Human Fibroblast Activation Protein Research Articles

Efficacy and safety evaluation of cross-reactive Fibroblast activation protein scFv-based CAR-T cells.

Fibroblast activation protein (FAP) overexpression on cancer-associated fibroblasts (CAFs) is associated with poor prognosis and worse clinical outcomes. Selective ablation of pro-tumorgenic FAP+ stromal cells with CAR-T cells may be a new therapeutic strategy. However, the clinical use of FAP-CAR T cells is suggested to proceed with caution for occasional poor efficacy and induction of on-target off-tumor toxicity (OTOT), including lethal osteotoxicity and cachexia. Hence, more investigations and preclinical trials are required to optimize the FAP-CAR T cells and to approve their safety and efficacy. In this study, we designed second-generation CAR T cells targeting FAP with 4-1BB as a co-stimulatory molecule, and tested their cytotoxicity against FAP-positive cells (hFAP-HT1080 cells and a variety of primary CAFs) in vitro and in Cell line-derived xenograft (CDX) and a patient-derived xenograft (PDX) model. Results showed that our FAP-CAR T cells were powerfully potent in killing human and murine FAP-positive tumor cells and CAFs in multiple types of tumors in BALB/c and C57BL/6 mice and in patient-derived xenografts (PDX) model. And they were proved to be biologically safe and exhibit low-level OTOT. Taken together, the human/murine cross-reactive FAP-CAR T cells were powerfully potent in killing human and murine FAP positive tumor cells and CAFs. They were biologically safe and exhibit low-level OTOT, warranting further clinical investigation into our FAP-CAR T cells.

Abstract 4167: Selective imaging of cancer-associated fibroblasts in the breast cancer microenvironment with near-infrared fluorescent probes

Abstract Tumors consist of a complex mixture of resident and infiltrating cells that can support their survival and evasion of treatment. Selective detection of genetically stable cells in the tumor microenvironment (TME) offers insight into tumor biology beyond the malignant cells, which are the focus of most molecular imaging approaches to cancer detection. Motivated by the goal of non-invasively understanding the primary breast TME, we have developed fluorescent molecular probes that target cancer-associated fibroblasts (CAFs), a major component of the stromal compartment of many tumors, and emit light in the near-infrared (NIR) spectral window, providing access to deeper tissues than visible probes. We synthesized and characterized a series of CAF-targeted antibody- and small molecule-based fluorescent imaging agents utilizing a highly hydrophilic NIR dye and ligands that target Fibroblast Activation Protein (FAP). FAP is a key cell-surface marker of breast cancer CAFs with an S1 phenotype, which are modulators of immunosuppression, metastatic spread, and are associated with worse outcomes, especially in HER2+ and triple-negative breast cancer. Modifications to the chemical structure of the linker and targeting ligands allowed us to alter the specificity and brightness of the CAF-probes in cellulo. Imaging of a co-implanted tumor model of SKBR3 human breast cancer cells and FAP expressing fibroblasts after administering our lead small-molecule CAF imaging agent revealed selective tumor targeting of CAF-rich tumors compared to cancer cell predominant tumors with favorable pharmacokinetics and tumor-to-background ratio (TBR) greater than 2. Similar imaging studies with MMTV-PyMT transgenic mice, an established murine model of breast cancer, showed analogous enhanced uptake in mammary tumors (TBR > 2) and clearance over 24 hours. NIR fluorescence also correlated with CAFs isolated from PyMT tumor tissue via flow cytometry, consistent with our hypothesis that this agent can specifically detect endogenous CAFs. This study identified a novel, bright, and water-soluble FAP-specific NIR agent for in vivo imaging, setting the stage for simultaneous optical imaging of malignant cells and activated fibroblasts in the TME to elucidate cancer-host interactions. Citation Format: Megan S. Michie, Junwei Du, Jiayu (Jennifer) Ye, Kathleen Duncan, Mikhail Y. Berezin, Samuel Achilefu. Selective imaging of cancer-associated fibroblasts in the breast cancer microenvironment with near-infrared fluorescent probes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4167.

Synthesis and Preliminary Study of 99mTc-Labeled HYNIC-FAPi for Imaging of Fibroblast Activation Proteins in Tumors.

Fibroblast activation protein (FAP) is an emerging target for cancer diagnosis. Different types of FAP inhibitor (FAPI)-based radiotracers have been developed and applied for tumor imaging. However, few FAPI tracers for single photon emission computed tomography (SPECT) imaging have been reported. SPECT imaging is less expensive and more widely distributed than positron emission tomography (PET), and thus, 99mTc-labeled FAPIs would be more available to patients in developing regions. Herein, we developed a FAPI-04-derived radiotracer, HYNIC-FAPi-04 (HFAPi), for SPECT imaging. 99mTc-HFAPi, with a radiochemical purity of >98%, was prepared using a kit formula within 30 min. The specificity of 99mTc-HFAPi for FAP was validated by a cell binding assay in vitro and SPECT/CT imaging in vivo. The binding affinity (Kd value) of 99mTc-HFAPi for human FAP and murine FAP was 4.49 and 2.07 nmol/L, respectively. SPECT/CT imaging in HT1080-hFAP tumor-bearing mice showed the specific FAP targeting ability of 99mTc-HFAPi in vivo. In U87MG tumor-bearing mice, 99mTc-HFAPi had a higher tumor uptake compared with that of HT1080-hFAP and 4T1-mFAP tumor models. Interestingly, 99mTc-HFAPi showed a relatively high uptake in some murine joints. 99mTc-HFAPi accumulated in tumor lesions with a high tumor-to-background ratio. A preliminary clinical study was also performed in breast cancer patients. Additionally, 99mTc-HFAPi exhibited an advantage over 18F-FDG in the detection of lymph node metastatic lesions in breast cancer patients, which is helpful in improving treatment strategies. In short, 99mTc-HFAPi showed excellent affinity and specificity for FAP and is a promising SPECT radiotracer for (re)staging and treatment planning of breast cancers.

An Antibody Targeting Fibroblast Activation Protein Simultaneously Fused to Interleukin-2 and Tumor Necrosis Factor Selectively Localizes to Neoplastic Lesions

The delivery of specific cytokine payloads to a neoplastic environment employing antibodies able to selectively accumulate at the tumor site represents an attractive strategy to stimulate an immune response to cancer. Whilst conventional antibody-cytokine fusions based on a single payload have shown potent anticancer activity, the concomitant delivery of two cytokine payloads may further improve the therapeutic outcome as the immune system typically adopts multiple signals to reinforce an antitumor strategy. We here describe a potency-matched dual-cytokine antibody fusion protein containing a tumor-targeting antibody fragment specific to human fibroblast activation protein (FAP), simultaneously linked to both interleukin-2 (IL2) and a tumor necrosis factor (TNF) mutant. The resulting fusion protein, termed IL2-7NP2-TNFmut, formed stable non-covalent trimers driven by the interaction of the tumor necrosis factor subunits. Both cytokine payloads retained their biological activity within the fusion protein, as shown by in vitro cellular assays. The tumor-targeting properties and the anticancer activity of IL2-7NP2-TNFmut were investigated in vivo in immunocompromised mice bearing SKRC52 cells transduced with human FAP. The fusion protein preferentially localized to the cancer site and induced partial tumor retardation.

Efficiency of Promoters of Human Genes FAP and CTGF at Organism Level in a Danio rerio Model.

The identification of tissue-specific promoters for gene therapeutic constructs is one of the aims of complex tumor therapy. The genes encoding the fibroblast activation protein (FAP) and the connective tissue growth factor (CTGF) can function in tumor-associated stromal cells but are practically inactive in normal adult cells. Accordingly, the promoters of these genes can be used to develop vectors targeted to the tumor microenvironment. However, the efficiency of these promoters within genetic constructs remains underexplored, particularly, at the organism level. Here, we used the model of Danio rerio embryos to study the efficiency of transient expression of marker genes under the control of promoters of the FAP, CTGF, and immediate early genes of Human cytomegalovirus (CMV). Within 96 h after the injection of vectors, the CTGF and CMV promoters provided similar equal efficiency of reporter protein accumulation. In the case of the FAP promoter, a high level of reporter protein accumulation was observed only in certain zebrafish individuals that were considered developmentally abnormal. Disturbed embryogenesis was the factor of changes in the exogenous FAP promoter function. The data obtained make a significant contribution to understanding the function of the human CTGF and FAP promoters within vectors to assess their potential in gene therapy.

Selection and Identification of an ssDNA Aptamer for Fibroblast Activation Protein.

As a type II transmembrane serine protease, fibroblast activation protein (FAP) is specifically expressed on the surface of fibroblasts associated with a variety of epithelial-derived malignancies such as pancreatic cancer, breast cancer, and colon cancer. It participates in the processes of tumorigenesis, progression, and immunosuppression. FAP constitutes an important target for tumor treatment; however, the current studies on FAP are mainly related to structural characteristics, enzymatic properties, and biological functions, and aptamers of FAP have not been investigated. In this work, by using recombinant human FAP as the target, five candidate aptamers, which are AptFAP-A1, AptFAP-A2, AptFAP-A3, AptFAP-A4, and AptFAP-A5, were selected by capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX), and their secondary structures were predicted to be mainly stem-loop. Moreover, the CE-laser-induced fluorescence (LIF) method was used to determine the equilibrium dissociation constant KD values between the FAP protein and candidate aptamers, and the KD value was in the low molar range. Finally, Cy5-labeled aptamers were co-incubated with human pancreatic cancer-associated fibroblasts highly expressing FAP protein, and confocal microscopy imaging showed that aptamer AptFAP-A4 had the highest affinities with the cells. The FAP aptamers screened in this study provide a promising direction for the development of rapid tumor diagnosis and targeted therapy.

Development and Characterization of Novel FAP-Targeted Theranostic Pairs: A Bench-to-Bedside Study.

Fibroblast activation protein (FAP) is among the most popular targets in nuclear medicine imaging and cancer theranostics. Several small-molecule moieties (FAPI-04, FAPI-46, etc.) are used for developing FAP-targeted theranostic agents. Nonetheless, the circulation time of FAP inhibitors is relatively short, resulting in rapid clearance via kidneys, low tumor uptake, and associated unsatisfactory treatment efficacy. To address the existing drawbacks, we engineered 3 peptides named FD1, FD2, and FD3 with different circulation times through solid-phase peptide synthesis. All the 3 reported peptides bind to human and murine FAP with single-digit nanomolar affinity measured by surface plasmon resonance. The diagnostic and therapeutic potential of the agents labeled with 68Ga and 177Lu was assessed in several tumor models exhibiting different levels of FAP expression. While radiolabeled FD1 was rapidly excreted from kidneys, radiolabeled FD2/FD3 have significantly prolonged circulation, increased tumor uptake, and decreased kidney accumulation. Our findings indicated that [68Ga]Ga-DOTA-FD1 positron emission tomography (PET) effectively detected FAP dynamics, whereas [177Lu]Lu-DOTA-FD2 and [177Lu]Lu-DOTA-FD3 exhibited remarkable therapeutic efficacy in FAP-overexpressing tumor models, including pancreatic cancer cell models characterized by abundant stroma. Moreover, a pilot translational investigation demonstrated that [68Ga]Ga-DOTA-FD1 had the capability to identify both primary and metastatic tumors with precision and distinction. In summary, we developed [68Ga]Ga-DOTA-FD1 for same-day PET imaging of FAP dynamics and [177Lu]Lu-DOTA-FD2 and [177Lu]Lu-DOTA-FD3 for effective radioligand therapy of FAP-overexpressing tumors.

Beta-endoproteolysis of the cellular prion protein by dipeptidyl peptidase-4 and fibroblast activation protein

The cellular prion protein (PrPC) converts to alternatively folded pathogenic conformations (PrPSc) in prion infections and binds neurotoxic oligomers formed by amyloid-βα-synuclein, and tau. β-Endoproteolysis, which splits PrPC into N- and C-terminal fragments (N2 and C2, respectively), is of interest because a protease-resistant, C2-sized fragment (C2Sc) accumulates in the brain during prion infections, seemingly comprising the majority of PrPSc at disease endpoint in mice. However, candidates for the underlying proteolytic mechanism(s) remain unconfirmed invivo. Here, a cell-based screen of protease inhibitors unexpectedly linked type II membrane proteins of the S9B serine peptidase subfamily to PrPC β-cleavage. Overexpression experiments in cells and assays with recombinant proteins confirmed that fibroblast activation protein (FAP) and its paralog, dipeptidyl peptidase-4 (DPP4), cleave directly at multiple sites within PrPC's N-terminal domain. For wild-type mouse and human PrPC substrates expressed in cells, the rank orders of activity were human FAP ~ mouse FAP > mouse DPP4 > human DPP4 and human FAP > mouse FAP > mouse DPP4 >> human DPP4, respectively. C2 levels relative to total PrPC were reduced in several tissues from FAP-null mice, and, while knockout of DPP4 lacked an analogous effect, the combined DPP4/FAP inhibitor linagliptin, but not the FAP-specific inhibitor SP-13786, reduced C2Sc and total PrPSc levels in two murine cell-based models of prion infections. Thus, the net activity of the S9B peptidases FAP and DPP4 and their cognate inhibitors/modulators affect the physiology and pathogenic potential of PrPC.

Development of Nectin4/FAP-targeted CAR-T cells secreting IL-7, CCL19, and IL-12 for malignant solid tumors.

Chimeric antigen receptor T (CAR-T) cell therapy has made significant advances for hematological malignancies but encounters obstacles in the treatment of solid tumors mainly due to tumor immunosuppressive microenvironment. Immunohistochemistry analysis was performed to examine the cellular expression of nectin cell adhesion molecule-4 (Nectin4) and fibroblast activation protein (FAP) in a variety of malignant solid tumors. Then, we engineered the fourth-generation Nectin4-targeted CAR-T (Nectin4-7.19 CAR-T) and FAP-targeted CAR-T (FAP-12 CAR-T) cells to evaluate their safety and efficacy in vitro and in vivo. In our study, we firstly demonstrated the aberrant overexpression of Nectin4 on both primary and metastatic solid tumors and FAP on cancer-associated fibroblasts. Then, we found that our fourth-generation Nectin4-7.19 CAR-T cells expressed IL-7 and CCL19 efficiently and exhibited superior proliferation, migration, and cytotoxicity compared to the second-generation Nectin4 CAR-T cells, while FAP-12 CAR-T cells exerted their ability of targeting both murine and human FAP effectively in vitro. In a fully immune-competent mouse model of metastatic colorectal cancer, lymphodepletion pretreated mice achieved complete remission with human Nectin4-targeted murine CAR-T (Nectin4 mCAR-T) cells. In the NSG mouse model of lung metastases, Nectin4-7.19 CAR-T cells eradicated metastatic tumors and prolonged survival in combination with FAP-12 CAR-T cells. These findings showed that Nectin4-7.19 CAR-T cells had potential therapeutic efficacy and exerted a synergistic role with FAP-12 CAR-T cells, further demonstrating that Nectin4 and FAP were able to serve as promising targets for safe and effective CAR-T therapy of malignant solid tumors.

Abstract A016: Selective delivery of fibroblast activation protein conjugated dual phosphoinositide 3-kinase–AKT Kinase-mTOR inhibitor associated with decreased tumor proliferation and on-target toxicity in high-grade soft-tissue sarcomas

Abstract Soft-tissue sarcomas (STS) are a group of lethal mesenchymal tumors that are a major cause of cancer related morbidity for those under the age of 20.PI3K inhibitors have been found to be effective against subset of STS but are associated with significant toxicities limiting their use.We have thought to test a selective fibroblast activation protein (FAP) targeted delivery of PI3K/AKT/mTOR inhibitor omipalisib to STS in pre-clinical setting as FAP is overwhelmingly expressed agressive soft tissue malignancies. Experimental Design: Patient derived fibrosarcomas (aSMA-,FAP+,pAKT+) were isolated by flow cytometry.FAP(-)HT1080 fibrosarcoma cell lines were used as a negative control whereas methylcoanthrene induced mouse fibrosarcoma transfected with human FAP cDNA were used as positive control.Cell expression of FAP,AKT,mTOR was compared using SDS Page,IHC,and flow cytometry.FAP ligand coupled to omipalisib(FAPL-PI3Ki)was then evaluated in-vitro and in small animal models. Results: Cell lines with known FAP expression demonstrated significant uptake of FAPL conjugated NIR fluorochrome.Wild type cells(hFAP-) showed no ligand binding both in flow cytometry and fluorescent microscopy(p<0.05).Escalating doses of FAP-PI3Ki suppressed AKT/mTOR activity in HT-1080 hFAP cells with 10 nM being the smallest effective dose.In hFAP+cell lines 10 nM of FAP-PI3Ki inhibited the AKT/mTOR signaling but inhibition was not observed in hFAP- cell lines demonstrating the need for FAP presence for FAPL-PI3Ki activity(p<0.05).Pan non-targeted PI3Ki inhibited the AKT/mTOR pathway in both hFAP+ and hFAP-cell lines.Densitometric analysis demonstrated that in hFAP+ cells,FAP-PI3Ki at above 10 nM suppresses the AKT/mTOR activity with ratio of pAKT/AKT and pmTOR/mTOR decreasing 11 and 4-fold respectively(p<0.05).FAP-PI3Ki activity significantly reduced when hFAP+ cells were competitively inhibited with free FAPL(4.6x increase in AKT activity,p<0.05).hFAP+ cells treated with FAP-PI3Ki had significant decrease in cell migration.In small animal models,SCID Mice injected with 25 umol/kg of either FAP-PI3Ki and non-targeted PI3Ki showed marked elevation of serum insulin levels in the non-targeted PI3Ki group (0.91 ng/mL vs 0.58 ng/mL,p<0.05).Xenograft tumor digest demonstrated mice treated with FAP-PI3Ki had Akt/mTOR pathway downregulated only in hFAP+ cell lines and this was evident in mice which received 10 nM or higher dosing(pAkt/Akt ratio of 1.1 WT mice vs 0.55 in hFAP+ mice,p<0.05).There was near complete suppression of the mTOR expression in the hFAP+ treatment group (pmTOR/mTOR 1.05 vs 0.087,p<0.05).There was no treatment related mortality in FAPL-PI3Ki group as compared to non-targeted PI3Ki delivery (p<0.05). Conclusion: Our study demonstrated that FAP conjugated modified omipalisib binds FAP expressing soft-tissue sarcomas both in-vitro and in xenograft models in concordance with FAP receptor density.FAP-PI3Ki induces PI3K/AkT/mTOR pathway inhibition similar to non-targeted PI3K inhibitors without significant systemic toxicities observed in pan nontargeted PI3K inhibitors. Citation Format: Feredun Azari, Gregory T. Kennedy, Ashley Chang, Bilal Nadeem, Azra Din, Isvita Marfatia, Steven Albelda, Philip Low, Madduri Srinivasarao, Ramesh Mukkamala, Neil T. Sullivan, Evgeniy Eruslanov, Sunil Singhal. Selective delivery of fibroblast activation protein conjugated dual phosphoinositide 3-kinase–AKT Kinase-mTOR inhibitor associated with decreased tumor proliferation and on-target toxicity in high-grade soft-tissue sarcomas [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A016.

Generation and in vivo validation of an IL-12 fusion protein based on a novel anti-human FAP monoclonal antibody

BackgroundIn this study, we describe the generation of a fully human monoclonal antibody (named ‘7NP2’) targeting human fibroblast activation protein (FAP), an antigen expressed in the microenvironment of different types...

Abstract 3317: Comparative biodistribution and radiotherapeutic efficacy of the fibroblast activation protein (FAP)-targeting agents FAP-2286 and FAPI-46

Abstract Background: FAP is a membrane-bound protease under investigation as a pan-cancer target given its limited expression in normal adult tissues but high expression on cancer-associated fibroblasts. FAP-2286 and FAPI-46 are radiotracers in clinical development that target FAP through different modalities: FAP-2286 employs a peptide macrocycle, whereas FAPI-46 is a quinoline-based small molecule. Here, the biochemical and cellular properties of FAP-2286 and FAPI-46 were evaluated, as well as their in vivo biodistribution and efficacy. Methods: FAP-2286 and FAPI-46 were assessed in biochemical and cellular assays for affinity to FAP and cellular uptake. Complexes of the compounds with gallium-68 (68Ga) or lutetium-177 (177Lu) were investigated in imaging and efficacy studies using the HEK-FAP xenograft mouse model. Results: FAP-2286 and FAPI-46 displayed potent affinity to human FAP by surface plasmon resonance with equilibrium dissociation constants of 1.1 and 0.04 nM, respectively. In addition, FAP-2286 and FAPI-46 inhibited human FAP protease activity with IC50 of 3.2 and 1.2 nM, respectively, and competitor binding to FAP-expressing cells with IC50 of 2.7 and 1.3 nM, respectively. In a PET imaging study, 68Ga-FAP-2286 or 68Ga-FAPI-46 resulted in comparable tumor uptake at 1 hour after injection (10 MBq; 10.6 vs 10.1 percent injected dose per gram [%ID/g]). In contrast, 177Lu-FAP-2286 and 177Lu-FAPI-46 had significant differences in tumor uptake at 24 hours (30 MBq; 15.8 vs 3.8 %ID/g, respectively; P=0.001) and 72 hours (16.4 vs 1.6 %ID/g respectively; P=0.002) after dosing as observed by SPECT imaging. Consistent with the SPECT imaging data, Alexa Fluor 488-derivatized FAP-2286 was retained in cells and secluded in endosomes out to 72 hours in vitro, whereas Alexa Fluor 488-derivatized FAPI-46 levels decreased over time starting at 8 hours, despite both showing a similar level of binding and initial internalization to HEK-FAP cells. FAP-2286 and FAPI-46 labeled with 177Lu demonstrated antitumor efficacy with mean tumor volumes (MTV) of 107 and 245 mm3, respectively, on day 9 after dosing compared with 952 mm3 for vehicle control (30 MBq; P<0.001). The suppressed tumor growth was maintained on day 23 for FAP-2286- but not for FAPI-46-treated animals (MTV 12 vs 1210 mm3; P<0.0001). At the end of the study (day 41), all mice treated with 177Lu-FAPI-46 had reached the endpoint MTV of >1500 mm³ with a median survival time (MST) of 27.5 days, whereas the MTV of mice treated with 177Lu-FAP-2286 was 106 mm3 with MST not being reached. Conclusions: In preclinical studies, the radiotherapeutic 177Lu-FAP-2286 showed longer tumor retention, resulting in greater tumor inhibition than 177Lu-FAPI-46. The phase 1/2 LuMIERE clinical trial (NCT04939610) will evaluate FAP-2286 as a therapeutic (177Lu-FAP-2286) and imaging (68Ga-FAP-2286) agent in multiple indications. Citation Format: Dirk Zboralski, Aileen Hoehne, Anne Bredenbeck, Matthias Paschke, Jan Lennart von Hacht, Jim Xiao, Andrew D. Simmons, Frank Osterkamp, Thomas Harding, Minh Nguyen. Comparative biodistribution and radiotherapeutic efficacy of the fibroblast activation protein (FAP)-targeting agents FAP-2286 and FAPI-46 [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 3317.

Abstract 4142: Fibroblast activation protein transmembrane receptor targeted near-infrared fluorochrome specifically identifies patient derived lung cancer associated fibroblasts in pre-clinical models

Abstract Objective: Cancer-associated fibroblasts (CAFs) are a key component of the tumor microenvironment and affect neoplastic progression by stromal remodeling, paracrine signaling, potentiating cancer cell proliferation, and immune system evasion. As such, they are a potential target for optimizing therapeutic and diagnostic strategies. The purpose of our study was to evaluate if Fibroblast Activation Protein (FAP) can be a suitable target for molecular imaging guided cancer localization during surgery because FAP is overwhelmingly expressed by CAFs. Methods: Patient derived lung adenocarcinoma FAP+/alpha SMA+ myofibroblasts (CAFs) were isolated by flow cytometry and cultured according to institutional best practices. Normal fibroblasts from 3T3 cell line were used as negative control whereas normal lung fibroblasts transfected with human FAP cDNA were used as positive control. Cell expression of FAP was compared using SDS Page analysis and flow cytometry. FAP ligand coupled to NIR S0456 fluorochrome (FAP-S0456) was then evaluated with in-vitro dose escalation, cell-toxicity study, fluorescence parameters, and immunofluorescence microscopy. Results: Patient derived lung cancer (LC) CAFs had lower FAP-S0456 dye binding expression compared to transfected fibroblasts (TF), while normal fibroblasts (FAP-, aSMA-) had no FAP-S0456 dye binding detected on FACS direct antibody analysis (MFI 1033 vs 244, p<0.05). SDS page immunofluorescence protein analysis with FAP-S0456 showed 5.33x relative expression of FAP in LC CAFs and transfected fibroblasts compared to normal controls on densitometric analysis (p<0.05). Both the LC CAFs and FAP transfected fibroblasts demonstrated targeted fluorochrome endosomal internalization at 798 nm wavelength with mean MFI of 418 (SD 41) compared to minimal uptake by normal fibroblasts (MFI 49 (SD 5.12)p<0.05). FAP-S0456 was internalized by FAP-expressing fibroblasts in a dose- and time-dependent manner, as compared to normal fibroblasts, which did not internalize the dye. Matrix co-culturing FAP+/GFP+ fibroblasts with A549 cells showed FAP-S0456 uptake in the GFP+ fibroblasts with no fluorescence observed in neoplastic cells. Evaluation of FAP-S0456 specificity for FAP transmembrane protein by competitive inhibition via 1000x free FAP ligand showed no uptake in the FAP expressing fibroblasts (LC CAF, FAP+ TF) with MFI similar to negative controls (normal fibroblasts, (39 vs 48, p=0.77). Conclusion: FAP transmembrane receptor is highly expressed in cancer associated fibroblasts and can serve as a potential target for FAP ligand conjugated near-infrared tracers. This study demonstrates proof of principle of utilizing FAP-S0456 in CAF in-vitro models and paves a path for pre-clinical diagnostic and therapeutic validation for surgical applications. Citation Format: Feredun Azari, Gregory Kennedy, Ashley Chang, Bilal Nadeem, Neil Sullivan, Emanuel Encarnado, Steven Albelda, Evgeniy Eruslanov, Sunil Singhal. Fibroblast activation protein transmembrane receptor targeted near-infrared fluorochrome specifically identifies patient derived lung cancer associated fibroblasts in pre-clinical models [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 4142.

Abstract LB527: A novel dimeric small molecule-radio conjugate targeting fibroblast activation protein with high and prolonged tumor uptake

Abstract Imaging procedures based on small molecule-radio conjugates targeting Fibroblast Activation Protein (FAP) are taking the spotlight in clinical practice for the diagnosis of a wide variety of cancer lesions. However, therapeutic applications of FAP-targeting radioligand therapeutics are limited by their short residence time in tumor lesions. We have recently described the discovery of OncoFAP, a novel ultra-high affinity ligand of FAP which has been already validated for PET imaging applications in more than twenty patients with solid tumors. In this abstract, we describe the development and in vivo characterization of BiOncoFAP, a dimeric FAP targeting small molecule based on OncoFAP. In particular, OncoFAP and BiOncoFAP displayed a superimposable affinity for recombinant human FAP (KD = 970 pM and 993 pM, respectively, measured by Fluorescence Polarization). Moreover, the two ligands exhibited a clear selectivity for the target, without significantly interacting with a wide panel of non-target proteins. BiOncoFAP was coupled to a DOTAGA chelator, suitable for radiolabeling with [177Lu]Lu for therapeutic applications. Cold [natLu]Lu-BiOncoFAP-DOTAGA showed an excellent stability in both mouse and human serum, with half-life longer than 5 days, supporting in vivo investigations. In a head-to-head in vivo biodistribution comparative study against [177Lu]Lu-OncoFAP-DOTAGA, [177Lu]Lu-BiOncoFAP-DOTAGA exhibited a more stable and prolonged residence time in FAP-positive tumors implanted in immunodeficient mice (~20% ID/g and ~200-to-1 tumor-to-blood ratio, 24 h after systemic administration). Notably, [177Lu]Lu-BiOncoFAP-DOTAGA did not significantly accumulate in healthy organs, thus showing an outstanding tumor-to-organ ratio (e.g., 12-to-1 tumor-to-kidney and 34-to-1 tumor-to-liver ratio, at the 24 h time point). These findings heighten BiOncoFAP as promising candidate for the development of anti-cancer radioligand therapeutics towards FAP-expressing tumor lesions. Citation Format: Samuele Cazzamalli, Andrea Galbiati, Aureliano Zana, Matilde Bocci, Jacopo Millul, Jacqueline Mock, Abdullah Elsayed, Dario Neri. A novel dimeric small molecule-radio conjugate targeting fibroblast activation protein with high and prolonged tumor uptake [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 LB527.

A Dimeric FAP-Targeting Small-Molecule Radioconjugate with High and Prolonged Tumor Uptake.

Imaging procedures based on small-molecule radioconjugates targeting fibroblast activation protein (FAP) have recently emerged as a powerful tool for the diagnosis of a wide variety of tumors. However, the therapeutic potential of radiolabeled FAP-targeting agents is limited by their short residence time in neoplastic lesions. In this work, we present the development and invivo characterization of BiOncoFAP, a new dimeric FAP-binding motif with an extended tumor residence time and favorable tumor-to-organ ratio. Methods: The binding properties of BiOncoFAP and its monovalent OncoFAP analog were assayed against recombinant human FAP. Preclinical experiments with 177Lu-OncoFAP-DOTAGA (177Lu-OncoFAP) and 177Lu-BiOncoFAP-DOTAGA (177Lu-BiOncoFAP) were performed on mice bearing FAP-positive HT-1080 tumors. Results: OncoFAP and BiOncoFAP displayed comparable subnanomolar dissociation constants toward recombinant human FAP in solution, but the bivalent BiOncoFAP bound more avidly to the target immobilized on solid supports. In a comparative biodistribution study, 177Lu-BiOncoFAP exhibited a more stable and prolonged tumor uptake than 177Lu-OncoFAP (∼20 vs. ∼4 percentage injected dose/g, respectively, at 24 h after injection). Notably, 177Lu-BiOncoFAP showed favorable tumor-to-organ ratios with low kidney uptake. Both 177Lu-OncoFAP and 177Lu-BiOncoFAP displayed potent antitumor efficacy when administered at therapeutic doses to tumor-bearing mice. Conclusion: 177Lu-BiOncoFAP is a promising candidate for radioligand therapy of cancer, with favorable invivo tumor-to-organ ratios, a long tumor residence time, and potent anticancer efficacy.

An ultra-high-affinity small organic ligand of fibroblast activation protein for tumor-targeting applications

We describe the development of OncoFAP, an ultra-high-affinity ligand of fibroblast activation protein (FAP) for targeting applications with pan-tumoral potential. OncoFAP binds to human FAP with affinity in the subnanomolar concentration range and cross-reacts with the murine isoform of the protein. We generated various fluorescent and radiolabeled derivatives of OncoFAP in order to study biodistribution properties and tumor-targeting performance in preclinical models. Fluorescent derivatives selectively localized in FAP-positive tumors implanted in nude mice with a rapid and homogeneous penetration within the neoplastic tissue. Quantitative in vivo biodistribution studies with a lutetium-177-labeled derivative of OncoFAP revealed a preferential localization in tumors at doses of up to 1,000 nmol/kg. More than 30% of the injected dose had already accumulated in 1 g of tumor 10 min after intravenous injection and persisted for at least 3 h with excellent tumor-to-organ ratios. OncoFAP also served as a modular component for the generation of nonradioactive therapeutic products. A fluorescein conjugate mediated a potent and FAP-dependent tumor cell killing activity in combination with chimeric antigen receptor (CAR) T cells specific to fluorescein. Similarly, a conjugate of OncoFAP with the monomethyl auristatin E-based Vedotin payload was well tolerated and cured tumor-bearing mice in combination with a clinical-stage antibody-interleukin-2 fusion. Collectively, these data support the development of OncoFAP-based products for tumor-targeting applications in patients with cancer.

An improved production and purification protocol for recombinant soluble human fibroblast activation protein alpha

Fibroblast activation protein alpha (FAP) is a cell-surface expressed type II glycoprotein that has a unique proteolytic activity. FAP has active soluble forms that retain the extracellular portion but lack the transmembrane domain and cytoplasmic tail. FAP expression is normally very low in adult tissue but is highly expressed by activated fibroblasts in sites of tissue remodelling. Thus, FAP is a potential biomarker and pharmacological target in liver fibrosis, atherosclerosis, cardiac fibrosis, arthritis and cancer. Understanding the biological significance of FAP by investigating protein structure, interactions and activities requires reliable methods for the production and purification of abundant pure and stable protein. We describe an improved production and purification protocol for His6-tagged recombinant soluble human FAP. A modified baculovirus expression construct was generated using the pFastBac1 vector and the gp67 secretion signal to produce abundant active soluble recombinant human FAP (residues 27–760) in insect cells. The FAP purification protocol employed ammonium sulphate precipitation, ion exchange chromatography, immobilised metal affinity chromatography and ultrafiltration. High purity was achieved, as judged by gel electrophoresis and specific activity. The purified 82 kDa FAP protein was specifically inhibited by a FAP selective inhibitor, ARI-3099, and was inhibited by zinc with an IC50 of 25 μM. Our approach could be adopted for producing the soluble portions of other type II transmembrane glycoproteins to study their structure and function.

The Latest Developments in Imaging of Fibroblast Activation Protein.

Fibroblast activation protein (FAP), a membrane-anchored peptidase, is highly expressed in cancer-associated fibroblasts in more than 90% of epithelial tumors and contributes to progression and worse prognosis of different cancers. Therefore, FAP is considered a promising target for radionuclide-based approaches for diagnosis and treatment of tumors and for the diagnosis of nonmalignant diseases associated with a remodeling of the extracellular matrix. Accordingly, a variety of quinolone-based FAP inhibitors (FAPIs) coupled to chelators were developed displaying specific binding to human and murine FAP with a rapid and almost complete internalization. Because of a high tumor uptake and a very low accumulation in normal tissues, as well as a rapid clearance from the circulation, a high contrast is obtained for FAPI PET/CT imaging even at 10 min after tracer administration. Moreover, FAPI PET/CT provides advantages over 18F-FDG PET/CT in several tumor entities for initial staging and detection of tumor recurrence and metastases, including peritonitis carcinomatosa.

Abstract A62: Inhibition of tumor growth by novel CART redirected against cells expressing high levels of fibroblast activation protein

Abstract Cancer-associated fibroblasts (CAFs) support tumor growth and metastasis of virtually all solid tumors, making them a potential “universal therapeutic target.” However, there are challenges to designing CAF targeted therapies. First, CAFs are heterogeneous, consisting of tumor-promoting and tumor-restraining populations. Second, molecular targets expressed strictly by tumor-promoting CAFs have yet to be defined. Nonetheless, Fibroblast Activation Protein (FAP) is selectively overexpressed on tumor-promoting CAFs. Moreover, deleting FAP+ cells inhibits tumor growth in preclinical models due to reductions in stroma-dependent signals that support tumor cell survival, growth, and progression and in stroma-derived immunosuppressive factors and can enhance efficacy of other therapies when given in combination. Prior studies in preclinical models suggest, however, that selective elimination of FAPhigh cells while sparing normal FAP+ cells that typically express lower levels of FAP may be necessary to avoid toxicity due to on-target, off-tumor effects. Thus, the aim of this study was to generate a FAP-chimeric antigen receptor (CAR) that selectively targets FAPhigh cells but that exhibits minimal activity against FAPlow cells for use in future clinical trials. We generated a FAP-CAR containing a scFv based on a monoclonal antibody (mAb) we generated that reacts with human and mouse FAP, the CD8 transmembrane region, and the CD3z and 41BB cytoplasmic domains. We show that primary human T cells transduced with this novel FAP-CAR exhibit strong reactivity against cells expressing high levels of human or mouse FAP but not FAPlow or FAP-negative cells. Moreover, these FAP-CART cells inhibited the growth of human FAP-negative A549 tumor xenografts infiltrated by FAP+ murine CAFs in NSG mice. Based on these characteristics, this novel FAP-CAR will be developed for use in clinical trials for human cancer patients. Citation Format: Estela Noguera-Ortega, Leslie Todd, James Monslow, Jing Sun, Steven M. Albelda, Ellen Pure. Inhibition of tumor growth by novel CART redirected against cells expressing high levels of fibroblast activation protein [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A62.

A Whole Cell Tumor Vaccine Expressing Human Fibroblast Activation Protein α Targets Cancer Cells and Cancer-Associated Fibroblasts

A Whole Cell Tumor Vaccine Expressing Human Fibroblast Activation Protein α Targets Cancer Cells and Cancer-Associated Fibroblasts