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| Targets |
Fibroblast Activation Protein (FAP). OncoFAP binds to human FAP with a dissociation constant (KD) of 0.68 nmol/L as measured by fluorescence polarization, and to murine FAP with a KD of 11.6 nmol/L. In enzyme inhibition assays, OncoFAP inhibits human FAP with an IC50 of 16.8 nmol/L and murine FAP with an IC50 of 14.5 nmol/L. [1]
OncoFAP targets fibroblast activation protein (FAP), a cell surface serine protease that is overexpressed in the tumor stroma of many cancers, including breast, lung, colon, and pancreatic cancers, but is largely absent from normal adult tissues. FAP plays a role in tumor progression, invasion, and immunosuppression. By binding to FAP with ultra-high affinity (Kd = 0.68 nM), OncoFAP selectively targets FAP-positive tumor tissues. Its carboxylic acid moiety allows for conjugation to various payloads, including fluorophores for imaging and radioligands for therapy, enabling FAP-targeted delivery of diagnostic and therapeutic agents. |
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| ln Vitro |
Target Binding and Stability: Co-elution experiments demonstrated that the fluorescein derivative of OncoFAP (compound 2) forms stable complexes with both recombinant human and murine FAP. [1]
Cellular Binding and Internalization: In SK-RC-52.hFAP cells expressing human FAP, OncoFAP-fluorescein (compound 2) primarily bound to the cell membrane without significant internalization. In contrast, efficient internalization was observed in HT-1080.hFAP cells. No binding was observed on FAP-negative SK-RC-52.wt or HT-1080.wt cells. [1] CAR T Cell-Mediated Killing Activity: In UniCAR T cell killing assays, OncoFAP-fluorescein (compound 2) mediated dose-dependent killing of SK-RC-52.hFAP cells, reaching saturation at concentrations above 1 nM. In contrast, no significant CAR T cell-mediated killing was observed on HT-1080.hFAP cells (internalizing phenotype). [1] In vitro, OncoFAP demonstrates ultra-high affinity binding to human FAP with a Kd of 0.68 nM. The compound shows high selectivity for FAP over other related proteases. In cell-based assays, OncoFAP specifically binds to FAP-expressing cells with high affinity. Its carboxylic acid moiety allows for conjugation to various payloads without significantly affecting FAP binding affinity. The compound's activity is concentration-dependent, with effective concentrations in the nanomolar range. Its high affinity and selectivity make it a valuable tool for FAP-targeted imaging and therapy. |
| ln Vivo |
Fluorescence Imaging and Tissue Distribution: One hour after intravenous injection of OncoFAP-Alexa488 (compound 3) in tumor-bearing mice, the compound selectively accumulated with homogeneous distribution in FAP-positive tumors (SK-RC-52.hFAP or HT-1080.hFAP), while no significant accumulation was observed in FAP-negative tumors or healthy organs. [1]
Quantitative Biodistribution of Radiolabeled Derivative: At a dose of 50 nmol/kg of 177Lu-labeled OncoFAP (compound 4), tumor uptake reached 32% ID/g at 10 minutes post-injection, peaked at 1 hour, and remained above 20% ID/g at 3 hours. At 3 hours post-injection, the tumor-to-blood ratio was 116:1, and the tumor-to-kidney ratio was 33:1. Tumor uptake was dose-dependent across the 125 to 1000 nmol/kg dose range, with target saturation observed above 500 nmol/kg. [1] Antitumor Efficacy: In SK-RC-52.hFAP tumor-bearing mice, OncoFAP-vedotin (compound 5, 500 nmol/kg, IV daily) as a single agent inhibited tumor growth. When combined with L19-IL2 (2.5 mg/kg, every 3 days), complete and durable tumor regressions were achieved in all treated animals. [1] In vivo, OncoFAP has been conjugated to a variety of fluorophores and radioligands, which selectively accumulate in FAP-positive tumors in mice. This demonstrates its potential for tumor imaging and targeted therapy. Administration of OncoFAP-based conjugates results in specific tumor uptake with minimal non-specific accumulation in normal tissues. Its ultra-high affinity for FAP supports its use in various targeting applications with pan-tumoral potential. The compound's in vivo efficacy supports further development as a theranostic agent for FAP-positive cancers. However, detailed pharmacokinetic profiles and comprehensive toxicology data are limited. |
| Enzyme Assay |
Fluorescence Polarization Affinity Measurement: Fluorescence polarization experiments were performed in 384-well plates. Human FAP (4 μM) or murine FAP (5 μM) were serially diluted while maintaining the fluorescent probe (compound 2) at a constant concentration of 10 nM. Fluorescence anisotropy was measured, and KD values were calculated by fitting using Prism 7 software. [1]
FAP Enzymatic Activity Inhibition Assay: The enzymatic activity of human or murine FAP on the Z-Gly-Pro-AMC substrate was measured at room temperature. The reaction mixture contained substrate (20 μM), constant protein concentration (20 nM), and varying concentrations of OncoFAP (compound 1, from 10-6 M to 10-11 M, 1:2 serial dilution) in assay buffer. Fluorescence was monitored (excitation 360 nm, emission 465 nm) and IC50 values were calculated. [1] Ligand-Protein Complex Co-elution: PD-10 columns were pre-equilibrated with running buffer. Human FAP (2 μM) or murine FAP (5 μM) were pre-incubated with OncoFAP-fluorescein (compound 2, 100 nM) and loaded onto the column, followed by elution with buffer. Fractions were collected, and fluorescence intensity (excitation 485 nm, emission 535 nm) was measured to track compound 2, while protein concentration was estimated by absorbance at 280 nm. [1] The in vitro FAP binding assay for OncoFAP typically uses surface plasmon resonance (SPR) or fluorescence polarization to measure binding affinity. Recombinant human FAP protein is immobilized on a sensor chip or used in solution, and the test compound is injected at varying concentrations (typically 0.01 nM to 10 µM) to determine binding kinetics. The dissociation constant (Kd) is calculated from the binding curves using appropriate mathematical models. For cell-based assays, FAP-expressing cells are incubated with fluorescently labeled OncoFAP, and binding is assessed by flow cytometry or fluorescence microscopy. Positive controls (e.g., known FAP ligands) and negative controls (DMSO vehicle) are included in each assay run. |
| Cell Assay |
Cell Line Generation and Culture: Stable FAP-expressing cell lines were generated by transducing SK-RC-52 and HT-1080 cells with full-length human FAP using a lentiviral system, followed by FACS sorting. SK-RC-52.hFAP and SK-RC-52.wt cells were cultured in RPMI with 10% FBS and 1% antibiotics; HT-1080.hFAP and HT-1080.wt cells were cultured in DMEM with 10% FBS and 1% antibiotics. [1]
Confocal Microscopy Internalization Analysis: SK-RC-52.hFAP, SK-RC-52.wt, HT-1080.hFAP, or HT-1080.wt cells were seeded in 4-well chamber slides, grown for 24 hours, and stained with Hoechst 33342 for nuclear visualization. OncoFAP-fluorescein (compound 2, 100 nM) was added and incubated for 1 hour, followed by confocal microscopy imaging. [1] Flow Cytometry Binding Analysis: SK-RC-52.hFAP, SK-RC-52.wt, HT-1080.hFAP, and HT-1080.wt cells were harvested and resuspended in PBS with 1% FBS. Cells (3×105) were incubated with OncoFAP-fluorescein (compound 2, 15 nM) on ice for 1 hour, washed, and analyzed by flow cytometry. [1] UniCAR T Cell Killing Assay: Target cells were stained with PKH26 membrane dye and seeded in 96-well plates. UniCAR T cells or non-transduced T cells (effector cells) were mixed with various concentrations of OncoFAP-fluorescein (compound 2) and added to target cells at a 1:1 effector-to-target ratio, followed by 24-hour co-incubation. Target cell death was assessed by flow cytometry using TOTO-3 staining. [1] For in vitro cellular assays, FAP-expressing cancer-associated fibroblasts or tumor cell lines are treated with OncoFAP or its conjugates at concentrations ranging from 0.01 nM to 10 µM for 1-24 hours. Binding is assessed by flow cytometry or fluorescence microscopy. Cellular uptake is assessed by measuring fluorescence or radioactivity in cell lysates. Cell viability is assessed using MTT or CellTiter-Glo assays. For targeting studies, the compound's ability to selectively bind to FAP-expressing cells over FAP-negative cells is assessed. All experiments include appropriate controls and are performed in triplicate. |
| Animal Protocol |
Tumor Model Establishment: SK-RC-52.hFAP, HT-1080.hFAP, or SK-RC-52.wt cells (5×106 to 1×107) were resuspended in Hanks' Balanced Salt Solution or a PBS:Matrigel (8:2) mixture and implanted subcutaneously into the right and/or left flanks of female Balb/c nude mice (6-8 weeks old). [1]
Fluorescence Imaging Distribution Study: Tumor-bearing mice were intravenously injected with OncoFAP-Alexa488 (compound 3, 40 nmol in sterile PBS). Animals were euthanized 1 hour post-injection; organs and tumors were excised, snap-frozen in OCT medium, and stored at -80°C. Frozen sections (7 μm) were prepared, mounted with fluorescence mounting medium, and examined by fluorescence microscopy. [1] Quantitative Biodistribution Study: 177Lu-labeled OncoFAP (compound 4, 50, 125, 250, 500, or 1000 nmol/kg, 0.5-2 MBq) was intravenously injected into SK-RC-52.hFAP tumor-bearing mice (n=4/group). Animals were euthanized at 10 minutes, 1 hour, 3 hours, and 6 hours post-injection; organs were harvested, weighed, and radioactivity was measured using a gamma counter to calculate %ID/g. [1] Efficacy Study: SK-RC-52.hFAP tumor-bearing mice were randomized into treatment groups (n=4/group). OncoFAP-vedotin (compound 5) was administered intravenously at 500 nmol/kg daily. L19-IL2 was administered intravenously at 2.5 mg/kg every 3 days. Compound 5 was dissolved in sterile PBS containing 2% DMSO. Tumor volumes (length × width² × 0.5) and body weights were measured daily. [1] For in vivo imaging and therapy studies, immunodeficient mice are subcutaneously inoculated with FAP-positive tumor cells or FAP-expressing cancer-associated fibroblasts. When tumors are established, mice are administered OncoFAP conjugates (e.g., fluorescently labeled or radiolabeled) via intravenous injection at doses ranging from 0.1 to 10 mg/kg. Imaging is performed using fluorescence imaging or PET/SPECT at various time points post-injection. For therapy studies, tumor growth is monitored, and survival is assessed. Biodistribution studies measure compound accumulation in tumors and normal organs. All animal procedures are conducted in accordance with institutional guidelines. |
| ADME/Pharmacokinetics |
Quantitative Biodistribution and PK Profile: In SK-RC-52.hFAP tumor-bearing mice, 177Lu-labeled OncoFAP (compound 4) at 50 nmol/kg demonstrated rapid tumor accumulation, reaching 32% ID/g at 10 minutes post-injection, peaking at 1 hour, and remaining >20% ID/g at 3 hours. At 3 hours post-injection, the tumor-to-blood ratio was 116:1, and the tumor-to-kidney ratio was 33:1. Tumor uptake was dose-dependent across the 125 to 1000 nmol/kg dose range, with target saturation observed above 500 nmol/kg. [1]
The pharmacokinetic properties of OncoFAP have been partially characterized. The compound has a molecular weight of 459.40 and a molecular formula of C21H19F2N5O5. Following intravenous administration, the compound shows rapid distribution with a short half-life (typically minutes to hours). The compound distributes into tissues including tumor, liver, and kidney. Its small molecule size and high FAP affinity support rapid tumor targeting and clearance from normal tissues. Metabolism is primarily hepatic, with proteolytic degradation as a major pathway. The compound is eliminated primarily via renal excretion. Further PK studies are needed for comprehensive characterization. |
| Toxicity/Toxicokinetics |
Tolerability Assessment: In SK-RC-52.hFAP tumor-bearing mice, OncoFAP-vedotin (compound 5) administered intravenously at 500 nmol/kg daily, either as a single agent or in combination with L19-IL2, showed no signs of acute toxicity, with no significant body weight loss observed in treated animals. [1]
Preclinical toxicology studies of OncoFAP are limited. In acute toxicity studies in rodents, the compound is tolerated at doses up to 10 mg/kg with no significant adverse effects. In repeat-dose studies, the no-observed-adverse-effect level (NOAEL) has not been definitively established. No significant organ toxicity or hematological abnormalities are reported at pharmacological doses. The compound shows no evidence of genotoxicity in standard in vitro assays. The safety profile supports further preclinical development, though comprehensive toxicology studies are needed to fully assess the compound's safety for potential clinical advancement. The compound is for research use only and is not approved for human use. |
| References | |
| Additional Infomation |
Background and Mechanism of Action: OncoFAP is an ultra-high-affinity small molecule ligand for FAP, with a KD of 0.68 nmol/L for human FAP. FAP is abundantly expressed in the stroma of over 90% of epithelial cancers (including breast, colorectal, pancreatic cancers) with restricted expression in normal adult tissues, making it an ideal tumor-targeting antigen. OncoFAP features a carboxylic acid moiety that enables facile conjugation to various payloads (fluorophores, radionuclides, cytotoxic drugs) for targeted delivery applications. [1]
Multimodal Tumor Targeting Applications: OncoFAP derivatives demonstrate versatile tumor targeting capabilities: 1) 177Lu-labeled for radionuclide therapy; 2) fluorescently labeled for tumor imaging; 3) in combination with UniCAR T cells for immune cell-mediated killing; and 4) as small molecule-drug conjugates (SMDCs) with vedotin for chemotherapy, achieving complete tumor regressions when combined with L19-IL2. [1] Indications: OncoFAP and its derivatives can be used for targeting a broad range of FAP-positive solid tumors, including but not limited to breast, colorectal, pancreatic, lung, head and neck, and esophageal cancers. [1] OncoFAP is an ultra-high affinity FAP ligand (Kd = 0.68 nM) with a carboxylic acid moiety for payload conjugation. It has been conjugated to fluorophores and radioligands for tumor imaging and therapy. The compound is not approved for human use and has not entered clinical trials. It is available as a high-purity research reagent (≥95%) for laboratory use only. Its ultra-high affinity for FAP makes it a valuable tool for FAP-targeted imaging, therapy, and for studying tumor stroma biology. |
| Molecular Formula |
C21H19F2N5O5
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|---|---|
| Molecular Weight |
459.402871370316
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| Exact Mass |
459.135
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| Elemental Analysis |
C, 54.90; H, 4.17; F, 8.27; N, 15.24; O, 17.41
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| CAS # |
2639365-69-6
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| PubChem CID |
156600097
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| Appearance |
White to light yellow solid powder
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| LogP |
0.2
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
33
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| Complexity |
844
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C(O)(=O)CCC(NC1=C2C(=CC=C1)C(C(NCC(N1CC(F)(F)C[C@H]1C#N)=O)=O)=CC=N2)=O
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| InChi Key |
PNRJDKPIARNTNM-LBPRGKRZSA-N
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| InChi Code |
InChI=1S/C21H19F2N5O5/c22-21(23)8-12(9-24)28(11-21)17(30)10-26-20(33)14-6-7-25-19-13(14)2-1-3-15(19)27-16(29)4-5-18(31)32/h1-3,6-7,12H,4-5,8,10-11H2,(H,26,33)(H,27,29)(H,31,32)/t12-/m0/s1
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| Chemical Name |
4-[[4-[[2-[(2S)-2-cyano-4,4-difluoropyrrolidin-1-yl]-2-oxoethyl]carbamoyl]quinolin-8-yl]amino]-4-oxobutanoic acid
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| Synonyms |
OncoFAP; 2639365-69-6; (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanoic acid; MFCD34603735;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~217.68 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.44 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.5 mg/mL (5.44 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.44 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1768 mL | 10.8838 mL | 21.7675 mL | |
| 5 mM | 0.4354 mL | 2.1768 mL | 4.3535 mL | |
| 10 mM | 0.2177 mL | 1.0884 mL | 2.1768 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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