Bioengineered ferritin-based lysosome-targeting chimera platform for tumor-targeted therapy

Domain: chemistry
Published: 2026-03-09
Editorial outcome: major_revision

Abstract
Lysosome-targeting chimeras (LYTACs) hold therapeutic potential by degrading pathogenesis-associated proteins. However, current LYTAC systems often require considerable effort for case-by-case construction and are devoid of a convenient and efficient modular platform. Here, we develop a modular LYTAC platform based on human heavy chain ferritin (HFn), leveraging its peptide-display function and TfR1-mediated lysosomal endocytosis. This system comprises a bioengineered HFn scaffold with enhanced TfR1 affinity and target-specific affibodies conjugated to the HFn via SpyTag-SpyCatcher system. Using this approach, HFn-LYTACs efficiently degrade epidermal growth factor receptor, epidermal growth factor receptor-2 and programmed death-ligand 1. Mechanistic studies indicate that the HFn-LYTAC platform mediates the degradation of POI via two distinct mechanisms: a TfR1-dependent endocytic pathway as well as the nanoparticle size and multivalent ligand effect of HFn-LYTAC. In vivo, HFn-LYTACs inhibit tumor growth with favorable safety. Therefore, the modular HFn-LYTAC platform represents a versatile, efficient, and promising strategy for tumor-targeted therapy.

Introduction
Targeted protein degradation (TPD) is becoming a promising therapeutic strategy. In contrast to the "occupancy-driven" pharmacological paradigm of traditional blocking drugs, TPDs employ the intrinsic protein degradation pathway of cells to achieve the degradation of disease-causing proteins through simple binding for therapeutic purposes1-5. Therefore, TPD technology is anticipated to facilitate the identification of "undruggable" molecules that are challenging to use with conventional small-molecule drugs2,6.

There are two primary categories of protein degradation pathways utilized: proteasome-dependent TPD and lysosomal-dependent TPD. Proteasome-dependent TPDs, such as proteolysis-targeting chimeras (PROTACs)7, molecular glues8 and proteolysis-targeting antibodies (PROTABs)9, recruit E3 ligases to promote the ubiquitination of target proteins for degradation via the ubiquitin–proteasome system. These degraders efficiently degrade pathogenic proteins, and many are being tested in clinical trials10. However, the reliance on specific E3 ligase subunits not only limits the application of these degraders to particular cell types but also may lead to resistance in cancer cells following chronic treatment11.

Recently, the emergence of lysosome-dependent TPD agents, including lysosome-targeting chimeras (LYTACs)12, autophagy-targeting chimeras (AUTACs)13 and autophagosome-tethering compounds (ATTECs)14, has further expanded the range of potential TPD targets by eliminating proteasome dependence. AUTACs and ATTECs facilitate the degradation of intracellular proteins or even damaged organelles via the autophagy pathway. LYTACs employ lysosomal-trafficking receptors to facilitate the degradation of both membrane proteins and extracellular proteins. Considering that the majority of drug targets are located at the cell surface or secreted, LYTACs are attracting increasing interest from scientists. A variety of antibody-based LYTACs12,15-18 or principally analogous LYTAC degraders19,20 have been developed. However, the preparation of antibody-based LYTACs generally requires sophisticated antibody engineering methodologies. Notably, LYTACs based on DNA aptamers or peptides have been developed and are capable of POI degradation. However, these degraders have limitations in terms of tissue persistence and require additional delivery systems for assistance21. Furthermore, the binding stability of short peptides is limited, and their performance needs to be enhanced through additional chemical modifications22. These factors restrict the construction and development of modular LYTAC systems. Therefore, it is imperative to develop a modular and easily constructed LYTAC platform.

Ferritin is a nanocage-like protein comprising 24 subunits and is widely expressed in humans. Human heavy chain ferritin (HFn) has been developed as a peptide display platform and nanodrug carrier for nanovaccine development and tumor therapy. HFn is capable of multivalent binding to the transferrin receptor (TfR1) and is subsequently endocytosed into lysosomes23. TfR1 is widely overexpressed in tumor cells and has been identified as a lysosomal-targeting receptor for developing TPD agents20,21,24,25. However, the binding of HFn to TfR1 and its subsequent internalization exhibit a threshold effect26 (Supplementary Fig. 4e, f), with HFn preferentially endocytosed by cells highly expressing TfR1, thereby ensuring the safety profile of HFn-LYTAC. Therefore, HFn is an ideal candidate for the construction of LYTACs.

Here, we designed a modular HFn-based LYTAC (HFn-LYTAC) platform for tumor therapy (Fig. 1). First, through structure-guided bioengineering, a HFn mutant with high affinity for TfR1 was screened, which serves as a universal modular nanoscaffold for LYTAC construction. With the SpyTag-SpyCatcher system, HFn-LYTACs were constructed via simple incubation to conjugate affibodies of POI to the surface of the HFn nanoscaffold. To validate the efficacy of the platform, we engineered HFn-LYTACs against three targets, including epidermal growth factor receptor (EGFR), epidermal growth factor receptor-2 (HER2), and programmed death-ligand 1 (PD-L1). All HFn-LYTACs effectively degraded the corresponding proteins, with degradation efficiencies exceeding 60%, and some even approaching 80% (determined via Western blotting). Mechanistic investigations indicate that HFn-LYTAC mediates the degradation of POI through two distinct mechanisms. The first mechanism involves the formation of a ternary complex among HFn-LYTAC, TfR1 and POI, leading to subsequent endocytosis and degradation of POI in lysosomes. The second mechanism involves the cellular trafficking of nanoparticles (HFn nanocages), as well as the multivalent interaction between HFn-LYTAC and POI, which promotes direct endocytosis and degradation. The second mechanism is independent of TfR1. Therapeutic experiments conducted in subcutaneous xenograft mice indicated that HFn-LYTAC effectively inhibited tumor progression and maintained favorable safety. In addition, our previous work demonstrated that HFn is able to traverse the blood‒brain barrier (BBB)23, which enables HFn-LYTAC to be a viable option in the treatment of glioblastoma. Therefore, the HFn-LYTAC platform is a powerful TPD tool for tumor membrane protein degradation. As a modular platform, the flexibility and superior safety of LYTAC facilitate its broader application in the medical field.

Results
Construction of HFn-LYTACs for EGFR degradation

First, the affinity of HFn for TfR1 was increased to acquire a modular scaffold for LYTAC construction. The complex structure of the HFn and TfR1 ectodomain from the cryoelectron microscopy data indicates that the N-terminal and BC loop regions of HFn interact with the apical domain of TfR127 (Fig. 2a, b). The structural reorganization induced by complex formation is constrained to the amino acid side chains in direct contact, leading to a diminished affinity between the two. Therefore, a series of HFn mutants were initially designed to increase their affinity for TfR1. The affinity between HFn and the TfR1 ectodomain obtained via surface plasmon resonance (SPR) was in the range of 10–7 M (Supplementary Table 1), which was consistent with the affinity between HFn and TfR1 on the cell surface28. Among the HFn residues involved in common contacts (T5, Q14, D15, E17, A18, A19, N21, R22, N25, R79, F81, Q83, K86, K87, and E116) with TfR1, D15, F81, Q83, N109, K119 and D123 were selected for mutation to increase the affinity for TfR1. These residues, located in the vicinity of the interaction region (Fig. 2a, b), had no or limited interactions with the TfR1 molecule. Therefore, they were selected for mutation to form additional hydrogen bonds between HFn and TfR1 (F81Y, N109R, D123E, and K119R) (Fig. 2c, Supplementary Fig. 1a) or strengthen the van der Waals forces within the hydrophobic microenvironment (D15L and Q83H) (Supplementary Fig. 1a). The wtHFn and HFn mutants were expressed in Escherichia coli BL21 (DE3) and purified by hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC). The affinity of the wtHFn and HFn mutants for TfR1 was subsequently determined via SPR (Supplementary Table 1). Among the mutants, variants HFn1 (D15L), HFn2 (F81Y) and HFn4 (N109R) exhibited enhanced affinity for TfR1, demonstrating a 2- to 5-fold increase compared with wtHFn. HFn6, an N109R/K119R mutant that presented a further increased affinity for TfR1 in the SPR experiment, exhibited a significantly reduced dissociation rate (koff), indicating a longer residence time on TfR1 (Fig. 2c).

Consequently, the HFn6 mutant was selected as the nanoscaffold for the development of HFn-LYTAC. The monomer of wtHFn comprises four α-helices (A-D helices) and one short E helix. The E-helix is located at the C-terminus and, following the self-assembly of a 24-mer HFn, is oriented toward the interior of the nanocage29. To mitigate the impact of the conjugated affibodies on the binding of HFn and TfR1, the SpyTag sequence was fused to the C-terminus of the HFn truncate with the E helix removed (Fig. 2d). The SPR results indicated that the fusion of the SpyTag sequence at the C-terminus of the HFn6-truncated variant (HFn6SpyTag) had a relatively modest effect on its affinity for TfR1 (Supplementary Table 1). The wtHFn truncated variant fused with the SpyTag sequence (wtHFnSpyTag) was also designed as a control.

Next, to demonstrate proof of concept, EGFR was chosen as a target protein to verify whether the HFn-LYTAC platform mediates membrane protein degradation. Affibodies against EGFR were obtained from previous studies30 and termed ZEGFR. A linker followed by the SpyCatcher domain was introduced to the C-terminus of ZEGFR for the construction of HFn-LYTAC, and a His tag was C-terminally fused to the SpyCatcher domain for purification (Fig. 2d).

Since HFn is a nanocage-like protein comprising 24 subunits, a single HFn nanocage is theoretically capable of coupling 24 binding affibodies. Consequently, we examined the influence of the conjugation ratio on EGFR degradation to develop HFn-LYTAC with optimal degradation efficiency (Fig. 2e). Specifically, HFn6SpyTag and ZEGFR-SpyCatcher were incubated at various molar ratios (ZEGFR-SpyCatcher: HFn6SpyTag ranging from 3:1 to 24:1) at room temperature for conjugation (HFn6-ZEGFR). The conjugation reaction was monitored by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) (Supplementary Fig. 1b). The ZEGFR-conjugated HFn6 monomer has a relatively high molecular weight, corresponding to the band with a molecular weight of approximately 40 kDa, as observed in the SDS‒PAGE image. Notably, no ZEGFR-SpyCatcher bands were observed at ZEGFR-SpyCatcher to HFn6SpyTag incubation ratios of less than 12:1, indicating complete conjugation of all the ZEGFRs to HFn6. As the incubation ratio increased, the intensity of the ZEGFR-SpyCatcher bands progressively intensified, suggesting a constrained additional coupling capacity of HFn6, potentially due to steric hindrance. Next, the EGFR degradation capacity of HFn6-ZEGFR at various incubation ratios was evaluated. U-87 MG cells, a glioblastoma cell line with high expression of both EGFR and TfR1, were incubated separately with different HFn6-ZEGFR samples for 24 hours, after which the surface levels of EGFR were quantified via flow cytometry. As the incubation ratio of ZEGFR-SpyCatcher and HFn6SpyTag increased, the clearance of surface EGFR on U-87 MG cells initially increased and subsequently decreased (Fig. 2f). HFn-LYTAC demonstrated optimal efficacy in surface EGFR clearance at ZEGFR : HFn6 incubation ratios of 12:1 and 15:1. This is because an increase in the number of ZEGFR molecules coupled to HFn enhances the chance of HFn-ZEGFR binding to EGFR on the cell surface, thereby improving its capacity to mediate clearance of surface EGFR. However, further elevation of the ZEGFR incubation ratio introduces steric hindrance, which could interfere with the interaction between HFn6 and TfR1. Moreover, at high ZEGFR incubation ratios, free ZEGFR in solution competitively inhibits HFn6-ZEGFR. Consequently, as the ZEGFR incubation ratio increases further, the clearance efficiency of HFn-ZEGFR toward surface EGFR gradually decreases. This specific conjugation ratio (ZEGFR : HFn6 = 12:1) was employed in subsequent experiments for EGFR degradation. We also fabricated ZEGFR-conjugated wtHFn (wtHFn-ZEGFR) at a molar ratio of 12:1 as a control. The constructed HFn6-ZEGFR and wtHFn-ZEGFR were characterized via transmission electron microscopy (TEM) and dynamic light scattering (DLS). Both wtHFn and HFn6 exhibited a uniform spherical morphology, whereas HFn6-ZEGFR and wtHFn-ZEGFR displayed spherical shapes with irregular protrusions (Supplementary Fig. 1f, g, h). The hydrodynamic sizes of both HFn6-ZEGFR and wtHFn-ZEGFR were greater than those of the ZEGFR-unconjugated form. These data indicate that ZEGFR was successfully conjugated to the surface of the HFn nanoscaffold.

Following the identification of the optimal coupling ratio, the HFn6-ZEGFR-mediated degradation of EGFR in different cell lines was thoroughly evaluated (Fig. 3a). U-87 MG and MDA-MB-231 cells were treated with HFn6-ZEGFR for 24 h, and the surface levels of EGFR were measured via flow cytometry with an orthogonal detection antibody. In U-87 MG and MDA-MB-231 cells, the surface EGFR levels were significantly reduced by an average of 61% and 75%, respectively (Fig. 3b, c). Equivalent doses of ZEGFR or ferritin (wtHFn and HFn6) resulted in only a minor reduction in cell surface EGFR levels (<15% in U-87 MG cells and <20% in MDA-MB-231 cells). However, wtHFn-ZEGFR also demonstrated comparable efficacy in the clearance of surface EGFR. Total EGFR degradation was further evaluated by Western blotting. In U-87 MG cells, treatment with HFn6-ZEGFR resulted in 62% degradation of total EGFR (Fig. 3d and Supplementary Fig. 2a), which aligns with observations from cell surface clearance studies. In contrast, wtHFn-ZEGFR induced only 35% degradation of total EGFR, which does not correspond with the findings of cell surface clearance. In MDA-MB-231 cells, HFn6-ZEGFR and wtHFn-ZEGFR caused 65% and 57% degradation of total EGFR, respectively. (Fig. 3e and Supplementary Fig. 2b). To investigate the reasons for the discrepancy between the results of cell surface EGFR clearance and total EGFR degradation in U-87 MG cells mediated by wtHFn-ZEGFR, confocal microscopy was used to visualize EGFR degradation (Fig. 3f, g and Supplementary Fig. 2n). After treatment with HFn6-ZEGFR or wtHFn-ZEGFR for 24 h, the cytoplasmic EGFR signal in the wtHFn-ZEGFR treatment group was 50% stronger than that in the HFn6-ZEGFR group (Supplementary Fig. 2o). However, the membrane EGFR signals in U-87 MG cells were reduced in both treatment groups (Supplementary Fig. 2p). Cytoplasmic proteins were isolated from U-87 MG cells treated with HFn-LYTAC using a membrane and cytosol protein extraction kit (Beyotime), and the expression levels of EGFR in the cytoplasm were analyzed by Western blotting. The results consistently exhibited higher EGFR levels in the wtHFn-ZEGFR treatment group compared to the HFn6-ZEGFR group (Supplementary Fig. 2q). These findings indicate that a portion of the EGFR on the cell membrane is internalized into the cell following its binding to wtHFn-ZEGFR but remains undegraded. This phenomenon will be further discussed in subsequent chapters. In addition, previous studies have reported that EGFR degradation results in reduced levels of p-AKT in its downstream signaling pathway31. This effect was confirmed following the treatment of U-87 MG cells with HFn6-ZEGFR. Owing to differences in total EGFR degradation capacity, the level of p-AKT in U-87 MG cells treated with wtHFn-ZEGFR was slightly greater than that in those treated with HFn6-ZEGFR (Supplementary Fig. 3a). Therefore, there is also a difference in their ability to inhibit cell proliferation. The IC50 value of HFn6-ZEGFR in U-87 MG cells was 24.2 nM, which was less than half that of wtHFn-ZEGFR (57.26 nM). Equivalent doses of HFn6 or ZEGFR did not demonstrate cytotoxic effects.(Supplementary Fig. 3b, c).

Expanding the target scope of the HFn-LYTAC

To explore the capacity of the HFn-LYTAC platform to degrade additional membrane targets, HER2 and PD-L1 were selected as the target proteins. The affibodies against HER2 and PD-L1 were derived from previous studies32,33 and termed PEPHER2 and FN3PD-L1, respectively. The protein sequence design of PEPHER2-Spycatcher and FN3PD-L1-Spycatcher for the construction of HFn-LYTAC was similar to that of ZEGFR-Spycatcher. The conjugation ratio of PEPHER2 and FN3PD-L1 to HFn6 was subsequently optimized. Similarly, owing to steric hindrance, HFn6 cannot conjugate with 24 affibodies (Supplementary Fig. 1c, d). The clearance of surface HER2 or PD-L1 by different incubation ratios of HFn-LYTAC exhibited a similar pattern to that of HFn6-ZEGFR (Fig. 2g, h). HFn-LYTAC demonstrated optimal surface HER2 clearance efficacy at a PEPHER2 to HFn6 conjugation ratio of 12:1. For PD-L1 target degradation, The optimal clearance efficiency of surface PD-L1 was achieved when FN3PD-L1 was conjugated with HFn6 at a ratio of 6:1. This may be because FN3PD-L1 has a relatively high molecular weight, thereby leading to enhanced steric hindrance effects. HFn6-PEPHER2 with a conjugation ratio of 12:1 and HFn6-FN3PD-L1 with a conjugation ratio of 6:1 were utilized for the following experiments. HFn6-PEPHER2 and HFn6-FN3PD-L1 were characterized via DLS and TEM (Supplementary Fig. 1f, h). Both HFn6-PEPHER2 and HFn6-FN3PD-L1 exhibited good dispersibility. Notably, HFn6-FN3PD-L1 has a relatively large hydrodynamic diameter, which is attributed to the largest molecular weight of the FN3PD-L1 ligand (Supplementary Fig. 1e). This observation suggests that, compared with other ligands, FN3PD-L1 has a more pronounced steric hindrance effect.

Furthermore, the HFn6-LYTAC-mediated degradation of HER2 and PD-L1 in different cell lines was evaluated (Fig. 3h, o). To substantiate the rationale for constructing the HFn6 mutant, we synthesized wtHFn-LYTAC as a control. For HER2 target degradation, SKBR3 and NCI-N87 cells were treated with HFn6-PEPHER2 for 24 h, after which the surface and total HER2 protein levels were quantified via flow cytometry and Western blotting, respectively. In SKBR3 and NCI-N87 cells, the levels of membrane HER2 decreased by more than 70%. (Fig. 3i, j), whereas the average degradation rates of total HER2 were 77% for SKBR3 cells and 63% for NCI-N87 cells (Fig. 3k, l and Supplementary Fig. 2c, d). Treatment with equivalent doses of PEPHER2 did not lead to a reduction in HER2 levels on the cell membrane or in the whole cell. However, no significant difference in HER2 degradation was observed between HFn6-PEPHER2 and wtHFn-PEPHER2, regardless of whether membrane HER2 or total HER2 was assessed. Confocal microscopy visualization of HER2 following treatment with HFn6-PEPHER2 or wtHFn-PEPHER2 revealed reduced HER2 signals, which was consistent with the findings from flow cytometry and Western blotting (Fig. 3m, n). The cytotoxic effects of HFn6-PEPHER2 and wtHFn-PEPHER2 were evaluated in SKBR3 cells. Neither HFn6 nor PEPHER2 demonstrated cytotoxicity, but both HFn6-PEPHER2 and wtHFn-PEPHER2 effectively inhibited the proliferation of SKBR3 cells. Given that they have the same HER2 degradation capacity, their IC50 values for SKBR3 cells are similar (Supplementary Fig. 3d, e). The IC50 of HFn6-PEPHER2 was 6.78 nM, and that of wtHFn-PEPHER2 was 9.29 nM. Moreover, HT1080 and MDA-MB-231 cells were used to evaluate the degradation of PD-L1 mediated by HFn6-FN3PD-L1 and wtHFn-FN3PD-L1. The average reduction rate of surface PD-L1 mediated by HFn6-FN3PD-L1 in both cell lines was approximately 50%, which was significantly higher than the 35% reduction rate mediated by wtHFn-FN3PD-L1 (Fig. 3p, q). The average degradation rates of total PD-L1 mediated by HFn6-FN3PD-L1 were greater than 65% for HT1080 cells and 60% for MDA-MB-231 cells, which were greater than those mediated by wtHFn-FN3PD-L1 (Fig. 3r, s and Supplementary Fig. 2e, f). Confocal microscopy visualization of PD-L1 following treatment with HFn6-FN3PD-L1 or wtHFn-FN3PD-L1 was consistent with the flow cytometry and Western blotting results (Fig. 3t, u). The degradation of HER2 and PD-L1 yielded distinct conclusions, whereas the degradation of PD-L1 mediated by HFn6-FN3PD-L1 was markedly greater than that mediated by wtHFn-FN3PD-L1, whereas there was no significant difference in the degradation of HER2 mediated by HFn6-PEPHER2 and wtHFn-PEPHER2. This phenomenon will be further investigated in mechanistic studies. Notably, the HFn-LYTACs developed in this study for three POIs did not lead to a decrease in total intracellular TfR1 levels (Supplementary Fig. 2g-l), which was attributed to the ability of TfR1 to be recycled back to the cell membrane34. Overall, these results for three different targets demonstrate the universality of the HFn-LYTAC platform for degrading cell-surface proteins (Supplementary Fig. 2m).

Principles and mechanisms of HFn-LYTAC-mediated target degradation

HER2 was selected as the model target to investigate the principles and mechanisms underlying protein degradation mediated by HFn-LYTAC. HFn6-PEPHER2 mediated over 50% degradation of HER2 following a 24-hour treatment. Within 24–48 hours after treatment with HFn6-PEPHER2, the total HER2 level in the SKBR3 cells remained low (Fig. 4a, Supplementary Fig. 4a). The degradation of HER2 in SKBR3 cells was found to be concentration dependent, with the concentration of HFn6-PEPHER2 required to achieve 50% degradation of total HER2 calculated to be 102.9 nM (Fig. 4b, Supplementary Fig. 4b). The HER2 level was restored to approximately 70% at 48 hours after HFn6-PEPHER2 washout, suggesting that the degradation of HER2 by HFn6-PEPHER2 is reversible (Supplementary Fig. 4c). Next, confocal microscopy was employed to visualize the process by which HFn6-PEPHER2 mediates HER2 internalization (Fig. 4c, Supplementary Fig. 5a). Following a 2-hour treatment with HFn6-PEPHER2, HER2 signaling was observed in SKBR3 cells and colocalized with HFn6-PEPHER2, indicating that HFn6-PEPHER2 associated with HER2 and facilitated its internalization. A 4-hour treatment with HFn6-PEPHER2 resulted in significant HER2 internalization. Notably, the internalized HER2 signals colocalized with lysosome-associated membrane protein 1 (LAMP1), suggesting that HER2 is internalized into lysosomes for degradation (Supplementary Fig. 5b).

To investigate the role of TfR1 in the degradation of target proteins mediated by HFn-LYTAC, a TfR1-knockdown SKBR3 cell line was established (Supplementary Fig. 4d). After TfR1 knockdown, the reduction of surface HER2 induced by HFn6-PEPHER2 was significantly inhibited. However, in cells transfected with nontargeting shRNA, the reduction of surface HER2 remained unaffected. (Fig. 4d). Moreover, coincubation with HFn6 at fivefold greater concentrations also significantly inhibited the degradation of HER2 mediated by HFn6-PEPHER2 (Fig. 4e). These data indicate that HFn-LYTAC is dependent on TfR1. However, the knockdown of TfR1 or competitive inhibition of HFn6 did not completely abolish the degradation effect of HFn-LYTAC, as HFn6-PEPHER2 still degraded 30–40% of HER2. Recent studies have indicated that the cellular trafficking of nanoparticles can be leveraged to design TPD agents35. HFn is a nanocage-like protein with an outer diameter of 12 nm. Consequently, we propose that the cellular trafficking of nanoparticles (HFn nanocages) facilitates the internalization and subsequent degradation of HER2. To test this hypothesis, ferritin derived from Pyrococcus furiosus (pFn), which does not interact with human TfR1 but possesses a similar nanostructure, was chosen for conjugation with PEPHER2. As anticipated, treatment of SKBR3 cells with pFn-PEPHER2 for 24 h led to less than 40% degradation of total HER2, which was more than 20% lower than that of HFn6-PEPHER2 and wtHFn-PEPHER2 (Fig. 4g). Furthermore, pFn-ZEGFR and pFn-FN3PD-L1 were constructed to evaluate the generalizability of the hypothesis. Treatment of U-87 MG cells with pFn-ZEGFR for 24 h resulted in an average 40% degradation of total EGFR (Fig. 4h), and pFn-FN3PD-L1 mediated the degradation of an average of 30% of total PD-L1 in HT1080 cells (Fig. 4i). Furthermore, upon reviewing the experiments regarding the conjugation ratio between the affibody and HFn6, the surface PD-L1 clearance efficiency achieved with a 1:6 molar ratio of HFn6 to affibody was significantly higher than that observed with a 1:3 ratio. Consequently, the multivalent binding of HFn-LYTAC with POI can also enhance its degradation effect. We believe that the nanoparticle size effects of HFn-LYTAC (cellular trafficking of nanoparticles) and the multivalent binding to POI form a synergistic effect, jointly mediating the degradation of POI. Collectively, these findings demonstrated that HFn-LYTAC mediated the degradation of the POI through two complementary mechanisms. The first involves simultaneous binding to TfR1 and POI, thereby facilitating endocytosis of the POI via TfR1-dependent pathways, resulting in its subsequent degradation in lysosomes. The second mechanism involves cellular trafficking of the nanoparticles and multivalent interactions between HFn-LYTAC and POI, which promote direct endocytosis and degradation. The advantage of the second mechanism lies in its independence from lysosomal endocytosis receptors, thereby broadening the application scope of HFn-LYTAC. However, its degradation efficiency remains limited, which may partially restrict therapeutic efficacy, but contributes to ensuring biological safety. The degradation efficiency of the second mechanism may be influenced by the size of the nanoparticle and the quantity of conjugated affibody molecules.

Interestingly, the EGFR degradation efficiency mediated by wtHFn-ZEGFR was comparable to that mediated by pFn-ZEGFR, suggesting that wtHFn-ZEGFR relies primarily on the second mechanism for EGFR degradation. To investigate the factors affecting the two degradation mechanisms, the affinity of all HFn-LYTACs for POI and TfR1 was determined via SPR (Supplementary Table 2). Owing to the multivalent binding effect, the affinity of HFn-LYTAC for the corresponding target (POI) was greater than two orders of magnitude greater than that of the free affibody. However, the conjugation of ZEGFR and FN3PD-L1 resulted in a diminished affinity of HFn6 for TfR1. This was especially the case for the HFn-LYTAC constructed on the basis of wtHFn, where its affinity for TfR1 was completely abrogated. A diminished affinity for TfR1 results in a compromised TfR1-dependent degradation mechanism, consequently reducing the efficiency of POI degradation. The conjugation of PEPHER2 had minimal influence on the binding affinity of HFn to TfR1, thereby allowing the TfR1-dependent degradation mechanism to remain functional. These data also indicate that when the affinity of HFn-LYTAC for TfR1 reaches 10–6 M, further increasing the affinity of HFn for TfR1 has a negligible effect on enhancing the degradation efficiency of HFn-LYTAC. Nevertheless, given the potential inhibitory effect of affibody conjugation on the affinity of HFn for TfR1, it is essential to design HFn mutants with enhanced affinity for TfR1.

To further investigate the contribution of the TfR1-dependent degradation mechanism to HFn6-LYTAC-mediated POI degradation, confocal microscopy was employed to visualize and analyze the subcellular distribution of TfR1 during HER2 internalization mediated by HFn6-PEPHER2. Following a 2- to 4-hour treatment of SKBR3 cells with HFn6-PEPHER2, both HER2 and TfR1 internalization were clearly observed, and internalized HER2 and TfR1 were significantly colocalized (Fig. 4f and Supplementary Fig. 5c, d). These findings indicate that the TfR1-dependent degradation mechanism plays a dominant role in the HER2 degradation process mediated by HFn6-PEPHER2. However, when competitive inhibition was achieved with HFn6 or when pFn was used to prevent binding to TfR1, HFn-LYTAC retained a weak ability to degrade POI (Fig. 4e, g-i). Therefore, the two mechanisms through which HFn-LYTAC mediates POI degradation constitute a dynamic process: the TfR1-dependent degradation mechanism plays a dominant role, and multivalent interaction-driven endocytosis is initiated only when HFn6 binding to TfR1 is blocked.

To investigate the internalization pathway of HFn6-LYTACS, chlorpromazine (CPZ), methyl-β-cyclodextrin (M-β-CD) or amiloride were utilized to inhibit clathrin- and caveolin-dependent endocytosis or micropinocytosis, respectively. In the CPZ pretreatment group, total HER2 degradation mediated by HFn6-PEPHER2 was significantly inhibited, whereas pretreatment with M-β-CD and amiloride had no effect on HER2 degradation. These findings indicate that HFn6-PEPHER2 is internalized into the cytoplasm via the clathrin-dependent endocytosis pathway (Fig. 4j). Pretreatment of SKBR3 cells with chloroquine inhibited the HFn6-PEPHER2-mediated degradation of HER2, confirming that the degradation promoted by LYTACs is dependent on lysosomes (Fig. 4k). The endocytic pathways involved in the multivalent binding-mediated endocytic degradation mechanism have also been explored. Inhibition of the clathrin-mediated endocytosis pathway by CPZ led to suppression of HER2 degradation mediated by pFn-PEPHER2, whereas pretreatment with M-β-CD and amiloride had no effect on HER2 degradation (Fig. 4l). Similarly, inhibition of lysosomal activity by chloroquine resulted in suppressed HER2 degradation mediated by HFn6-PEPHER2 (Fig. 4m).

In vivo antitumor efficacy and safety of the HFn-LYTAC platform

To evaluate the in vivo antitumor efficacy of HFn-LYTAC, HER2 was chosen as a model target, and the inhibitory effect of HFn6-PEPHER2 on the NCI-N87 xenograft tumor model was evaluated. The plasma half-life and tissue distribution of HFn6-PEPHER2 were first evaluated. Blood samples were collected at different time points following intravenous (i.v.) injection of Cy5.5-labeled HFn6-PEPHER2 or PEPHER2. Plasma was separated and the fluorescence intensity was detected. HFn6-PEPHER2 exhibited a prolonged circulation half-life of 131.8 min, which was more than tenfold longer than that of the PEPHER2 control group (9.3 min; Supplementary Fig. 7a). Given that the shedding of fluorescent groups could interfere with the accurate characterization of HFn6-PEPHER2, Western blotting was further utilized to directly evaluate its plasma half-life. The relative content of SpyCatcher in the plasma was detected and the plasma half-life of HFn6-PEPHER2 was determined to be 227 minutes (Fig. 5a). Tissue accumulation studies in the NCI-N87 xenograft mouse model demonstrated that 24 h after i.v. injection of HFn-PEPHER2, the average level of HFn6-PEPHER2 in tumors was over 5 times greater than that in the heart, spleen and lungs (Fig. 5b). The strong accumulation of HFn6-PEPHER2 in the liver and kidneys suggests that these organs are major sites of its metabolism. The low level of HFn6-PEPHER2 accumulation in the mouse brain indicates its capacity to cross the BBB. Given the nanoparticle properties of HFn6-PEPHER2, we investigated its uptake by monocytes and dendritic cells (Supplementary Fig. 6a). The uptake of HFn6 by SKBR3 cells was greater than that of HFn6-PEPHER2, a difference attributed to the dual-target binding mode of HFn6-PEPHER2. Compared with SKBR3 cells, DC2.4 cells had a slightly lower capacity for internalizing HFn6-PEPHER2. RAW 264.7 cells demonstrated robust endocytic activity toward both HFn6 and HFn6-PEPHER2, suggesting that a portion of HFn6-PEPHER2 may be taken up and cleared by monocytes following i.v. injection. Notably, the endocytosis of HFn6-PEPHER2 by RAW 264.7 cells and DC 2.4 cells did not significantly affect their proliferation (Supplementary Fig. 6b, c). Nevertheless, the multimerization of PEPHER2 displayed on HFn6 markedly enhances the binding affinity for HER2, enabling HFn6-PEPHER2 to rapidly engage HER2 after traversing the vasculature and reaching the tumor site, thereby facilitating detectable accumulation within the tumor (Fig. 5c). Compared with free PEPHER2, HFn6-PEPHER2 accumulated more persistently at the tumor site. This is attributed to the small molecular size of PEPHER2, which renders it susceptible to rapid clearance and degradation.

Next, the in vivo efficacy of HFn6-PEPHER2 in inhibiting tumor growth and its capacity to degrade POI were systematically evaluated (Fig. 5d). HFn6-PEPHER2 was administered at a dose of 30.6 mg/kg via the tail vein every 2 days. Owing to the large molecular weight of HFn-LYTACs (approximately 720 kDa on average), this dosage is equivalent to approximately 6 mg/kg of IgG (≈42 nmol/kg, on a molar basis of the entire HFn6-PEPHER2). After 7 administrations, tumor proliferation was significantly suppressed in the HFn6-PEPHER2 treatment group compared with the PBS control group (Fig. 5e, f). In contrast, equimolar doses of PEPHER2 and HFn6 exhibited no tumor suppressive activity. Two days after the final injection, the tumors were harvested, and HER2 levels in the tumor tissue were evaluated via flow cytometry and immunofluorescence. Significant HER2 degradation was observed in the HFn6-PEPHER2 treatment group (Fig. 5g, h). Furthermore, immunofluorescence analysis demonstrated that HFn6-PEPHER2 treatment did not alter the expression level of TfR1 in tumor tissues. (Fig. 5i).

Furthermore, the long-term safety of HFn-PEPHER2 after administration was also evaluated. The endotoxin level of purified protein was measured to not exceed 10 EU/mg, remaining within acceptable limits (Supplementary Fig. 1i). No weight loss was observed during the treatment process (Supplementary Fig. 7d). Histopathological analysis of the major organs in NCI-N87 xenograft mice after HFn6-PEPHER2 administration revealed that these organs retained typical physiological morphology, with no significant pathological abnormalities or lesions detected, including those in the liver, where HFn6-PEPHER2 was highly enriched (Fig. 5j). Prussian blue staining of liver tissue sections revealed that it does not significantly alter iron deposition levels in liver tissue, indicating that HFn6-PEPHER2 does not affect hemosiderin content (Supplementary Fig. 7c). Serum biochemical analysis indicated that HFn6-PEPHER2 administration did not cause liver or kidney toxicity in mice (Fig. 5k, l). Considering the physiological functions of ferritin and the role of TfR1 in erythropoiesis36,37, complete blood count (CBC) analysis was performed following treatment of the mice. No differences were observed among the PBS-, PEPHER2-, HFn6- or HFn6-PEPHER2-treated groups in terms of red blood cell (RBC) counts or hemoglobin (HGB) levels (Fig. 5m). No significant difference in reticulocyte (RET) content was observed in the peripheral blood of mice across the different treatment groups (Supplementary Fig. 7b).

HFn-LYTAC platform for the treatment of glioblastoma

More than 60% of glioblastomas overexpress EGFR, which promotes tumor growth, survival, angiogenesis, and invasion38. However, the presence of the BBB hinders the clinical efficacy of small molecule inhibitors and monoclonal antibodies that target EGFR39,40. Our previous work demonstrated that HFn can traverse the BBB23, making HFn-LYTAC a promising candidate for glioblastoma therapy. Consequently, the inhibitory effect of HFn6-ZEGFR on the U-87 MG orthotopic xenograft tumor mouse model was evaluated. To investigate the ability of HFn6-ZEGFR to cross the BBB, a transcytosis assay was performed using mouse brain endothelial cells. Both HFn6 and HFn6-ZEGFR effectively traverse the mouse BBB, and the transport rate is 3-fold greater than that of human light chain ferritin (LFn) (Fig. 6a, b). Moreover, an orthotopic xenograft mouse model of tumors was established using U-87 MG cells expressing luciferase. Fluorescence imaging of the brains of U-87 MG orthotopic xenograft mice demonstrated that HFn6-ZEGFR successfully traversed the mouse BBB and accumulated in glioblastoma. Although the establishment of orthotopic xenograft mice may partially compromise the integrity of the BBB, the accumulation of HFn6-ZEGFR in glioblastoma was more than twice that of ZEGFR 1 h after i.v. injection, suggesting that HFn6-ZEGFR possesses the ability to cross the BBB and specifically target brain tumors(Fig. 6c and Supplementary Fig. 7e). Next, the in vivo efficacy of HFn6-ZEGFR in inhibiting glioblastoma growth and its capacity to degrade POI were evaluated (Fig. 6d). HFn6-ZEGFR was intravenously injected at a dose of 10 mg/kg every two days. No weight loss was observed during the treatment process (Supplementary Fig. 7f). After seven administrations, tumor proliferation was significantly inhibited in the HFn6-ZEGFR treatment group compared with the PBS control group (Fig. 6e, f). In contrast, neither ZEGFR nor HFn6 demonstrated any tumor-suppressive activity. Two days after the final injection, the tumors were harvested for detection of EGFR degradation. Flow cytometry of tumor cells and immunofluorescence of tumor tissue revealed that EGFR was significantly degraded in the HFn6-ZEGFR treatment group (Fig. 6g, h). In the tumor tissue samples, the expression levels of TfR1 were not significantly different among the different treatment groups (Fig. 6h). In summary, these data indicate that the HFn-LYTAC platform has significant antitumor activity and good safety. Therefore, it is considered a promising drug for tumor treatment.

On the basis of the ability of HFn to target TfR1 and the advantages of peptide display platforms, we developed a modular targeted degradation platform capable of effectively degrading different membrane proteins on various tumor cell types. On the one hand, HFn-LYTAC facilitates the internalization of POI into lysosomes for degradation by concurrently recruiting TfR1 and POI. On the other hand, the cellular trafficking of the nanoparticles and the multivalent interaction of HFn-LYTAC with POI also promoted their internalization and degradation. In addition, enhancing the affinity of HFn for TfR1 facilitates the promotion of the TfR1-dependent degradation pathway, thereby increasing the efficiency of HFn-LYTAC in degrading POI. Considering the extensive high expression of TfR1 in tumor cells, HFn-LYTAC has significant potential for cancer therapy. Our findings provide preliminary evidence that HFn-LYTAC has antitumor activity and favorable safety.

A notable advantage of HFn-LYTAC lies in its modularity, making the construction process easy and straightforward. Two components, the TfR1 affinity-optimized HFn nanoscaffold and affibody of POI, can be easily obtained through prokaryotic expression and purification. The high stability and robust heat resistance of HFn render it particularly advantageous for purification and storage. By employing the SpyTag-SpyCatcher system, HFn-LYTACs targeting different POIs can be constructed by simply incubating the HFn nanoscaffold with the corresponding affibodies. In addition, HFn serves as an excellent nanomedicine carrier, facilitating the implementation of combination therapy for cancer. The ability of HFn to traverse the BBB makes HFn-LYTAC a viable option for the treatment of glioblastoma. The endocytosis of HFn is subjected to a threshold effect of TfR1 expression, thereby making it safer than antibodies. Overall, the HFn-LYTAC platform represents a promising approach for the targeted degradation of membrane proteins. Its efficient degradation capability, modular design, straightforward construction, and tumor-targeting potential support its broad applicability in cancer therapy and other biological fields.

Methods
Ethical regulations

All research complied with the relevant ethical regulations. All animal studies were performed according to the protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the Institute of Biophysics, Chinese Academy of Sciences (SYXK2024208). Mice were housed in groups of five per cage in a specific pathogen-free facility with controlled temperature (22-26 °C) and humidity (40-70%). The animals were kept on a 12-h light/dark cycle. The IACUC permits a maximum tumor size of 15 mm in diameter, in our work no mice exceeded this criterion. According to the IACUC guidelines, mice that experience weight loss exceeding 20% or display symptoms such as hunched posture, impaired locomotion, or respiratory distress should be promptly euthanized using CO2 gas. Otherwise, the mice were euthanized at the conclusion of the experiment.

Cloning, expression and purification of the recombinant protein

Human ferritin or ferritin mutants. The genes encoding human apo-H-chain ferritin (HFn), mutants 1 (D15L), 2 (F81Y), 3 (Q83H), 4 (N109R), 5 (D123E), and 6 (N109R/K119R), HFnSpyTag and HFn6SpyTag (HFn or HFn6 with SpyTag at the C-terminus) were designed, synthesized, optimized for Escherichia coli codon usage and cloned and inserted into the pET30a vector. The amino acid and nucleotide sequences of all proteins are provided in Supplementary Tables 3 and 4, respectively. For the purification of human ferritin and ferritin mutants, Escherichia coli BL21 (DE3) cells harboring recombinant plasmids were grown to an OD600 of 0.6 at 37 °C in 0.8 L of kanamycin-containing Luria–Bertani broth (LB) medium. Gene expression was induced by the addition of 0.4 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG), and the cells were further grown at 16 °C for 20 h. After cell harvesting, the pellet was suspended in 100 mM sodium phosphate (pH 8.0) and disrupted by a high-pressure cracker in the presence of 1 mM phenylmethylsulfonyl fluoride (PMSF) at 4 °C. The lysate was centrifuged, and the supernatant was heated at 62 °C for 15 min and then centrifuged to remove denatured proteins. Ammonium sulfate was added slowly to the supernatant with stirring to a final concentration of 0.7 M. The pellet was removed by centrifugation, and the supernatant was then loaded onto a Phenyl HP column (Cytiva). The ferritin eluant recovered was then precipitated using ammonium sulfate at 80% saturation. The pellet was resuspended and further polished with a HiLoad 16/600 Superdex 200 pg column (Cytiva) buffered with 10 mM HEPES, pH 7.0, and 50 mM NaCl. The eluant from the gel‒filtration column was concentrated and sterile-filtered before being stored at -80 °C. Purity was evaluated by SDS–PAGE, and the protein concentration was determined spectrophotometrically at 280 nm via the molar extinction coefficient (on a monomer basis) predicted from ProtParam on the basis of the amino acid sequence. (ProtParam software, http://www.expasy.org).

The human EGFR affibody ZEGFR. The sequence encoding the human EGFR affibody ZEGFR, followed by a 17 amino acid residue helix linker (17H, AEAAAKEAAAKEAAAKA), a SpyCatcher domain and a hexa-histidine tag at the C-terminus, was designed, synthesized, optimized for Escherichia coli codon usage and cloned and inserted into the pET30a vector. Escherichia coli BL21 (DE3) cells harboring recombinant plasmids were grown to an OD600 of 0.6 at 37 °C in 0.8 L of kanamycin-containing Luria–Bertani broth (LB) medium. Gene expression was induced by the addition of 0.4 mM IPTG, and the cells were further grown at 16 °C for 20 h. After cell harvesting, the pellet was suspended in 20 mM HEPES, 20 mM NaCl and 20 mM imidazole at pH 7.5 and disrupted by a high-pressure cracker in the presence of 1 mM PMSF. The lysate was centrifuged, and the supernatant was loaded onto a Ni-NTA (Cytiva, USA) column. The eluant from the Ni-NTA column was further polished with a HiLoad 16/600 Superdex 75 prep column (Cytiva) buffered with 10 mM HEPES, pH 7.0, and 50 mM NaCl. The eluant from the gel‒filtration column was concentrated and sterile-filtered before being stored at -80 °C. Purity was evaluated by SDS–PAGE; protein concentration was determined spectrophotometrically at 280 nm using a molar extinction coefficient predicted from ProtParam on the basis of amino acid sequence. (ProtParam software, http://www.expasy.org).

The human HER2 affibody PEPHER2. The sequence encoding the human tumor-targeting HER2 binding affibody PEPHER2 (J. Mol. Biol. 2008, 376, 1388-1402) followed by a 17 amino acid residue helix (17H, AEAAAKEAAAKEAAAKA), a SpyCatcher domain and a hexa-histidine tag at the C-terminus was designed, expressed and purified to homogeneity similar to that mentioned above. Purity was evaluated by SDS–PAGE.

The human PD-L1 affibody FN3PD-L1. The sequence encoding the PD-L1 affibody FN3PD-L1 (Protein Eng Des Sel. 2019 Dec 31;32(5):231-240), followed by a SpyCatcher domain, and a hexa-histidine tag at the C-terminus was designed, expressed and purified to homogeneity similar to that mentioned above. Purity was evaluated by SDS–PAGE.

Human TfR1. The gene encoding the ectodomain of human TfR1 (residues 88–760) was synthesized and inserted into pcDNA3.1(+) with the Kozak sequence (GCCACC), and the signal peptide from albumin was fused at the N-terminus. An HRV3C recognition sequence, an octa-histidine tag and a strep-tag were also placed at the C-terminus of the TfR1 gene. The expression plasmid was transiently transfected into HEK293T cells via Lipofectamine 2000 as a transfection agent. The cells were grown in DMEM (Thermo Fisher Scientific, Hampton, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C. The medium was replaced with fresh medium after 7 hours of transfection. After another 96 h, the supernatants were harvested, and TfR1 was purified via Ni-affinity and strep-tactin columns, followed by HRV3C cleavage to remove the affinity tags. The protein sample was further polished with a Ni-NTA column and a Superdex 200 Increase (10/300 GL, Cytiva) column to remove uncleaved protein, protein aggregates and trace amounts of miscellaneous proteins. The purified TfR1 ectodomain protein was stored at −80 °C in 10 mM HEPES and 150 mM NaCl, pH 7.5. The quality and quantity of the purified protein were evaluated via SDS–PAGE and UV/visible spectroscopy via the theoretical ε280 at 96,260 M−1 cm−1.

Endotoxin removal

Endotoxin removal was performed by the addition of 0.1% of the nonionic surfactant octylphenol ethoxylate (Triton X-114; Sigma‒Aldrich). Residual traces of Triton X-114 were removed with absorbant media (Bio-Beads; Bio-Rad). Endotoxin levels were examined with an endotoxin assay kit (GenScript), and the endotoxin levels in different batches were consistently approximately 0.01 EU/μg protein.

Protein labeling

For biotin labeling, HFn and TfR1 ectodomains were labeled with BMCC-Biotin (Pierce, Thermo Fisher) according to the manufacturer’s standard protocol. Briefly, protein was incubated with 5 mM TCEP in HEPES buffer at RT for 30 min, followed by TCEP removal with a HiTrap desalting column (Cytiva). A total of 1.5 mg/mL cysteine-exposed protein was mixed with a 20-fold molar excess of BMCC-Biotin in 20 mM HEPES, pH 7.0, 50 mM NaCl and incubated on ice overnight. The unreacted biotin solution was removed via HiTrap desalting chromatography, and biotin incorporation was estimated via the HABA (4´-hydroxyazobenzene-2-carboxylic acid) method.

For FITC and Cy5.5 labeling, FITC NHS ester (Thermo Scientific, 53027) or Cy5.5 NHS ester (MedChemExpress, HY-D0925) was dissolved in DMSO and subsequently added to NaHCO3 buffer (0.1 M, pH 8.5) containing 2 mg/mL purified protein. Cy5.5 was introduced at a molar ratio of 10:1 relative to the purified protein. The reaction mixture was incubated overnight at 4 °C with gentle agitation in the dark. Unbound dyes were removed via ultrafiltration centrifuge tubes, and the protein mixture was dialyzed into buffer consisting of 10 mM HEPES (pH 7.0) and 50 mM NaCl.

Construction and screening of HFn-LYTACs

On the basis of the interaction of SpyCatcher and SpyTag41, the affibodys (ZEGFR/PEPHER2/FN3PD-L1) were separately coupled to the surface of HFn6SpyTag to construct HFn-LYTAC for different targets. Specifically, HFn6SpyTag was mixed with SpyCatcher-ZEGFR/PEPHER2/FN3PD-L1 at molar ratios of 1:3, 1:6, 1:9, 1:12, 1:15, 1:18, 1:21 or 1:24 for 1 h at room temperature. SDS‒PAGE was used to evaluate the coupling efficiency. To screen for optimal coupling ratios, flow cytometry was used to detect the clearance of target proteins after treatment with different coupling ratios of HFn-LYTACs.

Biophysical characterization of HFn-LYTACs

Transmission electron microscopy (TEM):
For protein observation, the samples (1 μL, 0.05 mg/mL) were embedded in a Plasma Cleaner HPDC32G-treated copper grid, stained with 1% uranyl acetate for 1 min, and then imaged with a Tecnai Spirit 120 kV transmission electron microscope (FEI, U.S.A.).

Dynamic light scattering (DLS):
The samples were prepared in 10 mM HEPES and 50 mM NaCl buffer (25 μL, 0.2 mg/mL). DLS analysis was performed via a DynaPro Titan with a temperature-controlled microsampler (Wyatt Technology, U.S.A.) at 25 °C.

SPR assay

The interactions were measured via the SPR technique on a Biacore 8K instrument (Biacore, Uppsala, Sweden). Biotinylated proteins (Ligands, including TfR1, EGFR, HER2 or PD-L1) were immobilized on an SA Chip (GE Healthcare Europe GmbH) according to the manufacturer’s instructions. The SPR assay was performed at 16 °C at a flow rate of 30 μL/min. Analytes (HFn mutants, affibodies or HFn-LYTACs) were dissolved in 10 mM HEPES, pH 7.0, 50 mM NaCl, and 0.05% (vol/vol) Tween-20. The kon and koff rates as well as the dissociation constant (Kd) were estimated via Biacore8K multicycle kinetics evaluation software according to a 1:1-binding model. Fits are reported as black lines.

Cell lines

All cell lines were obtained directly from the American Type Culture Collection (ATCC). Cell line authentication was performed by the supplier prior to distribution according to standard quality control procedures. No additional authentication assays (such as STR profiling or karyotyping) were conducted by the authors. All cell cultures were routinely tested and confirmed to be free of mycoplasma contamination prior to use.

Adherent cells were cultured in 10-cm plates at 37 °C and 5% CO2. U-87 MG cells (ATCC) and MDA-MB-231 cells (ATCC) were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. SKBR3 cells (ATCC) were cultured in McCoy's 5a medium supplemented with 10% FBS and 1% penicillin/streptomycin. NCI-N87 cells (ATCC), HT1080 cells (ATCC) and HCT116 cells (ATCC) were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin.

Detection of membrane EGFR, HER2 and PD-L1 clearance via flow cytometry

The experiment was performed at 60% confluence of adherent cells (in a 12-well plate). The cells were incubated with 1 mL of complete growth medium supplemented with 0.3 μM HFn-LYTAC for 24 h. The cells were then trypsinized and harvested in 1.5 mL Eppendorf tubes. After being washed with ice-cold PBS+0.5% FBS three times (FACS buffer), the cells were incubated with an ABflo® 647-labeled primary antibody for 30 min on ice and washed again with FACS buffer three times. Flow cytometry was performed on a BD FACSCalibur, the number of cells collected for each sample exceeded 15,000 (Supplementary Fig. 8), and FlowJo software was used for analysis. The antibodies used included Alexa Fluor® 647 anti-EGFR (Abcam, ab307403, 1:500), ABflo® 647 anti-HER2 (ABclonal, A23183, 1:500), and ABflo® 647 anti-PD-L1 (ABclonal, A22305, 1:500) antibodies.

Detection of total EGFR, HER2 and PD-L1 degradation via Western blotting

The experiment was performed at 60% confluence of adherent cells (in a 24-well plate). The cells were incubated with 500 µl of complete growth medium supplemented with 0.3 μM HFn-LYTAC for 24 h. The cells were washed with ice-cold PBS three times and lysed with RIPA buffer (P0013B, Beyotime) according to the manufacturer’s instructions. The cell lysates were subsequently centrifuged at 16,000 ×g for 15 min at 4 °C. The supernatant was collected, and the protein concentration was determined via a BCA assay (Pierce). Equal amounts of lysate were separated by 10% SDS‒PAGE and transferred to polyvinylidene fluoride (PVDF) membranes blocked with 5% nonfat milk. After being washed with PBS, the membrane was incubated with primary antibody overnight at 4 °C and washed with PBS supplemented with 0.1% Tween 20 (PBST). The membrane was then incubated with an HRP-conjugated secondary antibody for 1 h at room temperature, washed three times with PBST, and developed with an enhanced chemiluminescence (ECL) substrate (Pierce). ImageJ software was used to quantify the band intensities. Unprocessed scans of the blots are provided in Source Data file. The antibodies used included anti-EGFR (Abcam, ab32077, 1:1000), anti-HER2 (Abcam, ab134182, 1:1000), anti-PD-L1 (Abcam, ab213524, 1:1000), anti-TfR1 (ABclonal, A22161, 1:1000), anti-β-tubulin (ABclonal, AC021, 1:5000), anti-GAPDH (ABclonal, AC002, 1:5000), HRP-conjugated goat anti-rabbit IgG (ABclonal, AS014, 1:2500), and HRP-conjugated goat anti-mouse IgG (ABclonal, AS003, 1:2500) antibodies.

Detection of EGFR, HER2 and PD-L1 degradation via confocal microscopy

The experiment was performed at 60% confluence of adherent cells (in a 20 mm glass bottom cell culture dish). The cells were incubated with 2 mL of complete growth medium supplemented with 0.3 μM HFn-LYTAC for 24 h. After being washed with ice-cold PBS, the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature and washed three times. The cells were then permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature, followed by three washes. The cells were blocked with 10% goat serum (homologous to secondary antibody) in PBS for 30 min at room temperature and incubated with primary antibody overnight at 4 °C. After being washed with PBS three times, the cells were incubated with secondary antibody for 1 h at room temperature, followed by three washes. The cells were finally incubated with DAPI for 10 min at room temperature and washed. The fluorescent signals were observed via confocal laser scanning microscopy (CLSM) (Olympus FV 3000RS, Tokyo, Japan). Images were acquired via 405 and 640 nm lasers. The antibodies used included anti-EGFR (Abcam, ab32077, 1:100), anti-HER2 (Abcam, ab134182, 1:100), anti-PD-L1 (Abcam, ab213524, 1:100), and Alexa Fluor® 647 goat anti-rabbit IgG H&L (Abcam, ab150079, 1:500) antibodies.

Cellular uptake

The cellular uptake of HFn-LYTAC was analyzed by flow cytometry and CLSM, and FITC-labeled HFn6 was used for the construction of HFn-LYTAC.

For flow cytometric analysis, cells (including SKBR3 cells, RAW 264.7 cells and DC2.4 cells) were cultured in 12-well plates. When the adherent cells reached 70–80% confluence, the cells were incubated with 100 nM FITC-HFn-LYTAC or FITC-HFn for 1 h. The cells were then trypsinized and harvested, flow cytometry was performed on a BD FACSCalibur, and FlowJo software was used for analysis.

For CLSM observation, SKBR3 cells were cultured in 20 mm glass bottom cell culture dishes. When the adherent cells reached 70–80% confluence, the cells were incubated with 300 nM FITC-HFn-LYTAC for 5 min, 1 h, 2 h or 4 h. After coincubation, the cells were washed with ice-cold PBS and fixed with 4% PFA in PBS for 10 min at room temperature. The cells were then permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature, followed by three washes. The cells were blocked with 10% goat serum in PBS for 30 min at room temperature and incubated with primary antibodies overnight at 4 °C. After being washed with PBS three times, the cells were incubated with secondary antibodies for 1 h at room temperature, followed by three washes. The cells were finally incubated with DAPI for 10 min at room temperature and washed. The fluorescent signals were observed via CLSM. Images were acquired via 405, 488, 561 and 640 nm lasers and processed via ImageJ. The antibodies used included anti-HER2 (rat-derived, Abcam, ab11710, 1:100), anti-LAMP1 (rabbit-derived, Abcam, ab278043, 1:50), anti-TfR1 (rabbit-derived, ABclonal, A22161, 1:100), Alexa Fluor® 647 goat anti-rabbit IgG H&L (Abcam, ab150079, 1:500) and Alexa Fluor® 568 goat anti-rat IgG H&L (Abcam, ab175476, 1:500) antibodies.

Cell viability assays

SKBR3 or U-87 cells were seeded in 96-well plates at a density of 800 cells per well. Following cell adherence (24 hours), the cells were treated with HFn6-LYTAC or wtHFn-LYTAC for 6 days. On day 3, the culture medium was replaced and supplemented with HFn-LYTAC. Following treatment, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37 °C for 1 hour. The absorbance was then measured at 450 nm via a microplate reader.

Lentivirus infection for TfR1 knockdown

TFRC-targeted shRNA (sense strand sequence: 5’-GCCCAGATGTTCTCAGATATTT-3’) were subcloned and inserted into the PLKO.1-puro vector to generate TfR1-knockdown (shTFRC) plasmid42. For the generation of lentiviral particles, HEK293T cells were transfected with shTFRC plasmids and cotransfected with helper plasmids (pMDLg/pRRE, pCMV-VSVG, and pRSV-Rev). Transfections were performed via the use of Fugene HD transfection reagent (Promega, E2311). After 48 h, the lentiviral supernatants were collected, filtered through 0.45 μm filters, and used to infect SKBR3 cells supplemented with polybrene (10 μg/mL). After lentiviral infection for 36–48 h, the cells were selected with puromycin (1 μg/mL).

In Vitro Transcytosis Assay

Mouse brain microvascular endothelial cells (bEnd.3) were used to establish an in vitro BBB model. Cells were seeded at a density of 1 × 105 cells/cm² onto gelatin-coated Transwell inserts with a pore size of 0.4 μm (24 mm diameter, Corning) and cultured for 48–72 h until confluence was achieved. The transcytosis assay was initiated when the trans-endothelial electrical resistance (TEER) reached 100 Ω·cm². FITC-labeled HFn6-ZEGFR, HFn6, HFn or LFn (100 μg) was added to the apical chamber. Transcytosis of ferritin-based proteins was assessed by collecting samples from the basal chamber at 120 min post-addition. The concentration of transcytosed ferritins in the basal chamber was quantified based on a pre-established linear relationship between protein concentration and FITC fluorescence intensity, measured using a Varioskan Flash spectral scanning multimode reader (ThermoFisher Scientific).

Animal experiments

Female NCG mice (6–8 weeks old) and male BALB/c-nude mice (6–8 weeks old) were purchased from GemPharmatech Co., Ltd. (Jiangsu). Female BALB/c mice (6–8 weeks old) were purchased from HFK Bioscience Co., Ltd. (Beijing). All animal studies were conducted with randomization and blinding experimental procedures.

In vivo half-life and tissue distribution of HFn-LYTACs

For the plasma half-life studies, healthy female BALB/c mice (6–8 weeks old) were i.v. injected with 10 mg/kg HFn6-PEPHER2 (n = 3, randomly grouped). Blood samples were collected at 5 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after injection. Plasma was obtained by centrifuging blood at 2000 ×g at 4 °C for 10 min. Subsequently, 1 µL of plasma was diluted in 10 µL of PBS and analyzed by Western blotting using an anti-SpyCatcher antibody (Bio-Rad, HCA379, 1:500). Prior to Western blotting, the stain-free gel was imaged to assess total protein levels. Protein band intensities were quantified via ImageJ. The half-life was calculated via the nonlinear regression function in GraphPad Prism (v.10.1.2).

For the biodistribution study of HFn6-PEPHER2 in NCI-N87 xenograft tumor mice, HFn6-PEPHER2 and free PEPHER2 were i.v. injected into NCI-N87-bearing female NCG mice at an equivalent PEPHER2 dose (10 mg/kg, n = 3 for each group). Twenty-four hours after injection, the mice were euthanized, and the tumors and various tissues (including the heart, liver, spleen, lung and kidney) were collected. The tumor and tissue samples were subsequently processed via a tissue grinder, and Western blotting analysis was carried out following the same protocol used for the plasma half-life studies.

For the biodistribution study of HFn6-PEPHER2 in U-87 MG orthotopic xenograft tumor mice. Cy5.5-coupled HFn6-ZEGFR and free ZEGFR were intravenously injected into U-87 MG-bearing mice at an equivalent ZEGFR dose (n = 3 for each group). At 1, 4, and 24 h postinjection, the mice were euthanized, and the brains containing tumor tissue were collected. Near-infrared fluorescence imaging was carried out with an IVIS® Lumina Series III system.

Evaluation of antitumor efficacy in a subcutaneous tumor model

To evaluate the antitumor efficacy of HFn6-PEPHER2, female NCG mice (6–8 weeks) were subcutaneously injected with 1×107 NCI-N87 cells per mouse to establish cell-derived xenograft tumor models. Once the tumor volume reached approximately 60 mm³ (day 5), the mice were randomly divided into four groups (n = 6) and injected with PBS, HFn6SpyTag, PEPHER2, or HFn6-PEPHER2 via the tail vein. The administered dose was 30.6 mg/kg for HFn6-PEPHER2 group (containing 10mg/kg PEPHER2 and 20.6 mg/kg HFn6SpyTag). The PEPHER2 and HFn6 groups received doses equivalent to their respective molar amounts in the HFn6-PEPHER2 group. The drugs were injected every two days for a total of 7 injections. The tumor volume and body weight of the mice in each group were recorded every two days. The tumor volume was calculated via the following formula: width2 × length × 0.5.

Evaluation of antitumor efficacy in an intracranial orthotopic glioblastoma model

To evaluate the antitumor efficacy of HFn6-ZEGFR, male Balb/c-nu mice (6-8 weeks old) were used to establish animal models of U-87 MG-luc-mCherry GBM. Briefly, tumor cells were intracranially injected into the right hemisphere of the mouse brain via a stereotaxic apparatus on day 0. Luciferase-catalyzed bioluminescence was utilized to characterize tumor growth. Specifically, 8 min after the intraperitoneal injection of D-luciferin (150 mg/kg), the animals were visualized with an IVIS Spectrum Imaging System (Caliper, USA), and the same procedure was repeated at the specified time. Imaging signals in cranial regions were quantified in units of mean photons per second per square centimeter per steradian (p/s/cm2/sr).

For the treatment experiments, the mice were randomly divided into four groups (n = 5) and injected with PBS, HFn6SpyTag, ZEGFR, or HFn6-ZEGFR via the tail vein. The administered dose was 30.6 mg/kg for HFn6-ZEGFR group (containing 10mg/kg ZEGFR and 20.6 mg/kg HFn6SpyTag). The ZEGFR and HFn6 groups received doses equivalent to their respective molar amounts in the HFn6-ZEGFR group. Each formulation was injected every 2 days for a total of 7 injections.

Detection of the expression levels of HER2 and EGFR by flow cytometry

The tumor tissue was isolated and cut into pieces. The tissue was then digested with NB4 tissue digestion solution containing DNase I and incubated in a shaker at 37 °C and 100 rpm for 1 hour. Next, DMEM was added to terminate the digestion, and the resulting suspension was filtered through 70-μm cell strainers (Corning, 352350) to obtain a single-cell suspension. The cells were washed with PBS containing 2% BSA and analyzed via the standard flow cytometry protocol with Alexa Fluor® 647-conjugated anti-EGFR (Abcam, ab307403, 1:500) and ABflo® 647-conjugated anti-HER2 (ABclonal, A23183, 1:500) antibodies.

HER2, EGFR and TfR1 expression levels were detected via CLSM

To analyze HER2 and EGFR degradation in tumors, the tumors (brains from U-87 MG-bearing mice) were embedded in optimal cutting temperature (OCT) compound and cut into 7 μm slices via a freezing microtome (Leica CM1950). Then, the tumor slices were treated with acetone to remove the OCT. After being washed with PBS 3 times, the tumor slices were blocked with 10% goat serum in PBS for 30 min at room temperature and incubated with primary antibodies overnight at 4 °C. After being washed with PBS three times, the cells were incubated with secondary antibodies for 1 h at room temperature, followed by three washes. The tumor slices were finally incubated with DAPI for 10 min at room temperature and washed. The immunofluorescence images were observed via CLSM.

In vivo safety of HFn-LYTACs

For histopathological and hemosiderin evaluation, the major organs of the mice in each group, including the heart, liver, lung, spleen, and kidney, were collected, embedded, sectioned, and stained with hematoxylin and eosin (H&E) to examine the effects of HFn-LYTACs on normal tissues. For hemosiderin evaluation, liver tissue sections were stained via the Prussian Blue Iron Stain Kit (Yeasen Biotechnology, Cat. No. 60533ES20) following the manufacturer's standard protocol.

For blood biochemistry and complete blood cell analysis, BALB/c mice were i.v. injected with PBS, HFn6, free binding affibodise or HFn-LYTACs. After 7 injections, the blood of each mouse was collected for complete blood cell analysis. For blood biochemistry analysis, after coagulation, the serum was separated via 6,000 ×g centrifugation. The levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and creatinine (CREA) in the serum were subsequently detected by corresponding assay kits obtained from the Institute of Biological Engineering of Nanjing Jianchen (Nanjing, China) according to the manufacturer’s protocol.

For reticulocyte analysis, BALB/c mice were i.v. injected with PBS, HFn6, free binding affibodies or HFn-LYTACs. After 7 injections, the blood of each mouse was collected in an EDTA anticoagulant tube (Solarbio, YA1461-1pk) and washed with PBS three times. The blood cells were fixed with 4% PFA in PBS for 10 min at room temperature. The cells were then permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature, followed by three washes. The cells were subsequently incubated with an anti-TfR1 antibody (ABclonal, A22161) at room temperature for 1 hour. The cells were then washed three times with PBS, followed by the addition of secondary antibody (Abcam, ab150079) and incubation at room temperature for 30 minutes. After another three washes with PBS, the cells were stained with PI staining solution at 4 °C for 30 minutes. Finally, the stained cells were analyzed via flow cytometry.

Data availability

The data supporting the findings of this study are available from the corresponding authors upon request. All the PDB structures are available in the PDB database (https://www.rcsb.org/). The data generated in this study are provided in the Source Data file. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study presents a modular LYTAC platform based on engineered heavy-chain ferritin (HFn) by optimizing its affinity for transferrin receptor 1 (TfR1) and leveraging the SpyTag-SpyCatcher system for targeted protein degradation. The platform demonstrates degradation efficiency and antitumor efficacy across multiple tumor models. The integration of HFn’s nanocage structure and TfR1 targeting provides a modular strategy for membrane protein degradation. However, several critical issues need to be addressed.\n1. In the Introduction section, the authors claimed that the TPD systems based on peptides or aptamers have limited tissue persistence. However, it seems that the HFn-LYTAC has very short half-life and not very promising tumor tissue accumulation. It is hard to see the advantages of this system compared with the recent published work of TransTAC and Pep-TAC.\n2. The authors proposed the possible degradation mechanisms of HFn-LYTAC in two aspect. Actually, the second mechanism through cellular trafficking of nanoparticles might cause unspecific degradation. This might not be the advantage compared with LTR-specific TPD.\n3. Since the fusion proteins are expressed in E. coli, it is necessary to measure the endotoxin level to avoid contamination.\n4. Since the HFn-LYTAC is a nanoparticle, the uptake by macrophages and other cells in the blood should be tested to explain why it has short half-life in the blood but can accumulate in the tumor tissue a little bit. Also, the in vitro effects of HFn-LYTAC on the proliferation of the tumor cells should be tested. By the way, EPR effects are highly questioned currently.\n5. The enhanced affinity of HFn6 for TfR1 via mutagenesis is a key innovation. However, the rationale for selecting specific residues (D15, F81, Q83, N109, K119, D123) in the TfR1-binding interface remains unclear. Please explicitly justify the selection of these residues (e.g., structural analysis, computational predictions, or prior literature). In Extended Data Fig. 1b, include computational data (e.g., binding free energy changes, hydrogen bond/electrostatic interactions) for each mutation. In Fig. 1b (mislabeled as Fig. 1c in the text), highlight the N109R and K119R mutation sites in the molecular docking interface, annotate bond energies, and provide the PDB ID of the HFn-TfR1 complex in the figure legend or Methods. Simplify Extended Data Fig. 1a: Use a single panel with “front view”; and “rotated 180° view”; to illustrate the overall binding mode, labeling key interacting residues.\n6. The authors propose that HFn-LYTAC mediates target protein degradation through two mechanisms: TfR1 dependent clathrin mediated endocytosis and multivalent interaction driven endocytosis. However, when exploring the multivalent mechanism of action, the authors only verified the dominance of the first mechanism through chlorpromazine (CPZ, clathrin inhibitor) experiments, and did not give a clear explanation for the experimental results of caveolin dependent endocytosis inhibitors (such as methyl-β-cyclodextrin, m-β-cd) and microcytosis inhibitors (amiloride). This leads to the following key questions not being answered: does multivalent interaction depend on caveolin or the microcytosis pathway? If not, is the multivalent interaction driven endocytosis through other unexplored mechanisms (such as non clathrin / non caveolin dependent pathways)? In addition, the specific contributions of the two mechanisms lack quantitative analysis.\n7. The optimal conjugation ratios vary significantly across targets (e.g., 12:1 for EGFR/HER2 vs. 6:1 for PD-L1). The authors analyzed whether it might be related to the size of HFn-LYTAC (Extended Data Fig. 3b), and whether it is related to the expression level of target proteins? It is suggested to supplement the analysis of the influence of target protein expression level on coupling efficiency, so as to provide guidance for subsequent target selection.\n8. Discrepancies between figure citations and text descriptions (e.g., Fig. 1c vs. Fig. 1b; missing Extended Data Fig. 3a discussion) reduce clarity. Ensure all figure citations align with the text (e.g., correct Fig. 1c to Fig. 1b; in the Results section). Describe Extended Data Fig. 3a in the main text. Also, there is no Fig. 1e.\n9. Extended Data Figure 7i, it seems that the tumor accumulation of HFn-ZEGFR is weaker than that of ZEGFR.", "reviewer_2": "In this work, Zhang et al. add to the scientific repertoire of lysosomal-degrading extracellular targeted protein degradation (eTPD) with a platform using an affinity-matured heavy chain ferritin complex conjugated to binders to POI via SpyTag/SpyCatcher. Use of ferritin is intriguing for target uptake and BBB penetration in the TPD field and the multimeric complex has some potential advantages. The authors performed well-constructed studies to investigate the clearance of multiple protein targets in vitro including assessments of clearance mechanism. While the declared modularity of the system still requires design and expression of each POI binder fusion, the in vivo data including tumor suppression and target degradation with an orthotopic brain tumor is exciting to see.\nMajor comments:\n1. The prose and figures frequently are mislabeled or unclear regarding POI removal from the cell surface (e.g., the flow method) versus degradation of POI (e.g., Western). These are both biologically meaningful but are quite different phenomena. This needs to be corrected and clear throughout the manuscript.\n2. Investigation of effects of the constructs on TfR biology could be improved. There is no quantification of effect on surface or total TfR levels after treatment. The authors do not consider the effect on reticulocytes nor discuss hemosiderin.\n3. Given the multimeric nature of the complexes, potential pathway activation by multimerization of POI should be investigated.\nMinor comments:\n1. 300 nM of HFn-LYTAC is a high concentration (with total in vivo doses ~30 mg/kg) implying lower potency versus some other eTPD approaches.\n2. The authors demonstrate target clearance in vitro, target clearance in vivo, and tumor growth inhibition in vivo, but they do not show data connecting the dots between target clearance and in vivo growth inhibition (such as pathway inhibition or specific growth inhibition in vitro, etc.).\n3. The authors describe their molecules as “LYTACs”, which have been described in the literature for ASGPR- and M6PR-mediated uptake, whereas other groups have used other platform names when driving uptake by receptors such as TfR, cytokine receptors, sortilin receptors, etc. The eTPD space is nascent thus nomenclature is evolving. The authors and editors should consider what nomenclature is most appropriate for these HFn constructs.\n4. The authors should point out that the BBB was not verified to be intact in their orthotopic U87 model.\n5. Figure legends/labels/descriptions need more details throughout. Test article (active and controls) concentration, timepoint, in vivo dose levels of all components for all experiments, y axis readout is often missing or unclear. Timepoints sometimes do not match between text and figure legend. Figure 4 are the n independent experiments or the number of animals per group? Figure 2 what is Control in flow? Is labeling on Figure 4i /Ext Data Fig 7i correct? Add more statistics to Fig. 4b.\n6. More methods need to be included, including method of measuring half-life, flow cytometry methods for xenografts, fluorescent labeling methods, SPR ligand species, catalog protein and antibody identities. Were antibodies used for flow measurement noncompetitive with the test articles? Clarification re: cross-reactivity to murine TfR and POI is important information for interpretation of in vivo results.\n7. The references for use of TfR for eTPD lists only one of three contemporaneous reports (doi.org/10.1038/s41586-024-07947-3, doi.org/10.1038/s41586-024-07948-2, doi.org/10.1038/s41467-024-52975-2).\n8. Tolerability or Safety is a more accurate description of the experiments than Biocompatibility or Biosafety.\n9. Conclusions might be clarified: In Fig. 3, multivalent POI binding without TfR binding reduces target ~36-50% and thus the addition of TfR binding increases degradation by ~13-30% - indicating multivalent POI binding may be the dominant mechanism with TfR-mediated uptake being secondary. In addition, the knockdown of TfR is not complete so it is not surprising that it did not abolish all degradation. Steric hindrance is a good hypothesis but not proven as the cause of reduced clearance/conjugation at higher binder ratios. HFn6 has the most but not the only koff reduction.", "reviewer_3": "This manuscript presents a new therapeutic strategy of modular mutant HFn-LYTAC for targeted degradation of pathological POI for cancer therapy. The novelty of the concept introduced here is noteworthy, but there are several critical concerns. In particular, data analysis/interpretation is often stretching, and several remarks/conclusions are made with speculations.\nBelow lists a few specific comments.\n1. The rationale for selecting the specific target residues (out of the listed candidates) to implement mutations to increase TfR1 binding affinity is not provided.\n2. TfR1 binding affinity of HFn6SpyTag was confirmed (Fig. E2b), but it is unclear whether the affinity is retained when affibodies are attached. To validate the authors’ hypothesis regarding the lack of EGFR degradation at high molar ratios, TfR1 binding affinity and cell uptake should be experimentally determined at the molar ratio range tested in this study.\n3. What are the molecular weights of three affibodies investigated in this manuscript?\n4. What are the error values (i.e., SD) and PDI for the DLS results in Fig. E3b?\n5. The authors concluded that cytoplasmic EGFR signals were significantly stronger when cells were treated with wtHFn-ZEGFR compared to HFn6-ZEGFR (Fig. 2d/E5) without quantitative and statistical analysis of experimental data.\n6. Statistical analysis is missing in Fig. E2.\n7. Are the confocal images in Fig. 3 representative? If so, how many independent cells were observed? Fig. 3c does not clearly show the colocalization of HFn6-PEPHER2/HER2 and HER2/LAMP1.\n8. The authors speculated cellular trafficking and multivalent interactions of HFn-LYTAC and POI as a mechanism of target protein degradation without convincing experimental validation.\n9. Quantitative comparisons made based entirely on WB analysis without statistical analysis do not convincingly support the authors’ conclusions.\n10. Have the authors compared HFn-LYTAC and respective affibodies at an equimolar of affibodies for their ability to degrade respective POI in vitro?\n11. The in vivo study in Fig. 4 lacks wtHFn-PEPHERs as a control. Based on the in vitro comparison in Fig. 3f, it is unclear whether the HFn mutation to improve TfR1 binding affinity is needed to enhance tumor delivery and POI degradation in vivo.\nOverall, it is unclear whether strong/enhanced TfR1 binding affinity is essential for the presented therapeutic strategy based on Fig. 3f and in vitro studies with PFn. Conventional synthetic nanoparticles decorated with high-density surface affibodies may work comparably."}, "author_response": "Response: We sincerely appreciate your valuable comments. We have revised the manuscript to clarify these issues.\n1. In the Introduction section, the authors claimed that the TPD systems based on peptides or aptamers have limited tissue persistence. However, it seems that the HFn-LYTAC has very short half-life and not very promising tumor tissue accumulation. It is hard to see the advantages of this system compared with the recent published work of TransTAC and Pep-TAC.\nResponse: We thank the reviewer for raising this important point, which allows us to critically reflect on the research significance of our manuscript.\nA previous study developed an LYTAC based on a TfR1-targeting peptide and a PD-L1 antibody-derived scFv fragment (TfR-LYTAC). However, its short plasma half-life limits tissue persistence. Administering TfR-LYTAC alone fails to effectively suppress tumors, and an additional delivery system is needed to improve therapeutic efficacy (Ref 1). This highlights the limited tissue persistence of peptide-based LYTACs. Although a direct comparison of plasma half-lives between HFn-LYTAC and TfR-LYTAC is not feasible (no relevant data are available for TfR-LYTAC), our results demonstrate that HFn6-PEP has a significantly HER2 extended plasma half-life compared with that of the free HER2-targeting protein PEP (20.2 HER2 kDa), with a 14-fold increase (131.8 min for HFn6-PEP and 9.3 min for PEP ) (Only HER2 HER2 for reviewer Fig. 1). Given that the shedding of fluorescent groups could interfere with the accurate characterization of HFn-LYTAC, we further utilized Western blotting to directly evaluate its in vivo behavior and accordingly updated the original dataset. Western blotting analysis further confirmed that HFn6-PEP has a half-life of ~227 min (Fig. 5a). In vivo HER2 distribution studies revealed that HFn6-PEP preferentially accumulated at the tumor site, HER2 with levels 10–16 times greater than those in major normal organs (Fig. 5b, c).\nThe half-life of HFn-LYTAC is shorter than that of TransTAC (16.6 days); however, increasing the dosing frequency allows HFn-LTYAC to achieve comparable POI degradation efficiency in vivo. Moreover, subsequent supplementary experiments suggest that rapid uptake by monocytes may contribute to the shortened circulation time (Supplementary Fig. 6a), providing a direction for future optimization.\nCompared with the two works mentioned by the reviewers, the feature of this study lies in the introduction of a plug-and-play modular LYTAC platform. The scaffold and POI ligands are designed separately, enabling easy large-scale preparation and modular assembly. The HFn6 scaffold serves as a universal platform, allowing LYTAC construction by simple incubation and conjugation with the target POI ligand—without additional chemical modifications, as required in Pep-TAC, which is based on peptides.\nDue to a change in the detection method, we have updated the in vivo characterization data for HFn-LYTAC. Below is the relevant excerpt from the revised manuscript:\n“The plasma half-life and tissue distribution of HFn6-PEP were first HER2 evaluated. ……Compared with free PEP , HFn6-PEP accumulated more persistently at HER2 HER2 the tumor site.”\nOnly for reviewer figure 1. Determination of the plasma half-life via Cy5.5-labeled HFn6- PEP . HER2 Figure 5. Evaluation of the in vivo antitumor efficacy and safety of the HFn-LYTAC platform. a, Plasma half-life of HFn6-PEP determined by Western blotting. b, Levels of HER2 HFn6-PEP in tumor, heart, liver, spleen, lung and kidney tissues determined by Western HER2 blotting. Samples were collected and prepared from each group 24 h after i.v. injection of HFn- PEP . HER2 c, Levels of PEP and HFn6-PEP in mouse tumor tissues determined by HER2 HER2 HER2 Western blotting. Samples were collected and prepared from each group at 1, 4, 8, and 24 hours after i.v. injection of HFn- PEP . HER2 Supplementary Figure 6. The uptake of HFn6-PEP by monocytes and dendritic HER2 cells. a, Uptake of HFn6 and HFn6-PEP in SKBR3 cells, RAW 264.7 cells and DC 2.4 HER2 cells was analyzed via flow cytometry.\nRef 1 Su, L.-Y. et al. Anti-tumor immunotherapy using engineered bacterial outer membrane vesicles fused to lysosome-targeting chimeras mediated by transferrin receptor. Cell Chemical Biology 31, 1219-1230.e1215.\n2. The authors proposed the possible degradation mechanisms of HFn-LYTAC in two aspects. Actually, the second mechanism through cellular trafficking of nanoparticles might cause unspecific degradation. This might not be the advantage compared with LTR-specific TPD.\nResponse: We thank the reviewer for raising this concern. In this study, we replaced HFn6 with Pyrococcus furiosus (pFn), which lacks TfR1 affinity, and demonstrated the existence of a second degradation mechanism. This mechanism represents a viable strategy for application in the field of targeted protein degradation and has been corroborated by previous studies (Ref 2). The second mechanism avoids the reliance on TfR1, thereby broadening the application scope of HFn-LYTAC. Although this mechanism lacks LTR targeting, it still relies on specific binding to POI to mediate the degradation of POI. Furthermore, the second mechanism results in relatively low degradation efficiency toward target proteins, which contributes to its safety profile to a certain extent. Relevant discussions have been added to the revised manuscript and are marked in red:\n“The second mechanism involves cellular trafficking of the nanoparticles and multivalent interactions between HFn-LYTAC and POI, which promote direct endocytosis and degradation. The advantage of the second mechanism lies in its independence from lysosomal endocytosis receptors, thereby broadening the application scope of HFn-LYTAC. However, its degradation efficiency remains limited, which may partially restrict therapeutic efficacy, but contributes to ensuring biological safety.”\nRef 2 Liu, Y., Liu, R., Dong, J. et al. Targeted protein degradation via cellular trafficking of nanoparticles. Nat. Nanotechnol. 20, 296–302 (2025).\n3. Since the fusion proteins are expressed in E. coli, it is necessary to measure the endotoxin level to avoid contamination.\nResponse: We thank the reviewer for this valuable suggestion. We supplemented endotoxin content testing for the proteins used in the in vivo experiments. The results indicated that the endotoxin content of the purified protein did not exceed 10 EU/mg, remaining within acceptable limits (Supplementary Fig. 1i). Relevant descriptions have been added to the revised manuscript and are marked in red:\n“The endotoxin level of purified protein was measured to not exceed 10 EU/mg, remaining within acceptable limits.”\nSupplementary Figure 1. i, Detection of endotoxin content in purified proteins for in vivo experiments.\n4. Since the HFn-LYTAC is a nanoparticle, the uptake by macrophages and other cells in the blood should be tested to explain why it has short half-life in the blood but can accumulate in the tumor tissue a little bit. Also, the in vitro effects of HFn-LYTAC on the proliferation of the tumor cells should be tested. By the way, EPR effects are highly questioned currently.\nResponse: We thank the reviewer for these valuable suggestions. We supplemented the experimental data on HFn-LYTAC endocytosis across multiple cell types, including mouse monocytes (RAW 264.7 cells), dendritic cells (DC 2.4 cells), and tumor cells (SKBR3 cells) (Supplementary Fig. 6a).\nFlow cytometry analysis revealed that the level of HFn-LYTAC internalized by DC 2.4 cells was slightly lower than that internalized by SKBR3 cells, whereas RAW 264.7 cells internalized 4.5 times more HFn-LYTAC than SKBR3 cells did. These findings suggest that HFn-LYTAC may be rapidly cleared by monocytes in circulation, potentially contributing to its short plasma half-life.\nNevertheless, the multimerization of PEP displayed on HFn6 markedly enhances the HER2 binding affinity for HER2, enabling HFn6-PEP to rapidly engage HER2 after traversing HER2 the vasculature and reaching the tumor site, thereby facilitating detectable accumulation within the tumor.\nAdditionally, we found that the endocytosis of HFn6-PEP by RAW 264.7 cells and DC HER2 2.4 cells did not significantly affect their proliferation (Supplementary Fig. 6b, c). Conversely, both HFn6-Z and HFn6-PEP effectively inhibited the proliferation of U-87 MG cells EGFR HER2 and SKBR3 cells (Supplementary Fig. 3b, d).\nWe also evaluated the inhibitory effect of wtHFn-LYTAC on cell proliferation. Owing to differences in total EGFR degradation capacity, wtHFn-Z EGFR exhibited a relatively weak cytotoxic effect, as evidenced by an IC value more than twice that of HFn6-Z (24.2 nM 50 EGFR for HFn6-Z and 57.26 nM for wtHFn-Z ). The IC values for the SKBR3 cells of EGFR EGFR 50 wtHFn-PEP and HFn6-PEP are similar because of their similar HER2 degradation HER2 HER2 capacities (6.78 nM for HFn6-PEP and 9.29 nM for wtHFn-PEP ). Neither HFn6 nor HER2 HER2 the POI ligand significantly inhibited cell proliferation (Supplementary Fig. 3c, e). Relevant descriptions have been added to pages 9 and 13 of the revised manuscript and are marked in red.\nRegarding the EPR effect, owing to the ongoing controversy, we have removed the relevant description and instead explained the accumulation of HFn-LYTAC at tumor sites through an active targeting mechanism:\n“Nevertheless, the multimerization of PEP displayed on HFn6 markedly enhances the HER2 binding affinity for HER2, enabling HFn6-PEP to rapidly engage HER2 after traversing HER2 the vasculature and reaching the tumor site, thereby facilitating detectable accumulation within the tumor.”\nSupplementary Figure 6. The uptake of HFn6-PEP by monocytes and dendritic cells. HER2 a, Uptake of HFn6 and HFn6-PEP in SKBR3 cells, RAW 264.7 cells and DC 2.4 cells was HER2 analyzed via flow cytometry. b, c, Cell viability assays of RAW 264.7 cells (b) and DC 2.4 cells (c) after treatment with gradient concentrations of HFn6 and HFn6-PEP . HER2 Supplementary Figure 3. HFn-LYTAC suppresses cell proliferation through targeted degradation of POI. b, Cell viability assays of U-87 MG cells after treatment with gradient concentrations of HFn6-Z and wtHFn-Z . c, Cell viability assays of U-87 MG cells after EGFR EGFR treatment with 300 nM HFn6-Z , wtHFn-Z or equivalent doses of HFn6 or Z . d, EGFR EGFR EGFR Cell viability assays of SKBR3 cells after treatment with gradient concentrations of HFn6- PEP and wtHFn-PEP . e, Cell viability assays of SKBR3 cells after treatment with 300 HER2 HER2 nM HFn6-PEP , wtHFn-PEP or equivalent doses of HFn6 or PEP . HER2 HER2 HER2\n5. The enhanced affinity of HFn6 for TfR1 via mutagenesis is a key innovation. However, the rationale for selecting specific residues (D15, F81, Q83, N109, K119, D123) in the TfR1-binding interface remains unclear. Please explicitly justify the selection of these residues (e.g., structural analysis, computational predictions, or prior literature). In Extended Data Fig. 1b, include computational data (e.g., binding free energy changes, hydrogen bond/electrostatic interactions) for each mutation. In Fig. 1b (mislabeled as Fig. 1c in the text), highlight the N109R and K119R mutation sites in the molecular docking interface, annotate bond energies, and provide the PDB ID of the HFn-TfR1 complex in the figure legend or Methods. Simplify Extended Data Fig. 1a: Use a single panel with “front view”; and “rotated 180° view”; to illustrate the overall binding mode, labeling key interacting residues.\nResponse: We thank the reviewer for the valuable comments and suggestions. The selection of HFn mutation sites was based on the analysis of the structure of the HFn and TfR1 ectodomain complex. The N-terminal and BC loop regions of HFn interact with the apical domain of TfR1 (Fig. 2a, b). The structural reorganization caused by complex formation is limited to the side chains that are in direct contact with amino acids, resulting in a relatively low affinity between the two. Therefore, it is theoretically possible to increase the affinity of HFn for TfR1 by strengthening the interaction between the side chains of amino acids at the binding interface of HFn and TfR1 or by introducing more amino acid residues involved in the interaction. Consequently, residues D15, F81, Q83, N109, K119 and D123, which are located in the vicinity of the interaction region and have no or limited interactions with the TfR1 molecule, were selected for mutation. These mutations were designed to either form additional hydrogen bonds between HFn and TfR1 (F81Y, D123E, N109R and K119R) (Fig. 2c and Supplementary Fig. 1a) or enhance van der Waals interactions within the hydrophobic microenvironment (D15L and Q83H) (Supplementary Fig. 1a). Relevant descriptions have been added to page 6 of the revised manuscript and are marked in red.\nThe structures of the HFn mutants were predicted via AlphaFold3 and superimposed onto the HFn molecule in the cryo-EM structure (PDB ID: 6H5I). The root-mean-square deviations (r.m.s.d.) in the positions of the carbon α atoms between the mutants and wtHFn ranged from 0.54 to 0.58 Å, indicating a high degree of structural similarity. In the N109R mutant, the amide group of R109 forms a hydrogen bond with the oxygen atom of E294 in TfR1, whereas in the K119R mutant, the amide group of R119 forms a hydrogen bond with the oxygen atom of Q197. The interaction interface and solvation free energy (ΔiG) gain upon interface formation, which is based on the HFn mutant–TfR1 complex structures, were predicted via PDBePISA software (Only for reviewer table 1). Unfortunately, we did not find any relationship between the solvation free energy gain upon formation of the interface (ΔiG) and the affinity obtained from the Biacore test, probably because the ΔiG value calculated with PDBePISA software does not include the effect of satisfactory hydrogen bonds and salt bridges across the interface, which is the mutation target in this experiment.\nThe predicted interface area between the HFn mutant and TfR1 seems to be consistent with the Biacore result. Compared with those of wtHFn, the interface areas between the two molecules of the complex increased in the HFn1, HFn2, HFn4 and HFn6 mutants and varied slightly in the HFn3 and HFn5 mutants. Those mutants whose interface area increased also achieved increased affinity in the Biacore test. HFn3 and HFn5, whose interface areas are comparable to that of wtHFn, have decreased affinities. We hypothesize that this phenomenon may be due to the inaccurate prediction of the structure by the Alpha Fold. Despite the enormous applications of alpha fold software in the field of structural prediction, its accuracy in predicting the protein flexible region still needs to be improved. Unfortunately, in our case, the HFn mutation sites lie in the flexible region. Experimental structure determination is time-consuming, and further computational prediction and computations based on a predicted structure are inaccurate. Therefore, we employed SPR technology to measure the actual affinity of the HFn mutants and TfR1 receptor to screen high-affinity proteins.\nWe sincerely appreciate the valuable suggestions made by the reviewers regarding the presentation of the figures. We have optimized the images and illustrations in the manuscript. The TfR1 molecule is shown in cyan, whereas wtHFn and its mutants as well as the related residues are presented in light blue and light brown, respectively. The residues for mutation are highlighted with red borders.\nFigure 2. Cryo-EM structure of the wtHFn and TfR1 ectodomain complex and the residues of wtHFn selected for mutation. a, Cryo-EM structure of the wtHFn and TfR1 ectodomain complex (PDB code: 6h5i) with a close-up view of the interacting region. b, wtHFn and TfR1 ectodomain complex after 180° rotation with a close-up view of the interacting region. c, Stick representation of the interaction regions of TfR1 and wtHFn or the HFn6 mutant. The TfR1 molecule is shown in cyan, whereas wtHFn and HFn6 are presented in light blue and light brown, respectively.\nSupplementary Figure 1. Construction and characterization of the HFn-LYTAC platform. c, Details of the interaction interfaces of the TfR1 ectodomain and wtHFn or HFn1-5 mutants. The TfR1 molecule is shown in cyan, whereas wtHFn and HFn mutants are presented in light blue and light brown, respectively.\nOnly for reviewer table 1. Predicted interaction interface and solvation free energy of HFn mutants–TfR1\nInterface ΔiG\nMutation site K (nM)\nd\narea (Å2) (kcal/mol)\nwtHFn ———— 113.0 884.0 -2.4\nHFn1 D15L 38.7 936.8 -4.7\nHFn2 F81Y 54.5 920.5 -3.3\nHFn3 Q83H 415.0 863.0 -2.9\nHFn4 N109R 29.8 941.1 -3.2\nHFn5 D123E 250.0 886.6 -3.6\nHFn6 N109R, K119R 20.0 961.8 -2.3\n6. The authors propose that HFn-LYTAC mediates target protein degradation through two mechanisms: TfR1 dependent clathrin mediated endocytosis and multivalent interaction driven endocytosis. However, when exploring the multivalent mechanism of action, the authors only verified the dominance of the first mechanism through chlorpromazine (CPZ, clathrin inhibitor) experiments, and did not give a clear explanation for the experimental results of caveolin dependent endocytosis inhibitors (such as methyl-β-cyclodextrin, m-β-cd) and microcytosis inhibitors (amiloride). This leads to the following key questions not being answered: does multivalent interaction depend on caveolin or the microcytosis pathway? If not, is the multivalent interaction driven endocytosis through other unexplored mechanisms (such as non clathrin / non caveolin dependent pathways)? In addition, the specific contributions of the two mechanisms lack quantitative analysis.\nResponse: We thank the reviewer for raising this important point. We have further elaborated on the results: treatment of cells with a caveolin-dependent endocytosis inhibitor (M-β-CD) or a macropinocytosis inhibitor (AMI) did not significantly affect HFn-LYTAC-mediated HER2 degradation, indicating that HFn-PEP does not enter cells via the caveolin-dependent HER2 endocytic or macropinocytic pathways (Fig. 4j).\nTo investigate the endocytic pathway involved in HER2 degradation mediated by the second mechanism, we constructed pFn-PEP , which does not interact with TfR1, and coincubated HER2 it with SKBR3 cells pretreated with various endocytosis inhibitors. The degradation of HER2 was then assessed. The results demonstrate that the second degradation mechanism also relies on clathrin-mediated endocytosis for the internalization and subsequent degradation of POI (Fig. 4l). In addition, both mechanisms require lysosomes for POI degradation (Fig. 4k, m).\nTo further investigate the contribution of the TfR1-dependent degradation mechanism to HFn6-LYTAC-mediated POI degradation, confocal microscopy was employed to visualize and analyze the subcellular distribution of TfR1 during HER2 internalization mediated by HFn6- PEP . Following a 2- to 4-hour treatment of SKBR3 cells with HFn6-PEP , both HER2 HER2 HER2 and TfR1 internalization were clearly observed, and the internalized HER2 and TfR1 were significantly colocalized (Fig. 4f and Supplementary Fig. 5d). This finding indicates that under our experimental conditions, the TfR1-dependent degradation mechanism plays a dominant role in the HER2 degradation process mediated by HFn6-PEP . However, when competitive HER2 inhibition was achieved with HFn6 or when pFn was used to prevent binding to TfR1, HFn-LYTAC retained a weak ability to degrade POI (Fig. 4e, g-i). Therefore, we propose that the two mechanisms through which HFn-LYTAC mediates POI degradation constitute a dynamic process: the TfR1-dependent degradation mechanism plays a dominant role, and multivalent interaction-driven endocytosis is initiated only when HFn6 binding to TfR1 is blocked.\nRelevant discussions have been added to the revised manuscript and are marked in red:\n“To further investigate the contribution of the TfR1-dependent degradation mechanism to HFn6-LYTAC-mediated POI degradation, ……the TfR1-dependent degradation mechanism plays a dominant role, and multivalent interaction-driven endocytosis is initiated only when HFn6 binding to TfR1 is blocked.”\nFigure 4. Mechanisms of HFn-LYTAC-mediated target degradation. e, Degradation of total HER2 in SKBR3 cells following 24 hours of coincubation with HFn6-PEP and HFn6. f, HER2 Representative fluorescence images of the colocalization of HER2 (purple) with TfR1 (red) and HFn-LYTAC (green) in SKBR3 cells via confocal microscopy after treatment with 300 nM HFn6-PEP for 0 min, 5 min, 2 h or 4 h. Scale bars, 20 μm. g-i, Comparison of the HER2 degradation efficiency of HER2 (g), EGFR (h), and PD-L1 (i) mediated by LYTACs constructed with HFn6, wtHFn, and pFn. j, Western blotting analysis of HER2 degradation in SKBR3 cells incubated with 300 nM HFn6-PEP for 24 h following the inhibition of HER2 different endocytosis pathways. k, Western blotting analysis of HER2 degradation in SKBR3 cells incubated with 300 nM HFn6-PEP for 24 h following the inhibition of lysosomes. HER2 l, Western blotting analysis of HER2 degradation in SKBR3 cells incubated with 300 nM pFn- PEP for 24 h following the inhibition of different endocytosis pathways. m, Western HER2 blotting analysis of HER2 degradation in SKBR3 cells incubated with 300 nM pFn-PEP HER2 for 24 h following the inhibition of lysosomes. Supplementary Figure 5. d, Representative line plot histograms of SKBR3 cells treated with 300 nM HFn6-PEP for 4 h. HER2\n7. The optimal conjugation ratios vary significantly across targets (e.g., 12:1 for EGFR/HER2 vs. 6:1 for PD-L1). The authors analyzed whether it might be related to the size of HFn-LYTAC (Extended Data Fig. 3b), and whether it is related to the expression level of target proteins? It is suggested to supplement the analysis of the influence of target protein expression level on coupling efficiency, so as to provide guidance for subsequent target selection.\nResponse: We thank the reviewer for raising this important point. Since all HFn6-LYTACs are constructed using the same HFn6 scaffold, variations in their diameters are attributed primarily to differences in the molecular weights and structures of the POI ligands.\nAlthough HFn6-FN3 has a relatively large diameter, we propose that a difference of 1– PD-L1 2 nm does not significantly influence the optimal conjugation ratio. Furthermore, analysis of POI and TfR1 expression levels across various cell lines from the CCLE database (Only for reviewer Table 2) revealed no clear correlation between POI expression levels and the optimal coupling ratio of HFn-LYTACs.\nTo further investigate this observation, we assessed the binding affinity of the HFn6:FN3 PD- L1 conjugates at conjugation ratios of 1:9 and 1:12 for TfR1 (Only for reviewer Fig. 2). The results demonstrated that neither conjugate exhibited notable affinity. These findings support the conclusion that the affinity of HFn-LYTAC for TfR1 plays a critical role in determining the optimal coupling ratio. As the number of attached POI ligands increases, steric hindrance reduces the affinity of HFn6 for TfR1. Consequently, while maintaining sufficient affinity for TfR1, increasing the number of POI ligands enhances the chance of HFn-LYTAC binding to the POI, thereby improving its degradation efficiency. However, further increases in the POI ligand incubation ratio introduce steric hindrance, which could interfere with the interaction between HFn6 and TfR1.\nMoreover, at various POI ligand incubation ratios, free POI ligands in solution competitively inhibited HFn-LYTAC. Owing to having the highest molecular weight among the ligands, FN3 has a more pronounced steric hindrance effect, resulting in a lower optimal PD-L1 conjugation ratio than those of Z and PEP . Relevant discussions have been added to the EGFR HER2 revised manuscript and are marked in red:\n“HFn-LYTAC demonstrated optimal EGFR degradation efficacy at Z to HFn6 EGFR incubation ratios of 12:1 and 15:1. This is because an increase in the number of Z EGFR molecules coupled to HFn enhances the chance of HFn-Z binding to EGFR on the cell EGFR surface, thereby improving its capacity to degrade EGFR. However, further elevation of the Z incubation ratio introduces steric hindrance, which could interfere with the interaction EGFR between HFn6 and TfR1. Moreover, at high Z incubation ratios, free Z in solution EGFR EGFR competitively inhibits HFn6-Z . Consequently, as the Z incubation ratio continues to EGFR EGFR rise, the degradation efficiency of HFn-Z toward EGFR gradually decreases.” EGFR\nOnly for reviewer table 2. Expression levels of POI and TfR1 in different cells.\nPOI TfR1\nCell Line POI Expression Value Expression value\n(log (TMP+1)) (log (TMP+1))\n2 2\nU-87 MG 5.10 8.33\nEGFR\nMDA-MB-231 5.72 7.10\nSKBR3 10.84 7.76\nHER2\nNCI-N87 11.76 7.17\nHT1080 5.04 7.25\nPD-L1\nMDA-MB-231 4.34 7.16\nOnly for reviewer figure 2. SPR sensorgrams of the TfR1-immobilized chip injected with\nHFn6-FN3 1:9 (a) and HFn6-FN3 1:12 (b).\nPD-L1 PD-L1\n8. Discrepancies between figure citations and text descriptions (e.g., Fig. 1c vs. Fig. 1b; missing Extended Data Fig. 3a discussion) reduce clarity. Ensure all figure citations align with the text (e.g., correct Fig. 1c to Fig. 1b; in the Results section). Describe Extended Data Fig. 3a in the main text. Also, there is no Fig. 1e.\nResponse: Thank you for raising these questions, and we apologize for the confusion. We have corrected the figure citations and provided an additional description for Extended Data Fig. 3a (now labeled Supplementary Fig. 1e in the revised manuscript).\n“Notably, HFn6-FN3 has a relatively large hydrodynamic diameter, which is attributed to PD-L1 the largest molecular weight of the FN3 ligand (Supplementary Fig. 1e). This observation PD-L1 suggests that, compared with other ligands, FN3 has a more pronounced steric hindrance PD-L1 effect.”\nSupplementary Figure 1. Construction and characterization of the HFn-LYTAC platform.\ne, SDS‒PAGE gel images of HFn-LYTACs utilized in both in vitro and in vivo studies.\n9. Extended Data Figure 7i, it seems that the tumor accumulation of HFn-ZEGFR is weaker than that of ZEGFR.\nResponse: Thank you for raising this question, and we apologize for the confusion. There is a labeling error in this figure: the labels of the HFn-Z group and the Z group are EGFR EGFR misplaced. This has been corrected.\nSupplementary Figure 7. Evaluation of the biocompatibility and targeting efficacy of HFn-LYTAC. d, Ex vivo fluorescence images of brains from U-87 MG orthotopic xenograft mice at 1 h, 4 h and 24 h after i.v. injection of Z or HFn6-Z . EGFR EGFR\n---------------------------------------------------------------------------------------------------\nReviewer #2 (Remarks to the Author):\nIn this work, Zhang et al. add to the scientific repertoire of lysosomal-degrading extracellular targeted protein degradation (eTPD) with a platform using an affinity-matured heavy chain ferritin complex conjugated to binders to POI via SpyTag/SpyCatcher. Use of ferritin is intriguing for target uptake and BBB penetration in the TPD field and the multimeric complex has some potential advantages. The authors performed well-constructed studies to investigate the clearance of multiple protein targets in vitro including assessments of clearance mechanism. While the declared modularity of the system still requires design and expression of each POI binder fusion, the in vivo data including tumor suppression and target degradation with an orthotopic brain tumor is exciting to see.\nResponse: We sincerely appreciate your recognition of our work. This has greatly assisted us in emphasizing the significance of our manuscript more effectively.\nMajor comments:\n1. The prose and figures frequently are mislabeled or unclear regarding POI removal from the cell surface (e.g., the flow method) versus degradation of POI (e.g., Western). These are both biologically meaningful but are quite different phenomena. This needs to be corrected and clear throughout the manuscript.\nResponse: We thank the reviewer for raising this important point, which will enhance the rigor of the article. In this study, fluorescently labeled antibodies and flow cytometry were utilized to quantify the expression level of the target protein (POI) on the surface of tumor cells, thereby evaluating the clearance efficacy of HFn-LYTACs on membrane-bound POI. Concurrently, Western blotting analysis was performed using whole-cell lysates to assess the intracellular degradation efficiency of total target protein mediated by HFn-LYTACs. We have updated the annotations in the figures and the corresponding descriptions in the manuscript to improve the accuracy and scientific rigor of the presented data.\n2. Investigation of effects of the constructs on TfR biology could be improved. There is no quantification of effect on surface or total TfR levels after treatment. The authors do not consider the effect on reticulocytes nor discuss hemosiderin.\nResponse: We thank the reviewer for raising this important point. In vitro experiments demonstrated that HFn-LYTAC did not significantly alter total TfR1 protein levels in cells, as confirmed by Western blotting (Supplementary Fig. 2g-l). For in vivo experiments, we supplemented the immunofluorescence experiment with tumor tissue sections to detect the expression level of TfR1 in tumor tissues. The results indicated that there was no statistically significant difference in TfR1 expression between the HFn-LYTAC treatment group and the PBS control group (Fig. 5i and Fig. 6h).\nPrussian blue staining of liver tissue sections revealed that hemosiderin did not significantly alter iron deposition levels in liver tissue, indicating that HFn6-PEP does not affect HER2 hemosiderin content (Supplementary Fig. 7a).\nIn addition, flow cytometry was used to quantify the proportion of reticulocytes (RET) in the peripheral blood of mice across different treatment groups. The results demonstrated that there was no statistically significant variation in reticulocyte levels among the groups (Supplementary Fig. 7b). The relevant results and discussions have been added to the revised manuscript.\nSupplementary Figure 2. The HFn-LYTAC platform promotes the degradation of various targets. g, h, Quantification of TfR1 levels from Fig. 3d, e, in U-87 MG cells (g) and MDA-MB-231 cells (h) treated with 300 nM HFn-LYTAC or an equivalent dose of HFn or Z . i, EGFR j, Quantification of the TfR1 level shown in Fig. 3k, l in SKBR3 cells (i) and NCI-N87 cells (j) treated with 300 nM HFn-LYTAC or an equivalent dose of HFn or PEP . k, l, Quantification HER2 of TfR1 levels from Fig. 3r in HT1080 cells (k) and MDA-MB-231 cells (l) treated with 300 nM HFn-LYTAC or an equivalent dose of HFn or FN3 . PD-L1 Figure 5. Evaluation of the in vivo antitumor efficacy and safety of the HFn-LYTAC platform. i, The expression level of TfR1 in tumor tissue was determined by immunofluorescence. Tumors were harvested on day 21. Scale bars, 100 μm.\nFigure 6. Evaluation of the HFn-LYTAC platform for the treatment of glioblastoma. h, H&E staining of brain tissue sections after treatment and IF imaging of tumor tissue were performed to assess the expression levels of EGFR and TfR1. Tumors were harvested on day 18. Scale bars, 40 μm.\nSupplementary Figure 7. Evaluation of the biocompatibility and targeting efficacy of HFn-LYTAC. a, Representative images of Prussian blue iron staining in liver tissues from different treatment groups. Scale bars, 200 μm. b, Flow cytometric analysis of reticulocytes in peripheral blood at 24 h after i.v. injection of different formulations.\n3. Given the multimeric nature of the complexes, potential pathway activation by multimerization of POI should be investigated.\nResponse: We thank the reviewer for raising this important point. Previous studies have reported that EGFR degradation results in reduced levels of p-AKT in its downstream signaling pathway (Ref 3). This effect was confirmed following the treatment of U-87 MG cells with HFn6-Z . Owing to differences in total EGFR degradation capacity, the level of p-AKT in EGFR U-87 MG cells treated with wtHFn-Z was slightly greater than that in those treated with EGFR HFn6-Z (Supplementary Fig. 3a). The relevant results and discussions have been added to EGFR the revised manuscript.\n“In addition, previous studies have reported that EGFR degradation results in reduced levels of p-AKT in its downstream signaling pathway31. This effect was confirmed following the treatment of U-87 MG cells with HFn6-Z . Owing to differences in total EGFR degradation EGFR capacity, the level of p-AKT in U-87 MG cells treated with wtHFn-Z was slightly greater EGFR than that in those treated with HFn6-Z (Supplementary Fig. 3a).” EGFR\nSupplementary Figure 3. a, Western blotting and quantification of EGFR and p-AKT in U-87 MG cells following incubation with 300 nM HFn-Z for 24 h. EGFR\nRef 3 Yao, N. et al. Discovery of a novel EGFR ligand DPBA that degrades EGFR and suppresses EGFR-positive NSCLC growth. Signal Transduction and Targeted Therapy 5, 214, doi:10.1038/s41392-020-00251-2 (2020).\nMinor comments:\n1. 300 nM of HFn-LYTAC is a high concentration (with total in vivo doses ~30 mg/kg) implying lower potency versus some other eTPD approaches.\nResponse: We thank the reviewer for raising this concern. Owing to the large molecular weight of HFn-LYTACs (approximately 720 kDa on average), its dosage in this study (total protein dose: ~30.6 mg/kg (10 mg/kg POI ligand + 20.6 mg/kg HFn6SpyTag)), when converted to an equivalent molar concentration (≈42 nmol/kg), corresponds to approximately 7 mg/kg of TransTAC or 65 μg/kg of PepTAC. Therefore, when compared on a molar dose basis, the dosage of HFn-LYTACs is relatively low (TransTAC: 10 mg/kg (Ref 4); PepTAC: 3.9 mg/kg (Ref 5)). Besides, the safety evaluation demonstrated that the administered dosage is well tolerated and poses no significant safety concerns. Relevant discussions have been added to page 23 of the revised manuscript and are marked in red.\nIn addition, endocytic uptake analysis of HFn-LYTACs revealed that RAW 264.7 cells can rapidly internalize HFn-LYTACs, suggesting that monocytes in circulation may influence the pharmacokinetic behavior of HFn-LYTACs. If the rapid clearance of HFn-LYTACs by monocytes can be mitigated, it may be possible to further reduce the required dosage.\nRef 4 Zhang, D., Duque-Jimenez, J., Facchinetti, F. et al. Transferrin receptor targeting chimeras for membrane protein degradation. Nature 638, 787–795 (2025).\nRef 5 Xiao, Y., He, Z., Li, W. et al. A covalent peptide-based lysosome-targeting protein degradation platform for cancer immunotherapy. Nat Commun 16, 1388 (2025).\n2. The authors demonstrate target clearance in vitro, target clearance in vivo, and tumor growth inhibition in vivo, but they do not show data connecting the dots between target clearance and in vivo growth inhibition (such as pathway inhibition or specific growth inhibition in vitro, etc.).\nResponse: We thank the reviewer for raising this important point. We investigated the in vitro effects of HFn-LYTACs on the proliferation of tumor cells.\nBoth HFn6-Z and HFn6-PEP effectively inhibited the proliferation of U-87 MG cells EGFR HER2 and SKBR3 cells (Supplementary Fig. 3b, d). We also evaluated the inhibitory effect of wtHFn-LYTAC on cell proliferation. Owing to differences in total EGFR degradation capacity, wtHFn- Z exhibited a relatively weak cytotoxic effect, as evidenced by an IC value more than EGFR 50 twice that of HFn6-Z (Supplementary Fig. 3c). Since the ability of wtHFn-PEP and EGFR HER2 HFn6-PEP to degrade HER2 is similar, their inhibitory effects on the proliferation of HER2 SKBR3 cells are also comparable. (Supplementary Fig. 3e). Relevant results and descriptions have been added to the revised manuscript and are marked in red:\n“The IC value of HFn6-Z in U-87 MG cells was 24.2 nM, which was less than half that 50 EGFR of wtHFn-Z (57.26 nM). Equivalent doses of HFn6 or Z did not demonstrate cytotoxic EGFR EGFR effects.”\n“Given that they have the same HER2 degradation capacity, their IC values for SKBR3 cells 50 are similar. The IC of HFn6-PEP was 6.78 nM, and that of wtHFn-PEP was 9.29 nM.” 50 HER2 HER2\nSupplementary Figure 3. HFn-LYTAC suppresses cell proliferation through targeted degradation of POI. b, Cell viability assays of U-87 MG cells after treatment with gradient concentrations of HFn6-Z and wtHFn-Z . c, Cell viability assays of U-87 MG cells after EGFR EGFR treatment with 300 nM HFn6-Z , wtHFn-Z or equivalent doses of HFn6 or Z . d, EGFR EGFR EGFR Cell viability assays of SKBR3 cells after treatment with gradient concentrations of HFn6- PEP and wtHFn-PEP . e, Cell viability assays of SKBR3 cells after treatment with 300 HER2 HER2 nM HFn6-PEP , wtHFn-PEP or equivalent doses of HFn6 or PEP . HER2 HER2 HER2\n3. The authors describe their molecules as “LYTACs”, which have been described in the literature for ASGPR- and M6PR-mediated uptake, whereas other groups have used other platform names when driving uptake by receptors such as TfRs, cytokine receptors, sortilin receptors, etc. The eTPD space is nascent; thus, nomenclature is evolving. The authors and editors should consider what nomenclature is most appropriate for these HFn constructs.\nResponse: We thank the reviewer for raising this important point. In the current publications, some TPDs are named according to lysosome-targeting receptors (e.g., TfR1—TransTAC; cytokine receptor—KineTAC), whereas others are designated based on their compositional elements (e.g., aptamer LYTAC for nucleic acid aptamers, Nano-LYTAC for polymeric nanomaterials, and peptide-based Pep-TAC). However, all such degraders facilitate targeted degradation by delivering the POI to the lysosome via lysosome-targeting receptor-mediated endocytosis. Consequently, this study retains the current naming convention.\n4. The authors should point out that the BBB was not verified to be intact in their orthotopic U87 model.\nResponse: We thank the reviewer for raising this important point. Since orthotopic tumors are constructed by puncturing brain tissue and then injecting tumor cells, there is currently no evidence to suggest that the blood‒brain barrier has not been disrupted. We have added supplementary explanations to the article as follows:\n“Moreover, an orthotopic xenograft mouse model of tumors was established using U-87 MG cells expressing luciferase. Fluorescence imaging of the brains of U-87 MG orthotopic xenograft mice demonstrated that HFn6-Z successfully traversed the mouse BBB and EGFR accumulated in glioblastoma. Although the establishment of orthotopic xenograft mice may partially compromise the integrity of the BBB, the accumulation of HFn6-Z in glioblastoma EGFR was more than twice that of Z 1 h after i.v. injection, suggesting that HFn6-Z possesses EGFR EGFR the ability to cross the BBB and specifically target brain tumors(Fig. 6c and Supplementary Fig. 7d).”\n5. Figure legends/labels/descriptions need more details throughout. Test article (active and controls) concentration, timepoint, in vivo dose levels of all components for all experiments, y axis readout is often missing or unclear. Timepoints sometimes do not match between text and figure legend. Figure 4 are the n independent experiments or the number of animals per group? Figure 2 what is Control in flow? Is labeling on Figure 4i /Ext Data Fig 7i correct? Add more statistics to Fig. 4b.\nResponse: We thank the reviewer for raising this question and apologize for the confusion. We have improved the figure legends to increase readability in the revised manuscript. In the results of the animal experiments, n represents the number of animals in each group. The flow cytometry results revealed that the control group represented the unstained control. There is a labeling error in Extended Data Fig. 7i, where the labels of the HFn-Z group and the Z group were misplaced. This has been corrected. (Supplementary Fig. 7d in the revised EGFR EGFR manuscript). For Fig. 4b in the original manuscript, given that the shedding of the fluorescent group could compromise the accuracy of in vivo characterization of HFn-LYTAC, we transitioned to Western blotting analysis for more reliable detection and accordingly updated the relevant data (Fig. 5a, b, c).\nSupplementary Figure 7. Evaluation of the biocompatibility and targeting efficacy of HFn-LYTAC. d, Ex vivo fluorescence images of brains from U-87 MG orthotopic xenograft mice at 1 h, 4 h and 24 h after i.v. injection of Z or HFn6-Z . EGFR EGFR\nFigure 5. Evaluation of the in vivo antitumor efficacy and safety of the HFn-LYTAC platform. a, Plasma half-life of HFn6-PEP determined by Western blotting. b, The levels HER2 of HFn6-PEP in tumor, heart, liver, spleen, lung and kidney tissues were determined by HER2 Western blotting. Samples were collected and prepared from each group 24 h after i.v. injection of HFn- PEP . HER2 c, The levels of PEP and HFn6-PEP in mouse tumor tissues were HER2 HER2 HER2 determined by Western blotting. Samples were collected and prepared from each group at 1, 4, 8, and 24 hours after i.v. injection of HFn- PEP . HER2\n6. More methods need to be included, including method of measuring half-life, flow cytometry methods for xenografts, fluorescent labeling methods, SPR ligand species, catalog protein and antibody identities. Were antibodies used for flow measurement noncompetitive with the test articles? Clarification re: cross-reactivity to murine TfR and POI is important information for interpretation of in vivo results.\nResponse: We thank the reviewer for raising this important point. We have supplemented the missing experimental methods as well as those corresponding to the newly added experiments. Since the binding site of the POI ligand to the POI remains unidentified, sequence analysis alone cannot confirm the absence of competitive binding between the flow cytometry antibody and the POI ligand. However, we established a control group in which cells were treated solely with the POI ligand. The results revealed no significant difference in the surface expression levels of POI between the untreated group and the POI ligand-treated group (Fig. 3b, c, i, j, p, q). These findings indicate that the POI ligand does not induce internalization or clearance of the POI from the cell surface and further support the conclusion that the flow cytometry antibody and the POI ligand do not compete for binding.\nImportantly, the POI ligand we selected does not interact with the murine POI. Furthermore, HFn does not bind to murine TfR1; rather, it traverses the blood‒brain barrier through its interaction with the TIM2 receptor expressed on mouse brain endothelial cells (Ref 6).\nFigure 3. The HFn-LYTAC platform promotes the degradation of various targets. b, c, Determination of surface EGFR degradation in U-87 MG cells (b) and MDA-MB-231 cells (c) via flow cytometry following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or Z (3.6 μM). i, j, Determination of surface HER2 degradation in EGFR SKBR3 cells (i) and NCI-N87 cells (j) via flow cytometry following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or PEP (3.6 μM). p,q, HER2 Determination of surface PD-L1 degradation in HT1080 cells (p) and MDA-MB-231 cells (q) via flow cytometry following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or FN3 (1.8 μM). PD-L1\nRef 6 Fan, K. et al. Ferritin nanocarrier traverses the blood brain barrier and kills glioma. ACS Nano 12, 4105-4115, DOI: 10.1021/acsnano.7b06969.\n7. The references for use of TfR for eTPD lists only one of three contemporaneous reports (doi.org/10.1038/s41586-024-07947-3, doi.org/10.1038/s41586-024-07948-2, doi.org/10.1038/s41467-024-52975-2).\nResponse: We thank the reviewer for raising this important point. We added these references to the Introduction section.\n“TfR1 is widely overexpressed in tumor cells and has been identified as a lysosomal-targeting receptor for developing TPD agents20,21,24,25.”\n8. Tolerability or Safety is a more accurate description of the experiments than Biocompatibility or Biosafety.\nResponse: We thank the reviewer for raising this important point. We have corrected the relevant descriptions in the manuscript.\n9. Conclusions might be clarified: In Fig. 3, multivalent POI binding without TfR binding reduces target ~36-50% and thus the addition of TfR binding increases degradation by ~13-30% - indicating multivalent POI binding may be the dominant mechanism with TfR-mediated uptake being secondary. In addition, the knockdown of TfR is not complete so it is not surprising that it did not abolish all degradation. Steric hindrance is a good hypothesis but not proven as the cause of reduced clearance/conjugation at higher binder ratios. HFn6 has the most but not the only koff reduction.\nResponse: We thank the reviewer for raising this important point. The specific contributions of these two mechanisms indeed require quantitative analysis. However, we believe that the contribution degrees of the two mechanisms are not simply linearly superimposed.\nTo further investigate the contribution of the TfR1-dependent degradation mechanism to HFn6-LYTAC-mediated POI degradation, confocal microscopy was employed to visualize and analyze the subcellular distribution of TfR1 during HER2 internalization mediated by HFn6- PEP . Following a 2- to 4-hour treatment of SKBR3 cells with HFn6-PEP , both HER2 HER2 HER2 and TfR1 internalization were clearly observed, and internalized HER2 and TfR1 were significantly colocalized (Fig. 4f and Supplementary Fig. 5d). This finding indicates that under our experimental conditions, the TfR1-dependent degradation mechanism plays a dominant role in the HER2 degradation process mediated by HFn6-PEP . However, when competitive HER2 inhibition was achieved with HFn6 or when pFn was used to prevent binding to TfR1, HFn-LYTAC retained a weak ability to degrade POI (Fig. 4e, g-i). Therefore, the two mechanisms through which HFn-LYTAC mediates POI degradation constitute a dynamic process: the TfR1- dependent degradation mechanism plays a dominant role, and multivalent interaction-driven endocytosis is initiated only when HFn6 binding to TfR1 is blocked.\nKnockdown of TfR1 has been reported to significantly impair cell growth and suppress clonogenic ability (Ref 7). In our experience, complete knockdown of TfR1 significantly affects cell viability, leading to cell death within two generations of passage. Under such circumstances, the results obtained are considered unreliable. Notably, TfR1-mediated HFn endocytosis has a threshold effect (Supplementary Fig. 4f), and knocking down TfR1 in 70% of cells is sufficient to significantly reduce HFn6 internalization by cells. On the basis of these findings, we propose that in TfR1-knockdown SKBR3 cells, HFn6-PEP primarily mediates HER2 degradation HER2 via the second mechanism.\nUnder conditions of high incubation ratios of HFn6 and POI ligand, the reduced degradation efficiency of POI by HFn-LTYAC can be attributed to excessive POI ligand coupling, which interferes with the binding of HFn6 to TfR1 and leads to a predominant reliance on the second degradation mechanism. Given that the second mechanism results in a lower POI degradation capacity than the first mechanism does, the overall degradation level of POI by HFn-LTYAC is significantly diminished under these conditions. Moreover, owing to steric hindrance, at a relatively high ratio of POI ligands to HFn6, not all POI ligands bind to HFn6 (Supplementary Fig. 1b, c, d). Free POI ligands in solution competitively inhibited HFn6-LYTAC. Consequently, as the POI ligand incubation ratio continues to increase, the degradation efficiency of HFn- LYTAC toward POI gradually decreases. Relevant discussions have been added to the revised manuscript and are marked in red:\n“HFn-LYTAC demonstrated optimal EGFR degradation efficacy at Z to HFn6 EGFR incubation ratios of 12:1 and 15:1. This is because an increase in the number of Z EGFR molecules coupled to HFn enhances the chance of HFn-Z binding to EGFR on the cell EGFR surface, thereby improving its capacity to degrade EGFR. However, further elevation of the Z incubation ratio introduces steric hindrance, which could interfere with the interaction EGFR between HFn6 and TfR1. Moreover, at high Z incubation ratios, free Z in solution EGFR EGFR competitively inhibits HFn6-Z . Consequently, as the Z incubation ratio continues to EGFR EGFR rise, the degradation efficiency of HFn-Z toward EGFR gradually decreases.” EGFR\nFigure 4. Mechanisms of HFn-LYTAC-mediated target degradation. e, Degradation of total HER2 in SKBR3 cells following 24 hours of coincubation with HFn6-PEP and HFn6. f, HER2 Representative fluorescence images of the colocalization of HER2 (purple) with TfR1 (red) and HFn-LYTAC (green) in SKBR3 cells via confocal microscopy after treatment with 300 nM HFn6-PEP for 0 min, 5 min, 2 h or 4 h. Scale bars, 20 μm. g-i, Comparison of the HER2 degradation efficiency of HER2 (g), EGFR (h), and PD-L1 (i) mediated by LYTACs constructed with HFn6, wtHFn, and pFn.\nSupplementary Figure 5. d, Representative line plot histograms of SKBR3 cells treated with 300 nM HFn6-PEP for 4 h. HER2\nSupplementary Figure 1. Construction and characterization of the HFn-LYTAC platform.\nb-d, SDS‒PAGE gel images of HFn6SpyTag incubated with SpyCatcher-Z (b), EGFR SpyCatcher-PEP (c) or SpyCatcher-FN3 (d) at various molar ratios for 1 h at room HER2 PD-L1 temperature.\nRef 7 Feng, G. et al. Knockdown of TFRC suppressed the progression of nasopharyngeal carcinoma by downregulating the PI3K/Akt/mTOR pathway. Cancer Cell Int 23, 185 (2023). https://doi.org/10.1186/s12935-023-02995-7\n---------------------------------------------------------------------------------------------------\nReviewer #3 (Remarks to the Author):\nThis manuscript presents a new therapeutic strategy of modular mutant HFn-LYTAC for targeted degradation of pathological POI for cancer therapy. The novelty of the concept introduced here is noteworthy, but there are several critical concerns. In particular, data analysis/interpretation is often stretching, and several remarks/conclusions are made with speculations. Below lists a few specific comments.\nResponse: We sincerely appreciate your recognition of our work. We have revised the manuscript to clarify these issues.\n1. The rationale for selecting the specific target residues (out of the listed candidates) to implement mutations to increase TfR1 binding affinity is not provided.\nResponse: We thank the reviewer for the valuable comments and suggestions. The selection of HFn mutation sites was based on the analysis of the structure of the HFn and TfR1 ectodomain complex. The N-terminal and BC loop regions of HFn interact with the apical domain of TfR1 (Fig. 2a, b). The structural reorganization caused by complex formation is limited to the side chains that are in direct contact with amino acids, resulting in a relatively low affinity between the two. Therefore, it is theoretically possible to increase the affinity of HFn for TfR1 by strengthening the interaction between the side chains of amino acids at the binding interface of HFn and TfR1 or by introducing more amino acid residues involved in the interaction. Consequently, residues D15, F81, Q83, N109, K119 and D123, which are located in the vicinity of the interaction region and have no or limited interactions with the TfR1 molecule, were selected for mutation. These mutations were designed to either form additional hydrogen bonds between HFn and TfR1 (F81Y, N109R, D123E, and K119R) (Fig. 2c, Supplementary Fig. 1a) or strengthen the van der Waals forces within the hydrophobic microenvironment (D15L and Q83H) (Supplementary Fig. 1a). Relevant discussions have been added to the revised manuscript and are marked in red:\n“The affinity between HFn and the TfR1 ectodomain obtained via surface plasmon resonance (SPR) was in the range of 10–7 M (Supplementary Table 1), which was consistent with the affinity between HFn and TfR1 on the cell surface28. Among the HFn residues involved in common contacts (T5, Q14, D15, E17, A18, A19, N21, R22, N25, R79, F81, Q83, K86, K87, and E116) with TfR1, D15, F81, Q83, N109, K119 and D123 were selected for mutation to increase the affinity for TfR1. These residues, located in the vicinity of the interaction region (Fig. 2a, b), had no or limited interactions with the TfR1 molecule. Therefore, they were selected for mutation to form additional hydrogen bonds between HFn and TfR1 (F81Y, N109R, D123E, and K119R) (Fig. 2c, Supplementary Fig. 1a) or strengthen the van der Waals forces within the hydrophobic microenvironment (D15L and Q83H) (Supplementary Fig. 1a).”\nFigure 2. Cryo-EM structure of the wtHFn and TfR1 ectodomain complex and the residues of wtHFn selected for mutation. a, Cryo-EM structure of the wtHFn and TfR1 ectodomain complex (PDB code: 6h5i) with a close-up view of the interacting region. b, wtHFn and TfR1 ectodomain complex after 180° rotation with a close-up view of the interacting region. c, Details of the interaction regions of TfR1 and wtHFn or the HFn6 mutant. The TfR1 molecule is shown in cyan, whereas wtHFn and HFn6 is presented in light blue and light brown, respectively.\nSupplementary Figure 1. Construction and characterization of the HFn-LYTAC platform. c, Details of the interaction interfaces of the TfR1 ectodomain and wtHFn or HFn1-5 mutants. The TfR1 molecule is shown in cyan, whereas wtHFn and HFn mutants are presented in light blue and light brown, respectively.\n2. TfR1 binding affinity of HFn6SpyTag was confirmed (Fig. E2b), but it is unclear whether the affinity is retained when affibodies are attached. To validate the authors’ hypothesis regarding the lack of EGFR degradation at high molar ratios, TfR1 binding affinity and cell uptake should be experimentally determined at the molar ratio range tested in this study.\nResponse: We thank the reviewer for the valuable comments. We employed Biacore technology to assess the binding affinity of HFn6 for both POI and TfR1 following its conjugation with POI ligands (HFn6: Z = 1:12, HFn6: PEP = 1:12, HFn6: FN3 = 1:6). The affinity EGFR HER2 PD-L1 of HFn6SpyTag for TfR1 is 2.17×10-8 M. Our findings demonstrate that POI ligand conjugation reduces the affinity of HFn6 for TfR1, especially in HFn6-Z and HFn6-FN3 EGFR PD-L1 (Supplementary Table 2, red marked). In addition, we assessed the binding affinity of the HFn6:FN3 conjugates at conjugation ratios of 1:9 and 1:12 for TfR1. The results PD-L1 demonstrated that neither conjugate exhibited notable affinity (Only for reviewer Fig. 2). These findings support the conclusion that the affinity of HFn-LYTAC for TfR1 plays a critical role in determining the optimal coupling ratio. As the number of attached POI ligands increases, steric hindrance reduces the affinity of HFn6 for TfR1. Consequently, while maintaining sufficient affinity for TfR1, increasing the number of POI ligands enhances the chance of HFn-LYTAC binding to the POI, thereby improving its degradation efficiency. However, further increases in the POI ligand incubation ratio introduce steric hindrance, which could interfere with the interaction between HFn6 and TfR1. Moreover, at various POI ligand incubation ratios, free POI ligands in solution competitively inhibited HFn-LYTAC.\nFor EGFR degradation, the solubility of HFn6-Z is lower than that of HFn6-PEP and EGFR HER2 HFn6-FN3 . As a result, protein precipitation was observed when HFn6 was incubated with PD-L1 high-molar-ratio Z (1:18–1:24), leading to the absence of EGFR degradation at incubation EGFR ratios of 1:21 and 1:24, as reported in the original manuscript. This issue can be resolved by reducing the protein concentration during incubation. The relevant data have been updated in the revised manuscript (Fig. 2f). Relevant discussions have been added to the revised manuscript and are marked in red:\n“HFn-LYTAC demonstrated optimal EGFR degradation efficacy at Z to HFn6 EGFR incubation ratios of 12:1 and 15:1. This is because an increase in the number of Z EGFR molecules coupled to HFn enhances the chance of HFn-Z binding to EGFR on the cell EGFR surface, thereby improving its capacity to degrade EGFR. However, further elevation of the Z incubation ratio introduces steric hindrance, which could interfere with the interaction EGFR between HFn6 and TfR1. Moreover, at high Z incubation ratios, free Z in solution EGFR EGFR competitively inhibits HFn6-Z . Consequently, as the Z incubation ratio continues to EGFR EGFR rise, the degradation efficiency of HFn-Z toward EGFR gradually decreases.” EGFR\nSupplementary Table 2. Determination of the affinity between HFn-LYTAC and TOI or TfR1 by SPR.\nAnalyte Ligand K (1/Ms) K (1/s) K (M)\non off D\nZ EGFR 5.59x105 9.07x10-4 1.62x10-9\nEGFR\nHFn6-Z EGFR 2.63x105 1.22x10-5 4.63x10-11\nEGFR\nHFn6-Z TfR1 5.08x103 8.38x10-3 1.65x10-6\nEGFR\nwtHFn-Z TfR1 N/A N/A N/A\nEGFR\nPEP HER2 1.71x106 1.45x10-3 8.44x10-10\nHER2\nHFn6-PEP HER2 1.41x106 1.83x10-7 1.30x10-13\nHER2\nHFn6-PEP TfR1 3.14x104 7.46x10-4 2.37x10-8\nHER2\nwtHFn-PEP TfR1 7.71x102 1.04 x 10-4 1.35x10-7\nHER2\nFN3 PD-L1 3.17x106 7.95x10-3 2.51x10-9\nPD-L1\nHFn6-FN3 PD-L1 2.15x106 6.29x10-5 2.93x10-11\nPD-L1\nHFn6-FN3 TfR1 2.41x103 1.05x10-2 4.38x10-6\nPD-L1\nwtHFn-FN3 TfR1 N/A N/A N/A\nPD-L1\nFigure 2. Construction of the HFn-LYTAC platform for target degradation. f, Determination of EGFR degradation in U-87 MG cells via flow cytometry following a 24-hour treatment with a mixture of HFn6SpyTag and SpyCatcher-Z at various molar ratios.\nEGFR\nOnly for reviewer figure 2. SPR sensorgrams of the TfR1-immobilized chip injected with\nHFn6-FN3 1:9 (a) and HFn6-FN3 1:12 (b).\nPD-L1 PD-L1\n3. What are the molecular weights of three affibodies investigated in this manuscript?\nResponse: We thank the reviewer for this valuable question. The original molecular weights of Z , PEP and FN3 are 6.68 kDa, 7.12 kDa and 11.07 kDa, respectively. For the EGFR HER2 PD-L1 construction of HFn-LYTAC, a linker followed by the SpyCatcher domain was introduced to the C-terminus of the POI ligands. Finally, the molecular weights of Z -SpyCatcher, EGFR PEP -SpyCatcher and FN3 -SpyCatcher are 19.9 kDa, 20.2 kDa and 23.2 kDa, HER2 PD-L1 respectively. The molecular weights of the proteins included in the text are listed in the corresponding protein sequence list (Supplementary Table 3).\n4. What are the error values (i.e., SD) and PDI for the DLS results in Fig. E3b?\nResponse: We thank the reviewer for this valuable point. We have supplemented the corresponding data (Supplementary Fig. 1g).\nDiameter (nm) PDI\nHFn6SpyTag 15.702±0.066 0.059±0.003\nHFn6-Z 19.232±0.096 0.151±0.010\nEGFR\nHFn6-PEP 18.530±0.111 0.192±0.012\nHER2\nHFn6-FN3 21.214±0.078 0.114±0.006\nPD-L1\nwtHFnSpyTag 14.608±0.068 0.116±0.007\nwtHFn-Z 19.378±0.097 0.121±0.006\nEGFR\nwtHFn-PEP 17.962±0.108 0.184±0.009\nHER2\nwtHFn-FN3 20.136±0.071 0.143±0.006\nPD-L1\nSupplementary Figure 1 g, Analysis of the size and polydispersity index (PDI) of HFnSpyTag and constructed HFn-LYTACs measured by DLS.\n5. The authors concluded that cytoplasmic EGFR signals were significantly stronger when cells were treated with wtHFn-ZEGFR compared to HFn6-ZEGFR (Fig. 2d/E5) without quantitative and statistical analysis of experimental data.\nResponse: We thank the reviewer for this valuable point. We have provided additional quantitative and statistical analyses of the confocal microscope images via ImageJ (Supplementary Fig. 2n, o, p). The results indicated that cytoplasmic EGFR signals were significantly stronger in cells treated with wtHFn-Z than in those treated with HFn6-Z . EGFR EGFR The relevant data have been updated in the revised manuscript.\nSupplementary Figure 2. Visualization of EGFR degradation in U-87 MG cells via confocal microscopy. n, Representative fluorescence images of U-87 MG cells after treatment with 300 nM HFn6-Z , wtHFn6-Z or an equivalent dose of Z for 24 h. EGFR was EGFR EGFR EGFR stainedwith an anti-EGFR antibody (magenta), and nuclei were stained with DAPI (blue). Scale bars, 60 μm. o, p, Corresponding quantitative data of cytoplasmic (o) and membrane (p) EGFR signals based on n. For o and p, the data are presented as the mean values ± s.d. of n = 15 cells. P values were determined by one-way ANOVA with Fisher’s LSD multiple comparisons test. NS, not significant.\n6. Statistical analysis is missing in Fig. E2.\nResponse: We thank the reviewer for this valuable point. We supplemented the differential analysis by comparing the incubation ratios corresponding to the best degradation efficiency and its neighboring ratios (Fig. 2f-h).\nFigure 2. Construction of the HFn-LYTAC platform for target degradation. f, Determination of EGFR degradation in U-87 MG cells by flow cytometry following a 24-hour treatment with a mixture of HFn6SpyTag and SpyCatcher-Z at various molar ratios. g, EGFR Determination of HER2 degradation in SKBR3 cells by flow cytometry following a 24-hour treatment with a mixture of HFn6SpyTag and SpyCatcher-PEP at various molar ratios. h, HER2 Determination of PD-L1 degradation in HT1080 cells via flow cytometry following a 24-hour treatment with mixtures of HFn6SpyTag and SpyCatcher-FN3 at various molar ratios.\nPD-L1\n7. Are the confocal images in Fig. 3 representative? If so, how many independent cells were observed? Fig. 3c does not clearly show the colocalization of HFn6-PEPHER2/HER2 and HER2/LAMP1.\nResponse: We thank the reviewer for this valuable point. We have updated the confocal images in the revised manuscript. We present large-field images in the supplementary information (Supplementary Fig. 5a). In the 4-hour HFn6-PEP treatment group, more than 25 cells HER2 exhibited HER2 internalization, accounting for approximately 70% of all cells in the field of view. Additionally, line plot histograms of representative cells demonstrated the colocalization of HFn6-PEP and HER2 (Supplementary Fig. 5b). Within the intracellular compartment, HER2 colocalization of HFn6-PEP , HER2, and LAMP1 (a lysosomal marker) was observed. The HER2 relevant data have been updated in the revised manuscript.\nSupplementary Fig. 5. Dynamic colocalization of HER2 with HFn6-PEP and LAMP1 HER2 in SKBR3 cells. a, Representative fluorescence images of the colocalization of HER2 (purple) with lysosomes (LAMP1, red) and HFn-LYTAC (green) in SKBR3 cells via confocal microscopy after treatment with 300 nM HFn6-PEP for 0 min, 5 min, 2 h or 4 h. Scale bars, HER2 40 μm. b, Representative line plot histograms of SKBR3 cells treated with 300 nM HFn6- PEP for 4 h. HER2\n8. The authors speculated cellular trafficking and multivalent interactions of HFn-LYTAC and POI as a mechanism of target protein degradation without convincing experimental validation.\nResponse: We thank the reviewer for this valuable comment. In this study, we constructed wtHFn-LYTAC using wild-type HFn as a control to verify the necessity of building HFn mutants with high TfR1 affinity. In the degradation experiments of EGFR and PD-L1, we found that the degradation ability of HFn6-LYTAC was greater than that of wtHFn-LYTAC. In addition, affinity measurements revealed that owing to steric hindrance, wtHFn-Z and EGFR wtHFn-FN3 exhibited no TfR1 affinity but still retained weak POI degradation ability. PD-L1 Therefore, we propose that HFn-LYTAC involves a non-TfR1-dependent mechanism for degrading POI. Since HFn has nanoscale properties and multiple POI ligands are conjugated on its surface, we propose that the cellular trafficking of nanoparticles and the multivalent binding of POI to HFn-LYTAC constitute the second mechanism by which HFn-LYTAC mediates POI degradation.\nTo validate this, we chose ferritin derived from Pyrococcus furiosus (pFn) to conjugate with the POI ligand and constructed pFn-LYTAC to demonstrate the second degradation mechanism. The chosen PFn does not interact with human TfR1 but possesses a nanostructure similar to that of HFn. As anticipated, pFN-LYTAC was capable of mediating the degradation of HER2, EGFR, and PD-L1, although its degradation efficacy was lower than that of HFn6-LYTAC (Fig. 4g, h, i). These results indicate that HFn-LYTAC can degrade POI through cellular internalization and multivalent binding, with this non-TfR1-dependent mechanism exhibiting reduced degradation potency relative to the TfR1-dependent degradation pathway. Furthermore, we utilized pFn-LYTAC to investigate the endocytic pathway on which the second degradation mechanism relies. The results demonstrate that the second degradation mechanism also relies on clathrin-mediated endocytosis for the internalization and subsequent degradation of POI (Fig. 4l). These figures are shown in Question 9. The relevant content has been supplemented and revised in the revised manuscript:\n“Recent studies have indicated that the cellular trafficking of nanoparticles can be leveraged to design TPD agents.……The endocytic pathways involved in the multivalent binding- mediated endocytic degradation mechanism have also been explored. Inhibition of the clathrin- mediated endocytosis pathway by CPZ led to suppression of HER2 degradation mediated by pFn-PEP , whereas pretreatment with M-β-CD and amiloride had no effect on HER2 HER2 degradation. Similarly, inhibition of lysosomal activity by chloroquine resulted in suppressed HER2 degradation mediated by HFn6-PEP .” HER2\n9. Quantitative comparisons made based entirely on WB analysis without statistical analysis do not convincingly support the authors’ conclusions.\nResponse: We thank the reviewer for this valuable point. Your comment contributed to improving the rigor and quality of our article. We have supplemented the relevant repeated experiments and statistical analysis in the revised manuscript.\nFigure 4. Mechanisms of HFn-LYTAC-mediated target degradation. e, Degradation of total HER2 in SKBR3 cells following 24 hours of coincubation with HFn6-PEP and HFn6. f, HER2 Representative fluorescence images of the colocalization of HER2 (purple) with TfR1 (red) and HFn-LYTAC (green) in SKBR3 cells via confocal microscopy after treatment with 300 nM HFn6-PEP for 0 min, 5 min, 2 h or 4 h. Scale bars, 20 μm. g-i, Comparison of the HER2 degradation efficiency of HER2 (g), EGFR (h), and PD-L1 (i) mediated by LYTACs constructed with HFn6, wtHFn, and pFn. j, Western blotting analysis of HER2 degradation in SKBR3 cells incubated with 300 nM HFn6-PEP for 24 h following the inhibition of HER2 different endocytosis pathways. k, Western blotting analysis of HER2 degradation in SKBR3 cells incubated with 300 nM HFn6-PEP for 24 h following the inhibition of lysosomes. HER2\n10. Have the authors compared HFn-LYTAC and respective affibodies at an equimolar of affibodies for their ability to degrade respective POI in vitro?\nResponse: We thank the reviewer for this valuable question. As shown in Figure 3 of the revised manuscript, all the experiments were conducted with equimolar amounts of POI ligands (affibodies) and HFn as controls to compare their abilities to degrade POI. These results demonstrated that POI ligands alone were insufficient to induce POI degradation. Additionally, we have provided the incubation concentration details of the POI ligands and HFn in the figure legends.\nFigure 3. The HFn-LYTAC platform promotes the degradation of various targets. a, Illustration of EGFR degradation mediated by HFn6-Z . b, c, Determination of surface EGFR degradation in U-87 MG cells (b) and MDA-MB-231 cells (c) via flow cytometry EGFR following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or Z (3.6 μM). d, e, Determination of total EGFR degradation in U-87 MG cells (d) EGFR and MDA-MB-231 cells (e) via western blotting following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or Z (3.6 μM). f, g, Visualization of EGFR EGFR degradation in U-87 MG cells (f) and MDA-MB-231 cells (g) via confocal microscopy following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or Z (3.6 μM). Scale bars, 20 μm. h, Illustration of HER2 degradation mediated by EGFR HFn6-PEP . i, j, Determination of surface HER2 degradation in SKBR3 cells (i) and NCI- HER2 N87 cells (j) via flow cytometry following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or PEP (3.6 μM). k, l, Determination of total HER2 HER2 degradation in SKBR3 cells (k) and NCI-N87 cells (l) via western blotting following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or PEP (3.6 HER2 μM). m, n, Visualization of HER2 degradation in SKBR3 cells (m) and NCI-N87 cells (n) via confocal microscopy following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or PEP (3.6 μM). Scale bars, 20 μm. o, Illustration of PD-L1 HER2 degradation mediated by HFn6-FN3 . p,q, Determination of surface PD-L1 degradation in PD-L1 HT1080 cells (p) and MDA-MB-231 cells (q) via flow cytometry following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or FN3 (1.8 μM). r, s, PD-L1 Determination of total PD-L1 degradation in HT1080 cells (r) and MDA-MB-231 cells (s) via western blotting following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or FN3 (1.8 μM). t,u, Visualization of PD-L1 degradation in HT1080 PD-L1 cells (t) and MDA-MB-231 cells (u) via confocal microscopy following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or FN3 (1.8 μM). Scale PD-L1 bars, 20 μm. For b, c, i, j, p and q, the data are presented as the mean values ± s.d. of n = 3 biological replicates. P values were determined via one-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) multiple comparisons test. NS, not significant. NT, no treatment group.\nThe icons in a, h and o were created with BioRender.com.\n11. The in vivo study in Fig. 4 lacks wtHFn-PEPHERs as a control. Based on the in vitro comparison in Fig. 3f, it is unclear whether the HFn mutation to improve TfR1 binding affinity is needed to enhance tumor delivery and POI degradation in vivo. Overall, it is unclear whether strong/enhanced TfR1 binding affinity is essential for the presented therapeutic strategy based on Fig. 3f and in vitro studies with PFn. Conventional synthetic nanoparticles decorated with high-density surface affibodies may work comparably.\nResponse: We thank the reviewer for this valuable point. A supplementary cell proliferation inhibition assay demonstrated that HFn6-PEP and wtHFn-PEP exhibited comparable HER2 HER2 inhibitory effects on SKBR3 cell proliferation, which was attributed to their similar HER2 degradation capacities (Supplementary Fig. 3d, e). These findings suggest that both HFn6- PEP and wtHFn-PEP may possess analogous therapeutic potential in vivo. However, HER2 HER2 this does not diminish the significance of designing HFn mutants with increased TfR1 affinity.\nFor Z and FN3 , our additional data confirm that enhanced TfR1 affinity is indeed EGFR PD-L1 necessary. Increased TfR1 affinity enhances the degradation efficiency of EGFR (also applicable to PD-L1) (Fig. 4h, i), thereby strengthening its inhibitory effect on tumor proliferation (Supplementary Fig. 3b, c). The design of the HFn6 mutant with enhanced TfR1 affinity makes it a universal scaffold for constructing LYTACs that target different POIs.\nWith respect to the development of targeted protein degraders through the conjugation of POI ligands with synthetic nanomaterials, the Shi team has achieved notable progress (Ref 2). However, as this type of degrader incorporates only POI antibodies, its POI degradation function relies on the second mechanism. Compared with HFn-LYTAC, this approach requires higher dosages and incurs greater costs to achieve comparable degradation efficacy.\nFurthermore, owing to the inherent ability of HFn to cross the blood‒brain barrier, HFn– LYTAC shows promising potential for targeted therapy of brain tumors.\nSupplementary Figure 3. HFn-LYTAC suppresses cell proliferation through targeted degradation of POI. b, Cell viability assays of U-87 MG cells after treatment with gradient concentrations of HFn6-Z and wtHFn-Z . c, Cell viability assays of U-87 MG cells after EGFR EGFR treatment with 300 nM HFn6-Z , wtHFn-Z or equivalent doses of HFn6 or Z . d, EGFR EGFR EGFR Cell viability assays of SKBR3 cells after treatment with gradient concentrations of HFn6- PEP and wtHFn-PEP . e, Cell viability assays of SKBR3 cells after treatment with 300 HER2 HER2 nM HFn6-PEP , wtHFn-PEP or equivalent doses of HFn6 or PEP . HER2 HER2 HER2\nFigure 4. Mechanisms of HFn-LYTAC-mediated target degradation. h, i, Comparison of the degradation efficiency of EGFR (h) and PD-L1 (i) mediated by LYTACs constructed with HFn6, wtHFn, and pFn.\nRef 2 Liu, Y., Liu, R., Dong, J. et al. Targeted protein degradation via cellular trafficking of nanoparticles. Nat. Nanotechnol. 20, 296–302 (2025)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In the revised version of the manuscript, the authors have addressed most of the issues raised in the first round of review. There are still several minor concerns need to be addressed.\n(1) The biodistribution of the LYTAC in the major organs (such as liver, heart, spleen, lung, kidney, and brain) and tumor tissues (better in s.c. inoculated tumor models) should be determined.\n(2) The serum half-life data shown in the rebuttal letter should be provided as the supplementary materials in the manuscript.\n(3) The \"x\" shown in supp Table 1 and Table 2 should be corrected into multiplication sign.", "reviewer_2": "The authors of this manuscript have made significant edits that improve the quality and clarity of the study. However, there are a few points raised in my prior review that could use additional improvement.\n• Major comment 1 (The prose and figures frequently are mislabeled or unclear regarding POI removal from the cell surface (e.g., the flow method) versus degradation of POI (e.g., Western). These are both biologically meaningful but are quite different phenomena. This needs to be corrected and clear throughout the manuscript)\nThe authors have made significant edits that help clarify measurements between surface removal of target (e.g., flow cytometry of intact cells) versus total degradation of target (e.g., Western analysis of lysed cells). However, there are many instances in the prose of the manuscript as well as figure labels and legends where the removal of target from the surface of the cell is equated with degradation of the target.\nWhen a surface receptor is endocytosed off the surface of the cell but is still intact and present in the endosome of the cell, it has not been degraded. It has only been removed from the cell surface. Unless experiments show that the surface-removed POI has in fact been degraded, it must not be described as degraded. It could be described as removed from the surface, surface depleted, or other phrasings, but not as degraded if it may still be a physically intact molecule that has been moved about within the cell.\nMoreover, the authors identify this phenomenon themselves, e.g., p. 10 “These findings indicate that a portion of the EGFR on the cell membrane is internalized into the cell… but remains undegraded”. Yet while the y axis label for Fig 3b correctly describes the measurement as “relative surface EGFR”, the figure legend describes 3b as measuring the the “surface EGFR degradation” and the prose on page 9 and 10 describes it as degradation as well.\nThere are many locations within the manuscript where surface removal is equated with degradation, such as Fig 2e cartoon text, many figure legends, pages 8, 9 10, 14, and more.\nRemoval of the POI from the cell surface is a valuable biological phenomenon to measure, it just needs to not be mischaracterized.\nI would ask the authors to thoroughly ensure that a protein is not described as “degraded” unless they have data showing it has been actually degraded versus potentially just endocytosed.\n• Minor comment 5 (Figure legends/labels/descriptions need more details throughout)\nThe authors have added many details through the manuscript that are very helpful. There are a few more experimental details that should be provided if not present. This is important for ensuring interpretability and reproducibility of the work.\ni. Fig. 4 g, h, i: timepoint?\nii. Fig. 6b: methods, time, concentration for transcytosis measurement?\niii. Supp. Fig. 2 o, p: how was membrane vs cytoplasmic protein determined?\niv. P. 9 (3 c) states there was not a statistically significant reduction of EGFR by ZEGFR or ferritin, but statistics are not provided comparing them to no treatment.\nv. When y axes are labeled as “relative” (e.g., Fig 3, 4, Supp. Fig. 2, 3 4), it would be more clear to say what it is relative to; e.g., relative to no treatment.\n• Minor comment 1 (300 nM of HFn-LYTAC is a high concentration)\nThe author’s addition on p. 28 is helpful, but should at least state that this dosage is equivalent on a MOLAR basis of the entire complex to 6 mg/kg IgG (if one were to calculate the molar equivalent amount of POI binders, it would be quite different).", "reviewer_3": "Most of the raised concerns are reasonably addressed. I have one recommendation. To enhance the reliability of the authors’ conclusion regarding comment #5, it would be ideal to selectively label intracellular EGFR, followed by quantitative analysis."}, "author_response": "Response: We sincerely appreciate your valuable comments. We have revised the manuscript to address these concerns.\n1. The biodistribution of the LYTAC in the major organs (such as liver, heart, spleen, lung, kidney, and brain) and tumor tissues (better in s.c. inoculated tumor models) should be determined.\nResponse: We thank the reviewer for this valuable suggestion. As suggested, we have performed additional expereiments and related results have been shown in Revised Fig. 5b.\nRevised Fig. 5 b, Levels of HFn6-PEP in tumor, heart, liver, spleen, lung, kidney and brain HER2 tissues determined by Western blotting. Samples were collected and prepared from each group 24 h after i.v. injection of HFn-PEP . The data are presented as the mean values ± s.d. of HER2 n = 3 mice per group.\n2. The serum half-life data shown in the rebuttal letter should be provided as the supplementary materials in the manuscript.\nResponse: We thank the reviewer for this valuable suggestion. The data has been added to the Supplementary Information (Supplementary Fig. 7a), and relevant descriptions have been added to the revised manuscript and are marked in red:\n“Blood samples were collected at different time points following intravenous (i.v.) injection of Cy5.5-labeled HFn6-PEP or PEP . Plasma was separated and the fluorescence HER2 HER2 intensity was detected. HFn6-PEP exhibited a prolonged circulation half-life of 131.8 min, HER2 which was more than tenfold longer than that of the PEP control group (9.3 min; HER2 Supplementary Fig. 7a).”\nSupplementary Fig. 7 a, Plasma half-life of HFn6-PEP and PEP was determined using HER2 HER2 Cy5.5-labeled PEP . The data are presented as the mean values ± s.d. of n = 3 mice per group. HER2\n3. The \"x\" shown in supp Table 1 and Table 2 should be corrected into multiplication sign.\nResponse: We thank the reviewer for raising this question, and we apologize for the oversight. The symbol “x” has been corrected into multiplication sign.\nSupplementary Table 1 Determination of the affinity between HFn mutants and TfR1 by SPR.\nMutation site k (1/Ms) k (1/s) K (nM)\non off d\nwtHFn ———— 6.93 × 104 7.84 × 10-3 113\nHFn1 D15L 9.04 × 104 3.50 × 10-3 38.7\nHFn2 F81Y 1.08 × 105 5.89 × 10-3 54.5\nHFn3 Q83H 2.17 × 104 9.00 × 10-3 415\nHFn4 N109R 9.31 × 104 2.77 × 10-3 29.8\nHFn5 D123E 6.07 × 104 1.52 × 10-2 250\nHFn6 N109R, K119R 6.16 × 103 1.23 × 10-4 20\nHFn6SpyTag N109R, K119R 2.79 × 104 6.05 × 10-4 21.7\nSupplementary Table 2 Determination of the affinity between HFn-LYTAC and TOI or TfR1 by SPR.\nAnalyte Ligand K (1/Ms) K (1/s) K (M)\non off D\nZ EGFR 5.59 × 105 9.07 × 10-4 1.62 × 10-9\nEGFR\nHFn6-Z EGFR 2.63 × 105 1.22 × 10-5 4.63 × 10-11\nEGFR\nHFn6-Z TfR1 5.08 × 103 8.38 × 10-3 1.65 × 10-6\nEGFR\nwtHFn-Z TfR1 N/A N/A N/A\nEGFR\nPEP HER2 1.71 × 106 1.45 × 10-3 8.44 × 10-10\nHER2\nHFn6-PEP HER2 1.41 × 106 1.83 × 10-7 1.30 × 10-13\nHER2\nHFn6-PEP TfR1 3.14 × 104 7.46 × 10-4 2.37 × 10-8\nHER2\nwtHFn-PEP TfR1 7.71 × 102 1.04 × 10-4 1.35 × 10-7\nHER2\nFN3 PD-L1 3.17 × 106 7.95 × 10-3 2.51 × 10-9\nPD-L1\nHFn6-FN3 PD-L1 2.15 × 106 6.29 × 10-5 2.93 × 10-11\nPD-L1\nHFn6-FN3 TfR1 2.41 × 103 1.05 × 10-2 4.38 × 10-6\nPD-L1\nwtHFn-FN3 TfR1 N/A N/A N/A\nPD-L1\n---------------------------------------------------------------------------------------------------\nReviewer #2 (Remarks to the Author):\nThe authors of this manuscript have made significant edits that improve the quality and clarity of the study. However, there are a few points raised in my prior review that could use additional improvement.\nResponse: We sincerely appreciate the reviewer's recognition of our revisions. We have revised the manuscript to address these points.\nMajor comment 1 (The prose and figures frequently are mislabeled or unclear regarding POI removal from the cell surface (e.g., the flow method) versus degradation of POI (e.g., Western). These are both biologically meaningful but are quite different phenomena. This needs to be corrected and clear throughout the manuscript)\nThe authors have made significant edits that help clarify measurements between surface removal of target (e.g., flow cytometry of intact cells) versus total degradation of target (e.g., Western analysis of lysed cells). However, there are many instances in the prose of the manuscript as well as figure labels and legends where the removal of target from the surface of the cell is equated with degradation of the target.\nWhen a surface receptor is endocytosed off the surface of the cell but is still intact and present in the endosome of the cell, it has not been degraded. It has only been removed from the cell surface. Unless experiments show that the surface-removed POI has in fact been degraded, it must not be described as degraded. It could be described as removed from the surface, surface depleted, or other phrasings, but not as degraded if it may still be a physically intact molecule that has been moved about within the cell.\nMoreover, the authors identify this phenomenon themselves, e.g., p. 10 “These findings indicate that a portion of the EGFR on the cell membrane is internalized into the cell… but remains undegraded”. Yet while the y axis label for Fig 3b correctly describes the measurement as “relative surface EGFR”, the figure legend describes 3b as measuring the the “surface EGFR degradation” and the prose on page 9 and 10 describes it as degradation as well.\nThere are many locations within the manuscript where surface removal is equated with degradation, such as Fig 2e cartoon text, many figure legends, pages 8, 9 10, 14, and more.\nRemoval of the POI from the cell surface is a valuable biological phenomenon to measure, it just needs to not be mischaracterized.\nI would ask the authors to thoroughly ensure that a protein is not described as “degraded” unless they have data showing it has been actually degraded versus potentially just endocytosed.\nResponse: We sincerely thank the reviewer for this vital clarification on the biological distinction between surface removal and degradation. We fully agree that equating these processes was biologically imprecise. We have made comprehensive revisions throughout the manuscript:\n1. All figure legends describing flow cytometry data (e.g., Figure 2, Figure 3 and Figure 4) now consistently employ the term “surface EGFR/HER2/PD-L1 clearance” and marked in red. Take the following figure as an example:\nRevised Fig. 3 b, c, Determination of surface EGFR clearance in U-87 MG cells (b) and MDA-MB-231 cells (c) via flow cytometry following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or Z (3.6 μM).\nEGFR\n2. References to “degradation” are reserved for WB/confocal data (e.g., Fig. 3)\n3. In the main text of the manuscript, all instances of the term “surface POI degradation” have been revised. For the description of flow cytometry results, the phrase \"reduction of surface POI\" or equivalent expressions are used to reflect decreased target levels on the cell surface. In the context of HFn-LYTAC's action on cell surface POIs, the term \"clearance\" is employed to more precisely convey the process of target protein internalization and subsequent removal from the cell membrane. Take the following text as an example:\n“In U-87 MG and MDA-MB-231 cells, the surface EGFR levels were significantly reduced by an average of 61% and 75%, respectively (Fig. 3b, c). Equivalent doses of Z or EGFR ferritin (wtHFn and HFn6) resulted in only a minor reduction in cell surface EGFR levels (<15% in U-87 MG cells and <20% in MDA-MB-231 cells). However, wtHFn-Z also EGFR demonstrated comparable efficacy in the clearance of surface EGFR.”\n• Minor comment 5 (Figure legends/labels/descriptions need more details throughout)\nThe authors have added many details through the manuscript that are very helpful. There are a few more experimental details that should be provided if not present. This is important for ensuring interpretability and reproducibility of the work.\ni. Fig. 4 g, h, i: timepoint?\nii. Fig. 6b: methods, time, concentration for transcytosis measurement?\niii. Supp. Fig. 2 o, p: how was membrane vs cytoplasmic protein determined?\niv. P. 9 (3 c) states there was not a statistically significant reduction of EGFR by ZEGFR or ferritin, but statistics are not provided comparing them to no treatment.\nv. When y axes are labeled as “relative” (e.g., Fig 3, 4, Supp. Fig. 2, 3 4), it would be more clear to say what it is relative to; e.g., relative to no treatment.\nResponse: We thank the reviewer for raising these important points, which will enhance the reliability of the manuscript. We have revised the relevant sections as follows:\ni. Fig. 4 g, h, i: timepoint?\nResponse: The figure legend has been updated to indicate that the concentration of all LYTACs was 300 nM, and the incubation time was 24 hours.\nFig. 4 g-i, Comparison of the degradation efficiency of HER2 (g), EGFR (h), and PD-L1 (i) after 24-hour treatment with LYTACs constructed by pFn, HFn6 and wtHFn at a concentration of 300 nM.\nii. Fig. 6b: methods, time, concentration for transcytosis measurement?\nResponse: We have supplemented the experimental methods of the transcytosis assay:\n“Mouse brain microvascular endothelial cells (bEnd.3) were used to establish an in vitro BBB model. Cells were seeded at a density of 1 × 105 cells/cm² onto gelatin-coated Transwell inserts with a pore size of 0.4 μm (24 mm diameter, Corning) and cultured for 48–72 h until confluence was achieved. The transcytosis assay was initiated when the trans-endothelial electrical resistance (TEER) reached 100 Ω·cm². FITC-labeled HFn6-Z , HFn6, HFn or LFn (100 μg) EGFR was added to the apical chamber. Transcytosis of ferritin-based proteins was assessed by collecting samples from the basal chamber at 120 min post-addition. The concentration of transcytosed ferritins in the basal chamber was quantified based on a pre-established linear relationship between protein concentration and FITC fluorescence intensity, measured using a Varioskan Flash spectral scanning multimode reader (ThermoFisher Scientific).”\niii. Supp. Fig. 2 o, p: how was membrane vs cytoplasmic protein determined?\nResponse: The quantification of membrane/cytoplasmic EGFR localization was performed in Image J through the following workflow:\n1. Cell identification: Individual cell boundaries were manually traced in bright-field images using the Polygon Selection tool, and corresponding masks (whole cell masks) were generated for further analysis.\n2. Membrane segmentation: The Morphological Filters plugin was applied with a radius of 3 pixels, and cell membrane masks were generated.\n3.Cytoplasm Isolation: Cytoplasmic masks were created by subtracting the membrane mask from the whole cell mask.\n4. Intensity quantification: Add the corresponding mask to the ROI manager and measure in the confocal image.\nThe analysis tools and parameter settings used for quantifying membrane/cytoplasmic EGFR have been detailed in the figure legend:\n“For o and p, the MFI of EGFR in the cytoplasm and membrane was quantified using the Morphological Filters plugin in ImageJ software (radius = 3 pixels). The data are presented as the mean values ± s.d. of n = 15 cells.”\niv. P. 9 (3 c) states there was not a statistically significant reduction of EGFR by Z or EGFR ferritin, but statistics are not provided comparing them to no treatment.\nResponse: As suggested, we have provided the comparative analysis between the untreated group (NT) and the POI ligand-treated group. The results are shown in Revised Fig. 3. Compared to the no treatment group, PEP or FN3 did not induce a significant reduction HER2 PD-L1 in surface HER2 or PD-L1, but the Z -treated group did exhibit reduced level of surface EGFR EGFR. The relevant descriptions in the original manuscript have been revised accordingly:\n“Equivalent doses of Z or ferritin (wtHFn and HFn6) resulted in only a minor reduction in EGFR cell surface EGFR levels (<15% in U-87 MG cells and <20% in MDA-MB-231 cells).”\nFurthermore, we compared the working concentrations of Z and flow cytometry antibody EGFR (Z : 3.6 μM; antibody: 0.014 nM) and, in conjunction with the high binding affinity of Z EGFR EGFR for EGFR (K = 1.62 × 10-9 M), concluded that the observed reduction in EGFR detection D signal is unlikely to be attributable to the competitive effect of Z on the detection antibody. EGFR\nRevised Fig. 3 The HFn-LYTAC platform promotes the degradation of various targets. b, c, Determination of surface EGFR clearance in U-87 MG cells (b) and MDA-MB-231 cells (c) via flow cytometry following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or Z (3.6 μM). i, j, Determination of surface HER2 clearance in EGFR SKBR3 cells (i) and NCI-N87 cells (j) via flow cytometry following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or PEP (3.6 μM). p, q, HER2 Determination of surface PD-L1 clearance in HT1080 cells (p) and MDA-MB-231 cells (q) via flow cytometry following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or FN3 (1.8 μM). For b, c, i, j, p and q, the data are presented as the mean PD-L1 values ± s.d. of n = 3 biological replicates. P values were determined via one-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) multiple comparisons test. NS, not significant. NT, no treatment group.\nv. When y axes are labeled as “relative” (e.g., Fig 3, 4, Supp. Fig. 2, 3 4), it would be more clear to say what it is relative to; e.g., relative to no treatment.\nResponse: We thank the reviewer for this valuable suggestion. We have revised all y-axis labels originally labeled as “Relative EGFR/HER2/PD-L1” to “EGFR/HER2/PD-L1 relative to NT (no treatment)”, as suggested. Take the following data as an example:\nFig. 3. b, c, Determination of surface EGFR clearance in U-87 MG cells (b) and MDA-MB- 231 cells (c) via flow cytometry following a 48-hour treatment with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or Z (3.6 μM). NT, no treatment. EGFR\n• Minor comment 1 (300 nM of HFn-LYTAC is a high concentration)\nThe author’s addition on p. 28 is helpful, but should at least state that this dosage is equivalent on a MOLAR basis of the entire complex to 6 mg/kg IgG (if one were to calculate the molar equivalent amount of POI binders, it would be quite different).\nResponse: We thank the reviewer for raising this important point. We have revised the manuscript to explicitly state the molar basis of HFn6-PEP :\nHER2\n“Owing to the large molecular weight of HFn-LYTACs (approximately 720 kDa on average), this dosage is equivalent to approximately 6 mg/kg of IgG (≈42 nmol/kg, on a molar basis of the entire HFn6-PEP ).” HER2\n---------------------------------------------------------------------------------------------------\nReviewer #3 (Remarks to the Author):\nMost of the raised concerns are reasonably addressed. I have one recommendation. To enhance the reliability of the authors’ conclusion regarding comment #5, it would be ideal to selectively label intracellular EGFR, followed by quantitative analysis.\nResponse: We sincerely appreciate your valuable recommendation. Since it is challenging to specifically label intracellular EGFR via immunofluorescence staining, we employed a membrane and cytosol protein extraction kit to isolate the cytoplasmic protein fraction from U- MG cells. Subsequently, the intracellular EGFR was detected using Western blotting. The relevant content has been incorporated into the manuscript and marked in red:\n“Cytoplasmic proteins were isolated from U-87 MG cells treated with HFn-LYTAC using a membrane and cytosol protein extraction kit (Beyotime), and the expression levels of EGFR in the cytoplasm were analyzed by Western blotting. The results consistently exhibited higher EGFR levels in the wtHFn-Z treatment group compared to the HFn6-Z group EGFR EGFR (Supplementary Fig. 2q).”\nSupplementary Fig. 2 q, Determination of cytoplasmic EGFR degradation in U-87 MG cells via Western blotting. After treating the cells with 300 nM HFn-LYTAC or an equivalent dose of HFn (300 nM) or Z (3.6 μM) for 48 hours, cytosolic proteins were extracted using the EGFR membrane and cytosol protein extraction kit and analyzed by Western blotting. The data are presented as the mean values ± s.d. of n = 3 biological replicates. P values were determined by one-way ANOVA with Fisher’s LSD multiple comparisons test. NS, not significant.\nReviewer #1 (Remarks to the Author):\nThe authors have addressed all the comments, and I have no further concerns.\nResponse: We sincerely appreciate your valuable comments.\n---------------------------------------------------------------------------------------------------\nReviewer #2 (Remarks to the Author):\nI am satisfied with the authors' edits to the manuscript.\nResponse: We sincerely appreciate your valuable comments.\nI do note there appears to be a typo of \"Her3\" on page 14, which I presume will be corrected to \"Her2\" during proofing.\nResponse: Thank you for your careful observation and sorry for the confusion. The typo \"Her3\" on page 14 has been corrected to \"HER2\" in the revised manuscript. Additionally, we have conducted a thorough proofreading of the entire manuscript to ensure no similar errors remain.\n---------------------------------------------------------------------------------------------------\nReviewer #3 (Remarks to the Author):\nThe raised concern has been addressed.\nResponse: We sincerely appreciate your valuable comments."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed all the comments, and I have no further concerns.", "reviewer_2": "I am satisfied with the authors' edits to the manuscript.\nI do note there appears to be a typo of \"Her3\" on page 14, which I presume will be corrected to \"Her2\" during proofing.", "reviewer_3": "The raised concern has been addressed."}, "author_response": "Response: We sincerely appreciate your valuable comments."}]

Decision letter