP-alpha signals localizing to the malignant cells. For endosialin, eNorthern analyses showed low to moderate RNA signals in many normal organs, whereas immunohistochemistry revealed endosialin in only some tissues, such as endometrium. Endosialin was detected at the RNA and protein level in sarcomas, notably malignant fibrous histiocytomas. Low to moderate endosialin RNA signals were found in epithelial cancer types for which immunostaining identifies expression in subsets of tumor capillaries or fibroblasts. These findings extend the FAP-alpha and endosialin profiling in silico to an unbiased tumor database and place both molecules in a novel context of endometrial biology and sarcoma subtyping. Our findings suggest that BioExpress can be searched directly for tumor stroma markers but may need prior enrichment for markers with narrow cellular representation, such as endosialin. Constructing databases from microdissected cancer tissues may be an essential step for tumor stroma-targeted therapies.

Adult↗
PubMed2004

PT-100, a small molecule dipeptidyl peptidase inhibitor, has potent antitumor effects and augments antibody-mediated cytotoxicity via a novel immune mechanism.

The amino boronic dipeptide, PT-100 (Val-boro-Pro), a dipeptidyl peptidase (DPP) inhibitor, has been shown to up-regulate gene expression of certain cytokines in hematopoietic tissue via a high-affinity interaction, which appears to involve fibroblast activation protein. Because fibroblast activation protein is also expressed in stroma of lymphoid tissue and tumors, the effect of PT-100 on tumor growth was studied in mice in vivo. PT-100 has no direct cytotoxic effect on tumors in vitro. Oral administration of PT-100 to mice slowed growth of syngeneic tumors derived from fibrosarcoma, lymphoma, melanoma, and mastocytoma cell lines. In WEHI 164 fibrosarcoma and EL4 and A20/2J lymphoma models, PT-100 caused regression and rejection of tumors. The antitumor effect appeared to involve tumor-specific CTL and protective immunological memory. PT-100 treatment of WEHI 164-inoculated mice increased mRNA expression of cytokines and chemokines known to promote T-cell priming and chemoattraction of T cells and innate effector cells. The role of innate activity was further implicated by observation of significant, although reduced, inhibition of WEHI 164 and A20/2J tumors in immunodeficient mice. PT-100 also demonstrated ability to augment antitumor activity of rituximab and trastuzumab in xenograft models of human CD20(+) B-cell lymphoma and HER-2(+) colon carcinoma where antibody-dependent cytotoxicity can be mediated by innate effector cells responsive to the cytokines and chemokines up-regulated by PT-100. Although CD26/DPP-IV is a potential target for PT-100 in the immune system, it appeared not to be involved because antitumor activity and stimulation of cytokine and chemokine production was undiminished in CD26(-/-) mice.

Administration, Oral↗