Endosome-phagophore linking assemblies for the degradation of membrane/extracellular proteins

Domain: chemistry
Published: 2025-12-22
Editorial outcome: major_revision

Abstract
Lysosome-targeting chimeras specifically degrade membrane/extracellular proteins with the specificity and potency highly dependent on the expression pattern and kinetics of lysosome-targeting receptors. Here we describe an endosome-phagophore linking assembly (EPLA), which is constructed by attaching a specific ligand for a protein of interest (POI) to an endosome surface-anchored phagophore-targeting peptide (ES-PTP) that consists of a pH-low insertion peptide and a phagophore-binding element. EPLAs degrade proteins by binding with POIs, being internalized into endosomes, inserting endosome membrane, anchoring the phagophore-targeting moiety onto endosome surface, and then triggering the spatial proximity of endosomes to phagophores. We show the modularity and generality of the EPLA technology by degrading membrane-bound PD-L1, RAGE, GRP94, PSMA, ICAM-1, p32, VCAM-1 and LRP1, and extracellular amyloid-β and tau. Our results establish a technology platform for autophagic degradation of the membrane/extracellular POIs, which may have broad implications for biochemical research and clinical therapeutics.

Introduction
Targeted protein degradation (TPD) technologies selectively sweep target components by leveraging endogenous cellular degradation machinery and either serendipitous or rationally designed degraders1. Cellular degradation machinery can be categorized into ubiquitin-proteasome (UPS), autophagy, and endosome-lysosome (EL) pathway2. The UPS-based TPD tools kidnap and escort proteins of interest (POIs) to ubiquitin E3 ligases for poly-ubiquitination and proteasomal breakdown. Based on the presence of cytoplasmic and transmembrane ubiquitin E3 ligases3, the UPS-based technologies can be applicable for degrading intracellular and membrane proteins. Proteolysis-targeting chimeras are currently the most established UPS-based technology with several compounds being tested in Phase 3 clinical trials. The autophagy-based degraders tether cytosol-localized target components including proteins, organelles, and pathogens to the nascent phagophore proteins, which are known as autophagy-related 8 (ATG8) family proteins, such as the microtubule-associated protein 1 light chain 3 (LC3)4–67. The EL-based degraders (e.g., lysosome-targeting chimera (LYTAC)), can induce the spatial proximity between membrane/extracellular POIs and cell surface lysosome-directing receptors for cell entry and lysosomal degradation via concurrent targeting POIs and the receptors8. These pioneering studies highlight the great potential of TPD platforms in widening the target spectrum to “undruggable” target components and opening alternative avenues for drug discovery9.

However, although an intense quest is increasingly spawned for new lysosome-sorting receptors, the degradation of membrane/extracellular POIs is challenged with a series of unresolved issues of LYTACs. First, the complicated design and synthesis are typically required to construct LYTACs to target certain lysosome-directional receptors. Second, the degradation efficacy is highly dependent on the expression levels and tissue distribution of lysosome-shuttling receptors10–12. Third, the degradation via bifunctional chimeras can be confounded by the potential hook effect13,14. Therefore, an alternative technological paradigm with a generalized and simple design is urgently needed to upgrade the TPD platforms for membrane/extracellular POIs.

Most membrane/extracellular proteins can enter cells via constitutive endocytosis (e.g., programmed death-ligand 1 (PD-L1)15) or can spread between cells via receptor-mediated endocytosis (e.g., the microtubule-associated protein tau16). Both the UPS pathway and the EL pathway can be used to degrade membrane/extracellular proteins. The UPS-based proteolysis-targeting antibodies use cell-surface E3 ubiquitin ligases to induce the internalization of target proteins3. LYTACs use cell surface lysosome-directing receptors for endocytosis of target proteins10. However, there has been so far no report on degradation tools that hijack constitutive endocytosis or intercellular spread mechanisms for POI internalization and subsequent degradation. Targeting only POIs rather than binding to two targets for cell entry holds the enormous potential to increase specificity of targeted degradation and to address the hook effects.

Only a small amount of endocytosed proteins can be transported from endosomes to lysosomes for degradation, most internalized proteins return to the plasma membrane and the extracellular space through recycling endosomes17–19. In the autophagy machinery of all eukaryotic cells, the open phagophores could selectively engulf intracellular damaged components including organelles, parts of the cytoplasm, and proteins and transport them to lysosomes for degradation20. Based on the selective phagocytosis and autophagy of the open phagophores, we hypothesized that the endosomal pathway and the autophagic machinery could be combined to establish an “endosome-phagophore-lysosome” non-traditional transport pathway for the degradation of membrane/extracellular proteins. We envisioned that after the entry of membrane/extracellular proteins into the endosomes through endocytosis, if the early endosomes containing membrane/extracellular POIs can be labeled with motifs targeting ATG8 family proteins, they would be specially swallowed by the open phagophores, thereby preventing their recycling and promoting their autophagic degradation. However, there has been so far no report on the autophagic depletion of membrane/extracellular proteins by labeling endosomes with phagophore-binding moieties and phagocytosing the labeled endosomes with phagophores.

Some functional peptides, such as pH-low insertion peptides (pHLIP)21, insert into the cell membrane under acidic pH conditions with their terminal groups anchored on the other side of the inserted membrane. Inspired by pHLIP’s function at acidic pH, here we present a generalized strategy to build an “endosome-phagophore-lysosome” non-traditional transport pathway by employing endosome-phagophore linking assemblies (EPLAs), for autophagic degradation of membrane/extracellular proteins. EPLAs are constructed by conjugating the POI-specific ligands to the N terminus of an endosome surface-anchored phagophore-targeting peptide (ES-PTP) that consists of a pHLIP and a C-terminal phagophore-binding element. EPLAs are expected to specially recognize and bind with membrane/extracellular POIs for cell entry, to insert into endosome membrane at acidic pH, and to label the endosome surface to target phagophores for protein degradation. This pHLIP-based endosome membrane insertion and endosome labeling technology may have broad implications for biochemical research. This generalized EPLA strategy could broaden the target spectrum of autophagy to membrane/extracellular POIs and may revolutionize the current TPD tool development landscape for the degradation of membrane/extracellular POIs.

Results
Based on the concept of conjugating POI-specific ligands to pHLIP N terminus and LC3-targeting motifs to pHLIP C terminus, we engineered endosomolytic peptides and pHLIP22–24 with N-terminal maleimide (Mal) and C-terminal LC3-targeting Q2425, to screen ES-PTPs and construct EPLAs (Fig. 1a and Supplementary Table 1). The canonical motifs of LC3-interacting region (LIR) are characterized with a core consensus sequence of [W/F/Y]-X1-X2-[I/L/V] (X1, X2 represent any amino acid). The Q24 peptide with a sequence of GIYVIWGPI was derived from the transmembrane (TM) domain of the mitochondrial-anchored ubiquitin-specific peptidase 30 (USP30), possesses the core consensus sequence of the canonical LIR motifs, and can bind with LC3 and accelerate autophagosome formation25. Besides the acceleration of autophagosome formation, Q24 does not require to be placed at the terminus, which endows Q24 the high applicability for different needs. However, Q24 may be not superior to other LIR motifs. For example, the addition of two LIR motifs at the N terminus of Q24 significantly facilitated the interaction with LC3 and the autophagosome membrane. Q24 could be upgraded or even replaced for more efficient degradation performance.

The interaction of engineered peptides with the lipid membrane in endosome lumen (acidic pH) was indicated by the hemoglobin release from red blood cells. Penetratin is the third helix of the Antennapedia homeodomain protein of Drosophila, residues 43–58, and has the ability to penetrate biological membranes26. MPG is a short peptide that contains both a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T-antigen for gene delivery27. HsPen and HsMPG were respectively derived from penetratin and MPG by substituting the Arg/Lys residues with histidine and/or adding N-terminal Phe-(Ser-Gly) for pH-dependent membrane perturbation for endosomal escape after cellular uptake23. Endosomolytic peptide L17E was derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin by substituting the leucine (residue 17) with glutamic acid for membrane perturbation and endosomal escape for cytosolic antibody delivery22. The further-engineered Mal-HsPen-Q24, Mal-HsMPG-Q24, and Mal-L17E-Q24 were used as control to compare with Mal-pHLIP-Q24. All engineered peptides showed higher release of hemoglobin at pH 6.0 (early endosome pH) and pH 5.5 (late endosome pH) than at pH 7.4 (Fig. 1b), suggesting the low plasma membrane damage at pH 7.4. In fact, both HsPen and HsMPG were engineered with the substitution of the Arg/Lys residues by histidine for pH-dependent membrane-perturbation activity to avoid random cell penetration23. L17E was proved to preferentially disrupt the negatively charged membranes (endosomal membranes) over the neutral membranes (plasma membranes)22. And pHLIP was reported to not damage red blood cells at concentrations up to 10 μM28. Our data are consistent with these previous reports. Mal-HsPen-Q24 and Mal-pHLIP-Q24 induced increased hemoglobin release even at pH 6.5, indicating the relatively higher pH responsivity than Mal-L17E-Q24 and Mal-HsMPG-Q24. While penetratin inserts its N terminus into lipid membrane29, pHLIP inserts its C terminus into lipid membrane at pH < 7.021. Therefore, Mal-pHLIP-Q24 was chosen as the ES-PTP to insert into endosome membrane at acidic pH to anchor LC3-targeting Q24 onto endosome surface for endosome labeling and phagophore targeting (Fig. 1c).

We next assessed the ability of ES-PTP (Mal-pHLIP-Q24) to form assemblies after conjugating with hydrophilic ligands of membrane/extracellular POIs. PD-L1 is a key immune checkpoint protein and is constitutively overexpressed across diverse malignancies. Its engagement with PD-1 on T lymphocytes transduces immunosuppressive signals to attenuate T-cell clonal expansion, thereby facilitating tumor immune evasion30. Besides, PD-L1 intracellular structure has been reported to promote tumor survival and proliferation31. Thus, PD-L1 degradation in tumor cells could increase activation of effector T cells and decrease activation of regulatory T cells for effective anti-tumor immune responses and even directly blocks tumor proliferation. Receptor for advanced glycation end products (RAGE) functions as a receptor for brain uptake and brain intercellular spread of amyloid-β (Aβ). RAGE expression is often elevated in brain regions affected by Alzheimer’s disease (AD) and is closely related to AD exacerbation including biochemical abnormalities and behavioral deficits32–34. RAGE degradation could reduce brain uptake and brain intercellular spread of pathological Aβ to potentially improve AD biochemical indicators and clinical symptoms. Thus, we chose PD-L1 and RAGE as model membrane POIs and Aβ as a model extracellular POI. H CVRARTR35 (CVR), ELKVLMEKEL36 (ELK) and RGTFEGKF37 (RGT) were selected as targeting ligands (Supplementary Table 2). Mal-pHLIP-Q24 was conjugated with cysteine-functionalized ligands and dialyzed in water to construct PD-L1-targeting CVR-EPLA, RAGE-targeting ELK-EPLA and Aβ-targeting RGT-EPLA. To fabricate assemblies that can insert endosome membranes but not link endosomes and phagophores, Mal-pHLIP was used to prepare CVR-pHLIP, ELK-pHLIP and RGT-pHLIP. Mal-pHLIP-Q24 and Mal-pHLIP were also mixed with distearyl acyl-phosphatidyl ethanolamine polyethyleneglycol to prepare non-targeting Ctrl-EPLA and Ctrl-pHLIP. To synthesize control formulations that can neither insert endosome membrane nor link endosomes and phagophores, nanoparticles (NPs) with surface display of only targeting ligands (CVR-NPs, ELK-NPs, RGT-NPs) were formed from the poly(lactic-co-glycolic acid)-poly(ε-carbobenzoxy-l-lysine) (PLGA-PLL) and maleimide polyethylene glycol (PEG) succinimidyl carboxymethyl ester (MAL-PEG-SCM)38.

PEGylated micelles were used as a control to assess the plasma protein adsorption. All the above formulations showed the normally distributed hydrodynamic diameters with the median diameters in the range of 48–177 nm and the polydispersity index in the range of 0.080–0.196 (Fig. 1d and Supplementary Fig. 1a), implying the possible formation of micelle-like structures. Although pHLIP does not damage red blood cells, its interaction with red blood cells at pH 7.4 could occupy the surface of 67% of the cells28. However, the possible micelle-like structures of EPLAs would reduce the contact of pHLIP with cells to reduce the possibility of plasma membrane damage. PEGylated micelles (~48 nm), non-targeting Ctrl-pHLIP (~89 nm) and Ctrl-EPLA (~97 nm) showed relatively smaller size than targeting assemblies (116–177 nm), which can be ascribed to the higher PLGA hydrophobicity and PEG hydrophilicity compared with Mal-pHLIP-Q24 and targeting peptides. While CVR-pHLIP and CVR-EPLA exhibited positive surface charges, ELK- and RGT-based assemblies showed negative potentials, which may be due to the different isoelectric points of these targeting ligands. In contrast, CVR-NPs showed negative zeta potentials, which may be due to the high surface display of hydrophilic PEG. All assemblies had high structure stability. ELK-EPLA, RGT-pHLIP and RGT-EPLA maintained their zeta potentials for a week (Supplementary Fig. 1b). Given the possible formation of micelle-like structures, the critical micelle concentrations were measured using pyrene-based fluorescence method39, and were measured in the range of ~1.2–6.8 nM (Fig. 1e and Supplementary Fig. 1c). Collectively, these data proved the successful synthesis of ES-PTP-based EPLAs. We assessed if EPLAs would adsorb plasma proteins to form protein corona40. The incubation with plasma transferred some plasma proteins to the assemblies and changed their zeta potentials (Fig. 1f–h and Supplementary Fig. 1d, e). However, the adsorption of plasma proteins by these assemblies was shown comparable to PEGylated micelles, implying that these assemblies may be “stealth” in the blood and have long blood circulation. CVR-EPLA and PEGylated micelles loaded with near-infrared fluorescent probe IR780 were constructed and intravenously injected to mice for the pharmacokinetics study. The blood circulation behaviors were explored by measuring IR780 concentration. CVR-EPLA and PEGylated micelles showed similar kinetic behaviors in the blood (Supplementary Fig. 1f), confirming the long blood circulation effects.

The proximity of PD-L1 (endosome) to phagophore (lysosome) CVR-EPLA is designed to target PD-L1 to form the CVR-EPLA/PD-L1 complex for internalization, to insert into endosome membrane to anchor phagophore-targeting Q24 onto endosome surface, and to recruit phagophores for the degradation of labeling endosomes (Fig. 2a). First, we examined the uptake of rhodamine-labeled CVR-EPLA in 4T1 mouse breast cancer cells (Fig. 2b). Non-targeting Ctrl-pHLIP was shown to be hardly internalized. However, both CVR-pHLIP and CVR-EPLA were shown to be efficiently internalized with the cellular fluorescence intensity ~12.6-fold and ~12.5-fold that for cells treated with Ctrl-pHLIP, suggesting the role of CVR in cellular uptake. The uptake of CVR-EPLA in 4T1 cells was significantly reduced by anti-PD-L1 antibody (by ~59.6%, all antibody information in Supplementary Table 3) and free CVR peptide (by ~42.5%) but not by free GRP94-targeting NG peptide (by ~1.4%, Fig. 2c), proving that CVR-EPLA may bind with PD-L1 for the specific internalization of both CVR-EPLA and PD-L1 into 4T1 cells. Clathrin (60.9%, chlorpromazine), macropinocytosis (42.2%, amiloride), lipid raft (39.6%, β-cyclodextrin) and caveolae (20.7%, nystatin) were involved in the internalization of CVR-EPLA into 4T1 cells, which was consistent with previous reports where the constitutive and continuous endocytic transport of PD-L1 was closely related to clathrin-mediated endocytosis41.

To study if early endosome can transport with PD-L1 after CVR-EPLA treatment, HEK293T human embryonic kidney cells were transfected to express EGFP-PD-L1 and BFP-Rab5 (an early endosome marker). At 4 h, the proportion of Rab5-co-localized EGFP-PD-L1 relative to total PD-L1 obviously increased compared with untreated cells (Fig. 2d), proving the internalization of PD-L1. At 12 h, the ratio of Rab5-co-localized EGFP-PD-L1 even further increased, which may be caused by the increased internalization of PD-L1 via CVR-EPLA, the partial degradation of total PD-L1, and the high co-localization of the remaining PD-L1 with Rab5. To explore if CVR-EPLA can translocate PD-L1-containing endosomes to phagophores and lysosomes, HEK293T cells were transfected to express EGFP-PD-L1, GFP-Rab5B (an early endosome marker), RFP-LC3 (a phagophore marker) and BFP-LAMP1 (a lysosome marker). In cells treated with Ctrl-pHLIP, Ctrl-EPLA, CVR-NPs, and CVR-pHLIP, EGFP-PD-L1 was primarily located at the cell membrane, and both EGFP-PD-L1 and early endosomes were observed to be located differently from phagophores and lysosomes (Fig. 2e). Without targeting any receptors or membrane proteins, the uptake of Ctrl-EPLA was low and then failed to induce the co-localization of endosomes and phagophores. However, in cells treated with CVR-EPLA, most of EGFP-PD-L1 was internalized and EGFP-PD-L1 and partial early endosomes were highly co-localized with both phagophores and lysosomes. The kinetics studies showed the gradual increase with time for the spatial proximity between the PD-L1-containing endosomes and phagophores (lysosomes) (Fig. 2f and Supplementary Fig. 2a). To investigate the endogenous fluorescence co-localization between PD-L1 and phagophores (lysosomes) after CVR-EPLA treatment, 4T1 cells were treated and then endogenous PD-L1, LC3 (phagophores) and LAMP1 (lysosomes) were labeled through immunofluorescence staining and the related antibodies (Supplementary Fig. 2b). In both untreated cells and cells and cells treated with Ctrl-pHLIP, Ctrl-EPLA and CVR-pHLIP, the endogenous PD-L1 was shown to be located both at cell membrane and in the cytoplasm but not co-localized with endogenous phagophores and lysosomes, which can be ascribed to the low uptake of Ctrl-pHLIP and Ctrl-EPLA and the absence of LC3-targeting Q24 in CVR-pHLIP. However, in cells treated with CVR-EPLA, most of PD-L1 was co-localized with phagophores with the decrease of PD-L1 at the cell membrane and some of PD-L1 was even co-localized with lysosomes. These data together proved the ability of CVR-EPLA to translocate PD-L1-containing endosomes to induce the spatial proximity of PD-L1-containing endosomes to phagophores (lysosomes).

To confirm if CVR-EPLA can transport together with PD-L1-containing endosomes to phagophores and lysosomes, 4T1 cells were treated with rhodamine-labeled CVR-EPLA for 4–8 h to investigate the co-localizations with endosomes, phagophores and lysosomes. Obvious increase was shown for the co-localizations of CVR-EPLA with endosomes, phagophores, and lysosomes from 4 h to 8 h (Supplementary Fig. 2c). The high co-localization of PD-L1 with Rab5 (Fig. 2d), the proximity of both PD-L1 and endosomes to phagophores and lysosomes (Fig. 2e and Supplementary Fig. 2b), and the high co-localizations of CVR-EPLA with endosomes, phagophores, and lysosomes (Supplementary Fig. 2c), collectively proved that CVR-EPLA can transport together with PD-L1-containing endosomes to phagophores and lysosomes for PD-L1 degradation. However, some complexes of CVR-EPLA and PD-L1 may still be released from endosomes into the cytoplasm for direct translocation to phagophores and degradation.

To study whether CVR-EPLA could eventually degrade PD-L1, 4T1 cells were first treated with different assemblies. The comparisons with Ctrl-pHLIP, Ctrl-EPLA, CVR-NPs, and CVR-pHLIP confirmed the indispensability of CVR, pHLIP and LC3-targeting Q24 in PD-L1 degradation in 4T1 cells (Fig. 3a and Supplementary Fig. 3a). Further, CVR-EPLA-induced PD-L1 degradation was dependent on the incubation concentration and time of CVR-EPLA (Fig. 3b, c and Supplementary Fig. 3b). CVR-pHLIP failed to regulate the levels of PD-L1 (Supplementary Fig. 3c–d), proving the necessity of targeting LC3 for PD-L1 elimination. In MDA-MB-231 human breast cancer cells, while CVR-pHLIP failed to decompose PD-L1, CVR-EPLA degraded PD-L1 in a concentration and time co-dependent manner (Supplementary Fig. 3e, f). And for the endogenous fluorescence co-localization between PD-L1 and LC3 (phagophores) in MDA-MB-231 cells, in both untreated cells and cells treated with CVR-NPs and CVR-pHLIP, PD-L1 was not co-localized with phagophores (Supplementary Fig. 3g). However, in cells treated with CVR-EPLA, significant co-localization was shown between PD-L1 and phagophores. These data proved the feasibility of using CVR-EPLA to degrading PD-L1 in human cells.

To explore the necessity of anchoring the Q24 terminus rather than the CVR terminus onto endosome surface and minimize peptide sequence interference, Q24-pHLIP-Mal was synthesized to construct Q24-pHLIP-CVR with CVR at pHLIP C terminus and Q24 at pHLIP N terminus, which is supposed to insert the CVR terminus across endosome membrane. In 4T1 cells, the PD-L1 degradation effect was induced by CVR-EPLA treatment rather than by Q24-pHLIP-CVR treatment (Supplementary Fig. 3h), confirming the effect of anchoring Q24 on LC3 binding. To further confirm the importance of anchoring the Q24-bearing terminus onto endosome surface, penetratin and its analog AP-2AL were introduced because penetratin slightly inserts its N terminus into lipid membrane and AP-2AL penetrates its C terminus into endosome membrane (Fig. 3d)29. Mal-HsPen-Q24 and Mal-AP-2AL-Q24 were synthesized based on penetratin and AP-2AL to separately construct CVR-HsPen-Q24 and CVR-AP-2AL-Q24. While CVR-HsPen-Q24 is expected to hide its Q24 in endosomes, CVR-AP-2AL-Q24 is expected to insert its C-terminal Q24 across the endosome membrane. Both CVR-AP-2AL-Q24 and CVR-EPLA downregulated the levels of PD-L1, but CVR-HsPen-Q24 did not show any comparable effect (Fig. 3e), proving the C-terminal directionality for endosome membrane insertion in CVR-EPLA-mediated PD-L1 degradation.

To explore the cellular mechanisms of CVR-EPLA-mediated PD-L1 degradation, 4T1 cells were treated with CVR-EPLA in the absence or presence of bafilomycin A1 (a lysosomal acidification inhibitor), chloroquine (an autophagy flux inhibitor), NH4Cl (an autophagy inhibitor), MG132 (a proteasome inhibitor), or rapamycin (a phagophore formation enhancer). Western blot analysis showed the elevated levels of LC3-II after treatments with bafilomycin A1, chloroquine, NH4Cl, and rapamycin but not MG132 (Supplementary Fig. 3i), confirming their autophagy-inhibiting or -enhancing effects. The changes of LC3-II levels reflect both formation and accumulation of phagophores. However, while increased formation of phagophores signifies the initiation of cellular degradation machinery, phagophore accumulation indicates the impaired phagophore-lysosome fusion and degradation. Chloroquine, bafilomycin A1 and NH4Cl suppress phagophore-lysosome fusion and then impedes lysosomal degradation of autophagic bodies5,42–45. Rapamycin is an autophagy enhancer that promotes phagophore generation43. Thus, these functional molecules could increase the levels of LC3-II, but chloroquine, bafilomycin A1 and NH4Cl inhibit the autophagic degradation, and rapamycin promotes the autophagic degradation. PD-L1 depletion was inhibited by bafilomycin A1, chloroquine, and NH4Cl and reinforced by rapamycin and carbamazepine at high concentration, whereas MG132 did not show any effects (Fig. 3f and Supplementary Fig. 3j), proving that CVR-EPLA degrades PD-L1 via the autophagic degradation machinery. In addition, treatment with the mixture of carbamazepine and CVR-EPLA was shown to successfully degrade ~80.1% of PD-L1 in 4T1 cells (Supplementary Fig. 3j), suggesting that the autophagy enhancers could be combined with EPLAs to further improve the degradation efficiency of EPLAs.

Atg5 is one of the major regulators of autophagy and is essential for the formation of LC3-II. Cells deficient of Atg5 are unable to form autophagosomes46. To prove the involvement of LC3 in EPLA-mediated protein degradation, MDA-MB-231 cells were transfected with a CRISPR-Cas9 plasmid targeting human Atg5 to knockdown Atg5 and then to reduce LC3-II. After the selection by puromycin for one week, the levels of LC3-II were significantly reduced (Supplementary Fig. 3k). Then MDA-MB-231 control cells and LC3-II-reduced cells were treated with CVR-EPLA for the evaluation of PD-L1 levels. CVR-EPLA failed to degrade PD-L1 in LC3-II-reduced cells (Fig. 3g), proving the involvement of LC3-II in CVR-EPLA-mediated PD-L1 degradation.

Considering the acidic feature of tumor microenvironment and the cell membrane insertion effect of pHLIP at acidic pH, we tested the PD-L1 degradation efficiency of CVR-EPLA on 4T1 cells at both neutral pH and acidic pH. It can be seen that there was no significant difference for the PD-L1 degradation effects of CVR-EPLA at both pH (Fig. 3h). There may exist a time-based competition between PD-L1-mediated CVR-EPLA endocytosis and pHLIP membrane insertion. However, even if pHLIP membrane insertion happens before CVR-EPLA endocytosis, CVR-EPLA endocytosis and following endosome labeling and PD-L1 degradation would happen because pHLIP does not induce endocytosis by itself. Therefore, CVR-EPLA showed similar PD-L1 degradation efficiency at neutral pH and acidic pH.

CVR-EPLA was proven to degrade PD-L1 by linking endosomes and phagophores. To explore whether CVR-EPLA damages endosomes, HEK293T cells were treated with different assemblies and calcein simultaneously because the membrane-impermeable calcein is generally sequestered in endosomes after cellular uptake. The co-incubation with CVR-NPs displayed obvious calcein puncta (Fig. 3i). However, cells treated with other assemblies showed calcein release from endosomes into the cytoplasm with an intracellular uniform calcein distribution. To confirm the endosome leakage, HEK293T cells were transfected with pmCherry-Gal3 and treated by these assemblies because the cytosolic galectin-3 (Gal3) could recognize and bind to β-galactosides at the luminal leaflet of damaged endosomes. CVR-NPs treatment did not change the homogeneous Gal3 distribution (Fig. 3i). However, cells treated with other assemblies showed the significant Gal3 puncta. Cathepsin B is a lysosome-specific cysteine endoproteinase and would be distributed into the cytosol once lysosome leakage happens. All above assemblies did not alter the distribution pattern of cathepsin B (Supplementary Fig. 3l), indicating the intactness of lysosomes. Although CVR-EPLA damages endosomes and may further activate autophagy and the endosomal complexes required for transport23, CVR-EPLA-mediated PD-L1 autophagic degradation is supposed to be more related to endosome labeling mediated endosome-phagophore linking than endosome repair and removal because CVR-pHLIP failed to degrade PD-L1. Collectively, these data proved that endosome leakage occurred in EPLA-mediated endosome membrane insertion and endosome-phagophore linking.

We first tested the feasibility of EPLAs to degrade Alzheimer’s disease-relevant membrane RAGE and extracellular Aβ oligomers. EPLAs are expected to specially bind with extracellular proteins to form the EPLA/protein/receptor ternary complex for internalization, to insert into endosome membrane to translocate phagophore-targeting ligands onto the endosome surface, and to recruit phagophores for the autophagic degradation of the labeling endosomes (Fig. 4a). RAGE-specific ELK-EPLA decomposed ~56.2% of RAGE in RAGE-upregulated bEnd.3 mouse brain endothelial cells (Supplementary Fig. 4a). Aβ oligomers can be internalized by RAGE-mediated endocytosis to propagate between brain parenchymal cells to promote the progression of Alzheimer’s disease32–34. The Aβ-specific RGT-EPLA is expected to form a ternary complex with Aβ oligomers and RAGE for cellular uptake. In RAGE-upregulated BV-2 mouse microglia, the uptake of Aβ oligomers was promoted by RGT-pHLIP and RGT-NPs (Fig. 4b, c and Supplementary Fig. 4b, c), which may be due to their polyvalent Aβ-binding effects. Notably, the levels of Aβ oligomers and RAGE in BV-2 microglia treated with RGT-EPLA were ~57% and ~58% of that in cells treated with RGT-pHLIP (Fig. 4c and Supplementary Fig. 4c), suggesting the degradation of Aβ oligomers and RAGE. To pre-exclude Aβ oligomer uptake difference between cells, BV-2 microglia were first treated with the mixture of Aβ oligomer and lysosomal inhibitor bafilomycin A1 and then treated with the mixture of Aβ oligomer, bafilomycin A1 and assemblies. In the presence of bafilomycin A1, BV-2 microglia showed similar Aβ oligomer uptake between RGT-EPLA and RGT-pHLIP (Supplementary Fig. 4d). However, in the absence of bafilomycin A1, the lower Aβ oligomer levels in cells with RGT-EPLA confirmed the degradation of Aβ oligomers. To explore if RGT-EPLA can translocate the internalized Aβ oligomers to phagophores and lysosomes, BV-2 microglia were transfected to express RFP-LC3 (a phagophore marker) and BFP-LAMP1 (a lysosome marker) and were co-incubated with unlabeled Aβ oligomers and different assemblies. In cells treated with Ctrl-pHLIP, Ctrl-EPLA, and RGT-pHLIP, FITC-labeled Aβ oligomers and early endosomes were located differently from phagophores and lysosomes (Fig. 4d and Supplementary Fig. 4e). However, in cells treated with RGT-EPLA, most of internalized Aβ oligomers and partial early endosomes were co-localized with phagophores and lysosomes. These data suggest that EPLA platforms can be used to degrade other extracellular and membrane POIs by inducing the spatial proximity between POI-containing endosomes and phagophores (lysosomes).

To endow the EPLA technology platforms with higher modularity and universality, we synthesized NP-based EPLA@NPs with surface display of POI-targeting ligands and ES-PTP using PLGA-PLL and MAL-PEG-SCM and through the ultrasonic emulsion evaporation technique, the succinimide–amino reaction chemistry, and the maleimide–thiol reaction chemistry (Fig. 4e). With solid PLGA-based NPs as the basic carrier and MAL-PEG-SCM as the linker, both hydrophilic and hydrophobic targeting ligands could be anchored on NP surface to broaden the application scope of EPLAs to more membrane and extracellular proteins. Direct conjugation with hydrophobic Mal-pHLIP-Q24 requires hydrophilic targeting ligands to form nanoscale micelle-like EPLAs. EPLA@NPs can also be freeze-dried for long storage and the commercialization potential. However, at the same mass concentration, the degradation performance of EPLAs may be better than EPLA@NPs due to the addition of PLGA and MAL-PEG-SCM in EPLA@NPs. To evaluate whether EPLA@NPs could decompose PD-L1, 4T1 cells were treated with CVR-EPLA@NPs. Substantial degradation of PD-L1 (~61.7%) was observed in cells treated with CVR-EPLA@NPs (Fig. 4f). There was no noticeable decrease for the levels of the control membrane protein, the low-density lipoprotein receptor-related protein 1 (LRP1, Fig. 4g), proving the high specificity of CVR-EPLA@NPs for PD-L1 degradation. We performed the proteomic profiling to further study the degradation specificity of CVR-EPLA. Compared with untreated cells, MDA-MB-231 cells treated with the peptide-based CVR-EPLA showed the up-regulation (107/8173, ~1.3%) and down-regulation (87/8173, ~1.1%) for a small amount of proteins (Supplementary Fig. 5a), which may be related to PD-L1 degradation and the designed “endosome-phagophore-lysosome” degradation pathway. While the level of the target protein PD-L1 was reduced, the levels of many membrane proteins including epidermal growth factor receptor (EGFR), low-density lipoprotein receptor (LDLR), CD44, mannose-6-phosphate receptor (M6PR), LRP1, and transferrin receptor (TFR) were not changed, proving the degradation specificity of CVR-EPLA. CVR-EPLA@NPs showed similar time-dependent intracellular distribution to the peptide-based CVR-EPLA with the gradually increased co-localization with early endosomes, phagophores, and lysosomes from 4 h to 8 h incubation in 4T1 cells (Supplementary Fig. 5b). The findings suggested the similar degradation mechanisms between the NP-based EPLAs and the peptide-based EPLAs. Lysosome-trafficking TFR has been exploited to design LYTACs for the degradation of membrane and extracellular proteins10,47. We synthesized PD-L1-targeting LYTACs (CVR-T12@NPs) by co-displaying CVR and TFR-targeting T12 on NPs because of the upregulated expression of TFR on 4T1 cells48,49. To compare the PD-L1 degradation efficiency, 4T1 cells were treated with either LYTACs (CVR-T12@NPs) or CVR-EPLA@NPs. The PD-L1 degradation was shown to be similar for both treatments (Supplementary Fig. 5c). However, if target cells only moderately express TFR, the degradation potency of LYTACs may decrease. Moreover, the inevitable expression of TFR on non-target cells, e.g., brain endothelial cells, could lead to off-target effects and toxicity.

The effectiveness of EPLA@NPs was further proved for other membrane proteins including glucose-regulated protein 94 (GRP94)50,51, prostate-specific membrane antigen (PSMA)52,53, and intercellular adhesion molecule-1 (ICAM-1)54. With the surface display of GRP94-specific ligand NGPTHEGGGGSVEN (NGP)55, NGP-EPLA@NPs induced the breakdown of ~50.1% of GRP94 in MDA-MB-231 cells (Fig. 4h and Supplementary Fig. 5d). Similarly, PSMA-targeting GRF-EPLA@NPs, with the surface modification of GRFLTGGTGRLLRIS (GRF)56, decomposed ~67.2% of PSMA in LNCaP human prostate cancer cells (Supplementary Fig. 5e). The last ICAM-1-targeting NNQ-EPLA@NPs was constructed with surface decoration of the peptide ligand NNQKIVNLKEKVAQLEA (NNQ)57 and was shown to induce the clearance of ~50.7% of ICAM-1 in 4T1 cells (Supplementary Fig. 5f). These data proved that EPLA@NPs can erode membrane proteins with high efficiency and specificity.

In the above design of ES-PTPs and EPLAs, LC3-targeting Q24 was derived from USP3025. LC3-targeting Q24 was changed to E16 (EDEVDGWLIIDLPDSY) to fabricate another type of ES-PTP (Mal-pHLIP-E16) and EPLAs to further verify the modularity of EPLA technology. E16 was derived from TP53INP2 and can bind with all six mammalian ATG8 family proteins, especially LC3 and GABA receptor-associated proteins on the autophagic membrane (Fig. 4i)58. The protein p32 is commonly upregulated at the tumor-associated cell membrane and is a promising cancer therapeutic target52,59. With the p32-targeting peptide ligand AKRGARSTA (AKR)60 at the surface, the E16-based AKR-EPLA@NPs eradicated ~64.1% of p32 in MDA-MB-231 cells (Fig. 4j and Supplementary Fig. 5g). Vascular cell adhesion molecule-1 (VCAM-1) is an endothelial cell membrane-bound sialoglycoprotein and mediates inflammatory cell adhesion and transmigration into damaged tissues, making it a possible therapeutic target for vascular diseases such as atherosclerosis61,62. The E16-based VHP-EPLA@NPs, with the surface display of VHPKQHRGGSKG (VHP)63, diminished ~57.7% of VCAM-1 in tumor-conditioned bEnd.3 mouse brain endothelial cells (Supplementary Fig. 5h). The above data further confirmed the modularity of EPLA technology and the involvement of the autophagy machinery in EPLA-based protein degradation.

Progression of Alzheimer’s disease can be characterized by the sequential spread and deposition of tau aggregates in a predictable pattern that correlates with clinical severity16. Targeted degradation of spreading tau may point to a potentially etiological treatment approach for tauopathies-including Alzheimer’s disease. We constructed tau-targeting VQI-EPLA@NPs by surface displaying of the tau-specific VQIINK (VQI)64 and ES-PTP (Mal-pHLIP-Q24). Based on the findings that LRP1 controls tau endocytosis and propagation to template tau misfolding and aggregation in naive cells16, the tau-specific VQI-EPLA@NPs is expected to specially form the ternary complex with tau and LRP1 for internalization into endosomes (Supplementary Fig. 6a). HEK293T cells were transfected using polyethyleneimine and the plasmid encoding pRK5-EGFP-Tau P301L to collect the soluble cell lysates as tau for tau uptake and degradation studies65. The expression of LRP1 in HA human astrocytes was confirmed (Supplementary Fig. 6b). The uptake of tau was shown to follow the expression pattern of LRP1 between cells (Supplementary Fig. 6c, d), implying the possible involvement of LRP1-mediated endocytosis. Further, the internalization of extracellular tau was shown to be dependent on the tau incubation concentration and time and could reach saturated uptake at 500 nM for 4 h (Supplementary Fig. 6e, f), also suggesting the receptor involvement in tau uptake or intercellular propagation.

Next, the uptake of Cy5-labeled different NPs was investigated in HA astrocytes. The co-existence of extracellular tau markedly enhanced the uptake of VQI-EPLA@NPs (Supplementary Fig. 6g). Meanwhile, in the presence of extracellular tau, the uptake of VQI-modified NPs and VQI-EPLA@NPs was higher than unmodified NPs and Ctrl-EPLA@NPs, both of which were endocytosed with a similar degree to VQI-EPLA@NPs without extracellular tau. These uptake data indicate that both extracellular tau and VQI modification play roles in mediating uptake and that the possible formation of the ternary complex of VQI, tau and LRP1 and LRP1-mediated endocytosis may work together to internalize tau and VQI-EPLA@NPs into endosomes. Finally, in SH-SY5Y human neuroblastoma cells, tau uptake was seen to be considerably increased by co-incubated VQI-EPLA@NPs after 8 h incubation (Supplementary Fig. 6h), which can be ascribed to the polyvalent tau-binding function of VQI-EPLA@NPs. SH-SY5Y cells were found to decompose the internalized tau by themselves. However, the co-incubation of VQI-EPLA@NPs dramatically degraded the internalized tau by ~91%, which was significantly greater than the value of ~54% in the absence of VQI-EPLA@NPs, proving the improved tau degradation by VQI-EPLA@NPs. Meanwhile, the reduced levels of LRP1 in the tau and VQI-EPLA@NPs co-incubation group (Supplementary Fig. 6i), also implied the possible formation of the ternary complex of VQI, tau and LRP1 and the concomitant LRP1 degradation.

Encouraged by the above findings, we further assessed whether CVR-EPLA could induce in vivo anti-tumor effects by targetedly depleting PD-L1 and inducing antitumor immunity. Balb/c immune-competent mice with subcutaneous 4T1 breast cancers were randomly grouped and injected intraperitoneally with antibody against mouse PD-L166 (clone No. 10F.9G2, BE0101, BioXcell, Lebanon, NH, USA, 100 μg per injection), or injected intravenously with different assemblies at 1.8 μmol kg−1 (Fig. 5a). The data on tumor growth curves, tumor weights and lung metastatic nodules showed that, anti-PD-L1, Ctrl-pHLIP, Ctrl-EPLA, CVR-NPs, and CVR-pHLIP failed to inhibit the tumor growth and metastasis (Fig. 5b–d), confirming the immunologically cold nature of 4T1 breast cancers and the low anti-tumor effects of control assemblies. However, CVR-EPLA markedly retarded 4T1 tumor growth and metastasis, proving the fruitful anti-tumor effects of CVR-EPLA. To explore the underlying therapeutic mechanisms of CVR-EPLA, the levels of PD-L1 and T cells in tumors were measured. While anti-PD-L1 and control assemblies did not alter the levels of PD-L1 in tumors, CVR-EPLA therapy prominently reduced the levels of tumor PD-L1 by ~69% (Fig. 5e), suggesting the crucial role of PD-L1 cytoplasmic region in tumor progression31,67. Further, the tumor-infiltrating CD4+ helper T cells and CD8+ cytotoxic T cells were elevated more evidently by CVR-EPLA therapy than by anti-PD-L1 antibody (Fig. 5f), which may partly explain the difference of their anti-tumor effects. For the possible side effects, mice treated with anti-PD-L1 antibody showed the shortest colon (Supplementary Fig. 7a), indicating the most severe colitis and the highest infiltration of inflammatory cells. Although some mice showed inherently high serum aspartate aminotransferase (AST) activity, the activity increase of alanine aminotransferase (ALT) and AST, indicating impaired liver functions, was only shown in anti-PD-L1-treated mice (Supplementary Fig. 7b). In addition, the proportions of CD4+ T cells and CD8+ T cells in liver and T helper 17 cells (CD3+CD4+IL17+) in spleen were increased by anti-PD-L1 antibody but not by CVR-EPLA (Supplementary Fig. 7c, d)68. According to the data on hematoxylin-eosin staining (Supplementary Fig. 7e), compared with untreated healthy tissues, three intravenous injections of CVR-EPLA did not cause any histological and morphological changes such as necrosis for the studied tissues. The data on TUNEL staining and proliferation marker Ki67 staining confirmed that, CVR-EPLA didn’t increase or decrease cell apoptosis (TUNEL+) and cell proliferation in all investigated mouse tissues, including heart, liver, spleen, lung, kidney and colon (Supplementary Fig. 7f, g). The Ki67 positive signals in colon sections indicated the presence of proliferative intestinal stem cells in the colon tissues69. These data collectively suggest that CVR-EPLA-mediated PD-L1 depletion may be more effective and safer than antibody-induced PD-L1 blockade in treating tumors.

We also studied whether CVR-EPLA@NPs could safely inhibit 4T1 tumor progression. Balb/c immune-competent mice bearing 4T1 tumors were injected intravenously with different NPs at 1.8 μmol kg-1 (Fig. 5g). While Ctrl-EPLA@NPs and CVR-NPs showed limited therapeutic effects, CVR-EPLA@NPs markedly retarded 4T1 tumor growth and lung metastasis (Fig. 5h–j), proving the feasibility of using CVR-EPLA@NPs for tumor therapy. Besides, CVR-EPLA@NPs efficiently reduced the levels of PD-L1 in 4T1 tumors by ~56.4% and elevated tumor infiltration of CD4+ T cells and CD8+ T cells (Fig. 5k, l). After treatments, all mice only showed restricted changes of colon lengths and liver and kidney functions (Supplementary Fig. 8). These data proved that CVR-EPLA@NPs could safely provoke tumor-specific immune responses by degrading tumor PD-L1.

LYTAC-mediated degradation of the membrane/extracellular POIs is limited by the expression pattern and kinetics of the co-opted lysosome-targeting receptors. Here we develop EPLAs by conjugating POI ligands and phagophore-targeting motifs separately to pHLIP N terminus and C terminus and self-assembling. EPLAs are proved to specially recognize and bind with membrane/extracellular POIs to enter cells, insert endosome membrane to anchor phagophore-binding ligands onto endosome surface, and mediate the spatial proximity between endosomes and phagophores to eliminate POIs. The EPLA technology can be applied to various membrane/extracellular POIs by virtue of ligand replacement, is verified through the degradation of membrane-bound PD-L1 and RAGE and extracellular Aβ, is further upgraded to EPLA@NPs by dual modification with POI-specific ligands and ES-PTP, and is finally confirmed by the degradation of membrane-bound PD-L1, GRP94, PSMA, ICAM-1, p32, VCAM-1 and LRP1 and extracellular tau. Besides the advantages of bypassing the hook issues and off-target effects, the EPLA-based technology holds great potential to overcome the issue of lysosome dysfunction in many diseases because the interaction with ATG8 proteins on the autophagic membrane could promote the formation of phagophores5,43, which is a distinctive advantage of harnessing the autophagy rather than the lysosomal machinery. However, some critical issues are still needed to be addressed to fully unveil the degradation mechanisms and unleash the translational potentials. First, how can EPLAs collapse to insert ES-PTP C terminus into endosome membrane? The critical micelle concentration to form EPLAs may change with pH to induce EPLA collapse. Second, could some complexes of EPLAs and POIs leak from endosomes and directly translocate to phagophores for POI degradation? Third, could some complexes of EPLAs and POIs be disassociated in endosomes and make EPLAs be recycled for more degradation? Fourth, due to the complex pathophysiological environments and the flexible and dynamic nature of peptide structures, POI-targeting ligands with higher specificity and affinity (e.g., monoclonal antibodies), ES-PTP with higher endosome-inserting and labeling ability, phagophore-targeting ligands with higher specificity and affinity (e.g., other LC3-specific ligands), autophagy enhancers (e.g., rapamycin and carbamazepine), and lysosome-enhancing compounds with the ability of boostingly lysosome biogenesis and function70, can be screened or combined to further strengthen the in vivo degradation potency of EPLAs. In addition, adjusting the incubation time and concentration may also be feasible to improve the degradation efficiency. In conclusion, we believe that this proof-of-concept study exhibit an enormous potential for pushing EPLA-based degraders toward clinical applications.

Methods
Cells and animals
HEK293T human embryonic kidney cells (Cat# ZQ0033), 4T1 mouse breast cancer cells (Cat# ZQ0201), bEnd.3 mouse brain endothelial cells (Cat# ZQ0090), BV-2 mouse microglia (Cat# ZQ0397), MDA-MB-231 human breast cancer cells (Cat# ZQ0118), LNCaP human prostate cancer cells (Cat# ZQ0039), and SH-SY5Y human neuroblastoma cells (Cat# ZQ0050) were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. HA human astrocytes (Cat# C1244) were obtained from WHELAB (Shanghai). Except for 4T1 cells and LNCaP cells that were cultured in RPMI-1640 mediums, all other cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 units mL−1 penicillin, and 100 μg mL−1 streptomycin and were maintained in a 37 °C incubator with 5% CO2.

Adult ICR mice (female, 3–8 week, and 20–25 g) and Balb/c immune-competent mice (female, 3–5 week, and 17–21 g) were purchased from Changzhou Cavens Experimental Animal Co., Ltd. The mice were group-housed (2–5) with same sex littermates under specific pathogen-free conditions, maintained in a temperature- and humidity-controlled room under a 12-h light/dark photoperiod, and provided ad libitum access to water and standard laboratory chow. For the establishment of 4T1 breast cancer mouse model, a million 4T1 cells were injected subcutaneously into the mammary fat pad of Balb/c immune-competent mice. All animal protocols used in this study are approved by the Animal Care and Use Committee of Soochow University (Protocol # 202310A0351, 202411A0780, 202507A0662, 202508A0235) and performed in accordance with the guidelines and ethical regulations of the Animal Care and Use Committee.

Measurements for the membrane perturbation effects
Fresh red blood cells were isolated from ICR mouse blood, washed three times using 150 mM NaCl and then resuspended in PBS 7.4 to achieve a concentration of ten million cells per mL. To detect if the engineered peptides could interact with lipid bilayer membrane to release hemoglobin from red blood cells, 10 μL of red blood cell suspension, 180 μL of 100 mM PBS with different pH, and 10 μL of 200 μM different peptides or 1% Triton X-100 (positive control) were mixed in 96-well plates. After 1 h incubation at 37 °C, the mixtures were centrifuged at 500 g for 5 min and the supernatants were monitored for the absorbance of the release hemoglobin at 492 nm. The membrane perturbation effects at different pH levels were defined as the relative hemoglobin release compared with 1% Triton X-100.

Construction of EPLAs and control formulations
ES-PTP (Mal-pHLIP-Q24, MW 5271.96, purity 96.55%, 2.8 mg) and the cysteine-bearing targeting ligands (CVR, MW 3021.32, purity 93.25%, 8 mg, 4.77-fold; ELK, MW 1334.64, purity 97.36%, 1.4 mg, 1.97-fold; or RGT, MW 1044.17, purity 95.05%, 1.14 mg, 2.01-fold) were dissolved in 490–560 μL DMF and stirred at room temperature for 4–24 h. Then the mixtures were injected into 5 mL of water and stirred for another 0.5 h at room temperature to fabricate assemblies, followed by 48 h dialysis in water using MWCO 3500 and 7000 dialysis bags. Finally, the liquid inside the dialysis bags was collected as CVR-EPLA, ELK-EPLA and RGT-EPLA, respectively. ES-PTP (Mal-pHLIP-Q24, 2.8 mg) was mixed with distearyl acyl-phosphatidyl ethanolamine polyethyleneglycol (MW 2000, 2.5 mg) in 500 μL DMF, injected into water, and dialyzed to collect the final Ctrl-EPLA. For the synthesis of Ctrl-pHLIP, CVR-pHLIP, ELK-pHLIP, and RGT-pHLIP, similar procedures were applied except that ES-PTP was replaced as Mal-pHLIP (MW 4272.73, purity 95.04%, 2.8 mg).

To construct rhodamine-labeled Ctrl-pHLIP, CVR-pHLIP and CVR-EPLA, rhodamine (20% weight of pHLIP or ES-PTP) was added before injecting the reaction mixture into water. Similar procedures were then applied.

PEGylated micelles were used as a control to investigate the plasma protein adsorption and were prepared by conjugating the succinimide-capped PLGA (MW 43000) to amine PEG carboxymethyl (MW 5000) and dialyzing in water. The liquid inside the dialysis bags was collected as PEGylated micelles.

For the synthesis of CVR-HsPen-Q24 and CVR-AP-2AL-Q24, similar procedures were applied except that ES-PTP was replaced as Mal-HsPen-Q24 (MW 4198.70, purity 90.64%, 2.4 mg) and Mal-AP-2AL-Q24 (MW 4019.73, purity 90.86%, 2.3 mg), respectively.

Considering the display of hydrophilic targeting peptides at the EPLA surface rather than PEG, which is often used to prolong the blood circulation time, the plasma protein adsorption and protein corona formation were investigated40. Briefly, 100 μL of freshly prepared EPLAs (0.4 mg mL−1 in PBS 7.4) was incubated with 100 μL PBS 7.4 or plasma at 37 °C for 1 h, followed by centrifugation at 70,000 g for 20 min at 4 °C. Then precipitate was either resuspended in PBS 7.4 for zeta potential measurement or lysed with protein lysis buffer on ice for 30 min and centrifuged at 12,000 g for 15 min at 4 °C for protein electrophoresis to evaluate the plasma protein adsorption.

The ultrasonic emulsion solvent evaporation technique was used38, to synthesize CVR-NPs, ELK-NPs, RGT-NPs, and LYTAC-mimetic CVR-T12@NPs. Briefly, 100 mg of the basic polymer PLGA-PLL was first dissolved in 2 mL of ethyl acetate. This organic solution was added dropwise to 4 mL of 2.5% polyvinyl alcohol under vortex and was immediately sonicated at 300 W for 3 × 10 s to form an oil-in-water emulsion. This emulsion was immediately poured into 100 mL of 0.3% polyvinyl alcohol and stirred overnight to evaporate ethyl acetate and solidify the emulsion into nanosuspension. This nanosuspension was purified from polyvinyl alcohol by centrifugation at 70,000 g for 20 min and ultrasonically resuspended in PBS 7.4. Subsequently, MAL-PEG-SCM (MW 3500, 11.2 mg per 100 mg PLGA-PLL, JenKem Technology, Beijing) was added and reacted with primary amines from hydrophilic PLL at the surface of nanosuspensions at room temperature for 1 h through the succinimide–amino reaction chemistry to introduce PEG and maleimide functional groups. The excess MAL-PEG-SCM was removed by centrifugation at 70,000 g for 20 min and the nanosuspension was resuspended and further decorated with an excess of the cysteine-bearing targeting ligands (CVR:T12 at 1:1 molar ratio for CVR-T12@NPs, 1.25-fold amount of MAL-PEG-SCM) at room temperature for 1 h through the maleimide–thiol reaction chemistry. Finally, the excess unreacted peptides were removed by centrifugation at 70,000 g for 20 min and the nanosuspension was resuspended in water and lyophilized for subsequent storage and characterization.

To prepare the NP-based EPLAs, the cysteine-functionalized pHLIP-Q24 (MW 5181.90, purity 92.61%) was mixed with cysteine-bearing targeting ligands (including PD-L1-targeting CVR, GRP94-targeting NGP, PSMA-targeting GRF, ICAM-1-targeting NNQ, and tau-targeting VQI) at a 1:1 molar ratio and 2.5-fold total peptides relative to the amount of MAL-PEG-SCM and then reacted with nanosuspensions with surface maleimide functional group to obtain the NP-based EPLAs. For the preparation of Cy5-labeled NP-based EPLAs, similar procedures were applied except that the solidified and purified nanosuspension was reacted with sulfo-cyanine 5 NHS ester (60 μg per 100 mg PLGA-PLL, DuoFluor Cat# D10017, MW 777.95, Wuhan, China) at room temperature for 1 h before the reaction with MAL-PEG-SCM. For the preparation of fluorescence probe doxorubicin (DOX)-labeled CVR-EPLA@NPs, similar procedures were applied except that 100 mg of PLGA-PLL and 10 mg of DOX were dissolved in 2 mL of ethyl acetate.

To prepare the NP- and E16-co-based EPLAs, the cysteine-functionalized pHLIP-E16 (MW 6043.67, purity ~80%) was mixed with cysteine-bearing targeting ligands (including p32-targeting AKR and VCAM-1-targeting VHP) at a 1:1 molar ratio and 2.5-fold total peptides relative to the amount of MAL-PEG-SCM were reacted with nanosuspensions with surface maleimide functional group to obtain the NP- and E16-co-based EPLAs.

Physicochemical characterization
The hydrodynamic diameters and zeta potentials were measured using a ZetaSizer Nano ZS90 and Zetasizer Software v7.13 (Malvern Panalytical). Considering the possible formation of micelle-like structures, there may exist a critical concentration to form these structures. Then the critical concentration was determined using the pyrene fluorescence probe method39,71. Briefly, 20 μL of pyrene ethanol solution (0.5 mg mL−1) was dried and then dissolved with 2 mL of different EPLAs at different concentrations. The mixtures were sonicated for 40 min, and followed by fluorescence quantification (335 nm excitation, 373 nm and 384 nm emission). The concentrations at the inflection point for the pyrene fluorescence intensity ratio (384 nm/373 nm)–EPLA concentration curves were defined as the critical concentrations to form micelle-like structures.

The uptake efficiency, specificity, and mechanisms of CVR-EPLA in 4T1 cells
4T1 cells were treated with rhodamine-labeled assemblies at 3.5 μg rhodamine mL−1 for 4 h and then measured for the internalized rhodamine using flow cytometry (FlowJo, 10.8.1, BD Life Sciences, OR, USA) to explore the cellular uptake efficiency of CVR-EPLA. To assess the uptake specificity and mechanisms of CVR-EPLA, 4T1 cells were pre-treated with anti-PD-L1 antibody (1 μL at original concentration), free CVR peptide (0.1 mg mL−1), free GRP94-targeting NG peptide (0.1 mg mL−1), or the indicated endocytosis inhibitors (25 μg mL−1 nystatin, 6.5 mg mL−1 β-cyclodextrin, 100 μg mL−1 amiloride, and 10 μg mL−1 chlorpromazine) for 1.5 h, incubated with the mixture of rhodamine-labeled CVR-EPLA and different inhibitors for 4 h, and then measured for the internalized rhodamine using flow cytometry.

The exogenous fluorescence co-localizations between EGFP-PD-L1 and BFP-Rab5
HEK293T cells were transfected with the 3:1 (mass ratio) complexes of polyethylenimine (PEI, 1 mg mL−1 and pH 6.9–7.1, Kyfora Bio Cat# 23966) and plasmids including pEGFP-N1/PD-L1 (addgene Cat# 121478) and BFP-Rab5 (addgene Cat# 49147) in serum-free DMEM for 24 h. The transfected cells were further incubated with 0.5 μM CVR-EPLA for various times, fixed with a 4% PFA solution for 15 min, and finally imaged using a confocal laser scanning microscope (NIS Viewer 5.21 and NIS-Elements Viewer 5.21.00, Nikon, Japan).

The exogenous fluorescence co-localizations between EGFP-PD-L1/GFP-Rab5B and RFP-LC3 (phagephores)/BFP-LAMP1 (lysosomes)
HEK293T cells were transfected with the 3:1 (mass ratio) complexes of PEI and plasmids, including pEGFP-N1/PD-L1, GFP-Rab5B (addgene Cat# 61802), pmRFP-LC3 (addgene Cat# 21075), and mTagBFP2-Lysosomes-20 (addgene Cat# 55308) in serum-free DMEM for 24 h. The transfected cells were further treated with 0.5 μM different assemblies for 12 h or incubated with 0.5 μM CVR-EPLA for various times, then fixed with a 4% PFA solution for 15 min, and finally imaged using a confocal laser scanning microscope.

The co-localizations between fluorescently labeled CVR-EPLA (or CVR-EPLA@NPs) and endogenous EEA1/LC3/LAMP1
4T1 cells were treated with rhodamine-labeled CVR-EPLA (4 μg rhodamine mL−1) or DOX-labeled CVR-EPLA@NPs (5 μg DOX mL−1) for 4 h and 8 h. Cells were then fixed with 4% PFA for 15 min, blocked with PBS 7.4 containing 4% serum and 0.4% Triton X-100 for 2 h, and incubated with the related primary antibodies. Cells were finally incubated with the green fluorescent secondary antibody for 2 h and imaged using a confocal laser scanning microscope. The images were semi-quantified using the software ImageJ (1.46r, NIH, USA).

The endogenous fluorescence co-localizations between PD-L1 and LC3/LAMP1
4T1 cells (or MDA-MB-231 cells) were treated with 0.5 μM different assemblies for 3 h, fixed with PBS 7.4 containing 4% PFA for 15 min, blocked with PBS 7.4 containing 4% serum and 0.4% Triton X-100 for 2 h, and incubated with the related primary antibodies. Cells were finally incubated with fluorescently labeled secondary antibody for 60 min and imaged using an ultra-high-resolution microscope (Leica TCS SP8).

PD-L1 degradation analysis via Western blot
For PD-L1 degradation, 4T1 cells (or MDA-MB-231 cells) in 6-well plates were treated with CVR-EPLA and CVR-pHLIP at different concentrations for 12 h or at 0.5 μM (10 μM for MDA-MB-231 cells) for various times. In another experiment, 4T1 cells in 6-well plates were treated with different assemblies at 0.5 μM for 12 h. To study CVR-EPLA-mediated PD-L1 degradation at different pH conditions, 4T1 cells were incubated with 0.5 μM CVR-EPLA at pH 7.4 or pH 6.0 for 12 h. To compare the PD-L1 degradation performance with LYTAC-mimetic CVR-T12@NPs, 4T1 cells were treated with CVR-EPLA@NPs and CVR-T12@NPs at 2 mg mL−1 for 12 h.

Treated cells were harvested on ice using a cell scraper and lysed via three freeze-thaw cycles and sonication in RIPA lysis buffer with protease inhibitors and phosphatase inhibitors. After centrifugation at 12,000 g for 10 min at 4 °C, the collected supernatant was analyzed for the levels of PD-L1 using western blot with the related antibody and software including GSCapture (GS-S900, Jingyi Technology, Guangzhou, China), Analysis (2.6.1.0, Clinx Science Instruments, Shanghai, China) and GraphPad Prism 9.0.0 (GraphPad Prism Inc., San Diego, CA, USA).

PD-L1 degradation mechanisms
To investigate the underlying mechanisms of CVR-EPLA-mediated PD-L1 degradation, 4T1 cells in 6-well plates were pre-treated with bafilomycin A1 (100 nM), chloroquine (20 μM), NH4Cl (10 mM), MG132 (5 μM), rapamycin (10 μM and 30 μM), or carbamazepine (50 μM) for 24 h, and were further treated with the mixtures of 0.5 μM CVR-EPLA and the above functional molecules for 12 h. To study the effect of Atg5 on CVR-EPLA-mediated PD-L1 degradation, MDA-MB-231 cells were transfected with the complexes of Lipofectamine 2000 (4 μL) and sgAtg5 plasmid (2 μg, Addgene Cat# 175023) in 200 μL of Opti-MEM medium for 5–6 h and further cultured in fresh complete medium for 48 h. Puromycin (10 μg mL−1) was added and changed every 2 days to select the positive cells. The successfully transfected cells were obtained after one week and treated with 0.5 μM CVR-EPLA for 12 h. Then the treated cells were analyzed for the levels of both PD-L1 and LC3-II using western blot with the related antibodies.

To inspect the endosome/lysosome leakage, the subcellular distribution patterns of calcein (internalized), mCherry-galectin-3 (addgene Cat# 85662, transfected by PEI) and cathepsin B were investigated. Briefly, HEK293T cells and mCherry-galectin-3-expressed HEK293T cells in 48-well plates were incubated with 0.5 μM different assemblies or their mixtures with 0.1 mg mL−1 calcein (Macklin, C866390) for 8 h. The treated cells were fixed with a 4% PFA solution for 15 min. For the detection of cathepsin B leakage, cells were stained with cathepsin B antibody overnight and an Alexa Flour 594 conjugated secondary antibody. Finally, cells were imaged using a confocal laser scanning microscope.

RAGE degradation analysis via Western blot
The human amyloid-β monomer 1-42 (Aβ1-42, DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA, Shanghai Royobiotech Co., Ltd) was prepared by dissolving Aβ powder in cold hexafluoroisopropanol to reach 1 mM, incubating 60 min at room temperature in a sealed state for complete solubilization, aliquoting on ice into centrifuge tubes, and evaporating hexafluoroisopropanol at room temperature for 12 h. The dried and transparent film was finally obtained as Aβ monomer and can be stored at −80 °C. To prepare Aβ oligomers, 44 μL of DMSO was added to dissolve the peptide membrane and form a 5 mM solution. Then the Opti-MEM™ (reduced serum medium, Gibco Cat# 31985070) was added to dilute the solution to a concentration of 100 μM. The solution was further incubated at 4 °C for 24 h and filtered through 0.22 μm membrane to obtain homogeneous Aβ oligomers, which were validated using western blot with Aβ-related antibody. RAGE-upregulated bEnd.3 mouse brain endothelial cells were established by incubating with 10 μM Aβ oligomers for 24 h and validated using western blot with RAGE-related antibody. For RAGE degradation, RAGE-upregulated bEnd.3 cells in 6-well plates were treated with 10 μM different assemblies, including RAGE-targeting ELK-EPLAs for 12 h. Then the treated cells were analyzed for the levels of RAGE using a western blot with the related antibody.

Aβ oligomer degradation analysis via western blot
RAGE-upregulated BV-2 microglia were formed by incubating with 10 μM Aβ oligomers for 24 h and validated using western blot with RAGE-related antibody. For Aβ uptake, RAGE-upregulated BV-2 microglia in 6-well plates were incubated with either the mixtures of 10 μM Aβ oligomers and different concentration RGT-pHLIP for 12 h or the mixture of 10 μM Aβ oligomers and 10 μM RGT-pHLIP for various times. For Aβ degradation, RAGE-upregulated BV-2 microglia in 6-well plates were treated with the mixtures of 10 μM Aβ oligomers and 10 μM different assemblies, including Aβ-targeting RGT-EPLA for 12 h. To pre-exclude Aβ oligomer uptake difference between cells, BV-2 microglia were first treated with 10 μM Aβ oligomer with or without 50 μM lysosomal inhibitor bafilomycin A1 for 24 h, and then incubated with the mixture of 10 μM Aβ oligomer and 10 μM RGT-pHLIP (or RGT-EPLA) with or without 50 μM bafilomycin A1 for 12 h. Then the treated cells were analyzed for the levels of both Aβ oligomers and RAGE using western blot with the related antibodies.

The co-localizations between Aβ/Rab5B and phagephore/lysosome
BV-2 microglia in 48-well plates were transfected with the complexes of Lipofectamine 2000 (Invitrogen Cat# 11668019) and plasmids including GFP-Rab5B, pmRFP-LC3, and mTagBFP2-Lysosomes-20 for 4 h. The transfected cells were used to establish RAGE-upregulated BV-2 microglia by incubating with 10 μM Aβ oligomers for 24 h. The transfected RAGE-upregulated BV-2 microglia were further treated with the mixtures of 10 μM different assemblies and either 10 μM FITC-labeled Aβ oligomers or unlabeled Aβ oligomers for 12 h. Then the cells were fixed with a 4% PFA solution for 15 min and finally imaged using a confocal laser scanning microscope.

Targeted degradation of other POIs using the NP-based EPLAs
The 4T1 cells in 6-well plates were treated with 2 mg mL−1 different NPs including PD-L1-targeting CVR-EPLA@NPs for 12 h to degrade PD-L1 and, LRP1 as a control membrane protein was also analyzed. MDA-MB-231 cells in 6-well plates were treated with 2 mg mL−1 different NPs, including GRP94-targeting NGP-EPLA@NPs for 12 h to degrade GRP94. LNCaP cells in 6-well plates were treated with 2 mg mL−1 different NPs including PSMA-targeting GRF-EPLA@NPs for 12 h to degrade PSMA. The 4T1 cells in 6-well plates were treated with 2 mg mL−1 different NPs, including ICAM-1-targeting NNQ-EPLA@NPs for 12 h to degrade ICAM-1. MDA-MB-231 cells in 6-well plates were treated with 2 mg mL−1 different NPs, including p32-targeting AKR-EPLA@NPs for 12 h to degrade p32. The bEnd.3 cells in 6-well plates were first incubated in MDA-MB-231 cell conditioned medium for 24 h to induce the expression of VCAM-152, and then were treated with 2 mg mL−1 different NPs, including VCAM-1-targeting VHP-EPLA@NPs for 12 h to degrade VCAM-1. The treated cells were analyzed for the levels of POIs using western blot with the related antibodies.

The uptake and degradation of EGFP-Tau in HA human astrocytes
To obtain the EGFP-Tau for the uptake and degradation experiments, HEK293T cells in 10-cm culture dishes were transfected with the 3:1 (mass ratio) complexes of PEI and plasmid pRK5-EGFP-Tau P301L (addgene Cat# 46908) in serum-free DMEM for 24 h. Then the medium was replaced with complete medium for another 48 h incubation. The transfected cells were centrifuged at 2800 g for 5 min. The cell precipitate was resuspended with water, rotated at 4 °C for 2 h and centrifuged at 12,000 g for 15 min to collect the supernatant as EGFP-Tau. EGFP-Tau in water was mixed with 5× PBS 7.4 to adjust the osmotic pressure. The BCA-measured concentration (~0.42 mg total protein mL−1) was converted into molar concentration (500 nM EGFP-Tau)72. Considering the role of LRP1 in tau internalization, the levels of LRP1 in HA astrocytes and HEK293T cells (negative control) were analyzed using western blot with the related antibody. Then, HA astrocytes in 6-well plates were incubated with 500 nM EGFP-Tau in serum-free DMEM for 4 h and were measured for the levels of EGFP-Tau using western blot with the related antibody or analyzed for GFP fluorescence intensity using flow cytometer. The EGFP-Tau uptake in HEK293T cells was used as a negative control. To obtain the optimum condition for EGFP-Tau uptake, HA astrocytes in 6-well plates were also incubated with EGFP-Tau in serum-free DMEM at different EGFP-Tau concentrations for 12 h or at 500 nM for various times and then were analyzed for the levels of EGFP-Tau using western blot with the related antibody.

To prove that the tau-targeting VQI-EPLA@NPs could be internalized into endosomes together with tau, HA astrocytes in 12-well plates were treated with Cy5-labeled different NPs, including tau-specific VQI-EPLA@NPs at 0.24 mg mL−1 in serum-free DMEM in the absence or presence of 500 nM EGFP-Tau for 2 h and then were analyzed for Cy5 fluorescence intensity using flow cytometer. To measure the uptake and degradation of extracellular EGFP-Tau, SH-SY5Y cells in 6-well plates were incubated with 500 nM tau with or without 2 mg mL−1 the NP-based VQI-EPLA@NPs in serum-free DMEM for 8 h and then incubated with fresh complete medium for another 4 h. The treated cells were analyzed for the levels of EGFP-Tau using western blot with the related antibody.

In vivo anti-tumor evaluation
Model mice with 4T1 breast cancers were constructed from Balb/c immune-competent mice and treated with either intraperitoneal anti-PD-L1 antibody (100 μg per injection on days 0, 3, 6, 9, and 12, 10F.9G2, BE0101, BioXcell) or intravenous NPs/assemblies (1.8 μmol kg−1 per injection on days 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18). Then the tumor volume (length × width2/2) was monitored every day. Mice were euthanized when the tumor volume exceeded 2000 mm³ or on day 20 to excise primary tissues for the therapeutic analysis. Before euthanasia, 100 μL of serum was harvested through the inner canthal vein and analyzed for the levels of aspartate aminotransferase, alanine aminotransferase, albumin, and urea nitrogen, to determine liver and kidney functions. The subcutaneous tumors were weighed and analyzed for the levels of tumor PD-L1 using western blot. The lungs were fixed with Bouin’s fixative (Sbjbio Cat# BL-G016) and imaged to count 4T1 metastatic nodules. The length of colons was measured to assess the immune-related colitis.

To detect tissue-infiltrating immune cells to explain the therapeutic effects and immune-related side effects, 4T1 tumors, livers, and spleens were completely minced, incubated in the solution of collagenase type 4 (Worthington Cat# LS004186) and DNase I (Sigma Cat# D5025) at a 37 °C shaker for 1 h for digestion, and filtered through a 70 μm strainer. After centrifugation at 350 g for 5 min at 4 °C, the cell precipitate was collected, resuspended, stained with antibodies against CD45, CD3, CD4, and CD8 for 4T1 tumors and livers and with antibodies against CD3, CD4, and Th17 for spleens, and analyzed using flow cytometer.

To further assess the biological safety through hematoxylin and eosin staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining and Ki67 staining, Balb/c immune-competent mice (female) were intravenously injected with CVR-EPLA or CVR-pHLIP at 1.8 μmol kg−1 per injection for three times on days 1, 3, and 5. Then mice were euthanized on day 7 to excise tissues for hematoxylin-eosin staining. TUNEL assay and Ki67 staining for all tissue sections were performed using a CF488 TUNEL cell apoptosis detection Kit (Servicebio, G1504-50T) according to manufacturer’s instructions or the related primary antibody and red fluorescent secondary antibody.

In vivo pharmacokinetics
Balb/c immune-competent mice were intravenously injected with near-infrared fluorescent probe IR780-loaded PEGylated micelles (PLGA-PEG), or CVR-EPLA at 0.75 mg IR780 kg−1. Blood samples (20 μL) were obtained through tail vein at different time points and immediately mixed with 180 μL DMSO to extract IR780. Then the mixtures were imaged using an IVIS imaging system (Lumina III, PerkinElmer). IR780 intensity was quantified using the software Living Image 4.2 (IVIS Lumina, PerkinElmer, USA) with the standard samples as control to obtain the blood concentration-time profiles.

Proteomic assay
MDA-MB-231 cells were treated with 0.5 μM CVR-EPLA for 12 h. The protocol for proteomic assay was described73. Briefly, the treated cells were collected and mixed with ice-cold acetone to precipitate the proteins and dissolve the lipids. Lipids were removed by centrifugation at 16,000 g, 4 °C for 15 min and the protein mixture was re-suspended, digested and centrifuged at 12,000 g for 10 min. The extracted peptides were then dissolved in 0.1% formic acid for mass spectrometry. The raw data were processed and analyzed by MaxQuant 1.6.5.0 (Max-Planck-Institute of Biochemistry, Martinsried, Germany). For analysis of proteomic data, the data were transformed and normalized using variance stabilizing normalization using the DEP 3.18 package of Bioconductor and DEP 2 from Github. The difference was analyzed using Limma 3.58.1. P value was set to be less than 0.05. Fold change value was set to be greater than 2.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Wang et al. reported an endosome surface-anchored phagophore-targeting peptide (ES-PTP; consisting of a pH-low\ninsertion peptide and a phagophore-binding peptide) for phagophore-based degradation of plasma membrane/extracellular\nproteins in combination with a ligand specific to endogenous cell-surface/extracellular POIs. The overall degradation of\nthese ES-PTP-based assemblies is promising and amenable to different proteins including PD-L1, RAGE, GRP94, PSMA,\nICAM-1, p32, VCAM-1, LRP1, amyloid-β and tau. Both peptide-based self-assemblies and nanoparticles surface-fabricated\nwith ES-PTP and POI ligands were employed and demonstrated to be effective in degrading targeted proteins with above\nstrategy, indicating its modularity and universality. However, the involvement of endosome for its “endosome-phagophore\nlinking” pathway (which is one of the most interesting features of the current study) as well as the comparison between the\npeptide assembly and NP-based system in view of degradation mechanism needs further experimental support. Please find\nmore details below.\n1. Line 147, Page 5: “However, the adsorption of plasma proteins by these assemblies was shown comparable to\nPEGylated micelles, implying that the plasma protein binding may not affect their pharmacokinetics.” Please add related\nreference or experimental results.\n2. For colocalization analysis, what’s the colocalization ratio change between PD-L1 and Rab5? What’s the difference\nbetween different peptides (e.g., CVR-pHLIP & CVR-EPLA) in view of endocytosis? Since internalization is the first step for\nfollowing degradation, it is critical to understand which component(s) is(are) responsible for it.\n3. A key control--conjugating POI-specific ligands to pHLIP C terminus and LC3-targeting motifs to pHLIP N terminus,\nis needed to confirm the expected effect of pHLIP for LC3 binding (which should be a direct and convincing control with\nminimal interference from the peptide sequence).\n4. Line 231, Page 9: “PD-L1 depletion was inhibited by bafilomycin A1, chloroquine, and NH4Cl and reinforced by\nrapamycin, whereas MG132 did not show any effects (Fig. 3f),” no significant difference was indicated/observed between\nCVR-EPLA and CVR-EPLA+Rapamycin? How comes the “reinforced by rapamycin”?\n5. Since endosome leakage was seen upon the incubation of CVR-EPLA, it is also possible that the released PD-L1-CVR-\nEPLA complex was translocated to phagophore directly for degradation rather than underwent endosome-phagophore\npathway? Currently, the involvement of endosome is mainly (1) the imaging result of Rab5B in Fig 2 (of note, only\ncolocalization between Rab5B and phagophore/lysosome were shown, with a ratio below 0.5; no data for PD-L1 and Rab5B\nwas found in the maintext nor the SI) and (2) the endosome leakage result in Fig. 3. Unfortunately, both results were not\nconvincing/direct enough to support the involvement of endosome for CVR-EPLA-mediated degradation. The reviewer think\nit seems more likely to be the release of the complex into cytosol for following translocation to phagophore.\n6. The effect of these endosomolytic agents on plasma membrane damage also needs to be illustrated/discussed.\n7. Line 286, Page 12: “Notably, the levels of Aβ oligomers and RAGE in BV-2 microglia treated with RGT-EPLA were ~57%\nand ~58% of that in cells treated with RGT-pHLIP (Fig. 4c and Extended Data 289 Fig. 4c), suggesting the degradation of Aβ\noligomers and RAGE.” It is not objective to conclude that degradation has occurred without pre-excluding the differences in\nendocytosis/uptake of Aβ oligomers into cells (i.e., by inhibiting the degradation of lysosome to check the cellular uptake).\n8. There was no obvious fluorescence from phagophore nor lysosome from the group of Ctril-pHLIP in Fig. 4? Similar\nphenomena were also seen from other control groups in Fig. 4. Please clarify the difference in these images to confirm the\naccuracy of colocalization ratio.\n9. Dose the NP-based EPLAs show same time-dependent cellular distribution (the colocalization with Rab5B, phagophore,\nand lysosome) as the peptide-based EPLAs?\n10. What is the requirement for the labeling ratio of ES-PTP and POI-targeting ligands onto NPs for optimal degradation\nefficacy?\n11. Is the immunodeficient BALB/c mouse the optimal animal model for PD-L1 antibodies?\n12. Why murine cell lines were employed for most cell-based experiments? Are there available POI-binding peptides for\nhuman cells, which is important for therapeutic potentials.", "reviewer_2": "Han and his colleagues have developed an endosome-phagophore linking assembly (EPLA) by inserting the endosome\nmembrane after internalization with a protein of interest (POI), anchoring a phagophore-targeting moiety onto endosome\nsurface for the treatment of cancer. The authors present several application examples in this study. However, this work\nsuffers from limited experimental depth and issues in both the practical utility of EPLA and experimental design.\n1. The introduction section, especially the third paragraph, fails to effectively highlight the innovation and significance of the\nstudy and needs to be substantially revised.\n2. For Fig. 1, multiple peptide abbreviations such as Hspen, LITE, and HSMPG are mentioned, but the authors did not\nprovide their sources or explain why they were selected. Additionally, the characteristics and advantages of Q24 were not\nadequately introduced, nor was the rationale for choosing it over other peptides.\n3. How about the intracellular uptake efficiency of EPLAs?\n4. In the Fig. 2B, exogenous co-localization was conducted. Although this provides partial support for the conclusion, it is\ninsufficient. Endogenous fluorescence co-localization, as well as intracellular uptake and co-localization at different time\npoints, should also be performed. Additionally, Fig. 2B lacks clear labeling—for example, it should indicate EGFP-PD-L1\ninstead of PD-L1.\n5. In the Fig. 2, the authors used fluorescence experiments to investigate the spatiotemporal regulation of EPLA, but this is\nnot sufficient. Techniques such as flow cytometry should be employed for further validation.\n6. In the Fig. 3, the order of panels A, B, and C lacks logic. It should be rearranged as C, A, and B.\n7. The key information of legends is missing, for example, the specific cell lines used are not clearly indicated.\n8. In Fig. 3A–C, the authors used the murine 4T1 cell line instead of human cell lines. The rationale for this choice is unclear.\nThe authors did not explain this in the results. Moreover, western blot and fluorescence results from human cell lines should\nbe provided.\n9. The mechanism of action is unclear. Although the authors state that EPLA degrades target proteins via an LC3-dependent\nautophagy-lysosomal pathway, they did not block LC3-dependent pathway to verify this mechanism.\n10. Fig. 5 should be moved to the supplementary results.\n11. In the Fig. 6, the authors used CVR-EPLA to evaluate its in vivo antitumor efficacy, but no experiments were conducted\nto assess its in vivo safety, which should be addressed.\n12. The authors emphasize that EPLA has certain advantages over traditional LYTACs, but no comparative degradation or\nfunctional experiments were provided. In the Fig. 3A, CVR-EPLA only degraded PD-L1 effectively at 1 μM. Therefore,\ncomparative experiments need to be included.\n13. How about the degradation specificity of CVR-EPLA? It is better to perform unbiased proteomics experiments to address\nthis issue.", "reviewer_3": "The manuscript has described an endosome-phagophore linking assembly (EPLA) for degradation of\nmembrane/extracellular proteins. The EPLA was built on pHLIP by modifying the N terminus of pHLIP with POI-specific\nligands and the C terminus of pHLIP with LC3-targeting Q24. Therefore, the EPLA can recognize and bind with\nmembrane/extracellular POIs for cell entry, to insert into endosome membrane at acidic pH, and to label the endosome\nsurface to target phagophores for protein degradation. They have demonstrated the EPLA technology by degrading\nmembrane-bound PD-L1, RAGE, GRP94, PSMA, ICAM-1, p32, VCAM-1 and LRP1, and extracellular amyloid-β and tau.\nAlthough it is an interesting work to broaden the target spectrum of autophagy to membrane/extracellular POIs,\nseveral\nimportant issues have to be addressed.\n1. What’s the biological consequence of degradation of PD-L1 or RAGE in cells?\n2. How about the degradation specificity of EPLA? It is necessary to perform global proteomic profiling using mass\nspectrometry.\n3. What is the internalization mechanism of EPLA? How specificity it is?\n4. Usually, the pHLIP would insert the cellular membrane at acidic pH. How about EPLA? The authors should test its cellular\nperformance at low pH, which mimics the tumor acidic environment.\n5. The degradation of extracellular amyloid-β and tau relies first on the membrane receptors to uptake. How can it be general\nto degrade other extracellular proteins without membrane receptors?\n6. Although the authors have verified the strategy with several proteins, all of them only achieved 50-60% degradation\nefficiency what might not be enough compared with other TPD reports. The degradation efficiency should be improved.\n7. For the degradation mechanism study, the bafilomycin A1, chloroquine, NH4Cl, and rapamycin induced the elevation of\nLC2-II level. Then, bafilomycin A1, chloroquine, and NH4Cl inhibited degradation, but rapamycin enhanced the degradation.\nWhat is the reason for this? What is the relation between LC2-II level and degradation?\n8. What is the performance difference for EPLA and NP-EPLA? Since EPLA already formed nanoparticle, what is the reason\nthe synthesis EPLA into another type of NP?\n9. For in vivo experiment, the antibody was injected intraperitoneally, while the EPLA was injected intravenously. What is\nreason for choosing different drug injection methods? Is it the reason for the side effect of antibody for colon shortening?\n10. Please use different terms to describe EPLA and NP-based EPLA. For example, in Figure 6, the drug treatment in figure\na looks like the same as that of figure h."}, "author_response": "Point-by-point response to the reviewers’ comments\nDear all reviewers,\nThank you. We deeply appreciate your patience for reviewing our manuscript. Your comments and\nquestions are really helpful for the improvement of our work. We have taken all these comments\nand questions into account in our work and improved our manuscript by making the related revisions.\nReviewer #1:\n1. Line 147, Page 5: “However, the adsorption of plasma proteins by these assemblies was shown\ncomparable to PEGylated micelles, implying that the plasma protein binding may not affect their\npharmacokinetics.” Please add related reference or experimental results.\nResponse to the reviewer: We are sorry for this unclear description. PEGylated micelles are widely\nthought to be “stealth” in the blood and have long blood circulation. Our assemblies showed similar\nplasma protein adsorption to PEGylated micelles, implying the comparably long blood circulation.\nWe have modified the description. “However, the adsorption of plasma proteins by these assemblies\nwas shown comparable to PEGylated micelles, implying that these assemblies may be “stealth” in\nthe blood and have long blood circulation”.\nAccording to this comment, we compared the pharmacokinetic behaviors of CVR-EPLA and\nPEGylated micelles after intravenous administration in mice. “CVR-EPLA and PEGylated micelles\nloaded with near-infrared fluorescent probe IR780 were constructed and intravenously injected to\nmice for the pharmacokinetics study. The blood circulation behaviors were explored by measuring\nIR780 concentration. CVR-EPLA and PEGylated micelles showed similar kinetic behaviors in the\nblood (Extended Data Fig. 1f), confirming the long blood circulation effects.”\nExtended Data Fig. 1. f, Blood IR780 concentration-time curves in Balb/c immune-competent mice\n(female) for intravenously injected different formulations (0.75 mg IR780 kg-1) were imaged using\nan IVIS imaging system and semi-quantified using the software Living Image 4.2 (n = 5).\n1 / 27\n2. For colocalization analysis, what’s the colocalization ratio change between PD-L1 and Rab5?\nWhat’s the difference between different peptides (e.g., CVR-pHLIP & CVR-EPLA) in view of\nendocytosis? Since internalization is the first step for following degradation, it is critical to\nunderstand which component(s) is(are) responsible for it.\nResponse to the reviewer: According to this comment, we evaluated cellular uptake of CVR-EPLA,\nassessed the effects of anti-PD-L1 antibody, free CVR peptide, free GRP94-targeting NG peptide,\nand various endocytosis inhibitors on the uptake of CVR-EPLA, and studied the co-localizations of\nPD-L1 with Rab5 following CVR-EPLA treatment.\n“First, we examined the uptake of rhodamine-labeled CVR-EPLA in 4T1 mouse breast cancer\ncells (Fig. 2b). Non-targeting Ctrl-pHLIP was shown to be hardly internalized. However, both CVR-\npHLIP and CVR-EPLA were shown to be efficiently internalized with the cellular fluorescence\nintensity ~12.6-fold and ~12.5-fold that for cells treated with Ctrl-pHLIP, suggesting the role of\nCVR in cellular uptake. The uptake of CVR-EPLA in 4T1 cells was significantly reduced by anti-\nPD-L1 antibody (by ~59.6%) and free CVR peptide (by ~42.5%) but not by free GRP94-targeting\nNG peptide (by ~1.4%, Fig. 2c), proving that CVR-EPLA may bind with PD-L1 for the specific\ninternalization of both CVR-EPLA and PD-L1 into 4T1cells. Clathrin (60.9%, chlorpromazine),\nmacropinocytosis (42.2%, amiloride), lipid raft (39.6%, β-cyclodextrin) and caveolae (20.7%,\nnystatin) were involved in the internalization of CVR-EPLA into 4T1 cells, which was consistent\nwith previous reports where the constitutive and continuous endocytic transport of PD-L1 was\nclosely related to clathrin-mediated endocytosis41.”\n“To study if early endosome can transport with PD-L1 after CVR-EPLA treatment, HEK293T\nhuman embryonic kidney cells were transfected to express EGFP-PD-L1 and BFP-Rab5 (an early\nendosome marker). At 4 h, the proportion of Rab5-co-localized EGFP-PD-L1 relative to total PD-\nL1 obviously increased compared with untreated cells (Fig. 2d), proving the internalization of PD-\nL1. At 12 h, the ratio of Rab5-co-localized EGFP-PD-L1 even further increased, implying that\npartial PD-L1 may have been degraded and the remaining PD-L1 were still highly co-localized with\nRab5.”\n2 / 27\nFig. 2. b, The uptake of rhodamine-labeled different assemblies in 4T1 cells after incubation at 3.5\nμg rhodamine mL-1 for 4 h was examined using a flow cytometry (n = 4). c, Cellular uptake of\nrhodamine-labeled CVR-EPLA (3.5 μg rhodamine mL-1 for 4 h) by 4T1 cells in the presence of anti-\nPD-L1 antibody (1 μL at original concentration), free CVR peptide (0.1 mg mL-1), free GRP94-\ntargeting NG peptide (0.1 mg mL-1), or the indicated endocytosis inhibitors (25 μg mL-1 nystatin,\n6.5 mg mL-1 β-cyclodextrin, 100 μg mL-1 amiloride, and 10 μg mL-1 chlorpromazine) was examined\nusing a flow cytometry (n = 4). d, The co-localizations of EGFP-PD-L1 with BFP-Rab5 (a marker\nof early endosomes) in HEK293T cells after treatments with 0.5 μM CVR-EPLA for 4 h and 12 h,\nwere imaged using confocal microscopy and semi-quantified using the software ImageJ (n = 4).\nScale bar, 10 μm.\n3. A key control--conjugating POI-specific ligands to pHLIP C terminus and LC3-targeting motifs\nto pHLIP N terminus, is needed to confirm the expected effect of pHLIP for LC3 binding (which\nshould be a direct and convincing control with minimal interference from the peptide sequence).\nResponse to the reviewer: According to this comment, we performed the new experiment. “To\nexplore the necessity of anchoring the Q24 terminus rather than the CVR terminus onto endosome\nsurface and minimize peptide sequence interference, Q24-pHLIP-Mal was synthesized to construct\nQ24-pHLIP-CVR with CVR at pHLIP C terminus and Q24 at pHLIP N terminus, which is supposed\nto insert the CVR terminus across endosome membrane. In 4T1 cells, the PD-L1 degradation effect\nwas induced by CVR-EPLA treatment rather than by Q24-pHLIP-CVR treatment (Extended Data\nFig. 3h), confirming the effect of anchoring Q24 on LC3 binding.”\n3 / 27\nExtended Data Fig. 3. h, The levels of PD-L1 in 4T1 cells following treatments with 0.5 μM\ndifferent assemblies for 12 h were evaluated using western blot (n = 3).\n4. Line 231, Page 9: “PD-L1 depletion was inhibited by bafilomycin A1, chloroquine, and NH Cl\n4\nand reinforced by rapamycin, whereas MG132 did not show any effects (Fig. 3f),” no significant\ndifference was indicated/observed between CVR-EPLA and CVR-EPLA+ Rapamycin? How comes\nthe “reinforced by rapamycin”?\nResponse to the reviewer: We are sorry for this inaccurate description. Previously, we used 10 μM\nrapamycin to promote autophagic degradation and PD-L1 degradation was only slightly enhanced\nbut without significant difference. According to this comment, we performed a new experiment with\n30 μM rapamycin and 50 μM carbamazepine. Both treatments significantly heightened the PD-L1\ndegradation efficiency to 69.4% and 80.1%, respectively. We modified the description in the revised\nmanuscript. “PD-L1 depletion was inhibited by bafilomycin A1, chloroquine, and NH Cl and\n4\nreinforced by rapamycin and carbamazepine at high concentration, whereas MG132 did not show\nany effects (Fig. 3f and Extended Data Fig. 3j), proving that CVR-EPLA degrades PD-L1 via the\nautophagic degradation machinery.”\n4 / 27\nExtended Data Fig. 3. j, The levels of PD-L1 in 4T1 cells after sequential treatments with 30 μM\nrapamycin (or 50 μM carbamazepine) for 24 h and with the mixture of 0.5 μM CVR-EPLA and\nrapamycin (or carbamazepine) for 12 h, were evaluated using western blot (n = 3).\n5. Since endosome leakage was seen upon the incubation of CVR-EPLA, it is also possible that the\nreleased PD-L1-CVR-EPLA complex was translocated to phagophore directly for degradation\nrather than underwent endosome-phagophore pathway? Currently, the involvement of endosome is\nmainly (1) the imaging result of Rab5B in Fig 2 (of note, only colocalization between Rab5B and\nphagophore/lysosome were shown, with a ratio below 0.5; no data for PD-L1 and Rab5B was found\nin the maintext nor the SI) and (2) the endosome leakage result in Fig. 3. Unfortunately, both results were not\nconvincing/direct enough to support the involvement of endosome for CVR-EPLA-\nmediated degradation. The reviewer think it seems more likely to be the release of the complex into\ncytosol for following translocation to phagophore.\nResponse to the reviewer: According to this comment, we first studied the co-localizations of PD-\nL1 with Rab5 following CVR-EPLA treatment and then studied the co-localizations of CVR-EPLA\nwith endosomes, phagophores and lysosomes.\n“To study if early endosome can transport with PD-L1 after CVR-EPLA treatment, HEK293T\nhuman embryonic kidney cells were transfected to express EGFP-PD-L1 and BFP-Rab5 (an early\nendosome marker). At 4 h, the proportion of Rab5-co-localized EGFP-PD-L1 relative to total PD-\nL1 obviously increased compared with untreated cells (Fig. 2d), proving the internalization of PD-\nL1. At 12 h, the ratio of Rab5-co-localized EGFP-PD-L1 even further increased, implying that\npartial PD-L1 may have been degraded and the remaining PD-L1 were still highly co-localized with\nRab5.”\n“To confirm if CVR-EPLA can transport together with PD-L1-containing endosomes to\nphagophores and lysosomes, 4T1 cells were treated with rhodamine-labeled CVR-EPLA for 4~8 h\nto investigate the co-localizations with endosomes, phagophores and lysosomes. Obvious increase\n5 / 27\nwas shown for the co-localizations of CVR-EPLA with endosomes, phagophores, and lysosomes\nfrom 4 h to 8 h (Extended Data Fig. 2c). The high co-localization of PD-L1 with Rab5 (Fig. 2d), the\nproximity of both PD-L1 and endosomes to phagophores and lysosomes (Fig. 2e and Extended Data\nFig. 2b), and the high co-localizations of CVR-EPLA with endosomes, phagophores, and lysosomes\n(Extended Data Fig. 2c), collectively proved that CVR-EPLA can transport together with PD-L1-\ncontaining endosomes to phagophores and lysosomes for PD-L1 degradation. However, some\ncomplexes of CVR-EPLA and PD-L1 may still be released from endosomes into the cytoplasm for\ndirect translocation to phagophores and degradation.”\nFig. 2. d, The co-localizations of EGFP-PD-L1 with BFP-Rab5 (a marker of early endosomes) in\nHEK293T cells after treatments with 0.5 μM CVR-EPLA for 4 h and 12 h, were imaged using\nconfocal microscopy and semi-quantified using the software ImageJ (n = 4). Scale bar, 10 μm.\nExtended Data Fig. 2. c, The co-localizations of rhodamine-labeled CVR-EPLA with endosomes,\nphagophores, and lysosomes in 4T1 cells, which were treated with rhodamine-labeled CVR-EPLA\nat 4 μg rhodamine mL-1 for 4 h or 8 h. Treated cells were incubated with primary antibodies for\nEEA1 (endosomes), LC3 (phagophores) and LAMP1 (lysosomes), stained with fluorescent labeled\nsecondary antibody, and observed using a confocal laser scanning microscope and semi-quantified\nusing ImageJ (n = 4–5). Scale bar, 10 μm.\n6. The effect of these endosomolytic agents on plasma membrane damage also needs to be\nillustrated/discussed.\n6 / 27\nResponse to the reviewer: We used pHLIP to construct EPLAs and used HsPen, HsMPG and L17E\nas the control. “The interaction of engineered peptides with the lipid membrane in endosome lumen\n(acidic pH) was indicated by the hemoglobin release from red blood cells.” The data at pH 7.4 can\nalso explain the effects of these peptides on plasma membrane damage. According to this comment,\nwe added the following discussion into the revised manuscript.\n“Penetratin is the third helix of the Antennapedia homeodomain protein of Drosophila, residues\n43-58, and has the ability to penetrate biological membranes26. MPG is a short peptide that contains\nboth a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain\nderived from the nuclear localization sequence of SV40 T-antigen for gene delivery27. HsPen and\nHsMPG were respectively derived from penetratin and MPG by substituting the Arg/Lys residues\nwith histidine and/or adding N-terminal Phe-(Ser-Gly) for pH-dependent membrane perturbation\n4\nfor endosomal escape after cellular uptake23. Endosomolytic peptide L17E was derived from the\ncationic and membrane-lytic spider venom peptide M-lycotoxin by substituting the leucine (residue\n17) with glutamic acid for membrane perturbation and endosomal escape for cytosolic antibody\ndelivery22. The further-engineered Mal-HsPen-Q24, Mal-HsMPG-Q24, and Mal-L17E-Q24 were\nused as control to compare with Mal-pHLIP-Q24. All engineered peptides showed higher release of\nhemoglobin at pH 6.0 (early endosome pH) and pH 5.5 (late endosome pH) than at pH 7.4 (Fig. 1b),\nsuggesting the low plasma membrane damage at pH 7.4. In fact, both HsPen and HsMPG were\nengineered with the substitution of the Arg/Lys residues by histidine for pH-dependent membrane-\nperturbation activity to avoid random cell penetration23. L17E was proved to preferentially disrupt\nthe negatively charged membranes (endosomal membranes) over the neutral membranes (plasma\nmembranes)22. And pHLIP was reported to not damage red blood cells at concentrations up to 10\nΜm28. Our data are consistent with these previous reports.”\n“Although pHLIP does not damage red blood cells, its interaction with red blood cells at pH\n7.4 could occupy the surface of 67% of the cells28. However, the possible micelle-like structures of\nEPLAs would reduce the contact of pHLIP with cells to reduce the possibility of plasma membrane\ndamage.”\n7. Line 286, Page 12: “Notably, the levels of Aβ oligomers and RAGE in BV-2 microglia treated\nwith RGT-EPLA were ~57% and ~58% of that in cells treated with RGT-pHLIP (Fig. 4c and\nExtended Data 289 Fig. 4c), suggesting the degradation of Aβ oligomers and RAGE.” It is not\nobjective to conclude that degradation has occurred without pre-excluding the differences in\nendocytosis/uptake of Aβ oligomers into cells (i.e., by inhibiting the degradation of lysosome to\ncheck the cellular uptake).\nResponse to the reviewer: According to this comment, we performed the new experiment with the\naddition of bafilomycin A1, which blocks lysosomal acidification and degradation activity. “To pre-\nexclude Aβ oligomer uptake difference between cells, BV-2 microglia were first treated with the\nmixture of Aβ oligomer and lysosomal inhibitor bafilomycin A1 and then treated with the mixture\n7 / 27\nof Aβ oligomer, bafilomycin A1 and assemblies. In the presence of bafilomycin A1, BV-2 microglia\nshowed similar Aβ oligomer uptake between RGT-EPLA and RGT-pHLIP (Extended Data Fig. 4d).\nHowever, in the absence of bafilomycin A1, the lower Aβ oligomer levels in cells with RGT-EPLA\nconfirmed the degradation of Aβ oligomers.”\nExtended Data Fig. 4. d, The intracellular levels of Aβ oligomers in BV-2 microglia after 24 h co-\nincubation with 10 μM Aβ oligomers (+/- 50 μM bafilomycin A1) and following 12 h co-incubation\nwith 10 μM Aβ oligomers (+/- 50 μM bafilomycin A1) and 10 μM RGT-pHLIP (or RGT-EPLA)\nwere measured using western blot (n = 3).\n8. There was no obvious fluorescence from phagophore nor lysosome from the group of Ctrl-pHLIP\nin Fig. 4? Similar phenomena were also seen from other control groups in Fig. 4. Please clarify the\ndifference in these images to confirm the accuracy of colocalization ratio.\nResponse to the reviewer: Phagosomes and lysosomes are constantly produced and consumed for\ncontinuous phagocytosis and degradation. However, their concentrations are relatively low under\nphysiological conditions. The obvious fluorescence of phagophores and lysosome in RGT-EPLA-\ntreated cells can be ascribed to the high uptake of RGT-EPLA and Q24-accelerated autophagosome\nformation.\nAccording to this comment, we checked the raw images (Extended Data Fig. 4e). Although\nsome images showed relatively stronger fluorescence of phagosomes and lysosomes for cells treated\nwith Ctrl-pHLIP, Ctrl-EPLA and RGT-pHLIP, the data about co-localization ratios are consistent\nwith the displayed data.\n8 / 27\nExtended Data Fig. 4. e, The co-localizations of the internalized FITC-labeled Aβ oligomers and\nGFP-Rab5B (a marker of early endosomes) with RFP-LC3 (a marker of phagophores) and BFP-\nLAMP1 (a marker of lysosomes, pseudo red) in RAGE-upregulated BV-2 microglia after 12 h co-\nincubation with the mixtures of 10 μM different assemblies and either 10 μM FITC-labeled Aβ\noligomers or unlabeled Aβ oligomers were imaged using confocal microscopy and semi-quantified\nfor confocal microscopy images using the software ImageJ (n = 3–5). Scale bar, 40 μm.\n9. Dose the NP-based EPLAs show same time-dependent cellular distribution (the colocalization\nwith Rab5B, phagophore, and lysosome) as the peptide-based EPLAs?\nResponse to the reviewer: According to this comment, PD-L1-targeting CVR-EPLA@NPs was\nstudied for the co-localizations with endosomes, phagophores and lysosomes. “CVR-EPLA@NPs\nshowed similar time-dependent intracellular distribution to the peptide-based CVR-EPLA with the\ngradually increased co-localization with early endosomes, phagophores, and lysosomes from 4 h to\n8 h incubation in 4T1 cells (Extended Data Fig. 5b). The findings suggested the similar degradation\nmechanisms between the NP-based EPLAs and the peptide-based EPLAs.”\n9 / 27\nExtended Data Fig. 5. b, The co-localizations of fluorescently labeled CVR-EPLA@NPs with\nendosomes, phagophores, and lysosomes in 4T1 cells, which were treated with fluorescently labeled\nCVR-EPLA@NPs at 5 μg probe (doxorubicin, DOX) mL-1 for 4 h or 8 h. Treated cells were\nincubated with primary antibodies for EEA1 (endosomes), LC3 (phagophores) and LAMP1\n(lysosomes), stained with fluorescent labeled secondary antibody, and observed using a confocal\nlaser scanning microscope and semi-quantified using ImageJ (n = 3). Scale bar, 10 μm.\n10. What is the requirement for the labeling ratio of ES-PTP and POI-targeting ligands onto NPs for\noptimal degradation efficacy?\nResponse to the reviewer: Based on the ratio of POI-targeting ligands to ES-PTP (1:1) in EPLAs,\nwe used the ligand ratio of 1:1 to display ES-PTP and POI-targeting ligands onto NPs to synthesize\nEPLA@NPs to prove our degradation concept. According to the positively related concentration-\ndependent CVR-EPLA-induced PD-L1 degradation (Fig. 3b and Extended Data Fig. 3b), we used\nexcessive POI-targeting ligands and ES-PTP to synthesize EPLA@NPs for the optimal degradation\nefficacy. However, the labeling ratio of ES-PTP and POI-targeting ligands may be further optimized\nfor EPLA@NPs.\n11. Is the immunodeficient BALB/c mouse the optimal animal model for PD-L1 antibodies?\nResponse to the reviewer: We are sorry for the unclear description. We used Balb/c immune-\ncompetent mice with 4T1 tumors to evaluate the effects of PD-L1-targeting therapy. According to\nthis comment, we detailed the information for mice in the revised Methods.\nThe growth and metastasis of 4T1 cells in Balb/c immune-competent mice are similar to\nclinical human breast cancer. PD-L1-targeting therapy functions by regulating immune cells, e.g.,\nT cells. While Balb/c immune-deficient mice are unable to produce effective immune responses due\nto the loss or dysfunction of immune cells, Balb/c immune-competent mice have been widely used\nto evaluate the therapeutic effects of PD-L1 antibodies1-3.\n1. Huang L. et al. Mild photothermal therapy potentiates anti-PD-L1 treatment for\nimmunologically cold tumors via an all-in-one and all-in-control strategy. Nat Commun 10,\n4871 (2019).\n10 / 27\n2. Tu J. et al. Nintedanib enhances the efficacy of PD-L1 blockade by upregulating MHC-I and\nPD-L1 expression in tumor cells. Theranostics 12, 747-766 (2022).\n3. Lee Y. et al. Hyaluronic acid-bilirubin nanomedicine-based combination\nchemoimmunotherapy. Nat Commun 14, 4771 (2023).\n12. Why murine cell lines were employed for most cell-based experiments? Are there available POI-\nbinding peptides for human cells, which is important for therapeutic potentials.\nResponse to the reviewer: To evaluate the in vivo antitumor immunotherapeutic effects of PD-L1-\ntargeting EPLAs, immune-competent mice should be used. We selected Balb/c mice with 4T1 breast\nc breast\ncancers as the animal model. Therefore, we used the murine 4T1 cell line for most PD-L1-targeting\ncell-based experiments.\nIn fact, the degradation performance of NGP-EPLA@NPs for GRP94 and AKR-EPLA@NPs\nfor p32 was evaluated in MDA-MB-231 human breast cancer cells.\nIn addition, the ligands including PD-L1-targeting CVR, ICAM-1-targeting NNQ, and VCAM-\n1-targeting VHP have been widely used for human cells1-3. Therefore, our EPLA platforms can be\nbroadly used for human cells for therapeutic potentials.\n1. Gurung S. et al. Phage display-identified PD-L1-binding peptides reinvigorate T-cell activity\nand inhibit tumor progression. Biomaterials 247, 119984 (2020).\n2. Yang Y. et al. Inflammation-targeting polymeric nanoparticles deliver sparfloxacin and\ntacrolimus for combating acute lung sepsis. J. Control. Release 321, 463-474 (2020).\n3. Afrin H. et al. Detection of Anticancer Drug-Induced Cardiotoxicity Using VCAM1-Targeted\nNanoprobes. ACS Appl Mater Interfaces 14, 37566-37576 (2022).\nReviewer #2:\n1. The introduction section, especially the third paragraph, fails to effectively highlight the\ninnovation and significance of the study and needs to be substantially revised.\nResponse to the reviewer: Thank the reviewer for recognizing the innovation and significance of\nthe study. We sincerely appreciate your valuable suggestions regarding our manuscript. According\nto this comment, we have substantially revised the introduction, especially the third paragraph.\n“Most membrane/extracellular proteins can enter cells via constitutive endocytosis (e.g.,\nprogrammed death-ligand 1 (PD-L1)15) or can spread between cells via receptor-mediated\nendocytosis (e.g., the microtubule-associated protein tau16). Both the UPS pathway and the EL\npathway can be used to degrade membrane/extracellular proteins. The UPS-based proteolysis-\ntargeting antibodies use cell-surface E3 ubiquitin ligases to induce the internalization of target\nproteins3. LYTACs use cell surface lysosome-directing receptors for endocytosis of target proteins10.\nHowever, there has been so far no report on degradation tools that hijack constitutive endocytosis\nor intercellular spread mechanisms for POI internalization and subsequent degradation. Targeting\nonly POIs rather than binding to two targets for cell entry holds the enormous potential to increase\nspecificity of targeted degradation and to address the hook effects.”\n“Only a small amount of endocytosed proteins can be transported from endosomes to\n11 / 27\nlysosomes for degradation, most internalized proteins return to the plasma membrane and the\nextracellular space through recycling endosomes17-19. In the autophagy machinery of all eukaryotic\ncells, the “open” phagophores could selectively engulf intracellular damaged components including\norganelles, parts of the cytoplasm, and proteins and transport them to lysosomes for degradation20.\nBased on the selective phagocytosis and autophagy of the “open” phagophores, we hypothesized\nthat the endosomal pathway and the autophagic machinery could be combined to establish an\n“endosome-phagophore-lysosome” non-traditional transport pathway for the degradation of\nmembrane/extracellular proteins. We envisioned that after the entry of membrane/extracellular\nproteins into the endosomes through endocytosis, if the early endosomes containing\nmembrane/extracellular POIs can be labeled with motifs targeting ATG8 family proteins, they would\nbe specially swallowed by the “open” phagophores, thereby preventing their recycling and\npromoting their autophagic degradation. However, there has been so far no report on the autophagic\ndepletion of membrane/extracellular proteins by labeling endosomes with phagophore-binding\nmoieties and phagocytosing the labeled endosomes with phagophores.”\n“Some functional peptides, such as pH-low insertion peptides (pHLIP)21, insert into the cell\nmembrane under acidic pH conditions with their terminal groups anchored on the other side of the\ninserted membrane. Inspired by pHLIP’s function at acidic pH, here we present a generalized\nstrategy to build an “endosome-phagophore-lysosome” non-traditional transport pathway by\nemploying endosome-phagophore linking assemblies (EPLAs), for autophagic degradation of\nmembrane/extracellular proteins. EPLAs are constructed by conjugating the POI-specific ligands to\nthe N terminus of an endosome surface-anchored phagophore-targeting peptide (ES-PTP) that\nconsists of a pHLIP and a C-terminal phagophore-binding element. EPLAs are expected to specially\nrecognize and bind with membrane/extracellular POIs for cell entry, to insert into endosome\nmembrane at acidic pH, and to label the endosome surface to target phagophores for protein\ndegradation. This pHLIP-based endosome membrane insertion and endosome labeling technology\nmay have broad implications for biochemical research. This generalized EPLA strategy could\nbroaden the target spectrum of autophagy to membrane/extracellular POIs and may revolutionize\nthe current TPD tool development landscape for the degradation of membrane/extracellular POIs.”\n2. For Fig. 1, multiple peptide abbreviations such as Hspen, LITE, and HSMPG are mentioned, but\nthe authors did not provide their sources or explain why they were selected. Additionally, the\ncharacteristics and advantages of Q24 were not adequately introduced, nor was the rationale for\nchoosing it over other peptides.\nResponse to the reviewer: We are sorry for this unclear description. According to this comment,\nwe provided the related information in the revised manuscript.\n“Penetratin is the third helix of the Antennapedia homeodomain protein of Drosophila, residues\n43-58, and has the ability to penetrate biological membranes26. MPG is a short peptide that contains\nboth a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain\n12 / 27\nderived from the nuclear localization sequence of SV40 T-antigen for gene delivery27. HsPen and\nHsMPG were respectively derived from penetratin and MPG by substituting the Arg/Lys residues\nwith histidine and/or adding N-terminal Phe-(Ser-Gly)4 for pH-dependent membrane perturbation\nfor endosomal escape after cellular uptake23. Endosomolytic peptide L17E was derived from the\ncationic and membrane-lytic spider venom peptide M-lycotoxin by substituting the leucine (residue\n17) with glutamic acid for membrane perturbation and endosomal escape for cytosolic antibody\ndelivery22. The further-engineered Mal-HsPen-Q24, Mal-HsMPG-Q24, and Mal-L17E-Q24 were\nused as control to compare with Mal-pHLIP-Q24.”\n“The canonical motifs of LC3-interacting region (LIR) are characterized with a core consensus\nsequence of [W/F/Y]-X1-X2-[I/L/V] (X1, X2 represent any amino acid). The Q24 peptide with a\nsequence of GIYVIWGPI was derived from the transmembrane (TM) domain of the mitochondrial-\nanchored ubiquitin-specific peptidase 30 (USP30), possesses the core consensus sequence of the\ncanonical LIR motifs, and can bind with LC3 and accelerate autophagosome formation25. Besides\nthe acceleration of autophagosome formation, Q24 does not require to be placed at the terminus,\nwhich endows Q24 the high applicability for different needs. However, Q24 may be not superior to\nother LIR motifs. For example, the addition of two LIR motifs at the N terminus of Q24 significantly\nfacilitated the interaction with LC3 and the autophagosome membrane. Q24 could be upgraded or\neven replaced for more efficient degradation performance.”\n3. How about the intracellular uptake efficiency of EPLAs?\nResponse to the reviewer: According to this comment, we evaluated the uptake of CVR-EPLA in\n4T1 cells. “First, we examined the uptake of rhodamine-labeled CVR-EPLA in 4T1 mouse breast\ncancer cells (Fig. 2b). Non-targeting Ctrl-pHLIP was shown to be hardly internalized. However,\nboth CVR-pHLIP and CVR-EPLA were shown to be efficiently internalized with the cellular\nfluorescence intensity ~12.6-fold and ~12.5-fold that for cells treated with Ctrl-pHLIP, suggesting\nthe role of CVR in cellular uptake.”\nFig. 2. b, The uptake of rhodamine-labeled different assemblies in 4T1 cells after incubation at 3.5\nμg rhodamine mL-1 for 4 h was examined using a flow cytometry (n = 4).\n13 / 27\n4. In the Fig. 2B, exogenous co-localization was conducted. Although this provides partial support\nfor the conclusion, it is insufficient. Endogenous fluorescence co-localization, as well as\nintracellular uptake and co-localization at different time points, should also be performed.\nAdditionally, Fig. 2B lacks clear labeling—for example, it should indicate EGFP-PD-L1 instead of\nPD-L1.\nResponse to the reviewer: We are sorry for the insufficient proof and unclear labeling. According\nto this comment, we first studied the co-localizations of PD-L1 with Rab5. We further investigated\nthe endogenous fluorescence co-localization of PD-L1 with phagophores and lysosomes and the co-\nlocalizations of CVR-EPLA with endosomes, phagophores and lysosomes by immunofluorescence\nstaining, to prove that CVR-EPLA can transport together with PD-L1-containing endosomes to\nphagophores and lysosomes for PD-L1 degradation. We also improved the labeling for all data in\nthe revised manuscript.\n“To study if early endosome can transport with PD-L1 after CVR-EPLA treatment, HEK293T\nhuman embryonic kidney cells were transfected to express EGFP-PD-L1 and BFP-Rab5 (an early\nendosome marker). At 4 h, the proportion of Rab5-co-localized EGFP-PD-L1 relative to total PD-\nL1 obviously increased compared with untreated cells (Fig. 2d), proving the internalization of PD-\nL1. At 12 h, the ratio of Rab5-co-localized EGFP-PD-L1 even further increased, implying that\npartial PD-L1 may have been degraded and the remaining PD-L1 were still highly co-localized with\nRab5.”\n“To investigate the endogenous fluorescence co-localization between PD-L1 and phagophores\n(lysosomes) after CVR-EPLA treatment, 4T1 cells were treated and then endogenous PD-L1, LC3\n(phagophores) and LAMP1 (lysosomes) were labeled through immunofluorescence staining and the\nrelated antibodies (Extended Data Fig. 2b). In both untreated cells and cells treated with Ctrl-pHLIP,\nCtrl-EPLA and CVR-pHLIP, the endogenous PD-L1 was shown to be located both at cell membrane\nand in the cytoplasm but not co-localized with endogenous phagophores and lysosomes, which can\nbe ascribed to the low uptake of Ctrl-pHLIP and Ctrl-EPLA and the absence of LC3-targeting Q24\nin CVR-pHLIP. However, in cells treated with CVR-EPLA, most of PD-L1 was co-localized with\nphagophores with the decrease of PD-L1 at the cell membrane and some of PD-L1 was even co-\nlocalized with lysosomes.”\n“To confirm if CVR-EPLA can transport together with PD-L1-containing endosomes to\nphagophores and lysosomes, 4T1 cells were treated with rhodamine-labeled CVR-EPLA for 4~8 h\nto investigate the co-localizations with endosomes, phagophores and lysosomes. Obvious increase\nwas shown for the co-localizations of CVR-EPLA with endosomes, phagophores, and lysosomes\nfrom 4 h to 8 h (Extended Data Fig. 2c). The high co-localization of PD-L1 with Rab5 (Fig. 2d), the\nproximity of both PD-L1 and endosomes to phagophores and lysosomes (Fig. 2e and Extended Data\nFig. 2b), and the high co-localizations of CVR-EPLA with endosomes, phagophores, and lysosomes\n(Extended Data Fig. 2c), collectively proved that CVR-EPLA can transport together with PD-L1-\ncontaining endosomes to phagophores and lysosomes for PD-L1 degradation. However, some\n14 / 27\ncomplexes of CVR-EPLA and PD-L1 may still be released from endosomes into the cytoplasm for\ndirect translocation to phagophores and degradation.”\nFig. 2. d, The co-localizations of EGFP-PD-L1 with BFP-Rab5 (a marker of early endosomes) in\nHEK293T cells after treatments with 0.5 μM CVR-EPLA for 4 h and 12 h, were imaged using\nconfocal microscopy and semi-quantified using the software ImageJ (n = 4). Scale bar, 10 μm.\nExtended Data Fig. 2. b, The co-localizations of PD-L1 with LC3 (phagophores) and LAMP1\n(lysosomes) in 4T1 cells, which were treated with 0.5 μM different assemblies for 3 h. Treated cells\nwere incubated with primary antibodies for PD-L1, LC3 (phagophores) and LAMP1 (lysosomes),\nstained with fluorescent labeled secondary antibody, and observed using a confocal laser scanning\nmicroscope. Scale bar, 10 μm. c, The co-localizations of rhodamine-labeled CVR-EPLA with\nendosomes, phagophores, and lysosomes in 4T1 cells, which were treated with rhodamine-labeled\nCVR-EPLA at 4 μg rhodamine mL-1 for 4 h or 8 h. Treated cells were incubated with primary\nantibodies for EEA1 (endosomes), LC3 (phagophores) and LAMP1 (lysosomes), stained with\n15 / 27\nfluorescent labeled secondary antibody, and observed using a confocal laser scanning microscope\nand semi-quantified using ImageJ (n = 4–5). Scale bar, 10 μm.\n5. In the Fig. 2, the authors used fluorescence experiments to investigate the spatiotemporal\nregulation of EPLA, but this is not sufficient. Techniques such as flow cytometry should be\nemployed for further validation.\nResponse to the reviewer: Thank the reviewer for this very valuable comment. According to this\ncomment, we added the flow cytometry data of the uptake efficiency, specificity and mechanisms\nof CVR-EPLA in 4T1 cells (Fig. 2b–c). We also added the data of the co-localizations of PD-L1\nwith Rab5 following CVR-EPLA treatment (Fig. 2d), the endogenous fluorescence co-localization\nof PD-L1 with phagophores and lysosomes (Extended Data Fig. 2b), and the endogenous\nfluorescence co-localizations of peptide-based and NP-based CVR-EPLA with endosomes,\nphagophores and lysosomes (Extended Data Fig. 2c and Extended Data Fig. 5b). We think that these\nadded data may be sufficient to reveal the intracellular EPLA spatiotemporal distribution to prove\nthat, CVR-EPLA can transport together with PD-L1-containing endosomes to phagophores and\nlysosomes for PD-L1 degradation.\n6. In the Fig. 3, the order of panels A, B, and C lacks logic. It should be rearranged as C, A, and B.\nResponse to the reviewer: According to this comment, we have rearranged the order of panels (Fig.\n3a–c) and the related descriptions.\n7. The key information of legends is missing, for example, the specific cell lines used are not clearly\nindicated.\nResponse to the reviewer: We apologized for the omission of the key information of legends.\nAccording to this comment, we have specified the details including cell lines in the revised legends.\n8. In Fig. 3A–C, the authors used the murine 4T1 cell line instead of human cell lines. The rationale\nfor this choice is unclear. The authors did not explain this in the results. Moreover, western blot and\nfluorescence results from human cell lines should be provided.\nResponse to the reviewer: To evaluate the in vivo antitumor immunotherapeutic effects of PD-L1-\ntargeting EPLAs, immune-competent mice should be used. We selected Balb/c mice with 4T1 breast\ncancers as the animal model. Therefore, we used the murine 4T1 cell line for most PD-L1-targeting\ncell-based experiments.\nPD-L1-targeting ligand CVR can be used for targeting PD-L1 in human cells1. According to\nthis comment, we studied the degradation performance of CVR-EPLA for PD-L1 and the co-\nlocalizations of PD-L1 with LC3 following CVR-EPLA treatment in MDA-MB-231 cells.\n“In MDA-MB-231 human breast cancer cells, while CVR-pHLIP failed to decompose PD-L1,\nCVR-EPLA degraded PD-L1 in a concentration and time co-dependent manner (Extended Data Fig.\n16 / 27\n3e–f). And for the endogenous fluorescence co-localization between PD-L1 and LC3 (phagophores)\nin MDA-MB-231 cells, in both untreated cells and cells treated with CVR-NPs and CVR-pHLIP,\nPD-L1 was not co-localized with phagophores (Extended Data Fig. 3g). However, in cells treated\nwith CVR-EPLA, significant co-localization was shown between PD-L1 and phagophores. These\ndata proved the feasibility of using CVR-EPLA to degrading PD-L1 in human cells.”\n1. Gurung S. et al. Phage display-identified PD-L1-binding peptides reinvigorate T-cell activity\nand inhibit tumor progression. Biomaterials 247, 119984 (2020).\nExtended Data Fig. 3. e-f, The levels of PD-L1 in MDA-MB-231 cells after treatments with CVR-\nEPLA and CVR-pHLIP at different concentrations for 12 h or at 0.5 μM for various times were\nevaluated using western blot. g, The co-localizations of PD-L1 with LC3 (phagophores) in MDA-\nMB-231 cells, which were treated with different assemblies for 3 h. Treated cells were incubated\nwith primary antibodies for PD-L1 and LC3 (phagophores), stained with fluorescent labeled\nsecondary antibody, and observed using a confocal laser scanning microscope. Scale bar, 10 μm.\n9. The mechanism of action is unclear. Although the authors state that EPLA degrades target proteins\nvia an LC3-dependent phagophore-lysosomal pathway, they did not block LC3-dependent pathway\nto verify this mechanism.\nResponse to the reviewer: According to this comment, we performed a new experiment. “Atg5 is\none of the major regulators of autophagy and is essential for the formation of LC3-II. Cells deficient\n17 / 27\nof Atg5 are unable to form autophagosomes46. To prove the involvement of LC3 in EPLA-mediated\nprotein degradation, MDA-MB-231 cells were transfected with a CRISPR-Cas9 plasmid targeting\nhuman Atg5 to knockdown Atg5 and then to reduce LC3-II. After the selection by puromycin for\none week, the levels of LC3-II were significantly reduced (Extended Data Fig. 3k). Then MDA-\nMB-231 control cells and LC3-II-reduced cells were treated with CVR-EPLA for the evaluation of\nPD-L1 levels. CVR-EPLA failed to degrade PD-L1 in LC3-II-reduced cells (Fig. 3g), proving the\ninvolvement of LC3-II in CVR-EPLA-mediated PD-L1 degradation.”\nFig. 3. g, The levels of PD-L1 in MDA-MB-231 cells (+/- Atg5) following treatments with 0.5 μM\nCVR-EPLA for 12 h were evaluated using western blot (n = 3).\nExtended Data Fig. 3. k, The levels of LC3-II following transfection with a CRISPR-Cas9 plasmid\ntargeting human Atg5, puromycin selection for one week, and treatment with 0.5 μM CVR-EPLA\nfor 12 h were evaluated using western blot (n = 3).\n10. Fig. 5 should be moved to the supplementary results.\nResponse to the reviewer: According to this comment, we have moved Fig. 5 to the supplementary\nresults as Extended Data Fig. 6 and modified the related citations.\n18 / 27\n11. In the Fig. 6, the authors used CVR-EPLA to evaluate its in vivo antitumor efficacy, but no\nexperiments were conducted to assess its in vivo safety, which should be addressed.\nResponse to the reviewer: We are sorry our unclear data display. Previously, we studied the in vivo\nsafety and displayed the data in the original Extended Data Fig. 6 (Extended Data Fig. 7 in the\nrevised manuscript). The colitis, impaired liver functions, and liver and spleen T cell infiltration\nwere shown in mice treated with anti-PD-L1 antibodies but not in mice treated with CVR-EPLA\n(Extended Data Fig. 7a–d).\nAccording to this comment, we performed hematoxylin and eosin staining for tissues excised\nfrom CVR-EPLA-treated mice. “According to the data on hematoxylin-eosin staining (Extended\nData Fig. 7e), compared with untreated healthy tissues, three intravenous injections of CVR-EPLA\ndidn’t cause any histological and morphological changes such as necrosis for the studied tissues.”\nExtended Data Fig. 7. e, Hematoxylin-eosin staining was performed for various organs from Balb/c\nimmune-competent mice (female), which were intravenously injected with CVR-EPLA or CVR-\npHLIP (1.8 μmol kg-1 per injection) three times on days 0, 2, and 4 and euthanized on day 7. Scale\nbars, 50 µm.\n12. The authors emphasize that EPLA has certain advantages over traditional LYTACs, but no\ncomparative degradation or functional experiments were provided. In the Fig. 3A, CVR-EPLA only\ndegraded PD-L1 effectively at 1 μM. Therefore, comparative experiments need to be included.\nResponse to the reviewer: “Lysosome-targeting TFRC has been exploited to design LYTACs for\nthe degradation of membrane and extracellular proteins10,47. We synthesized PD-L1-targeting\nLYTACs (CVR-T12@NPs) by co-displaying CVR and TFRC-targeting T12 on NPs because of the\nupregulated expression of TFRC on 4T1 cells48,49. To compare the PD-L1 degradation efficiency,\n4T1 cells were treated with either LYTACs (CVR-T12@NPs) or CVR-EPLA@NPs. The PD-L1\ndegradation was shown to be similar for both treatments (Extended Data Fig. 5c). However, if target\ncells only moderately express TFRC, the degradation potency of LYTACs may decrease. Moreover,\nthe inevitable expression of TFRC on non-target cells, e.g., brain endothelial cells, could lead to off-\ntarget effects and toxicity.”\n19 / 27\nExtended Data Fig. 5. c, The levels of PD-L1 in 4T1 cells following treatments with CVR-\nT12@NPs or CVR-EPLA@NPs at 2 mg mL-1 for 12 h were evaluated using western blot (n = 3).\n13. How about the degradation specificity of CVR-EPLA? It is better to perform unbiased\nproteomics experiments to address this issue.\nResponse to the reviewer: Previously, we used western blot to study the degradation specificity of\nCVR-EPLA by setting the membrane protein LRP1 as the control. According to this comment, we\nperformed the proteomic profiling to further study the degradation specificity of CVR-EPLA.\n“We performed the proteomic profiling to further study the degradation specificity of CVR-\nEPLA. Compared with untreated cells, MDA-MB-231 cells treated with CVR-EPLA showed the\nup-regulation (107/8173, ~1.3%) and down-regulation (87/8173, ~1.1%) for a small amount of\nproteins (Extended Data Fig. 5a), which may be related to PD-L1 degradation and the designed\n“endosome-phagophore-lysosome” degradation pathway. However, the levels of many membrane\nproteins including epidermal growth factor receptor (EGFR), low-density lipoprotein receptor\n(LDLR), CD44, mannose-6-phosphate receptor (M6PR), LRP1, and transferrin receptor (TFRC)\nwere not changed, proving the degradation specificity of CVR-EPLA.”\n20 / 27\nExtended Data Fig. 5. a, Quantitative proteomic analysis showing the specificity of CVR-EPLA\nin degrading the PD-L1 in MDA-MB-231 cells. MDA-MB-231 cells were incubated with 0.5 μM\nCVR-EPLA or PBS (as a control) at 37 °C for 12 h. Each experiment was performed in three\nreplicates, and the unpaired t test was used to calculate the P values followed by Benjamini–\nHochberg adjustment. Proteins with adjusted P-values less than 0.05 (y-axis) and fold changes\ngreater than 2 (x-axis) were considered to be significantly changed.\nReviewer #3:\n1. What’s the biological consequence of degradation of PD-L1 or RAGE in cells?\nResponse to the reviewer: According to this comment, we added the biological consequence of\ndegradation of PD-L1 or RAGE in target cells in the revised manuscript.\n“PD-L1 is a key immune checkpoint protein and is constitutively overexpressed across diverse\nmalignancies. Its engagement with PD-1 on T lymphocytes transduces immunosuppressive signals\nto attenuate T-cell clonal expansion, thereby facilitating tumor immune evasion30. Besides, PD-L1\nintracellular structure has been reported to promote tumor survival and proliferation31. Thus, PD-L1\ndegradation in tumor cells could increase activation of effector T cells and decrease activation of\nregulatory T cells for effective anti-tumor immune responses and even directly blocks tumor\nproliferation.”\n“Receptor for advanced glycation end products (RAGE) functions as a receptor for brain\nuptake and brain intercellular spread of amyloid-β (Aβ). RAGE expression is often elevated in brain\nregions affected by Alzheimer’s disease (AD) and is closely related to AD exacerbation including\nbiochemical abnormalities and behavioral deficits32-34. RAGE degradation could reduce brain\nuptake and brain intercellular spread of pathological Aβ to potentially improve AD biochemical\nindicators and clinical symptoms.”\n2. How about the degradation specificity of EPLA? It is necessary to perform global proteomic\nprofiling using mass spectrometry.\nResponse to the reviewer: Previously, we used western blot to study the degradation specificity of\nCVR-EPLA by setting the membrane protein LRP1 as the control. According to this comment, we\nperformed the proteomic profiling to further study the degradation specificity of CVR-EPLA.\n“We performed the proteomic profiling to further study the degradation specificity of CVR-\nEPLA. Compared with untreated cells, MDA-MB-231 cells treated with CVR-EPLA showed the\nup-regulation (107/8173, ~1.3%) and down-regulation (87/8173, ~1.1%) for a small amount of\nproteins (Extended Data Fig. 5a), which may be related to PD-L1 degradation and the designed\n“endosome-phagophore-lysosome” degradation pathway. However, the levels of many membrane\nproteins including epidermal growth factor receptor (EGFR), low-density lipoprotein receptor\n(LDLR), CD44, mannose-6-phosphate receptor (M6PR), LRP1, and transferrin receptor (TFRC)\nwere not changed, proving the degradation specificity of CVR-EPLA.”\n21 / 27\nExtended Data Fig. 5. a, Quantitative proteomic analysis showing the specificity of CVR-EPLA\nin degrading the PD-L1 in MDA-MB-231 cells. MDA-MB-231 cells were incubated with 0.5 μM\nCVR-EPLA or PBS (as a control) at 37 °C for 12 h. Each experiment was performed in three\nreplicates, and the unpaired t test was used to calculate the P values followed by Benjamini–\nHochberg adjustment. Proteins with adjusted P-values less than 0.05 (y-axis) and fold changes\ngreater than 2 (x-axis) were considered to be significantly changed.\n3. What is the internalization mechanism of EPLA? How specificity it is?\nResponse to the reviewer: According to this comment, we assessed the effects of anti-PD-L1\nantibody, free CVR peptide, free GRP94-targeting NG peptide, and various endocytosis inhibitors\non the uptake of CVR-EPLA in 4T1 cells.\n“The uptake of CVR-EPLA in 4T1 cells was significantly reduced by anti-PD-L1 antibody (by\n~59.6%) and free CVR peptide (by ~42.5%) but not by free GRP94-targeting NG peptide (by ~1.4%,\nFig. 2c), proving that CVR-EPLA may bind with PD-L1 for the specific internalization of both\nCVR-EPLA and PD-L1 into 4T1cells. Clathrin (60.9%, chlorpromazine), macropinocytosis (42.2%,\namiloride), lipid raft (39.6%, β-cyclodextrin) and caveolae (20.7%, nystatin) were involved in the\ninternalization of CVR-EPLA into 4T1 cells, which was consistent with previous reports where the\nconstitutive and continuous endocytic transport of PD-L1 was closely related to clathrin-mediated\nendocytosis41.”\n22 / 27\nFig. 2. c, Cellular uptake of rhodamine-labeled CVR-EPLA (3.5 μg rhodamine mL-1 for 4 h) by 4T1\ncells in the presence of anti-PD-L1 antibody (1 μL at original concentration), free CVR peptide (0.1\nmg mL-1), free GRP94-targeting NG peptide (0.1 mg mL-1), or the indicated endocytosis inhibitors\n(25 μg mL-1 nystatin, 6.5 mg mL-1 β-cyclodextrin, 100 μg mL-1 amiloride, and 10 μg mL-1\nchlorpromazine) was examined using a flow cytometry (n = 4).\n4. Usually, the pHLIP would insert the cellular membrane at acidic pH. How about EPLA? The\nauthors should test its cellular performance at low pH, which mimics the tumor acidic environment.\nResponse to the reviewer: According to this comment, we compared PD-L1 degradation efficiency\nfor CVR-EPLA on 4T1 cells at both neutral pH and acidic pH. “Considering the acidic feature of\ntumor microenvironment and the cell membrane insertion effect of pHLIP at acidic pH, we tested\nthe PD-L1 degradation efficiency of CVR-EPLA on 4T1 cells at both neutral pH and acidic pH. It\ncan be seen that, there was no significant difference for the PD-L1 degradation effects of CVR-\nEPLA at both pH (Fig. 3h). There may exist a time-based competition between PD-L1-mediated\nCVR-EPLA endocytosis and pHLIP membrane insertion. However, even if pHLIP membrane\ninsertion happens before CVR-EPLA endocytosis, CVR-EPLA endocytosis and following\nendosome labeling and PD-L1 degradation would happen because pHLIP does not induce\nendocytosis by itself. Therefore, CVR-EPLA showed similar PD-L1 degradation efficiency at\nneutral pH and acidic pH.”\nFig. 3. h, The levels of PD-L1 in 4T1 cells following treatments with 0.5 μM CVR-EPLA at pH 7.4\n23 / 27\nor at pH 6.0 for 12 h were evaluated using western blot (n = 4).\n5. The degradation of extracellular amyloid-β and tau relies first on the membrane receptors to\nuptake. How can it be general to degrade other extracellular proteins without membrane receptors?\nResponse to the reviewer: We could divide extracellular proteins without membrane receptors into\ntwo categories. Category 1: proteins that can be internalized into acidic endosome compartments\nvia clathrin-mediated endocytosis, macropinocytosis, lipid raft and caveolae-mediated endocytosis;\nThese proteins could be degraded using the tailor-made EPLAs with both ES-PTP and POI-targeting\nligands. Category 2: proteins that cannot be internalized. These proteins can also be degraded using\nthe tailor-made EPLAs but with the addition of ligands that could trigger cellular internalization.\nTherefore, extracellular proteins that cannot bind with membrane receptors, can also be degraded.\n6. Although the authors have verified the strategy with several proteins, all of them only achieved\n50-60% degradation efficiency what might not be enough compared with other TPD reports. The\ndegradation efficiency should be improved.\nResponse to the reviewer: Thanks for this comment. We first summarized some potential strategies\nthat may improve the degradation efficiency in the revised manuscript. “…POI-targeting ligands\nwith higher specificity and affinity (e.g., monoclonal antibodies), ES-PTP with higher endosome\ninserting and labeling ability, phagophore-targeting ligands with higher specificity and affinity (e.g.,\nother LC3-specific ligands), autophagy enhancers (e.g., rapamycin and carbamazepine), and\nlysosome enhancing compounds with the ability of boosting lysosome biogenesis and function62,\ncan be screened or combined to further strengthen the in vivo degradation potency of EPLAs. In\naddition, adjusting the incubation time and concentration may also be feasible to improve the\ndegradation efficiency.”\nAccording to this comment, we performed a new experiment to use rapamycin and\ncarbamazepine to boost the degradation efficiency of EPLAs. “In addition, treatment with the\nmixture of carbamazepine and CVR-EPLA was shown to successfully degrade ~80.1% of PD-L1\nin 4T1 cells (Extended Data Fig. 3j), suggesting that the autophagy enhancers could be combined\nwith EPLAs to further improve the degradation efficiency of EPLAs.”\n24 / 27\nExtended Data Fig. 3. j, The levels of PD-L1 in 4T1 cells after sequential treatments with 30 μM\nrapamycin (or 50 μM carbamazepine) for 24 h and with the mixture of 0.5 μM CVR-EPLA and\nrapamycin (or carbamazepine) for 12 h, were evaluated using western blot (n = 3).\n7. For the degradation mechanism study, the bafilomycin A1, chloroquine, NH Cl, and rapamycin\n4\ninduced the elevation of LC2-II level. Then, bafilomycin A1, chloroquine, and NH Cl inhibited\n4\ndegradation, but rapamycin enhanced the degradation. What is the reason for this? What is the\nrelation between LC2-II level and degradation?\nResponse to the reviewer: We are sorry for this unclear description. According to this comment,\nwe added the related information in the revised manuscript. “The changes of LC3-II levels reflect\nboth formation and accumulation of phagophores. However, while increased formation of\nphagophores signifies the initiation of cellular degradation machinery, phagophore accumulation\nindicates the impaired phagophore-lysosome fusion and degradation. Chloroquine, bafilomycin A1\nand NH Cl suppress phagophore-lysosome fusion and then impedes lysosomal degradation of\n4\nautophagic bodies5,42-45. Rapamycin is an autophagy enhancer that promotes phagophore\ngeneration43. Thus, these functional molecules could increase the levels of LC3-II, but chloroquine,\nbafilomycin A1 and NH Cl inhibit the autophagic degradation, and rapamycin promotes the\n4\nautophagic degradation.”\n8. What is the performance difference for EPLA and NP-EPLA? Since EPLA already formed\nnanoparticle, what is the reason the synthesis EPLA into another type of NP?\nResponse to the reviewer: We are sorry for poorly explaining the reason for designing EPLA@NPs.\nPreviously, we explained the reason as “to endow the EPLA technology platforms with higher\nmodularity and universality”.\nAccording to this comment, we added the detailed reasons in the revised manuscript. “With\nsolid PLGA-based NPs as the basic carrier and MAL-PEG-SCM as the linker, both hydrophilic and\n25 / 27\nhydrophobic targeting ligands could be anchored on NP surface to broaden the application scope of\nEPLAs to more membrane and extracellular proteins. Direct conjugation with hydrophobic Mal-\npHLIP-Q24 requires hydrophilic targeting ligands to form nanoscale micelle-like EPLAs.\nEPLA@NPs can also be freeze-dried for long storage and the commercialization potential. However,\nat the same mass concentration, the degradation performance of EPLAs may be better than\nEPLA@NPs due to the addition of PLGA and MAL-PEG-SCM in EPLA@NPs.”\n9. For in vivo experiment, the antibody was injected intraperitoneally, while the EPLA was injected\nintravenously. What is reason for choosing different drug injection methods? Is it the reason for the\nside effect of antibody for colon shortening?\nResponse to the reviewer: In preclinical studies, both intraperitoneal route and intravenous route\nhave been widely used for PD-L1 antibody-mediated tumor treatments. Treatments with immune\ncheckpoint inhibitors are often associated with serious immune-related adverse effects. In one study\nabout intravenously delivering PD-L1 antibody to treat 4T1 mouse breast cancers, both colitis and\nmouse death were reported for PD-L1 antibody dose over 60 μg per injection (five doses and every\n2 days)1. Therefore, colon shortening cannot be ascribed to intraperitoneal route because\nintravenous PD-L1 antibody also induces colitis. More importantly, for PD-L1 antibody, the\nintraperitoneal route was reported to be more effective than the intravenous route in reducing\nsubcutaneous tumors2. Therefore, we chose the intraperitoneal injection route for PD-L1. At the\ndose of 100 μg per injection (five doses), shown was colon shortening but not mouse death.\n1. Li Y. et al. Covalent LYTAC Enabled by DNA Aptamers for Immune Checkpoint Degradation\nTherapy. J. Am. Chem. Soc. 145, 24506-24521 (2023).\n2. Lee C. K. et al. Anti-PD-L1 F(ab) Conjugated PEG-PLGA Nanoparticle Enhances Immune\nCheckpoint Therapy. Nanotheranostics 6, 243-255 (2022).\n10. Please use different terms to describe EPLA and NP-based EPLA. For example, in Figure 6, the\ndrug treatment in figure a looks like the same as that of figure h.\nResponse to the reviewer: According to this comment, we re-named NP-based EPLA as Ctrl-\nEPLA@NPs and CVR-EPLA@NPs.\n26 / 27\n27 / 27\nPoint-by-point response to the reviewers’ comments\nDear all reviewers,\nThank you. We deeply appreciate your patience for reviewing our manuscript. Your comments and\nquestions are really helpful for the improvement of our work. We have taken all these comments\nand questions into account in our work and improved our manuscript by making the related revisions.\nReviewer #1:\n1. For the response to question 5 from Reviewer 1, “To study if early endosome can transport with\nPD-L1 after CVR-EPLA treatment, HEK293T human embryonic kidney cells were transfected to\nexpress EGFP-PD-L1 and BFP-Rab5 (an early endosome marker). At 4 h, the proportion of Rab5-\nco-localized EGFP-PD-L1 relative to total PD-L1 obviously increased compared with untreated\ncells (Fig. 2d), proving the internalization of PD-L1. At 12 h, the ratio of Rab5-co-localized EGFP-\nPD-L1 even further increased, implying that partial PD-L1 may have been degraded and the\nremaining PD-L1 were still highly co-localized with Rab5.” How can “the ratio of Rab5-co-\nlocalized EGFP-PD-L1 even further increased” implied “partial PD-L1 may have been degraded”?\nIt may also be caused by increased internalization of PD-L1 via CVR-EPLA? Please check this\ndescription.\nResponse to the reviewer: We are sorry for the inaccurate description. We also totally agree with\nthe reviewer’s comment that the increase of the ratio of Rab5-co-localized EGFP-PD-L1 relative to\ntotal PD-L1 may also be caused by the increased internalization of PD-L1 via CVR-EPLA.\nAccording to the data of Fig. 3a–c, the 12 h incubation with CVR-EPLA reduced the levels of\ntotal PD-L1 by degrading PD-L1. So we think that the increase of the ratio of Rab5-co-localized\nEGFP-PD-L1 relative to total PD-L1 at 12 h may also be caused by the partial degradation of total\nPD-L1.\nWe have improved the description in the revised manuscript. “To study if early endosome can\ntransport with PD-L1 after CVR-EPLA treatment, HEK293T human embryonic kidney cells were\ntransfected to express EGFP-PD-L1 and BFP-Rab5 (an early endosome marker). At 4 h, the\nproportion of Rab5-co-localized EGFP-PD-L1 relative to total PD-L1 obviously increased\ncompared with untreated cells (Fig. 2d), proving the internalization of PD-L1. At 12 h, the ratio of\nRab5-co-localized EGFP-PD-L1 even further increased, which may be caused by the increased\ninternalization of PD-L1 via CVR-EPLA, the partial degradation of total PD-L1, and the high co-\nlocalization of the remaining PD-L1 with Rab5.”\nReviewer #3:\n1. The authors performed quantitative proteomic analysis to verify the specificity of CVR-EPLA in\ndegrading the PD-L1 in MDA-MB-231 cells. However, in Extended Data Fig. 5a, we only see some\n1 / 3\nunchanged membrane proteins, where is the target PD-L1?\nResponse to the reviewer: We are sorry for omitting the labeling of the target protein PD-L1. We\nhave added the labeling of the target protein PD-L1 in Extended Data Fig. 5a and improved the\ndescription in the revised manuscript.\n“While the level of the target protein PD-L1 was reduced, the levels of many membrane\nproteins including epidermal growth factor receptor (EGFR), low-density lipoprotein receptor\n(LDLR), CD44, mannose-6-phosphate receptor (M6PR), LRP1, and transferrin receptor (TFRC)\nwere not changed, proving the degradation specificity of CVR-EPLA.”\nExtended Data Fig. 5. a, Quantitative proteomic analysis showing the specificity of CVR-EPLA\nin degrading the PD-L1 in MDA-MB-231 cells. MDA-MB-231 cells were incubated with 0.5 μM\nCVR-EPLA or PBS (as a control) at 37 °C for 12 h. Each experiment was performed in three\nreplicates, and the unpaired t test was used to calculate the P values followed by Benjamini–\nHochberg adjustment. Proteins with adjusted P-values less than 0.05 (y-axis) and fold changes\ngreater than 2 (x-axis) were considered to be significantly changed.\n2. Only HE staining was performed to demonstrate the biosafety in vivo, which might be insufficient.\nOther routine tests such as TUNEL and Ki67 should also be performed.\nResponse to the reviewer: According to this comment, we performed TUNEL and Ki67 staining\nfor tissues excised from CVR-EPLA-treated mice. “The data on TUNEL staining and proliferation\nmarker Ki67 staining confirmed that, CVR-EPLA didn’t increase or decrease cell apoptosis\n(TUNEL+) and cell proliferation in all investigated mouse tissues including heart, liver, spleen, lung,\nkidney and colon (Extended Data Fig. 7f–g). The Ki67 positive signals in colon sections indicated\nthe presence of proliferative intestinal stem cells in the colon tissues69.”\n2 / 3\nExtended Data Fig. 7. f-g, TUNEL assay staining (green, f) and immunofluorescence staining of\nKi67 (red, g) in tissue sections from Balb/c immune-competent mice (female), which were\nintravenously injected with CVR-EPLA or CVR-pHLIP (1.8 μmol kg-1 per injection) three times on\ndays 0, 2, and 4 and euthanized on day 7. Nuclei were counterstained with DAPI (blue).\n3 / 3"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed most of the reviewers’ comments and provided substantial experimental evidence\nin support of their responses. One minor point remains, as detailed below.\nFor the response to question 5 from Reviewer 1, “To study if early endosome can transport with PD-L1 after CVR-EPLA\ntreatment, HEK293T human embryonic kidney cells were transfected to express EGFP-PD-L1 and BFP-Rab5 (an early\nendosome marker). At 4 h, the proportion of Rab5-co-localized EGFP-PD-L1 relative to total PDL1 obviously increased\ncompared with untreated cells (Fig. 2d), proving the internalization of PDL1. At 12 h, the ratio of Rab5-co-localized EGFP-\nPD-L1 even further increased, implying that partial PD-L1 may have been degraded and the remaining PD-L1 were still\nhighly co-localized with Rab5.” How can “the ratio of Rab5-co-localized EGFP-PD-L1 even further increased” implied\n“partial PD-L1 may have been degraded”? It may also be caused by increased internalization of PD-L1 via CVR-EPLA?\nPlease check this description.", "reviewer_2": "All my concerns have been addressed.", "reviewer_3": "The authors have well addressed most of the reviewers' questions and comments. However, two issues still need further\nverfication.\n1. The authors performed quantitative proteomic analysis to verify the specificity of CVR-EPLA in degrading the PD-L1 in\nMDA-MB-231 cells. Howevere, in Extended Data Fig. 5a, we only see some unchanged membrane proteins, where is the\ntarget PD-L1?\n2. Only HE staining was performed to demonstrate the biosafety in vivo, which might be insufficient. Other routine tests such\nas TUNEL and Ki67 should also be performed."}, "author_response": "Point-by-point response to the reviewers’ comments\nDear all reviewers,\nThank you. We deeply appreciate your patience for reviewing our manuscript. Your comments and\nquestions are really helpful for the improvement of our work. We have taken all these comments\nand questions into account in our work and improved our manuscript by making the related revisions.\nReviewer #1:\n1. For the response to question 5 from Reviewer 1, “To study if early endosome can transport with\nPD-L1 after CVR-EPLA treatment, HEK293T human embryonic kidney cells were transfected to\nexpress EGFP-PD-L1 and BFP-Rab5 (an early endosome marker). At 4 h, the proportion of Rab5-\nco-localized EGFP-PD-L1 relative to total PD-L1 obviously increased compared with untreated\ncells (Fig. 2d), proving the internalization of PD-L1. At 12 h, the ratio of Rab5-co-localized EGFP-\nPD-L1 even further increased, implying that partial PD-L1 may have been degraded and the\nremaining PD-L1 were still highly co-localized with Rab5.” How can “the ratio of Rab5-co-\nlocalized EGFP-PD-L1 even further increased” implied “partial PD-L1 may have been degraded”?\nIt may also be caused by increased internalization of PD-L1 via CVR-EPLA? Please check this\ndescription.\nResponse to the reviewer: We are sorry for the inaccurate description. We also totally agree with\nthe reviewer’s comment that the increase of the ratio of Rab5-co-localized EGFP-PD-L1 relative to\ntotal PD-L1 may also be caused by the increased internalization of PD-L1 via CVR-EPLA.\nAccording to the data of Fig. 3a–c, the 12 h incubation with CVR-EPLA reduced the levels of\ntotal PD-L1 by degrading PD-L1. So we think that the increase of the ratio of Rab5-co-localized\nEGFP-PD-L1 relative to total PD-L1 at 12 h may also be caused by the partial degradation of total\nPD-L1.\nWe have improved the description in the revised manuscript. “To study if early endosome can\ntransport with PD-L1 after CVR-EPLA treatment, HEK293T human embryonic kidney cells were\ntransfected to express EGFP-PD-L1 and BFP-Rab5 (an early endosome marker). At 4 h, the\nproportion of Rab5-co-localized EGFP-PD-L1 relative to total PD-L1 obviously increased\ncompared with untreated cells (Fig. 2d), proving the internalization of PD-L1. At 12 h, the ratio of\nRab5-co-localized EGFP-PD-L1 even further increased, which may be caused by the increased\ninternalization of PD-L1 via CVR-EPLA, the partial degradation of total PD-L1, and the high co-\nlocalization of the remaining PD-L1 with Rab5.”\nReviewer #3:\n1. The authors performed quantitative proteomic analysis to verify the specificity of CVR-EPLA in\ndegrading the PD-L1 in MDA-MB-231 cells. However, in Extended Data Fig. 5a, we only see some\n1 / 3\nunchanged membrane proteins, where is the target PD-L1?\nResponse to the reviewer: We are sorry for omitting the labeling of the target protein PD-L1. We\nhave added the labeling of the target protein PD-L1 in Extended Data Fig. 5a and improved the\ndescription in the revised manuscript.\n“While the level of the target protein PD-L1 was reduced, the levels of many membrane\nproteins including epidermal growth factor receptor (EGFR), low-density lipoprotein receptor\n(LDLR), CD44, mannose-6-phosphate receptor (M6PR), LRP1, and transferrin receptor (TFRC)\nwere not changed, proving the degradation specificity of CVR-EPLA.”\nExtended Data Fig. 5. a, Quantitative proteomic analysis showing the specificity of CVR-EPLA\nin degrading the PD-L1 in MDA-MB-231 cells. MDA-MB-231 cells were incubated with 0.5 μM\nCVR-EPLA or PBS (as a control) at 37 °C for 12 h. Each experiment was performed in three\nreplicates, and the unpaired t test was used to calculate the P values followed by Benjamini–\nHochberg adjustment. Proteins with adjusted P-values less than 0.05 (y-axis) and fold changes\ngreater than 2 (x-axis) were considered to be significantly changed.\n2. Only HE staining was performed to demonstrate the biosafety in vivo, which might be insufficient.\nOther routine tests such as TUNEL and Ki67 should also be performed.\nResponse to the reviewer: According to this comment, we performed TUNEL and Ki67 staining\nfor tissues excised from CVR-EPLA-treated mice. “The data on TUNEL staining and proliferation\nmarker Ki67 staining confirmed that, CVR-EPLA didn’t increase or decrease cell apoptosis\n(TUNEL+) and cell proliferation in all investigated mouse tissues including heart, liver, spleen, lung,\nkidney and colon (Extended Data Fig. 7f–g). The Ki67 positive signals in colon sections indicated\nthe presence of proliferative intestinal stem cells in the colon tissues69.”\n2 / 3\nExtended Data Fig. 7. f-g, TUNEL assay staining (green, f) and immunofluorescence staining of\nKi67 (red, g) in tissue sections from Balb/c immune-competent mice (female), which were\nintravenously injected with CVR-EPLA or CVR-pHLIP (1.8 μmol kg-1 per injection) three times on\ndays 0, 2, and 4 and euthanized on day 7. Nuclei were counterstained with DAPI (blue).\n3 / 3"}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors have addressed my comments and concerns."}, "author_response": ""}]

Decision letter