Synthesis and Isolation of Relevant Compounds
All commercially available compounds were purchased from suppliers worldwide. Solvents used for synthesis were obtained from local suppliers, while high-performance liquid chromatography (HPLC)-grade solvents were purchased from Fisher Scientific. Synthetic routes and chemical structures for all relevant compounds are provided in Supporting Information Section 2. Core PhoPEx structures were synthesized according to a previously reported method48. Compound quality was evaluated using ultrahigh-performance liquid chromatography–mass spectrometry (UPLC-MS) and high-resolution mass spectrometry (HRMS). The analytical data are provided in Supporting Information Section S6.
Proteins and Antibodies
FAP (C14G) was purchased from Novoprotein. DPPIV (HY-P70017), PREP (HY-P703584), ALB (HY-P1956A) and ALP (HY-P2818) were purchased from MCE. CTSB (C6286) was purchased from Sigma. PD-L1 (PD1-H5229 and PD1-H82E5) was obtained from ACROBiosystems. Sibrotuzumab was purchased from WuXi Biologics. The following antibodies were used: rabbit monoclonal anti-FAP antibody from Abcam (ab207178, clone EPR20021; 1:50); mouse monoclonal anti-pan-cytokeratin antibody from ZSGB-BIO (ZM-0069, clone AE1/AE3; 1:100); mouse monoclonal anti-CK5/CK6 antibody from ZSGB-BIO (ZM-0313, clone OT1F8; 1:100); mouse monoclonal anti-CK7 antibody from ZSGB-BIO (ZM-0071, clone UMAB161; 1:100); and rabbit monoclonal anti-PD-L1 antibody from Abcam (ab205921, clone 28-8; 1:250).
Measurement of Chemical Reaction Rates
A stock solution was prepared by dissolving the purified substrate in dimethylformamide (DMF) and diluting it to concentrations ranging from 2 mM to 0.2 mM. Each solution was analyzed by UPLC-MS with ultraviolet detection, and peak areas were integrated at the maximum absorption wavelength (λmaximum). Measurements were repeated three times to generate a standard curve. For reactivity assays, each 300-μl reaction mixture contained 1.5 mM substrate, 6 mM tetramethylguanidine and 22.5 mM Tyr-OMe-NAc (15 equivalents) and was incubated at 37 °C. At defined time points, 30-μl aliquots were collected, vortexed, centrifuged and quenched with 30 μl of 40 mM formic acid in acetonitrile. Quenched samples were analyzed by UPLC-MS, and compound concentrations were calculated using the standard curve. Pseudo-first-order kinetics were used to determine second-order rate constants. For the faster-reacting substrates 11 and 12, the reaction conditions were adjusted to 1 mM substrate, 1 mM tetramethylguanidine and 2 mM Tyr-OMe-NAc (2 equivalents) to directly model second-order reaction kinetics. Stability was evaluated in pH 9.5 phosphate-buffered saline (PBS) containing 1.5 mM substrate using the same analytical procedure.
In Vitro Compound Release Assays
For experimental samples, compound stock solutions were diluted in PBS and mixed with protein solutions at the appropriate ratio (protein:small molecule, 4:1 or 5:1). Samples were incubated at 37 °C, and aliquots were collected at the indicated time points. Compound concentrations were determined using a pre-established standard curve. For control samples, compound stock solutions were diluted to the same concentration in PBS and incubated under identical conditions without protein. Three independently prepared samples were analyzed for each condition. Fluorescent compounds were quantified using a microplate reader. Compounds without strong characteristic absorbance, including MMAE, were quantified by UPLC-MS.
Human Lymph Node Staining and Immunohistochemistry
Human lymph node tissue excised by a clinician was cleaned to remove surrounding adipose tissue and bisected using a surgical blade. One half was paraffin-embedded, sectioned into 4-μm slices and stained with hematoxylin and eosin for histological analysis. For immunohistochemistry (IHC), tissue sections were deparaffinized in xylene, rehydrated through a graded ethanol series and subjected to microwave antigen retrieval in citrate buffer (pH 6.0). The other half of the tissue was immersed in a fluorescent compound solution and stirred at 37 °C for 40 min. The tissue was then washed three times with PBS, with each wash performed for 10 min at 37 °C with stirring. Finally, the tissue was imaged using a near-infrared fluorescence imaging system. The use of patient-derived lymph nodes was approved by the Institutional Review Board of the Cancer Hospital of the Chinese Academy of Medical Sciences (approval number 2001.25/328–5274).
Radiolabeling and PET/SPECT-CT Imaging
Radiolabeling and imaging procedures were performed according to previously published methods49. Briefly, the appropriate label molecule (10 nmol) was combined with the corresponding radionuclide under the specified reaction conditions. The mixture was heated at 90–95 °C for 10–15 min and then cooled to room temperature. The reaction product was purified by filtration through a Sep-Pak Light C18 cartridge preconditioned with deionized water to remove unbound radionuclide. The radiolabeled compound was eluted with ethanol, diluted with saline and used for subsequent experiments. When necessary, ethanol was removed by bubbling nitrogen through the sample using a Termovap sample concentrator before dilution. Radiochemical yield was measured using a radiometer, and radiochemical purity was determined by radio-HPLC. For PET/SPECT-CT imaging, radiolabeled compounds were administered to mice through the tail vein. Animals were anesthetized with isoflurane in oxygen 10 min before each imaging time point. Imaging was performed using a Mediso nanoScan PET 122S system (Mediso) or an InliView-3000B PET/SPECT/CT system (Novel Medical). Standard PET or SPECT data acquisition and image reconstruction procedures were used. Images and quantitative data were analyzed with Interview Fusion software (v.3.09.008.0000) and NMSoft-AIWS software (v.1.8).
Tumor Xenograft Models and Therapeutic Studies
For FAP-targeted cell-derived xenograft (CDX) models, HT-1080, HT-1080-FAP or MC38-FAP cells were inoculated into mice in PBS. HT-1080-FAP and HT-1080 cells were administered at 5 × 106 cells per mouse, while MC38-FAP cells were administered at 5 × 105 cells per mouse into 4- to 6-week-old female C57BL/6 mice. For PD-L1-targeted CDX models, 5 × 106 HT-1080 cells in PBS were inoculated into 6- to 8-week-old female BNDG mice.
Patient-derived xenograft (PDX) models were established using 6- to 8-week-old BNDG mice matched to the sex of the patients who provided the tumor specimens. Primary tumor tissue was implanted subcutaneously to establish the P1 generation. When tumors reached 1,000–1,500 mm3, they were harvested, cut into approximately 3-mm pieces and transplanted by trocar into the forelimb axilla of recipient mice to establish the P2 generation. Subsequent passages were performed using the same procedure. The final generation used for treatment underwent PET imaging and IHC to confirm target expression. The use of patient-derived tissue for PDX model development was approved by the Institutional Review Board of the Cancer Hospital of the Chinese Academy of Medical Sciences (approval number 2001.25/328–5274) and the Beijing Cancer Hospital Institutional Review Board (approval numbers 25/328-5274 and 2026KT67).
All animal studies were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Peking University (approval number CCME-LiuZB-2) and the Institutional Animal Care and Use Committee of the China Brain Institute, Beijing (approval number CIBR-IACUC-071). Tumor volume was calculated using the formula: volume = (length × width2)/2. Tumor burden did not exceed 10% of mouse body weight, and body weight loss did not exceed 25% of the starting body weight, in accordance with the limits specified in the IACUC protocols.
Cell Culture and Fluorescence Imaging
HT1080-FAP cells customized by WuXi AppTec were cultured in Eagle’s minimum essential medium containing 10% fetal bovine serum (FBS), 1% antibiotic–antimycotic and 4 μg ml−1 blasticidin S. HT1080 cells from WuXi AppTec were cultured in minimum essential medium containing 10% FBS and 1% antibiotic–antimycotic. U87MG-FAP and U87MG-Vector cells from Pyrotech Biotechnology were cultured in minimum essential medium containing 10% FBS, 1% antibiotic–antimycotic and 2 μg ml−1 puromycin. MC38-FAP cells from WuXi AppTec were cultured in Dulbecco’s modified Eagle medium containing 10% FBS, 1% antibiotic–antimycotic and 3 μg ml−1 puromycin. All cell lines were routinely tested for mycoplasma contamination and maintained in a humidified incubator at 37 °C with 5% CO2.
For fluorescence imaging assays, cells were seeded in eight-well confocal dishes and incubated with the indicated compounds, including 10 μM FAPI-PhoPEx-MeRho for 24 h, compound 3 and 2 μM FAPI-VC-MeRho. Fluorescence was imaged at room temperature using a Nikon A1R-si laser-scanning confocal microscope. Fluorescence intensity was quantified with ImageJ.
Half-Maximal Inhibitory Concentration Assays
Two cell types were seeded in 96-well plates at a density of 5,000 cells per well and cultured for 12 h at 37 °C. Complete culture medium was used to prepare serial drug dilutions. After removal of the original medium, the drug solutions were added to the cells. Following the compound-specific treatment period, the drug solution was removed, and the cells were gently washed with PBS. Fresh drug-free complete medium was added, and the cells were cultured for an additional 24 h. Cell viability was then assessed using the Cell Counting Kit-8 (CCK-8) assay to determine half-maximal inhibitory concentration (IC50) values.
Biodistribution Analysis
Biodistribution was evaluated for each compound using four groups of mice, with six mice euthanized at each designated time point—2, 6, 24, 48, 72 and 120 h after injection—for tissue collection. Following intravenous administration through the tail vein, blood, liver and tumor tissues were collected and weighed. Tissues were homogenized for 30 min at 4 °C in RIPA lysis buffer containing PMSF. For approximately 500–900 mg of tissue, 1 ml of RIPA buffer containing 100 μl of 100 mM PMSF per milliliter of buffer was used. After homogenization, 2 ml of RIPA buffer, 2 ml of methanol and 1 ml of acetonitrile were added. Samples were sonicated for 10 min and centrifuged at 4,000 rpm for 10 min. A 5-ml aliquot of each supernatant was evaporated using a centrifugal concentrator. The residue was reconstituted in 800 μl of water:methanol:acetonitrile (1:1:1, v/v/v) and filtered through a 0.22-μm membrane. MMAE was quantified by UPLC-MS based on integrated mass-spectrometry signals (Supplementary Fig. 9). The area under the curve (AUC) for each group of six mice was calculated from payload concentrations measured at the six indicated time points.
Cryo-Electron Microscopy
For single-particle cryo-electron microscopy (cryo-EM), FAP protein at 0.4 mg ml−1 was incubated with a 20-fold molar excess of enantiomerically pure FAPI.ahMe-MMAE at 4 °C for 2 h. The resulting complexes were applied to graphene oxide-coated grids for cryo-EM analysis. Data acquisition, single-particle image processing, three-dimensional reconstruction and model building were performed as described in Supplementary Tables 1 and 2 and Supplementary Figure 22.
Surface Plasmon Resonance Binding Assay
Surface plasmon resonance (SPR) experiments were performed using a Biacore 8K+ instrument (Cytiva) equipped with a Series S Sensor Chip SA (Cytiva, 29104992). Biotinylated PD-L1 was captured on the sensor chip through a streptavidin–biotin interaction at a flow rate of 30 μl min−1. Analytes were injected at six sequentially increasing concentrations ranging from 5.12 to 500 nM using approximately 2.5-fold serial dilutions and single-cycle kinetics. Each concentration was injected at a flow rate of 30 μl min−1, with 150 s of association and 600 s of dissociation. A buffer blank was injected under identical conditions before each analyte. Binding curves were fitted to a 1:1 binding model using Biacore evaluation software. Each analyte was tested in triplicate.
Study Design and Reporting Summary
Additional information about the study design is available in the Nature Portfolio Reporting Summary linked to this article.
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