Multibody-Enabled Lysosome-Targeting Drug Conjugates for Target Protein Degradation and Combination Therapy

Multibody-Enabled Lysosome-Targeting Drug Conjugates for Target Protein Degradation and Combination Therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Multibody-Enabled Lysosome-Targeting Drug Conjugates for Target Protein Degradation and Combination Therapy Lixin Zhang, Luping Chen, Shuai Yao, Xiaoli Tian, li ping, Pan Tan, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8651044/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Current lysosome-targeting chimeras (LYTACs) and antibody-drug conjugates (ADCs) face inherent limitations, including reliance on monospecific binders and target-dependent internalization efficiency. Here, we report a versatile therapeutic platform that overcomes these constraints by engineering a novel class of multivalent, multispecific binding proteins, termed Multibodies . Using the fungal immunomodulatory protein LZ-8 as a scaffold, we developed a lysosome-targeting chimera-drug conjugates (LYTAC-DCs) system capable of mediating the degradation of multiple membrane proteins while enabling site-specific drug release. While wild-type LZ-8 demonstrated potent lysosomal targeting, it induced significant off-target toxicity and lysosomal dysfunction. Through rational protein engineering, we identified a key residue (Y84) responsible for promiscuous receptor binding and generated an optimized variant, LZ-8-2.3 (Y84A), which eliminated toxicity while preserving efficient lysosomal trafficking. The resulting LYTAC-DC platform mediated high-fidelity degradation of EGFR, PD-L1, and HER2 across diverse cancer models, concurrently delivering cytotoxic (e.g., MMAE) or immunogenic (e.g., doxorubicin) payloads. Efficacy was validated in patient-derived organoids and murine xenografts, including against osimertinib-resistant lung cancer. Furthermore, AI-assisted directed evolution enabled the development of non-chimeric lysosome-targeting drug conjugates (LYTA-DCs), highlighting the modularity and engineerability of the Multibody scaffold. Our work establishes a unified and programmable strategy for targeted protein degradation and drug delivery, significantly expanding the therapeutic landscape beyond conventional LYTAC and ADC technologies. Health sciences/Medical research/Preclinical research Biological sciences/Cancer Multibody LZ-8 LYTAC-DC LYTA-DC Targeted protein degradation Antibody-drug conjugate Lysosome targeting Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights Proposes and validates the novel concept of "Mu l tibody," establishing a modular lysosome-targeting drug design platform that encompasses both chimera-dependent (LYTAC-DC) and chimera-independent (LYTA-DC) strategies, overcoming the limitations of conventional LYTACs and ADCs. Identifies the fungal protein LZ-8 as a potent yet off-target-prone lysosome-targeting scaffold, and engineers the key variant LZ-8-2.3 through rational design. This variant retains efficient endocytosis and lysosomal trafficking while eliminating off-target degradation and lysosomal dysfunction mediated by the Y84 residue. The developed LYTAC-DC platform demonstrates robust versatility, mediating high-fidelity degradation of multiple cancer-related membrane proteins, including EGFR, PD-L1, and HER2, with concurrent delivery of cytotoxic or immunogenic small-molecule payloads, as validated across in vitro , organoid, and in vivo models. Converges targeting and degradation functions into a single molecule via AI-assisted directed evolution, enabling the development of non-chimeric LYTA-DCs and showcasing the unique evolvability and engineerability of the Mutibody scaffold for "all-in-one" smart drug design. The platform shows promise in overcoming clinical resistance. LZ-8-2.3-based LYTAC-DC effectively inhibits the growth of osimertinib-resistant NSCLC models and demonstrates potent anti-tumor activity in patient-derived tumor organoids harboring triple EGFR mutations (Del19/T790M/C797S). Main The endolysosomal system represents a critical pathway for targeted therapeutic intervention 1 , 2 , serving as a natural cellular mechanism for the degradation of extracellular and membrane proteins, as well as for the intracellular delivery of therapeutic agents 3 , 4 , 5 , 6 , 7 , 8 , 9 . Lysosome-targeting chimeras (LYTAC) exemplify this approach by employing bispecific molecules that simultaneously engage a cell-surface lysosome-targeting receptor and a disease-relevant target protein, directing the latter for lysosomal degradation. However, current LYTAC platforms face inherent limitations, including restricted target specificity, reliance on specific lysosome-targeting receptors (e.g., IGF2R), and variable internalization efficiency, which collectively constrain their broad applicability 10 . Concurrently, antibody-drug conjugates (ADCs) exploit receptor-mediated endocytosis for precise drug delivery but are fundamentally limited by the intrinsic internalization kinetics of their target antigens 11 , 12 , posing significant challenges for targeting poorly internalizing receptors. A common constraint across these advanced therapeutic modalities is their dependence on conventional antibodies or nanobodies, which are typically monospecific and monovalent 13 , 14 , 15 , 16 . This molecular architecture restricts multi-target engagement and complicates the integration of additional functionalities, such as synergistic payload delivery or combinatorial degradation strategies. To overcome these constraints, we developed a flexible and multidimensional platform that integrates targeted protein degradation with site-specific drug release via the endolysosomal pathway. Inspired by natural mechanisms for clearing heterologous proteins 17 , we identified the fungal immunomodulatory protein LZ-8 as a multivalent and multispecific lysosome-targeting scaffold. Initial characterization confirmed its potent endocytic activity and broad receptor interactions. However, the native protein induced significant lysosomal dysfunction and off-target degradation, limiting its therapeutic utility. Through rational protein engineering, we generated an optimized variant, LZ-8-2.3 (Y84A), which eliminated these adverse effects while retaining efficient lysosomal trafficking. This engineered Mutibody (LZ-8-2.3) serves as the core component of our Lysosome-Targeting Chimeric Drug Conjugates (LYTAC-DC) platform, enabling simultaneous, high-fidelity degradation of diverse oncogenic membrane proteins and concurrent delivery of cytotoxic or immunogenic payloads. This study establishes the LYTAC-DC platform as a unified strategy to address the key limitations of conventional LYTAC and ADC technologies, offering a modular and engineerable framework for next-generation targeted therapies. Results LZ-8 as a Potent but Flawed Lysosome-Targeting Scaffold Current LYTAC tools, primarily antibody-based conjugates, not only limit the further conjugation of small-molecule drugs but also increase the inherent complexity and instability of drug synthesis. To address this, we explored novel recombinant protein-based LYTAC tools, as these not only can be easily mass-produced but also facilitate subsequent small-molecule drug conjugation. Inspired by the innate ability of cells to clear heterologous proteins via the lysosomal pathway, we selected four heterologous proteins previously reported to induce endocytosis, including fungal (LZ-8) 18 , camelid (7D12) 19 , bacterial (STxB-TDP) 20 , and viral (TAT) 21 . These proteins were genetically fused with fluorescent protein EGFP for tracking. Confocal microscopy and flow cytometry results revealed that the fungal-derived LZ-8 exhibited exceptionally stronger intracellular internalization capabilities compared to other 3 proteins (Extended Data Fig. 1 a-b). LZ-8 (PDB ID:3F3H) forms a dimer through its N-terminal α-helical dimerization arm, with a C-terminal immunoglobulin-like domain (Extended Data Fig. 1 c). Preliminary screen showed that LZ-8 induced broad-spectrum cytotoxicity in multiple cell lines in a dose-dependent manner (Extended Data Fig. 1 d-g). Based on the relatively lower cytotoxicity at 500 nM in HeLa cell line (viability > 85%), this concentration was used for the subsequent experiments as the maximum dose. Trypan blue quenching assays confirmed that LZ-8-EGFP entered HeLa cells rather than remaining on the cell membrane (Extended Data Fig. 1 h). Time- and dose-dependent analyses demonstrated that LZ-8-EGFP efficiently bound to cells and accumulated intracellularly (Extended Data Fig. 1 i-k). This property was common across multiple fungal-derived immunomodulatory protein family members (Extended Data Fig. 1 l-m) 22 . To evaluate LZ-8’s lysosomal targeting efficiency as a LYTAC tool, multi-organelle co-localization analysis revealed that LZ-8-EGFP primarily localized to the endosomal network adjacent to the cis-Golgi apparatus (Extended Data Fig. 2 a). Time-dependent co-localization experiments with early endosome marker EEA1 and late endosome marker RAB7 confirmed that LZ-8-EGFP effectively completed endosome maturation and entered LAMP1-positive lysosomes (Extended Data Fig. 2 c-e). In HeLa cells overexpressing LAMP1-GFP, LZ-8-mCherry showed strong punctate co-localization with LAMP1-GFP (Fig. 1 b); while the degree of co-localization with lysosomal probe LysoTracker increased over time (Extended Data Fig. 2 b). Dual-fluorescence pulse-chase experiments demonstrated that the early accumulated LZ-8-EGFP was quenched, which could be reversed by BafA1 treatment, while sustained endocytic flux led to intracellular accumulation of LZ-8-mCherry (Extended Data Fig. 2 f-h). TFEB overexpression significantly suppressed LZ-8 accumulation by enhancing lysosomal biogenesis, suggesting that increased lysosomal degradation capacity aids intracellular clearance of LZ-8 (Extended Data Fig. 2 i). Collectively, LZ-8 drives persistent cellular internalization and efficient lysosomal targeting. Building on LZ-8’s lysosomal targeting properties, we constructed a recombinant LYTAC system by fusing LZ-8 and the various nanobodies against different targets, aiming to trigger enhanced degradation of target proteins (Fig. 1 c). For the degradation of extracellular model protein EGFP, LZ-8-Nb EGFP , a fusion protein of LZ-8 and EGFP-targeting nanobody, was constructed. Significant intracellular EGFP enrichment in HeLa cells was detected after 30 minutes incubation with LZ-8-Nb EGFP , indicating that LZ-8-Nb EGFP efficiently captured EGFP in the medium and mediated its internalization. The intracellular EGFP level was markedly decreased after removal of EGFP from the culture for 3 hours, indicating rapid degradation of EGFP after internalization. However, the presence of lysosome inhibitor BafA1 inhibited the degradation process, confirming the fact that the degradation is lysosome-dependent (Extended Data Fig. 3 a). For membrane bound proteins, we first tested degradation of EGFR by fusing LZ-8 to an EGFR-targeting nanobody, 7D12(Extended Data Fig. 3 b). Although previous studies have shown that LZ-8 itself can bind EGFR and mediate its degradation 18 , which was also confirmed by our results (Extended Data Fig. 3 c); the fusion protein LZ-8-7D12 induced significant lysosome-dependent EGFR degradation in HeLa and A431 cells at the concentration as low as 20 nM. As the lysosomal inhibitor BafA1 treatment attenuated the degradation, confirming its lysosome dependent activity (Fig. 1 d-e,Extended Data Fig. 3 d-e). To further verify the effect of LZ-8, PD-L1 (highly expressed in MDA-MB-231 cells) and HER2 (highly expressed in SKBR3 cells) were selected as non-LZ-8-associated targets (Extended Data Fig. 3 f). After incubation of the LZ-8 with MDA-MB-231 and SKBr3 cells respectively, Western blot (WB) analysis showed that LZ-8 at doses below 500 nM did not induce PD-L1 degradation, but triggered HER2 degradation, suggesting HER2, like EGFR, is also a potential receptor for LZ-8 (Extended Data Fig. 3 g). It was then demonstrated that LZ-8-KN035 (PD-L1 nanobody fusion) and LZ-8-11A4 (HER2 nanobody fusion) induced specific degradation of PD-L1 and HER2, respectively, indicating that LZ-8-mediated LYTACs significantly enhance degradation of both non-LZ-8 receptor targets and LZ-8 receptor-associated targets (Extended Data Fig. 3 h). The high efficiency of LZ-8 as a LYTAC tool further motivated us to investigate the feasibility of enhancing drug delivery through establishing LYTAC-drug conjugate(LYTAC-DC). To prepare LYTAC-DC, we introduced a cysteine residue at the C-terminus of LZ-8-7D12, which was successively conjugated with vcMMAE (a cytotoxic small molecule containing a maleimide group) via an addition reaction (Extended Data Fig. 3 i) 23 . The construct of LZ-8-7D12-MMAE efficiently binds to EGFR and LZ-8 receptors, entering lysosomes through LZ-8 receptor-mediated endocytosis to degrade EGFR. Concurrently, the protein degradation releases MMAE, inducing apoptosis by disrupting microtubule assembly. MTT assays show that both LZ-8-7D12-MMAE and LZ-8-MMAE effectively induced cell death of A431 cells that express high level of EGFR, but exhibited lower activity in HeLa cells (with moderate EGFR expression) (Fig. 1 f, Extended Data Fig. 3 j). It is noteworthy that LZ-8 itself, not just LZ-8-MMAE or LZ-8-7D12-MMAE, also showed significant cell toxicity toward human embryonic lung MRC-5 cells (EGFR-positive), suggesting its potential off-target effect (Extended Data Fig. 3 k). Since LZ-8 binds to multiple receptors including EGFR and induces their degradation, we investigated whether LZ-8-KN035 could cause EGFR degradation in MDA-MB-231 and HeLa cells. Western blot results confirmed that LZ-8-KN035 significantly induced EGFR degradation (Fig. 1 g, Extended Data Fig. 3 l), indicating that the off-target effect of LZ-8 was mediated by its receptor. Similarly, LZ-8-11A4 induced strong EGFR degradation in SKBR3 cells, further supporting the conclusion that LZ-8 induces off-target effect on protein degradation, which limited its utility as a LYTAC-DC tool (Extended Data Fig. 3 m). LZ-8, as a LYTAC-DC tool, induces lysosomal dysfunction After observing the high efficiency but significant off-target effects of LZ-8, we further investigated the impact of its persistent high endocytic flux on lysosomal function. Previous studies suggest that sustained high endocytic flux may impair lysosomal function 24 , potentially reducing the efficiency of lysosome-dependent targeted protein degradation. Utilizing the lysosomal function probe DQ-BSA-Red (where lysosomal function correlates with red fluorescence intensity) 25 , confocal experiments revealed that prolonged LZ-8 treatment significantly reduced lysosomal function (Fig. 2 a). Consistently, ubiquitinated substrates showed dose-dependent accumulation in LZ-8/MG132 co-treated cells, indicating impaired lysosomal degradation(Extended Data Fig. 4 a) 26 . To elucidate the molecular mechanism of the LZ-8-induced lysosome toxicity, we systematically analyzed key regulators of the endolysosomal pathway. Western blot exhibited that LZ-8 treatment specifically downregulated lysosomal membrane protein LAMP1, lysosomal enzyme transport receptor IGF2R, and transcription regulator TFEB, while early/late endosome markers remained unaffected (Fig. 2 b, Extended Data Fig. 4 b). TFEB is a transcriptional regulator critical for lysosomal biogenesis and requires nuclear translocation for activation 27 . We examined the effect of LZ-8 on TFEB nuclear transport in TFEB-GFP-overexpressing cells. Confocal results showed that LZ-8 did not significantly affect TFEB nuclear translocation (Extended Data Fig. 4 c), indicating that LZ-8 does not interfere with TFEB-mediated lysosomal biogenesis. LAMP1 and IGF2R are essential for lysosomal function: LAMP1 maintains lysosomal membrane stability, while IGF2R transports mannose-6-phosphate-tagged lysosomal enzyme precursors (e.g., CTSB) into lysosomes for maturation 28 , 29 . Immunostaining revealed that LZ-8 significantly altered the intracellular distribution of LAMP1 and IGF2R, where LAMP1 was changed from a diffuse pattern to perinuclear aggregation, while IGF2R was translocated from perinuclear regions to the cell membrane (Fig. 3 c). Flow cytometry further confirmed LZ-8-induced downregulation of membrane LAMP1 and upregulation of membrane IGF2R (Extended Data Fig. 4 d-e). Time-dependent immunofluorescence also showed IGF2R membrane redistribution (Extended Data Fig. 4 f). It should be noted that a drug-induced IGF2R membrane redistribution has not been reported previously. Similar LZ-8 effects on LAMP1 and IGF2R downregulation were also observed in NCI-H226 and SMMC-7721 cells (Extended Data Fig. 4 g), indicating a common mechanism of LZ-8 on lysosome toxicity, with reversibility in HeLa cells (Extended Data Fig. 4 h). Given the critical roles of LAMP1 and IGF2R in lysosomal function, we further investigated the mechanism underlying their LZ-8-induced downregulation. qPCR and transcriptomic analyses showed that LZ-8 did not transcriptionally downregulate IGF2R or LAMP1 (Extended Data Fig. 4 i). Additionally, transcriptomic data revealed no significant downregulation of lysosome-related genes, further supporting the conclusion that LZ-8 does not affect TFEB function (Extended Data Fig. 4 j). Cycloheximide (CHX) inhibition experiments demonstrated that LZ-8 treatment reduced LAMP1 and IGF2R half-lives, suggesting post-translational regulation (Extended Data Fig. 4 k-l). Pretreatment with lysosome inhibitor BafA1, but not proteasome inhibitor MG132, inhibited LZ-8-mediated LAMP1 downregulation, confirming that LZ-8 reduces LAMP1 via lysosomal degradation (Extended Data Fig. 4 m). Surprisingly, LZ-8-induced IGF2R downregulation was not inhibited by either inhibitor. To find out the underlying mechanism of IGF2R’s membrane redistribution, we hypothesized that IGF2R downregulation might occur via exocytosis, therefore examined the transporters of VPS35, SNX5, and SNX3 that regulate IGF2R intracellular trafficking. WB experiments showed no significant changes in expression of any of the transporters (Extended Data Fig. 4 n), suggesting that LZ-8 does not alter IGF2R-associated trafficking machinery. Given IGF2R’s role in lysosomal enzyme maturation, we speculated that LZ-8 might impair the maturation of enzymes like cathepsin B (CTSB). WB confirmed that LZ-8 induced time-dependent downregulation of mature CTSB (Fig. 2 d), linking IGF2R dysregulation to impaired lysosomal enzyme maturation and dysfunction. Lysosomal function depends on acidic intra-lysosomal pH 30 . We hypothesized that LZ-8’s persistent endocytic flux might neutralize lysosomal acidity. Lysotracker (indicating lysosomal quantity) and LysoSensor (indicating lysosomal acidity) assays showed that LZ-8 treatment significantly increased Lysotracker intensity (but not LysoSensor) over 12–24 hours, suggesting lysosomal pH elevation (Extended Data Fig. 4 o-q). Lysosomal dysfunction can block autophagic flux. WB of autophagy markers LC3-II/p62 and LC3B-GFP-mCherry overexpression confirmed impaired clearance, resulting in autophagosome accumulation in LZ-8-treated cells (Fig. 2 E-F, Extended Data Fig. 4 r) 31 . These multi-dimensional findings demonstrate that LZ-8 disrupts LAMP1/IGF2R-mediated lysosomal acidification and enzyme maturation, ultimately causing lysosomal dysfunction (mechanistic summary in Fig. 2 g). Y84 is the key residue of LZ-8 as Multibody To elucidate the interaction mechanisms of LZ-8 with potential target proteins and optimize its utility as a LYTAC tool, we systematically investigated its receptor-binding spectrum and critical functional residues for receptor binding (Fig. 3 a). Using EGFP-labeled pull-down combined with LC/MS screening of HeLa cell membrane protein interactomes (Extended Data Fig. 5 a), LZ-8 was found to primarily bind to cell surface receptors L1CAM and EGFR (screening thresholds: protein abundance > 1×10^10, Log2 enrichment > 2) (Fig. 3 b-c). Further validation using an ELISA library comprising 11 membrane protein receptors confirmed the high affinity of LZ-8-EGFP (EC50 < 10 nM) to α5β1 integrin, c-MET, EGFR, L1CAM, HER3, and PD1, in which L1CAM, PD1, and α5β1 were newly identified as high-affinity receptors of LZ-8 (Fig. 3 d, Extended Data Fig. 5 b) 32 . To verify the ELISA results further, the credibility of the ELISA was confirmed by further BLI affinity assays and cellular colocalization experiments, which were performed using selected targets: EGFR with high affinity and PD-L1 with low affinity (Extended Data Fig. 5 c-d). We found that dimeric LZ-8 induced HeLa cells aggregation, whereas monomeric 7D12 did not, indicating that LZ-8 engages membrane receptors via monomeric binding (Extended Data Fig. 5 e-f). Although earlier studies proposed that LZ-8 interacts with EGFR primarily through residue K41 18 , our molecular docking predicted that Y84 residue in the LZ-8 monomer forms a critical hydrogen bond network with the receptors (Extended Data Fig. 5 g). The mutant of Y84A (LZ-8-2.3) exhibited > 95% reduction in binding efficiency to HeLa cells, as well as SMMC-7721, H226, and MDA-MB-231 cells, in flow cytometry assays, whereas the K41A mutant (previously reported) showed minor impact on HeLa cell affinity (Fig. 3 e, Extended Data Fig. 5 h-j). AlphaFold2.3 structural predictions indicated that the Y84A mutation does not alter LZ-8’s overall conformation (Fig. 3 f); while MTT assays showed that Y84A mutation significantly reduced its tumor cell inhibitory activity and normal cell (MRC-5) toxicity (Fig. 3 g, Extended Data Fig. 5 k). LZ-8’s crystal structure revealed that the phenolic hydroxyl group of the Y84 side chain is solvent-exposed and protrudes from the molecular surface (Extended Data Fig. 6a). Intriguingly, the monomeric structure of LZ-8 resembles that of nanobodies, prompting us to propose a simplified model of LZ-8-receptor interactions (Extended Data Fig. 6b-d). ELISA further demonstrated that the Y84A mutant barely retained its affinity to the six high-affinity receptors identified by wild-type LZ-8 (Fig. 3 h). Treatment with the Y84A mutant (LZ-8-2.3) did not downregulate lysosomal functional markers IGF2R and LAMP1 in HeLa cells, with similar observations in four additional cell lines (Fig. 3 i, Extended Data Fig. 6e-f). Furthermore, no abnormal accumulation of autophagy flux-related proteins (LC3-II and p62) or changes in lysosomal quantity (LysoTracker) were observed (Extended Data Fig. 6g-h), confirming that the Y84A mutation abolished the toxic effect of LZ-8 on lysosomes. In a HeLa xenograft model, wild-type LZ-8 (1 mg/kg) significantly suppressed tumor growth, whereas Y84A (1 mg/kg) showed no efficacy (Extended Data Fig. 6i-l). Nevertheless, histological examinations failed to detect pathological signs, and no significant changes in body weight upon LZ-8 group treatment (Extended Data Fig. 6m-n). These results indicate that although LZ-8 engages multiple receptors, including EGFR, with high affinity, but had no pathological consequences. In comparison, Y84 residue is central to its receptor-binding activity. The Y84A mutant retains partial receptor affinity while eliminating lysosomal toxicity and cytotoxicity. The Y84A-Multibody enables lysosome-safe and target-specific LYTAC-DC Based on the low toxicity to lysosomal function, we moved forward to validate the feasibility of Y84A (LZ-8-2.3) as a lysosome-targeting chimera (LYTAC) tool, as it minimal off-target effects (Fig. 4 a). To demonstrate modular degradation capabilities, we conjugated the model protein Nb EGFP with LZ-8-2.3 and confirmed that LZ-8-2.3-Nb EGFP efficiently mediated lysosome-dependent degradation of extracellular proteins. This degradation was blocked by lysosomal inhibitor BafA1, validating its lysosome-specific function (Extended Data Fig. 7a). In EGFR-positive HeLa cells, LZ-8-2.3 alone did not induce significant EGFR degradation, demonstrating its low off-target activity (Extended Data Fig. 7b). The EGFR-targeting LYTAC construct (LZ-8-2.3-7D12) achieved robust EGFR degradation in HeLa and A431 cells. Covalent conjugation of LZ-8-2.3 with 7D12 showed significantly higher efficiency compared to non-covalent mixtures (LZ-8-2.3 + 7D12) (Fig. 4 b, Extended Data Fig. 7c). Dose-response analyses revealed a differential effect of LZ-8-2.3-7D12 promoted degradation of EGFR, with a DC 50 of 55.3 nM (D max = 55.46%) in HeLa cells and DC 50 of 11.87 nM (D max = 70.07%) in A431 cells. Its efficacy in HeLa cells significantly surpassed that of the positive control IGF2-7D12 (Extended Data Fig. 7d) 33 . Time-course immunoblotting demonstrated sustained EGFR degradation by LZ-8-2.3-7D12 within 6 hours (Extended Data Fig. 7e). To visualize EGFR internalization and degradation, we performed whole-cell immunofluorescence imaging and membrane-specific flow cytometry. Both methods confirmed that LZ-8-2.3-7D12 significantly induced EGFR internalization and degradation, with efficacy exceeding that of IGF2-7D12 (Extended Data Fig. 7f-g). Pre-treatment with lysosomal inhibitor BafA1 ceased EGFR degradation in both HeLa and A431 cells, further validating lysosome-dependent mechanisms (Fig. 4 c, Extended Data Fig. 7h). To assess lysosomal targeting efficiency, we created construct of EGFP fused to LZ-8-2.3, LZ-8-2.3-7D12, respectively. Confocal microscopy revealed that LZ-8-2.3 exhibited reduced fluorescence intensity compared to wild-type LZ-8, indicating lower lysosomal burden and minimized risk of lysosomal dysfunction (Extended Data Fig. 7i). Additionally, LZ-8-2.3-7D12 showed time-dependent co-localization with lysosomal probe LysoTracker, confirming efficient lysosomal trafficking (Extended Data Fig. 7i). To evaluate efficacy in complex tumor models, we cultured 3D HeLa cell spheroids (> 200 µm in diameter) (Extended Data Fig. 7j). Dose-response experiments demonstrated that LZ-8-2.3-7D12 efficiently degraded EGFR in these spheroids, highlighting its potential for penetrating solid tumors (Extended Data Fig. 7k). Pre-treatment with LZ-8-2.3 or 7D12 partially reduced EGFR degradation, indicating that the activity of LZ-8-2.3-7D12 depends on both components (Extended Data Fig. 7l).To ensure lysosomal safety, we compared the effects of LZ-8-2.3-7D12 with wild-type LZ-8-7D12 and the positive control IGF2-7D12. While wild-type LZ-8-7D12 induced significant downregulation of IGF2R alongside EGFR degradation, LZ-8-2.3-7D12 degraded EGFR without affecting IGF2R levels, confirming its lysosomal safety (Fig. 4 d, Extended Data Fig. 7m). To further validate whether the platform is compatible with other targets, we constructed a PD-L1-targeting LYTAC by fusing LZ-8-2.3 with KN035. Western blotting results indicated that LZ-8-2.3-KN035 effectively degraded PD-L1 in MDA-MB-231 cells (Extended Data Fig. 8a). Dose-dependent degradation assays showed that LZ-8-2.3-KN035 outperformed the control IGF2-KN035 (Fig. 4 e, Extended Data Fig. 8b). Immunoblotting confirmed that LZ-8-2.3-KN035 selectively degrades PD-L1 without non-specific degradation of wild-type LZ-8 receptors (e.g., EGFR) or downregulation of lysosomal markers like IGF2R (Fig. 4 f). Flow cytometry further validated PD-L1-specific degradation and minimal off-target effects on EGFR (Extended Data Fig. 8c-d). Co-localization with LysoTracker confirmed that LZ-8-2.3-KN035 retains efficient lysosomal targeting (Extended Data Fig. 8e). Successful HER2 degradation using LZ-8-2.3-11A4 further supports the versatility of LZ-8-2.3 as a broad-spectrum LYTAC tool (Extended Data Fig. 8f-h). Given the co-upregulation of multiple membrane proteins in cancer progression, we engineered dual-target LYTACs 34 . The construct 7D12-LZ-8-2.3-KN035 successfully and efficiently co-degraded EGFR and PD-L1 in MDA-MB-231 cells(Extended Data Fig. 8i-k). Similarly, the EGFR/HER2 dual-target tool (7D12-LZ-8-2.3-11A4) co-degraded both receptors (Extended Data Fig. 8l-m), demonstrating the platform’s capability for multiplexed protein intervention. To develop LYTAC-DCs, we introduced a reactive cysteine residue at the C-terminus of LZ-8-2.3 and conjugated it with maleimide-functionalized MMAE via addition reactions. Mass spectrometry confirmed the successful synthesis of LZ-8-2.3-7D12-MMAE and control conjugates (Extended Data Fig. 9a). The LYTAC-DC platform leverages LZ-8-2.3’s lysosomal targeting to internalize and degrade membrane proteins (e.g., EGFR, PD-L1) while releasing MMAE to disrupt microtubule assembly and induce apoptosis. The immunoblot results showed that MMAE conjugation did not affect the ability of LZ-8-2.3-7D12 to induce EGFR degradation, laying the foundation for its subsequent efficient delivery of MMAE(Extended Data Fig. 9b). MTT assays demonstrated that LZ-8-2.3-7D12-MMAE potently inhibited proliferation of EGFR-high A431 tumor cells, with efficacy significantly exceeding LZ-8-2.3-MMAE or 7D12-MMAE (Fig. 4 g). Cytotoxicity assays in other cells confirmed that LYTAC-DC activity correlates with target expression, which largely minimized off-target toxicity (Extended Data Fig. 9c-f). Recent studies have demonstrated that EGFR protein degradation holds promise for treating clinically relevant EGFR mutation-driven cancers such as non-small cell lung cancer (NSCLC) and overcoming TKI resistance 3 . We hypothesized that simultaneous EGFR degradation and small-molecule drug delivery could significantly suppress NSCLC progression. To test this, we selected the NSCLC cell line H1975, which carries the L858R/T790M double mutation in EGFR and is sensitive to the third-generation TKI inhibitor osimertinib. Through in vitro dose-gradient culture with osimertinib, we successfully generated osimertinib-resistant H1975 cells (H1975/GR; Extended Data Fig. 9g-h). Dose-gradient experiments with LZ-8-2.3-7D12 showed efficient EGFR degradation in both 2D and 3D cultures of H1975 and H1975/GR cells (Extended Data Fig. 9i-j). Further cell viability assays revealed that LZ-8-2.3-7D12 effectively inhibited the growth of H1975/GR cells compared to osimertinib (Extended Data Fig. 9k). However, at 500 nM, the inhibitory efficiency of LZ-8-2.3-7D12 against osimertinib-resistant H1975/GR cells remained significantly lower than that reported for TransTAC tools 3 , potentially due to limited efficacy and incomplete EGFR degradation by LZ-8-2.3-7D12 in H1975/GR cells. Conjugation with the cytotoxic small molecule MMAE markedly enhanced the ability of LZ-8-2.3-7D12-MMAE to suppress H1975/GR cell viability, achieving nearly complete killing of osimertinib-resistant lung cancer cells at 500 nM, whereas LZ-8-2.3-7D12 or osimertinib at this concentration showed limited activity at this concentration(Extended Data Fig. 9l). In recent years, patient-derived tumor organoids have been increasingly used to evaluate antitumor drug activity, better reflecting in vivo efficacy 35 . We further assessed the effect of LZ-8-2.3-7D12-MMAE on targeting LD1-0025-411169 cells, a patient-derived lung cancer cell line harboring triple EGFR mutations (Del19, T790M, and C797S), which confer resistance to all currently approved third-generation TKIs 3 . We first established a patient-derived xenograft (PDX) model using LD1-0025-411169 cells with confirmed TKI resistance and validated their osimertinib resistance in vivo (Extended Data Fig. 9m). The PDX-derived organoids (PDXO) cultured in ultra-low attachment 96-well plates exhibited robust proliferation, confirming their suitability for drug efficacy evaluation(Extended Data Fig. 9n). As expected, LZ-8-2.3-7D12-MMAE significantly inhibited the viability of LD1-0025-411169 organoids at low concentrations, outperforming both LZ-8-2.3-MMAE and 7D12-MMAE (Extended Data Fig. 9o). These results further demonstrate the dual mechanism of overcoming resistance through lysosome-targeted protein degradation and enhancing efficacy via synergistic cytotoxic payload delivery. To explore the versatility of the LYTAC-DC platform, we extended our targeting strategy to other types of small-molecule drugs. Doxorubicin (DOX), a chemotherapeutic anthracycline agent, is known to exhibit significantly lower toxicity compared to MMAE. Additionally, low-dose DOX can induce immunogenic cell death (ICD), leading to the release of damage-associated molecular patterns (DAMPs) and tumor-associated antigens, thereby potentiating immunotherapy 36 . We selected DOXO-EMCH (a maleimide-containing DOX prodrug) as an alternative to vcMMAE and conjugated it with LZ-8-2.3-7D12-Cys to generate a novel conjugate, LZ-8-2.3-7D12-DOX (Extended Data Fig. 9p) 37 . Similar to the MMAE conjugate, LZ-8-2.3-7D12-DOX binds membrane protein receptors, undergoes internalization and lysosomal degradation, and releases DOX to induce CRT translocation (Fig. 4 i). Consistent with the expectations, flow cytometry results showed that LZ-8-2.3-7D12-DOX effectively induced CRT exposure and significantly enhanced antitumor activity of DOX in both A431 and HeLa cells compared to LZ-8-2.3-7D12 (Fig. 4 j, Extended Data Fig. 9q-r). To evaluate the antitumor efficacy of the LZ-8-2.3–based LYTAC-DC platform in vivo , we first established a subcutaneous xenograft model using A431 cells, an aggressive skin cancer line known for rapid progression and poor clinical control. Based on the potent cytotoxicity of LZ-8-2.3-7D12-MMAE observed in vitro , we administered the conjugate intravenously at 3 mg/kg every two days for four doses (Extended Data Fig. 10a). Treatment with LZ-8-2.3-7D12-MMAE significantly suppressed tumor growth compared with LZ-8-2.3-7D12 alone, as measured by both tumor volume and weight (Fig. 4 h, Extended Data Fig. 10b–c). Notably, LZ-8-2.3-7D12—a degradation-only LYTAC—showed limited efficacy against highly malignant A431 tumors, consistent with our in vitro viability data (Fig. 4 g). No significant body weight loss was observed, indicating a favorable safety profile in vivo (Extended Data Fig. 10d). Furthermore, TUNEL apoptosis staining, H&E histology and anti-EGFR immunohistochemistry revealed markedly increased apoptosis and EGFR degradation in tumors treated with LZ-8-2.3-7D12-MMAE compared to PBS or LZ-8-2.3-7D12 groups, suggesting improved prognostic potential (Extended Data Fig. 10e-f). We next asked whether LZ-8-2.3-7D12-MMAE could also exhibit efficacy in a triple-negative breast cancer model, MDA-MB-231, which showed limited response in vitro . Mice bearing MDA-MB-231 xenografts received 1.5 mg/kg LZ-8-2.3-7D12-MMAE every three days for eight doses (Extended Data Fig. 10g). The conjugate significantly inhibited tumor growth relative to LZ-8-2.3-MMAE or 7D12-MMAE controls (Fig. 4 i, Extended Data Fig. 10h–i). Treatment was well tolerated, with no notable changes in body weight or organ pathology (Extended Data Fig. 10j, k). Although lung metastases were observed—consistent with the high metastatic potential of MDA-MB-231 cells—conjugate treatment effectively suppressed pulmonary tumor growth (Extended Data Fig. 10i). Enhanced apoptosis by TUNEL and reduced proliferation by Ki67 staining further supported the therapeutic benefit of LZ-8-2.3-7D12-MMAE in this model. Together, these results demonstrate that LZ-8-2.3-7D12-MMAE exerts robust antitumor activity in vivo against both highly sensitive and less responsive tumor types, with no overt systemic toxicity. These findings underscore the translational potential of the LYTAC-DC platform and support further development of LZ-8-2.3–based drug conjugates for a broad range of therapeutic targets. AI-Assisted Evolution of Non-Chimeric LYTA-DCs Finally, we hypothesized that directed evolution could ameliorate the non-specific interaction of LZ-8 and enhance its selectivity towards a given receptor, aiming at targeted protein degradation and small-molecule drug delivery without requiring a chimera ligand (Fig. 5 a). Notably, LZ-8 possesses a canonical immunoglobulin-like domain at its C-terminus and shares high structural similarity with non-antibody scaffold proteins like monobody, which can be engineered to bind specific target protein receptors 38 . Compared to traditional antibodies or ligands, engineering LZ-8 into a novel non-antibody scaffold offers several advantages: its inherent multi-receptor binding capability, lack of disulfide bonds (facilitating correct folding and stability in the reductive cytosolic environment), relatively small molecular weight (~ 14 kDa), and the multivalency conferred by its dimeric form. Therefore, developing LZ-8 as a novel non-antibody scaffold protein for LYTA-DC drug design holds substantial potential. To validate this concept, we selected EGFR as the target and employed AI-assisted protein directed evolution to enhance LZ-8's affinity for EGFR (Fig. 5 b). First, we used a molecular docking model of LZ-8 with receptor EGFR and identified 29 potential beneficial single-site mutations. The mutant proteins were dually validated using ELISA and flow cytometry assays. Unexpectedly, though N103S and I106Q demonstrated significant enhancement of EGFR binding in both assays, majority of the mutants displayed discrepancies between ELISA and flow cytometry analyses (Extended Data Fig. 11a-b). Subsequently, we utilized the sequence-structure protein language model ProSST model to combine the beneficial mutations of N103S and I106Q along with other mutants, generated 18 multi-site mutants 39 . ELISA and flow cytometry analyses showed significant synergistic enhancement in specific interaction with EGFR, with no apparent epistatic effects (Extended Data Fig. 11c-d). Flow cytometry analysis of cell surface EGFR internalization revealed that the top 6–7 selected multi-site mutants significantly enhanced EGFR internalization (Fig. 5 c, Extended Data Fig. 11e). Immunoblotting further confirmed that multi-site mutants significantly enhanced EGFR degradation efficiency (Extended Data Fig. 11f-g).Some of the multi-site mutants also exhibited differential affinities in Hela and Mada-mb-231 cells (Extended Data Fig. 11h). Cytotoxicity assays found increased toxicity of some mutants in comparison to WT LZ-8, potentially linked to mutation introduced non-specific binding (Extended Data Fig. 11i). Four multi-site mutants with low toxicity were selected for ELISA analysis. Results showed similar binding characteristics of all four protein mutants, which significantly enhanced affinity for EGFR, while retained certain binding capability to other receptors like HER3 and c-Met (Fig. 5 d, Extended Data Fig. 11j). Nevertheless, we went on to establish four LZ-8 mutant-MMAE conjugates through the introduced C-termini cysteine Cell viability assays showed that multiple 11(N102S; I105Q)-MMAE conjugates exhibited higher cytotoxicity than the WT LZ-8-MMAE (Fig. 5 e) Next, we focused on the Y84A mutant for further directed evolution. We introduced the Y84A mutation into the mutants of all 30 generated a new set of single-site mutants on the Y84A background. Surprisingly, the evolutionary trajectory on the Y84A background differed almost completely from that on the wild-type LZ-8, indicating that Y84 is critically involved in the evolutionary pathways (Extended Data Fig. 12a-b). Particularly, three mutants at the L9 position (e.g., L9G, L9N, L9A) almost completely abolished the EGFR affinity of the Y84A mutant, while these mutations had minimal impact on the EGFR affinity of wild-type LZ-8 (Extended Data Fig. 11a-b). Multi-target ELISA assays and broad receptor profiling via flow cytometry on Hela cells confirmed that the L9 mutations caused widespread inactivation of the Y84A mutant (Extended Data Fig. 12c-d). The LZ-8 crystal structure reveals that residue L9 is located internally within the dimerization arm, not directly involved in receptor interaction, but is crucial for dimerization 40 . Competitive ELISA showed that pre-saturation binding with wild-type LZ-8 almost completely blocked subsequent binding of Y84A, whereas pre-saturation with Y84A caused no significant steric hindrance to subsequent wild-type LZ-8 binding (Extended Data Fig. 12e). Therefore, we conclude that the high-affinity binding of LZ-8 to cell surface receptors primarily depends on the monomer containing Y84, while the lower-affinity binding of the Y84A mutant relies on dimerization. LZ-8 likely utilizes two parallel, non-interfering interaction modes with receptors: high-affinity monomeric binding mediated by Y84 and low-affinity dimeric binding mediated by L9-dependent dimerization, with prioritized monomeric binding. Discussion This study has established a unified and engineerable LYTAC-DC platform that synergistically integrates targeted protein degradation in lysosome with precision drug delivery by leveraging a novel class of binding proteins—Multibodies. Using the identified fungal-derived protein LZ-8 as a prototype, we demonstrate that polyvalent, polyspecific targeting scaffolds can overcome fundamental limitations of conventional lysosome-targeting chimeras and antibody-drug conjugates, with regard to target specificity, internalization efficiency, and payload versatility. Through rational engineering, we mitigated off-target toxicity of the WT LZ-8 via Y84A mutation, yielding a high-fidelity LYTAC and LYTAC-DC system capable of degrading diverse cancer-related membrane proteins while concurrently delivering cytotoxic or immunogenic payloads. Moreover, AI-assisted evolution enabled the development of non-chimeric LYTA-DCs, further highlighting the modularity and adaptability of the Multibody concept. The molecular mechanisms of the LZ-8 binding to receptors are highlighted through combined double mutation of Y84 and L9, which provides valuable information for further rational design of specific mutibodies. Recent studies, including the degrader-drug conjugates (DDCs) 41 and the intratumoural vaccination chimera (iVAC) 42 , alongside this work, collectively underscore the broad potential of exploiting the lysosomal pathway for synergistic "degradation-plus" therapies. Our Multibody-LYTAC-DC platform shares the core commonality of hijacking the endolysosomal pathway with these cutting-edge strategies, yet it is fundamentally distinguished by its implementation route and mode of functional integration. The DDC strategy enhances the delivery efficiency of traditional antibody-drug conjugates by recruiting specific endocytic receptors (e.g., LDLR), while iVAC combines target degradation with specific immune activation through covalent binding and antigen presentation. In contrast, this study develops a unified platform featuring multi-valent targeting, efficient degradation, payload delivery, and AI-assisted directed evolution from a structurally unique and engineerable fungal protein scaffold (LZ-8). Its core advantages lie in: First, the Multibody itself serves as a multifunctional "engine," avoiding complex multi-component assembly and simplifying design. Second, a single point mutation (Y84A) is sufficient to abolish off-target toxicity while retaining efficient lysosomal targeting, demonstrating excellent tunability. Third, and most importantly, through AI-assisted evolution, we successfully converged degradation and targeting functions into a single, non-chimeric LYTA-DC molecule. This points towards a new direction for developing smaller, more stable, and more tumor-penetrant "smart" single-agent therapeutics. These properties not only make our platform suitable for "degradation + chemotherapy" enhanced killing but also establish a unique and flexible foundation for its future expansion into multi-dimensional synergistic therapies, such as "degradation + immunomodulation." In summary, the Multibody platform has demonstrated unique properties and advantages in comparison to LYTAC-DC platform. To advance the concept towards clinical applications, future efforts will focus on optimizing Multibody specificity and reducing immunogenicity, broadening the repertoire of targetable receptors, and evaluating combination therapies across heterogeneous cancers diseases. The integration of AI-assisted design and directed protein evolution will accelerate the development of next-generation lysosome-targeting therapeutics, potentially enabling personalized and adaptive treatment strategies. The novel concept of Multibody is expected to add a new dimension to proceed the progress in specified and personalized therapy for diseases, as the transformative tool is far from restricted to cancer treatment, but can be adapted to treatment of protein-accumulation diseases including the A-beta-deposition induced Alzheimers disease. Declarations Animal Ethics Statement All animal experiments described in this study were conducted in accordance with the relevant guidelines and regulations for the care and use of laboratory animals. The experimental protocols involving non-human vertebrates were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of East China University of Science and Technology. All efforts were made to minimize animal suffering and to reduce the number of animals used. Supporting Information Further information on research design is available in the Reporting Summary linked to this article. Declaration of Interests The authors declare no competing interests. Author Contributions J.Z., L.H., F.T., and L.Z. conceptualized and supervised the project. J.Z., F.W., P.T., L.H., F.T. and L.Z. provided funding support. L.C. designed and performed the main experiments. S.Y., Y.G., Y.W., S.C. and D.L. participated in the experiments. Q.S. performed in the Protein small molecule drug coupling experiment. P.T. and J.H. performed in the Mutant directed evolution design. X.T. performed in vivo experiments on mice. L.C., X.T., J.Z., J.L., W.W. and L.O. wrote the manuscript. J.L., L.F., W.H., F.W., J.Z., Y.Z., F.T. and L.Z. edited the manuscript. Acknowledgments This work was supported by the National Natural Science Foundation of China (32121005 and 32327801 to L.Z.,32571667 to J.Z., and 22422705 to F.T.), the National Key Research and Development Program of China (2020YFA0907800 to L.Z., 2024YFA0917603 to L.H. and 2020YFA0907200 to J.Z.), Shanghai Municipal Science and Technology Major Project (to L.H.), Shanghai Science and Technology Commission (24HC2820200 to L.Z., 24ZR1417000 to J.Z.), the Computational Biology Key Program of Shanghai Science and Technology Commission (23JS1400600 to L.H.), Shanghai Municipal Education Commission (2024AIZD015 to L.H.), Shanghai Jiao Tong University Scientific and Technological Innovation Funds (21X010200843 to L.H.), Open Project Funding of the State Key Laboratory of Bioreactor Engineering, the 111 Project (B18022 to L.Z.), Science and Technology Innovation Key R&D Program of Chongqing (CSTB2022TIADSTX0017 to L.H., CSTB2024TIAD-STX0032 to P.T.), the Fundamental Research Funds for the Central Universities (L.Z.).and also by Zhejiang Fonow Medicine Co., Ltd. (Grant No. F100-42106L to F.W.). We are grateful to Tsingke Biotechnology Co., Ltd. for technical support in mRNA transcriptome sequencing, Shenzhen Kangti Life Technology Co., Ltd. for assistance with BLI-based affinity measurements, and Shanghai OE Biotech Co., Ltd. for protein gel LC/MS analysis. We are grateful to Professor Huizhan Zhang (East China University of Science and Technology) for constructive discussions regarding this study. We are grateful to Siwu Guo (Shanghai Beautiful Life Medical Technology Co., Ltd.), Yinan Wang (Shanghai Beautiful Life Medical Technology Co., Ltd.) and Zhenwei Wang (Shanghai Beautiful Life Medical Technology Co., Ltd.) for technical support in vivo experiments on mice . Data Availability Statement All data reported in this paper will be shared by the lead contact upon request. Code availability The code of the ProSST used in this work can be found in https://github.com/ai4protein/ProSST. 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Technology","correspondingAuthor":false,"prefix":"","firstName":"Simin","middleName":"","lastName":"Chen","suffix":""},{"id":581809109,"identity":"4dd975c2-68ec-4f06-b284-d0c5f159b19c","order_by":11,"name":"Dong-Yuan Lv","email":"","orcid":"","institution":"East China University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Dong-Yuan","middleName":"","lastName":"Lv","suffix":""},{"id":581809110,"identity":"ad727005-7832-47e4-8018-cb50857ad25f","order_by":12,"name":"Wei Wu","email":"","orcid":"","institution":"Shanghai Zhenge Biotechnology Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wu","suffix":""},{"id":581809111,"identity":"a71bf631-5eea-423b-96e6-de112526be42","order_by":13,"name":"Liming Ouyang","email":"","orcid":"","institution":"East China University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Liming","middleName":"","lastName":"Ouyang","suffix":""},{"id":581809112,"identity":"379f5eca-f686-4442-a494-23d6b4162518","order_by":14,"name":"Liqiang Fan","email":"","orcid":"","institution":"East China University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Liqiang","middleName":"","lastName":"Fan","suffix":""},{"id":581809113,"identity":"0cb81e5e-4529-4bdb-9cf2-4565c4f6303d","order_by":15,"name":"Wei Huang","email":"","orcid":"","institution":"University of Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Huang","suffix":""},{"id":581809114,"identity":"d244bf66-d752-47df-8345-8d5bcfba0124","order_by":16,"name":"Fujun Wang","email":"","orcid":"","institution":"Zhejiang Fonow Medicine Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Fujun","middleName":"","lastName":"Wang","suffix":""},{"id":581809115,"identity":"a9a907a8-2c1c-404d-b8ec-ee9553986e0d","order_by":17,"name":"Yuzheng Zhao","email":"","orcid":"https://orcid.org/0000-0001-5400-0135","institution":"East China University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yuzheng","middleName":"","lastName":"Zhao","suffix":""},{"id":581809116,"identity":"c76110eb-4341-4060-8f1b-00288b178c26","order_by":18,"name":"Jian Zhao","email":"","orcid":"","institution":"East China University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Zhao","suffix":""},{"id":581809117,"identity":"8869ce4c-c01a-4f2e-9c25-b1396e73325c","order_by":19,"name":"Jingyu Zhang","email":"","orcid":"","institution":"East China University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Jingyu","middleName":"","lastName":"Zhang","suffix":""},{"id":581809118,"identity":"0606c13b-a4ff-467e-84b8-4f5c1d9b8ce1","order_by":20,"name":"Liang Hong","email":"","orcid":"https://orcid.org/0000-0003-0107-336X","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Hong","suffix":""},{"id":581809119,"identity":"fc1a2673-d11c-4b41-872b-5c46e9f86fa2","order_by":21,"name":"Feng Tang","email":"","orcid":"https://orcid.org/0000-0002-2156-7271","institution":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2026-01-20 16:13:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8651044/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8651044/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102939988,"identity":"33edf0a6-d997-4dfd-9e55-1274b5ee48a6","added_by":"auto","created_at":"2026-02-18 16:58:54","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114379,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of Lz-8 as a LYTAC-DC. \u003cstrong\u003ea. \u003c/strong\u003eSchematic diagram illustrating the mechanism of lysosome-targeting chimaera–drug conjugate (LYTAC-DC). LYTAC-DC binds to membrane proteins via specific targeting ligands (e.g., nanobodies), undergoes internalization mediated by lysosome-targeting molecules, and promotes degradation of the target receptors in lysosomes. During this process, the conjugated small molecule drug is released and exerts its therapeutic effect, leading to both degradation of the cell surface receptor and cytotoxic drug release, ultimately resulting in cancer cell death. \u003cstrong\u003eb.\u003c/strong\u003e HeLa cells transiently expressing EGFP-Lamp1 were treated with 500 nM Lz-8-mCherry for 0, 6, 12, or 24 h, fixed, and imaged by confocal microscopy. Nuclei were counterstained with DAPI. Right panels show magnified views of boxed regions. Scale bars: 50 μm. \u003cstrong\u003ec.\u003c/strong\u003e Schematic representation of Lz-8-nanobody–mediated degradation of target receptors (including soluble extracellular or membrane proteins) through Lz-8 receptor binding–induced endocytosis and subsequent lysosomal degradation. \u003cstrong\u003ed. \u003c/strong\u003eWestern blot analysis of EGFR expression in HeLa cells treated for 24 h with Lz-8-7D12 at indicated concentrations (0, 20, 100, and 500 nM). e. Western blot of EGFR in HeLa cells treated with 500 nM Lz-8-7D12 for 6 h with or without 50 nM bafilomycin A1 (BafA1). \u003cstrong\u003ef.\u003c/strong\u003e Viability of A431 cells treated for 24 h with 100 nM of the indicated proteins (Lz-8, 7D12, Lz-8-8D12, Lz-8-MMAE, 7D12-MMAE, or Lz-8-7D12-MMAE), as measured by MTT assay. Data were analyzed using GraphPad Prism 8.0.2 and are presented as mean ± SD. \u003cstrong\u003eg.\u003c/strong\u003e Western blot analysis of EGFR and PD-L1 expression in MDA-MB-231 cells treated for 24 h with Lz-8-KN035 at the indicated concentrations (0, 20, 100, and 500 nM).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8651044/v1/69af1dec6059dce38e81a2cd.jpg"},{"id":102939985,"identity":"d61cdd24-3329-468d-adff-4199c93cfaf8","added_by":"auto","created_at":"2026-02-18 16:58:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":105157,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Lz-8 on lysosomal function in HeLa cells. \u003cstrong\u003ea. \u003c/strong\u003eDegradation of DQ-BSA in HeLa cells treated with 500 nM Lz-8 for 0, 6, 12, or 24 h. Cells were fixed and imaged by confocal microscopy. Enlarged views of boxed regions are shown on the right.\u003cstrong\u003e b. \u003c/strong\u003eWestern blot analysis of IGF2R and LAMP1 expression in HeLa cells treated with 500 nM Lz-8 for the indicated durations (0, 6, 12, or 24 h).\u003cbr\u003e\n \u003cstrong\u003ec.\u003c/strong\u003e HeLa cells treated with 500 nM Lz-8 for 6 h were fixed and co-stained with antibodies against LAMP1 or IGF2R, followed by incubation with Alexa Fluor 488-conjugated secondary antibodies. Confocal images are shown, with enlarged views of boxed regions presented on the right. \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee. \u003c/strong\u003eWestern blot analysis of mature CTSB, LC3-I/II, and p62 in HeLa cells treated with 500 nM Lz-8 for 0, 6, 12, or 24 h. \u003cstrong\u003ef.\u003c/strong\u003e HeLa cells transiently expressing mCherry-GFP-LC3B were treated with 500 nM Lz-8 for 6 h, fixed, and imaged by confocal microscopy. Arrows indicate green puncta, suggesting unmetabolized GFP signals due to impaired lysosomal function. \u003cstrong\u003eg. \u003c/strong\u003eProposed model of Lz-8–induced lysosomal dysfunction: (i) Lz-8 binds cell surface receptors and induces sustained internalization; (ii) continuous endosomal-lysosomal fusion leads to lysosomal pH neutralization and LAMP1 downregulation; (iii) Lz-8 mediates IGF2R depletion in the trans-Golgi network and enhances its anterograde trafficking to the plasma membrane; (iv) collectively, lysosomal acidification defects, LAMP1 deficiency, and IGF2R-mediated mistrafficking of enzymes result in lysosomal dysfunction.All cells in panels a, c, and f were counterstained with DAPI. Scale bars: 50 μm.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8651044/v1/9aa5ba8883cad19f513f7f8d.jpg"},{"id":102939986,"identity":"9a594f88-3d33-4ed8-9d3f-2371ba26541f","added_by":"auto","created_at":"2026-02-18 16:58:54","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":226730,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of Lz-8 receptors and design of a mutant Lz-8. \u003cstrong\u003ea. \u003c/strong\u003eSchematic overview of the strategy used to identify Lz-8 receptors and critical binding residues. Membrane proteins from HeLa cells were isolated by subcellular fractionation and incubated with anti-EGFP nanobody-conjugated agarose beads. Potential Lz-8-binding receptors were captured and eluted, followed by detection via SDS-PAGE, liquid chromatography–mass spectrometry (LC-MS), and ELISA. Key residues were predicted through molecular docking and validated by mutagenesis. b, \u003cstrong\u003ec. \u003c/strong\u003eAbundances of candidate Lz-8 receptors in HeLa cells identified by co-immunoprecipitation (Co-IP) and LC-MS (b), and their enrichment in membrane extracts pulled down with Lz-8-EGFP-bound anti-EGFP beads (c). \u003cstrong\u003ed.\u003c/strong\u003e ELISA results showing binding between Lz-8-EGFP and selected candidate receptors. \u003cstrong\u003ee.\u003c/strong\u003e Internalization of EGFP into HeLa cells after 6 h treatment with 500 nM Lz-8-EGFP or its mutant form, as quantified by mean fluorescence intensity (MFI) using live-cell flow cytometry. \u003cstrong\u003ef.\u003c/strong\u003e Surface hydrophobicity representations of the predicted structures of Lz-8 and the mutant Lz-8-2.3 (Y84A), as predicted by AlphaFold2. \u003cstrong\u003eg.\u003c/strong\u003e Viability of MRC-5 cells treated with 500 or 2500 nM Lz-8 or Lz-8-2.3 for 48 h, assessed by MTT assay. \u003cstrong\u003eh.\u003c/strong\u003e ELISA measuring binding of 20 nM Lz-8-EGFP or Lz-8-2.3-EGFP to α5β1, c-MET, EGFR, L1CAM, HER3, and PD-1. \u003cstrong\u003ei. \u003c/strong\u003eWestern blot analysis of IGF2R and EGFR expression in HeLa cells treated with 500 nM Lz-8 or Lz-8-2.3 for 24 h. Statistical significance was determined by one-way ANOVA with multiple comparisons: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001; ns, not significant. Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8651044/v1/b8156df9000ce757a6c7325b.jpg"},{"id":102939990,"identity":"2be6765f-cdc3-49b3-8ef0-31b08b47aefc","added_by":"auto","created_at":"2026-02-18 16:58:55","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":95387,"visible":true,"origin":"","legend":"\u003cp\u003eApplication of Lz-8-2.3 (Y84A) in developing a lysosome-sparing LYTAC-DC.\u003cbr\u003e\n \u003cstrong\u003ea.\u003c/strong\u003e Schematic comparison of receptor degradation mediated by Lz-8-2.3-Nb versus Lz-8-Nb. The Lz-8-2.3-based chimera addresses three limitations of Lz-8-Nb: (i) off-target effects, (ii) lysosomal dysfunction, and (iii) cytotoxicity. \u003cstrong\u003eb.\u003c/strong\u003e Western blot analysis of EGFR in HeLa cells treated for 24 h with 500 nM of the indicated proteins (Lz-8-2.3, 7D12, or Lz-8-2.3 + 7D12). \u003cstrong\u003ec. \u003c/strong\u003eWestern blot of EGFR in HeLa cells treated with 100 nM Lz-8-2.3-7D12 for 12 h with or without 50 nM bafilomycin A1 (BafA1).\u003cbr\u003e\n \u003cstrong\u003ed.\u003c/strong\u003e Western blot of IGF2R and EGFR in HeLa cells treated for 24 h with Lz-8-2.3-7D12 at the indicated concentrations (0, 20, 100, or 500 nM).\u003cstrong\u003ee.\u003c/strong\u003e Western blot of PD-L1 in MDA-MB-231 cells treated for 24 h with Lz-8-2.3-KN035 at the indicated concentrations (0 to 100 nM). \u003cstrong\u003ef.\u003c/strong\u003e Western blot of IGF2R, EGFR, and PD-L1 in MDA-MB-231 cells treated for 24 h with Lz-8-2.3-KN035 (0 to 500 nM). \u003cstrong\u003eg.\u003c/strong\u003e Viability of A431 (EGFR-high) cells treated for 48 h with indicated concentrations (0 to 500 nM) of Lz-8-2.3-7D12, Lz-8-2.3-MMAE, 7D12-MMAE, or Lz-8-2.3-7D12-MMAE, as measured by MTT assay.\u003cstrong\u003e H-i.\u003c/strong\u003e Tumor volume measured every 2-3 days throughout the treatment period. \u003cstrong\u003ej.\u003c/strong\u003e Molecular design of the LYTAC-DC construct comprising three functional modules: an engineered lysosome-targeting ligand (Lz-8-2.3), an EGFR-specific nanobody (7D12), and doxorubicin (DOX) covalently linked. The proposed mechanism includes: (i) specific EGFR binding via 7D12; (ii) Lz-8-2.3 receptor-mediated endocytosis and lysosomal trafficking; (iii) lysosomal degradation of EGFR and release of DOX; (iv) dual action through sustained EGFR degradation and DOX-induced immunogenic cell death marked by calreticulin surface exposure. \u003cstrong\u003ek.\u003c/strong\u003e Percentage of calreticulin (CRT)-positive HeLa cells after 24 h treatment with 500 nM DOX, Lz-8-2.3-7D12, or indicated concentrations (20–500 nM) of Lz-8-2.3-7D12-DOX, as determined by flow cytometry using anti-CRT antibody and Alexa Fluor 488-conjugated secondary antibody. Statistical significance was determined by one-way ANOVA: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001; ns, not significant. Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8651044/v1/53c036f1cb06fc9da991d64c.jpg"},{"id":102963825,"identity":"da2d7537-2846-49ec-b84d-df261c6f957d","added_by":"auto","created_at":"2026-02-19 04:20:39","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":62691,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Schematic representation of the LYTAC-DC system. The left panel illustrates the chimeric design of LYTAC-DC as established in this study; the right panel depicts targeted protein degradation and drug delivery mediated by the multivalent and multi-target binding capability of the multibody construct. \u003cstrong\u003eb.\u003c/strong\u003e Workflow of AI-assisted directed evolution for enhancing multibody affinity toward target proteins. The process involved: docking the multibody with the target protein; performing in silico single-point saturation mutagenesis using a large language model; selecting top 29 single mutations and constructing EGFP-tagged variants for validation via flow cytometry (cellular level) and ELISA (molecular level); converting affinity results into scores for model fine-tuning; subsequently combining beneficial mutations; and finally outputting top candidates for functional assessment in the LYTAC-DC context. \u003cstrong\u003ec.\u003c/strong\u003e Cell surface EGFR levels in A431 cells incubated with 100 nM Lz-8 or its multipoint mutants for 1 h, stained with APC-conjugated anti-EGFR antibody and analyzed by flow cytometry. Mean fluorescence intensity (MFI) reflects EGFR expression. Statistical significance was calculated by one-way ANOVA compared to control or wild-type (WT): ****p \u0026lt; 0.0001. \u003cstrong\u003ed.\u003c/strong\u003e ELISA binding curves of Lz-8-EGFP or multipoint mutants-EGFP at indicated concentrations (0–50 nM) to EGFR. EC₅₀ values were derived using GraphPad Prism.\u003cstrong\u003e e.\u003c/strong\u003e Viability of A431 cells treated with 100 nM MMAE-conjugated multipoint mutants of Lz-8 for 24 h, assessed by MTT assay. Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8651044/v1/5a25a0d78b37edcf185c8f98.jpg"},{"id":102965446,"identity":"77f2a936-f0e5-42c6-9fbd-f42b947d9d78","added_by":"auto","created_at":"2026-02-19 04:31:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1527699,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8651044/v1/8141a01f-4c2f-4e77-8315-cdc82a599f7c.pdf"},{"id":102964174,"identity":"94f9de7e-adad-4354-9b8d-da136cb32f45","added_by":"auto","created_at":"2026-02-19 04:21:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6694575,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFig.docx","url":"https://assets-eu.researchsquare.com/files/rs-8651044/v1/23aed80f500ff076472a1e75.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Multibody-Enabled Lysosome-Targeting Drug Conjugates for Target Protein Degradation and Combination Therapy","fulltext":[{"header":"Highlights","content":"\u003cul start=\"50\"\u003e\n \u003cli\u003e\u003cstrong\u003eProposes and validates the novel concept of \u0026quot;Mu\u003c/strong\u003e\u003cstrong\u003el\u003c/strong\u003e\u003cstrong\u003etibody,\u0026quot;\u003c/strong\u003e establishing a modular lysosome-targeting drug design platform that encompasses both chimera-dependent (LYTAC-DC) and chimera-independent (LYTA-DC) strategies, overcoming the limitations of conventional LYTACs and ADCs.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eIdentifies the fungal protein LZ-8 as a potent yet off-target-prone lysosome-targeting scaffold, and engineers the key variant LZ-8-2.3 through rational design.\u003c/strong\u003e This variant retains efficient endocytosis and lysosomal trafficking while eliminating off-target degradation and lysosomal dysfunction mediated by the Y84 residue.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eThe developed LYTAC-DC platform demonstrates robust versatility,\u003c/strong\u003e mediating high-fidelity degradation of multiple cancer-related membrane proteins, including EGFR, PD-L1, and HER2, with concurrent delivery of cytotoxic or immunogenic small-molecule payloads, as validated across \u003cem\u003ein vitro\u003c/em\u003e, organoid, and\u003cem\u003ein vivo\u003c/em\u003e models.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eConverges targeting and degradation functions into a single molecule via AI-assisted directed evolution,\u003c/strong\u003e enabling the development of non-chimeric LYTA-DCs and showcasing the unique evolvability and engineerability of the Mutibody scaffold for \u0026quot;all-in-one\u0026quot; smart drug design.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eThe platform shows promise in overcoming clinical resistance.\u003c/strong\u003e LZ-8-2.3-based LYTAC-DC effectively inhibits the growth of osimertinib-resistant NSCLC models and demonstrates potent anti-tumor activity in patient-derived tumor organoids harboring triple EGFR mutations (Del19/T790M/C797S).\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Main","content":"\u003cp\u003eThe endolysosomal system represents a critical pathway for targeted therapeutic intervention\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, serving as a natural cellular mechanism for the degradation of extracellular and membrane proteins, as well as for the intracellular delivery of therapeutic agents\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Lysosome-targeting chimeras (LYTAC) exemplify this approach by employing bispecific molecules that simultaneously engage a cell-surface lysosome-targeting receptor and a disease-relevant target protein, directing the latter for lysosomal degradation. However, current LYTAC platforms face inherent limitations, including restricted target specificity, reliance on specific lysosome-targeting receptors (e.g., IGF2R), and variable internalization efficiency, which collectively constrain their broad applicability\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Concurrently, antibody-drug conjugates (ADCs) exploit receptor-mediated endocytosis for precise drug delivery but are fundamentally limited by the intrinsic internalization kinetics of their target antigens\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, posing significant challenges for targeting poorly internalizing receptors.\u003c/p\u003e \u003cp\u003eA common constraint across these advanced therapeutic modalities is their dependence on conventional antibodies or nanobodies, which are typically monospecific and monovalent\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. This molecular architecture restricts multi-target engagement and complicates the integration of additional functionalities, such as synergistic payload delivery or combinatorial degradation strategies. To overcome these constraints, we developed a flexible and multidimensional platform that integrates targeted protein degradation with site-specific drug release via the endolysosomal pathway.\u003c/p\u003e \u003cp\u003eInspired by natural mechanisms for clearing heterologous proteins\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, we identified the fungal immunomodulatory protein LZ-8 as a multivalent and multispecific lysosome-targeting scaffold. Initial characterization confirmed its potent endocytic activity and broad receptor interactions. However, the native protein induced significant lysosomal dysfunction and off-target degradation, limiting its therapeutic utility. Through rational protein engineering, we generated an optimized variant, LZ-8-2.3 (Y84A), which eliminated these adverse effects while retaining efficient lysosomal trafficking. This engineered Mutibody (LZ-8-2.3) serves as the core component of our Lysosome-Targeting Chimeric Drug Conjugates (LYTAC-DC) platform, enabling simultaneous, high-fidelity degradation of diverse oncogenic membrane proteins and concurrent delivery of cytotoxic or immunogenic payloads. This study establishes the LYTAC-DC platform as a unified strategy to address the key limitations of conventional LYTAC and ADC technologies, offering a modular and engineerable framework for next-generation targeted therapies.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLZ-8 as a Potent but Flawed Lysosome-Targeting Scaffold\u003c/h2\u003e \u003cp\u003eCurrent LYTAC tools, primarily antibody-based conjugates, not only limit the further conjugation of small-molecule drugs but also increase the inherent complexity and instability of drug synthesis. To address this, we explored novel recombinant protein-based LYTAC tools, as these not only can be easily mass-produced but also facilitate subsequent small-molecule drug conjugation. Inspired by the innate ability of cells to clear heterologous proteins via the lysosomal pathway, we selected four heterologous proteins previously reported to induce endocytosis, including fungal (LZ-8)\u003csup\u003e18\u003c/sup\u003e, camelid (7D12)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, bacterial (STxB-TDP)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and viral (TAT)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. These proteins were genetically fused with fluorescent protein EGFP for tracking. Confocal microscopy and flow cytometry results revealed that the fungal-derived LZ-8 exhibited exceptionally stronger intracellular internalization capabilities compared to other 3 proteins (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b). LZ-8 (PDB ID:3F3H) forms a dimer through its N-terminal α-helical dimerization arm, with a C-terminal immunoglobulin-like domain (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Preliminary screen showed that LZ-8 induced broad-spectrum cytotoxicity in multiple cell lines in a dose-dependent manner (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-g). Based on the relatively lower cytotoxicity at 500 nM in HeLa cell line (viability\u0026thinsp;\u0026gt;\u0026thinsp;85%), this concentration was used for the subsequent experiments as the maximum dose. Trypan blue quenching assays confirmed that LZ-8-EGFP entered HeLa cells rather than remaining on the cell membrane (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Time- and dose-dependent analyses demonstrated that LZ-8-EGFP efficiently bound to cells and accumulated intracellularly (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei-k). This property was common across multiple fungal-derived immunomodulatory protein family members (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003el-m)\u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate LZ-8\u0026rsquo;s lysosomal targeting efficiency as a LYTAC tool, multi-organelle co-localization analysis revealed that LZ-8-EGFP primarily localized to the endosomal network adjacent to the cis-Golgi apparatus (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Time-dependent co-localization experiments with early endosome marker EEA1 and late endosome marker RAB7 confirmed that LZ-8-EGFP effectively completed endosome maturation and entered LAMP1-positive lysosomes (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-e). In HeLa cells overexpressing LAMP1-GFP, LZ-8-mCherry showed strong punctate co-localization with LAMP1-GFP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb); while the degree of co-localization with lysosomal probe LysoTracker increased over time (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Dual-fluorescence pulse-chase experiments demonstrated that the early accumulated LZ-8-EGFP was quenched, which could be reversed by BafA1 treatment, while sustained endocytic flux led to intracellular accumulation of LZ-8-mCherry (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-h). TFEB overexpression significantly suppressed LZ-8 accumulation by enhancing lysosomal biogenesis, suggesting that increased lysosomal degradation capacity aids intracellular clearance of LZ-8 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). Collectively, LZ-8 drives persistent cellular internalization and efficient lysosomal targeting.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBuilding on LZ-8\u0026rsquo;s lysosomal targeting properties, we constructed a recombinant LYTAC system by fusing LZ-8 and the various nanobodies against different targets, aiming to trigger enhanced degradation of target proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). For the degradation of extracellular model protein EGFP, LZ-8-Nb\u003csup\u003eEGFP\u003c/sup\u003e, a fusion protein of LZ-8 and EGFP-targeting nanobody, was constructed. Significant intracellular EGFP enrichment in HeLa cells was detected after 30 minutes incubation with LZ-8-Nb\u003csup\u003eEGFP\u003c/sup\u003e, indicating that LZ-8-Nb\u003csup\u003eEGFP\u003c/sup\u003e efficiently captured EGFP in the medium and mediated its internalization. The intracellular EGFP level was markedly decreased after removal of EGFP from the culture for 3 hours, indicating rapid degradation of EGFP after internalization. However, the presence of lysosome inhibitor BafA1 inhibited the degradation process, confirming the fact that the degradation is lysosome-dependent (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). For membrane bound proteins, we first tested degradation of EGFR by fusing LZ-8 to an EGFR-targeting nanobody, 7D12(Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Although previous studies have shown that LZ-8 itself can bind EGFR and mediate its degradation\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, which was also confirmed by our results (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec); the fusion protein LZ-8-7D12 induced significant lysosome-dependent EGFR degradation in HeLa and A431 cells at the concentration as low as 20 nM. As the lysosomal inhibitor BafA1 treatment attenuated the degradation, confirming its lysosome dependent activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-e,Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-e). To further verify the effect of LZ-8, PD-L1 (highly expressed in MDA-MB-231 cells) and HER2 (highly expressed in SKBR3 cells) were selected as non-LZ-8-associated targets (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). After incubation of the LZ-8 with MDA-MB-231 and SKBr3 cells respectively, Western blot (WB) analysis showed that LZ-8 at doses below 500 nM did not induce PD-L1 degradation, but triggered HER2 degradation, suggesting HER2, like EGFR, is also a potential receptor for LZ-8 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). It was then demonstrated that LZ-8-KN035 (PD-L1 nanobody fusion) and LZ-8-11A4 (HER2 nanobody fusion) induced specific degradation of PD-L1 and HER2, respectively, indicating that LZ-8-mediated LYTACs significantly enhance degradation of both non-LZ-8 receptor targets and LZ-8 receptor-associated targets (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe high efficiency of LZ-8 as a LYTAC tool further motivated us to investigate the feasibility of enhancing drug delivery through establishing LYTAC-drug conjugate(LYTAC-DC). To prepare LYTAC-DC, we introduced a cysteine residue at the C-terminus of LZ-8-7D12, which was successively conjugated with vcMMAE (a cytotoxic small molecule containing a maleimide group) via an addition reaction (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The construct of LZ-8-7D12-MMAE efficiently binds to EGFR and LZ-8 receptors, entering lysosomes through LZ-8 receptor-mediated endocytosis to degrade EGFR. Concurrently, the protein degradation releases MMAE, inducing apoptosis by disrupting microtubule assembly. MTT assays show that both LZ-8-7D12-MMAE and LZ-8-MMAE effectively induced cell death of A431 cells that express high level of EGFR, but exhibited lower activity in HeLa cells (with moderate EGFR expression) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). It is noteworthy that LZ-8 itself, not just LZ-8-MMAE or LZ-8-7D12-MMAE, also showed significant cell toxicity toward human embryonic lung MRC-5 cells (EGFR-positive), suggesting its potential off-target effect (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek). Since LZ-8 binds to multiple receptors including EGFR and induces their degradation, we investigated whether LZ-8-KN035 could cause EGFR degradation in MDA-MB-231 and HeLa cells. Western blot results confirmed that LZ-8-KN035 significantly induced EGFR degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el), indicating that the off-target effect of LZ-8 was mediated by its receptor. Similarly, LZ-8-11A4 induced strong EGFR degradation in SKBR3 cells, further supporting the conclusion that LZ-8 induces off-target effect on protein degradation, which limited its utility as a LYTAC-DC tool (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003em).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLZ-8, as a LYTAC-DC tool, induces lysosomal dysfunction\u003c/h3\u003e\n\u003cp\u003eAfter observing the high efficiency but significant off-target effects of LZ-8, we further investigated the impact of its persistent high endocytic flux on lysosomal function. Previous studies suggest that sustained high endocytic flux may impair lysosomal function\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, potentially reducing the efficiency of lysosome-dependent targeted protein degradation. Utilizing the lysosomal function probe DQ-BSA-Red (where lysosomal function correlates with red fluorescence intensity)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, confocal experiments revealed that prolonged LZ-8 treatment significantly reduced lysosomal function (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Consistently, ubiquitinated substrates showed dose-dependent accumulation in LZ-8/MG132 co-treated cells, indicating impaired lysosomal degradation(Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the molecular mechanism of the LZ-8-induced lysosome toxicity, we systematically analyzed key regulators of the endolysosomal pathway. Western blot exhibited that LZ-8 treatment specifically downregulated lysosomal membrane protein LAMP1, lysosomal enzyme transport receptor IGF2R, and transcription regulator TFEB, while early/late endosome markers remained unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). TFEB is a transcriptional regulator critical for lysosomal biogenesis and requires nuclear translocation for activation\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. We examined the effect of LZ-8 on TFEB nuclear transport in TFEB-GFP-overexpressing cells. Confocal results showed that LZ-8 did not significantly affect TFEB nuclear translocation (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), indicating that LZ-8 does not interfere with TFEB-mediated lysosomal biogenesis. LAMP1 and IGF2R are essential for lysosomal function: LAMP1 maintains lysosomal membrane stability, while IGF2R transports mannose-6-phosphate-tagged lysosomal enzyme precursors (e.g., CTSB) into lysosomes for maturation\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Immunostaining revealed that LZ-8 significantly altered the intracellular distribution of LAMP1 and IGF2R, where LAMP1 was changed from a diffuse pattern to perinuclear aggregation, while IGF2R was translocated from perinuclear regions to the cell membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Flow cytometry further confirmed LZ-8-induced downregulation of membrane LAMP1 and upregulation of membrane IGF2R (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-e). Time-dependent immunofluorescence also showed IGF2R membrane redistribution (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). It should be noted that a drug-induced IGF2R membrane redistribution has not been reported previously. Similar LZ-8 effects on LAMP1 and IGF2R downregulation were also observed in NCI-H226 and SMMC-7721 cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg), indicating a common mechanism of LZ-8 on lysosome toxicity, with reversibility in HeLa cells (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eGiven the critical roles of LAMP1 and IGF2R in lysosomal function, we further investigated the mechanism underlying their LZ-8-induced downregulation. qPCR and transcriptomic analyses showed that LZ-8 did not transcriptionally downregulate IGF2R or LAMP1 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). Additionally, transcriptomic data revealed no significant downregulation of lysosome-related genes, further supporting the conclusion that LZ-8 does not affect TFEB function (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej). Cycloheximide (CHX) inhibition experiments demonstrated that LZ-8 treatment reduced LAMP1 and IGF2R half-lives, suggesting post-translational regulation (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek-l). Pretreatment with lysosome inhibitor BafA1, but not proteasome inhibitor MG132, inhibited LZ-8-mediated LAMP1 downregulation, confirming that LZ-8 reduces LAMP1 via lysosomal degradation (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003em). Surprisingly, LZ-8-induced IGF2R downregulation was not inhibited by either inhibitor. To find out the underlying mechanism of IGF2R\u0026rsquo;s membrane redistribution, we hypothesized that IGF2R downregulation might occur via exocytosis, therefore examined the transporters of VPS35, SNX5, and SNX3 that regulate IGF2R intracellular trafficking. WB experiments showed no significant changes in expression of any of the transporters (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003en), suggesting that LZ-8 does not alter IGF2R-associated trafficking machinery. Given IGF2R\u0026rsquo;s role in lysosomal enzyme maturation, we speculated that LZ-8 might impair the maturation of enzymes like cathepsin B (CTSB). WB confirmed that LZ-8 induced time-dependent downregulation of mature CTSB (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), linking IGF2R dysregulation to impaired lysosomal enzyme maturation and dysfunction. Lysosomal function depends on acidic intra-lysosomal pH\u003csup\u003e30\u003c/sup\u003e. We hypothesized that LZ-8\u0026rsquo;s persistent endocytic flux might neutralize lysosomal acidity. Lysotracker (indicating lysosomal quantity) and LysoSensor (indicating lysosomal acidity) assays showed that LZ-8 treatment significantly increased Lysotracker intensity (but not LysoSensor) over 12\u0026ndash;24 hours, suggesting lysosomal pH elevation (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eo-q). Lysosomal dysfunction can block autophagic flux. WB of autophagy markers LC3-II/p62 and LC3B-GFP-mCherry overexpression confirmed impaired clearance, resulting in autophagosome accumulation in LZ-8-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003er)\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. These multi-dimensional findings demonstrate that LZ-8 disrupts LAMP1/IGF2R-mediated lysosomal acidification and enzyme maturation, ultimately causing lysosomal dysfunction (mechanistic summary in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg).\u003c/p\u003e\n\u003ch3\u003eY84 is the key residue of LZ-8 as Multibody\u003c/h3\u003e\n\u003cp\u003eTo elucidate the interaction mechanisms of LZ-8 with potential target proteins and optimize its utility as a LYTAC tool, we systematically investigated its receptor-binding spectrum and critical functional residues for receptor binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eUsing EGFP-labeled pull-down combined with LC/MS screening of HeLa cell membrane protein interactomes (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), LZ-8 was found to primarily bind to cell surface receptors L1CAM and EGFR (screening thresholds: protein abundance\u0026thinsp;\u0026gt;\u0026thinsp;1\u0026times;10^10, Log2 enrichment\u0026thinsp;\u0026gt;\u0026thinsp;2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-c). Further validation using an ELISA library comprising 11 membrane protein receptors confirmed the high affinity of LZ-8-EGFP (EC50\u0026thinsp;\u0026lt;\u0026thinsp;10 nM) to α5β1 integrin, c-MET, EGFR, L1CAM, HER3, and PD1, in which L1CAM, PD1, and α5β1 were newly identified as high-affinity receptors of LZ-8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb)\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. To verify the ELISA results further, the credibility of the ELISA was confirmed by further BLI affinity assays and cellular colocalization experiments, which were performed using selected targets: EGFR with high affinity and PD-L1 with low affinity (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-d). We found that dimeric LZ-8 induced HeLa cells aggregation, whereas monomeric 7D12 did not, indicating that LZ-8 engages membrane receptors via monomeric binding (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-f). Although earlier studies proposed that LZ-8 interacts with EGFR primarily through residue K41\u003csup\u003e18\u003c/sup\u003e, our molecular docking predicted that Y84 residue in the LZ-8 monomer forms a critical hydrogen bond network with the receptors (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). The mutant of Y84A (LZ-8-2.3) exhibited\u0026thinsp;\u0026gt;\u0026thinsp;95% reduction in binding efficiency to HeLa cells, as well as SMMC-7721, H226, and MDA-MB-231 cells, in flow cytometry assays, whereas the K41A mutant (previously reported) showed minor impact on HeLa cell affinity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh-j). AlphaFold2.3 structural predictions indicated that the Y84A mutation does not alter LZ-8\u0026rsquo;s overall conformation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef); while MTT assays showed that Y84A mutation significantly reduced its tumor cell inhibitory activity and normal cell (MRC-5) toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ek). LZ-8\u0026rsquo;s crystal structure revealed that the phenolic hydroxyl group of the Y84 side chain is solvent-exposed and protrudes from the molecular surface (Extended Data Fig.\u0026nbsp;6a). Intriguingly, the monomeric structure of LZ-8 resembles that of nanobodies, prompting us to propose a simplified model of LZ-8-receptor interactions (Extended Data Fig.\u0026nbsp;6b-d). ELISA further demonstrated that the Y84A mutant barely retained its affinity to the six high-affinity receptors identified by wild-type LZ-8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTreatment with the Y84A mutant (LZ-8-2.3) did not downregulate lysosomal functional markers IGF2R and LAMP1 in HeLa cells, with similar observations in four additional cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei, Extended Data Fig.\u0026nbsp;6e-f). Furthermore, no abnormal accumulation of autophagy flux-related proteins (LC3-II and p62) or changes in lysosomal quantity (LysoTracker) were observed (Extended Data Fig.\u0026nbsp;6g-h), confirming that the Y84A mutation abolished the toxic effect of LZ-8 on lysosomes. In a HeLa xenograft model, wild-type LZ-8 (1 mg/kg) significantly suppressed tumor growth, whereas Y84A (1 mg/kg) showed no efficacy (Extended Data Fig.\u0026nbsp;6i-l). Nevertheless, histological examinations failed to detect pathological signs, and no significant changes in body weight upon LZ-8 group treatment (Extended Data Fig.\u0026nbsp;6m-n). These results indicate that although LZ-8 engages multiple receptors, including EGFR, with high affinity, but had no pathological consequences. In comparison, Y84 residue is central to its receptor-binding activity. The Y84A mutant retains partial receptor affinity while eliminating lysosomal toxicity and cytotoxicity.\u003c/p\u003e\n\u003ch3\u003eThe Y84A-Multibody enables lysosome-safe and target-specific LYTAC-DC\u003c/h3\u003e\n\u003cp\u003eBased on the low toxicity to lysosomal function, we moved forward to validate the feasibility of Y84A (LZ-8-2.3) as a lysosome-targeting chimera (LYTAC) tool, as it minimal off-target effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eTo demonstrate modular degradation capabilities, we conjugated the model protein Nb\u003csup\u003eEGFP\u003c/sup\u003e with LZ-8-2.3 and confirmed that LZ-8-2.3-Nb\u003csup\u003eEGFP\u003c/sup\u003e efficiently mediated lysosome-dependent degradation of extracellular proteins. This degradation was blocked by lysosomal inhibitor BafA1, validating its lysosome-specific function (Extended Data Fig.\u0026nbsp;7a). In EGFR-positive HeLa cells, LZ-8-2.3 alone did not induce significant EGFR degradation, demonstrating its low off-target activity (Extended Data Fig.\u0026nbsp;7b). The EGFR-targeting LYTAC construct (LZ-8-2.3-7D12) achieved robust EGFR degradation in HeLa and A431 cells. Covalent conjugation of LZ-8-2.3 with 7D12 showed significantly higher efficiency compared to non-covalent mixtures (LZ-8-2.3\u0026thinsp;+\u0026thinsp;7D12) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, Extended Data Fig.\u0026nbsp;7c). Dose-response analyses revealed a differential effect of LZ-8-2.3-7D12 promoted degradation of EGFR, with a DC\u003csub\u003e50\u003c/sub\u003e of 55.3 nM (D\u003csub\u003emax\u003c/sub\u003e = 55.46%) in HeLa cells and DC\u003csub\u003e50\u003c/sub\u003e of 11.87 nM (D\u003csub\u003emax\u003c/sub\u003e = 70.07%) in A431 cells. Its efficacy in HeLa cells significantly surpassed that of the positive control IGF2-7D12 (Extended Data Fig.\u0026nbsp;7d)\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Time-course immunoblotting demonstrated sustained EGFR degradation by LZ-8-2.3-7D12 within 6 hours (Extended Data Fig.\u0026nbsp;7e). To visualize EGFR internalization and degradation, we performed whole-cell immunofluorescence imaging and membrane-specific flow cytometry. Both methods confirmed that LZ-8-2.3-7D12 significantly induced EGFR internalization and degradation, with efficacy exceeding that of IGF2-7D12 (Extended Data Fig.\u0026nbsp;7f-g). Pre-treatment with lysosomal inhibitor BafA1 ceased EGFR degradation in both HeLa and A431 cells, further validating lysosome-dependent mechanisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, Extended Data Fig.\u0026nbsp;7h).\u003c/p\u003e \u003cp\u003eTo assess lysosomal targeting efficiency, we created construct of EGFP fused to LZ-8-2.3, LZ-8-2.3-7D12, respectively. Confocal microscopy revealed that LZ-8-2.3 exhibited reduced fluorescence intensity compared to wild-type LZ-8, indicating lower lysosomal burden and minimized risk of lysosomal dysfunction (Extended Data Fig.\u0026nbsp;7i). Additionally, LZ-8-2.3-7D12 showed time-dependent co-localization with lysosomal probe LysoTracker, confirming efficient lysosomal trafficking (Extended Data Fig.\u0026nbsp;7i). To evaluate efficacy in complex tumor models, we cultured 3D HeLa cell spheroids (\u0026gt;\u0026thinsp;200 \u0026micro;m in diameter) (Extended Data Fig.\u0026nbsp;7j). Dose-response experiments demonstrated that LZ-8-2.3-7D12 efficiently degraded EGFR in these spheroids, highlighting its potential for penetrating solid tumors (Extended Data Fig.\u0026nbsp;7k). Pre-treatment with LZ-8-2.3 or 7D12 partially reduced EGFR degradation, indicating that the activity of LZ-8-2.3-7D12 depends on both components (Extended Data Fig.\u0026nbsp;7l).To ensure lysosomal safety, we compared the effects of LZ-8-2.3-7D12 with wild-type LZ-8-7D12 and the positive control IGF2-7D12. While wild-type LZ-8-7D12 induced significant downregulation of IGF2R alongside EGFR degradation, LZ-8-2.3-7D12 degraded EGFR without affecting IGF2R levels, confirming its lysosomal safety (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, Extended Data Fig.\u0026nbsp;7m).\u003c/p\u003e \u003cp\u003eTo further validate whether the platform is compatible with other targets, we constructed a PD-L1-targeting LYTAC by fusing LZ-8-2.3 with KN035. Western blotting results indicated that LZ-8-2.3-KN035 effectively degraded PD-L1 in MDA-MB-231 cells (Extended Data Fig.\u0026nbsp;8a). Dose-dependent degradation assays showed that LZ-8-2.3-KN035 outperformed the control IGF2-KN035 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, Extended Data Fig.\u0026nbsp;8b). Immunoblotting confirmed that LZ-8-2.3-KN035 selectively degrades PD-L1 without non-specific degradation of wild-type LZ-8 receptors (e.g., EGFR) or downregulation of lysosomal markers like IGF2R (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Flow cytometry further validated PD-L1-specific degradation and minimal off-target effects on EGFR (Extended Data Fig.\u0026nbsp;8c-d). Co-localization with LysoTracker confirmed that LZ-8-2.3-KN035 retains efficient lysosomal targeting (Extended Data Fig.\u0026nbsp;8e). Successful HER2 degradation using LZ-8-2.3-11A4 further supports the versatility of LZ-8-2.3 as a broad-spectrum LYTAC tool (Extended Data Fig.\u0026nbsp;8f-h). Given the co-upregulation of multiple membrane proteins in cancer progression, we engineered dual-target LYTACs\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The construct 7D12-LZ-8-2.3-KN035 successfully and efficiently co-degraded EGFR and PD-L1 in MDA-MB-231 cells(Extended Data Fig.\u0026nbsp;8i-k). Similarly, the EGFR/HER2 dual-target tool (7D12-LZ-8-2.3-11A4) co-degraded both receptors (Extended Data Fig.\u0026nbsp;8l-m), demonstrating the platform\u0026rsquo;s capability for multiplexed protein intervention.\u003c/p\u003e \u003cp\u003eTo develop LYTAC-DCs, we introduced a reactive cysteine residue at the C-terminus of LZ-8-2.3 and conjugated it with maleimide-functionalized MMAE via addition reactions. Mass spectrometry confirmed the successful synthesis of LZ-8-2.3-7D12-MMAE and control conjugates (Extended Data Fig.\u0026nbsp;9a). The LYTAC-DC platform leverages LZ-8-2.3\u0026rsquo;s lysosomal targeting to internalize and degrade membrane proteins (e.g., EGFR, PD-L1) while releasing MMAE to disrupt microtubule assembly and induce apoptosis. The immunoblot results showed that MMAE conjugation did not affect the ability of LZ-8-2.3-7D12 to induce EGFR degradation, laying the foundation for its subsequent efficient delivery of MMAE(Extended Data Fig.\u0026nbsp;9b). MTT assays demonstrated that LZ-8-2.3-7D12-MMAE potently inhibited proliferation of EGFR-high A431 tumor cells, with efficacy significantly exceeding LZ-8-2.3-MMAE or 7D12-MMAE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). Cytotoxicity assays in other cells confirmed that LYTAC-DC activity correlates with target expression, which largely minimized off-target toxicity (Extended Data Fig.\u0026nbsp;9c-f).\u003c/p\u003e \u003cp\u003eRecent studies have demonstrated that EGFR protein degradation holds promise for treating clinically relevant EGFR mutation-driven cancers such as non-small cell lung cancer (NSCLC) and overcoming TKI resistance\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. We hypothesized that simultaneous EGFR degradation and small-molecule drug delivery could significantly suppress NSCLC progression. To test this, we selected the NSCLC cell line H1975, which carries the L858R/T790M double mutation in EGFR and is sensitive to the third-generation TKI inhibitor osimertinib. Through in vitro dose-gradient culture with osimertinib, we successfully generated osimertinib-resistant H1975 cells (H1975/GR; Extended Data Fig.\u0026nbsp;9g-h). Dose-gradient experiments with LZ-8-2.3-7D12 showed efficient EGFR degradation in both 2D and 3D cultures of H1975 and H1975/GR cells (Extended Data Fig.\u0026nbsp;9i-j). Further cell viability assays revealed that LZ-8-2.3-7D12 effectively inhibited the growth of H1975/GR cells compared to osimertinib (Extended Data Fig.\u0026nbsp;9k). However, at 500 nM, the inhibitory efficiency of LZ-8-2.3-7D12 against osimertinib-resistant H1975/GR cells remained significantly lower than that reported for TransTAC tools\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, potentially due to limited efficacy and incomplete EGFR degradation by LZ-8-2.3-7D12 in H1975/GR cells. Conjugation with the cytotoxic small molecule MMAE markedly enhanced the ability of LZ-8-2.3-7D12-MMAE to suppress H1975/GR cell viability, achieving nearly complete killing of osimertinib-resistant lung cancer cells at 500 nM, whereas LZ-8-2.3-7D12 or osimertinib at this concentration showed limited activity at this concentration(Extended Data Fig.\u0026nbsp;9l).\u003c/p\u003e \u003cp\u003eIn recent years, patient-derived tumor organoids have been increasingly used to evaluate antitumor drug activity, better reflecting \u003cem\u003ein vivo\u003c/em\u003e efficacy\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. We further assessed the effect of LZ-8-2.3-7D12-MMAE on targeting LD1-0025-411169 cells, a patient-derived lung cancer cell line harboring triple EGFR mutations (Del19, T790M, and C797S), which confer resistance to all currently approved third-generation TKIs\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. We first established a patient-derived xenograft (PDX) model using LD1-0025-411169 cells with confirmed TKI resistance and validated their osimertinib resistance \u003cem\u003ein vivo\u003c/em\u003e (Extended Data Fig.\u0026nbsp;9m). The PDX-derived organoids (PDXO) cultured in ultra-low attachment 96-well plates exhibited robust proliferation, confirming their suitability for drug efficacy evaluation(Extended Data Fig.\u0026nbsp;9n). As expected, LZ-8-2.3-7D12-MMAE significantly inhibited the viability of LD1-0025-411169 organoids at low concentrations, outperforming both LZ-8-2.3-MMAE and 7D12-MMAE (Extended Data Fig.\u0026nbsp;9o). These results further demonstrate the dual mechanism of overcoming resistance through lysosome-targeted protein degradation and enhancing efficacy via synergistic cytotoxic payload delivery.\u003c/p\u003e \u003cp\u003eTo explore the versatility of the LYTAC-DC platform, we extended our targeting strategy to other types of small-molecule drugs. Doxorubicin (DOX), a chemotherapeutic anthracycline agent, is known to exhibit significantly lower toxicity compared to MMAE. Additionally, low-dose DOX can induce immunogenic cell death (ICD), leading to the release of damage-associated molecular patterns (DAMPs) and tumor-associated antigens, thereby potentiating immunotherapy\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. We selected DOXO-EMCH (a maleimide-containing DOX prodrug) as an alternative to vcMMAE and conjugated it with LZ-8-2.3-7D12-Cys to generate a novel conjugate, LZ-8-2.3-7D12-DOX (Extended Data Fig.\u0026nbsp;9p)\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Similar to the MMAE conjugate, LZ-8-2.3-7D12-DOX binds membrane protein receptors, undergoes internalization and lysosomal degradation, and releases DOX to induce CRT translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). Consistent with the expectations, flow cytometry results showed that LZ-8-2.3-7D12-DOX effectively induced CRT exposure and significantly enhanced antitumor activity of DOX in both A431 and HeLa cells compared to LZ-8-2.3-7D12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej, Extended Data Fig.\u0026nbsp;9q-r).\u003c/p\u003e \u003cp\u003eTo evaluate the antitumor efficacy of the LZ-8-2.3\u0026ndash;based LYTAC-DC platform \u003cem\u003ein vivo\u003c/em\u003e, we first established a subcutaneous xenograft model using A431 cells, an aggressive skin cancer line known for rapid progression and poor clinical control. Based on the potent cytotoxicity of LZ-8-2.3-7D12-MMAE observed \u003cem\u003ein vitro\u003c/em\u003e, we administered the conjugate intravenously at 3 mg/kg every two days for four doses (Extended Data Fig.\u0026nbsp;10a). Treatment with LZ-8-2.3-7D12-MMAE significantly suppressed tumor growth compared with LZ-8-2.3-7D12 alone, as measured by both tumor volume and weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh, Extended Data Fig.\u0026nbsp;10b\u0026ndash;c). Notably, LZ-8-2.3-7D12\u0026mdash;a degradation-only LYTAC\u0026mdash;showed limited efficacy against highly malignant A431 tumors, consistent with our \u003cem\u003ein vitro\u003c/em\u003e viability data (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). No significant body weight loss was observed, indicating a favorable safety profile in vivo (Extended Data Fig.\u0026nbsp;10d). Furthermore, TUNEL apoptosis staining, H\u0026amp;E histology and anti-EGFR immunohistochemistry revealed markedly increased apoptosis and EGFR degradation in tumors treated with LZ-8-2.3-7D12-MMAE compared to PBS or LZ-8-2.3-7D12 groups, suggesting improved prognostic potential (Extended Data Fig.\u0026nbsp;10e-f).\u003c/p\u003e \u003cp\u003eWe next asked whether LZ-8-2.3-7D12-MMAE could also exhibit efficacy in a triple-negative breast cancer model, MDA-MB-231, which showed limited response \u003cem\u003ein vitro\u003c/em\u003e. Mice bearing MDA-MB-231 xenografts received 1.5 mg/kg LZ-8-2.3-7D12-MMAE every three days for eight doses (Extended Data Fig.\u0026nbsp;10g). The conjugate significantly inhibited tumor growth relative to LZ-8-2.3-MMAE or 7D12-MMAE controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei, Extended Data Fig.\u0026nbsp;10h\u0026ndash;i). Treatment was well tolerated, with no notable changes in body weight or organ pathology (Extended Data Fig.\u0026nbsp;10j, k). Although lung metastases were observed\u0026mdash;consistent with the high metastatic potential of MDA-MB-231 cells\u0026mdash;conjugate treatment effectively suppressed pulmonary tumor growth (Extended Data Fig.\u0026nbsp;10i). Enhanced apoptosis by TUNEL and reduced proliferation by Ki67 staining further supported the therapeutic benefit of LZ-8-2.3-7D12-MMAE in this model.\u003c/p\u003e \u003cp\u003eTogether, these results demonstrate that LZ-8-2.3-7D12-MMAE exerts robust antitumor activity \u003cem\u003ein vivo\u003c/em\u003e against both highly sensitive and less responsive tumor types, with no overt systemic toxicity. These findings underscore the translational potential of the LYTAC-DC platform and support further development of LZ-8-2.3\u0026ndash;based drug conjugates for a broad range of therapeutic targets.\u003c/p\u003e\n\u003ch3\u003eAI-Assisted Evolution of Non-Chimeric LYTA-DCs\u003c/h3\u003e\n\u003cp\u003eFinally, we hypothesized that directed evolution could ameliorate the non-specific interaction of LZ-8 and enhance its selectivity towards a given receptor, aiming at targeted protein degradation and small-molecule drug delivery without requiring a chimera ligand (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Notably, LZ-8 possesses a canonical immunoglobulin-like domain at its C-terminus and shares high structural similarity with non-antibody scaffold proteins like monobody, which can be engineered to bind specific target protein receptors\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Compared to traditional antibodies or ligands, engineering LZ-8 into a novel non-antibody scaffold offers several advantages: its inherent multi-receptor binding capability, lack of disulfide bonds (facilitating correct folding and stability in the reductive cytosolic environment), relatively small molecular weight (~\u0026thinsp;14 kDa), and the multivalency conferred by its dimeric form. Therefore, developing LZ-8 as a novel non-antibody scaffold protein for LYTA-DC drug design holds substantial potential.\u003c/p\u003e \u003cp\u003eTo validate this concept, we selected EGFR as the target and employed AI-assisted protein directed evolution to enhance LZ-8's affinity for EGFR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). First, we used a molecular docking model of LZ-8 with receptor EGFR and identified 29 potential beneficial single-site mutations. The mutant proteins were dually validated using ELISA and flow cytometry assays. Unexpectedly, though N103S and I106Q demonstrated significant enhancement of EGFR binding in both assays, majority of the mutants displayed discrepancies between ELISA and flow cytometry analyses (Extended Data Fig.\u0026nbsp;11a-b). Subsequently, we utilized the sequence-structure protein language model ProSST model to combine the beneficial mutations of N103S and I106Q along with other mutants, generated 18 multi-site mutants\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. ELISA and flow cytometry analyses showed significant synergistic enhancement in specific interaction with EGFR, with no apparent epistatic effects (Extended Data Fig.\u0026nbsp;11c-d). Flow cytometry analysis of cell surface EGFR internalization revealed that the top 6\u0026ndash;7 selected multi-site mutants significantly enhanced EGFR internalization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, Extended Data Fig.\u0026nbsp;11e). Immunoblotting further confirmed that multi-site mutants significantly enhanced EGFR degradation efficiency (Extended Data Fig.\u0026nbsp;11f-g).Some of the multi-site mutants also exhibited differential affinities in Hela and Mada-mb-231 cells (Extended Data Fig.\u0026nbsp;11h). Cytotoxicity assays found increased toxicity of some mutants in comparison to WT LZ-8, potentially linked to mutation introduced non-specific binding (Extended Data Fig.\u0026nbsp;11i). Four multi-site mutants with low toxicity were selected for ELISA analysis. Results showed similar binding characteristics of all four protein mutants, which significantly enhanced affinity for EGFR, while retained certain binding capability to other receptors like HER3 and c-Met (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, Extended Data Fig.\u0026nbsp;11j). Nevertheless, we went on to establish four LZ-8 mutant-MMAE conjugates through the introduced C-termini cysteine Cell viability assays showed that multiple 11(N102S; I105Q)-MMAE conjugates exhibited higher cytotoxicity than the WT LZ-8-MMAE (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee)\u003c/p\u003e \u003cp\u003eNext, we focused on the Y84A mutant for further directed evolution. We introduced the Y84A mutation into the mutants of all 30 generated a new set of single-site mutants on the Y84A background. Surprisingly, the evolutionary trajectory on the Y84A background differed almost completely from that on the wild-type LZ-8, indicating that Y84 is critically involved in the evolutionary pathways (Extended Data Fig.\u0026nbsp;12a-b). Particularly, three mutants at the L9 position (e.g., L9G, L9N, L9A) almost completely abolished the EGFR affinity of the Y84A mutant, while these mutations had minimal impact on the EGFR affinity of wild-type LZ-8 (Extended Data Fig.\u0026nbsp;11a-b). Multi-target ELISA assays and broad receptor profiling via flow cytometry on Hela cells confirmed that the L9 mutations caused widespread inactivation of the Y84A mutant (Extended Data Fig.\u0026nbsp;12c-d). The LZ-8 crystal structure reveals that residue L9 is located internally within the dimerization arm, not directly involved in receptor interaction, but is crucial for dimerization\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Competitive ELISA showed that pre-saturation binding with wild-type LZ-8 almost completely blocked subsequent binding of Y84A, whereas pre-saturation with Y84A caused no significant steric hindrance to subsequent wild-type LZ-8 binding (Extended Data Fig.\u0026nbsp;12e). Therefore, we conclude that the high-affinity binding of LZ-8 to cell surface receptors primarily depends on the monomer containing Y84, while the lower-affinity binding of the Y84A mutant relies on dimerization. LZ-8 likely utilizes two parallel, non-interfering interaction modes with receptors: high-affinity monomeric binding mediated by Y84 and low-affinity dimeric binding mediated by L9-dependent dimerization, with prioritized monomeric binding.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study has established a unified and engineerable LYTAC-DC platform that synergistically integrates targeted protein degradation in lysosome with precision drug delivery by leveraging a novel class of binding proteins\u0026mdash;Multibodies. Using the identified fungal-derived protein LZ-8 as a prototype, we demonstrate that polyvalent, polyspecific targeting scaffolds can overcome fundamental limitations of conventional lysosome-targeting chimeras and antibody-drug conjugates, with regard to target specificity, internalization efficiency, and payload versatility. Through rational engineering, we mitigated off-target toxicity of the WT LZ-8 via Y84A mutation, yielding a high-fidelity LYTAC and LYTAC-DC system capable of degrading diverse cancer-related membrane proteins while concurrently delivering cytotoxic or immunogenic payloads. Moreover, AI-assisted evolution enabled the development of non-chimeric LYTA-DCs, further highlighting the modularity and adaptability of the Multibody concept. The molecular mechanisms of the LZ-8 binding to receptors are highlighted through combined double mutation of Y84 and L9, which provides valuable information for further rational design of specific mutibodies.\u003c/p\u003e \u003cp\u003eRecent studies, including the degrader-drug conjugates (DDCs)\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003eand the intratumoural vaccination chimera (iVAC)\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, alongside this work, collectively underscore the broad potential of exploiting the lysosomal pathway for synergistic \"degradation-plus\" therapies. Our Multibody-LYTAC-DC platform shares the core commonality of hijacking the endolysosomal pathway with these cutting-edge strategies, yet it is fundamentally distinguished by its implementation route and mode of functional integration. The DDC strategy enhances the delivery efficiency of traditional antibody-drug conjugates by recruiting specific endocytic receptors (e.g., LDLR), while iVAC combines target degradation with specific immune activation through covalent binding and antigen presentation. In contrast, this study develops a unified platform featuring multi-valent targeting, efficient degradation, payload delivery, and AI-assisted directed evolution from a structurally unique and engineerable fungal protein scaffold (LZ-8). Its core advantages lie in: First, the Multibody itself serves as a multifunctional \"engine,\" avoiding complex multi-component assembly and simplifying design. Second, a single point mutation (Y84A) is sufficient to abolish off-target toxicity while retaining efficient lysosomal targeting, demonstrating excellent tunability. Third, and most importantly, through AI-assisted evolution, we successfully converged degradation and targeting functions into a single, non-chimeric LYTA-DC molecule. This points towards a new direction for developing smaller, more stable, and more tumor-penetrant \"smart\" single-agent therapeutics. These properties not only make our platform suitable for \"degradation\u0026thinsp;+\u0026thinsp;chemotherapy\" enhanced killing but also establish a unique and flexible foundation for its future expansion into multi-dimensional synergistic therapies, such as \"degradation\u0026thinsp;+\u0026thinsp;immunomodulation.\"\u003c/p\u003e \u003cp\u003eIn summary, the Multibody platform has demonstrated unique properties and advantages in comparison to LYTAC-DC platform. To advance the concept towards clinical applications, future efforts will focus on optimizing Multibody specificity and reducing immunogenicity, broadening the repertoire of targetable receptors, and evaluating combination therapies across heterogeneous cancers diseases. The integration of AI-assisted design and directed protein evolution will accelerate the development of next-generation lysosome-targeting therapeutics, potentially enabling personalized and adaptive treatment strategies. The novel concept of Multibody is expected to add a new dimension to proceed the progress in specified and personalized therapy for diseases, as the transformative tool is far from restricted to cancer treatment, but can be adapted to treatment of protein-accumulation diseases including the A-beta-deposition induced Alzheimers disease.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003eAnimal Ethics Statement All animal experiments described in this study were conducted in accordance with the relevant guidelines and regulations for the care and use of laboratory animals. The experimental protocols involving non-human vertebrates were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of East China University of Science and Technology. All efforts were made to minimize animal suffering and to reduce the number of animals used.\u003c/p\u003e\u003ch2\u003eSupporting Information\u003c/h2\u003e\n\u003cp\u003eFurther information on research design is available in the Reporting Summary linked to this article.\u003c/p\u003e\n\u003ch2\u003eDeclaration of Interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eJ.Z., L.H., F.T., and L.Z. conceptualized and supervised the project. J.Z., F.W., P.T., L.H., F.T. and L.Z. provided funding support. L.C. designed and performed the main experiments. S.Y., Y.G., Y.W., S.C. and D.L. participated in the experiments. Q.S. performed in the Protein small molecule drug coupling experiment. P.T. and J.H. performed in the Mutant directed evolution design. X.T. performed \u003cem\u003ein vivo\u003c/em\u003e experiments on mice. L.C., X.T., J.Z., J.L., W.W. and L.O. wrote the manuscript. J.L., L.F., W.H., F.W., J.Z., Y.Z., F.T. and L.Z. edited the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32121005 and 32327801 to L.Z.,32571667 to J.Z., and 22422705 to F.T.), the National Key Research and Development Program of China (2020YFA0907800 to L.Z., 2024YFA0917603 to L.H. and 2020YFA0907200 to J.Z.), Shanghai Municipal Science and Technology Major Project (to L.H.), Shanghai Science and Technology Commission (24HC2820200 to L.Z., 24ZR1417000 to J.Z.), the Computational Biology Key Program of Shanghai Science and Technology Commission (23JS1400600 to L.H.), Shanghai Municipal Education Commission (2024AIZD015 to L.H.), Shanghai Jiao Tong University Scientific and Technological Innovation Funds (21X010200843 to L.H.), Open Project Funding of the State Key Laboratory of Bioreactor Engineering, the 111 Project (B18022 to L.Z.), Science and Technology Innovation Key R\u0026amp;D Program of Chongqing (CSTB2022TIADSTX0017 to L.H., CSTB2024TIAD-STX0032 to P.T.), the Fundamental Research Funds for the Central Universities (L.Z.).and also by Zhejiang Fonow Medicine Co., Ltd. (Grant No. F100-42106L to F.W.). We are grateful to Tsingke Biotechnology Co., Ltd. for technical support in mRNA transcriptome sequencing, Shenzhen Kangti Life Technology Co., Ltd. for assistance with BLI-based affinity measurements, and Shanghai OE Biotech Co., Ltd. for protein gel LC/MS analysis. We are grateful to Professor Huizhan Zhang (East China University of Science and Technology) for constructive discussions regarding this study. We are grateful to Siwu Guo (Shanghai Beautiful Life Medical Technology Co., Ltd.), Yinan Wang (Shanghai Beautiful Life Medical Technology Co., Ltd.) and Zhenwei Wang (Shanghai Beautiful Life Medical Technology Co., Ltd.) for technical support in vivo experiments on mice .\u003c/p\u003e\n\u003ch2\u003eData Availability Statement\u003c/h2\u003e\n\u003cp\u003eAll data reported in this paper will be shared by the lead contact upon request.\u003c/p\u003e\n\u003ch2\u003eCode availability\u003c/h2\u003e\n\u003cp\u003eThe code of the ProSST used in this work can be found in https://github.com/ai4protein/ProSST.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCullen PJ, Steinberg F (2018) To degrade or not to degrade: mechanisms and significance of endocytic recycling. 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Nature. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41586-025-09903-1\u003c/span\u003e\u003cspan address=\"10.1038/s41586-025-09903-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Multibody, LZ-8, LYTAC-DC, LYTA-DC, Targeted protein degradation, Antibody-drug conjugate, Lysosome targeting","lastPublishedDoi":"10.21203/rs.3.rs-8651044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8651044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCurrent lysosome-targeting chimeras (LYTACs) and antibody-drug conjugates (ADCs) face inherent limitations, including reliance on monospecific binders and target-dependent internalization efficiency. Here, we report a versatile therapeutic platform that overcomes these constraints by engineering a novel class of multivalent, multispecific binding proteins, termed \u003cb\u003eMultibodies\u003c/b\u003e. Using the fungal immunomodulatory protein LZ-8 as a scaffold, we developed a lysosome-targeting chimera-drug conjugates (LYTAC-DCs) system capable of mediating the degradation of multiple membrane proteins while enabling site-specific drug release. While wild-type LZ-8 demonstrated potent lysosomal targeting, it induced significant off-target toxicity and lysosomal dysfunction. Through rational protein engineering, we identified a key residue (Y84) responsible for promiscuous receptor binding and generated an optimized variant, LZ-8-2.3 (Y84A), which eliminated toxicity while preserving efficient lysosomal trafficking. The resulting LYTAC-DC platform mediated high-fidelity degradation of EGFR, PD-L1, and HER2 across diverse cancer models, concurrently delivering cytotoxic (e.g., MMAE) or immunogenic (e.g., doxorubicin) payloads. Efficacy was validated in patient-derived organoids and murine xenografts, including against osimertinib-resistant lung cancer. Furthermore, AI-assisted directed evolution enabled the development of non-chimeric lysosome-targeting drug conjugates (LYTA-DCs), highlighting the modularity and engineerability of the Multibody scaffold. Our work establishes a unified and programmable strategy for targeted protein degradation and drug delivery, significantly expanding the therapeutic landscape beyond conventional LYTAC and ADC technologies.\u003c/p\u003e","manuscriptTitle":"Multibody-Enabled Lysosome-Targeting Drug Conjugates for Target Protein Degradation and Combination Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-18 16:58:32","doi":"10.21203/rs.3.rs-8651044/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"eb0ab448-1e7c-4186-9a54-41b116e30127","owner":[],"postedDate":"February 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61898739,"name":"Health sciences/Medical research/Preclinical research"},{"id":61898740,"name":"Biological sciences/Cancer"}],"tags":[],"updatedAt":"2026-02-18T16:58:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-18 16:58:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8651044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8651044","identity":"rs-8651044","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}