2q24.2

DPPIV,FAPA,FAPalpha,SIMP

The relatively selective expression of FAP in tumors and/or tumor-associated stromal cells and its presumed direct role in various aspects of cancer progression including the re-modeling of the tumor microenvironment make FAP attractive as a therapeutic target.
As mentioned above, FAP has both collagenase and dipeptidase activities which can degrade gelatin, collagen, and other substrates of dipeptidase, and can promote tumor growth, migration, invasion, metastasis, and ECM degradation. Therefore, it was hypothesized that selective blocking of the enzymatic activity of FAP can be a strategy to prevent tumor development. Val-boroPro, also known as Talabostat (PT100), is a broad S9 protease inhibitor and currently is the only non-specific FAP inhibitor that has been tested in cancer patients in clinical trials (Bainbridge et al., 2017) It was well tolerated in a phase I study in solid tumor patients who were receiving myelosuppressive chemotherapy and it accelerated neutrophil recovery. However, despite the promising results in phase I tests, PT100 failed in phase II clinical trials performed in patients with metastatic colon cancer, non-small cell lung cancer, and melanoma (Busek et al., 2018; Jiang et al., 2016). There is still no consistent conclusion to explain why the drug was not successful during the phase II trial, but reports suggest that the enzymatic activity of FAP has little to do with increased tumor growth. Another explanation for this failure is that serine proteases can function as tumor suppressors (Jiang et al., 2016). However, after this disappointing clinical outcome of PT100, studies concentrated on the investigation of FAP inhibitor antibodies. An inhibitory scFv antibody, named E3 was identified, which could modify the FAP-mediated rearrangement of the fibronectin fibers in vitro, but its effect on cancer growth still needs to be determined (Zhang et al., 2013). In another study, rabbits were immunized with recombinant murine FAP to develop polyclonal anti-FAP antibodies. The antibodies significantly inhibited murine FAP dipeptidyl peptidase activity in vitro and slowed tumor growth in a mouse xenotransplantation model (Cheng et al., 2002).
131-labeled monoclonal antibody (131I-mAbF19) was the first monoclonal antibody against FAP that was used in patients with hepatic metastases from colorectal carcinoma. The 131I-mAbF19 was administered by intravenous injection and no toxicity was observed (Jiang et al., 2016). Sibrotuzumab, a humanized version of the F19 antibody, was tested in phase I clinical trial in metastatic cancer patients. It was found to be non-toxic, did not accumulate in healthy tissues, and was successful in targeting the tumor stroma. However, no therapeutic response was observed in either phase I or the subsequent phase II trial in metastatic colorectal cancer; moreover, some of the patients developed anti-human antibodies (Busek et al., 2018). Despite these disappointing results, the search for more efficient FAP antibodies continues. Novel human-mouse cross-reactive antibodies, ESC11, and ESC14 labeled with the radionuclide (177) Lu were designed and tested for their capacity to accumulate in tumors. In a melanoma xenograft model, (177)Lu-labeled ESC11 was found to be specific to tumor tissue, suggesting these antibodies can be potent radioimmunoconjugates or antibody-drug conjugates for diagnostic and therapeutic use in patients with FAP-expressing tumors (Fischer et al., 2012).
To selectively activate apoptotic pathways in tumor cells, a bispecific antibody (RG7386) binding to both FAP and TNFRSF10B (DR5, Death Receptor 5) was designed. The binding of this antibody to FAP on stromal cells caused a hyperclustering of DR5 which resulted in the induction of apoptosis in tumor cells in in vitro co-culture systems and reduced tumor growth in vivo. When combined with irinotecan, a chemotherapeutic drug to inhibit topoisomerase I, an enzyme involved in DNA replication, this bispecific antibody led to complete tumor eradication in a colorectal cancer mouse model (Brünker et al., 2016). As another approach, a dimeric anti-FAP-TNF protein was designed to activate TNF receptor signaling only when bound to FAP. The protein delayed tumor growth and enhanced macrophage recruitment to the tumor in immunodeficient mice xenografted with FAP-transfected fibrosarcoma HT1080 cells (Bauer et al., 2006, 2004). Other studies have aimed for specific stimulation of local antitumor immune response using FAP targeting antibodies. For instance, in a 3D heterotypic spheroid model containing colon cancer cells, fibroblasts, and peripheral blood mononuclear cells, a bispecific antibody targeting FAP and CD3E (CD3 Cluster of Differentiation 3 epsilon) resulted in a decrease in FAP expressing fibroblasts and this inhibitory effect was more pronounced when variant IL2 (IL-2v) was applied simultaneously (Herter et al., 2017). Hornig et al. reported the construction of a bispecific FAP-CD3 antibody by using the FAP targeting antibodies MO33, MO36, and a costimulatory ligand B7.2. The B7.2 containing antibody was next combined with another costimulatory ligand 4-1BBL fused to an anti-endoglin antibody to induce T cell activation and the release of IL-2 and IFNγ in the presence of target cells expressing both FAP and ENG (endoglin). By the costimulation of CD28 and 4-1BB, the overall T-cell population was strongly increased in activation-experienced memory phenotype accompanied by a decrease in naive phenotype (Hornig et al., 2012). In another study, an antibody cytokine fusion protein (scFv_RD_IL-15), composed of an antibody moiety targeting FAP, an extended IL-15Rα sushi domain (RD) and IL15 was generated to improve the efficiency of IL15. The fusion protein exhibited antibody-mediated specific binding and cytokine activity in both soluble and targeted form and improved the antitumor effect of IL15 in a mouse B16F10/lung metastasis model (Kermer et al., 2012).
Anti-FAP antibody fragments were also immobilized on liposomes to create nanocarriers for therapeutically active agents to specifically target tumor tissue. Immunoliposomes presenting single-chain Fv molecules targeting FAP were able to bind to FAP-expressing cells and were internalized. When loaded with the fluorescent dye DY-676-COOH, these immunoliposomes could be used for the visualization of FAP+ cells in vitro and in a mouse xenograft model (Rüger et al., 2014). The immunoliposomes were also used to detect the metastatic spread of tumors. Bispecific immunliposomes recognizing FAP and endoglin exhibited a higher binding to target stromal cells compared to monospecific liposomes that targeted FAP or endoglin separately. More importantly, these bispecific immunoliposomes showed robust interaction with target cells and upon loading with doxorubicin, showed enhanced cytotoxicity (Rabenhold et al., 2015). Tansi et al. designed bispecific liposomes containing single-chain antibody fragments specific for FAP and ERBB2 (HER2, Human Epidermal Growth Factor Receptor 2) to target both stromal and transformed cells. In a mouse breast cancer model, these bispecific immunoliposomes accumulated in fibroblasts, perivascular cells, as well as in HER2+ tumor elements and were successful in delivering the encapsulated therapeutic cargo to tumor cells and tumor stromal fibroblasts (Tansi et al., 2017). In summary, although the available studies are promising, preclinical and clinical experiments are needed to investigate the efficiency of diagnostic and therapeutic effects of the FAP antibody conjugates in patients with FAP-expressing tumors.
FAP is also a candidate target for DNA vaccines for cancer treatment. An oral DNA vaccine was constructed to target FAP, which could successfully suppress primary tumor cell growth and metastasis of multidrug-resistant murine colon and breast carcinoma. Furthermore, tumor tissue of Fap-vaccinated mice revealed markedly decreased collagen type I expression and up to 70% greater uptake of chemotherapeutic drugs. More importantly, Fap-vaccinated mice treated with chemotherapy showed prolonged lifespan and marked suppression of tumor growth, with half of the animals completely overcoming a tumor cell challenge (Loeffler et al., 2006). Similarly, in in vivo models of melanoma, carcinoma, and lymphoma, tumor growth was inhibited by immunization against FAP using dendritic cells transfected with FAP mRNA. The magnitude of the antitumor response was comparable to that of vaccination against tumor cell-expressed antigens (Lee et al., 2005). To enhance the immunogenicity of the mRNA-translated FAP product, a lysosomal targeting signal derived from LAMP1 (Lysosome-Associated Membrane Protein-1) was fused to the COOH terminus of FAP to redirect the translated product into the MHC class II presentation pathway. Dendritic cells transfected with mRNA encoding the FAP-LAMP fusion product was shown to stimulate CD4+ and CD8+ T-cell responses (Fassnacht et al., 2005).
Cancer immunotherapy by chimeric antigen receptor-modified T (CAR-T) cells has shown robust clinical efficacy for hematological malignancies. Nonetheless, challenges remain for the use of CAR-T cell therapy to treat solid tumors (Yu et al., 2017). In 2013, Kakarla et al. constructed a FAP-CAR, based on an anti-FAP antibody MO36. The resulting FAP-specific T cells recognized and killed FAP+ cancer target cells as determined by proinflammatory cytokine release and target cell lysis. In an A549 lung cancer model, the adoptive transfer of FAP-specific T cells significantly reduced FAP+ stromal cells, with a concomitant decrease in tumor growth. Combining these FAP-specific T cells with T cells that targeted the EPHA2 (EPH Receptor A2) antigen on the A549 cancer cells significantly enhanced overall antitumor activity and conferred a survival advantage (Kakarla et al., 2013). In another study, CD8+ human T cells were retrovirally transduced with a CAR based on the anti-FAP F19 antibody. When contacted with FAP+ mesothelioma cells, these CAR-T cells released IFNγ and specifically lysed the target cells in vitro. In an in vivo mesothelioma model, these CAR-T cells delayed tumor growth and significantly prolonged the survival of mice (Schuberth et al., 2013). Wang et al. developed a retroviral CAR construct specific for mouse Fap, comprising of a single-chain Fv FAP with the CD8A (CD8α) hinge and transmembrane regions, and the human CD247 (CD3ζ) and 4-1BB activation domains. The transduced muFAP-CAR mouse T cells secreted IFNγ and killed FAP-expressing 3T3 target cells specifically. In in vivo studies, these CAR-T cells reduced the number of FAP+ stromal fibroblasts and leukocytes, thus slowing tumor growth in several syngeneic mouse models. To a large extent, the observed effects were dependent on the augmentation of endogenous CD8+ T cell antitumor responses (L. C. S. Wang et al., 2014). On the other hand, Tran et al. showed that FAP-targeting CAR-T cells may lead to severe side effects. Two CAR constructs were generated using the scFv from FAP-specific monoclonal antibodies FAP5 and sibrotuzumab (F19; humanized monoclonal antibody directed against FAP). The resulting CAR-T cells were capable of specific degranulation and could produce effector cytokines in the presence of FAP-expressing cell lines. Nonetheless, their effect on tumor growth in a broad panel of mouse models was limited. More importantly, the injection of FAP5-CAR-T cells led to morbidity and mortality in most of the mice. Severe bone marrow hypocellularity and cachexia caused by the targeting of FAP+ osteogenic cells, including BMSC and possibly mesenchymal stromal cells in other organs were observed in FAP5-CAR-T cell treated animals. Similar toxic effects were observed in a pancreatic adenocarcinoma model rich in FAP expressing stroma (Busek et al., 2018; Tran et al., 2013). Tran et al. reported that some tumor stromal cells may simply be multipotent MSCs recruited into the tumor microenvironment. Thus, the promising strategy of targeting these normal tumor components must be approached with great care to avoid potentially life-threatening collateral damage to essential regenerative cells (Tran et al., 2013). Therefore, the use of FAP as a universal target antigen in cell-based immunotherapy should be further investigated particularly in light of the life-threatening side-effects.
The design of the FAP-activated prodrugs is another approach for tumor therapy. A non-toxic prodrug can be selectively activated to a highly potent form by the enzymatic activity of FAP (Busek et al., 2018; Puré and Blomberg, 2018). As cytotoxic drugs, melittin, doxorubicin, and thapsigargin were used as activable prodrugs coupled to FAP substrates (Akinboye et al., 2016; Brennen et al., 2012a, 2012b; S. Huang et al., 2011; Ke et al., 2017; LeBeau et al., 2009). Additionally, by linking multiple drugs with a FAP-cleavable linker enhanced efficacy could be achieved (Ke et al., 2017). Such a concept of FAP-mediated activation can also be applied to imaging techniques for cancer detection by using reporter constructs (Bainbridge et al., 2017; Feng et al., 2017). However, no clinical success has been reported with FAP based prodrug formulations.

Breast cancer

FAP was identified in the reactive stroma of breast cancer and epithelial tumor cells in ductal carcinomas (Kelly et al., 2012). However, data on the possible association of FAP expression with breast cancer survival are conflicting (Ariga et al., 2001; Jia et al., 2014; Jung et al., 2015).

Oral squamous cell carcinoma

FAP is a negative prognostic marker in oral squamous cell carcinoma (H. Wang et al., 2014).

Basal cell carcinoma, squamous cell carcinoma of the skin

Enhanced expression of FAP was detected in fibroblasts located close to cancer cells. Although FAP expression was absent in benign epithelial tumors, its positivity in the stroma was reported to be a useful criterion for differentiating between morpheaform/infiltrative basal cell carcinomas and FAP-negative desmoplastic trichoepithelioma (Busek et al., 2018).

Gastric cancer

FAP+ cells were shown to be accumulated in gastric cancer tissues (Song et al., 2017). Enhanced stromal FAP was associated with worse survival (Wen et al., 2017) as well as low tumor cell differentiation, more advanced TNM stage, serosal invasion, and poor survival (Shan et al., 2012). In intestinal-type gastric cancer, enhanced stromal FAP expression was associated with the presence of liver and lymph node metastases (Okada et al., 2003).

Melanoma

FAP expression was detected in in 30% of benign melanocytic nevi (a subset of melanocytes), but the expression was not detectable in malignant melanoma cells in melanoma tissues (Huber et al., 2006, 2003). A positive correlation was detected between ECM composition and inflammatory cell infiltration and the number of FAP+ stromal cells (Samaniego et al., 2013). On the other hand, there are also studies in which several human melanoma cell lines were shown to express FAP, which in turn, contributed to their invasiveness in vitro (Aoyama and Chen, 1990; Monsky et al., 1994; Tulley and Chen, 2014).

Esophageal cancer

FAP is expressed in cancer cells as well as in premalignant metaplastic cells of the esophagus in both adenocarcinoma and squamous cell carcinoma (Busek et al., 2018).

Colorectal cancer

Wikberg et al. reported that stromal FAP expression is common in colorectal cancer, and FAP expression was high in the tumor center, but not the tumor front. A high FAP expression in the tumor center was suggested as a negative prognostic factor (Wikberg et al., 2013). In patients with stage IV tumors, high FAP was associated with worse survival (Henry et al., 2007).

Pancreatic adenocarcinoma

FAP+ cells were reported to enhance pancreatic ductal adenocarcinoma progression (Lo et al., 2017). Its expression in carcinoma cells was associated with larger tumor size, presence of a fibrotic focus, perineural invasion, and a worse prognosis (Shi et al., 2012). Although stromal FAP expression was shown to be correlated with reduced survival and lymph node metastasis (Cohen et al., 2008; Kawase et al., 2015; Lo et al., 2017; Patsouras et al., 2015; Shi et al., 2012), Park et al. claimed an association between a lower number of FAP+ fibroblasts and a decreased overall survival (Park et al., 2017).

Hepatocellular carcinoma

In hepatocellular carcinoma, the over-expression of FAP has been reported in CAFs rather than hepatocellular carcinoma cells. Zou et al. showed that FAP can be induced in hepatocellular cancer cells under hypoxia and its expression was correlated with poor clinical outcomes (Zou et al., 2018). FAP expression was detected especially in tumors with abundant fibrous stroma (Kim et al., 2014).

Non-small cell lung cancer

FAP was found to be highly expressed in cancer stroma and it was also a predictor of poor survival of non-small cell lung cancer patients (Liao et al., 2013)

Mesothelioma

FAP expression has been detected in all subtypes (Schuberth et al., 2013).

FAP expression was not detected in multiple myeloma cells, but its expression was found in osteoclasts, endothelial cells, adipocytes, and fibrotic stroma (Ge et al., 2006).

Renal cancer

Stromal FAP expression was shown to be associated with worse survival in clear cell renal cell carcinoma and FAP was defined as a marker of aggressiveness and metastasis (Errarte et al., 2016; López et al., 2016).

Prostate cancer

The expression of FAP in stromal cells was reported (Tuxhorn et al., 2002).

Cervical cancer

Enhanced expression of FAP was found in cervical cancer cells and subepithelial stromal cells in some of the micro-invasive and all of the invasive carcinomas (Jin et al., 2003).

Ovarian carcinoma

FAP+ cells were detected in cancer cells in 21% of tumors and stromal positivity was detected in 61% of tumors (Mhawech-Fauceglia et al., 2015). FAP expressing malignant cells were present in malignant pleural and peritoneal effusions and associated with worse survival (Zhang et al., 2007). FAP+ cells were also found to be positively correlated with tumor stage and negative FAP expression was reported to be associated with longer disease-free survival (Zhang et al., 2015).

Thyroid cancer

In aggressive papillary thyroid carcinomas, FAP was found to be one of the upregulated genes (Fluge et al., 2006). FAP expression in the peritumoral and intratumoral stromal compartment in medullary thyroid carcinoma was shown to be correlated with the degree of desmoplasia and presence of lymph node metastases (Koperek et al., 2007).

Glioma

Enhanced FAP expression was detected in glioblastoma; however, the overall FAP quantity was not found to be associated with patient survival (Busek et al., 2016).

Sarcomas

FAP expression was detected in malignant cells in fibrosarcomas, leiomyosarcoma, malignant fibrous histiocytoma (Rettig et al., 1988), low-grade myofibroblastic sarcoma, fibroblastic areas in osteosarcomas, osteoid osteoma (Dohi et al., 2009), and in osteosarcoma (Ding et al., 2014). In osteosarcoma, enhanced FAP expression was associated with a more advanced clinical stage, presence of metastasis, high histological grade, and a worse progression-free and overall survival (Yuan et al., 2013).