FAP Expression Research Articles

Visualization of fibroblast activation after myocardial infarction using 68Ga-FAPI-PET

Abstract Introduction Fibroblast activation protein (FAP) alpha specifically expressed by activated fibroblasts has been shown to be significantly involved in processes of tissue healing after injury, including fibrotic remodeling after myocardial infarction (MI). Positron-emission tomography (PET) with the tracer FAP-inhibitor (FAPI) is a novel diagnostic tool primarily described in oncologic patients for tumor stroma detection. We present data from a pilot study investigating patients after MI who underwent subsequent 68Ga-FAPI-PET for assessment of potential FAP-related myocardial remodeling. Methods In this retrospective study we analyzed a cohort of 10 post-MI patients who underwent 68Ga-FAPI-PET imaging with respect to cardiac tracer pattern. Standardized maximum and mean uptake values and total volume of tracer enrichment (fibroblast activation volume, FAV) of the infarcted area were calculated. A visual grading scale was established to assess the level of agreement between maximum localized tracer uptake and segments belonging to the supply area of the culprit vessel. Tracer uptake was correlated to clinical variables, with readers blinded to the clinical characteristics. Results Focal 68Ga-FAPI uptake was detected in all 10/10 patients after MI. According to our grading system for visual agreement of polar map segments and culprit lesion, we observed a very good (in 37.5% of patients) and a moderate (62.5% of patients) match between tracer uptake and the culprit lesion, respectively. We demonstrated a negative correlation of FAV uptake and left ventricular systolic function (R2=−0.69, p<0.05) and a very strong positive correlation of FAV and peak creatinine kinase level (R2=0.90, p<0.01). Conclusion Increased myocardial 68Ga-FAPI uptake, serving as a surrogate for FAP expression after MI, corresponds well to the supply area of the culprit coronary vessel as well as to biomarker levels of myocardial injury. This imaging technique represents a highly promising, novel bio-signal in the assessment of myocardial injury and consecutive remodeling. Large controlled studies are warranted to further evaluate the prognostic impact of this novel imaging technique. Funding Acknowledgement Type of funding sources: None.

Targeting fibroblast activation protein in newly diagnosed squamous cell carcinoma of the oral cavity \u2013 initial experience and comparison to [18F]FDG PET/CT and MRI

PurposeWhile [18F]-fluorodeoxyglucose ([18F]FDG) is the standard for positron emission tomography/computed tomography (PET/CT) imaging of oral squamous cell carcinoma (OSCC), diagnostic specificity is hampered by uptake in inflammatory cells such as neutrophils or macrophages. Recently, molecular imaging probes targeting fibroblast activation protein α (FAP), which is overexpressed in a variety of cancer-associated fibroblasts, have become available and might constitute a feasible alternative to FDG PET/CT.MethodsTen consecutive, treatment-naïve patients (8 males, 2 females; mean age, 62 ± 9 years) with biopsy-proven OSCC underwent both whole-body [18F]FDG and [68Ga]FAPI-04 (FAP-directed) PET/CT for primary staging prior to tumor resection and cervical lymph node dissection. Detection of the primary tumor, as well as the presence and number of lymph node and distant metastases was analysed. Intensity of tracer accumulation was assessed by means of maximum (SUVmax) and peak (SUVpeak) standardized uptake values. Histological work-up including immunohistochemical staining for FAP served as standard of reference.Results[18F]FDG and FAP-directed PET/CT detected all primary tumors with a SUVmax of 25.5 ± 13.2 (FDG) and 20.5 ± 6.4 (FAP-directed) and a SUVpeak of 16.1 ± 10.3 ([18F]FDG) and 13.8 ± 3.9 (FAP-directed), respectively. Regarding cervical lymph node metastases, FAP-directed PET/CT demonstrated comparable sensitivity (81.3% vs. 87.5%; P = 0.32) and specificity (93.3% vs. 81.3%; P = 0.16) to [18F]FDG PET/CT. FAP expression on the cell surface of cancer-associated fibroblasts in both primary lesions as well as lymph nodes metastases was confirmed in all samples.ConclusionFAP-directed PET/CT in OSCC seems feasible. Future research to investigate its potential to improve patient staging is highly warranted.

O44: WOUND HEALING INFLAMMATORY MARKERS PREDICT PROGNOSIS AND SURVIVAL IN EARLY BREAST CANCER

Abstract Introduction Cancer is likened to a non-healing wound. There is limited evidence on the expression of wound healing tissue inflammatory markers, CD68(pan-macrophage marker), HO-1(tumour cell marker) and FAP(cancer-associated fibroblast marker) in human breast cancer. Method In 201 invasive breast cancer and 58 DCIS patients, CD68+TAM expression, tumour HO-1 and fibroblast FAP expression, quantified by immunohistochemistry(dichotomised: high/present vs low/absent), was correlated with tumour factors (grade, proliferation(Ki67), ER, HER2); demographic factors, behavioural factors (smoking, alcohol) and survival status(DFS, OS) Result High CD68+macrophage expression was increased in invasive breast cancer, compared to DCIS, and normal tissue distant from the tumour(59%,41%and 6% respectively; p<0.001).In invasive cancer,CD68+TAM expression increased with increasing tumour grade(Grade 1:42%, Grade 2:58%, Grade 3:72%; p=0.006), high Ki67(71%vs.47%; p=0.004), ER negativity(79.4%vs.55.4%; p=0.01) and HER2(HER2 positive 81.8% vs. HER2 negative 56.3%; p=0.03). CD68+TAM expression was higher in high compared to low/intermediate grade DCIS(44% % vs. 31% p=0.52). CD68+TAM expression was increased in patients who self-reported alcohol intake(non-drinker 43% vs. drinker 62%; p=0.01). HO-1 was associated with shorter DFS(HR:3.22,p=0.027) and OS(HR:2.86,p=0.029).FAP fibroblast expression correlated with longer DFS (HR:0.296,p=0.029) and OS (HR:0.271,p=0.008). Conclusion Tumour inflammation as assessed by CD68+TAM expression shows utility in identifying aggressive breast cancer sub-types. The association reported between CD68+TAM density and alcohol intake suggests a possible mechanism for alcohol as a risk factor for breast cancer. The prognostic value of HO-1 and FAP expression demonstrated here suggests a functional role of these wound healing markers in breast cancer. HO-1:Heme-oxygenase-1; FAP:Fibroblast activation protein; TAM:Tumour associated macrophage; DCIS: Ductal carcinoma in situ Take-home message Wound healing pathways of inflammation may be implicated in early breast cancer development

Stromal FAP is an independent poor prognosis marker in non-small cell lung adenocarcinoma and associated with p53 mutation

ObjectivesFibroblasts regulate tumor growth and immune surveillance. Here, we study FAP, PDGFβR and α-SMA fibroblast markers in a well-annotated clinical cohort of non–small-cell lung cancer (NSCLC) for analyses of associations with immune cell infiltration, mutation status and survival. Materials and MethodsA well-annotated NSCLC cohort was subjected to IHC analyses of stromal expression of FAP, PDGFβR and α-SMA and of stromal CD8 density. Fibroblast markers-related measurements were analyzed with regard to potential associations with CD8 density, cancer genetic driver mutations, survival and PD-L1 expression in the whole NSCLC cohort and in subsets of patients. ResultsHigh stromal FAP expression was identified as an independent poor prognostic marker in the whole study population (HR 1.481; 95 % CI, 1.012–2.167, p = 0.023) and in the histological subset of adenocarcinoma (HR 1.720; 95 % CI, 1.126–2.627, p = 0.012). Among patients with adenocarcinoma, a particularly strong association of FAP with poor survival was detected in patients with low stromal CD8 infiltration, and in other subpopulations identified by specific clinical characteristics; elderly patients, females, non-smokers and patients with normal ECOG performance status. α-SMA expression was negatively associated with CD8 infiltration in non-smokers, but none of the fibroblast markers expression was associated with CD8 density in the whole study population. Significant associations were detected between presence of p53 mutations and high α-SMA (p = 0.003) and FAP expression (p < 0.001). ConclusionThe study identifies FAP intensity as a candidate independent NSCLC prognostic biomarker. The study also suggests continued analyses of the relationships between genetic driver mutations and the composition of tumor stroma, as well as continued probing of marker-defined fibroblasts as NSCLC subset-specific modifiers of immune surveillance and outcome.

High Expression of FAP in Colorectal Cancer Is Associated With Angiogenesis and Immunoregulation Processes.

Fibroblast activation protein α (FAP) plays an important role in tissue remodeling and helps tumor cells invade surrounding tissue. We sought to investigate FAP as a prognostic molecular marker in colorectal cancer (CRC) using immunohistochemical and transcriptomic data. FAP expression and clinicopathological information were obtained from The Cancer Genome Atlas data set. The association of FAP expression and tissue cellular heterogeneity landscape was explored using the xCell method. We evaluated FAP protein expression in a cohort of 92 CRCs and 19 non-tumoral tissues. We observed that FAP was upregulated in tumors both at the mRNA and protein levels, and its expression was associated with advanced stages, poor survival, and consensus molecular subtype 4. FAP expression was also associated with angiogenesis and collagen degradation. We observed an enrichment in immune-cell process–related genes associated with FAP overexpression. Colorectal cancers with high FAP expression display an inflamed phenotype enriched for macrophages and monocytes. Those tumors showed enrichment for regulatory T cell populations and depletion of TH1 and natural killer T cells, pointing to an immunosuppressive environment. Colorectal cancers with high levels of stromal FAP are associated with aggressive disease progression and survival. Our results suggest that FAP plays additional roles in tumor progression such as modulation of angiogenesis and immunoregulation in the tumor microenvironment.

Fibroblast imaging of hepatic carcinoma with 68Ga-FAPI-04 PET/CT: a pilot study in patients with suspected hepatic nodules.

68Ga-FAPI-04 is a rapidly evolving PET tracer for whole-body imaging in a variety of cancers. We aimed to evaluate the diagnostic performance of 68Ga-FAPI-04 for detecting and characterizing hepatic nodules in patients with suspected carcinoma. Twenty-five patients showing suspicious hepatic lesions for malignancy underwent 68Ga-FAPI-04 PET. The maximum and mean standardised uptake values (SUVmax, SUVmean) were measured for all detected lesions and normal hepatic tissues, respectively. The target-to-background ratio (TBR) was calculated by dividing the lesion SUVmax with the SUVmean of non-tumour liver tissue. Lesion uptake value was correlated with the in vitro hepatic FAP expression determined by immunohistochemistry (IHC). In total, 17 patients who underwent surgery or biopsy were recruited for the final analysis. A total of 28 intrahepatic malignant lesions were detected in 16 patients; the mean SUVmax was 8.36 ± 4.21 (range 2.21 to 15.86), and mean TBR was 13.15 ± 9.48 (range 2.79 to 38.12) in all detected lesions (n= 28). One benign patient showed negligible hepatic uptake (SUVmax= 0.47), whereas 75% of the primary intrahepatic hepatocellular carcinoma (HCC) lesions (n= 6) showed prominent FAP expression, 12.5% of the lesions (n= 1) showed moderate expression in stromal cells, and one showed negligible expression. 68Ga-FAPI-04 showed high sensitivity in detecting hepatic malignancies, particularly in poorly differentiated forms with concordantly elevated FAP expression.

Immunohistochemical study of fibroblast activation protein and α-smooth muscle actin expression and distribution in triple-negative breast cancer

Background: Triple-negative breast cancer constitutes 10-20% of all breast cancers, and are more frequent in younger patients with a poor prognostic outcome. The tumor microenvironment is formed of activated fibroblasts (FAP) which are known as cancer-associated fibroblasts, which can be detected by fibroblast activation protein [FAP] and α-smooth muscle actin [α-SMA]. Aim: The aim of the work is the evaluation of the expression and distribution of FAP and α-SMA in triple-negative breast cancer. Material and Methods: This study was carried on 100 paraffin blocks from pathologically proved triple-negative invasive breast cancer patients and subjected to immunostaining of SMA and FAP antibodies, Evaluation of antibodies expression according to its distribution in tumor margin and in the tumor center. Results: There were significant differences between FAP as well as SMA expression in the tumor center and tumor margin, where FAP and SMA expression in the tumor center was positively correlated with tumor size and grade. In contrast to tumor margin, FAP and SMA expression were negatively correlated with the tumor size and lymph node metastasis. Conclusion: We speculated that high FAP expression and α-SMA expression in the tumor center, but not the tumor margin, is correlated with poor patients clinicopathological parameters.

Fibroblastic FAP promotes intrahepatic cholangiocarcinoma growth via MDSCs recruitment

Desmoplasia is a hallmark of intrahepatic cholangiocarcinoma (ICC), which constitutes a barrier to infiltration of lymphocyte, but not myeloid cells. Given that dense desmoplastic stroma has been reported to be a barrier to infiltration of lymphocyte, but not myeloid cells. We here investigated whether fibroblastic FAP influenced ICC progression via non-T cell-related immune mechanisms. We demonstrated fibroblastic FAP expression was critical for STAT3 activation and CCL2 production, and ICC-CAFs were the primary source of CCL2 in human ICC microenvironment by using ICC-Fbs from six ICC patients. Fibroblastic knockdown of FAP significantly impaired the ability of ICC-CAFs to promote ICC growth, MDSCs infiltration and angiogenesis, which was restored by adding exogenous CCL2. Furthermore, interestingly, the tumor-promoting effect of fibroblastic FAP is dependent on MDSCs via secretion of CCL2, as depletion of Gr-1+ cells reversed the restoring effects of exogenous CCL2 on tumor growth and angiogenesis. In vitro migration assay confirmed that exogenous CCL2 could rescue the impaired ability of ICC-Fbs to attract Gr-1+ cells caused by fibroblastic FAP knockdown. In contrast, fibroblastic FAP knockdown had no effect on ICC cell proliferation and apoptotic resistance. Depletion MDSCs by anti-Gr-1 monoclonal antibody in subcutaneous transplanted tumor model abrogated tumor promotion by ICC-CAFs suggested that the pro-tumor function of Fibroblastic FAP relied on MDSCs. Mechanical, flow cytometry and chamber migration assay were conducted to find Fibroblastic FAP was required by the ability of ICC-CAFs to promote MDSC migration directly. Moreover, fibroblastic FAP knockdown had no effect on cell proliferation and apoptotic resistance. Here, we revealed the T-cell independent mechanisms underlying the ICC-promoting effect of fibroblastic FAP by attracting MDSCs via CCL2, which was mainly attributed to the ability of FAP to attract MDSCs and suggests that specific targeting fibroblastic FAP may represent a promising therapeutic strategy against ICC.

P715Deletion of fibroblast activation protein decreases experimental atherosclerotic plaque formation and vulnerability

Abstract Background Fibroblast activation protein (FAP) is a serine protease that is upregulated in sites of tissue remodeling, including arthritis, tumors and atherosclerosis. We have reported that FAP degrades type I collagen in human thin-cap fibroatheromata; its expression is enhanced in advanced human plaques and induced by inflammation. However, the role of endogenous FAP in atherosclerosis remains unknown. Purpose To investigate the effects of constitutive Fap loss-of-function on atherosclerotic plaque formation and vulnerability. Methods and results Male 8-week-old Apoe−/− Fap+/+ and Apoe−/− Fap−/− mice were fed a high-cholesterol diet (1.25% chol) for 12 weeks. En face analyses of thoracoabdominal aortae using Oil Red O (ORO) revealed decreased plaques in Apoe−/− Fap−/− mice (5.7±0.5%; n=21) compared to Apoe−/− Fap+/+ mice (10.7±0.7%; n=24; p&lt;0.0001). In parallel, ORO analyses of serial aortic root cross sections showed diminished plaques in Fap-deficient mice (18.4±3.4% vs 27.6±2.1%). As a surrogate of plaque vulnerability, fibrous cap thickness was increased in Apoe−/− Fap−/− mice (65±6 mm vs 35±3 mm; p&lt;0.01), whereas necrotic core size, plaque macrophages (CD68) and T cells (CD3) accumulation, as well as VCAM1 expression did not differ. These changes were independent of plasma triglycerides, total and LDL-cholesterol levels. Plasma of Fap-deficient mice showed decreased FAP activity compared to Fap wildtype controls. Notably, second harmonics generation in cross sections of aortic root plaques showed that the deposition and density of fibrillar collagens was enhanced in Fap-deficient (25.5±4.4%) compared to control plaques (13.8±2.5%; p&lt;0.05). Consistently, Fap deletion led to an accumulation of uncleaved pre-COL3A1, a proteolytic target of FAP. Conclusions Constitutive Fap deletion decreases experimental atherosclerosis and features of plaque vulnerability. Thus, inhibition of FAP expression or activity may be a promising therapeutic target in atherosclerosis. Acknowledgement/Funding Swiss National Science Foundation, Swiss Heart Foundation

P11.41 Comparison of fibroblast activation protein expression and localization in glioblastomas and brain metastases

Abstract BACKGROUND Fibroblast activation protein (FAP) is a transmembrane serine protease that is frequently upregulated in the tumor microenvironment. In several cases, FAP protein itself and/or FAP expressing stromal cells have been shown to contribute to cancer progression and to be associated with more aggressive cancer behaviour and shorter patient survival. The aim of this study was to determine FAP expression in glioblastomas and brain metastases and to identify the cell types that express FAP in the microenvironment of these malignancies. MATERIAL AND METHODS FAP enzymatic activity and protein concentration were determined in samples from patients with brain metastases, glioblastomas and pharmacoresistant epilepsy (control non-tumorous brain tissue) by an enzymatic assay using a specific fluorogenic substrate and ELISA, respectively. Immunohistochemical labelling with antibodies against FAP and markers of astroglia, epithelial cancer cells and mesenchymal stromal cells was performed to characterize FAP expressing cells. RESULTS FAP was significantly upregulated in the majority of glioblastomas and brain metastases in comparison to non-tumorous brain tissue. In glioblastomas, FAP was localized perivascularly and in mesenchymal cells, and in part of the tumors also in the glioma cells. In brain metastases, FAP positivity was abundantly present in the stroma and predominantly co-localised with markers of mesenchymal stromal cells (TE-7, SMA, PDGFRbeta, NG2), but there was no overlap between FAP and markers of epithelial cancer cells (EpCAM, pancytokeratin). CONCLUSION FAP is upregulated in the microenvironment of human glioblastomas and brain metastases compared to non-tumorous brain tissue. In glioblastomas, FAP is expressed in part of the glioma cells, in pericytes and mesenchymal stromal cells, whereas no positivity in cancer cells and more abundant FAP+ stroma was detected in brain metastases. The selective expression of FAP in these brain tumors may be useful for the visualization and possibly therapeutic targeting of their tumor microenvironment. GRANT SUPPORT Ministry of Health of the Czech Republic, grant No. 15-31379A, Progres Q28/LF1, 2015064 LM EATRIS and the project,Center for Tumor Ecology - Research of the Cancer Microenvironment Supporting Cancer Growth and Spread” (reg. n. CZ.02.1.01/0.0/0.0/16_019/0000785) supported by the Operational Programme Research, Development and Education.

Fibroblast activation protein restrains adipogenic differentiation and regulates matrix-mediated mTOR signaling

Obesity is a risk factor for multiple diseases, including diabetes, cardiovascular disease, and cancer. Within obese adipose tissue, multiple factors contribute to creating a disease-promoting environment, including metabolic dysfunction, inflammation, and fibrosis. Recent evidence points to fibrotic responses, particularly extracellular matrix remodeling, in playing a highly functional role in the pathogenesis of obesity. Fibroblast activation protein plays an essential role in remodeling collagen-rich matrices in the context of fibrosis and cancer. We observed that FAP-null mice have increased weight compared to wild-type controls, and so investigated the role of FAP in regulating diet-induced obesity. Using genetically engineered mouse models and in-vitro cell-derived matrices, we demonstrate that FAP expression by pre-adipocytes restrains adipogenic differentiation. We further show that FAP-mediated matrix remodeling alters lipid metabolism in part by regulating mTOR signaling. The impact of FAP on adipogenic differentiation and mTOR signaling together confers resistance to diet-induced obesity. The critical role of ECM remodeling in regulating obesity offers new potential targets for therapy.

Abstract 1095: Macrophage is a novel and rich source of cancer-associated fibroblasts in the tumor microenvironment

Abstract Background: Cancer-associated fibroblasts (CAF) are highly heterogeneous and their origins are largely unknown. Recently, we revealed that bone marrow-derived macrophages (BMDM) can further differentiate into myofibroblasts locally at the inflammatory site in a Smad3-dependent manner, but its potential role in the tumor microenvironment (TME) is still unexplored. Thus, we hypothesize that tumor-associated macrophages (TAM) may be able to transit into CAF, termed TAM to CAF transition (TAM-CAF), in TME for cancer promotion. Here, we are the first study to discover TAM-CAF as a common phenomenon in cancer. Methods: The evidence of TAM-CAF was confirmed on tumor biopsies from several cancer types including non-small cell lung carcinoma (NSCLC) tissue microarray. The occurrence pattern of TAM-CAF was determined in LysM-Cre/Rosa26-tdTomato mice with syngeneic lung carcinoma LLC by the fate-mapping study. The pathogenic role of TAM-CAF was examined by adoptive transferring of TAM-CAF cells onto LLC-bearing NOD-SCID mice. The regulatory mechanism was further elucidated on Smad3-WT/KO mice in vivo and BMDM in vitro. Results: Interestingly, we observed myofibroblast marker expressing TAM (α-SMA+CD68+) in the tumor biopsies of liver, kidney and lung cancers, which contributed to ~40% of the total CAF and positively associated with the NSCLC mortality. Fate mapping study confirmed the existence of macrophage-derived CAF (α-SMA+ tdTomato+) in LLC-tumor, where the contribution of TAM-CAF on CAF production was progressively increased throughout the tumorigenesis. Furthermore, adoptive transferring of TAM-CAF cells largely promotes tumor progression in LLC-bearing NOD/SCID mice associated with a marked increase of angiogenesis in the TME. Mechanistically, we found that phosphorylation of Smad3 is significantly associated with CAF production in NSCLC-TME (n=120), supported by the dramatic reduction of TAM-CAF in LLC-tumor and BMDM after deletion of Smad3. More importantly, we identified a conserved Smad3 binding site on the 5’UTR of FAP gene by ECR browser and ChIP assay in vitro; revealing its direct regulation on FAP expression at transcriptional level during TAM-CAF. Conclusion: We are the first study to uncover TAM as a rich source of CAF, which is commonly occurred in TME for cancer promotion via a Smad3-dependent mechanism. Thus, TAM-CAF may represent a novel and precision therapeutic target for cancer. Acknowledgments: This study was supported by Lui Che Woo Institute of Innovative Medicine (CARE program), Research Grants Council of Hong Kong (GRF 14117815, 14121816, 14163317, C7018-16G, TRS T12-402/13N), Health and Medical Research Fund (03140486, 14152321), Innovation and Technology Fund of Hong Kong (ITS/068/18), Direct Grant for Research CUHK (2017.002). Citation Format: Philip Chiu-Tsun Tang, Patrick Ming-Kuen Tang, Jeff Yat-Fai Chung, Xiao-Ru Huang, Ka-Fai TO, Hui-Yao LAN. Macrophage is a novel and rich source of cancer-associated fibroblasts in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1095.

Abstract A096: The potential role of fibroblast activation protein as a natural killer cell immune checkpoint

Abstract Fibroblast activation protein (FAP) is a type II transmembrane serine protease that functions as both a dipeptidyl peptidase and an endopeptidase. FAP is minimally expressed in normal pancreas but overexpressed in 90% of pancreatic ductal adenocarcinoma (PDAC) specimens. A meta-analysis of PDAC studies demonstrated elevated tumor FAP expression is associated with worse clinical outcomes. While immunotherapy offers remarkable results for certain cancer types, it has been largely ineffective in PDAC. This lack of efficacy may be attributed to the dense stromal fibrosis, comprised largely of pancreatic stellate cells (PSCs), that is characteristic of PDAC lesions. Here we demonstrate that human NK cell line (NK92) is activated by and kill PSCs. Upon direct contact with PSCs, NK92 cells upregulate FAP. FAP expression by NK92 cells is associated with an inactivation phenotype. Talabostat, a non-specific inhibitor of FAP, enhances NK92 killing of PSCs in vitro and enhances PDAC tumor clearance in vivo. This suggests that FAP may be a novel NK cell immune checkpoint that can be pharmacologically modulated to enhance NK cell antitumor activity. Citation Format: Allison O'Connell, Shangzi Wang, Louis M. Weiner. The potential role of fibroblast activation protein as a natural killer cell immune checkpoint [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A096.

Abstract 4552: Tumor-restricted immune modulation by multispecific molecules from the DARPin toolbox

Abstract During the last years, immune-modulating drugs became an important cornerstone in the treatment of cancer patients. In particular, the PD1/PDL-1 and CTLA-4 antagonists revolutionized the field. However, only a limited number of patients benefit from these antagonistic molecules and more combination therapies are on the way to increase the number of patients benefiting from these novel therapies. Among the combinations, drugs that are T-cell or myeloid cell agonists belonging to the TNFR-superfamily show first promising clinical results. However, systemic immune activation bears the risk of severe side effects that will not allow using these powerful drugs at an effective dose. We have developed a new class of DARPin molecules that enable tumor-restricted immune cell activation of TNFR-superfamily agonists in the tumor only, thereby preventing systemic immune-activation. We generated DARPin molecules that bind with high affinity to TNFR-superfamily members (CD134, CD137 and CD40) and DARPin molecules that bind to tumor-specific antigens such as HER2 and EGFR or targets restricted to the tumor stroma compartment like FAP and extra-domain B of fibronectin (ED-B). Using these building blocks from our DARPin toolbox, we constructed a variety of multi-specific molecules consisting of a TNFR-superfamily receptor targeting DARPin molecule and a tumor-localizing DARPin molecule. In reporter cell assays the multi-specific DARPin molecules activate the respective TNF-superfamily receptor only in the presence of a cell expressing the tumor-localizer; e.g. the FAP-CD134 molecule activates CD134 only in the presence of a stromal cell expressing FAP and not in its absence. Moreover, activation of CD134 was dependent on the density of FAP expression on cells, showing that CD134 only becomes activated if a certain level of the tumor-localizing target is expressed. These finding were confirmed in experiments with primary immune cells where we see immune-cell activation only upon binding to the tumor-localizing target. This could be shown for multiple combinations (&amp;gt;8) and supports the concept that multi-specific DARPin molecules are powerful drug-candidates allowing tumor-restricted immune cell activation. Citation Format: Ulrike Fiedler, Christian Reichen, Joanna Taylor, Patricia Schildknecht, Sophie Barsin, Clara Metz, Anja Schlegel, Simon Fontaine, Denis Villemagne, Julia Ahlskog, Yvonne Kaufmann, Alexander Link, Nicolo Rigamonti, Julia Hepp, Michael T. Stumpp. Tumor-restricted immune modulation by multispecific molecules from the DARPin toolbox [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4552.

Targeting fibroblast activation protein in cancer - Prospects and caveats.

Fibroblast activation protein (FAP, seprase) is a serine protease with post-proline dipeptidyl peptidase and endopeptidase enzymatic activity. FAP is upregulated in several tumor types, while its expression in healthy adult tissues is scarce. FAP molecule itself and FAP+ stromal cells play an important although probably context-dependent and tumor type-specific pathogenetic role in tumor progression. We provide an overview of FAP expression under both physiological and pathological conditions with focus on human malignancies. We also review and critically analyze the results of studies which used various strategies for the therapeutic targeting of FAP including the use of low molecular weight inhibitors, FAP activated prodrugs, anti-FAP antibodies and their conjugates, FAP-CAR T cells, and FAP vaccines. A unique enzymatic activity and selective expression in tumor microenvironment make FAP a promising therapeutic target. A better understanding of its role in individual tumor types, careful selection of patients, and identification of suitable combinations with currently available anticancer treatments will be critical for a successful translation of preclinically tested approaches of FAP targeting into clinical setting.

981P - Reactive stroma mediates CD8+ T cell spatial distribution and function in ovarian cancer

Background: Close proximity between cytotoxic T cells and tumor cells is key to effective immunotherapy. Ovarian cancer exhibits diverse immune phenotypes with distinct prevalence and spatial localization of CD8+ T cells. This study is aimed to characterize the molecular mechanisms orchestrating the localization and function of CD8+ T Cells in ovarian cancer. Methods: CD8 IHC and RNAseq were performed on 277 ovarian tumor tissues from ICON7 phase 3 trial. CD8 T-cells in tumor vs. stromal area was assessed by digital pathology. A Random Forest regression model was constructed to identify molecular features associated with enumeration or spatial localization of CD8+ T cells. In situ validation was performed by MHCI IHC and FAP RNAish. Functional role of ovarian fibroblasts was characterized by ex vivo T cell function assays. Results: We identified three main immune phenotypes, including T-cell infiltrated, T-cell excluded, and immune desert. The immune phenotypes are highly associated with prognosis and the molecular subtypes of ovarian cancer. The T-cell infiltrated phenotype is denoted by high expression of T-effector signatures and antigen presentation machinery. The T-cell excluded phenotype showed similar expression of T-effector signatures as the T-cell infiltrated phenotype, however, most of the CD8+ T-cells were excluded from the tumor bed. The T-cell excluded phenotype showed high expression of the reactive stroma signatures (i.e. FAP), and low expression of class I antigen presentation genes. Lastly, the immune desert phenotype featured low prevalence of CD8+ T-cells, and high expression of neuroendocrine and metabolic pathways. In situ analysis confirmed the two key molecular features associated with the T-cell excluded phenotype: 1) loss of the MHC I expression in the tumor compartment, and 2) high FAP expression in CAFs. Co-culturing of ovarian fibroblast cells with T-cells resulted in reduced T-cell activation and proliferation. Conclusions: Our study uncovered key molecular mechanisms mediating the interplay between CD8+ T cell localization and function in ovarian cancer. Our findings underscore the potential of targeting reactive stroma as a novel therapeutic strategy to optimize immunotherapy for ovarian cancer patients. Legal entity responsible for the study: Genentech Funding: Genentech Disclosure: All authors have declared no conflicts of interest.

Fibroblast activation protein alpha expression identifies activated fibroblasts after myocardial infarction

IntroductionFibroblast activation protein α (FAP) is a membrane-bound serine protease expressed by activated fibroblasts during wound healing in the skin. Expression of FAP after myocardial infarction (MI) and potential effects on cardiac wound healing are largely unknown. MethodsMI was induced in rats and FAP expression was analyzed at 3, 7 and 28days post-MI by microarray, Western blot and immunohistochemistry. In human hearts after MI, a FAP+ fibroblast population was identified, and characterized by immunohistochemistry for prolyl-4-hydroxylase β, α-smooth muscle actin, Thy-1 and vimentin. Signaling pathways leading to FAP expression were studied in human cardiac fibroblasts by Western blot and ELISA using TGFβ1, TGF-beta type I-receptor (TGFbR1)-inhibitor SB431542 or the MAPK-inhibitor U0126 as well as siRNA targeting SMAD2 and SMAD3. Finally, fibroblasts were assayed for FAP-dependent migration (modified Boyden-chamber), proliferation (BrdU-assay) and gelatinolytic activity by gelatin zymography. ResultsIn rats, FAP expression was increased after MI especially in the peri-infarct area peaking at 7days post-MI. Co-localization analysis identified the majority of FAP+ cells as activated proto-myofibroblasts and myofibroblasts. Concordantly, FAP+ fibroblasts were abundant in ischemic tissue of human hearts after MI, but not in healthy control hearts. In vitro, FAP was induced by TGFβ1 via the canonical SMAD2/SMAD3 pathway. Depletion of FAP in fibroblasts reduced migratory capacity, while proliferation was not affected. Gelatin zymography revealed gelatinase activity by fibroblast-derived FAP. ConclusionIn this study, we show for the first time the expression of FAP in activated fibroblasts after MI and its activation by TGFβ1. Effects of FAP on fibroblast migration and gelatinolytic activity indicate a potential role in cardiac wound healing and remodeling.

Major proteomic changes associated with amyloid-induced biofilm formation in Pseudomonas aeruginosa PAO1.

The newly identified functional amyloids in Pseudomonas (Fap) are associated with increased aggregation and biofilm formation in the opportunistic pathogen P. aeruginosa; however, whether this phenomenon can be simply ascribed to the mechanical properties of the amyloid fibrils remains undetermined. To gain a deeper understanding of the Fap-mediated biofilm formation, the physiological consequences of Fap expression were investigated using label-free protein quantification. The functional amyloids were found to not solely act as inert structural biofilm components. Their presence induced major changes in the global proteome of the bacterium. These included the lowered abundance of classical virulence factors such as elastase B and the secretion system of alkaline protease A. Amyloid-mediated biofilm formation furthermore increased abundance of the alginate and pyoverdine synthesis machinery, which turned P. aeruginosa PAO1 into an unexpected mucoid phenotype. The results imply a significant impact of functional amyloids on the physiology of P. aeruginosa with subsequent implications for biofilm formation and chronic infections.

Abstract 2592: Tumor targeting and pharmacodynamics of the novel targeted immunocytokine FAP-IL2v in a tumor-bearing Rhesus monkey

Abstract FAP-IL2v is a novel monomeric FAP-targeted immunocytokine where a single, engineered IL-2 variant (IL-2v) with abolished IL-2Ralpha (CD25) binding is fused to the C-terminus of an high affinity anti-FAP antibody (1). FAP-IL2v was designed to selectively bind to the tumor thereby avoiding the systemic toxicity inherent to systemic administration of cytokines. The goal of this study was to assess the tumor targeting and pharmacodynamic properties of FAP-IL2v in an appropriate tumor bearing animal model. Explanted tumor xenografts in mice do not have high expression of FAP and lack the complete immune system which is important to assess the safety, efficacy, and biodistribution of these targeted immunocytokines. Therefore, we elected to use monkeys with spontaneous tumors for this study. Herein, we report a first of kind study in monkeys with spontaneous tumors to better understand tumor targeting and pharmacodynamics of FAP-IL2v. Three monkeys with a history of cancer and suspected relapse were identified. Immunohistochemistry was done on archival tumor tissue to assess target (FAP) expression and FDG-PET performed to detect metastatic lesions. Based on the positive FAP expression on tumor tissue and detectable tumor lesions and lesion locations from FDG-PET scans, a rhesus monkey with metastatic breast cancer was selected for further experimentation with FAP-IL2v. The monkey was administered with 0.5 mg/kg of FAP-IL2v mixed with tracer amounts of 89Zr labeled FAP-IL2v for assessment of tumor targeting using PET imaging. Baseline and on-treatment blood samples and tumor biopsies were collected after single-administration for assessment of pharmacodynamic effects. The study revealed that FAP-IL2v selectively targets and accumulates in the tumor lesions with minimal uptake in the lungs, spleen and lymphoid tissues (major sink organs and potential risk factors). All three FDG-avid lesions showed accumulation of FAP-IL2v over 6 days with peak uptake of SUV= 3.1 at day 3. After single administration, transient lymphocytosis and peripheral expansion of immune cells were observed. Analyses of tumor biopsies suggested increased immune cell infiltration. No signs of distress or cytokine release were observed during the treatment period. In conclusion, using a monkey with spontaneous tumors, we demonstrated that the FAP-IL2v targeted immunocytokine can selectively be delivered to tumor and induce intended changes in immune microenvironment without major adverse events. (1) C. Klein et al., Tumor-targeted, engineered IL-2 variant (IL-2v)-based immunocytokines for the immunotherapy of cancer, AACR 2013, Poster 486 Citation Format: Stefan Evers, Pradeep K. Garg, J. Mark Cline, Sudha Garg, Greg Dugan, Anne Freimoser-Grundschober, Natalie D. Keirstead, Christian Klein, Tapan Nayak. Tumor targeting and pharmacodynamics of the novel targeted immunocytokine FAP-IL2v in a tumor-bearing Rhesus monkey. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2592. doi:10.1158/1538-7445.AM2014-2592

Circulating concentrations of fibroblast activation protein α in apparently healthy individuals and patients with acute coronary syndrome as assessed by sandwich ELISA

Fibroblast activation protein α (FAP) is a membrane glycoprotein with dipeptidyl-peptidase and collagenase activity and is expressed in cancer, arthritis, and atherosclerotic plaques. We hypothesized that FAP can be measured quantitatively in the circulation and provide prognostic information in acute coronary syndrome (ACS). We assessed the performance of a commercially available FAP ELISA and the pre-analytic characteristics of the marker. We determined FAP concentrations in EDTA plasma samples from 101 apparently healthy blood donors and 407 patients with ACS. Patients were followed for 12 months regarding all-cause mortality and non-fatal myocardial infarction (MI). FAP was stable at room temperature (for 1 day) and 4°C (3 days) and resistant to 3 freeze/thaw cycles. Recovery of recombinant human FAP ranged from 78 to 103% and serial dilutions of spiked samples resulted in measurements within 91 to 120% of expected values. Patients with ACS had lower plasma FAP concentrations compared with blood donors [median (25th-75th percentiles): 84 (69-101) ng/mL vs. 108 (87-124) ng/mL, P < 0.001]. Patients presenting with FAP concentrations in the first quartile had a 3.0-fold higher risk of death (95% confidence interval 1.4-6.2) compared with patients in the second to fourth quartiles (P = 0.004). FAP concentration was not related to the risk of MI. Our study is the first to associate FAP with prognosis in ACS. The favorable pre-analytic characteristics of FAP will facilitate future studies of the marker in other disease settings associated with altered FAP expression.