A binding-to-release strategy for targeted anticancer drug delivery

Targeted drug conjugates, such as antibody–drug conjugates (ADCs), represent a transformative therapeutic modality in oncology^1,2,3. Although their clinical success is undeniable—with the market projected to reach US$26 billion^8,9,10,11—the field remains constrained by a fundamental limitation: a strong dependency on efficient receptor-mediated endocytosis and lysosomal trafficking for payload release^12,13 (Fig. 1a, left). This endocytic prerequisite creates a dual bottleneck. First, it restricts the druggable target landscape to a small fraction of the human membrane proteome (about 180 of more than 2,000 proteins^4,5,6,7) that undergo efficient internalization, funnelling development into intense competition on a handful of antigens such as HER2 and TROP2^11,14. Second, it subjects drug delivery to the cumulative inefficiency of the multi-step lysosomal trafficking pathway, resulting in only a fraction of the internalized conjugates successfully releasing their payload^15,16. Although recent evidence suggests that extracellular payload release may also contribute to ADC efficacy in some settings, the dominant pattern of the field and optimization efforts remain centred on endocytosis^13,17. Consequently, the vast majority of tumour-specific cell surface antigens with poor internalization efficiency remain an untapped ‘blue ocean’, representing a substantial opportunity for expanding the therapeutic reach of drug conjugates.

The case of FAP exemplifies this untapped potential. As a pan-cancer antigen highly expressed on cancer-associated fibroblasts with minimal normal tissue expression¹⁸, its remarkable targeting potential is clinically validated by the diagnostic success of FAPI-based radiopharmaceuticals across more than 28 cancer types¹⁹. However, the development of FAP-targeted drug conjugates, such as the clinical candidate OMTX705²⁰, has been hampered by the low internalization efficiency of FAP²¹, as evidenced by a best response of only disease stabilization in 26% of patients²². This impasse underscores the unmet need for a drug release pattern that bypasses the endocytic machinery.

Inspired by the principles of ligand-directed chemistry^23,24, which uses target binding to enable proximity-induced ligation^25,26 (Fig. 1b), we developed a binding-to-release (BTR) strategy for endocytosis-independent drug delivery. Our core design involves engineering a conjugate with a new linker that remains stable in circulation. However, on ligand–receptor engagement, the linker can release the payload when positioned in proximity to nucleophilic residues within the binding pocket of the target. This proximity-induced reaction triggers the efficient, endocytosis-independent release of a membrane-permeable cytotoxic payload directly at the tumour site (Fig. 1a, right), thereby circumventing the endocytic bottleneck.

To translate this concept into reality, a chemical tool that combines circulatory stability with rapid, proximity-triggered cleavage is essential. Although sulfur(VI) fluoride exchange (SuFEx) chemistry offers a promising balance of stability and reactivity^27,28, we initially sought to integrate a self-immolative phenol-based scaffold—a structure widely used to mediate efficient drug release²⁹—by developing a sulfur–phenol exchange (SuPEx) system. However, unlike SuFEx, SuPEx—even with p-nitrophenol as the leaving group—showed negligible tyrosine reactivity in tubes (Extended Data Fig. 1a). Inspired by the review in ref. ³⁰, we substituted sulfur (S) with phosphorus (P) as the central electrophilic atom and developed the phosphorus(V)–phenol exchange (PhoPEx) reaction (Fig. 1c), and this replacement enhanced the tyrosine reaction rate in vitro. Therefore, we engineered the first-generation PhoPEx linker, using p-hydroxybenzyl alcohol as the leaving group and conjugated it to a FAP-targeting ligand (FAPI) to evaluate its BTR ability. This design was based on molecular docking predictions indicating the proximity of reactive nucleophilic residues to the FAPI binding pocket within FAP (Fig. 1d). In vitro, PhoPEx-based BTR conjugates bearing caged fluorescent reporters (coumarin or MeRho) demonstrated FAP-dependent payload release after 24 h of co-incubation, a response absent in SuPEx-based conjugates and phosphate-buffered saline (PBS) controls (Fig. 1e,f). This specificity was confirmed in cellular models: HT1080-FAP exhibited pronounced fluorescence activation after 24 h, whereas HT1080 controls showed minimal signal (Fig. 1g).

To investigate whether the BTR mechanism bypasses endocytosis, we compared the efficacy of conjugates bearing membrane-permeable (monomethyl auristatin E, MMAE) and comparatively impermeable (monomethyl auristatin F, MMAF) payloads. Although the internalization-to-release (ITR) Val-Cit-linker mediated cell killing in HT1080-FAP cells with both MMAE and MMAF, the PhoPEx-based BTR conjugate induced potent cytotoxicity only with MMAE; the MMAF-conjugate was ineffective (Fig. 1h). This disparity indicates that the BTR platform operates by an extracellular, non-internalizing release mechanism, as only the membrane-permeable MMAE could diffuse into cells to exert its effect. We acknowledge that this mechanism inherently necessitates the use of payloads with high membrane permeability. But this requirement aligns with the prevailing trend in ADC development², driven by the success of agents such as DS8201 (Enhertu), in which highly permeable payloads such as DXd and MMAE are prioritized for their enhanced efficacy, particularly against heterogeneous tumours³¹. Furthermore, for a stromal target such as FAP, which is often expressed on cancer-associated fibroblasts rather than directly on cancer cells, an extracellular release mechanism offers a distinct advantage. The released, permeable drug can diffuse throughout the tumour microenvironment, potentially achieving a broader distribution and mitigating the challenge of tumour heterogeneity compared with an internalization-dependent approach (Supplementary Fig. 15). Tail vein administration of the MMAE-loaded BTR conjugate (compound 1, Extended Data Fig. 5b) elicited potent and specific tumour growth inhibition in HT1080-FAP xenografts, with negligible efficacy in FAP-negative HT1080 tumours (Fig. 1i–k). These initial results demonstrated that the first-generation PhoPEx linker enabled FAP-specific payload release without endocytosis. However, its suboptimal reaction efficiency still remained incompatible with the short circulatory half-life of small-molecule agents, necessitating the development of more reactive PhoPEx linkers.

Given the suboptimal reaction kinetics of the first-generation linker, we systematically pursued structural optimization of the PhoPEx scaffold to enhance its reactivity while preserving stability. Based on the electronic (electron-withdrawing and electron-donating) effects of substituents, we introduced various functional groups at different positions on the benzene ring of the leaving group, designing and synthesizing a total of 15 PhoPEx structures (Extended Data Fig. 1c). Reactivity was assessed by their reaction with the tyrosine analogue (Tyr-OMe-NAc) in DMF with tetramethylguanidine (TMG) as a catalyst, whereas stability was evaluated by measuring hydrolysis rates in PBS buffer at pH 9.5 (Fig. 2a). The SuFEx structure was used as a benchmark for parallel comparison of both reactivity and stability. Using second-order kinetic fitting (Extended Data Fig. 1b), we obtained the reaction rate constants (k_react) and hydrolysis rate constants (k_hydro) for all 15 PhoPEx structures as well as the SuFEx reference (Fig. 2b and Extended Data Fig. 1e–j). When plotted with SuFEx as the baseline, PhoPEx structures falling within the red region exhibited reactivity comparable to SuFEx while demonstrating higher stability (Fig. 2c). Among these, the para-amide and meta-fluoro substitution performed best, with reaction rate constants of 4.35 × 10⁻³ M⁻¹ s⁻¹ and 3.92 × 10⁻³ M⁻¹ s⁻¹, respectively (SuFEx: 3.99 × 10⁻³ M⁻¹ s⁻¹), and hydrolysis rate constants of 3.07 × 10⁻⁸ M⁻¹ s⁻¹ and 2.28 × 10⁻⁸ M⁻¹ s⁻¹, respectively (SuFEx: 9.51 × 10⁻⁸ M⁻¹ s⁻¹) (Extended Data Fig. 1k).

Incorporation of the evolved meta-fluoro-substituted PhoPEx linker (compound 2, Extended Data Fig. 5b) markedly improved reactivity over the parent compound 1, resulting in a shortened reaction half-life of 3.5 h for better pharmacokinetic matching with small molecules, while largely preserving hydrolysis stability (Fig. 2f). In parallel, we prepared targeted covalent radiopharmaceuticals incorporating the evolved PhoPEx linkers (PhoPEx-FAPI-mF and PhoPEx-FAPI-pCONH) as well as the unsubstituted one (PhoPEx-FAPI-H), allowing for a comparison in vivo. Positron emission tomography–computed tomography (PET-CT) imaging showed that the PhoPEx-FAPI-mF achieved about two-fold higher tumour uptake in both SUV_max and SUV_mean compared with the unsubstituted counterpart, indicating enhanced reaction efficiency within living systems (Fig. 2d,e). For PhoPEx-FAPI-pCONH, it demonstrated rapid and irreversible covalent binding to the FAP protein (Fig. 2i, left) and exhibited comparable tumour uptake against SuFEx-FAPI (Supplementary Figs. 8 and 24), suggesting that PhoPEx chemistry is comparable to SuFEx chemistry. The covalency led to higher tumour uptake and retention compared with the non-covalent FAPI04 (Extended Data Fig. 2f–i and Supplementary Fig. 11).

Having established the PhoPEx-based BTR strategy and its proximity-induced cleavage mechanism in model systems, we next asked whether this approach functions in clinical samples. To address this, we developed an activatable fluorescent probe, FAPI-P-Cy5-QSY21. On binding to FAP, the probe releases the quencher QSY21, restoring Cy5 fluorescence^32,33 (Extended Data Fig. 3a). FAPI-P-Cy5-QSY21 showed FAP-specific fluorescence enhancement in vitro, in cells and in mouse models (Extended Data Fig. 3b–e). We then evaluated its performance on clinical specimens. Intraoperative assessment of lymph node metastasis in medullary thyroid carcinoma at present relies on subjective selection of a few nodes for frozen section analysis, which limits diagnostic accuracy. Given that FAP co-localizes with tumours in this cancer³⁴, we proposed that incubating all resected lymph nodes with the probe and measuring fluorescence intensity could objectively determine FAP expression in metastases (Extended Data Fig. 3f). After optimizing probe concentration, incubation time and temperature (Extended Data Fig. 4d–f and Supplementary Fig. 5), we bisected lymph nodes (typically <5 mm in diameter), disrupted their capsules, incubated them with 2 μM probe at 37 °C for 40 min and washed three times with PBS. This protocol produced two distinct fluorescence intensity populations (Supplementary Fig. 6). Subsequent FAP immunohistochemistry (IHC) showed that nodes with higher fluorescence intensity were mostly FAP-positive, whereas those with lower intensity were predominantly FAP-negative (Extended Data Fig. 3g). Quantitative analysis showed that the average radiant efficiency in FAP-positive nodes was 2.9-fold higher than in FAP-negative nodes (Extended Data Fig. 3h). Together, these results demonstrate that the PhoPEx-based BTR strategy is effective in clinical specimens.

With the BTR platform validated, we then used meta-fluoro-substituted PhoPEx linker to continue evaluating whether BTR would meet the critical parameters for a controlled-release system—specificity and efficiency—and exploring its potential advantages compared with the classical internalization-to-release (ITR) pattern (exemplified by the Val-Cit dipeptide linker³⁵ used in the approved ADCs) when both are applied to the FAP target. Specificity and efficiency are two parameters that are central to controlled-release systems: high specificity minimizes off-target effects, reduces systemic toxicity and increases the maximum tolerated dose (MTD); high efficiency ensures sufficient drug release within tumour tissues, enhancing efficacy and lowering the minimum effective dose. At the molecular level, FAP-BTR-SMDC (compound 5) exhibited high binding selectivity for FAP protein, showing more than 1,000-fold selectivity against homologous proteins (Fig. 3b). Even in the presence of 100-fold equivalents of nucleophilic amino acids, 10-fold equivalents of other proteins (for example, albumin, alkaline phosphatase), or in human serum (Extended Data Fig. 5d,e), payload release—ranging from fluorescent molecules (compound 3) to cytotoxic agents such as the microtubule inhibitor MMAE (compound 5) and the TOPI inhibitor exatecan (compound 4)—occurred rapidly and specifically only on addition of FAP protein (Fig. 3c,d, Extended Data Fig. 5b and Supplementary Figs. 16, 25 and 26). Notably, Gly-Pro dipeptide, a common FAP-release substrate, can be non-specifically cleaved by homologues, such as DPPIV and PREP, as well as hydrolases, such as CTSB³⁶ (Extended Data Fig. 5f,g). At the cellular level, FAP-BTR-SMDC (compound 5) demonstrated enhanced cytotoxicity against FAP-positive cells, with IC₅₀ values of 498.9 nM in U87MG-Vector and 9.0 nM in U87MG-FAP, suggesting release specificity (Fig. 3e and Supplementary Fig. 14). Compared with the ITR Val-Cit-linker, compound 3 (BTR PhoPEx linker) released more than eight-fold higher fluorescent signal at the same time point under equivalent incubation conditions, indicating better release efficiency (Fig. 3f,g). In animal models, tumour analysis following intravenous administration of 1.0 mg kg⁻¹ FAP-BTR-SMDC (compound 5) showed that MMAE concentrations in HT1080-FAP tumours were 34.5-fold higher than in HT1080 tumours at 24 h (Fig. 3i), and MMAE exposure (area under the curve from 0 to 120 h, AUC_0–120 h) in HT1080-FAP tumours was 5.9-fold higher for FAP-BTR-SMDC (compound 5) than for FAP-ITR-SMDC (Val-Cit-linker, equal moles of MMAE), and comparable to FAP-ITR-ADC (FAP-targeted sibrotuzumab coupling with VC-MMAE through cysteine and maleimide, drug-to-antibody ratio (DAR) of 3.5, Extended Data Fig. 6b–d, equivalent dose of MMAE) (Fig. 3m). Single photon emission computed tomography with computed tomography (SPECT-CT) imaging showed additional uptake of FAP-BTR-SMDC in the liver. Thus, liver and blood were used as reference normal tissues to assess nonspecific release (Fig. 3h and Supplementary Fig. 10). Both in blood and in liver, nonspecific release in non-target organs was lower for FAP-BTR-SMDC, with tumour-to-blood and tumour-to-liver ratios reaching 39.7 and 358.1, respectively—14.7- and 55.1-fold higher than those of FAP-ITR-SMDC, and 3.6- and 58.7-fold higher than those of FAP-ITR-ADC (Fig. 3j–l,n and Supplementary Fig. 27). These results indicate that the PhoPEx linker operating through the BTR mechanism showed improved release specificity and efficiency compared with the internalization-dependent Val-Cit-linker on FAP protein. Moreover, despite the different pharmacokinetic profiles, the FAP-BTR-SMDC achieved tumour drug exposure comparable to that of the antibody-based FAP-ITR-ADC, with higher tumour-to-normal tissue ratios.

We then tested whether release specificity and efficiency would translate into a broadened therapeutic window. In acute toxicity studies, in which the MTD was defined as the highest dose that did not cause body weight loss exceeding 20%, the MTD of the FAP-BTR-SMDCs was 7.5-fold molar dose higher than that of the FAP-ITR-SMDCs, indicating a reduction in systemic toxicity (Fig. 4a). In cell-derived xenograft (CDX) model efficacy studies, at equivalent doses, FAP-BTR-SMDCs demonstrated greater tumour growth inhibition. A single intravenous administration of FAP-BTR-SMDCs (6.2 mg kg⁻¹) resulted in complete response in 100% of mice (8 out of 8) within 30 days, accompanied by stable body weight and 100% survival (Fig. 4b–f and Supplementary Figs. 13 and 29). In patient-derived xenograft (PDX) models, we evaluated FAP-BTR-SMDC against FAP-ITR-SMDC and a reference FAP-ITR-ADC. In a sarcoma PDX (Fig. 4h–j), in which FAP is expressed directly on tumour cells, FAP-BTR-SMDC (dosed at 6.2 mg kg⁻¹, about 15% of its MTD) showed better anti-tumour efficacy than FAP-ITR-SMDC and achieved efficacy slightly better than the FAP-ITR-ADC (dosed at 22.2 mg kg⁻¹, approximately 50% of its MTD; Extended Data Fig. 6e). In a pancreatic adenosquamous carcinoma (PASC) PDX (Fig. 4k–m)—a clinically aggressive, treatment-refractory malignancy characterized by a dense desmoplastic stroma that limits the penetration of large molecules³⁷—FAP is expressed on cancer-associated fibroblasts rather than on tumour cells. In this model, FAP-BTR-SMDC demonstrated better efficacy compared with the FAP-ITR-ADC. Terminal tumour weight measurements and photos (Extended Data Fig. 7e) of tumours indicated the improved efficacy of FAP-BTR-SMDC (tumour weight: FAP-BTR-SMDC group 0.19 ± 0.07 g compared with ADC group 1.32 ± 0.41 g). Apart from these models, FAP-BTR-SMDC also showed anti-tumour activity in MC38-FAP CDX (Extended Data Fig. 7b), hypopharyngeal PDX (Extended Data Fig. 7f–j) and periampullary carcinoma PDX models (Extended Data Fig. 7k–n), with efficacy correlating with FAP expression levels. Collectively, these data from both toxicity tolerance and treatment efficacy dimensions indicate that the BTR platform broadens the therapeutic window.

The rational design of BTR strategy on FAP has demonstrated considerable potential, while also revealing vast, untapped chemical opportunities at the protein-binding interface. mRNA display is a high-throughput screening technology that can create ultralarge libraries (typically exceeding 10¹³ unique members) and can support highly efficient direct in vitro screening³⁸ (Fig. 5a). In previous work, researchers reported the identification of covalent macrocyclic peptides containing SuFEx through mRNA display screening³⁹. Given the mechanistic similarities between PhoPEx and SuFEx chemistry, we directly substituted the SuFEx-based unnatural amino acid in the cyclic peptide with an unnatural amino acid incorporating the PhoPEx release structure and conducted systematic evaluation (Fig. 5b). Experimental results showed that the payload release rate reached its maximum when the linker contained four carbon atoms, with a rate constant of 3.25 h⁻¹, enabling complete release of the fluorescent molecule within 1 h (Fig. 5c,d). This preliminarily suggested the feasibility of screening PhoPEx-based peptide–drug conjugates (PDCs) by mRNA display.

Furthermore, we performed a bioinformatic analysis of all ligand–protein co-crystal structures in the PDBbind database^40,41,42 (v2020.R1, 19,037 entries), which showed that 17,967 structures (94.4%) contain at least one tyrosine residue within 10 Å of the ligand. PD-L1 was selected as the second target because of its clinical importance and poor internalization. According to a reported cyclic peptide ligand⁴³ (PDB: 9MAP), we introduced PhoPEx at either a Gly or a Leu position (Fig. 5e,f). Substitution at Gly reduced binding affinity to 0.54 ± 0.11 μM, whereas substitution at Leu maintained high affinity (8.4 ± 2.2 nM) (Extended Data Fig. 8b and Supplementary Fig. 28). The high-affinity variant (Leu) enabled better PhoPEx-mediated release of coumarin, MeRho and MMAE in vitro (Fig. 5g–i). In vivo, in HT1080 tumour-bearing mice, which expressed PD-L1 (Extended Data Fig. 8d), the PD-L1-BTR-SMDC conjugate showed more tumour-specific MMAE release than PD-L1-ITR-SMDC and could be blocked by the peptide BMS-986189 (Fig. 5j). The PD-L1-BTR-SMDC, therefore, showed better anti-tumour efficacy in a PDX model of gastric adenocarcinoma (Fig. 5k–m and Extended Data Fig. 8c). These findings suggest that the BTR strategy holds promise for extension to other important therapeutic targets with versatile routes, demonstrating broad potential applicability.

In this study, we first developed the PhoPEx chemistry, optimized its reactivity and stability, and preliminarily studied its mechanism (Supplementary Fig. 17). Based on PhoPEx chemistry, we developed the BTR strategy, which enables payload release independently of endocytosis. Using FAP as a model target with poor internalization²¹ (Extended Data Fig. 5h,i), we optimized the PhoPEx linker and identified Tyr745 as the key nucleophilic residue for the proximity-induced reaction. The platform was successfully applied to SMDCs, activatable fluorescent probes, and—on a second target—a PD-L1-targeted conjugate, demonstrating its adaptability.

The BTR strategy is intended as a complementary approach to the conventional ITR strategy. ITR is highly effective for targets with efficient endocytosis (for example, HER2, TROP2), but faces inherent limitations for most of the membrane proteins that internalize poorly. Recognizing this gap, several recent approaches have explored non-internalizing or extracellular release mechanisms^{13,17,44,45,46}, including releasing payload through extracellular cathepsin cleavage, by tumour-microenvironment proteases, or by developing a two-component click-to-release system using an exogenous trigger to cleave the linker. Our BTR platform uses a single-agent, proximity-induced chemical reaction directly triggered by target binding, without relying on enzymatic activity or exogenous co-administration.

Compared with intracellular payload release, extracellular release of a membrane-permeable payload could offer potential advantages, including better drug distribution within the tumour microenvironment and the ability to kill neighbouring antigen-negative cells (bystander effect). At the same time, it could in principle raise off-tumour toxicity concerns. Yet, this risk is manageable, as it is suggested that the amount of payload released in the tumour represents only a small fraction of the total administered dose⁴⁷, and the side effects caused by the minor leakage from the tumour may be insignificant. Moreover, potential mitigation strategies include optimizing payload permeability, adopting fractionated dosing regimens and implementing rigorous haematological monitoring, which are also available in preclinical or clinical settings. Looking forward, the PhoPEx-based BTR platform operates through a direct binding-induced mechanism that transcends the cellular uptake limitations of conventional approaches. Thus, the BTR strategy provides a complementary framework for targeting antigens that are not efficiently addressed by ITR, expanding the scope of conjugate therapies.

Synthesis and isolation of all related compounds

All available compounds were purchased from commercial suppliers around the world. All solvents for synthesis were purchased from local suppliers, and solvents of high-performance liquid chromatography (HPLC)-grade were purchased from Fisher Scientific. The synthetic routes and chemicals used for all related compounds are shown in Supplementary Information section 2 (Core PhoPEx structure was prepared following a reported method⁴⁸). Both UPLC-mass spectrometry and high-resolution mass spectrometry were used to assess the quality of all compounds and are provided in Supplementary Information section S6.

Proteins and antibodies

FAP (C14G) were purchased from Novoprotein. DPPIV (HY-P70017), PREP (HY-P703584), ALB (HY-P1956A) and ALP (HY-P2818) were purchased from MCE. CTSB (C6286) were purchased from Sigma. PD-L1 (PD1-H5229 and PD1-H82E5) were purchased from ACRObiosystems. Sibrotuzumabs were purchased from WuXi Biologics. Rabbit monoclonal anti-FAP antibody was purchased from Abcam, ab207178, clone EPR20021, 1:50; mouse monoclonal anti-CK (pan) 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.

Chemical reaction rate detection

The purified substrate was dissolved in DMF to prepare a stock solution, then diluted to a concentration gradient (2 mM to 0.2 mM). Each solution was analysed by UPLC-MS with ultraviolet detection; peak areas were integrated at λ_max, and the process was repeated in triplicate to generate a standard curve. For reactivity assays, each reaction mixture (300 μl) contained 1.5 mM substrate, 6 mM tetramethylguanidine and 22.5 mM Tyr-OMe-NAc (15 equiv.) at 37 °C. At timed intervals, aliquots (30 μl) were withdrawn, vortexed, centrifuged and quenched with 30 μl of 40 mM formic acid in acetonitrile. Quenched samples were analysed by UPLC-MS; concentrations were determined from the standard curve. Pseudo-first-order kinetics were fitted to obtain second-order rate constants. For the faster substrates (nos. 11 and 12), conditions were adjusted to 1 mM substrate, 1 mM tetramethylguanidine and 2 mM Tyr-OMe-NAc (2 equiv.), and second-order kinetics were directly simulated. Stability evaluation was performed in pH 9.5 PBS containing 1.5 mM substrate, using the same detection method as in the reactivity assay.

Detection of release experiments in vitro

For the experimental group, the molecular stock solution was diluted with PBS and then mixed with the protein solution at an appropriate ratio (protein:small molecule = 4:1 or 5:1). The mixture was incubated at 37 °C, and aliquots were withdrawn at designated time points. The concentration of the molecule was determined using a pre-established standard curve. For the control group, the molecular stock solution was diluted with PBS to the same concentration and incubated under identical conditions without protein. Each condition was performed in three independently prepared samples. Fluorescent molecules were detected using a microplate reader; molecules lacking strong characteristic absorption (for example, MMAE) were quantified by UPLC-MS.

Human lymph node staining and IHC

Human lymph node tissue excised by clinicians was cleaned to remove the surrounding adipose tissue and bisected with a surgical blade. One half was paraffin-embedded, sectioned into 4 µm slices and stained with haematoxylin and eosin for histology. For IHC, sections were deparaffinized in xylene, rehydrated through graded ethanol and subjected to antigen retrieval by microwave treatment in citrate buffer (pH 6.0). The other half was immersed in a fluorescent molecule solution, agitated at 37 °C for 40 min, then washed three times with PBS (10 min at 37 °C with agitation for each wash). Finally, the tissue was imaged using a near-infrared fluorescence imaging system. The use of patients’ lymph nodes was approved by the Institutional Review Board of the Cancer Hospital, Chinese Academy of Medical Sciences, under approval no. 25/328–5274.

Procedures for radiolabelling and PET/SPECT-CT imaging

The radiolabelling and imaging were in accordance with previous literature⁴⁹. For radiolabelling, the reaction mixture was prepared in 0.2 M sodium acetate buffer (pH 4.0–4.5) containing the appropriate labelling molecule (10 nmol) and the corresponding radionuclide (¹⁷⁷Lu, ⁶⁸Ga or ⁸⁶Y). The mixture was heated at 90–95 °C for 10–15 min and then allowed to cool. To purify the mixture, the cooled reaction product was filtered through a pre-conditioned Sep-Pak Light C18 cartridge using deionized water, with the aim of removing unbound radionuclides. The radiolabelled compound was subsequently eluted with ethanol. This eluate was then diluted with saline for use in follow-up experiments. Where needed, ethanol was eliminated by nitrogen blowing using a Termovap sample concentrator before the dilution step. Evaluation of radiochemical yield was conducted using a radioactivity meter, and radiochemical purity was analysed by radio-HPLC. PET/SPECT-CT imaging for animals was performed on a Mediso nanoScan PET 122S system (Mediso) or InliView-3000B PET/SPECT/CT imaging system (Novel Medical) with mice injected with compounds through the tail vein. Mice were anaesthetized with isoflurane in oxygen 10 min before each imaging time point. Standard data acquisition and image reconstruction of the PET or SPECT data were performed. Data analysis was performed on Interview Fusion software (v.3.09.008.0000) and NMSoft-AIWS software (v.1.8).

Tumour xenograft construction and treatment experiments

For CDX models targeting FAP, HT-1080 or HT-1080-FAP cells (5 × 10⁶ in PBS) were implanted subcutaneously into the right flank of 6–8-week-old female NU/NU nude mice, whereas MC38-FAP cells (5 × 10⁵ in PBS) were implanted into 4–6-week-old female C57BL/6 mice. For CDX models targeting PD-L1, HT-1080 cells (5 × 10⁶ in PBS) were implanted into 6–8-week-old female BNDG mice.

For all PDX models, BNDG mice aged 6–8 weeks were used, with the sex matched to that of the patient from whom the tumour specimen was obtained. Primary patient tumour tissue was directly implanted subcutaneously to generate the P1 generation. Once the P1 tumour had grown to 1,000–1,500 mm³, it was harvested and cut into fragments of approximately 3 mm in diameter, which were then implanted by puncture into the forelimb axilla of recipient mice to establish the P2 generation. Subsequent passages were performed in a similar manner. The final generation used for treatment was subjected to PET imaging and IHC to evaluate target expression. The use of patients’ tissues for PDX modelling was approved by the Institutional Review Board of the Cancer Hospital, Chinese Academy of Medical Sciences, under approval no. 25/328-5274, and by the Institutional Review Board of Beijing Cancer Hospital, under approval no. 2026KT67.

All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Peking University, under approval no. CCME-LiuZB-2, and by the Institutional Animal Care and Use Committee of Chinese Institute for Brain Research, Beijing, under approval no. CIBR-IACUC-071. The tumour volume was computed using the formula: volume = (length × width²)/2. In none of the experiments did the tumour burden surpass 10% of mouse weight and 25% weight loss compared with the original weight (according to the limits defined by the IACUC protocol).

Cell culture

The 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 (WuXi AppTec) were cultured in minimum essential medium containing 10% FBS, 1% antibiotic–antimycotic. The U87MG-FAP and U87MG-Vector (both from Pyrotech Biotechnology) were cultured in minimum essential medium containing 10% FBS, 1% antibiotic–antimycotic and 2 μg ml⁻¹ puromycin. The MC38-FAP cells (WuXi AppTec) were cultured in Dulbecco’s modified Eagle medium containing 10% FBS, 1% antibiotic–antimycotic and 3 μg ml⁻¹ puromycin. All cells were regularly tested for mycoplasma contamination and cultured or incubated (as in cell-based assays) in a 5% CO₂ incubator at 37 °C.

For the fluorescent assay, cells were seeded in eight-well confocal dishes and incubated with different compounds (FAPI-PhoPEx-MeRho at 10 μM for 24 h, compound 3 and FAPI-VC-MeRho at 2 μM). Fluorescence was observed at room temperature using a Nikon A1R-si Laser scanning confocal microscope. The fluorescence intensity was measured using ImageJ.

Half-maximal inhibitory concentration

Initially, two types of cells were seeded into a 96-well plate at a density of 5,000 cells per well and cultured in an incubator maintained at 37 °C for 12 h. A series of drug solutions with concentration gradients was then prepared using complete cell culture medium. After aspirating the original medium, the drug solutions were added for treatment. Following 6 h (compound 5, mF substitution) to 12 h (compound 1, no substitution) of incubation, the drug solutions were removed, and the cells were gently washed with PBS. Fresh drug-free complete medium was then added, and the cells were cultured for an additional 24 h. Finally, cell viability was assessed using the CCK-8 reagent.

Biological distribution experiment

The biological distribution experiments of each compound were evaluated by four groups of mice, each group containing 6 mice, which were euthanized at 2, 6, 24, 48, 72, and 120 h after injection for tissue collection. After tail vein administration of the drug, blood, liver and tumour tissues were collected and weighed. RIPA lysis buffer and PMSF were added, and the tissues were homogenized at 4 °C for 30 min (for about 500–900 mg tissue: 1 ml RIPA buffer containing 100 μl of 100 mM PMSF per ml of buffer). After lysis, 2 ml RIPA buffer, 2 ml methanol and 1 ml acetonitrile were added. The sample was sonicated for 10 min and centrifuged at 4,000 rpm for 10 min. A 5 ml aliquot of 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. Finally, MMAE was quantified by UPLC-MS based on mass spectrometry signal integration (Supplementary Fig. 9). The AUC for each group (6 mice) was calculated based on the payload concentrations determined by the aforementioned assays at the six indicated time points.

Cryo-EM experiment

For single-particle cryo-EM analysis, FAP protein at a concentration of 0.4 mg ml⁻¹ was incubated with a 20-fold molar excess of enantiomerically pure FAPI-oMe-MMAE for 2 h at 4 °C. The resulting complex was then prepared for cryo-EM using graphene-oxide-coated grids. Data collection was performed, followed by single-particle data processing, which ultimately led to three-dimensional reconstruction and model building (Supplementary Tables 1 and 2 and Supplementary Fig. 22).

Surface plasmon resonance experiment

Surface plasmon resonance (SPR) experiments were performed on a Biacore 8K+ (Cytiva) using a Series S Sensor Chip SA (Cytiva, 29104992). Biotinylated PD-L1 was captured onto the chip by streptavidin–biotin interaction at a flow rate of 30 μl min⁻¹. Analytes were then injected in a single-cycle kinetics at six consecutive increasing concentrations (5.12–500 nM, approximately 2.5-fold serial dilution). Injection parameters consisted of a constant flow rate of 30 μl min⁻¹, 150 s association and 600 s dissociation per concentration. Before each analyte injection, a buffer blank was injected under identical conditions. The resultant binding curves were fitted to a 1:1 binding model using Biacore Evaluation Software for each dataset. Each analyte was tested in triplicate.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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Author notes

1. These authors contributed equally: Zihao Wen, Mengxin Xu, Zijun Yan, Ziren Kong

Authors and Affiliations

1. Beijing National Laboratory for Molecular Sciences, Cross-disciplinary Center for f-Elements (CCFE), Radiochemistry and Radiation Chemistry Key Laboratory of Fundamental Science, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, China

   Zihao Wen, Zijun Yan, Junyi Chen, Yuedan Zheng, Zichen Gu & Zhibo Liu

2. Changping Laboratory, Beijing, China

   Mengxin Xu, Ziren Kong, Pei Liu, Xi-Yang Cui, Tianyi Cen, Xinwei Li, Changlun Wang & Zhibo Liu

3. Department of Head and Neck Surgery, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

   Ziren Kong

4. Pyrotech (Beijing) Biotechnology, Beijing, China

   Yupeng Wang

5. Key Laboratory of Carcinogenesis and Translational Research of Ministry of Education, Key Laboratory for Research and Evaluation of Radiopharmaceuticals of National Medical Products Administration, Department of Nuclear Medicine, Peking University Cancer Hospital and Institute, Beijing, China

   Yanzhao Liu & Zhibo Liu

6. Key Laboratory of Carcinogenesis and Translational Research of Ministry of Education, Department of Hepatopancreatobiliary Surgery, Peking University Cancer Hospital and Institute, Beijing, China

   Da Xu

7. Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

   Qiang Xu

8. Department of Nuclear Medicine, State Key Laboratory of Common Mechanism Research for Major Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

   Yaping Luo

9. Peking University–Tsinghua University Center for Life Sciences, Peking University, Beijing, China

   Zhibo Liu

10. Peking University Chengdu Academy for Advanced Interdisciplinary Biotechnologies, Chengdu, China

    Zhibo Liu

11. Laboratory for Synthetic Chemistry and Chemical Biology Limited, Hong Kong Science Park, Hong Kong, China

    Zhibo Liu

Contributions

Z.L. conceived the study. Z.W., assisted by Z.Y., Y. Liu, Y.Z., X.-Y.C. and Z.G., performed material synthesis, characterization and chemical analysis. Z.W., assisted by M.X. and J.C., performed radiosynthesis, PET imaging and data analysis. Z.W., assisted by M.X. and Y.W., performed cell studies. Z.W., assisted by Z.Y., performed the cryo-EM experiments. Z.W., assisted by Z.K., performed the lymph-node detection experiments. P.L. performed the dynamic calculations. Z.W., assisted by M.X., Z.Y., T.C., X.L. and C.W., performed the CDX therapeutic experiments. Z.W., assisted by M.X., Z.Y., D.X., Y. Luo and Q.X., performed the PDX therapeutic experiments. Z.W., assisted by M.X., performed the SPR experiments. Z.W. and Z.L. analysed the data. Z.W. and Z.L. wrote the manuscript with input from all authors. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Zhibo Liu.

Competing interests

Z.L., Z.W. and X.-Y.C. are co-inventors on a relevant patent application (PCT/CN2025/075553) filed by Peking University. Z.L. is a co-founder of and scientific advisor for BoomRay Pharmaceuticals. The remaining authors declare no competing interests.

Peer review information

Nature thanks Li Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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