FAP-expressing Cells Research Articles

Magnetic Resonance Imaging of Fibroblast Activation Protein Using a Targeted Gadolinium-Based Contrast Agent.

The aim of this study was to synthesize a quinoline-based MRI contrast agent, Gd-DOTA-FAPI04, and assess its capacity for targeting fibroblast activation protein (FAP)-positive tumors in vivo. Gd-DOTA-FAPI04 was synthesized by attaching a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complex of gadolinium(III) to FAP inhibitor FAPI04. The longitudinal relaxation time (T1) of the contrast agent was measured using a Siemens Prisma 3.0T MR system, and the CCK-8 assay was performed to evaluate its potential cytotoxicity. Male nude mice bearing tumors grown from FAP-expressing fibrosarcoma cells were divided into experimental (n = 4) and control (n = 4) groups, and T1-weighted image enhancement was measured at different times (0, 10, 30, 60, 90, and 120 min) postinjection of Gd-DOTA-FAPI04. The control group received an additional preinjection of excess FAPI04. FAP expression in tumor tissue was investigated by using immunohistochemistry with an anti-FAP antibody. The longitudinal relaxivities of gadodiamide and Gd-DOTA-FAPI04 were measured to be 3.734 mM-1 s-1 and 5.323 mM-1 s-1, respectively. The CCK-8 assay demonstrated that Gd-DOTA-FAPI04 has minimal toxicity to cultured human fibrosarcoma cells. In vivo MRI showed that peak accumulation of Gd-DOTA-FAPI04 in FAP-expressing tumors occurred 1 h postinjection and could be blocked by preinjection of excess FAPI04. Immunohistochemical analysis of harvested tumor tissue supported the above findings. Gd-DOTA-FAPI04 is a promising contrast agent for in vivo imaging of FAP.

Harnessing the Potential of FAP-IL-12mut TMEkine™ for Targeted and Enhanced Anti-tumor Responses.

While cancer immunotherapy has yielded encouraging outcomes in hematological malignancies, it has faced challenges in achieving the same level of effectiveness in numerous solid tumors, primarily because of the presence of immune-suppressive tumor microenvironments (TMEs). The immunosuppressive qualities of the TME have generated considerable interest, making it a focal point for treatments aimed at enhancing immune responses and inhibiting tumor progression. Fibroblast activation protein (FAP), an attractive candidate for targeted immunotherapy, is prominently expressed in the TME of various solid tumors. Interleukin-12 (IL-12), recognized as a key mediator of immune responses, has been explored as a potential candidate for cancer treatment. Nevertheless, initial efforts to administer IL-12 systemically demonstrated limited efficacy and notable side effects, emphasizing the necessity for innovation. To address these concerns, our molecules incorporated specific IL-12 mutations, called IL-12mut, which reduced toxicity. This study explored the therapeutic potential of the FAP-IL-12mut TMEkine™-a novel immunotherapeutic agent selectively engineered to target FAP-expressing cells in preclinical cancer models. Our preclinical results, conducted across diverse murine cancer models, demonstrated that FAP-IL-12mut significantly inhibits tumor growth, enhances immune cell infiltration, and promotes a shift toward a cytotoxic immune activation profile. These findings suggest that FAP-IL-12mut could offer effective cancer treatment strategies.

In vitro and in vivo analyses of eFAP: a novel FAP-targeting small molecule for radionuclide theranostics and other oncological interventions

BackgroundFibroblast activation protein (FAP), a transmembrane serine protease overexpressed by cancer-associated fibroblasts in the tumor stroma, is an interesting biomarker for targeted radionuclide theranostics. FAP-targeting radiotracers have demonstrated to be superior to [18F]FDG PET/CT in various solid cancers. However, these radiotracers have suboptimal tumor retention for targeted radionuclide therapy (TRT). We aimed to develop a novel FAP-targeting pharmacophore with improved pharmacokinetics by introducing a substitution at the 8-position of (4-quinolinoyl)-glycyl-2-cyanopyrrolidine, which allows for conjugation of a chelator, dye, or other payloads.ResultsHere we showed the synthesis of DOTA-conjugated eFAP-6 and sulfo-Cyanine5-conjugated eFAP-7. After chemical characterization, the uptake and specificity of both tracers were determined on FAP-expressing cells. In vitro, [111In]In-eFAP-6 demonstrated a superior affinity and a more rapid, although slightly lower, peak uptake than gold standard [111In]In-FAPI-46. Confocal microscopy demonstrated a quick FAP-mediated internalization of eFAP-7. Studies with HT1080-huFAP xenografted mice confirmed a more rapid uptake of [177Lu]Lu-eFAP-6 vs. [177Lu]Lu-FAPI-46. However, tumor retention at 24 h post injection of [177Lu]Lu-eFAP-6 was lower than that of [177Lu]Lu-FAPI-46, hereby currently limiting its use for TRT.ConclusionThe superior affinity and faster tumor accumulation of eFAP-6 over FAPI-46 makes it a suitable compound for radionuclide imaging. After further optimization, the eFAP series has great potential for various oncological interventions, including fluorescent-guided surgery and effective targeted radionuclide theranostics.

Mechanistic Characterization of Cancer-associated Fibroblast Depletion via an Antibody-Drug Conjugate Targeting Fibroblast Activation Protein.

The direct elimination of FAP-expressing CAFs disrupts the cross-talk with cancer cells leading to a proinflammatory response and alterations in the immune microenvironment and antitumor immune response.

Abstract 1331: MiNK-215, an IL-15 armored FAP-targeting CAR iNKT cell therapy, effectively treats human organoid models of treatment-refractory MSS colorectal cancer (CRC) liver metastases

Abstract The presence of liver metastases in patients with microsatellite-stable (MSS) or mismatch repair proficient (pMMR) colorectal cancer (CRC) is associated with poor response to current pharmacological treatments, including immune checkpoint blockade (ICB). The tumor microenvironment (TME) of CRC liver metastases is characterized by a highly immunosuppressive phenotype that includes poor immunogenicity, a paucity of dendritic cells, elimination of tumor-specific effector T cells, and an abundance of immunosuppressive intrahepatic Kupffer cells and myeloid-derived suppressor cells. This underscores the urgent need for innovative therapeutic approaches targeted specifically to patients with CRC liver metastases. Here we describe MiNK-215, an IL-15 armored FAP-targeting CAR iNKT cell therapy, derived from the AgenT-797 allogeneic iNKT platform, as a potential novel therapeutic approach for patients with CRC liver metastases. To better model ICB refractory human MSS CRC liver metastases, we developed an ex vivo human organoid model that recapitulates the histological and immunological features of human disease. Organoids were comprised of MSS CRC cells engineered to express nuclear GFP and HLA-A2-NY-ESO-1, hepatocytes, Kupffer cell-enriched non-parenchymal cells, and HLA-matched peripheral blood mononuclear cells (PBMCs) supplemented with NY-ESO-1-reactive T cells. Organoid models of CRC lung metastases, which are traditionally more responsive to ICB therapy were also developed to compare the models to clinical experience and further validate their usefulness. Models were treated with Fc-enhanced anti-CTLA-4 (botensilimab) and anti-PD-1 (balstilimab) antibodies, or MiNK-215 to assess tumor killing and immune activation. Botensilimab and balstilimab enhanced T cell activation and cytotoxicity of CRC tumor cells in the absence of “normal” liver cells and in lung organoids. However, they were less effective in organoid models of CRC with liver metastases consistent with clinical observations. In contrast, MiNK-215 potently enhanced tumor killing by T cells in models of CRC with liver metastases. Tumor killing in CRC liver organoid models by MiNK-215 was associated with depletion of immune suppressive FAP-expressing stellate cells and increased CD8+ T cell infiltration. Our findings demonstrate that in treatment-refractory CRC-liver metastatic organoid models, MiNK-215 overcomes the limitations of ICB therapy to home to sites of disease, reprogram the TME, recruit tumor-reactive T cells and enhance tumor killing. Ex-vivo human CRC liver and lung metastatic organoid models are a novel platform that can be leveraged to rapidly identify new therapeutic approaches for potential clinical evaluation. Citation Format: Shanmugarajan Krishnan, Bishnu Joshi, Justin Keith, Ryan Frazier, Barbara Kalinowska, Jin San Choi, Stephanie Sanders, Shannon Boi, Olivier Le Tonqueze, Nick Kushner, Kah Teong Soh, Robert Stein, Tyler J. Curiel, Marc A. van Dijk, Enoch Kim, Eleni Chantzoura, Nils-Petter Rudqvist, Dhan Chand. MiNK-215, an IL-15 armored FAP-targeting CAR iNKT cell therapy, effectively treats human organoid models of treatment-refractory MSS colorectal cancer (CRC) liver metastases [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 1331.

Immunotheranostic target modules for imaging and navigation of UniCAR T-cells to strike FAP-expressing cells and the tumor microenvironment

BackgroundChimeric antigen receptor (CAR) T-cells are a promising approach in cancer immunotherapy, particularly for treating hematologic malignancies. Yet, their effectiveness is limited when tackling solid tumors, where immune cell infiltration and immunosuppressive tumor microenvironments (TME) are major hurdles. Fibroblast activation protein (FAP) is highly expressed on cancer-associated fibroblasts (CAFs) and various tumor cells, playing an important role in tumor growth and immunosuppression. Aiming to modulate the TME with increased clinical safety and effectiveness, we developed novel small and size-extended immunotheranostic UniCAR target modules (TMs) targeting FAP.MethodsThe specific binding and functionality of the αFAP-scFv TM and the size-extended αFAP-IgG4 TM were assessed using 2D and 3D in vitro models as well as in vivo. Their specific tumor accumulation and diagnostic potential were evaluated using PET studies after functionalization with a chelator and suitable radionuclide.ResultsThe αFAP-scFv and -IgG4 TMs effectively and specifically redirected UniCAR T-cells using 2D, 3D, and in vivo models. Moreover, a remarkably high and specific accumulation of radiolabeled FAP-targeting TMs at the tumor site of xenograft mouse models was observed.ConclusionsThese findings demonstrate that the novel αFAP TMs are promising immunotheranostic tools to foster cancer imaging and treatment, paving the way for a more convenient, individualized, and safer treatment of cancer patients.Graphical

Abstract 13851: CAR-T Cells: A Potential Novel Strategy for Mitigating Fibrosis in Duchenne Muscular Dystrophy

Introduction: Chimeric Antigen T (CAR)-T cells have proven effective preclinically for mitigating cardiac fibrosis in hypertensive cardiomyopathy. Hypothesis: We assessed whether this approach could be applied to Duchenne Muscular Dystrophy (DMD) by targeting Fibroblast Activating Protein (FAP)-expressing activated fibroblasts in cardiac and skeletal muscle fibrosis. Methods: A protocol for the lymphodepletion conditioning of mice was developed to optimize the dosing of cyclophosphamide. Next, immune-magnetically selected T cells were retrovirally transduced to target FAP (FAP-CAR) and quality-controlled by co-culture killing assays using FAP-expressing HEK target cells. Finally, lymphodepleted D2. mdx mice were intravenously injected with 10 7 FAP-CAR T cells (n=9) or 10 7 GFP-T cells (n=9) or Bovine Serum Albumin (BSA, n=8). Cardiac and skeletal muscle end points were assessed 2 and 5 weeks after treatment. Results: In vitro , the engineered CAR-T cells expressed activation markers (CD69, PD-1) and released large amounts of granzyme, perforin and interferon γ upon exposure to FAP-expressing target cells. One IP injection of cyclophosphamide (3mg) 4 days before treatment was safe and induced a lymphodepletion which enabled robust in vivo expansion of FAP-CAR T cell assessed by flow cytometry in lymphoid organs and blood: 10 5 , 2.10 6 and 10 7 detected CAR-T cells at day 3, 5 and 7 post-injections, respectively. This was paralleled by a more than a 2-fold decrease in endogenous FAP expression in skeletal muscles, as assessed by PCR at day 5 post-injections, and measurable FAP-CAR levels in the spleen compared with GFP-T cell controls. In vivo , FAP-CAR T cells significantly improved LV function with an EF change from baseline (Mean±SEM) of 33.1% ± 6.7% in FAP-CAR vs. 3.7% ± 6.4% and -9.6% ± 7.9% in GFP-T and BSA ( p <0.037 and p <0.005, respectively) at 2 weeks. The benefit was maintained at 5 weeks post-treatment. Several markers of fibrosis, particularly collagens 1 and 3, were also down-regulated in heart and muscles. Conclusions: FAP-CAR-T cells might represent a new therapeutic option for mitigating fibrosis in DMD, likely in conjunction with approaches aimed at addressing the root cause of the disease through a re-expression of a functional dystrophin.

Fibroblast Activation Protein-Targeted Photodynamic Therapy of Cancer-Associated Fibroblasts in Murine Models for Pancreatic Ductal Adenocarcinoma.

Patients with pancreatic ductal adenocarcinoma (PDAC) have a dismal 5 year survival of 9%. One important limiting factor for treatment efficacy is the dense tumor-supporting stroma. The cancer-associated fibroblasts in this stroma deposit excessive amounts of extracellular matrix components and anti-inflammatory mediators, which hampers the efficacy of chemo- and immunotherapies. Systemic depletion of all activated fibroblasts is, however, not feasible nor desirable and therefore a local approach should be pursued. Here, we provide a proof-of-principle of using fibroblast activation protein (FAP)-targeted photodynamic therapy (tPDT) to treat PDAC. FAP-targeting antibody 28H1 and irrelevant control antibody DP47GS were conjugated to the photosensitizer IRDye700DX (700DX) and the chelator diethylenetriaminepentaacetic acid. In vitro binding and cytotoxicity were evaluated using the fibroblast cell-line NIH-3T3 stably transfected with FAP. Biodistribution of 111In-labeled antibody-700DX constructs was determined in mice carrying syngeneic tumors of the murine PDAC cell line PDAC299, and in a genetically engineered PDAC mouse model (CKP). Then, tPDT was performed by exposing the subcutaneous or the spontaneous PDAC tumors to 690 nm light. Induction of apoptosis after treatment was assessed using automated analyses of immunohistochemistry for cleaved caspase-3. 28H1-700DX effectively bound to 3T3-FAP cells and induced cytotoxicity upon exposure to 690 nm light, whereas no binding or cytotoxic effects were observed for DP47GS-700DX. Although both 28H1-700DX and DP47GS-700DX accumulated in subcutaneous PDAC299 tumors, autoradiography demonstrated that only 28H1-700DX reached the tumor core. On the contrary, control antibody DP47GS-700DX was only present at the tumor rim. In CKP mice, both antibodies accumulated in the tumor, but tumor-to-blood ratios of 28H1-700DX were higher than that of the control. Notably, in vivo FAP-tPDT caused upregulation of cleaved caspase-3 staining in both subcutaneous and in spontaneous tumors. In conclusion, we have shown that tPDT is a feasible approach for local depletion of FAP-expressing stromal cells in murine models for PDAC.

Fibroblast activation protein as a potential theranostic target in brain metastases of diverse solid tumours

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.

Translational assessment of a DATA-functionalized FAP inhibitor with facile 68Ga-labeling at room temperature.

The present study aims at evaluating the preclinical and the clinical performance of [68Ga]Ga-DATA5m.SA.FAPi, which has the advantage to be labeled with gallium-68 at room temperature. [68Ga]Ga-DATA5m.SA.FAPi was assessed in vitro on FAP-expressing stromal cells, followed by biodistribution and in vivo imaging on prostate and glioblastoma xenografts. Moreover, the clinical assessment of [68Ga]Ga-DATA5m.SA.FAPi was conducted on six patients with prostate cancer, aiming on investigating, biodistribution, biokinetics, and determining tumor uptake. [68Ga]Ga-DATA5m.SA.FAPi is quantitatively prepared in an instant kit-type version at room temperature. It demonstrated high stability in human serum, affinity for FAP in the low nanomolar range, and high internalization rate when associated with CAFs. Biodistribution and PET studies in prostate and glioblastoma xenografts revealed high and specific tumor uptake. Elimination of the radiotracer mainly occurred through the urinary tract. The clinical data are in accordance with the preclinical data concerning the organ receiving the highest absorbed dose (urinary bladder wall, heart wall, spleen, and kidneys). Different to the small-animal data, uptake of [68Ga]Ga-DATA5m.SA.FAPi in tumor lesions is rapid and stable and tumor-to-organ and tumor-to-blood uptake ratios are high. The radiochemical, preclinical, and clinical data obtained in this study strongly support further development of [68Ga]Ga-DATA5m.SA.FAPi as a diagnostic tool for FAP imaging.

Fibroblast Activation Protein-Targeting Minibody-IRDye700DX for Ablation of the Cancer-Associated Fibroblast with Photodynamic Therapy.

Fibroblast activation protein (FAP), expressed on cancer-associated fibroblasts, is a target for diagnosis and therapy in multiple tumour types. Strategies to systemically deplete FAP-expressing cells show efficacy; however, these induce toxicities, as FAP-expressing cells are found in normal tissues. FAP-targeted photodynamic therapy offers a solution, as it acts only locally and upon activation. Here, a FAP-binding minibody was conjugated to the chelator diethylenetriaminepentaacetic acid (DTPA) and the photosensitizer IRDye700DX (DTPA-700DX-MB). DTPA-700DX-MB showed efficient binding to FAP-overexpressing 3T3 murine fibroblasts (3T3-FAP) and induced the protein's dose-dependent cytotoxicity upon light exposure. Biodistribution of DTPA-700DX-MB in mice carrying either subcutaneous or orthotopic tumours of murine pancreatic ductal adenocarcinoma cells (PDAC299) showed maximal tumour uptake of 111In-labelled DTPA-700DX-MB at 24 h post injection. Co-injection with an excess DTPA-700DX-MB reduced uptake, and autoradiography correlated with FAP expression in the stromal tumour region. Finally, in vivo therapeutic efficacy was determined in two simultaneous subcutaneous PDAC299 tumours; only one was treated with 690 nm light. Upregulation of an apoptosis marker was only observed in the treated tumours. In conclusion, DTPA-700DX-MB binds to FAP-expressing cells and targets PDAC299 tumours in mice with good signal-to-background ratios. Furthermore, the induced apoptosis indicates the feasibility of targeted depletion of FAP-expressing cells with photodynamic therapy.

Cell-specific expression of the FAP gene is regulated by enhancer elements.

Fibroblast activation protein (FAP) is an integral membrane serine protease that acts as both dipeptidyl peptidase and collagenase. In recent years, FAP has attracted considerable attention due to its specific upregulation in multiple types of tumor cell populations, including cancer cells in various cancer types, making FAP a potential target for therapy. However, relatively few papers pay attention to the mechanisms driving the cell-specific expression of the FAP gene. We found no correlation between the activities of the two FAP promoter variants (short and long) and the endogenous FAP mRNA expression level in several cell lines with different FAP expression levels. This suggested that other mechanisms may be responsible for specific transcriptional regulation of the FAP gene. We analyzed the distribution of known epigenetic and structural chromatin marks in FAP-positive and FAP-negative cell lines and identified two potential enhancer-like elements (E1 and E2) in the FAP gene locus. We confirmed the specific enrichment of H3K27ac in the putative enhancer regions in FAP-expressing cells. Both the elements exhibited enhancer activity independently of each other in the functional test by increasing the activity of the FAP promoter variants to a greater extent in FAP-expressing cell lines than in FAP-negative cell lines. The transcription factors AP-1, CEBPB, and STAT3 may be involved in FAP activation in the tumors. We hypothesized the existence of a positive feedback loop between FAP and STAT3, which may have implications for developing new approaches in cancer therapy.

Fibroblast Activation Protein-Targeted Radioligand Therapy for Treatment of Solid Tumors.

Fibroblast activation protein (FAP) has received increasing attention as an oncologic target because of its prominent expression in solid tumors but virtual absence from healthy tissues. Most radioligand therapies (RLTs) targeting FAP, however, suffer from inadequate tumor retention or clearance from healthy tissues. Herein we report a FAP-targeted RLT comprising an FAP6 ligand conjugated to DOTA and an albumin binder (4-p-iodophenylbutyric acid, or IP) for enhanced pharmacokinetics. We evaluated the performance of the resulting FAP6-IP-DOTA conjugate in 4 tumor models, 3 of which express FAP only on cancer-associated fibroblasts, that is, analogously to human tumors. Methods: Single-cell RNA-sequencing data were analyzed from 34 human breast, ovarian, colorectal, and lung cancers to quantify FAP-overexpressing cells. FAP6-DOTA conjugates were synthesized with or without an albumin binder (IP) and investigated for binding to human FAP-expressing cells. Accumulation of 111In- or 177Lu-labeled conjugates in KB, HT29, U87MG, and 4T1 murine tumors was also assessed by radioimaging or biodistribution analyses. Radiotherapeutic potency was quantitated by measuring tumor volumes versus time. Results: Approximately 5% of all cells in human tumors overexpressed FAP (cancer-associated fibroblasts comprised ∼77% of this FAP-positive subpopulation, whereas ∼2% were cancer cells). FAP6 conjugates bound to FAP-expressing cells with high affinity (dissociation constant, ∼1 nM). 177Lu-FAP6-IP-DOTA achieved an 88-fold higher tumor dose than 177Lu-FAP6-DOTA and improved all tumor-to-healthy-organ ratios. Single doses of 177Lu-FAP6-IP-DOTA suppressed tumor growth by about 45% in all tested tumor models without causing reproducible toxicities. Conclusion: We conclude that 177Lu-FAP6-IP-DOTA constitutes a promising candidate for FAP-targeted RLT of solid tumors.

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.

Assaying fibroblast activation protein (FAP) expression in vivo and in vitro for possible targeting with chimeric antigen receptor (CAR) T cells

Fibroblast activation protein (FAP) is a marker of activated fibroblasts in various human cardiovascular diseases. FAP is of special interest because it is expressed exclusively in activated, pathogenic fibroblasts and has been proposed as a target for chimeric antigen receptor (CAR) T cell ablation therapy. It has been shown previously that fibroblasts from patients with hypertrophic and dilated cardiomyopathy express FAP, while fibroblasts from non‐failing hearts did not. The aim of the project was to examine FAP expression in other human cardiac diseases by obtaining heart samples from patients who suffered diseases characterized by cardiac fibrosis so that we could determine which cardiac diseases might be amenable to FAP CAR T therapy. In addition, we sought to characterize an in vitro model of FAP expression to aid in the ability to assess the effectiveness of engineered T cells to kill FAP‐expressing cells. Heart tissue from patients diagnosed with various cardiac disorders, including idiopathic giant cell myocarditis, COVID‐19, and sarcoidosis were stained for FAP expression and compared to non‐failing, control hearts. The results showed significant FAP expression in all cardiac specimens with interstitial fibrosis compared to non‐failing hearts. Notably, samples from patients who died from COVID‐19 exhibited significant fibrosis and FAP expression. This suggests that there may be a large and growing population that may be amenable to anti‐fibrotic therapy. In order to develop an in vitro model of FAP expression to assay anti‐FAP CAR T cell activity, we treated IMR‐90 human fibroblasts with TGF‐B, a known activator of fibrosis, and assayed for FAP expression. TGF‐B exposure resulted in an increase in FAP expression 24‐hours after treatment. Our observation of fibroblast restricted FAP expression in multiple different cardiovascular diseases supports the potential use of FAP CAR T cell therapy as an anti‐fibrotic treatment in multiple heart diseases.

New Fully Automated Preparation of High Apparent Molar Activity 68Ga-FAPI-46 on a Trasis AiO Platform.

A large number of applications for fibroblast activation protein inhibitors (FAPI)-based PET agents have been evaluated in conditions ranging from cancer to non-malignant diseases such as myocardial infarction. In particular, 68Ga-FAPI-46 was reported to have a high specificity and affinity for FAP-expressing cells, a fast and high accumulation in tumor lesions/injuries together with a fast body clearance when investigated in vivo. Due to the increasing interest in the use of the agent both preclinically and clinically, we developed an automated synthesis for the production of 68Ga-FAPI-46 on a Trasis AiO platform. The new synthetic procedure, which included the processing of the generator eluate using a strong cation exchange resin and a final purification step through an HLB followed by a QMA cartridge, yielded 68Ga-FAPI-46 with high radiochemical purity (>98%) and apparent molar activity (271.1 ± 105.6 MBq/nmol). Additionally, the in vitro and in vivo properties of the product were assessed on glioblastoma cells and mouse model. Although developed for the preparation of 68Ga-FAPI-46 for preclinical use, our method can be adapted for clinical production as a reliable alternative to the manual (i.e., cold kit) or modular systems preparations already described in the literature.

18F-labeled tracers targeting fibroblast activation protein

BackgroundCancer-associated fibroblasts are found in the stroma of epithelial tumors. They are characterized by overexpression of the fibroblast activation protein (FAP), a serine protease which was already proven as attractive target for chelator-based theranostics. Unfortunately, the value of gallium-68 labeled tracers is limited by their batch size and the short nuclide half-life. To overcome this drawback, radiolabeling with aluminum fluoride complexes and 6-fluoronicotinamide derivatives of the longer-lived nuclide fluorine-18 was established. The novel compounds were tested for their FAP-specific binding affinity. Uptake and binding competition were studied in vitro using FAP expressing HT-1080 cells. HEK cells transfected with the closely related dipeptidyl peptidase-4 (HEK-CD26) were used as negative control. Small animal positron emission tomography imaging and biodistribution experiments were performed in HT-1080-FAP xenografted nude mice. [18F]AlF-FAPI-74 was selected for PET/CT imaging in a non-small cell lung cancer (NSCLC) patient.ResultsIn vitro, 18F-labeled FAPI-derivatives demonstrated high affinity (EC50 = < 1 nm to 4.2 nm) and binding of up to 80% to the FAP-expressing HT1080 cells while no binding to HEK-CD26 cells was observed. While small animal PET imaging revealed unfavorable biliary excretion of most of the 18F-labeled compounds, the NOTA bearing compounds [18F]AlF-FAPI-74 and -75 achieved good tumor-to-background ratios, as a result of their preferred renal excretion. These two compounds showed the highest tumor accumulation in PET imaging. The organ distribution values of [18F]AlF-FAPI-74 were in accordance with the small animal PET imaging results. Due to its less complex synthesis, fast clearance and low background values, [18F]AlF-FAPI-74 was chosen for clinical imaging. PET/CT of a patient with metastasized non-small cell lung cancer (NSCLC), enabled visualization of the primary tumor and its metastases at the hepatic portal and in several bones. This was accompanied by a rapid clearance from the blood pool and low background in healthy organs.Conclusion[18F]AlF-labeled FAPI derivatives represent powerful tracers for PET. Owing to an excellent performance in PET imaging, FAPI-74 can be regarded as a promising precursor for [18F]AlF-based FAP-imaging.

Abstract 2273: Selection of first-in-human clinical dose range for the tumor-targeted 4-1BB agonist MP0310 (AMG 506) using a pharmacokinetic/pharmacodynamics modeling approach

Abstract Following the clinical success of checkpoint inhibitors, cancer immunotherapy is rapidly expanding into combination treatments to enhance response rates and duration. Engagement of co-stimulatory molecules from the tumor necrosis factor receptor (TNFR) superfamily, including 4-1BB, may be a promising approach to enhance the benefits of cancer immunotherapy. Agonistic antibodies against the costimulatory receptor 4-1BB (CD137) have been shown to effectively enhance the anti-tumor activity of checkpoint inhibitors and other agents in preclinical animal models. However, the clinical development of 4-1BB agonistic antibodies has met with limited success thus far. Anti-4-1BB monoclonal antibodies have either been reported to cause significant dose-limiting hepatotoxicity or demonstrated limited efficacy as single agent therapeutics. We generated a DARPin® therapeutic candidate, MP0310 (AMG 506), which comprises domains binding to 4-1BB and fibroblast activation protein (FAP). MP0310 triggers 4-1BB activation only if bound and clustered via FAP which is abundantly expressed by cancer associated fibroblasts present in many solid tumors. In vitro functional assays indicate that MP0310 is a potent T cell co-stimulator in the presence of FAP-expressing cells. In vivo activity of tumor-targeted 4-1BB agonism was assessed in the HT-29 colon carcinoma xenograft model in PBMC humanized mice in combination with a T cell engager. Consistent with tumor targeting, MP0310 enhanced intra-tumoral CD8 T cell expansion while showing only limited systemic activity. We used a translational pharmacokinetic-pharmacodynamic (PK-PD) modeling approach to integrate in vitro and in vivo data to support the estimation of a minimal anticipated biological effect level (MABEL) and the relevant dose range for first in human (FIH) studies. In addition to a PK model describing the MP0310 concentrations over time in mouse and monkey, direct and indirect response models were used to describe receptor occupancy (RO), intra-tumoral CD8 T cell infiltration and peripheral CD8 count kinetics. Predictions from the combined PK and PD models using average intra-tumoral and peripheral drug concentrations (Cav) provide a MABEL dose with minimal expected systemic PD effects at 20% RO as well as the anticipated therapeutic dose range in humans. In conclusion, our PK/PD-modeling approach provides support for the selection of a safe starting dose for MP0310 (AMG506) in patients. It provides a rationale for selecting the maximum tested dose, and may help reduce the number of cancer patients receiving sub-therapeutic doses. MP0310 (AMG 506) is currently being evaluated in a Ph1 clinical study. Citation Format: Alexander Link, Laurent Juglair, Heïdi Poulet, Guy Lemaillet, Christian Reichen, Patricia Schildknecht, Ivana Tosevski, Joanna Robinson, Niina Veitonmäki, Jörg Herbst, Keith Dawson, Christof Zitt, Camila de Almeida, Rik de Greef, Victor Levitsky, Michael T. Stumpp, Hong Ji, Elmar Vom Baur. Selection of first-in-human clinical dose range for the tumor-targeted 4-1BB agonist MP0310 (AMG 506) using a pharmacokinetic/pharmacodynamics modeling approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2273.

Targeting Fibroblast Activation Protein: Radiosynthesis and Preclinical Evaluation of an 18F-Labeled FAP Inhibitor.

Fibroblast activation protein (FAP) has emerged as an interesting molecular target used in the imaging and therapy of various types of cancers. 68Ga-labeled chelator-linked FAP inhibitors (FAPIs) have been successfully applied to PET imaging of various tumor types. To broaden the spectrum of applicable PET tracers for extended imaging studies of FAP-dependent diseases, we herein report the radiosynthesis and preclinical evaluation of an 18F-labeled glycosylated FAPI ([18F]FGlc-FAPI). Methods: An alkyne-bearing precursor was synthesized and subjected to click chemistry-based radiosynthesis of [18F]FGlc-FAPI by 2-step 18F-fluoroglycosylation. FAP-expressing HT1080hFAP cells were used to study competitive binding to FAP, cellular uptake, internalization, and efflux of [18F]FGlc-FAPI in vitro. Biodistribution studies and in vivo small-animal PET studies of [18F]FGlc-FAPI compared with [68Ga]Ga-FAPI-04 were conducted in nude mice bearing HT1080hFAP tumors or U87MG xenografts. Results: [18F]FGlc-FAPI was synthesized with a 15% radioactivity yield and a high radiochemical purity of more than 99%. In HT1080hFAP cells, [18F]FGlc-FAPI showed specific uptake, a high internalized fraction, and low cellular efflux. Compared with FAPI-04 (half maximal inhibitory concentration [IC50] = 32 nM), the glycoconjugate, FGlc-FAPI (IC50 = 167 nM), showed slightly lower affinity for FAP in vitro, whereas plasma protein binding was higher for [18F]FGlc-FAPI. Biodistribution studies revealed significant hepatobiliary excretion of [18F]FGlc-FAPI; however, small-animal PET studies in HT1080hFAP xenografts showed higher specific tumor uptake of [18F]FGlc-FAPI (4.5 percentage injected dose per gram of tissue [%ID/g]) than of [68Ga]Ga-FAPI-04 (2 %ID/g). In U87MG tumor-bearing mice, both tracers showed similar tumor uptake, but [18F]FGlc-FAPI showed a higher tumor retention. Interestingly, [18F]FGlc-FAPI demonstrated high specific uptake in bone structures and joints. Conclusion: [18F]FGlc-FAPI is an interesting candidate for translation to the clinic, taking advantage of the longer half-life and physical imaging properties of 18F. The availability of [18F]FGlc-FAPI may allow extended PET studies of FAP-related diseases, such as cancer, but also arthritis, heart diseases, or pulmonary fibrosis.

Design and Development of 99mTc-Labeled FAPI Tracers for SPECT Imaging and 188Re Therapy.

Most epithelial tumors recruit fibroblasts and other nonmalignant cells and activate them into cancer-associated fibroblasts. This often leads to overexpression of the membrane serine protease fibroblast-activating protein (FAP). It has already been shown that DOTA-bearing FAP inhibitors (FAPIs) generate high-contrast images with PET/CT scans. Since SPECT is a lower-cost and more widely available alternative to PET, 99mTc-labeled FAPIs represent attractive tracers for imaging applications in a larger number of patients. Furthermore, the chemically homologous nuclide 188Re is available from generators, which allows FAP-targeted endoradiotherapy. Methods: For the preparation of 99mTc-tricarbonyl complexes, a chelator was selected whose carboxylic acids can easily be converted into various derivatives in the finished product, enabling a platform strategy based on the original tracer. The obtained 99mTc complexes were investigated in vitro by binding and competition experiments on FAP-transfected HT-1080 (HT-1080-FAP) or on mouse FAP-expressing (HEK-muFAP) and CD26-expressing (HEKCD26) HEK cells and characterized by planar scintigraphy and organ distribution studies in tumor-bearing mice. Furthermore, a first-in-humans application was done on 2 patients with ovarian and pancreatic cancer, respectively. Results:99mTc-FAPI-19 showed specific binding to recombinant FAP-expressing cells with high affinity. Unfortunately, liver accumulation, biliary excretion, and no tumor uptake were observed on planar scintigraphy for a HT-1080-FAP-xenotransplanted mouse. To improve the pharmacokinetic properties, hydrophilic amino acids were attached to the chelator moiety of the compound. The resulting 99mTc-labeled FAPI tracers revealed excellent binding properties (≤45% binding; >95% internalization), high affinity (half-maximal inhibitory concentration, 6.4-12.7 nM), and significant tumor uptake (≤5.4% injected dose per gram of tissue) in biodistribution studies. The lead candidate 99mTc-FAPI-34 was applied for diagnostic scintigraphy and SPECT of patients with metastasized ovarian and pancreatic cancer for follow-up to therapy with 90Y-FAPI-46. 99mTc-FAPI-34 accumulated in the tumor lesions, as also shown on PET/CT imaging using 68Ga-FAPI-46. Conclusion:99mTc-FAPI-34 represents a powerful tracer for diagnostic scintigraphy, especially when PET imaging is not available. Additionally, the chelator used in this compound allows labeling with the therapeutic nuclide 188Re, which is planned for the near future.