Studies on single domain antibodies VHH against FAPα as potential cancer therapeutics

Unlike heterogeneous tumor cells, cancer-associated fibroblasts (CAF) are genetically more stable which serve as a reliable target for tumor immunotherapy. Fibroblast activation protein (FAP) which is restrictively expressed in tumor cells and CAF in vivo and plays a prominent role in tumor initiation, progression, and metastasis can function as a tumor rejection antigen. In the current study, we have constructed artificial FAP(+) stromal cells which mimicked the FAP(+) CAF in vivo. We immunized a breast cancer mouse model with FAP(+) stromal cells to perform immunotherapy against FAP(+) cells in the tumor microenvironment. By forced expression of FAP, we have obtained FAP(+) stromal cells whose phenotype was CD11b(+)/CD34(+)/Sca-1(+)/FSP-1(+)/MHC class I(+). Interestingly, proliferation capacity of the fibroblasts was significantly enhanced by FAP. In the breast cancer-bearing mouse model, vaccination with FAP(+) stromal cells has significantly inhibited the growth of allograft tumor and reduced lung metastasis indeed. Depletion of T cell assays has suggested that both CD4(+) and CD8(+) T cells were involved in the tumor cytotoxic immune response. Furthermore, tumor tissue from FAP-immunized mice revealed that targeting FAP(+) CAF has induced apoptosis and decreased collagen type I and CD31 expression in the tumor microenvironment. These results implicated that immunization with FAP(+) stromal cells led to the disruption of the tumor microenvironment. Our study may provide a novel strategy for immunotherapy of a broad range of cancer.

CAR T cell therapies have demonstrated considerable potency in treating hematologic cancers, but only limited efficacy in eliminating solid tumors. One reason for this discrepancy may derive from the immunologic and physical barriers created by infiltration of cancer associated fibroblasts (CAFs) into solid tumors. These CAFs, which can comprise 15-85% of the stromal cells in a tumor mass, are correlated with poor patient survival and can reduce CAR T cell efficacies by secreting immunosuppressive cytokines, stimulating tumor cell proliferation, and depositing fibrotic barriers to CAR T cell penetration. Here, we are pursuing a method to suppress or eliminate CAF activities in the tumor microenvironment (TME). A potential CAR T cell target that is uniquely expressed on CAF surfaces is fibroblast activation protein (FAP). In this study, we have employed a novel, highly specific FAP targeting small molecule to direct our universal CAR T cells to CAFs. For this purpose, we have designed a CAR that contains an otherwise classical CAR except the extracellular scFv specifically binds fluorescein. Upon addition of a bispecific adaptor comprised of fluorescein linked via a short spacer to our FAP ligand, a bridge is formed between the anti-fluorescein CAR on the T cell and FAP on the CAF, resulting in formation of an immunologic synapse between the CAR T cell and CAF that triggers CAF destruction and CAR T cell proliferation. Importantly, this CAR T cell design also permits simultaneous administration of a second bispecific adaptor (i.e. fluorescein linked via a short spacer to a cancer-specific ligand) that can enable the same CAR T cell to concurrently kill adjacent cancer cells. To test whether this universal CAR T cell can eliminate both cancer cells and CAFs, we have implanted KB cells (i.e. a cell line that creates an immunologically “cold” folate receptor (FR) expressing solid tumor) into NSG mice and quantitated the CAR T cell’s toxicity in the presence of one or both bispecific adapters. While administration of the universal CAR T cells followed by intravenous injection of an FR-targeting bispecific adaptor achieved significant anti-tumor efficacy, co-injection of a FAP-targeted bispecific adaptor enhanced this efficacy without apparent toxicity. Analyses of tumor masses from the therapy further revealed that co-administration of the FAP-targeted bispecific adaptor not only promoted CAF elimination but also enhanced CAR T infiltration and activation. Importantly, a similar improvement in anti-tumor efficacy could be readily demonstrated in a second immunologically “cold” PSMA-expressing solid tumor model. Taken together, we conclude that the bispecific adaptor/universal CAR T approach offers a unique opportunity to concurrently eradicate cancer cells and tumor-supporting CAFs in a manner that can improve the overall performance of the CAR T cells in solid tumors. Citation Format: Bo Huang, Suilan Zheng, Haiyan Chu, Ramesh Mukkamala, Suresh K. Bowroju, Yashapal Singh, Sudarsan R. Kasireddy, Md Sazzadul Bari, Madduri Srinivasarao, Laurie Beitz, Byoung Ryu, Michael Jensen, Andrew M. Scharenberg, Philip S. Low. Dual targeting of tumor cells and cancer associated fibroblasts enhances CAR T efficacy in solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4099.

Oncolytic virotherapy represents a promising therapeutic strategy for cancers that do not respond well to immune checkpoint inhibitors. Oncolytic viruses (OV) preferentially replicate in and lyse tumor cells, leading to systemic immune stimulation. Studies have shown that hypersialylation promotes tumor growth and metastasis, as well as the suppression of immune cells. Cancer-associated fibroblasts (CAFs) are known to facilitate tumor invasion and angiogenesis and maintain immunosuppressive microenvironment in solid tumors. We are developing vvDD-Sial-FAP/CD3, an oncolytic vaccinia virus expressing a membrane-bound sialidase, to remove sialic acids from the cell surface glycans, and fibroblast activation protein (FAP)-targeted T cell engager, to eliminate glycoimmune checkpoint and tumor stroma, respectively. vvDD-Sial-FAP/CD3 is an engineered vaccinia virus of Western Reserve (WR) strain with: (1) an insertional disruption of the viral thymidine kinase (TK) gene with the sialidase and FAP/CD3 transgenes. TK is an essential enzyme for the pyrimidine synthesis pathway; viral TK gene deletion thus results in selective replication of virus in rapidly dividing cancerous cells with high intracellular nucleotide pools, and (2) a deletion of the vaccinia growth factor (VGF) genes for greater dependence on the cell cycling status of the cancer cells. Our results showed that sialidase expressed from vvDD-Sial-FAP/CD3 efficiently cleaves the sialic acids from the cell surface and the Fc fused to the sialidase induced antibody-dependent cell-mediated cytotoxicity (ADCC) using an ADCC reporter assay. In vitro efficacy studies were conducted using HCT-116 human colon cancer cells mixed with FAP-expressing normal human dermal fibroblasts or FAP-positive HCC1143 human breast cancer cells in the presence of human peripheral blood mononuclear cells. In both tumor models, vvDD-Sial-FAP/CD3 induced activation of both CD4+ and CD8+ T cells, as measured by the upregulation of CD69 and CD25 markers and increased granzyme B release, which resulted in enhanced cell killing. Using A549 co-cultured with CAFs microtissues, we showed that vvDD-Sial-FAP/CD3 spread efficiently within the tumor spheroids. In addition, vvDD-Sial-FAP/CD3 increased tumor-infiltrated lymphocytes, leading to enhanced cell killing. Taken together, our results have demonstrated potent anti-tumor and -stroma effects induced by vvDD-Sial-FAP/CD3 in multiple cancer models. In vivo efficacy of vvDD-Sial-FAP/CD3, either alone or in combination with checkpoint inhibitor or NK cell therapy, is being evaluated. Citation Format: Winnie M. Chan, Mariya Viskovska, Micaela McArthur, Xue Mary Yu, Wei Liang, Zane Norman, Xiaotong Song, Nancy T. Chang, Haiyan Jiang. Glycoimmune checkpoint and tumor stroma-targeted oncolytic vaccinia virus for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3300.

Background: The tumor and its microenvironment have dynamic interactions and signaling that strongly affect tumor progression. Cancer associated fibroblasts (CAFs) are activated fibroblasts and thought to be an important player in the tumor microenvironment. Although there are several indicators of CAF, fibroblast activation protein (FAP) is unique in its selectivity for CAFs in comparison with other markers. The aim of this study is to investigate CAFs expressing FAP and its relationship to cancer metastasis in esophageal cancer. Methods: Sections of paraffin-embedded resected primary human esophageal cancer specimens from 2008 through 2010 in Okayama University hospital were stained with antibody directed against FAP. Overall percentage of stromal FAP staining of the primary tumor was assessed semi-quantitatively (0, 1, 2, 3, 4, 5) and staining intensity was also graded (none, weak, intermediate, strong). Survival time and time to recurrence data were analyzed using Kaplan-Meier plots, log-rank tests, and Cox proportional hazards models. Results: 49 patients with resected specimens were available for study with 26 (53.1%) stage I, 8 (16.3%) stage II, 14 (28.6%) stage III, and 1 (2.0%) stage IV patients. All patients were divided to 2 groups, FAP high group and FAP low group, which are 25 patients (51%) and 24 (49%) respectively. The cutoff for subgroups was defined at the median value. FAP (immune) expression at tumoral stroma was a significant predictive factor for lymph node metastasis (p<0.01) and vessel invasion (p<0.01). In survival analysis of DFS (disease free survival) and OS (overall survival), FAP high stroma was associated with shorter period to recurrence (p<0.05) and death (p<0.05) than those of FAP low stroma. Conclusions: Our clinicopathological analysis indicates that patients whose esophageal carcinoma have high levels of stromal FAP expression are more likely to have an aggressive disease progression and a potential development of metastases and recurrence. Although therapeutic strategies targeting the tumor cells have been generally inadequate in esophageal carcinomas yet, here we announce that a stroma-targeted therapy should be considered. Now we are ongoing to evaluate metastatic potential of CAFs in vitro and vivo, in order to elucidate the function of FAP expressing CAFs and, in future to establish a novel therapeutic strategy. Citation Format: Hajime Kashima, Kazuhiro Noma, Yuki Katsura, Takuya Kato, Ryoichi Katsube, Takayuki Ninomiya, Toshiaki Ohara, Hiroshi Tazawa, Shunsuke Kagawa, Yasuhiro Shirakawa, Toshiyoshi Fujiwara. Correlation of FAP(fibroblast activation protein)-expressing cancer associated fibroblasts (CAFs) and tumor metastasis in esophageal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1560.

Brain metastases are a very common and serious complication of oncological diseases. Despite the vast progress in multimodality treatment, brain metastases significantly decrease the quality of life and prognosis of patients. Therefore, identifying new targets in the microenvironment of brain metastases is desirable. Fibroblast activation protein (FAP) is a transmembrane serine protease typically expressed in tumour-associated stromal cells. Due to its characteristic presence in the tumour microenvironment, FAP represents an attractive theranostic target in oncology. However, there is little information on FAP expression in brain metastases. In this study, we quantified FAP expression in samples of brain metastases of various primary origin and characterised FAP-expressing cells. We have shown that FAP expression is significantly higher in brain metastases in comparison to non-tumorous brain tissues, both at the protein and enzymatic activity levels. FAP immunopositivity was localised in regions rich in collagen and containing blood vessels. We have further shown that FAP is predominantly confined to stromal cells expressing markers typical of cancer-associated fibroblasts (CAFs). We have also observed FAP immunopositivity on tumour cells in a portion of brain metastases, mainly originating from melanoma, lung, breast, and renal cancer, and sarcoma. There were no significant differences in the quantity of FAP protein, enzymatic activity, and FAP+ stromal cells among brain metastasis samples of various origins, suggesting that there is no association of FAP expression and/or presence of FAP+ stromal cells with the histological type of brain metastases. In summary, we are the first to establish the expression of FAP and characterise FAP-expressing cells in the microenvironment of brain metastases. The frequent upregulation of FAP and its presence on both stromal and tumour cells support the use of FAP as a promising theranostic target in brain metastases.

Targeting solid tumors with CAR T cells has proven to be more difficult due to their heterogenous target antigen expression, antigen escape, and due to their hostile tumor microenvironment (TME). Mesothelin represents a promising surface tumor antigen, since it has been associated with tumor invasion and is highly expressed on various cancer types, including pancreatic adenocarcinoma. As clinical trials of mesothelin targeting CAR have yet to show efficacy, we hypothesized that tumor stromal cells such as cancer-associated fibroblasts (CAFs) may play a role in this resistance. To provide a more favorable TME for CAR T cells, we generated a bicistronic lentiviral vector encoding a mesothelin CAR along with a secreted T cell engaging molecule (TEAM) that targets fibroblast activation protein (FAP), which is expressed by CAFs. We termed our constructs CARTEAM, and in this case, mesoFAP. We have assessed the activation and proliferative capacity of these CARTEAM through in vitro assays. We showed that TEAMs secreted by CAR T cells bind their appropriate target antigen by adding supernatant from CARTEAM to target cells expressing FAP. In a co-culture assay using a transwell system we demonstrated the cytotoxic effect of secreted TEAM interacting and recruiting bystander T cells against CAFs. In a real-time cell analysis (RTCA) co-culture assay with a pancreatic cancer cell line (AsPC1) and FAP expressing CAFs, we showed cell death of AsPC1 upon CAR recognition and cell death of CAFs through TEAM-mediated recruitment of bystander T cells and CART cells. We show in these co-culture systems, mimicking tumor and TME, that our CARTEAM construct is superior in the elimination of both cancer cell line and CAF, in comparison to control constructs, including mesothelin targeting CAR T cells (meso CAR) and meso CAR T cells secreting an unspecific CD19 TEAM (mesoCD19). We also used acoustic force microscopy to evaluate the additive effect of the TEAM molecule secreted to binding to tumor cells by mesothelin CAR T cells. In vivo experiments of subcutaneously injected tumor cells admixed with CAFs, show superior tumor control when treated with CARTEAM in comparison to control constructs. Based on these data, we demonstrate both the effective in vitro elimination of CAFs and pancreatic cancer cells through the application of CARTEAM and control of pancreatic tumor growth in vivo. Our studies provide a deeper insight into a dual targeting strategy using a novel CAR T cell secreting a TEAM against pancreatic cancer and its tumor microenvironment. Citation Format: Marc Wehrli, Adam Kuo, Rebecca Larson, Irene Scarfò, Amanda Bouffard, Korneel Grauwet, Mark Leick, Andrea Schmidts, Stefanie Bailey, Tamina Kienka, Michael Kann, Sonika Vatsa, Harrison Silva, Kathleen Gallagher, Max Jan, Bryan Choi, David Ting, Marcela Maus. Mesothelin CAR T cells secreting FAP specific T cell engaging molecule (TEAM) target pancreatic cancer and its tumor microenvironment (TME) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 569.

<div>Abstract<p>Cancer-associated fibroblasts (CAFs) are generally associated with poor clinical outcome. CAFs support tumor growth in a variety of ways and can suppress antitumor immunity and response to immunotherapy. However, a precise understanding of CAF contributions to tumor growth and therapeutic response is lacking. Discrepancies in this field of study may stem from heterogeneity in the composition and function of fibroblasts in the tumor microenvironment. Furthermore, it remains unclear whether CAFs directly interact with and suppress T cells. Here, mouse and human breast tumors were used to examine stromal cells expressing fibroblast activation protein (FAP), a surface marker for CAFs. Two discrete populations of FAP<sup>+</sup> mesenchymal cells were identified on the basis of podoplanin (PDPN) expression: a FAP<sup>+</sup>PDPN<sup>+</sup> population of CAFs and a FAP<sup>+</sup>PDPN<sup>−</sup> population of cancer-associated pericytes (CAPs). Although both subsets expressed extracellular matrix molecules, the CAF transcriptome was enriched in genes associated with TGFβ signaling and fibrosis compared with CAPs. In addition, CAFs were enriched at the outer edge of the tumor, in close contact with T cells, whereas CAPs were localized around vessels. Finally, FAP<sup>+</sup>PDPN<sup>+</sup> CAFs suppressed the proliferation of T cells in a nitric oxide–dependent manner, whereas FAP<sup>+</sup>PDPN<sup>−</sup> pericytes were not immunosuppressive. Collectively, these findings demonstrate that breast tumors contain multiple populations of FAP-expressing stromal cells of dichotomous function, phenotype, and location.</p></div>

<div>Abstract<p>Cancer-associated fibroblasts (CAFs) are generally associated with poor clinical outcome. CAFs support tumor growth in a variety of ways and can suppress antitumor immunity and response to immunotherapy. However, a precise understanding of CAF contributions to tumor growth and therapeutic response is lacking. Discrepancies in this field of study may stem from heterogeneity in the composition and function of fibroblasts in the tumor microenvironment. Furthermore, it remains unclear whether CAFs directly interact with and suppress T cells. Here, mouse and human breast tumors were used to examine stromal cells expressing fibroblast activation protein (FAP), a surface marker for CAFs. Two discrete populations of FAP<sup>+</sup> mesenchymal cells were identified on the basis of podoplanin (PDPN) expression: a FAP<sup>+</sup>PDPN<sup>+</sup> population of CAFs and a FAP<sup>+</sup>PDPN<sup>−</sup> population of cancer-associated pericytes (CAPs). Although both subsets expressed extracellular matrix molecules, the CAF transcriptome was enriched in genes associated with TGFβ signaling and fibrosis compared with CAPs. In addition, CAFs were enriched at the outer edge of the tumor, in close contact with T cells, whereas CAPs were localized around vessels. Finally, FAP<sup>+</sup>PDPN<sup>+</sup> CAFs suppressed the proliferation of T cells in a nitric oxide–dependent manner, whereas FAP<sup>+</sup>PDPN<sup>−</sup> pericytes were not immunosuppressive. Collectively, these findings demonstrate that breast tumors contain multiple populations of FAP-expressing stromal cells of dichotomous function, phenotype, and location.</p></div>

Adoptive T-cell therapy has had considerable success in effectuating anti-tumor responses, however complete eradication of bulky solid tumors is rarely observed. This limited efficacy can in part be attributed to the tumor associated stroma; an immunosuppressive microenvironment that greatly decreases the efficacy of the T-cell product. Specifically, cancer associated fibroblasts (CAFs); the central component of the tumor stroma, secrete inhibitory factors and nutrient depleting enzymes that are detrimental to effector T cell function. In addition, CAFs secrete components of the extra-cellular matrix that contributes to desmoplasia and tumor growth. Recent reports also suggest a more pivotal role of the CAFs in regulating the self-renewing cancer stem cell (CSC) niche. CAFs express fibroblast activation protein alpha (FAP); a membrane bound serine protease, in a number of a solid tumors making it an attractive immunotherapeutic target. We hypothesized that targeting CAFs with FAP-specific T cells will destroy the ‘tumor promoting haven’, resulting in significant antitumor effects. To test this hypothesis, we successfully generated FAP-specific T cells using a second-generation chimeric antigen receptor (CAR) specific for FAP. A prototypical CAR combines the antigen specificity of an antibody with the signaling function of a T cell. The resulting genetically engineered FAP-specific T cells recognized and killed human as well as murine FAP-positive target cells ex vivo. To analyze the effects of targeting the tumor associated stroma in vivo we utilized 1) a locoregional model using the FAP negative lymphoblastoid cell line (LCL) tumor and 2) a systemic model using the FAP negative A549 lung adenocarcinoma. In the local model, mice receiving a subcutaneous admixture of FAP-specific T cells and LCL showed a significant decrease in tumor engraftment and growth. Both untreated and control T cell treated mice showed progressive tumor growth with a concomitant induction of murine FAP rich supportive stroma. In our systemic model, the FAP negative non-small cell lung carcinoma (NSCLC) A549 cell line induced the expression of a murine FAP positive reactive stroma in the lungs when injected intravenously through the tail vein. Treatment with FAP-specific T cells showed a significant decrease in murine FAP expression in the lungs with a concomitant decrease in tumor growth and improved survival. Thus targeting FAP in the tumor stroma alone, resulted in a significant anti-tumor effect both in our local and systemic models. Given the reciprocal relationship between tumor cells and CAFs, we hypothesized that co-targeting the tumor cells and CAFs would result in enhanced anti-tumor response than targeting either alone. Erythropoietin-producing hepatocellular carcinoma-A2 (EphA2) CAR positive T cells were used to target the A549 tumor cells. EphA2-specific T cells when administered together with FAP-T cells, resulted in a significant decrease in tumor growth and increased survival compared to mice that received either EphA2 or FAP-T cells alone. Our research therefore underscores the importance of targeting the tumor associated stroma in addition to tumor targeting for a more efficacious therapeutic response. This abstract is also presented as Poster A91. Citation Format: Sunitha Kakarla, Kevin Chow, Melinda Mata, Xiao-Tong Song, Meng-Fen Wu, Hao Liu, Lisa Wang, David Rowley, Klaus Pfizenmaier, Stephen Gottschalk. Genetically engineered T cells redirected against cancer-associated fibroblasts to potentiate immunotherapeutic effects. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR16.

Identifizierung und Charakterisierung FAP-spezifischer Liganden für die zielgerichtete Diagnostik und Therapie maligner Tumoren

Fibroblast activation protein (FAP) is a post-prolyl proteolytic enzyme that is prominently expressed in cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME) and in activated fibroblasts that contribute to tissue remodeling in chronic inflammatory diseases, such as Crohn’s disease (CD). Interestingly, FAP exhibits low expression and activity in healthy tissues, making it a promising diagnostic and therapeutic target for cancer and chronic inflammatory diseases. Unfortunately, FAP’s functional role in these pathological conditions remains poorly understood, and current tools and methods for studying FAP are often characterized by a lack of selectivity. Additionally, research has primarily focused on FAP mRNA and protein expression, often overlooking its enzymatic activity, which is critical for understanding its biological functions. These limitations highlight the urgent need for innovative, highly selective tools and methods to detect FAP and elucidate its role in disease mechanisms. Hence, the goal of this doctoral research was to develop and validate novel FAP-selective tools and methods to enhance our understanding of FAP’s biological function in cancer and Crohn’s disease. To achieve this, we established an in-house recombinant human FAP (rhFAP) production system to ensure a robust supply of high-quality active enzyme for downstream evaluation and validation of novel FAP-targeting tools and compounds. We produced and purified a highly specific antibody against FAP and developed fluorescent FAP-targeting activity-based probes (ABPs) that enable selective, high-affinity visualization of FAP’s enzymatic activity. Furthermore, we evaluated novel [18F]-labeled FAP inhibitors ([18F]-FAPIs) for PET/CT imaging as a non-invasive diagnostic approach for tumors and fibrotic diseases in which FAP plays a role. With these tools in hand, we gained new insights into the expression and function of FAP in the TME, with a particular focus on its activity and role in natural killer (NK) cells and its interaction with CAFs. Our findings suggest that FAP is involved in NK cell activity and the activation status within the TME. In addition, we used these FAP-specific tools to investigate its function in CD, revealing its involvement in chronic inflammation and intestinal fibrosis. Overall, this research not only offers new insights into FAP’s role in the TME and CD, but also provides the scientific community with a comprehensive toolkit for the selective detection and functional analysis of FAP. By filling critical knowledge gaps in FAP’s biological function, our findings pave the way for novel diagnostic and therapeutic strategies targeting FAP in oncology, Crohn’s disease and beyond.

Carcinomas represent complex neo-organs in which extensive communication networks between surrounding non-transformed endothelial, neural, inflammatory and immune cells provide biochemical signals that govern the behavior of malignant epithelial cells. Moreover, the initiation, progression and metastatic spread of malignant cells is in many human carcinomas associated with desmoplasia. Desmoplasia is typically associated with poor prognosis and in many contexts, confers resistance to therapy. Desmoplasia is characterized by activation of mesenchymal-derived stromal cells such as adipocytes and cancer associated fibroblasts (CAFs) and dynamic remodeling of the extracellular matrix that underlies the dramatic physical differences of tumor tissue compared to corresponding normal tissue that result in further alternations in biochemical signals, contribute to a pro-tumorigenic metabolic milieu as well as alterations in biomechanical signals. Recent studies indicate that fibroblasts also play a critical role in regulating inflammatory and immune cell infiltration, intra-tumoral migration and function in primary tumors and metastatic disease. Recent studies have established that the stromal cell component is heterogeneous with subpopulations of CAFs and specific CAF-mediated pathways that either restrain or promote tumor progression. Genetic and immune-based therapy approaches have identified a subset of Fibroblast Activation Protein (FAP)-expressing tumor promoting CAFs in pre-clinical models of multiple types of carcinoma and FAP -expression correlates with worse survival of patients with a variety of carcinomas including breast cancer. Studies demonstrating that FAP+ CAFs promote tumorigenesis through both immune-dependent and immune-independent mechanisms in pre-clinical models will be presented that support efforts to develop FAP+ cell-targeted therapies in combination with cancer cell targeted therapies. In an effort to refine our approach to therapeutically targeting stroma-dependent mechanisms from a cellular to a molecular level, we have also identified the proteolytic activity of FAP, a cell surface serine protease, as a critical molecular pathway by which FAP+ cells exert their pro-tumorogenic effects. We previously demonstrated in models of lung, colon, and pancreatic cancer, that FAP-null mice are protected from tumorigenesis, progression and metastasis. We recently expanded these results to pre-clinical models of breast cancer and investigated whether stroma-dependent mechanisms contribute to the increased risk of breast cancer incidence and/or progression associated with obesity. Using the spontaneous MMTV-neu murine breast cancer model, we found that FAP-null mice showed delayed primary tumorigenesis. In vitro spheroid formation assays revealed a role for FAP+/+ cells in supporting tumorigenesis, while experiments using primary stromal cell-derived matrices (CDMs) also established a role for FAP-mediated matrix remodeling in promoting cancer cell growth. Taken together, these data suggest that stromal cell-expressed FAP has multiple pro-tumorigenic functions in the context of early breast cancer. Citation Format: E Pure. Stromal Cells and Matrix Remodeling as Essential Regulators of the Tumor Microenvironment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PL1.

Rationale: The recent success of chimeric antigen receptor (CAR) T cell therapy in treating leukemia has brought new enthusiasm to this strategy. While the majority of CAR research has been primarily focused on targeting tumor cells, our group is developing a new CAR construct to target Fibroblast Activation Protein (FAP), a molecule that is highly and selectively expressed on cancer-associated fibroblasts (CAFs). It is well known that these CAFs play an important role in tumor development and tumor progression. Thus, we hypothesized that the immune-mediated destruction of CAFs by adoptively transferred FAP-CAR T cells would inhibit tumor growth in pre-clinical tumor models. Experimental procedures: The 73.3 anti-mouse FAP hybridoma was sequenced to obtain the single chain Fv to insert into our existing CAR construct with the 4-1BB and CD3ζ intracellular signaling domains. Different combinations of FAP-CAR constructs were synthesized and transduced into human T cells first to screen for optimal structure-activity response. The best two FAP-CAR constructs were then selected to transduce into mouse T cells for in vitro cytotoxicity and cytokine assays, as well as in vivo studies. To evaluate the in vivo efficacy, 2 tumor cell lines, AE17 (mouse mesothelioma) and TC1 (mouse lung cancer), were inoculated subcutaneously into flanks of syngeneic C57Bl6 mice. When the flank tumors reached 150 mm3, 10 million CAR T cells were injected through tail vein and tumors were then monitored. Tumor tissues were collected at the end of the study to evaluate the effect of CAR T cells on tumor microenvironment by flow cytometry and/or by immunohistochemistry. Results: All 8 different combinations of anti-mouse FAP CAR constructs expressed on human T cells showed target-specific activities against 3T3parental (FAP-null) versus 3T3FAP cells, in terms of cytotoxicity and cytokine production. The two best FAP-CAR constructs were then transduced into mouse T cells and re-tested for cytotoxicity and cytokine production against 3T3parental and 3T3-FAP fibroblasts, and again showed target-specific killing and cytokine production. When FAP-CAR T cells were adoptively transferred into tumor bearing mice, tumor growth inhibition (~50%) was observed in both flank tumor models while control T cells did not show any efficacy. No toxicity was detected in the animal studies. We found that FAP-CAR T cell-treated tumors had significantly more apoptotic and less proliferating tumor cells, less matrix (collagen, fibronectin), less M2 macrophages and fewer blood vessels, in comparison with the untreated and control T cell-treated tumors. To assure specificity, similar in vivo studies were also carried out in FAP knockout mice. In the KO mice, all the anti-tumor activity of FAP-CAR T cells was lost. Conclusion: Here, we demonstrate the feasibility of inhibiting tumor growth by targeting tumor stroma, instead of tumor cells, with adoptively transferred CAR T cells. We detected no safety issues with these stroma-targeted CAR T cells. We also showed that the anti-tumor effects induced by our T cells were specific to FAP. We are currently evaluating different CAR constructs against human FAP with a goal of conducting a clinical trial. This abstract is also presented as Poster A5. Citation Format: Liang-Chuan S. Wang, Albert Lo, John Scholler, Carl H. June, Ellen Pure, Steven M. Albelda. Fibroblast activation protein as a universal target for chimeric antigen receptor T cell therapy in solid tumors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR14.

SY40-03: The FAP+ stromal cell: Cancer-induced immune suppression and cachexia

Fibroblast activation protein (FAP) is a protease selectively expressed in a number of extracranial cancers especially in cancer associated fibroblasts (CAF). CAF play an important role in the pathogenesis of malignant tumors through extracellular matrix remodeling, secretion of growth factors and cytokines, and promotion of angiogenesis and intratumoral immunosuppression. Glioblastomas (GBMs) have a unique composition of the tumor microenvironment and whether CAF are present in these tumors remains controversial. We have recently shown that FAP is upregulated in a subgroup of GBMs and is expressed in both cancer and stromal cells. The role of FAP+ stromal cells in GBM is however unknown. FAP positive stromal cells were isolated using magnetic activated cell sorting (MACS), and cultured in pericyte media. Expression of FAP and other phenotypic markers was assessed by immunocytochemistry. Conditioned media were prepared and used as a chemoattractant in a transwell migration assay. Cell growth was analyzed using the XTT assay, angiogenesis was evaluated using a 3D angiogenesis assay in type I collagen gel. A cytokine array was used to analyze soluble mediators released by FAP+ stromal cells. FAP+ stromal cells were isolated from several human GBMs and characteristically expressed mesenchymal (TE-7, PDGFR, SMA), but not endothelial (vWF) or glial (GFAP) markers. Conditioned media from these FAP+ cells enhanced the migration of glioma and endothelial cells and enhanced endothelial sprouting in a 3D angiogenesis assay. Part of the conditioned media had a mild growth promoting effect on endothelial and glioma cells. Using cytokine arrays, we identified several chemokines, growth factors and angiogenic factors produced by these FAP+ stromal cells. Our data demonstrate that FAP+ mesenchymal cells analogous to cancer associated fibroblasts participate on multidirectional interactions among cell subpopulations in human glioblastomas mediated, at least in part, by soluble factors. These FAP+ stromal cells may contribute to the invasive growth and neoangiogenesis in glioblastoma. Ministry of Health of CR grant 15-31379A, Progres Q28/1LFUK and grant LM2015064 of the EATRIS-CZ.