Cisplatin (cat. no. HY-17394; CAS no. 15663-27-1) was purchased from MedChemExpress. Human anti-CD26 polyclonal antibody (cat. no. 29403-1-AP), human anti-FAP polyclonal antibody (cat. no. 11779-1-AP), human anti-caspase-3 polyclonal antibody (cat. no. 19677-1-AP), HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibody (cat. no. SA00001-2) and human anti-cleaved caspase 3 polyclonal antibody (cat. no. 25128-1-AP) were purchased from Proteintech Group, Inc. Pomalidomide-PEG2-FAP2286: FAP-D was obtained from Shanghai Apeptide Co., Ltd. Protein extraction kit (cat. no. R0018M), Cell-Counting Kit-8 (CCK-8; cat. no. c0038), radioimmunoprecipitation assay buffers (cat. no. P0013B) and bicinchoninic acid (BCA) protein concentration assay kit (cat. no. P0012) were purchased from Beyotime Institute of Biotechnology. NCI-H1299 cells (cat. no. SCSP-589) were purchased from National Collection of Authenticated Cell Cultures. All other reagents, chemicals and cell lines were local.

Molecular docking was performed using AutoDock Vina 1.2.0 (19). Concerning the docking parameters of the receptor, the center coordinates of the docking box (center X, center Y and center Z) were set to (38.034, 0.53 and 69.455). The number of grid points in each direction of XYZ was set to 60×60×60, the docking accuracy exhaustiveness was 25 and the output binding conformation was set to 100. The docking results were visualized using PyMOL (version 2.5.0) (20) and LigPlot⁺(version 2.3) (21). The structure of human FAP alpha was obtained from Protein Data Bank (https://www.rcsb.org/structure/1Z68).

NCI-H1299 cells (5×10⁵ cells/ml) were cultured in 96-well plates. FAP-D was added to each well (Final concentrations: 0.01, 0.05, 0.1, 0.25, 0.5, 1, 5, 10 and 20 µM), and cells were cultured at 37°C for 12 h. Then, proteins were extracted from cells for western blotting. The protocols assessing the effects of FAP-D on protein degradation in H460 cells and effects of time on protein degradation were similar.

H1299 cells (1×10⁵ cells/ml) were cultured in 96-well plates. FAP-D was added to each well (final concentrations: 0.5, 1, 5, 10 and 20 µM), and cells were cultured at 37°C for 24 h. Then, the culture medium was discarded, and cell viability was determined according to the standard CCK-8 assay protocol using the EnVision microplate reader (Revvity, Inc.). For half-maximal inhibitory concentration (IC₅₀) assessment, FAP-D (0 or 5 µM) was added to each well, and cells were cultured at 37°C for 12 h. The culture medium was discarded, and cisplatin was added to each well (final concentrations: 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, and 500 µM), after which cells were cultured at 37°C for 12 h. After discarding the culture medium, cell viability was determined according to the standard CCK-8 assay protocol using the EnVision microplate reader. Volume of CCK-8 used was 20 µl and the solutions were incubated at 37°C for 2 h. The absorbance at 450 nm of solutions cultured without drugs was defined as 100%. The protocols of effects of FAP-D and cisplatin + FAP-D on cell viabilities of H460 cells were similar. Unless otherwise stated, all experiments were repeated three times.

H1299 or H460 cells were cultured in serum-free medium without/with cisplatin (5 µM, 24 h) or cisplatin (5 µM) + FAP-D (5 µM; cells were cultured with FAP-D for 12 h and then with cisplatin for 12 h). Cells were then cultured to confluence and scratched with a cell scraper. The debris was removed via washes with the culture medium. The cells were subsequently cultured in an incubator for 24 h. The images were acquired by microscopy and analyzed using ImageJ software (version 1.52a; National Institutes of Health).

The samples were prepared according as follows: cells were lysed using the radioimmunoprecipitation assay buffers and the solutions were centrifuged (14,000 × g 10 min). The protein concentrations were obtained using BCA assay. The samples were separated through 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (transfer current set to 250 mA). The membranes were blocked in 5% milk at room temperature for 1 h, and then were incubated with primary antibodies (FAP antibody: Dilution, 1:1,000; CD26 antibody: Dilution, 1:1,500; caspase-3 antibody: Dilution, 1:1,000; and cleaved caspase 3 antibody: Dilution, 1:1,000) at 4°C. Membranes were then washed with Tris-buffered saline with Tween-20 (0.05%) buffer and incubated with horseradish peroxidase-modified secondary antibodies (1:10,000) at room temperature for 1 h. Lastly, the membranes were imaged through Amersham™ ImageQuant™ 800 western blot imaging systems. The densitometric analysis were performed from ImageJ software (1.52a; National Institutes of Health)

The animal experiments were approved by the institutional animal ethics committee of Kunming Medical University (approval no. kmmu20241355; Kunming, China). BALB/c mice were purchased from the Department of Experimental Zoology, Kunming Medical University (Kunming, China). All clinical procedures were conducted in accordance with the relevant provisions of the Declaration of Helsinki. The studies were additionally conducted in accordance with local legislation and institutional requirements. Body weights of mice were ~18–19 g (healthy, 5–6 weeks old). Mice were fed at 25°C in the standard animal experiment lab and the humidity were maintained at 53%. The feeds and water were replaced every 3 days. The light/dark cycle was set as 12 h light/12 h dark. For biosafety experiments, BALB/c mice were randomly divided into two groups (n=5/group): PBS (control) group and FAP-D group. Mice in the FAP-D group were first anesthetized with 3% isoflurane via inhalation and then intraperitoneally injected with FAP-D (3 mg/kg, once daily for 7 days). Mice in the control group were first anesthetized with 3% isoflurane via inhalation and then intraperitoneally injected with phosphate-buffered saline (PBS). After treatment, mice were sacrificed via spinal dislocation, and the major organs were collected for analysis. For intervention experiments, nude BALB/c mice were randomly divided into three groups (n=6/group): PBS (control) group, cisplatin group, and cisplatin + FAP-D group. The mice were first subcutaneously injected with H1299 cells (5×10⁶ cells, 100 µl, in the right sides of the mice back) and monitored for 10 days to permit tumor growth. After tumor formation, mice in the cisplatin group were first anesthetized with 3% isoflurane via inhalation and then intratumorally injected with cisplatin (20 mg/kg, once every 3 days, 3 times). Then, the body weight and volume were recorded every 3 days. After treatment, mice were sacrificed via spinal dislocation, and the tumors were weighed and then sliced or mashed for further experiments. Mice in the cisplatin + FAP-D group were first anesthetized with 3% isoflurane via inhalation and then intratumorally injected with FAP-D (1.5 mg/kg, once every 3 days, three times), followed by an intraperitoneal injection of cisplatin (20 mg/kg, once every 3 days, three times) after 12 h. Mice were euthanized after 21 days or upon reaching tumor volume ≥2.000 mm³ or >20% body weight loss, with tumors processed for analysis. The other protocols were similar.

Statistical analysis was performed using GraphPad Prism 9.0 (Dotmatics) and Origin 9 software (OriginLab Corporation). All data are determined from at least three independent experiments and presented as the mean ± standard deviation (SD). Statistical significance was assessed by ANOVA followed by Dunnett post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

FAP-D consisted of three components: Pomalidomide, the FAP-binding peptide FAP 2286, and a linker (Diethylene glycol, Fig. 1A). The results of high-performance liquid chromatography and mass spectrometry are presented in Figs. S1 and S2. Molecular docking exhibited that several hydrophobic interactions and hydrogen bonds were formed between FAP-D and FAP (Figs. 1A and S3). Specifically, Arg123, Tyr124 and Gln547 in FAP formed hydrogen bonds with oxygen atoms in FAP-D. Trp623, Leu571, Ala554, Val552, Ile367, Phe350, Phe579, Gly349, Phe351, Val352, Ala207 and Ile398 in FAP formed hydrophobic interactions with FAP-D. The binding energy between FAP-D and FAP was −10.484 kcal/mol. These results suggested the binding force between FAP-D and FAP was strong. Western blotting demonstrated that FAP degradation increased as the FAP-D concentration increased (Figs. 1B and S4). Conversely, CD26 was not degraded by FAP-D, supporting the selectivity of FAP-D. In addition, FAP was degraded by FAP-D within 12 h (Figs. 1C and S5). These findings indicate that FAP-D is an ideal FAP degrader.

Considering the degradation performance of FAP-D, its effect on the cisplatin sensitivity of H1299 cells was explored. First, the effects of FAP-D on H1299 cell viability were studied. As presented in Fig. 2A, the viability of H1299 cells was reduced from 100 to 80% in response to increasing FAP-D concentration, suggesting that FAP degradation has little effect on cell viability. Second, FAP degradation was found to reduce the IC₅₀ of cisplatin in H1299 cells (1.36 µM vs. 5.71 µM, Fig. 2B), indicating enhanced cisplatin sensitivity. In addition, the mobility of H1299 cells treated with cisplatin + FAP-D was reduced (Fig. 2C and D). This result suggested that the extracellular matrix, which is responsible for proliferation and migration, was destroyed by FAP-D, leading to reduced cell viability. Lastly, the expression of cleaved caspase 3 (a major index of apoptosis) was increased by treatment with cisplatin + FAP-D (Figs. 2E and S6). These results reveal that the cisplatin sensitivity of H1299 cells was enhanced by FAP-D, resulting in strengthened apoptosis. Similarly, these results were confirmed through H460 cells (Figs. S7A-E and S8).

The in vivo therapeutic effects of cisplatin + FAP-D were then studied. First, the potential in vivo toxicity of FAP-D was investigated. As presented in Fig. 3, no damage was observed in the major organs (heart, liver, spleen, lungs and kidneys) of mice treated with FAP-D, indicating its favorable biocompatibility.

After confirming the biocompatibility of FAP-D, the in vivo therapeutic effects of cisplatin + FAP-D were systematically examined. As illustrated in Fig. 4A, the tumor volumes of mice treated with cisplatin alone were smaller than those in control mice. Interestingly, tumor growth was inhibited in mice pre-treated with FAP-D, as confirmed by tumor weight (Fig. 4B). Moreover, H&E and TUNEL staining revealed that apoptosis was enhanced in the tumors of mice co-treated with FAP-D (Fig. 4C and D). In addition, no significant changes in body weights were observed (Fig. S9). These results reveal that apoptosis was enhanced by FAP-D in tumors.