A Novel Bispecific Antibody Targeting Frizzled7 and PD-L1 Reverses Immunotherapy Resistance in Non-Small Cell Lung Cancer by Reprogramming the Tumor Microenvironment

A Novel Bispecific Antibody Targeting Frizzled7 and PD-L1 Reverses Immunotherapy Resistance in Non-Small Cell Lung Cancer by Reprogramming the Tumor Microenvironment | 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 A Novel Bispecific Antibody Targeting Frizzled7 and PD-L1 Reverses Immunotherapy Resistance in Non-Small Cell Lung Cancer by Reprogramming the Tumor Microenvironment Shuyang Mao, Xiaofan Zhang, Zitong Wang, Pan Zhou, Zixuan Wang, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9337927/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Immunotherapy efficacy in non-small cell lung cancer (NSCLC) remains limited, largely due to aberrant activation of the Wnt/β-Catenin pathway. However, the precise role of its receptor Frizzled7 (Fzd7) in immune checkpoint inhibitor (ICI)-resistance remains unexplored. Here, we elucidate the role of Fzd7 in shaping the immunosuppressive tumor microenvironment (TME) and develop a novel bispecific antibody (named SHH-YN-Bi) that co-targets Fzd7 and programmed cell death ligand 1 (PD-L1) to overcome ICI-resistance. This study reveals that Fzd7 is remarkably enriched in human NSCLC tissues and correlates with PD-L1 expression and immune exclusion. Targeting Fzd7 sensitizes NSCLC to Atezolizumab in multiple orthotopic NSCLC models. SHH-YN-Bi was generated via “Knob-into-Hole”, demonstrating effective dual binding to Fzd7 and PD-L1 and superior tumor targeting compared with Atezolizumab. We show that SHH-YN-Bi concurrently recruits and activates CD8 + T cells in a basic leucine zipper transcriptional factor ATF-like 3 (Batf3) + conventional type 1 dendritic cells (cDC1s)-dependent manner to reprogram the TME, yielding robust antitumor activity in ICI-resistant NSCLC orthotopic models. Furthermore, scRNA-seq analysis uncovered potent correlation between Fzd7 and cancer-associated fibroblast (CAF)-mediated extracellular matrix (ECM) remodeling, and SHH-YN-Bi treatment induced a phenotypic switch of CAFs from a highly oncogenic to a normal state, associated with elevated CD8 + T-cell infiltration. SHH-YN-Bi overcomes ICI-resistance in NSCLC by simultaneously blocking the oncogenic Wnt3a/Fzd7/β-Catenin signaling and PD-1/PD-L1 axis. Our findings highlight the critical role of Fzd7 in immunosuppressive TME, particularly in CAF-mediated ECM remodeling, and offer a innovative approach for NSCLC treat. Biological sciences/Cancer Biological sciences/Immunology Health sciences/Oncology NSCLC Frizzled7 PD-L1 Wnt/β-Catenin signaling pathway Tumor microenvironment Immunotherapy Cancer-associated fibroblast Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Since the U.S. Food and Drug Administration (FDA) first approved Nivolumab and Pembrolizumab for the treatment of advanced NSCLC, ICI therapy has progressively transformed the treatment paradigm of NSCLC from chemotherapy to immunotherapy [ 1 , 2 ]. However, a substantial proportion of patients still do not derive clinical benefit [ 1 ]. Among these patients, those with “hot tumors” who initially respond to ICIs may develop acquired resistance due to the upregulation of alternative immune checkpoints (ICPs), loss of tumor antigen expression or presentation, induction of T-cell exhaustion, or metabolic dysregulation [ 3 , 4 ]. In contrast, the majority of NSCLCs are classified as “cold tumors,” characterized by low tumor mutational burden (TMB), low PD-L1 expression, and limited immune cell infiltration. These tumors exhibit primary resistance to ICIs [ 1 , 5 ]. These observations highlight that immunotherapy resistance represents a major obstacle limiting the clinical benefit for NSCLC patients. Given the significant impact of non-immune components in the TME on the response to ICIs, tumors with high TMB and intermediate/high PD-L1 expression still paradoxically display abundant infiltration of CAFs, regulatory T cells (Tregs), and tumor-associated macrophages (TAMs). These factors contribute to an immunosuppressive TME and lead to CD8 + T-cell dysfunction [ 1 , 6 – 8 ]. To overcome ICI-resistance, anti-PD-(L)1 antibodies are commonly combined with other immunostimulatory or immunogenic therapeutic modalities [ 9 ]. Therefore, simultaneous targeting of the TME and ICPs is a promising approach to reverse ICI resistance. The Wnt/β-Catenin pathway is an oncogenic pathway with immunomodulatory properties [ 10 – 15 ], modulating PD-L1 expression and fostering a non-T cell-inflamed TME [ 11 , 16 – 19 ]. Fzd7 is a key receptor in the Wnt/β-Catenin pathway, exhibits upregulated expression in various malignancies, including NSCLC. Transcriptomic analysis in this study revealed that high Fzd7 expression is linked to stronger Wnt/β-Catenin signaling and weaker immune infiltration in NSCLC (Figure. 1A-C). SHH002-hu1, a full-length humanized antibody targeting Fzd7 developed by us previously, has demonstrated significant anti-tumor activity in triple-negative breast cancer (TNBC) and NSCLC [ 20 , 21 ]. Furthermore, an NK-cell engager (NKCE) derived from SHH002-hu1, designated SHH002-hu1-MICA, has shown significant anti-tumor effects against TNBC cancer stem cells (TNBCSCs), highlighting its potential as a therapeutic agent capable of both inhibiting oncogenic signaling and enhancing immune cell infiltration [ 22 ]. Given that Fzd7 represents a promising cooperative target for tumor-targeted therapy and immunotherapy, the precise mechanism by which Fzd7 contributes to the immunosuppressive TME remains to be elucidated. This study employed fluorescence in situ hybridization (FISH) and Tyramide Signal Amplification-based multiplex fluorescence immunohistochemistry (TSA mIHC) to analyze human NSCLC tissue microarrays (TMAs), confirming that Fzd7 is remarkably enriched in NSCLC tissues and correlated with PD-L1 expression. The combination treatment of SHH002-hu1 and Atezolizumab showed significant tumor suppression in multiple orthotopic models. These findings indicate potential synergistic anti-tumor effects between blocking Fzd7 and PD-L1. Herein, we further developed a novel bispecific antibody (BsAb), SHH-YN-Bi, that simultaneously targets Fzd7 and PD-L1, demonstrating effective dual binding to Fzd7 and PD-L1 and superior tumor targeting over Atezolizumab. Subsequent investigations revealed that SHH-YN-Bi exerted potent anti-tumor effect through blocking oncogenic Wnt3a/Fzd7/β-Catenin signaling and PD-1/PD-L1 axis. Moreover, scRNA-seq analysis indicated that Fzd7 is highly correlated with ECM remodeling in CAFs, and SHH-YN-Bi treatment induced a phenotypic switch of CAFs from a highly oncogenic to a normal state, associated with elevated CD8 + T-cell infiltration. Our study elucidates the role of Fzd7 in promoting Batf3 + cDC1 deficiency through the Wnt3a/Fzd7/β-Catenin/ATF3 axis, and uncovers the involvement of Fzd7 in CAF-mediated ECM remodeling. Addressing the critical clinical challenge of ICI-resistance in NSCLC, SHH-YN-Bi represents a novel therapeutic strategy that integrates Wnt targeted therapy and immunotherapy. Results High Fzd7 Expression in NSCLC is Positively Associated with PD-L1 Level and Reduced Immune Cell Infiltration Fzd7, a key receptor of the Wnt/β-Catenin pathway, is significantly overexpressed in NSCLC [21]. Based on transcriptomic data analysis from TCGA lung cancer cohort, the patients were stratified into Fzd7-low and Fzd7-high groups based on expression cut-offs of 4.12 and 6.23, respectively (Figure. 1A). Single-sample gene set enrichment analysis (ssGSEA) revealed that high Fzd7 expression was associated with significantly lower enrichment scores across multiple immune-related biological processes and immune cell markers (Figure. 1B). Further analysis indicated that genes associated with Wnt/β-Catenin signaling, such as ATF3 , CD44 , and CD274 , were upregulated in Fzd7-high tumors, whereas genes related to immune cell infiltration, including CD8A , CCL4 , and Batf3 , were consistently downregulated (Figure. 1C). RNAscope FISH analysis on a human NSCLC TMA (HLugA150CS03) confirmed elevated Fzd7 mRNA levels in tumor tissues compared to adjacent normal tissues (Figure. 1D, E; Supplementary Figure. S1A). Correlation analysis further revealed a significant positive association between Fzd7 and PD-L1 mRNA expression levels (Figure. 1F; Supplementary Figure. S1B). mIHC analysis of NSCLC TMA (HLugA150CS03, HLugA060PG02) revealed significantly higher proportions of tumor cells with high or moderate Fzd7 expression, as well as increased frequencies of Fzd7 and PD-L1 double-positive cells in tumor tissues compared to paracancerous tissues (Figure. 1G-I; Supplementary Figure. S1C, D). Moreover, the expression of Fzd7, nuclear-β-catenin, and total-β-catenin each correlated positively with PD-L1 protein levels in tumors (Supplementary Figure. S1E-G). Notably, in CD34 + humanized NSG mouse bearing orthotopic A549/H1975 tumors, regions with high Fzd7 expression exhibited significantly fewer CD8 + T cells and CD141 + cDC1s (Figure. 1J-M). These findings indicate that in NSCLC, Fzd7 overexpression is closely associated with aberrant activation of the Wnt/β-Catenin pathway, and drives PD-L1 upregulation, thereby contributing to an immunosuppressive TME characterized by reduced immune infiltration and a deficiency in Batf3 + cDC1-dependent CD8 + T cells. SHH002-hu1 Enhances Atezolizumab Sensitivity to NSCLC by Blocking the Wnt/β-Catenin pathway Since the FDA approval of Atezolizumab for second-line or later treatment of advanced NSCLC [1, 23], α-PD-L1-based immunotherapies have gained considerable attention in clinical research. However, not all patients with high PD-L1 expression benefit from ICI therapy, primarily due to immune exclusion in the TME and dysfunction of effector T cells [1, 24]. To address these challenges, multiple combination immunotherapy approaches have been explored [25–27]. This study employed two Atezolizumab-insensitive human NSCLC cell lines, A549 and H1975 [28], along with murine LLC cells, which are also associated with a low immune infiltration TME [29]. Western blot analysis confirmed that both Fzd7 siRNA and SHH002-hu1 effectively reversed the Wnt3a-induced upregulation of PD-L1 and Fzd7 in A549, H1975, and LLC cells (Figure. 2A). Given the central role of DCs in anti-tumor immunity, CD34 + humanized NSG mouse were employed to enable stable reconstitution of a human immune system containing lymphocytes and DCs [28]. Orthotopic A549/H1975-luc and LLC-luc tumors were established in CD34 + humanized NSG mouse and C57BL/6 mouse, respectively (Figure. 2B), and tumor growth was dynamically monitored using in vivo bioluminescent imaging (Figure. 2C, D; Supplementary Figure. S2A). As shown in Figure. 2E, F and Supplementary Figure. S2B, Atezolizumab monotherapy was insensitive against all three cell lines in vivo , whereas SHH002-hu1 alone, or in combination with Atezolizumab, significantly inhibited tumor progression. Notably, the combination therapy exhibited superior efficacy compared to SHH002-hu1 monotherapy. Furthermore, mIHC of tumor tissues (Figure. 2G-J; Supplementary Figure. S2C, D) revealed that SHH002-hu1 and the combination treatment markedly reduced β-catenin and PD-L1 expression and suppressed β-catenin nuclear translocation. In contrast, Atezolizumab exerted no effect on either β-catenin or PD-L1 levels. Collectively, these findings demonstrate that SHH002-hu1 restores sensitivity of NSCLC to Atezolizumab by targeting the Wnt/β-Catenin signaling pathway. Design and Characterization of a BsAb Targeting Fzd7 and PD-L1 Given that the Wnt/β-Catenin signaling pathway regulates PD-L1 expression, the prevalence of Fzd7/PD-L1 double-positive cells in NSCLC, and the enhanced in vivo sensitivity to Atezolizumab upon Fzd7 inhibition, we engineered a BsAb, SHH-YN-Bi, capable of simultaneously binding Fzd7 and PD-L1 using the Konb-into-Hole technology (Figure. 3A). The H-chain HC1 (Human IgG1 Knob, T366W) was constructed based on the Atezolizumab H-chain sequence, while the H-chain HC2 (Human IgG1 Hole, T366S, L368A, Y407V) was based on the SHH002-hu1 H-chain sequence. The L-chain consisted of LC1 (Human Lambda) from Atezolizumab and LC2 (Human Kappa) from SHH002-hu1. All four chains were co-expressed in ExpiCHO-S cells, and the resulting BsAb was purified by protein A affinity chromatography. SDS-PAGE analysis of the purified SHH-YN-Bi confirmed the correct expression of the H-chain (50 kDa) and L-chain (25 kDa) at the expected molecular weights, assembled into a complete protein with an approximate molecular mass of 150 kDa (Figure. 3B). HPLC-MS analysis of the deglycosylated protein showed a measured molecular weight of 144,565.8 Da, within 4 Da of the theoretical mass (144,562.3 Da), and 89% of molecules exhibited correct chain pairing. Primary post-translational modifications (PTMs) were predominantly limited to G0F and G1F glycoforms on the H-chains, with no detectable alterations in complementarity-determining region (CDR) integrity (Supplementary Figure. S3A). SEC-HPLC indicated a purity of approximately 95% for the purified SHH-YN-Bi (Supplementary Figure. S3B), while CE-SDS under non-reducing and reducing conditions revealed purities of 93.24% and 94.71%, respectively (Supplementary Figure. S3C). DSF analysis demonstrated favorable thermal stability, with melting temperatures (Tm1: 67.00°C, Tm2: 74.80°C) approaching those of Herceptin (Tm1: 68.40°C, Tm2: 79.20°C) (Supplementary Figure. S3D). cIEF further revealed a charge variant profile consisting of a main peak (34.06%, pI 8.55), an acidic peak (33.46%, pI 8.02), and a basic peak (32.47%, pI 9.22) (Supplementary Figure. S3E). These results collectively support the correct assembly, structural integrity, and promising developability of SHH-YN-Bi. To evaluate the affinity of SHH-YN-Bi, SPR assays demonstrated that SHH-YN-Bi exhibits extremely high affinity for rhFzd7 ( ka (1/Ms): 1.47 × 10⁵, kd (1/s): 3.94 × 10⁻⁴, KD (M): 2.69 × 10⁻⁹) and rhPD-L1 ( ka (1/Ms): 1.48 × 10⁵, kd (1/s): 4.87 × 10⁻⁶, KD (M): 3.29 × 10⁻¹¹) (Figure. 3C). To confirm that SHH-YN-Bi retains the binding activity of its parental antibodies, binding to CHO-K1 cells overexpressing Fzd7 or PD-L1 was assessed by FCM. SHH-YN-Bi bound both CHO-K1-OE-Fzd7 (EC50 = 4.483 nM) and CHO-K1-OE-PD-L1 (EC50 = 5.535 nM) in a dose-dependent manner, with no significant reduction in potency compared to SHH002-hu1 (EC50 = 2.314 nM for Fzd7) and Atezolizumab (EC50 = 1.892 nM for PD-L1) (Figure. 3D). FCM further confirmed that SHH-YN-Bi competitively inhibited Wnt3a binding to CHO-K1-OE-Fzd7 cells in a dose-dependent manner (IC50 = 6.861 nM), with potency comparable to that of SHH002-hu1 (IC50 = 6.467 nM). Similarly, SHH-YN-Bi inhibited PD-1 binding to CHO-K1-OE-PD-L1 (IC50 = 8.226 nM), with activity slightly lower than Atezolizumab (IC50 = 4.491 nM) (Figure. 3E). The therapeutic potential of SHH-YN-Bi in NSCLC relies on its ability to simultaneously engage both Fzd7 and PD-L1. As shown in Fig. 3F, when rhFzd7 was immobilized on an NTA sensor chip and 1000 nM SHH-YN-Bi was injected as the analyte, a significant binding signal emerged at approximately 500 seconds. Subsequent injection of 1000 nM rhPD-L1 resulted in a further significant binding signal around 700 seconds, demonstrating that rhPD-L1 can bind to the preformed SHH-YN-Bi-rhFzd7 complex. To investigate the binding ability in tumor cells, SHH-YN-Bi was evaluated in A549, H1975, and LLC cells. In A549 and LLC cells (high Fzd7, low PD-L1), SHH-YN-Bi exhibited effective binding mediated by the highly expressed target (Figure. 3G; Supplementary Figure. S3F). In contrast, in H1975 cells (high Fzd7, high PD-L1), SHH-YN-Bi demonstrated enhanced binding relative to its parental monoclonal antibodies (Figure. 3G). These findings indicate that SHH-YN-Bi effectively engages tumor cells with either dual-high target expression or predominant single-target expression, highlighting its functional adaptability across heterogeneous antigen TME. The specific targeting capability of SHH-YN-Bi to NSCLC tissues is essential for its therapeutic efficacy. We previously demonstrated that SHH002-hu1 specifically binds to Fzd7-positive xenograft tumor tissues [21]. This study further evaluated the targeting specificity of Atezolizumab and SHH-YN-Bi in A549 xenograft models. Following administration of NIRB-labeled Atezolizumab and SHH-YN-Bi, fluorescent signals rapidly distributed throughout the body. By 8 hours post-injection, A549 xenografts were clearly delineated by fluorescence, with detectable signals persisting up to 32 hours (Figure. 3H, I). In contrast, the blocking group exhibited no significant fluorescence accumulation at the tumor site, confirming that tumor targeting was specifically mediated by Atezolizumab and SHH-YN-Bi. Notably, the tumor/normal tissue fluorescence ratio in the SHH-YN-Bi group was higher than that in the Atezolizumab group at 12 hours (Figure. 3I(c)), featuring superior targeting capability compared with Atezolizumab. These findings establish a critical foundation for subsequent evaluation of the anti-tumor efficacy of SHH-YN-Bi. SHH-YN-Bi Suppresses the Migration and Invasion of NSCLCs in vitro Activation of the Wnt/β-Catenin pathway is closely associated with tumor cell migration and invasion [30]. SHH-YN-Bi potently inhibited the migratory and invasive capabilities of A549, H1975, and LLC cells in transwell assays. Treatment with 100 nM SHH-YN-Bi significantly reduced Wnt3a-induced cell migration (Figure. 4A, B), with efficacy comparable to that of SHH002-hu1 or in combination with Atezolizumab. Notably, in the LLC model, SHH-YN-Bi exhibited greater inhibitory activity than the combination therapy. In contrast, Atezolizumab had no effect on Wnt3a-driven migration in any of the cell lines tested. Similarly, SHH-YN-Bi significantly suppressed the invasion of Wnt3a-stimulated cells across all models (Figure. 4C, D), achieving inhibition levels equivalent to SHH002-hu1 or combination treatment, while Atezolizumab showed no activity. These results demonstrate that SHH-YN-Bi effectively suppresses migration and invasion in NSCLC cells. To further investigate whether SHH-YN-Bi suppresses NSCLC cell migration and invasion through inhibition of the Wnt/β-Catenin signaling pathway, we assessed the expression of canonical Wnt pathway components in A549, H1975, and LLC cells. As shown in Fig. 4E, Wnt3a stimulation significantly increased levels of total β-catenin, nuclear β-catenin, total LRP6, phosphorylated LRP6 (LRP6-Pi), VEGFA, c-Myc, and CD44. In contrast, treatment with either SHH002-hu1 or SHH-YN-Bi markedly suppressed the Wnt3a-induced upregulation of these components, whereas Atezolizumab exerted no effect. Consistently, in a TCF/LEF luciferase reporter assay, both SHH002-hu1 and SHH-YN-Bi, but not Atezolizumab, suppressed Wnt3a-induced Wnt/β-catenin pathway activation in A549, H1975, and LLC cells (Figure. 4F). These findings demonstrate that SHH-YN-Bi attenuates NSCLC cell migration and invasion by targeting Fzd7 and inhibiting Wnt/β-Catenin pathway activation. SHH-YN-Bi Potentiates Effector T cells Cytotoxicity by Conjugation with Tumor cells SHH-YN-Bi enhances T cell-mediated tumor cell killing by simultaneously engaging Fzd7 and PD-L1 on NSCLC cells. We next evaluated whether SHH-YN-Bi enhances T cell cytotoxicity against NSCLC cells. In co-culture systems containing DiO-labeled (Ex 484 nm/Em 501 nm) T cells and Dil-labeled (Ex 549 nm/Em 565 nm) tumor cells, SHH002-hu1, Atezolizumab, combination therapy, and SHH-YN-Bi progressively reduced tumor cell viability at multiple time points (1 h, 6 h, 12 h) compared to Wnt3a or Control treatments (Supplementary Figure. S4A-C). The combination and SHH-YN-Bi groups exhibited the most pronounced tumor cell clearance, outperforming either monotherapy. Using the CytoTox 96 Non-Radioactive Cytotoxicity Assay, we confirmed that Wnt3a impaired T cell cytotoxic function, whereas all therapeutic interventions restored tumor cell killing (Figure. 4G). To further evaluate its blocking effect on the PD-1/PD-L1 axis, an NFAT-luciferase reporter gene system was employed. SHH-YN-Bi effectively disrupted the PD-1/PD-L1 interaction with an IC50 of 2.74 nM, although Atezolizumab exhibited greater potency (IC50 = 0.77 nM) (Figure. 4H). Once again, the combination and SHH-YN-Bi groups induced the highest levels of target cell lysis, demonstrating that dual targeting of Fzd7 and PD-L1 most effectively reverses Wnt-mediated T cell dysfunction and enhances anti-tumor immunity. SHH-YN-Bi Induces CD8 + T cell Activation by Recovering Batf3 + cDC1s Function The anti-tumor activity of ICIs is primarily mediated through the reactivation of pre-existing, DC-primed tumor-reactive T cells [31–33]. In contrast, Wnt/β-Catenin pathway activation in tumors induces the expression of PD-L1 and the transcriptional repressor ATF3, which suppresses CCL4 production. This reduction in CCL4 impairs the recruitment and function of Batf3 + cDC1s, thereby compromising CD8 + T cell priming and culminating in an immunosuppressive TME deficient in effective CD8 + T cells [19]. To investigate whether targeting Fzd7 enhances therapeutic sensitivity in NSCLC by promoting Batf3 + cDC1-mediated priming and infiltration of CD8 + T cells, we employed Batf3 -KO mouse, allocating them into Control, SHH002-hu1, and SHH-YN-Bi treatment groups (Figure. 5A). Following 18 days of treatment, no significant differences in tumor growth kinetics were observed across the experimental groups (Figure. 5B). Moreover, mIHC analysis revealed that, compared to the Control group, SHH002-hu1 treatment did not significantly increase the intratumoral infiltration or activation status of CD8 + T cells (Figure. 5C, D). These results collectively indicate that the ability of targeting Fzd7 to enhance CD8 + T cell infiltration and activation in NSCLC tumors depends on the restoration of Batf3 + cDC1 function. To clarify how targeting Fzd7 affects immune infiltration, western blot analysis showed that SHH002-hu1 or Fzd7 siRNA reduced active β-catenin levels and suppressed Wnt3a-induced pathway activation. SHH002-hu1 markedly decreased ATF3 expression (Supplementary Figure. S5A), and SHH-YN-Bi showed similar effects, while Atezolizumab had no impact (Supplementary Figure. S5B), indicating that Fzd7 targeting downregulates ATF3 via Wnt/β-Catenin inhibition. ChIP assays revealed that ATF3 binding to the Ccl4 promoter was significantly reduced by Fzd7 siRNA or SHH002-hu1 compared to the Wnt3a group in A549, H1975, and LLC cells (Supplementary Figure. S5C). SHH-YN-Bi also attenuated ATF3 binding, whereas Atezolizumab showed no significant effect (Figure. 5E). ELISA results showed increased CCL4 expression after Fzd7 siRNA or SHH002-hu1 treatment compared to Control and Wnt3a groups (Supplementary Figure. S5D); SHH-YN-Bi similarly upregulated CCL4, whereas Atezolizumab had no significant effect (Figure. 5F). In summary, SHH-YN-Bi restores Batf3 + cDC1 recruitment and CD8 + T cell infiltration by disrupting the Wnt3a/Fzd7/β-Catenin/ATF3 axis. Anti-tumor Efficacy of SHH-YN-Bi in NSCLC through Promoting CD8 T cells Infiltration SHH-YN-Bi enhances CD8 + T cells cytotoxicity through dual mechanisms: blocking PD-1/PD-L1 interaction and targeting Fzd7 to suppress the Wnt/β-Catenin pathway. Evaluation of the in vivo anti-tumor efficacy of SHH-YN-Bi demonstrated its potent suppression of tumor growth in A549, H1975, and LLC orthotopic models. The anti-tumor activity of SHH-YN-Bi was superior to that of combination therapy (Supplementary Figure. S6A; Figure. 6A). Furthermore, mIHC analysis confirmed that SHH-YN-Bi exerts its anti-tumor effect by inhibiting both the expression and nuclear translocation of β-catenin, leading to downregulation of PD-L1 expression (Figure. 6B; Supplementary Figure. S6B). In immunotherapy, activation and infiltration of CD8 + T cells are critically dependent on cDC1s. These cells capture tumor antigens within the TME and migrate to the tumor‑draining lymph nodes (tdLNs) along a chemokine gradient, where they prime naïve CD8 + T cells for subsequent homing back to the TME to mediate cytotoxic functions [34, 35]. Among these activated populations, CD39 + CD8 + T cells are recognized as a key subset exhibiting tumor-reactive specificity [36]. To investigate how SHH-YN-Bi exerts excellent anti-tumor effect in vivo , we performed mIHC analysis on PanCK + tumor regions. The results revealed that SHH002-hu1, combination therapy, and SHH-YN-Bi all significantly increased the densities of CD8 + T cells and CD141 + /CD103 + cDC1s, as well as the frequency of activated CD39 + CD8 + T cells, compared to the Control and Atezolizumab groups (Figure. 6C, D; Supplementary Figure. S6C-F). This enhancement was more pronounced with combination therapy and SHH-YN-Bi. In summary, SHH-YN-Bi demonstrates superior in vivo anti-tumor efficacy relative to combination therapy. This enhanced activity stems from its dual targeting, reflecting a systemic boost in anti-tumor immunity rather than a simple additive effect. SHH-YN-Bi Promotes CD8 T Cell Infiltration by Conquering CAF-Mediated ECM Barrier With the deepening understanding of the TME, the mechanisms of therapy resistance mediated by CAFs have become increasingly clear. Accordingly, targeting CAFs has emerged as a key strategy in current anti-tumor therapeutics [37]. The transition of normal fibroblasts into CAFs within the TME is primarily driven by two mechanisms: the release of tumorigenic signals from tumor cells, and the intrinsic activation of oncogenic pathways, such as Wnt/β-Catenin pathway in CAFs to maintain their phenotype [38]. We confirmed that CM from A549 cells could induce the transdifferentiation of normal MRC-5 fibroblasts into CAFs (Figure. 7A). Notably, mIHC evaluation of CAF markers (COL1A1, FAP, and α-SMA) revealed that A549 CM pre-incubated with either SHH002-hu1 or SHH-YN-Bi lost this transdifferentiation capacity. In contrast, A549 CM treated with Atezolizumab retained the ability to induce CAF transformation (Figure. 7B, C). To further evaluate the impact of SHH-YN-Bi on CD8 + T cell infiltration via its action on CAFs, we established a co-culture infiltration model (Figure. 7D). The results demonstrated that targeting either Fzd7 or PD-L1 alone enhanced CD8 + T cell infiltration, with the most pronounced effect observed when both CAFs and tumor cells were simultaneously targeted (Figure. 7E, F). Notably, SHH-YN-Bi exhibited the greatest efficacy, indicating that it promotes CD8 + T cell infiltration and cytotoxicity through concurrent suppression of the Wnt/β-Catenin pathway in CAFs and blockade of the PD-1/PD-L1-mediated immunosuppressive axis. ECM remodeling and deposition mediated by CAFs represents a critical mechanism underlying the low survival rate of NSCLC patients [39]. To further investigate the effect of SHH-YN-Bi on CAFs, the A549 CM induced CAFs were then treated with 100 nM of Atezolizumab or SHH-YN-Bi for 24 hours and subjected to scRNA-seq analysis. Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction and clustering analysis revealed that the induced MRC-5 cells could be divided into 23 distinct clusters (Figure. 7G). Cell type identification using ACTA2 (α-SMA), FAP , COL1A1 , and COL2A1 as markers confirmed that clusters 0–22 were all identified as CAFs (Supplementary Figure. S7A). Further analysis of key genes in the Wnt/β-Catenin signaling pathway ( Fzd7 , CTNNB1 , APC ) in CAFs (Supplementary Figure. S7B), with adjustment for multiple comparisons, showed that Fzd7 was significantly highly expressed in clusters 8 and 20. Subsequently, using AddModuleScore to evaluate the activity of the Wnt/β-Catenin pathway-related gene set and the ECM deposition gene set, we found that clusters 8 and 20 exhibited significantly higher scores in both assessments (Figure. 7H, I). These two clusters also showed a significant correlation with each other (Supplementary Figure. S7C-E). Analysis of the top differentially expressed genes in clusters 8 and 20 (Supplementary Figure. S7F-H), combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, indicated that both clusters were closely associated with ECM deposition (Figure. 7J). Collectively, these findings show that Wnt pathway activation in CAFs drives the robust ECM deposition characteristic of the matrix-depositing CAF phenotype. Through Gene Set Variation Analysis (GSVA) analysis, we found that SHH-YN-Bi treatment significantly suppressed the Wnt pathway compared to the Control or Atezolizumab groups (Figure. 7K, Supplementary Figure. S7I). This finding was further supported by GSEA multi-gene enrichment analysis (Supplementary Figure. S7J, K). Notably, SHH-YN-Bi also markedly reduced the expression of ECM deposition-related genes, including COL1A1 , COL1A2 , COL3A1 , FN1 , and LAMC1 , relative to the Control (Figure. 7L). Pseudotemporal trajectory analysis further revealed that SHH-YN-Bi significantly suppressed the developmental progression of clusters 8 and 20 (Figure. 7M). Further analysis demonstrates that SHH-YN-Bi, through Fzd7 binding and Wnt/β-Catenin inhibition, downregulates multiple CAF-expressed genes critical for ECM remodeling and deposition (Supplementary Figure. S8). This effect may extend to the suppression of CAF-mediated ECM deposition and the downregulation of promigratory enzymes, such as MMP2 and MMP14. Western blot analysis confirmed that SHH-YN-Bi inhibits ECM deposition and reduces the expression of the metastasis-associated marker MMP2 in CAFs through the inhibition of Wnt/β-Catenin signaling (Supplementary Figure. S7L). Collectively, these findings indicate that Fzd7 is highly correlated with ECM remodeling in CAFs, and SHH-YN-Bi not only has the potential to suppress malignant metastasis in NSCLC but may also contribute to remodeling the ECM to promote immune cell infiltration. Discussion Lung cancer, particularly NSCLC, remains a significant clinical challenge [40–42]. Although ICIs have transformed the therapeutic landscape, their efficacy is frequently constrained by primary or acquired resistance [1, 43]. The non-T cell-inflamed TME mediated by the Wnt/β-Catenin pathway represents the underlying mechanism for ICI-resistance in NSCLC. Specifically, aberrant activation of the Wnt pathway not only exerts intrinsic oncogenic functions but also fosters an immunosuppressive TME by upregulating PD-L1, impairing the recruitment of Batf3+ cDC1s, and promoting a highly oncogenic CAF phenotype [13, 17, 38, 44]. Consequently, therapeutic targeting of the Wnt/β-Catenin pathway may not only directly suppress tumor progression but also offer a promising strategy to overcome ICI-resistance by restoring CD8+ T cell infiltration into the TME. The role of Fzd7 inhibition in counteracting Wnt/β-Catenin-mediated tumor progression is well-established, as demonstrated by our previous studies with SHH002-hu1 [20–22, 45, 46]. Batf3+ cDC1s act as a critical bridge in anti-tumor immunity by capturing antigens in the TME, migrating to the tdLN to activate CD8+ T cells, and finally guiding the primed T cells to home back to the tumor site for cytotoxic killing [34, 35, 47]. Our findings reveal the critical relationship between Fzd7 and PD-L1 and demonstrate that targeting Fzd7 sensitizes NSCLC to Atezolizumab treatment. Based on these, we designed SHH-YN-Bi, exhibits superior targeting capability compared with Atezolizumab. SHH-YN-Bi suppresses the Wnt/β-Catenin pathway, blocks PD-L1 to relieve immunosuppression, and demonstrates robust anti-tumor efficacy in multiple orthotopic NSCLC models. Mechanistically, we uncovered that targeting Fzd7 restores Batf3+ cDC1-dependent CD8+ T-cell infiltration by interrupting the Wnt3a/Fzd7/β-Catenin/ATF3 axis. As a first-in-class agent combining Wnt inhibition with ICP blockade, SHH-YN-Bi showcases the potential of simultaneously targeting the TME and ICPs to overcome ICI-resistance. With increasing understanding of the TME, it has become clear that the dense and highly organized physical barrier formed by CAFs and the ECM constitutes a major obstacle to immune cell infiltration and effector function [48]. CAFs exhibit distinct functional heterogeneity, with specific subtypes mediating divergent biological roles. Specifically, matrix CAFs (mCAFs) are primarily responsible for ECM remodeling and deposition [49, 50]. Herein, we show that MRC-5-derived CAFs induced by A549 CM predominantly adopt an mCAF phenotype. Furthermore, Fzd7 expression in these mCAFs is positively correlated with ECM deposition. Notably, SHH-YN-Bi effectively attenuates CAF-mediated ECM remodeling and deposition by inhibiting the Wnt/β-Catenin pathway in mCAFs, thereby promoting CD8+ T cell infiltration into the TME. Our study further reveals that suppression of the Wnt/β-Catenin pathway in CAFs downregulates the expression of promigratory enzymes such as MMP2. This dual regulation underscores the capacity of SHH-YN-Bi to concomitantly foster an immunopermissive TME while restraining the metastatic potential of tumors. Intriguingly, in the immune infiltration assays, administration of Atezolizumab in the presence of CAFs also enhanced CD8+ T cell infiltration. However, PD-L1 expression is low in MRC-5-derived CAFs induced by CM alone (Fig. S9 A, B). Based on this observation, we hypothesize that upregulation of PD-L1 in CAFs likely depends on sustained non-cell-autonomous signaling from tumor cells. As shown in Fig. S9 C-E, PD-L1 expression in CAFs is significantly elevated upon prolonged stimulation by A549 cells compared to stimulation with CM alone. Collectively, this study originally reveals the involvement of Fzd7 in CAF-mediated ECM remodeling, and these findings support the dual role of SHH-YN-Bi as both a classical inhibitor of an oncogenic pathway and an immunomodulatory agent that promotes immune cell recruitment and function within the TME. In summary, SHH-YN-Bi effectively abrogates the PD-1/PD-L1-mediated immunosuppression while simultaneously inhibiting the oncogenic activity of the Wnt/β-Catenin pathway. Furthermore, it enhances the immune infiltration of CD8+ T cells through two distinct mechanisms: by restoring the recruitment of Batf3+ cDC1s to the TME, and by disrupting the CAF-derived immunosuppressive barrier as well as attenuating CAF-mediated ECM remodeling and deposition (Fig. 8). Importantly, emerging evidence indicates that targeting PD-L1 on DCs is essential for achieving optimal therapeutic efficacy. Therefore, SHH-YN-Bi, through its bispecific design, not only blocks PD-L1 but also effectively bridges DCs and tumor cells, thereby establishing a novel DC-tumor cell engager (DCE) modality. This approach may overcome the limitations of traditional monocyte-derived dendritic cell (moDC) vaccines, such as suboptimal antigen presentation and impaired lymph node trafficking, thus promoting a more robust in situ immune response [34, 50]. Ultimately, our work for the first time elucidates the role of Fzd7 in shaping the immunosuppressive TME via Wnt3a/Fzd7/β-Catenin axis, particularly in CAF-mediated ECM remodeling. SHH-YN-Bi represents a promising therapeutic strategy for overcoming immunotherapy resistance in NSCLC, addressing a critical unmet clinical need in NSCLC patient management. Methods Ethics The cancer TMA (HLugA150CS03 and HLugA060PG02) were from Shanghai Outdo Biotech and approval from Shanghai Outdo Biotech Ethics Committee (SHYJS-CP-1901005 and SHYJS-CP-1904007). All human PBMCs were isolated from donors following written informed consent and approval from the Medical Ethics Committee Shanghai First Maternity and Infant Hospital (KS22174). All animal experiments were conducted in accordance with protocols approved by the Animal Ethics Committee of Shanghai University of Medicine and Health Sciences (2024-GZR-16-321281198802248253). Cell culture and transfection The human NSCLC cell line A549 (RRID: CVCL_0023), murine Lewis lung carcinoma cell line LLC (RRID: CVCL_4358), human T-cell line TALL-104 (RRID: CVCL_2771), and human lung fibroblast cell line MRC-5 (RRID: CVCL_0440) were obtained from the American Type Culture Collection (ATCC, New York, USA) and cultured in DMEM (Gibco, Grand Island, USA), supplemented with 10% (v/v) FBS (Transgen, Beijing, China), 100 µg/mL penicillin, and 100 µg/mL streptomycin (Gibco). The human NSCLC cell line H1975 (RRID: CVCL_1511) was obtained from the ATCC and cultured in RPMI 1640 (Gibco), supplemented with 10% (v/v) FBS, 100 µg/mL penicillin, and 100 µg/mL streptomycin. The Chinese hamster ovary cell line CHO (RRID: CVCL_0213) was obtained from the European Collection of Authenticated Cell Cultures (ECACC, Porton Down, UK), and the human PD-L1 cell line CHO-K1-OE-PD-L1 (Cat# B22301201, Biointron, Shanghai, China) was cultured in DMEM, supplemented with 10% (v/v) FBS, 100 µg/mL penicillin, and 100 µg/mL streptomycin. The human Fzd7-overexpressing cell line CHO-K1-OE-Fzd7 was generated by transient transfection of a full-length human Fzd7 plasmid (Sangon Biotech, Shanghai, China), and cultured in DMEM, supplemented with 10% (v/v) FBS, 100 µg/mL penicillin and 100 µg/mL streptomycin. The Chinese hamster ovary cell line ExpiCHO-S (RRID: CVCL_5J31) was obtained from Gibco and maintained in ExpiCHO expression medium. HEK293-PD-L1-OS8 and Jurkat-PD-1-NFAT-luc were obtained from Biointron and cultured in RPMI 1640 (Gibco), supplemented with 10% (v/v) FBS, 100 µg/mL penicillin, and 100 µg/mL streptomycin. Fzd7 -specific small interfering RNA (siRNA) was synthesized by Genepharma (Shanghai, China). The nucleotide target sequence for Fzd7 (siFzd7) was: 5’-GCACCAUCAUGAAACACGATT-3’. Then, transfection was performed using GP-transfect-Mate (Genepharma). BsAb constructure The light-chain (LC) and heavy-chain (HC) gene sequences of SHH002-hu1 and Atezolizumab were synthesized by Biointron. To construct SHH-YN-Bi, site-directed mutagenesis was performed on the original expression vectors of SHH002-hu1 and Atezolizumab to introduce specific mutations into the constant region of the heavy chain 3 (CH3), generating the knob (T366W) and hole (T366S, L368A, Y407V) variants in accordance with Knob-into-Hole (KIH) technology. Expression, purification and identification of SHH-YN-Bi For SHH-YN-Bi expression, ExpiCHO-S cells were co-transfected with four IgG expression plasmids (pcDNA3.4 vector) encoding LC1, LC2, HC knob, and HC hole at a 1:1:1:1 molar ratio. SHH-YN-Bi was purified from culture supernatants by protein A affinity chromatography (GE Healthcare, Buckinghamshire, UK) and characterized using reducing and non-reducing SDS-PAGE, followed by size-exclusion chromatography-HPLC (SEC-HPLC) and capillary electrophoresis-SDS (CE-SDS) assays. The complete molecular structure of SHH-YN-Bi was analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS), capillary isoelectric focusing (cIEF), and differential scanning fluorimetry (DSF) to assess its charge heterogeneity and conformational stability. Binding affinity and kinetic analysis The binding kinetics of SHH-YN-Bi to rhFzd7-His (Sangon Biotech) and rhPD-L1-Fc (R&D Systems, USA) were measured using the ForteBio Octet Red96 system (PALL, USA). Briefly, SHH-YN-Bi was captured by anti-human IgG Fc antibodies immobilized on a CM5 sensor chip (Cytiva). Subsequently, rhFzd7-His or rhPD-L1-Fc was injected at varying concentrations into running buffer (KB buffer: 0.1% BSA + 0.025% Tween 20 dissolved in PBS, pH 7.2), and real-time binding responses were recorded. Sensorgrams were generated at each concentration, and the association rate constant ( ka ) and dissociation rate constant ( kd ) were determined by global fitting analysis. The equilibrium dissociation constant (KD) was then calculated as the ratio kd / ka . To demonstrate whether SHH-YN-Bi can simultaneously bind rhFzd7-His and rhPD-L1-Fc, a surface plasmon resonance (SPR) assay was performed using a Biacore T200 instrument (Cytiva). After immobilizing rhFzd7 on an NTA sensor chip (Cytiva), SHH-YN-Bi was first injected, followed by injection of rhPD-L1. Flow-cytometry (FCM)-based binding and competition assays To evaluate the binding capacity of SHH-YN-Bi to CHO-K1-OE-Fzd7 and CHO-K1-OE-PD-L1 cells, the following experimental groups were established: Isotype Control (anti-HEL human IgG1; Biointron), SHH002-hu1/Atezolizumab, and SHH-YN-Bi. Antibodies were serially diluted across ten concentration gradients (0-100 nM). Cells were harvested, resuspended, and incubated with diluted antibodies for 30 min at 4°C in 2% FBS/PBS. After incubation, cells were washed and subsequently incubated with FITC-conjugated goat anti-human IgG (Sangon Biotech) for 20 min at 4°C. Fluorescence signals were acquired using a NovoCyte Advanteon Dx flow cytometer (Agilent, California, USA), and data were analyzed using FlowJo software (version 10). The binding affinity for A549, H1975, and LLC cells was measured using the same method as described above. To assess the competitive binding of SHH-YN-Bi to CHO-K1-OE-Fzd7 and CHO-K1-OE-PD-L1 cells in the presence of rhWnt3a and rhPD-1 (MedChemExpress, Jersey, USA), FITC-labeled rhWnt3a and rhPD-1 were first prepared using a FITC conjugation kit (Sangon Biotech). Briefly, saturating concentrations of FITC-rhWnt3a and FITC-rhPD-1 were added to suspensions of CHO-K1-OE-Fzd7 or CHO-K1-OE-PD-L1 cells, respectively. Serially diluted antibodies were then added to the mixtures, and the samples were incubated for 20 min at 4°C. Finally, fluorescence was measured using FCM. TOP/FOP-FLASH luciferase reporter assay The β-catenin/TCF-mediated transcriptional activity was evaluated using a dual-luciferase reporter system following transient transfection of A549, H1975, and LLC cells with the TOP/FOP-FLASH luciferase reporter plasmid and a Renilla luciferase Control plasmid for normalization. Briefly, cells were seeded into 96-well plates and transfected with the TOP or FOP-FLASH plasmid using TransIntro EL Transfection Reagent (Transgen, Beijing, China). Four hours post-transfection, Wnt3a (200 ng/mL) was added to stimulate pathway activation. After an additional 12 h, cells were treated with antibodies (100 nM). Luciferase activities were measured 24 h after transfection using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA), and firefly luciferase signals were normalized to Renilla values. NFAT report assays HEK293-PD-L1-OS8 and Jurkat-PD-1-NFAT-luc cells were harvested from T75 cell culture flasks, resuspended in RPMI 1640 medium without FBS, and adjusted to final densities of 5×10 5 cells/mL and 1×10 6 cells/mL, respectively. Antibody dilutions were prepared in RPMI 1640 medium with a top concentration of 100 nM, followed by five-fold serial dilutions across seven dose levels. Cells were seeded into 96-well plates at 2×10 4 cells per well in a volume of 40 µL. Then, 20 µL of each antibody dilution was added to the designated wells. The plates were incubated at 37°C in a 5% CO₂ incubator for 6 hours. After incubation, cells were equilibrated to room temperature for 5–10 min. An equal volume (100 µL/well) of KeyTec Ultra Luciferase Detection Reagent was added to each well, and the plate was shaken for 10 min to ensure complete cell lysis prior to luminescence measurement using a luminometer. In vivo dynamics and targeting capability by near infrared (NIR) imaging Five-week-old female BALB/c-nude mouse were purchased from the Shanghai Laboratory Animal Research Center (Shanghai, China). A549-Luc cells were implanted into the lung parenchyma to establish orthotopic tumor models. Upon confirmation of tumor formation, mouse were randomly assigned to two groups (n = 5 per group). An NIRB-NHS fluorescence probe (Keyuandi Biotechnology, Shanghai, China) was conjugated with SHH-YN-Bi to generate the NIRB-SHH-YN-Bi fluorescent probe. In addition, free SHH-YN-Bi (2.5 µmol/kg) was co-administered with NIRB-SHH-YN-Bi (50 nmol/kg) to evaluate competitive binding and target specificity in vivo . After intravenous injection, fluorescence images were acquired at predetermined time points using an IVIS Spectrum CT imaging system (PerkinElmer, USA). Tumor-to-normal tissue ratios (T/N ratios) were calculated based on region-of-interest (ROI) analysis. The same experimental procedure was performed for the Atezolizumab Control group. Western blot assay The whole cell proteins were extracted from A549, H1975, LLC, and CAF cells using RIPA buffer (Beyotime, Shanghai, China), and nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce Biotechnology, Rockford, USA). Western blotting was performed with primary antibodies against phospho-LRP6 (Ser1490) (Cat# abs140173, Absin), LRP6 (Cat# abs174285, Absin), β-catenin (Cat# 9562, CST), active β-catenin (Cat# 4270, CST), PD-L1 (Cat# abs155263, Absin), c-Myc (Cat# 9402, CST), ATF3 (Cat# 18665, CST), Fzd7 (Cat# ab64636, Abcam), CD44 (Cat# 37259, CST), VEGFA (Cat# abs149552, Absin), MMP2 (Cat# abs158236, Absin), COL1A1 (Cat# abs118788, Absin), FAP (Cat# abs134422, Absin), α-SMA (Cat# abs120451, Absin), and β-actin (Cat# 8457, CST). Subsequently, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized using enhanced chemiluminescence detection (Immobilon ECL Ultra Western HRP Substrate, Millipore, Burlington, MA, USA). ATF3 - specific ChIP assay ChIP assays were performed using the ChIP Assay Kit from Merck. When cell density reached 80–90%, A549, H1975, or LLC cells were crosslinked with 1% formaldehyde and lysed for 10 minutes at 37°C. The crosslinking was quenched with 10× glycine, followed by PBS washing. Cells were then collected and incubated on ice for 15 minutes in cell lysis buffer containing protease inhibitors, with resuspension every 5 minutes. Chromatin was sheared by sonication. The lysates were incubated with ATF3-specific or Control IgG antibodies. DNA-protein complexes were captured using Protein A/G magnetic beads, eluted with ChIP elution buffer containing Proteinase K, and further purified. The isolated DNA was analyzed by qPCR. qPCR Reaction System: DNA 1.6 µL + Forward primer 0.2 µL + Reverse primer 0.2 µL + Nuclease-free Water 3 µL + 2 × PerfectStart Green qPCR SuperMix 5 µL. Ccl4 primer (Human): Forward primer 5’-AAGGTGAGCAGGTGGGTTAG-3’; Reverse primer 5’-TGGCTGGTTTGACAGTTGCT-3’. Ccl4 primer (mouse): Forward primer 5’-CTCAGCCCTGATGCTTCTCAC-3’; Reverse primer 5’-AGAGGGGCAGGAAATCTGAAC-3’. Elisa assay For the ELISA-based CCL4 assays, after treating A549, H1975, or LLC cells with Wnt3a and antibodies, the cell supernatants were collected using sterile tubes and centrifuged at 2200 rpm for 20 minutes. Following centrifugation, the supernatants were transferred to new sterile tubes. The concentrations of CCL4 were measured using the MIP-1β/CCL4 ELISA Kit (animaluni, Shanghai, China). Biotinylated human CCL4 or mouse CCL4 antigens were coated onto 96-well plates. After the biotinylated antigens bound to CCL4, SABC was added and incubated at 37°C for 30 minutes to form immune complexes. TMB substrate solution was then added, and the plates were incubated in the dark at 37°C for 10–20 minutes. The reaction was stopped by adding 50 µL of stop solution per well, and the absorbance was measured at 450 nm using a microplate reader. Cytotoxicity assay The CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, USA) was used to measure lactate dehydrogenase (LDH) released from damaged NSCLC cells. Except for the blank Control group, other groups were pre-treated with 200 ng/mL Wnt3a for 12 hours. TALL-104 cells were co-cultured with A549 or H1975 cells at effector-to-target (E:T) ratios of 10:1, 5:1, and 2.5:1 in 96-well plates using DMEM containing 1% FBS and 100 nM of the corresponding antibodies. Similarly, activated mouse CD8⁺ T cells were co-cultured with LLC cells under the same conditions for 6 hours at 37°C. Control groups included spontaneous LDH release from effector or target cells alone, as well as target cell maximum LDH release. The percentage of specific cell lysis was calculated using the formula: 100 × (Experimental LDH Release – Effector Spontaneous LDH Release – Target Spontaneous LDH Release) / (Target Maximum LDH Release – Target Spontaneous LDH Release). Cell migration and invasion assay Here, except for the blank Control group, other groups were pre-treated with 200 ng/mL Wnt3a for 12 hours. 4×10 4 of A549, H1975, or LLC cells suspended in serum-free medium were plated into the upper wells of a 24-well transwell chamber (Millipore, Billerica, USA), coated with Matrigel (Corning, Bedford, USA), and then treated with antibodies. At 24h later, non-invasive cells on the upper layer were removed, whereas the invaded cells were fixed and stained. Images were taken using an Olympus inverted microscope; the invaded cells were counted using the Image-Pro-Plus program and invasion percentages were quantified based on the untreated Control. The migration assay was performed without Matrigel coating. Immuno co-culture assay To evaluate the SHH-YN-Bi-mediated cytotoxic effects of TALL-104 or mouse CD8⁺ T cells on NSCLC cells, DiO-labeled (Ex 484 nm/Em 501 nm) TALL-104 and mouse CD8⁺ T cells and Dil-labeled (Ex 549 nm/Em 565 nm) A549, H1975, and LLC cells were co-cultured with 100 nM SHH-YN-Bi. Cellular status was subsequently observed using an inverted fluorescence microscope at 1 h, 6 h, and 12 h after treatment. Animal studies SPF-grade female CD34 + humanized non-obese diabetic-scid gamma (NSG) mouse and C57BL/6 mouse aged 5–6 weeks were purchased from Shanghai Model Organisms Center, Inc. and maintained under sterile conditions. All animal experiments involved in this study were reviewed and approved by the Scientific Research Ethics Committee of Shanghai University of Medicine & Health Sciences before implementation. Orthotopic tumor models of A549, H1975, and LLC were established. When the average bioluminescence signal intensity in tumor-bearing mouse reached approximately 1.5 × 10⁵, the mouse were administered the following treatments via tail vein injection: SHH002-hu1 (5 mg/kg), Atezolizumab (5 mg/kg), SHH002-hu1 (5 mg/kg) + Atezolizumab (5 mg/kg), and SHH-YN-Bi (5 mg/kg). All groups were dosed once every three days. Bioluminescence signal intensity was monitored by intraperitoneal injection of 15 mg/mL D-luciferin (10 µL/g). After the luciferin signal stabilized, the bioluminescence intensity in tumor-bearing mouse was measured and recorded using a small animal in vivo optical 3D imaging system. Twelve-week-old female Batf3 -KO mouse were also purchased from Shanghai Model Organisms Center, Inc. After successful establishment of the orthotopic LLC tumor model, the mouse were administered PBS, SHH002-hu1 (5 mg/kg), or SHH-YN-Bi (5 mg/kg) via tail vein injection. Bioluminescence signal intensity in the tumor-bearing mouse was recorded using the same method described above after drug administration. NSCLC tumor tissues analysis The human NSCLC TMA (HLugA150CS03, HLugA060PG02) was purchased from Shanghai Outdo Biotech Co., Ltd. Fluorescence in situ hybridization (FISH) was performed using the Boshide Custom Fzd7 FISH Detection Kit and Boshide Custom PD-L1 (CD274) FISH Detection Kit. TSA-based multi-color immunohistochemistry (mIHC) was performed on HLugA150CS03 and HLugA060PG02 TMAs using antibodies against Fzd7, PD-L1, CK, β-catenin, and c-Myc. Formalin-fixed paraffin-embedded (FFPE) sections were prepared from the NSCLC tissues obtained from the aforementioned animal experiments. These sections were subsequently subjected to hematoxylin and eosin (HE) staining and mIHC. The antibodies used in this section included: anti-Fzd7, anti-PD-L1, anti-β-catenin, anti-CD3, anti-CD8, anti-CD39, anti-CD141, anti-CD103, and anti-PanCK. CAF associated mIHC To generate cancer cell conditioned media (CM), A549 cancer cells at 70–80% confluency were treated with SHH-YN-Bi for 24 h. The cells were then washed with PBS and the media was replaced with fresh media supplemented with 5% FBS for 48 h. The media was centrifuged for 5 min at 1200 rpm, and the supernatant (CM) was used for stimulation of fibroblasts. MRC5 fibroblasts were cultured in a 6-well glass-bottom plate and grown to 50% confluency, then growth arrested for 48 h. The fibroblasts were then co-cultured with cancer cell CM for 48 h. The fibroblasts were then stained for mIHC. The antibodies used in this section were: anti-COL1A1, anti-FAP, anti-α-SMA. CD8 + T cells isolation Mouse CD8⁺ T cells were isolated from C57BL/6 mouse using the EasySep™ mouse CD8⁺ T Cell Isolation Kit (STEMCELL Technologies). Spleens were harvested and mechanically dissociated in PBS containing 2% FBS, then passed through a 70 µm strainer. The resulting suspension was centrifuged at 1200 rpm for 10 minutes. After discarding the supernatant, the cell pellet was resuspended in 0.5-2 mL RoboSep™ Buffer (STEMCELL). Subsequently, 20 µL of mouse FcR Blocking Reagent and 50 µL of the Isolation Cocktail were added, followed by incubation at room temperature for 10 minutes. Then, 125 µL/mL of RapidSpheres™ were added and incubated for 5 minutes at room temperature. The cell suspension was diluted with 1 mL RoboSep™ Buffer, and CD8⁺ T cells were positively selected using a STEMCELL Separator. Finally, the isolated cells were activated for 2 hours with in vivo anti-mouse CD3 Recombinant mAb (STARTER) and in vivo anti-mouse CD28 mAb (STARTER) at appropriate concentrations. To prepare the magnetic bead buffer for isolating human CD8⁺ T cells, 2 mM EDTA and 2.5 g BSA standard protein were separately dissolved in 50 mL PBS, thoroughly mixed, filtered through a 0.22 µm filter, and then added to 400 mL PBS to obtain a final volume of 500 mL. Human Peripheral Blood Mononuclear Cells (PBMCs) were resuspended in 1 mL of the prepared bead buffer, and 1 × 10⁷ cells were transferred to a 1.5 mL microcentrifuge tube, followed by centrifugation at 300 × g for 5 minutes. After discarding the supernatant, the cell pellet was resuspended in 40 µL bead buffer. For CD8⁺ T cell isolation, 20 µL of CD8-Biotin antibody was added and incubated, followed by the addition of 20 µL anti-Biotin MicroBeads and thorough mixing. The mixtures were incubated for 15 minutes at 4°C protected from light, and magnetic separation was performed using the OctoMACS system (Miltenyi Biotec). CD8 + T cells infiltration assay After 48 hours of co-incubation with rhFzd7, the maturation of DCs (Isolated from human PBMC) was assessed by FCM based on the expression of CD80/CD86 markers. Mature DCs loaded with Fzd7 antigen were then co-cultured with CD8⁺ T cells (stained with Dil) for 48 hours at a DC:T cell ratio of 1:3 to generate adoptive T cells. Following the procedure of the aforementioned Transwell assay, 4 × 10⁴ MRC-5 cells were diluted in complete medium and mixed with an equal volume of Matrigel to form an ECM barrier. A total of 5 × 10⁴ A549 or H1975 cells (stained with DiO) were seeded into the upper chamber of an ultra-low attachment 24-well plate and treated with the respective antibodies according to the experimental groups. Then, 5 × 10⁴ adoptive T cells were added to the upper chamber. After 24 hours, the 24-well plate was examined under a fluorescence microscope to evaluate T cell infiltration. Single-cell RNA sequencing and analysis Following the induction of MRC5 into CAFs using A549-CM, the resulting CAFs were divided into three experimental groups (n = 3): Control, Atezolizumab (100 nM), and SHH-YN-Bi (100 nM). After a 24-hour incubation in 5% FBS DMEM, cells were collected via PBS washing and centrifugation (1200 rpm, 5 min, twice). Single-cell suspensions meeting quality criteria (viability > 80%, > 1×10 6 cells/group) were used for library preparation. Single-cell isolation was achieved using Gel Bead-in-Emulsions (GEMs), followed by post-GEM-RT cleanup, cDNA amplification, and quality Control. Finally, sequencing was carried out on a DNBSEQ-T7 platform with a PE100/150 protocol. Data analysis was performed using the Majorbio Cloud (www.majorbio.com). Statistical analysis All results were presented as mean ± s.d., and all experiments were independently repeated at least three times with at least three independent biological samples. Student’s t-test and ANOVA were used to determine significant differences. GraphPad Prism ( V.10.0 ) was used for specific comparisons throughout the manuscript, with P values indicated in figures. A P value of < 0.05 was considered statistically significant. In the direct image presentation, the results from the same experiment were selected for presentation. Declarations Competing interests The authors declare that they have no competing interests. Author Contribution Shuyang Mao: Writing – review & editing, Methodology, Conceptualization, Writing – original draft, Investigation. Xiaofan Zhang: Funding acquisition, Software, Formal analysis, Data curation. Zitong Wang: Formal analysis, Data curation. Pan Zhou: Investigation, Data curation. Zixuan Wang: Investigation, Validation. Yijun Zhao: Investigation. Jiayi Ye: Investigation. Guoqing Wan: Formal analysis. Tong Wang: Data curation. Gangyi Hu: Data curation. Kexin Zhu: Data curation. Yuxia Liu: Investigation. Lin Yang: Funding acquisition, Conceptualization. Xiaofei Zhang: Funding acquisition, Supervision. Gang Huang: Funding acquisition, Conceptualization, Supervision. Qingqing Huang: Formal analysis, Conceptualization. Wei Xie: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. All authors reviewed the manuscript. Acknowledgements Thanks all support from Shanghai Key Laboratory of Molecular Imaging & Digital and Intelligent Empowerment Biomedical Innovation Center, Pudong Gongli Hospital, School of Pharmacy, Shanghai University of Medicine and Health Sciences. This article was supported by the Natural Science Foundation of Shanghai (23ZR1427400); Key Clinical Program of Shanghai Municipal Health Commission (20214Y0516); Construction project of Shanghai Key Laboratory of Molecular Imaging (18DZ2260400); National Natural Science Foundation of China (82203714, 32300295). 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Xie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYDACZiD+AMQGIA4PsVoYZ5CmBaSLhyQtBseZj0nb/Dkcbc5/gPHB2zYGeXNCWiSb2dKkc9sO5+6ckcBsOLeNwXBnAwEt/Mw8ZrdzGw7nbrjBwCbN28aQYHCAgBY2Zv5vty3+ALWcP8D+mygtQFvYbjOwAbUcSGBjJkoL0C/mP3vb0oEOS2yWnHNOwnADIS0G5w8/NvjxxxrosMMHP7wps5EnaAsSYGwAEhLEqx8Fo2AUjIJRgBsAAGkUPjEwXK61AAAAAElFTkSuQmCC","orcid":"","institution":"Shanghai University of Medicine and Health Sciences","correspondingAuthor":true,"prefix":"","firstName":"Wei","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2026-04-07 01:09:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9337927/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9337927/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108090437,"identity":"951c23d5-a3df-4743-a99f-abfa516150bd","added_by":"auto","created_at":"2026-04-29 09:13:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11843125,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh Fzd7 Levels are Linked to Low Immune Infiltration in NSCLC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eStratification of the TCGA lung cancer cohort into Fzd7 low (\u0026lt; 4.12) and Fzd7 high (\u0026gt; 6.23) expression groups based on a Bayesian Gaussian Mixture Model. \u003cstrong\u003eB \u003c/strong\u003eComparison of immune signature enrichment scores between Fzd7 low and Fzd7 high groups using ssGSEA. \u003cstrong\u003eC\u003c/strong\u003e Expression of significantly altered Wnt/β-Catenin immune-related genes in Fzd7 low versus Fzd7 high groups. Data are presented as Log₂(TPM). \u003cstrong\u003eD \u003c/strong\u003eRepresentative FISH Images of Fzd7(red) and PD-L1(green) in Lung Cancer TMA, bar = 200 μm. \u003cstrong\u003eE\u003c/strong\u003eDifferential Fzd7 mRNA expression between tumor and paired adjacent tissues, detected by RNAscope FISH. \u003cstrong\u003eF \u003c/strong\u003ePositive correlation of Fzd7 with PD-L1 mRNA in NSCLC tumors, assessed by Pearson correlation analysis (\u003cem\u003eP \u003c/em\u003e= 0.011). \u003cstrong\u003eG \u003c/strong\u003eRepresentative mIHC Images of CK(purple), Fzd7(green), PD-L1(blue), β-catenin(red) and c-Myc(yellow) in Lung Cancer TMA, bar = 200 μm. \u003cstrong\u003eH \u003c/strong\u003eFzd7 high/medium expression between tumor and paired adjacent tissues, detected by TSA mIHC. \u003cstrong\u003eI \u003c/strong\u003eDistribution of Fzd7⁺ PD-L1⁺ cells in tumor and paired adjacent tissues. \u003cstrong\u003eJ, K \u003c/strong\u003eSpatial expression of Fzd7, CD8, and CD141 in orthotopic A549/H1975 xenograft tumors detected by mIHC, bar = 20 μm. \u003cstrong\u003eL, M\u003c/strong\u003e Correlation analysis of Fzd7, CD8, and CD141, n = 3. Results are presented as mean ± SD. Paired t tests and ANOVA was used to determine significant differences, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9337927/v1/be6ccd48c0bea8f372f3e4f9.png"},{"id":108182293,"identity":"4b8cb2fa-2844-4a5a-8c9f-05ce0db0539e","added_by":"auto","created_at":"2026-04-30 08:59:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17728151,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSHH002-hu1 Enhances the Therapeutic Sensitivity of Atezolizumab in NSCLC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eSuppression of Wnt3a (200 ng/mL)-induced Fzd7 and PD-L1 upregulation by Fzd7 siRNA and SHH002-hu1 (100 nM) in A549, H1975, and LLC cells. Representative Western blots are shown. \u003cstrong\u003eB \u003c/strong\u003e\u003cem\u003eIn vivo\u003c/em\u003e animal models and drug administration. \u003cstrong\u003eC, D\u003c/strong\u003e \u003cem\u003eIn vivo\u003c/em\u003e bioluminescence imaging of orthotopic A549/H1975-luc tumors in CD34\u003csup\u003e+\u003c/sup\u003e humanized NSG mouse following treatment with PBS (100 μL), SHH002-hu1 (100 nM), Atezolizumab (100 nM), or SHH002-hu1 and Atezolizumab combination (n = 5).\u003cstrong\u003e E, F \u003c/strong\u003eTumor growth curves monitored by total radiance (10\u003csup\u003e5\u003c/sup\u003ep/s/cm\u003csup\u003e2\u003c/sup\u003e/sr) in A549 and H1975 models across different treatment groups. \u003cstrong\u003eG, H \u003c/strong\u003eExpression of PD-L1 and β-catenin in orthotopic A549/H1975 xenograft tumors detected by HE and mIHC. Staining showed HE (purple), PD-L1 (red) and β-catenin (green) merged with DAPI-stained nuclei (blue), bar = 20 μm. \u003cstrong\u003eI, J \u003c/strong\u003emIHC analysis of β-catenin and PD-L1 expression in A549 and H1975 orthotopic tumor tissues across different treatment groups. Results are presented as mean ± SD. \u003cem\u003eP\u003c/em\u003e values were determined by ANOVA, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9337927/v1/e0ea26aeb20043530e53b481.png"},{"id":108090441,"identity":"75b2ccf5-244b-4713-98ef-0cb8a97a0008","added_by":"auto","created_at":"2026-04-29 09:13:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6936974,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration and Characterization of SHH-YN-Bi\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eSchematic diagram of SHH-YN-Bi construction (KIH). \u003cstrong\u003eB\u003c/strong\u003e SDS-PAGE analysis of SHH-YN-Bi. \u003cstrong\u003eC \u003c/strong\u003eSet of sensorgrams of rhFzd7 (ranged from 1.5625 to 200 nM) or rhPD-L1 (ranged from 3.125 to 200 nM) binding with SHH-YN-Bi. \u003cstrong\u003eD\u003c/strong\u003e (a) Binding affinity of SHH002-hu1 and SHH-YN-Bi to CHO-K1-OE-Fzd7 cells. Dose-response curves from FCM are shown, with fitted EC50 values of 2.314 nM and 4.483 nM, respectively. (b) Binding affinity of Atezolizumab and SHH-YN-Bi to CHO-K1-OE-PD-L1 cells. Dose-response curves from FCM are shown, with fitted EC50 values of 1.892 nM and 5.535 nM, respectively. \u003cstrong\u003eE \u003c/strong\u003e(a) Competitive binding assay for Wnt3a/Fzd7 blockade. CHO-K1-OE-Fzd7 were incubated with Wnt3a-FITC and increasing concentration of indicated antibodies. Dose-response curves from FCM are shown, with fitted IC50 values of 6.467 nM and 6.861 nM, respectively. (b) Competitive binding assay for PD-1/PD-L1 blockade. CHO-K1-OE-PD-L1 were incubated with fluorescently labeled PD-1 (PD-1-FITC) together with increasing concentrations of Atezolizumab, SHH-YN-Bi, and Isotype-Control. Dose-response curves from FCM are shown, with fitted IC50\u003csub\u003e \u003c/sub\u003evalues of 4.491 nM and 8.226 nM, respectively. \u003cstrong\u003eF \u003c/strong\u003eSet of sensorgrams of rhFzd7 binding with SHH-YN-Bi and rhPD-L1. 1000 nM rhPD-L1 was injected after 1000 nM SHH-YN-Bi was flowed over the 100μg/mL rhFzd7-immobilized NTA sensor chip. \u003cstrong\u003eG\u003c/strong\u003e Binding affinity of SHH002-hu1, Atezolizumab and SHH-YN-Bi to A549/H1975 cells. \u003cstrong\u003eH, I \u003c/strong\u003e(a)\u003cstrong\u003e \u003c/strong\u003eNIRB imaging assay to evaluate the bio-distribution of NIRB-Atezolizumab and NIRB-SHH-YN-Bi in A549 bearing nude mouse. (b) In blocking experiments, free Atezolizumab and SHH-YN-Bi inhibited the probes from binding to the tumor sites. Tumor/normal tissue ratios calculated at 12 h post-injection of probe groups into orthotopic A549 tumors in nude mouse from the region of interest. Results are presented as mean ± SD. \u003cem\u003eP\u003c/em\u003e values were determined by ANOVA and Paired t tests, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9337927/v1/3bd5cc43052b08cc5e8b94f9.png"},{"id":108182701,"identity":"e21c61de-b5af-4b68-bdb0-a5067d974263","added_by":"auto","created_at":"2026-04-30 08:59:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":14233887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSHH-YN-Bi Suppresses Tumor Progression and Enhances Effector T Cell Cytotoxicity by Inhibiting Wnt/β-Catenin Pathway and Blocking PD-1/PD-L1 Interaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u0026nbsp;Representative images of migration assays in A549, H1975, and LLC cells under the indicated treatment conditions, bar = 100 μm. \u003cstrong\u003eB\u003c/strong\u003e Quantification of cell migration. Migration rates were normalized to Wnt3a group (set as 100%) and are presented as mean ± SD, n = 3, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003eC\u003c/strong\u003e Representative images of invasion assays in A549, H1975, and LLC cells, bar = 100 μm. \u003cstrong\u003eD\u003c/strong\u003e Quantification of cell invasion. Invasion rates were normalized to Wnt3a group (set as 100%) and are presented as mean ± SD, n = 3, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003eE\u003c/strong\u003e Western blot analysis of the Wnt/β-Catenin signaling associated targets (total β-catenin, nuclear β-catenin, total LRP6, LRP6-Pi, VEGFA, c-Myc, and CD44) under the indicated treatments. \u003cstrong\u003eF\u003c/strong\u003e A549, H1975, and LLC cells were treated under indicated conditions and assayed using a TCF/LEF luciferase reporter. Data are presented as relative luciferase units (RLU, fold change versus untreated Control) and mean ± SD, n = 3, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003eG\u003c/strong\u003e Cytotoxicity assay to assess the TALL-104/mouse CD8\u003csup\u003e+\u003c/sup\u003e T cells-mediated killing of NSCLC cells. Data were presented as mean ± SD, n = 3, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003eH \u003c/strong\u003eNFAT reporter assay of PD-1/PD-L1 blockade. SHH-YN-Bi (IC50\u003csub\u003e \u003c/sub\u003e= 2.74 nM) and Atezolizumab (IC50\u003csub\u003e \u003c/sub\u003e= 0.77 nM) effectively disrupted PD-1/PD-L1 interaction.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9337927/v1/171be6dec71caa46731a9489.png"},{"id":108181591,"identity":"e688e520-34ee-4857-b722-7a2cbc30837a","added_by":"auto","created_at":"2026-04-30 08:58:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8985287,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Restoration of Anti-tumor T Cell Activation and Infiltration by SHH-YN-Bi Depends on the Recruitment of Batf3\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cDC1s\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003e\u003cem\u003eIn vivo\u003c/em\u003e\u0026nbsp;bioluminescence imaging of orthotopic LLC-luc tumors in \u003cem\u003eBatf3\u003c/em\u003e-KO C57BL/6 mouse following treatment with PBS (100 μL), SHH002-hu1 (100 nM) and SHH-YN-Bi (100 nM) (n = 5). \u003cstrong\u003eB \u003c/strong\u003eTumor growth curves monitored by total radiance (10\u003csup\u003e4\u003c/sup\u003ep/s/cm\u003csup\u003e2\u003c/sup\u003e/sr) in LLC models. \u003cstrong\u003eC\u003c/strong\u003e HE and mIHC staining to detect the infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells and CD103\u003csup\u003e+\u003c/sup\u003e cDC1 cells in orthotopic LLC xenografted tumors. Staining showed HE (purple), CD3 (blue), CD8 (green), CD39 (red), CD103 (yellow) and PanCK (purple) merged with DAPI-stained nuclei (blue), bar = 20 μm. \u003cstrong\u003eD\u003c/strong\u003e Quantitative analysis of CD3\u003csup\u003e+\u003c/sup\u003e cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells and CD39\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells relative infiltration rate. Data were presented as the mean ± SD. \u003cstrong\u003eE\u003c/strong\u003e ChIP analysis of ATF3 binding to \u003cem\u003eCcl4\u003c/em\u003e promoter region. qPCR was performed to calculate fold enrichment relative to IgG Control. Data were presented as mean ± SD, n = 3, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003eF\u003c/strong\u003e ELISA analysis of CCL4 secretion in NSCLC cells under indicated conditions. Data were presented as mean ± SD, n = 3, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9337927/v1/046338a76f4462ef8f60de8b.png"},{"id":108181500,"identity":"afa8afc6-e4b4-4fc0-aaf6-432f7dfcf231","added_by":"auto","created_at":"2026-04-30 08:58:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":11327114,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSHH-YN-Bi Restores Immune Infiltration in NSCLC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eTumor growth curves monitored by total radiance (10\u003csup\u003e5\u003c/sup\u003ep/s/cm\u003csup\u003e2\u003c/sup\u003e/sr) in A549, H1975, and LLC models across different treatment groups. \u003cstrong\u003eB\u003c/strong\u003e Quantitative analysis of PD-L1 and β-catenin. Data were presented as the mean ± SD, n = 3, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. \u003cstrong\u003eC \u003c/strong\u003eHE and mIHC staining to detect the infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells and CD141\u003csup\u003e+\u003c/sup\u003e cDC1s in orthotopic A549 xenografted tumors. Staining showed HE (purple), CD3 (red), CD8 (purple), CD39 (green), CD141/CD103 (yellow) and PanCK (orange) merged with DAPI-stained nuclei (blue), bar = 20 μm. \u003cstrong\u003eD\u003c/strong\u003e Quantitative analysis of CD3\u003csup\u003e+\u003c/sup\u003e cells, CD8\u003csup\u003e+\u003c/sup\u003e T cells, CD39\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells and CD141\u003csup\u003e+\u003c/sup\u003e cDC1s relative infiltration rate. Data were presented as the mean ± SD, n = 3, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-9337927/v1/a26505e25d440267e7879b35.png"},{"id":108090445,"identity":"644b1eb9-ab80-450e-9d4e-aa1b7d3e2907","added_by":"auto","created_at":"2026-04-29 09:13:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10899971,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSHH-YN-Bi Disrupts the ECM Remodeling and Deposition by CAFs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA \u003c/strong\u003eConversion of MRC-5 fibroblasts to CAFs by A549 CM. \u003cstrong\u003eB \u003c/strong\u003eInduction inhibitory effects of different treatments assessed by mIHC. Representative images show reduced expression of COL1A1 (green), FAP (yellow) and α-SMA (purple) merged with DAPI-stained nuclei (blue), bar = 20 μm. \u003cstrong\u003eC \u003c/strong\u003eQuantitative analysis of COL1A1, FAP and α-SMA relative MFI. Data were presented as the mean ± SD, n = 3, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. \u003cstrong\u003eD \u003c/strong\u003eImmune infiltration assay of CD8\u003csup\u003e+\u003c/sup\u003e T cells. \u003cstrong\u003eE \u003c/strong\u003eRepresentative images of co-culture models showing A549/H1975 cells (red) and CD8\u003csup\u003e+\u003c/sup\u003e T cells (green) under different treatment conditions. Drugs were applied to CAFs alone or to both CAFs and tumor cells. \u003cstrong\u003eF\u003c/strong\u003e Quantitative analysis of CD8\u003csup\u003e+\u003c/sup\u003e T cells relative infiltration rate. Data were presented as the mean ± SD, n = 3, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. \u003cstrong\u003eG\u003c/strong\u003e UMAP clustering reveals 23 distinct clusters of MRC-5-derived CAFs across Control, Atezolizumab, and SHH-YN-Bi treatment groups. \u003cstrong\u003eH \u003c/strong\u003eWnt/β-Catenin pathway gene set scoring in CAFs under different treatment conditions. \u003cstrong\u003eI\u003c/strong\u003e ECM deposition gene set scoring in CAFs under different treatment conditions. \u003cstrong\u003eJ \u003c/strong\u003eKEGG pathway enrichment analysis of clusters 8 and 20. \u003cstrong\u003eK\u003c/strong\u003e GSVA reveals pathway suppression following SHH-YN-Bi treatment. \u003cstrong\u003eL \u003c/strong\u003eVolcano plot of differentially expressed genes (DEGs): SHH-YN-Bi vs. Control. \u003cstrong\u003eM \u003c/strong\u003ePseudotime analysis reveals the developmental trajectories of clusters 8 and 20 across treatment groups.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-9337927/v1/96e4c3330c3e2b1ae8bd9348.png"},{"id":108181799,"identity":"02d6989f-3168-4019-9752-ff517e15d504","added_by":"auto","created_at":"2026-04-30 08:58:56","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":7957161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration depicting the role of SHH-YN-Bi in NSCLC\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-9337927/v1/8ae7fc7327268475524140c2.png"},{"id":109080981,"identity":"7bb85913-95a4-4d29-9fba-2ecbf32b012c","added_by":"auto","created_at":"2026-05-12 11:38:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":83946934,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9337927/v1/00b9f0e8-289c-44b4-90d2-619be4199680.pdf"},{"id":108181911,"identity":"2837880e-e84a-4d68-b397-bf094056aae4","added_by":"auto","created_at":"2026-04-30 08:59:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2802712,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"supplementarydata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9337927/v1/e81d36142612e4ed0c721149.pdf"},{"id":108090439,"identity":"dcd95980-af31-41ac-b016-71248d4a2be6","added_by":"auto","created_at":"2026-04-29 09:13:53","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":527267,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-9337927/v1/110ea4259fb4fac1a015f2c6.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Novel Bispecific Antibody Targeting Frizzled7 and PD-L1 Reverses Immunotherapy Resistance in Non-Small Cell Lung Cancer by Reprogramming the Tumor Microenvironment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSince the U.S. Food and Drug Administration (FDA) first approved Nivolumab and Pembrolizumab for the treatment of advanced NSCLC, ICI therapy has progressively transformed the treatment paradigm of NSCLC from chemotherapy to immunotherapy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, a substantial proportion of patients still do not derive clinical benefit [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among these patients, those with \u0026ldquo;hot tumors\u0026rdquo; who initially respond to ICIs may develop acquired resistance due to the upregulation of alternative immune checkpoints (ICPs), loss of tumor antigen expression or presentation, induction of T-cell exhaustion, or metabolic dysregulation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In contrast, the majority of NSCLCs are classified as \u0026ldquo;cold tumors,\u0026rdquo; characterized by low tumor mutational burden (TMB), low PD-L1 expression, and limited immune cell infiltration. These tumors exhibit primary resistance to ICIs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These observations highlight that immunotherapy resistance represents a major obstacle limiting the clinical benefit for NSCLC patients.\u003c/p\u003e \u003cp\u003eGiven the significant impact of non-immune components in the TME on the response to ICIs, tumors with high TMB and intermediate/high PD-L1 expression still paradoxically display abundant infiltration of CAFs, regulatory T cells (Tregs), and tumor-associated macrophages (TAMs). These factors contribute to an immunosuppressive TME and lead to CD8\u003csup\u003e+\u003c/sup\u003e T-cell dysfunction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. To overcome ICI-resistance, anti-PD-(L)1 antibodies are commonly combined with other immunostimulatory or immunogenic therapeutic modalities [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, simultaneous targeting of the TME and ICPs is a promising approach to reverse ICI resistance.\u003c/p\u003e \u003cp\u003eThe Wnt/β-Catenin pathway is an oncogenic pathway with immunomodulatory properties [\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], modulating PD-L1 expression and fostering a non-T cell-inflamed TME [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Fzd7 is a key receptor in the Wnt/β-Catenin pathway, exhibits upregulated expression in various malignancies, including NSCLC. Transcriptomic analysis in this study revealed that high Fzd7 expression is linked to stronger Wnt/β-Catenin signaling and weaker immune infiltration in NSCLC (Figure. 1A-C). SHH002-hu1, a full-length humanized antibody targeting Fzd7 developed by us previously, has demonstrated significant anti-tumor activity in triple-negative breast cancer (TNBC) and NSCLC [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, an NK-cell engager (NKCE) derived from SHH002-hu1, designated SHH002-hu1-MICA, has shown significant anti-tumor effects against TNBC cancer stem cells (TNBCSCs), highlighting its potential as a therapeutic agent capable of both inhibiting oncogenic signaling and enhancing immune cell infiltration [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Given that Fzd7 represents a promising cooperative target for tumor-targeted therapy and immunotherapy, the precise mechanism by which Fzd7 contributes to the immunosuppressive TME remains to be elucidated.\u003c/p\u003e \u003cp\u003eThis study employed fluorescence in situ hybridization (FISH) and Tyramide Signal Amplification-based multiplex fluorescence immunohistochemistry (TSA mIHC) to analyze human NSCLC tissue microarrays (TMAs), confirming that Fzd7 is remarkably enriched in NSCLC tissues and correlated with PD-L1 expression. The combination treatment of SHH002-hu1 and Atezolizumab showed significant tumor suppression in multiple orthotopic models. These findings indicate potential synergistic anti-tumor effects between blocking Fzd7 and PD-L1. Herein, we further developed a novel bispecific antibody (BsAb), SHH-YN-Bi, that simultaneously targets Fzd7 and PD-L1, demonstrating effective dual binding to Fzd7 and PD-L1 and superior tumor targeting over Atezolizumab. Subsequent investigations revealed that SHH-YN-Bi exerted potent anti-tumor effect through blocking oncogenic Wnt3a/Fzd7/β-Catenin signaling and PD-1/PD-L1 axis. Moreover, scRNA-seq analysis indicated that Fzd7 is highly correlated with ECM remodeling in CAFs, and SHH-YN-Bi treatment induced a phenotypic switch of CAFs from a highly oncogenic to a normal state, associated with elevated CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration. Our study elucidates the role of Fzd7 in promoting Batf3\u003csup\u003e+\u003c/sup\u003e cDC1 deficiency through the Wnt3a/Fzd7/β-Catenin/ATF3 axis, and uncovers the involvement of Fzd7 in CAF-mediated ECM remodeling. Addressing the critical clinical challenge of ICI-resistance in NSCLC, SHH-YN-Bi represents a novel therapeutic strategy that integrates Wnt targeted therapy and immunotherapy.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eHigh Fzd7 Expression in NSCLC is Positively Associated with PD-L1 Level and Reduced Immune Cell Infiltration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFzd7, a key receptor of the Wnt/β-Catenin pathway, is significantly overexpressed in NSCLC [21]. Based on transcriptomic data analysis from TCGA lung cancer cohort, the patients were stratified into Fzd7-low and Fzd7-high groups based on expression cut-offs of 4.12 and 6.23, respectively (Figure. 1A). Single-sample gene set enrichment analysis (ssGSEA) revealed that high Fzd7 expression was associated with significantly lower enrichment scores across multiple immune-related biological processes and immune cell markers (Figure. 1B). Further analysis indicated that genes associated with Wnt/β-Catenin signaling, such as \u003cem\u003eATF3\u003c/em\u003e, \u003cem\u003eCD44\u003c/em\u003e, and \u003cem\u003eCD274\u003c/em\u003e, were upregulated in Fzd7-high tumors, whereas genes related to immune cell infiltration, including \u003cem\u003eCD8A\u003c/em\u003e, \u003cem\u003eCCL4\u003c/em\u003e, and \u003cem\u003eBatf3\u003c/em\u003e, were consistently downregulated (Figure. 1C). RNAscope FISH analysis on a human NSCLC TMA (HLugA150CS03) confirmed elevated Fzd7 mRNA levels in tumor tissues compared to adjacent normal tissues (Figure. 1D, E; Supplementary Figure. S1A). Correlation analysis further revealed a significant positive association between Fzd7 and PD-L1 mRNA expression levels (Figure. 1F; Supplementary Figure. S1B). mIHC analysis of NSCLC TMA (HLugA150CS03, HLugA060PG02) revealed significantly higher proportions of tumor cells with high or moderate Fzd7 expression, as well as increased frequencies of Fzd7 and PD-L1 double-positive cells in tumor tissues compared to paracancerous tissues (Figure. 1G-I; Supplementary Figure. S1C, D). Moreover, the expression of Fzd7, nuclear-β-catenin, and total-β-catenin each correlated positively with PD-L1 protein levels in tumors (Supplementary Figure. S1E-G). Notably, in CD34\u003csup\u003e+\u003c/sup\u003e humanized NSG mouse bearing orthotopic A549/H1975 tumors, regions with high Fzd7 expression exhibited significantly fewer CD8\u003csup\u003e+\u003c/sup\u003e T cells and CD141\u003csup\u003e+\u003c/sup\u003e cDC1s (Figure. 1J-M). These findings indicate that in NSCLC, Fzd7 overexpression is closely associated with aberrant activation of the Wnt/β-Catenin pathway, and drives PD-L1 upregulation, thereby contributing to an immunosuppressive TME characterized by reduced immune infiltration and a deficiency in Batf3\u003csup\u003e+\u003c/sup\u003e cDC1-dependent CD8\u003csup\u003e+\u003c/sup\u003e T cells.\u003c/p\u003e\n\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003eSHH002-hu1 Enhances Atezolizumab Sensitivity to NSCLC by Blocking the Wnt/β-Catenin pathway\u003c/h2\u003e\n \u003cp\u003eSince the FDA approval of Atezolizumab for second-line or later treatment of advanced NSCLC [1, 23], α-PD-L1-based immunotherapies have gained considerable attention in clinical research. However, not all patients with high PD-L1 expression benefit from ICI therapy, primarily due to immune exclusion in the TME and dysfunction of effector T cells [1, 24]. To address these challenges, multiple combination immunotherapy approaches have been explored [25–27].\u003c/p\u003e\n \u003cp\u003eThis study employed two Atezolizumab-insensitive human NSCLC cell lines, A549 and H1975 [28], along with murine LLC cells, which are also associated with a low immune infiltration TME [29]. Western blot analysis confirmed that both Fzd7 siRNA and SHH002-hu1 effectively reversed the Wnt3a-induced upregulation of PD-L1 and Fzd7 in A549, H1975, and LLC cells (Figure. 2A). Given the central role of DCs in anti-tumor immunity, CD34\u003csup\u003e+\u003c/sup\u003e humanized NSG mouse were employed to enable stable reconstitution of a human immune system containing lymphocytes and DCs [28]. Orthotopic A549/H1975-luc and LLC-luc tumors were established in CD34\u003csup\u003e+\u003c/sup\u003e humanized NSG mouse and C57BL/6 mouse, respectively (Figure. 2B), and tumor growth was dynamically monitored using \u003cem\u003ein vivo\u003c/em\u003e bioluminescent imaging (Figure. 2C, D; Supplementary Figure. S2A). As shown in Figure. 2E, F and Supplementary Figure. S2B, Atezolizumab monotherapy was insensitive against all three cell lines \u003cem\u003ein vivo\u003c/em\u003e, whereas SHH002-hu1 alone, or in combination with Atezolizumab, significantly inhibited tumor progression. Notably, the combination therapy exhibited superior efficacy compared to SHH002-hu1 monotherapy. Furthermore, mIHC of tumor tissues (Figure. 2G-J; Supplementary Figure. S2C, D) revealed that SHH002-hu1 and the combination treatment markedly reduced β-catenin and PD-L1 expression and suppressed β-catenin nuclear translocation. In contrast, Atezolizumab exerted no effect on either β-catenin or PD-L1 levels. Collectively, these findings demonstrate that SHH002-hu1 restores sensitivity of NSCLC to Atezolizumab by targeting the Wnt/β-Catenin signaling pathway.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eDesign and Characterization of a BsAb Targeting Fzd7 and PD-L1\u003c/h3\u003e\n\u003cp\u003eGiven that the Wnt/β-Catenin signaling pathway regulates PD-L1 expression, the prevalence of Fzd7/PD-L1 double-positive cells in NSCLC, and the enhanced \u003cem\u003ein vivo\u003c/em\u003e sensitivity to Atezolizumab upon Fzd7 inhibition, we engineered a BsAb, SHH-YN-Bi, capable of simultaneously binding Fzd7 and PD-L1 using the Konb-into-Hole technology (Figure. 3A). The H-chain HC1 (Human IgG1 Knob, T366W) was constructed based on the Atezolizumab H-chain sequence, while the H-chain HC2 (Human IgG1 Hole, T366S, L368A, Y407V) was based on the SHH002-hu1 H-chain sequence. The L-chain consisted of LC1 (Human Lambda) from Atezolizumab and LC2 (Human Kappa) from SHH002-hu1. All four chains were co-expressed in ExpiCHO-S cells, and the resulting BsAb was purified by protein A affinity chromatography.\u003c/p\u003e\n\u003cp\u003eSDS-PAGE analysis of the purified SHH-YN-Bi confirmed the correct expression of the H-chain (50 kDa) and L-chain (25 kDa) at the expected molecular weights, assembled into a complete protein with an approximate molecular mass of 150 kDa (Figure. 3B). HPLC-MS analysis of the deglycosylated protein showed a measured molecular weight of 144,565.8 Da, within 4 Da of the theoretical mass (144,562.3 Da), and 89% of molecules exhibited correct chain pairing. Primary post-translational modifications (PTMs) were predominantly limited to G0F and G1F glycoforms on the H-chains, with no detectable alterations in complementarity-determining region (CDR) integrity (Supplementary Figure. S3A). SEC-HPLC indicated a purity of approximately 95% for the purified SHH-YN-Bi (Supplementary Figure. S3B), while CE-SDS under non-reducing and reducing conditions revealed purities of 93.24% and 94.71%, respectively (Supplementary Figure. S3C). DSF analysis demonstrated favorable thermal stability, with melting temperatures (Tm1: 67.00°C, Tm2: 74.80°C) approaching those of Herceptin (Tm1: 68.40°C, Tm2: 79.20°C) (Supplementary Figure. S3D). cIEF further revealed a charge variant profile consisting of a main peak (34.06%, pI 8.55), an acidic peak (33.46%, pI 8.02), and a basic peak (32.47%, pI 9.22) (Supplementary Figure. S3E). These results collectively support the correct assembly, structural integrity, and promising developability of SHH-YN-Bi.\u003c/p\u003e\n\u003cp\u003eTo evaluate the affinity of SHH-YN-Bi, SPR assays demonstrated that SHH-YN-Bi exhibits extremely high affinity for rhFzd7 (\u003cem\u003eka\u003c/em\u003e (1/Ms): 1.47 × 10⁵, \u003cem\u003ekd\u003c/em\u003e (1/s): 3.94 × 10⁻⁴, \u003cem\u003eKD\u003c/em\u003e (M): 2.69 × 10⁻⁹) and rhPD-L1 (\u003cem\u003eka\u003c/em\u003e (1/Ms): 1.48 × 10⁵, \u003cem\u003ekd\u003c/em\u003e (1/s): 4.87 × 10⁻⁶, \u003cem\u003eKD\u003c/em\u003e (M): 3.29 × 10⁻¹¹) (Figure. 3C). To confirm that SHH-YN-Bi retains the binding activity of its parental antibodies, binding to CHO-K1 cells overexpressing Fzd7 or PD-L1 was assessed by FCM. SHH-YN-Bi bound both CHO-K1-OE-Fzd7 (EC50 = 4.483 nM) and CHO-K1-OE-PD-L1 (EC50 = 5.535 nM) in a dose-dependent manner, with no significant reduction in potency compared to SHH002-hu1 (EC50 = 2.314 nM for Fzd7) and Atezolizumab (EC50 = 1.892 nM for PD-L1) (Figure. 3D). FCM further confirmed that SHH-YN-Bi competitively inhibited Wnt3a binding to CHO-K1-OE-Fzd7 cells in a dose-dependent manner (IC50 = 6.861 nM), with potency comparable to that of SHH002-hu1 (IC50 = 6.467 nM). Similarly, SHH-YN-Bi inhibited PD-1 binding to CHO-K1-OE-PD-L1 (IC50 = 8.226 nM), with activity slightly lower than Atezolizumab (IC50 = 4.491 nM) (Figure. 3E).\u003c/p\u003e\n\u003cp\u003eThe therapeutic potential of SHH-YN-Bi in NSCLC relies on its ability to simultaneously engage both Fzd7 and PD-L1. As shown in Fig.\u0026nbsp;3F, when rhFzd7 was immobilized on an NTA sensor chip and 1000 nM SHH-YN-Bi was injected as the analyte, a significant binding signal emerged at approximately 500 seconds. Subsequent injection of 1000 nM rhPD-L1 resulted in a further significant binding signal around 700 seconds, demonstrating that rhPD-L1 can bind to the preformed SHH-YN-Bi-rhFzd7 complex. To investigate the binding ability in tumor cells, SHH-YN-Bi was evaluated in A549, H1975, and LLC cells. In A549 and LLC cells (high Fzd7, low PD-L1), SHH-YN-Bi exhibited effective binding mediated by the highly expressed target (Figure. 3G; Supplementary Figure. S3F). In contrast, in H1975 cells (high Fzd7, high PD-L1), SHH-YN-Bi demonstrated enhanced binding relative to its parental monoclonal antibodies (Figure. 3G). These findings indicate that SHH-YN-Bi effectively engages tumor cells with either dual-high target expression or predominant single-target expression, highlighting its functional adaptability across heterogeneous antigen TME.\u003c/p\u003e\n\u003cp\u003eThe specific targeting capability of SHH-YN-Bi to NSCLC tissues is essential for its therapeutic efficacy. We previously demonstrated that SHH002-hu1 specifically binds to Fzd7-positive xenograft tumor tissues [21]. This study further evaluated the targeting specificity of Atezolizumab and SHH-YN-Bi in A549 xenograft models. Following administration of NIRB-labeled Atezolizumab and SHH-YN-Bi, fluorescent signals rapidly distributed throughout the body. By 8 hours post-injection, A549 xenografts were clearly delineated by fluorescence, with detectable signals persisting up to 32 hours (Figure. 3H, I). In contrast, the blocking group exhibited no significant fluorescence accumulation at the tumor site, confirming that tumor targeting was specifically mediated by Atezolizumab and SHH-YN-Bi. Notably, the tumor/normal tissue fluorescence ratio in the SHH-YN-Bi group was higher than that in the Atezolizumab group at 12 hours (Figure. 3I(c)), featuring superior targeting capability compared with Atezolizumab. These findings establish a critical foundation for subsequent evaluation of the anti-tumor efficacy of SHH-YN-Bi.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSHH-YN-Bi Suppresses the Migration and Invasion of NSCLCs\u003c/strong\u003e \u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eActivation of the Wnt/β-Catenin pathway is closely associated with tumor cell migration and invasion [30]. SHH-YN-Bi potently inhibited the migratory and invasive capabilities of A549, H1975, and LLC cells in transwell assays. Treatment with 100 nM SHH-YN-Bi significantly reduced Wnt3a-induced cell migration (Figure. 4A, B), with efficacy comparable to that of SHH002-hu1 or in combination with Atezolizumab. Notably, in the LLC model, SHH-YN-Bi exhibited greater inhibitory activity than the combination therapy. In contrast, Atezolizumab had no effect on Wnt3a-driven migration in any of the cell lines tested. Similarly, SHH-YN-Bi significantly suppressed the invasion of Wnt3a-stimulated cells across all models (Figure. 4C, D), achieving inhibition levels equivalent to SHH002-hu1 or combination treatment, while Atezolizumab showed no activity. These results demonstrate that SHH-YN-Bi effectively suppresses migration and invasion in NSCLC cells.\u003c/p\u003e\n\u003cp\u003eTo further investigate whether SHH-YN-Bi suppresses NSCLC cell migration and invasion through inhibition of the Wnt/β-Catenin signaling pathway, we assessed the expression of canonical Wnt pathway components in A549, H1975, and LLC cells. As shown in Fig.\u0026nbsp;4E, Wnt3a stimulation significantly increased levels of total β-catenin, nuclear β-catenin, total LRP6, phosphorylated LRP6 (LRP6-Pi), VEGFA, c-Myc, and CD44. In contrast, treatment with either SHH002-hu1 or SHH-YN-Bi markedly suppressed the Wnt3a-induced upregulation of these components, whereas Atezolizumab exerted no effect. Consistently, in a TCF/LEF luciferase reporter assay, both SHH002-hu1 and SHH-YN-Bi, but not Atezolizumab, suppressed Wnt3a-induced Wnt/β-catenin pathway activation in A549, H1975, and LLC cells (Figure. 4F). These findings demonstrate that SHH-YN-Bi attenuates NSCLC cell migration and invasion by targeting Fzd7 and inhibiting Wnt/β-Catenin pathway activation.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eSHH-YN-Bi Potentiates Effector T cells Cytotoxicity by Conjugation with Tumor cells\u003c/h3\u003e\n\u003cp\u003eSHH-YN-Bi enhances T cell-mediated tumor cell killing by simultaneously engaging Fzd7 and PD-L1 on NSCLC cells. We next evaluated whether SHH-YN-Bi enhances T cell cytotoxicity against NSCLC cells. In co-culture systems containing DiO-labeled (Ex 484 nm/Em 501 nm) T cells and Dil-labeled (Ex 549 nm/Em 565 nm) tumor cells, SHH002-hu1, Atezolizumab, combination therapy, and SHH-YN-Bi progressively reduced tumor cell viability at multiple time points (1 h, 6 h, 12 h) compared to Wnt3a or Control treatments (Supplementary Figure. S4A-C). The combination and SHH-YN-Bi groups exhibited the most pronounced tumor cell clearance, outperforming either monotherapy. Using the CytoTox 96 Non-Radioactive Cytotoxicity Assay, we confirmed that Wnt3a impaired T cell cytotoxic function, whereas all therapeutic interventions restored tumor cell killing (Figure. 4G).\u003c/p\u003e\n\u003cp\u003eTo further evaluate its blocking effect on the PD-1/PD-L1 axis, an NFAT-luciferase reporter gene system was employed. SHH-YN-Bi effectively disrupted the PD-1/PD-L1 interaction with an IC50 of 2.74 nM, although Atezolizumab exhibited greater potency (IC50 = 0.77 nM) (Figure. 4H). Once again, the combination and SHH-YN-Bi groups induced the highest levels of target cell lysis, demonstrating that dual targeting of Fzd7 and PD-L1 most effectively reverses Wnt-mediated T cell dysfunction and enhances anti-tumor immunity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSHH-YN-Bi Induces CD8\u003c/strong\u003e \u003csup\u003e \u003cstrong\u003e+\u003c/strong\u003e \u003c/sup\u003e \u003cstrong\u003eT cell Activation by Recovering Batf3\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e \u003cstrong\u003ecDC1s Function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe anti-tumor activity of ICIs is primarily mediated through the reactivation of pre-existing, DC-primed tumor-reactive T cells [31–33]. In contrast, Wnt/β-Catenin pathway activation in tumors induces the expression of PD-L1 and the transcriptional repressor ATF3, which suppresses CCL4 production. This reduction in CCL4 impairs the recruitment and function of Batf3\u003csup\u003e+\u003c/sup\u003e cDC1s, thereby compromising CD8\u003csup\u003e+\u003c/sup\u003e T cell priming and culminating in an immunosuppressive TME deficient in effective CD8\u003csup\u003e+\u003c/sup\u003e T cells [19].\u003c/p\u003e\n\u003cp\u003eTo investigate whether targeting Fzd7 enhances therapeutic sensitivity in NSCLC by promoting Batf3\u003csup\u003e+\u003c/sup\u003e cDC1-mediated priming and infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells, we employed \u003cem\u003eBatf3\u003c/em\u003e-KO mouse, allocating them into Control, SHH002-hu1, and SHH-YN-Bi treatment groups (Figure. 5A). Following 18 days of treatment, no significant differences in tumor growth kinetics were observed across the experimental groups (Figure. 5B). Moreover, mIHC analysis revealed that, compared to the Control group, SHH002-hu1 treatment did not significantly increase the intratumoral infiltration or activation status of CD8\u003csup\u003e+\u003c/sup\u003e T cells (Figure. 5C, D). These results collectively indicate that the ability of targeting Fzd7 to enhance CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration and activation in NSCLC tumors depends on the restoration of Batf3\u003csup\u003e+\u003c/sup\u003e cDC1 function.\u003c/p\u003e\n\u003cp\u003eTo clarify how targeting Fzd7 affects immune infiltration, western blot analysis showed that SHH002-hu1 or Fzd7 siRNA reduced active β-catenin levels and suppressed Wnt3a-induced pathway activation. SHH002-hu1 markedly decreased ATF3 expression (Supplementary Figure. S5A), and SHH-YN-Bi showed similar effects, while Atezolizumab had no impact (Supplementary Figure. S5B), indicating that Fzd7 targeting downregulates ATF3 \u003cem\u003evia\u003c/em\u003e Wnt/β-Catenin inhibition. ChIP assays revealed that ATF3 binding to the \u003cem\u003eCcl4\u003c/em\u003e promoter was significantly reduced by Fzd7 siRNA or SHH002-hu1 compared to the Wnt3a group in A549, H1975, and LLC cells (Supplementary Figure. S5C). SHH-YN-Bi also attenuated ATF3 binding, whereas Atezolizumab showed no significant effect (Figure. 5E). ELISA results showed increased CCL4 expression after Fzd7 siRNA or SHH002-hu1 treatment compared to Control and Wnt3a groups (Supplementary Figure. S5D); SHH-YN-Bi similarly upregulated CCL4, whereas Atezolizumab had no significant effect (Figure. 5F). In summary, SHH-YN-Bi restores Batf3\u003csup\u003e+\u003c/sup\u003e cDC1 recruitment and CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration by disrupting the Wnt3a/Fzd7/β-Catenin/ATF3 axis.\u003c/p\u003e\n\u003ch3\u003eAnti-tumor Efficacy of SHH-YN-Bi in NSCLC through Promoting CD8 T cells Infiltration\u003c/h3\u003e\n\u003cp\u003eSHH-YN-Bi enhances CD8\u003csup\u003e+\u003c/sup\u003e T cells cytotoxicity through dual mechanisms: blocking PD-1/PD-L1 interaction and targeting Fzd7 to suppress the Wnt/β-Catenin pathway. Evaluation of the \u003cem\u003ein vivo\u003c/em\u003e anti-tumor efficacy of SHH-YN-Bi demonstrated its potent suppression of tumor growth in A549, H1975, and LLC orthotopic models. The anti-tumor activity of SHH-YN-Bi was superior to that of combination therapy (Supplementary Figure. S6A; Figure. 6A). Furthermore, mIHC analysis confirmed that SHH-YN-Bi exerts its anti-tumor effect by inhibiting both the expression and nuclear translocation of β-catenin, leading to downregulation of PD-L1 expression (Figure. 6B; Supplementary Figure. S6B). In immunotherapy, activation and infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells are critically dependent on cDC1s. These cells capture tumor antigens within the TME and migrate to the tumor‑draining lymph nodes (tdLNs) along a chemokine gradient, where they prime naïve CD8\u003csup\u003e+\u003c/sup\u003e T cells for subsequent homing back to the TME to mediate cytotoxic functions [34, 35]. Among these activated populations, CD39\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells are recognized as a key subset exhibiting tumor-reactive specificity [36]. To investigate how SHH-YN-Bi exerts excellent anti-tumor effect \u003cem\u003ein vivo\u003c/em\u003e, we performed mIHC analysis on PanCK\u003csup\u003e+\u003c/sup\u003e tumor regions. The results revealed that SHH002-hu1, combination therapy, and SHH-YN-Bi all significantly increased the densities of CD8\u003csup\u003e+\u003c/sup\u003e T cells and CD141\u003csup\u003e+\u003c/sup\u003e/CD103\u003csup\u003e+\u003c/sup\u003e cDC1s, as well as the frequency of activated CD39\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells, compared to the Control and Atezolizumab groups (Figure. 6C, D; Supplementary Figure. S6C-F). This enhancement was more pronounced with combination therapy and SHH-YN-Bi. In summary, SHH-YN-Bi demonstrates superior \u003cem\u003ein vivo\u003c/em\u003e anti-tumor efficacy relative to combination therapy. This enhanced activity stems from its dual targeting, reflecting a systemic boost in anti-tumor immunity rather than a simple additive effect.\u003c/p\u003e\n\u003ch3\u003eSHH-YN-Bi Promotes CD8 T Cell Infiltration by Conquering CAF-Mediated ECM Barrier\u003c/h3\u003e\n\u003cp\u003eWith the deepening understanding of the TME, the mechanisms of therapy resistance mediated by CAFs have become increasingly clear. Accordingly, targeting CAFs has emerged as a key strategy in current anti-tumor therapeutics [37]. The transition of normal fibroblasts into CAFs within the TME is primarily driven by two mechanisms: the release of tumorigenic signals from tumor cells, and the intrinsic activation of oncogenic pathways, such as Wnt/β-Catenin pathway in CAFs to maintain their phenotype [38]. We confirmed that CM from A549 cells could induce the transdifferentiation of normal MRC-5 fibroblasts into CAFs (Figure. 7A). Notably, mIHC evaluation of CAF markers (COL1A1, FAP, and α-SMA) revealed that A549 CM pre-incubated with either SHH002-hu1 or SHH-YN-Bi lost this transdifferentiation capacity. In contrast, A549 CM treated with Atezolizumab retained the ability to induce CAF transformation (Figure. 7B, C). To further evaluate the impact of SHH-YN-Bi on CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration \u003cem\u003evia\u003c/em\u003e its action on CAFs, we established a co-culture infiltration model (Figure. 7D). The results demonstrated that targeting either Fzd7 or PD-L1 alone enhanced CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration, with the most pronounced effect observed when both CAFs and tumor cells were simultaneously targeted (Figure. 7E, F). Notably, SHH-YN-Bi exhibited the greatest efficacy, indicating that it promotes CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration and cytotoxicity through concurrent suppression of the Wnt/β-Catenin pathway in CAFs and blockade of the PD-1/PD-L1-mediated immunosuppressive axis.\u003c/p\u003e\n\u003cp\u003eECM remodeling and deposition mediated by CAFs represents a critical mechanism underlying the low survival rate of NSCLC patients [39]. To further investigate the effect of SHH-YN-Bi on CAFs, the A549 CM induced CAFs were then treated with 100 nM of Atezolizumab or SHH-YN-Bi for 24 hours and subjected to scRNA-seq analysis. Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction and clustering analysis revealed that the induced MRC-5 cells could be divided into 23 distinct clusters (Figure. 7G). Cell type identification using \u003cem\u003eACTA2\u003c/em\u003e (α-SMA), \u003cem\u003eFAP\u003c/em\u003e, \u003cem\u003eCOL1A1\u003c/em\u003e, and \u003cem\u003eCOL2A1\u003c/em\u003e as markers confirmed that clusters 0–22 were all identified as CAFs (Supplementary Figure. S7A). Further analysis of key genes in the Wnt/β-Catenin signaling pathway (\u003cem\u003eFzd7\u003c/em\u003e, \u003cem\u003eCTNNB1\u003c/em\u003e, \u003cem\u003eAPC\u003c/em\u003e) in CAFs (Supplementary Figure. S7B), with adjustment for multiple comparisons, showed that Fzd7 was significantly highly expressed in clusters 8 and 20. Subsequently, using AddModuleScore to evaluate the activity of the Wnt/β-Catenin pathway-related gene set and the ECM deposition gene set, we found that clusters 8 and 20 exhibited significantly higher scores in both assessments (Figure. 7H, I). These two clusters also showed a significant correlation with each other (Supplementary Figure. S7C-E). Analysis of the top differentially expressed genes in clusters 8 and 20 (Supplementary Figure. S7F-H), combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, indicated that both clusters were closely associated with ECM deposition (Figure. 7J). Collectively, these findings show that Wnt pathway activation in CAFs drives the robust ECM deposition characteristic of the matrix-depositing CAF phenotype.\u003c/p\u003e\n\u003cp\u003eThrough Gene Set Variation Analysis (GSVA) analysis, we found that SHH-YN-Bi treatment significantly suppressed the Wnt pathway compared to the Control or Atezolizumab groups (Figure. 7K, Supplementary Figure. S7I). This finding was further supported by GSEA multi-gene enrichment analysis (Supplementary Figure. S7J, K). Notably, SHH-YN-Bi also markedly reduced the expression of ECM deposition-related genes, including \u003cem\u003eCOL1A1\u003c/em\u003e, \u003cem\u003eCOL1A2\u003c/em\u003e, \u003cem\u003eCOL3A1\u003c/em\u003e, \u003cem\u003eFN1\u003c/em\u003e, and \u003cem\u003eLAMC1\u003c/em\u003e, relative to the Control (Figure. 7L). Pseudotemporal trajectory analysis further revealed that SHH-YN-Bi significantly suppressed the developmental progression of clusters 8 and 20 (Figure. 7M). Further analysis demonstrates that SHH-YN-Bi, through Fzd7 binding and Wnt/β-Catenin inhibition, downregulates multiple CAF-expressed genes critical for ECM remodeling and deposition (Supplementary Figure. S8). This effect may extend to the suppression of CAF-mediated ECM deposition and the downregulation of promigratory enzymes, such as MMP2 and MMP14. Western blot analysis confirmed that SHH-YN-Bi inhibits ECM deposition and reduces the expression of the metastasis-associated marker MMP2 in CAFs through the inhibition of Wnt/β-Catenin signaling (Supplementary Figure. S7L). Collectively, these findings indicate that Fzd7 is highly correlated with ECM remodeling in CAFs, and SHH-YN-Bi not only has the potential to suppress malignant metastasis in NSCLC but may also contribute to remodeling the ECM to promote immune cell infiltration.\u003c/p\u003e"},{"header":"Discussion","content":"Lung cancer, particularly NSCLC, remains a significant clinical challenge [40–42]. Although ICIs have transformed the therapeutic landscape, their efficacy is frequently constrained by primary or acquired resistance [1, 43]. The non-T cell-inflamed TME mediated by the Wnt/β-Catenin pathway represents the underlying mechanism for ICI-resistance in NSCLC. Specifically, aberrant activation of the Wnt pathway not only exerts intrinsic oncogenic functions but also fosters an immunosuppressive TME by upregulating PD-L1, impairing the recruitment of Batf3+ cDC1s, and promoting a highly oncogenic CAF phenotype [13, 17, 38, 44]. Consequently, therapeutic targeting of the Wnt/β-Catenin pathway may not only directly suppress tumor progression but also offer a promising strategy to overcome ICI-resistance by restoring CD8+ T cell infiltration into the TME.\n\nThe role of Fzd7 inhibition in counteracting Wnt/β-Catenin-mediated tumor progression is well-established, as demonstrated by our previous studies with SHH002-hu1 [20–22, 45, 46]. Batf3+ cDC1s act as a critical bridge in anti-tumor immunity by capturing antigens in the TME, migrating to the tdLN to activate CD8+ T cells, and finally guiding the primed T cells to home back to the tumor site for cytotoxic killing [34, 35, 47]. Our findings reveal the critical relationship between Fzd7 and PD-L1 and demonstrate that targeting Fzd7 sensitizes NSCLC to Atezolizumab treatment. Based on these, we designed SHH-YN-Bi, exhibits superior targeting capability compared with Atezolizumab. SHH-YN-Bi suppresses the Wnt/β-Catenin pathway, blocks PD-L1 to relieve immunosuppression, and demonstrates robust anti-tumor efficacy in multiple orthotopic NSCLC models. Mechanistically, we uncovered that targeting Fzd7 restores Batf3+ cDC1-dependent CD8+ T-cell infiltration by interrupting the Wnt3a/Fzd7/β-Catenin/ATF3 axis. As a first-in-class agent combining Wnt inhibition with ICP blockade, SHH-YN-Bi showcases the potential of simultaneously targeting the TME and ICPs to overcome ICI-resistance.\n\nWith increasing understanding of the TME, it has become clear that the dense and highly organized physical barrier formed by CAFs and the ECM constitutes a major obstacle to immune cell infiltration and effector function [48]. CAFs exhibit distinct functional heterogeneity, with specific subtypes mediating divergent biological roles. Specifically, matrix CAFs (mCAFs) are primarily responsible for ECM remodeling and deposition [49, 50]. Herein, we show that MRC-5-derived CAFs induced by A549 CM predominantly adopt an mCAF phenotype. Furthermore, Fzd7 expression in these mCAFs is positively correlated with ECM deposition. Notably, SHH-YN-Bi effectively attenuates CAF-mediated ECM remodeling and deposition by inhibiting the Wnt/β-Catenin pathway in mCAFs, thereby promoting CD8+ T cell infiltration into the TME. Our study further reveals that suppression of the Wnt/β-Catenin pathway in CAFs downregulates the expression of promigratory enzymes such as MMP2. This dual regulation underscores the capacity of SHH-YN-Bi to concomitantly foster an immunopermissive TME while restraining the metastatic potential of tumors. Intriguingly, in the immune infiltration assays, administration of Atezolizumab in the presence of CAFs also enhanced CD8+ T cell infiltration. However, PD-L1 expression is low in MRC-5-derived CAFs induced by CM alone (Fig. S9 A, B). Based on this observation, we hypothesize that upregulation of PD-L1 in CAFs likely depends on sustained non-cell-autonomous signaling from tumor cells. As shown in Fig. S9 C-E, PD-L1 expression in CAFs is significantly elevated upon prolonged stimulation by A549 cells compared to stimulation with CM alone. Collectively, this study originally reveals the involvement of Fzd7 in CAF-mediated ECM remodeling, and these findings support the dual role of SHH-YN-Bi as both a classical inhibitor of an oncogenic pathway and an immunomodulatory agent that promotes immune cell recruitment and function within the TME.\n\nIn summary, SHH-YN-Bi effectively abrogates the PD-1/PD-L1-mediated immunosuppression while simultaneously inhibiting the oncogenic activity of the Wnt/β-Catenin pathway. Furthermore, it enhances the immune infiltration of CD8+ T cells through two distinct mechanisms: by restoring the recruitment of Batf3+ cDC1s to the TME, and by disrupting the CAF-derived immunosuppressive barrier as well as attenuating CAF-mediated ECM remodeling and deposition (Fig. 8). Importantly, emerging evidence indicates that targeting PD-L1 on DCs is essential for achieving optimal therapeutic efficacy. Therefore, SHH-YN-Bi, through its bispecific design, not only blocks PD-L1 but also effectively bridges DCs and tumor cells, thereby establishing a novel DC-tumor cell engager (DCE) modality. This approach may overcome the limitations of traditional monocyte-derived dendritic cell (moDC) vaccines, such as suboptimal antigen presentation and impaired lymph node trafficking, thus promoting a more robust in situ immune response [34, 50]. Ultimately, our work for the first time elucidates the role of Fzd7 in shaping the immunosuppressive TME via Wnt3a/Fzd7/β-Catenin axis, particularly in CAF-mediated ECM remodeling. SHH-YN-Bi represents a promising therapeutic strategy for overcoming immunotherapy resistance in NSCLC, addressing a critical unmet clinical need in NSCLC patient management."},{"header":"Methods","content":"\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003eEthics\u003c/h2\u003e\n \u003cp\u003eThe cancer TMA (HLugA150CS03 and HLugA060PG02) were from Shanghai Outdo Biotech and approval from Shanghai Outdo Biotech Ethics Committee (SHYJS-CP-1901005 and SHYJS-CP-1904007). All human PBMCs were isolated from donors following written informed consent and approval from the Medical Ethics Committee Shanghai First Maternity and Infant Hospital (KS22174). All animal experiments were conducted in accordance with protocols approved by the Animal Ethics Committee of Shanghai University of Medicine and Health Sciences (2024-GZR-16-321281198802248253).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eCell culture and transfection\u003c/h2\u003e\n \u003cp\u003eThe human NSCLC cell line A549 (RRID: CVCL_0023), murine Lewis lung carcinoma cell line LLC (RRID: CVCL_4358), human T-cell line TALL-104 (RRID: CVCL_2771), and human lung fibroblast cell line MRC-5 (RRID: CVCL_0440) were obtained from the American Type Culture Collection (ATCC, New York, USA) and cultured in DMEM (Gibco, Grand Island, USA), supplemented with 10% (v/v) FBS (Transgen, Beijing, China), 100 µg/mL penicillin, and 100 µg/mL streptomycin (Gibco). The human NSCLC cell line H1975 (RRID: CVCL_1511) was obtained from the ATCC and cultured in RPMI 1640 (Gibco), supplemented with 10% (v/v) FBS, 100 µg/mL penicillin, and 100 µg/mL streptomycin. The Chinese hamster ovary cell line CHO (RRID: CVCL_0213) was obtained from the European Collection of Authenticated Cell Cultures (ECACC, Porton Down, UK), and the human PD-L1 cell line CHO-K1-OE-PD-L1 (Cat# B22301201, Biointron, Shanghai, China) was cultured in DMEM, supplemented with 10% (v/v) FBS, 100 µg/mL penicillin, and 100 µg/mL streptomycin. The human Fzd7-overexpressing cell line CHO-K1-OE-Fzd7 was generated by transient transfection of a full-length human Fzd7 plasmid (Sangon Biotech, Shanghai, China), and cultured in DMEM, supplemented with 10% (v/v) FBS, 100 µg/mL penicillin and 100 µg/mL streptomycin. The Chinese hamster ovary cell line ExpiCHO-S (RRID: CVCL_5J31) was obtained from Gibco and maintained in ExpiCHO expression medium. HEK293-PD-L1-OS8 and Jurkat-PD-1-NFAT-luc were obtained from Biointron and cultured in RPMI 1640 (Gibco), supplemented with 10% (v/v) FBS, 100 µg/mL penicillin, and 100 µg/mL streptomycin.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eFzd7\u003c/em\u003e-specific small interfering RNA (siRNA) was synthesized by Genepharma (Shanghai, China). The nucleotide target sequence for \u003cem\u003eFzd7\u003c/em\u003e (siFzd7) was: 5’-GCACCAUCAUGAAACACGATT-3’. Then, transfection was performed using GP-transfect-Mate (Genepharma).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eBsAb constructure\u003c/h2\u003e\n \u003cp\u003eThe light-chain (LC) and heavy-chain (HC) gene sequences of SHH002-hu1 and Atezolizumab were synthesized by Biointron. To construct SHH-YN-Bi, site-directed mutagenesis was performed on the original expression vectors of SHH002-hu1 and Atezolizumab to introduce specific mutations into the constant region of the heavy chain 3 (CH3), generating the knob (T366W) and hole (T366S, L368A, Y407V) variants in accordance with Knob-into-Hole (KIH) technology.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eExpression, purification and identification of SHH-YN-Bi\u003c/h2\u003e\n \u003cp\u003eFor SHH-YN-Bi expression, ExpiCHO-S cells were co-transfected with four IgG expression plasmids (pcDNA3.4 vector) encoding LC1, LC2, HC knob, and HC hole at a 1:1:1:1 molar ratio. SHH-YN-Bi was purified from culture supernatants by protein A affinity chromatography (GE Healthcare, Buckinghamshire, UK) and characterized using reducing and non-reducing SDS-PAGE, followed by size-exclusion chromatography-HPLC (SEC-HPLC) and capillary electrophoresis-SDS (CE-SDS) assays. The complete molecular structure of SHH-YN-Bi was analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS), capillary isoelectric focusing (cIEF), and differential scanning fluorimetry (DSF) to assess its charge heterogeneity and conformational stability.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003eBinding affinity and kinetic analysis\u003c/h2\u003e\n \u003cp\u003eThe binding kinetics of SHH-YN-Bi to rhFzd7-His (Sangon Biotech) and rhPD-L1-Fc (R\u0026amp;D Systems, USA) were measured using the ForteBio Octet Red96 system (PALL, USA). Briefly, SHH-YN-Bi was captured by anti-human IgG Fc antibodies immobilized on a CM5 sensor chip (Cytiva). Subsequently, rhFzd7-His or rhPD-L1-Fc was injected at varying concentrations into running buffer (KB buffer: 0.1% BSA + 0.025% Tween 20 dissolved in PBS, pH 7.2), and real-time binding responses were recorded. Sensorgrams were generated at each concentration, and the association rate constant (\u003cem\u003eka\u003c/em\u003e) and dissociation rate constant (\u003cem\u003ekd\u003c/em\u003e) were determined by global fitting analysis. The equilibrium dissociation constant (KD) was then calculated as the ratio \u003cem\u003ekd\u003c/em\u003e/\u003cem\u003eka\u003c/em\u003e. To demonstrate whether SHH-YN-Bi can simultaneously bind rhFzd7-His and rhPD-L1-Fc, a surface plasmon resonance (SPR) assay was performed using a Biacore T200 instrument (Cytiva). After immobilizing rhFzd7 on an NTA sensor chip (Cytiva), SHH-YN-Bi was first injected, followed by injection of rhPD-L1.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003eFlow-cytometry (FCM)-based binding and competition assays\u003c/h2\u003e\n \u003cp\u003eTo evaluate the binding capacity of SHH-YN-Bi to CHO-K1-OE-Fzd7 and CHO-K1-OE-PD-L1 cells, the following experimental groups were established: Isotype Control (anti-HEL human IgG1; Biointron), SHH002-hu1/Atezolizumab, and SHH-YN-Bi. Antibodies were serially diluted across ten concentration gradients (0-100 nM). Cells were harvested, resuspended, and incubated with diluted antibodies for 30 min at 4°C in 2% FBS/PBS. After incubation, cells were washed and subsequently incubated with FITC-conjugated goat anti-human IgG (Sangon Biotech) for 20 min at 4°C. Fluorescence signals were acquired using a NovoCyte Advanteon Dx flow cytometer (Agilent, California, USA), and data were analyzed using FlowJo software (version 10). The binding affinity for A549, H1975, and LLC cells was measured using the same method as described above.\u003c/p\u003e\n \u003cp\u003eTo assess the competitive binding of SHH-YN-Bi to CHO-K1-OE-Fzd7 and CHO-K1-OE-PD-L1 cells in the presence of rhWnt3a and rhPD-1 (MedChemExpress, Jersey, USA), FITC-labeled rhWnt3a and rhPD-1 were first prepared using a FITC conjugation kit (Sangon Biotech). Briefly, saturating concentrations of FITC-rhWnt3a and FITC-rhPD-1 were added to suspensions of CHO-K1-OE-Fzd7 or CHO-K1-OE-PD-L1 cells, respectively. Serially diluted antibodies were then added to the mixtures, and the samples were incubated for 20 min at 4°C. Finally, fluorescence was measured using FCM.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003e\u003cstrong\u003eTOP/FOP-FLASH luciferase reporter assay\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eThe β-catenin/TCF-mediated transcriptional activity was evaluated using a dual-luciferase reporter system following transient transfection of A549, H1975, and LLC cells with the TOP/FOP-FLASH luciferase reporter plasmid and a Renilla luciferase Control plasmid for normalization. Briefly, cells were seeded into 96-well plates and transfected with the TOP or FOP-FLASH plasmid using TransIntro EL Transfection Reagent (Transgen, Beijing, China). Four hours post-transfection, Wnt3a (200 ng/mL) was added to stimulate pathway activation. After an additional 12 h, cells were treated with antibodies (100 nM). Luciferase activities were measured 24 h after transfection using the Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA), and firefly luciferase signals were normalized to Renilla values.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003eNFAT report assays\u003c/h2\u003e\n \u003cp\u003eHEK293-PD-L1-OS8 and Jurkat-PD-1-NFAT-luc cells were harvested from T75 cell culture flasks, resuspended in RPMI 1640 medium without FBS, and adjusted to final densities of 5×10\u003csup\u003e5\u003c/sup\u003e cells/mL and 1×10\u003csup\u003e6\u003c/sup\u003e cells/mL, respectively. Antibody dilutions were prepared in RPMI 1640 medium with a top concentration of 100 nM, followed by five-fold serial dilutions across seven dose levels. Cells were seeded into 96-well plates at 2×10\u003csup\u003e4\u003c/sup\u003e cells per well in a volume of 40 µL. Then, 20 µL of each antibody dilution was added to the designated wells. The plates were incubated at 37°C in a 5% CO₂ incubator for 6 hours. After incubation, cells were equilibrated to room temperature for 5–10 min. An equal volume (100 µL/well) of KeyTec Ultra Luciferase Detection Reagent was added to each well, and the plate was shaken for 10 min to ensure complete cell lysis prior to luminescence measurement using a luminometer.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003edynamics and targeting capability by near infrared (NIR) imaging\u003c/strong\u003e\u003c/p\u003e Five-week-old female BALB/c-nude mouse were purchased from the Shanghai Laboratory Animal Research Center (Shanghai, China). A549-Luc cells were implanted into the lung parenchyma to establish orthotopic tumor models. Upon confirmation of tumor formation, mouse were randomly assigned to two groups (n = 5 per group). An NIRB-NHS fluorescence probe (Keyuandi Biotechnology, Shanghai, China) was conjugated with SHH-YN-Bi to generate the NIRB-SHH-YN-Bi fluorescent probe. In addition, free SHH-YN-Bi (2.5 µmol/kg) was co-administered with NIRB-SHH-YN-Bi (50 nmol/kg) to evaluate competitive binding and target specificity \u003cem\u003ein vivo\u003c/em\u003e. After intravenous injection, fluorescence images were acquired at predetermined time points using an IVIS Spectrum CT imaging system (PerkinElmer, USA). Tumor-to-normal tissue ratios (T/N ratios) were calculated based on region-of-interest (ROI) analysis. The same experimental procedure was performed for the Atezolizumab Control group.\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003eWestern blot assay\u003c/h2\u003e The whole cell proteins were extracted from A549, H1975, LLC, and CAF cells using RIPA buffer (Beyotime, Shanghai, China), and nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce Biotechnology, Rockford, USA). Western blotting was performed with primary antibodies against phospho-LRP6 (Ser1490) (Cat# abs140173, Absin), LRP6 (Cat# abs174285, Absin), β-catenin (Cat# 9562, CST), active β-catenin (Cat# 4270, CST), PD-L1 (Cat# abs155263, Absin), c-Myc (Cat# 9402, CST), ATF3 (Cat# 18665, CST), Fzd7 (Cat# ab64636, Abcam), CD44 (Cat# 37259, CST), VEGFA (Cat# abs149552, Absin), MMP2 (Cat# abs158236, Absin), COL1A1 (Cat# abs118788, Absin), FAP (Cat# abs134422, Absin), α-SMA (Cat# abs120451, Absin), and β-actin (Cat# 8457, CST). Subsequently, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized using enhanced chemiluminescence detection (Immobilon ECL Ultra Western HRP Substrate, Millipore, Burlington, MA, USA).\u003cbr\u003e\u003cstrong\u003eATF3\u003c/strong\u003e \u003cstrong\u003e-\u003c/strong\u003e \u003cstrong\u003especific ChIP assay\u003c/strong\u003e\u003cbr\u003e\n \u003cp\u003eChIP assays were performed using the ChIP Assay Kit from Merck. When cell density reached 80–90%, A549, H1975, or LLC cells were crosslinked with 1% formaldehyde and lysed for 10 minutes at 37°C. The crosslinking was quenched with 10× glycine, followed by PBS washing. Cells were then collected and incubated on ice for 15 minutes in cell lysis buffer containing protease inhibitors, with resuspension every 5 minutes. Chromatin was sheared by sonication. The lysates were incubated with ATF3-specific or Control IgG antibodies. DNA-protein complexes were captured using Protein A/G magnetic beads, eluted with ChIP elution buffer containing Proteinase K, and further purified. The isolated DNA was analyzed by qPCR.\u003c/p\u003e\n \u003cp\u003eqPCR Reaction System: DNA 1.6 µL + Forward primer 0.2 µL + Reverse primer 0.2 µL + Nuclease-free Water 3 µL + 2 × PerfectStart Green qPCR SuperMix 5 µL. \u003cem\u003eCcl4\u003c/em\u003e primer (Human): Forward primer 5’-AAGGTGAGCAGGTGGGTTAG-3’; Reverse primer 5’-TGGCTGGTTTGACAGTTGCT-3’.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eCcl4\u003c/em\u003e primer (mouse): Forward primer 5’-CTCAGCCCTGATGCTTCTCAC-3’; Reverse primer 5’-AGAGGGGCAGGAAATCTGAAC-3’.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003eElisa assay\u003c/h2\u003e For the ELISA-based CCL4 assays, after treating A549, H1975, or LLC cells with Wnt3a and antibodies, the cell supernatants were collected using sterile tubes and centrifuged at 2200 rpm for 20 minutes. Following centrifugation, the supernatants were transferred to new sterile tubes. The concentrations of CCL4 were measured using the MIP-1β/CCL4 ELISA Kit (animaluni, Shanghai, China). Biotinylated human CCL4 or mouse CCL4 antigens were coated onto 96-well plates. After the biotinylated antigens bound to CCL4, SABC was added and incubated at 37°C for 30 minutes to form immune complexes. TMB substrate solution was then added, and the plates were incubated in the dark at 37°C for 10–20 minutes. The reaction was stopped by adding 50 µL of stop solution per well, and the absorbance was measured at 450 nm using a microplate reader.\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003eCytotoxicity assay\u003c/h2\u003e The CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, USA) was used to measure lactate dehydrogenase (LDH) released from damaged NSCLC cells. Except for the blank Control group, other groups were pre-treated with 200 ng/mL Wnt3a for 12 hours. TALL-104 cells were co-cultured with A549 or H1975 cells at effector-to-target (E:T) ratios of 10:1, 5:1, and 2.5:1 in 96-well plates using DMEM containing 1% FBS and 100 nM of the corresponding antibodies. Similarly, activated mouse CD8⁺ T cells were co-cultured with LLC cells under the same conditions for 6 hours at 37°C. Control groups included spontaneous LDH release from effector or target cells alone, as well as target cell maximum LDH release. The percentage of specific cell lysis was calculated using the formula: 100 × (Experimental LDH Release – Effector Spontaneous LDH Release – Target Spontaneous LDH Release) / (Target Maximum LDH Release – Target Spontaneous LDH Release).\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003eCell migration and invasion assay\u003c/h2\u003e Here, except for the blank Control group, other groups were pre-treated with 200 ng/mL Wnt3a for 12 hours. 4×10\u003csup\u003e4\u003c/sup\u003e of A549, H1975, or LLC cells suspended in serum-free medium were plated into the upper wells of a 24-well transwell chamber (Millipore, Billerica, USA), coated with Matrigel (Corning, Bedford, USA), and then treated with antibodies. At 24h later, non-invasive cells on the upper layer were removed, whereas the invaded cells were fixed and stained. Images were taken using an Olympus inverted microscope; the invaded cells were counted using the Image-Pro-Plus program and invasion percentages were quantified based on the untreated Control. The migration assay was performed without Matrigel coating.\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\"\u003e\n \u003ch2\u003eImmuno co-culture assay\u003c/h2\u003e To evaluate the SHH-YN-Bi-mediated cytotoxic effects of TALL-104 or mouse CD8⁺ T cells on NSCLC cells, DiO-labeled (Ex 484 nm/Em 501 nm) TALL-104 and mouse CD8⁺ T cells and Dil-labeled (Ex 549 nm/Em 565 nm) A549, H1975, and LLC cells were co-cultured with 100 nM SHH-YN-Bi. Cellular status was subsequently observed using an inverted fluorescence microscope at 1 h, 6 h, and 12 h after treatment.\u003cbr\u003e\n \u003cdiv id=\"Sec23\"\u003e\n \u003ch2\u003eAnimal studies\u003c/h2\u003e SPF-grade female CD34\u003csup\u003e+\u003c/sup\u003e humanized non-obese diabetic-scid gamma (NSG) mouse and C57BL/6 mouse aged 5–6 weeks were purchased from Shanghai Model Organisms Center, Inc. and maintained under sterile conditions. All animal experiments involved in this study were reviewed and approved by the Scientific Research Ethics Committee of Shanghai University of Medicine \u0026amp; Health Sciences before implementation. Orthotopic tumor models of A549, H1975, and LLC were established. When the average bioluminescence signal intensity in tumor-bearing mouse reached approximately 1.5 × 10⁵, the mouse were administered the following treatments \u003cem\u003evia\u003c/em\u003e tail vein injection: SHH002-hu1 (5 mg/kg), Atezolizumab (5 mg/kg), SHH002-hu1 (5 mg/kg) + Atezolizumab (5 mg/kg), and SHH-YN-Bi (5 mg/kg). All groups were dosed once every three days. Bioluminescence signal intensity was monitored by intraperitoneal injection of 15 mg/mL D-luciferin (10 µL/g). After the luciferin signal stabilized, the bioluminescence intensity in tumor-bearing mouse was measured and recorded using a small animal \u003cem\u003ein vivo\u003c/em\u003e optical 3D imaging system.\u003cbr\u003e\n \u003cp\u003eTwelve-week-old female \u003cem\u003eBatf3\u003c/em\u003e-KO mouse were also purchased from Shanghai Model Organisms Center, Inc. After successful establishment of the orthotopic LLC tumor model, the mouse were administered PBS, SHH002-hu1 (5 mg/kg), or SHH-YN-Bi (5 mg/kg) \u003cem\u003evia\u003c/em\u003e tail vein injection. Bioluminescence signal intensity in the tumor-bearing mouse was recorded using the same method described above after drug administration.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\"\u003e\n \u003ch2\u003eNSCLC tumor tissues analysis\u003c/h2\u003e The human NSCLC TMA (HLugA150CS03, HLugA060PG02) was purchased from Shanghai Outdo Biotech Co., Ltd. Fluorescence in situ hybridization (FISH) was performed using the Boshide Custom Fzd7 FISH Detection Kit and Boshide Custom PD-L1 (CD274) FISH Detection Kit. TSA-based multi-color immunohistochemistry (mIHC) was performed on HLugA150CS03 and HLugA060PG02 TMAs using antibodies against Fzd7, PD-L1, CK, β-catenin, and c-Myc.\u003cbr\u003e\n \u003cp\u003eFormalin-fixed paraffin-embedded (FFPE) sections were prepared from the NSCLC tissues obtained from the aforementioned animal experiments. These sections were subsequently subjected to hematoxylin and eosin (HE) staining and mIHC. The antibodies used in this section included: anti-Fzd7, anti-PD-L1, anti-β-catenin, anti-CD3, anti-CD8, anti-CD39, anti-CD141, anti-CD103, and anti-PanCK.\u003c/p\u003e\n \u003cdiv id=\"Sec25\"\u003e\n \u003ch2\u003eCAF associated mIHC\u003c/h2\u003e To generate cancer cell conditioned media (CM), A549 cancer cells at 70–80% confluency were treated with SHH-YN-Bi for 24 h. The cells were then washed with PBS and the media was replaced with fresh media supplemented with 5% FBS for 48 h. The media was centrifuged for 5 min at 1200 rpm, and the supernatant (CM) was used for stimulation of fibroblasts. MRC5 fibroblasts were cultured in a 6-well glass-bottom plate and grown to 50% confluency, then growth arrested for 48 h. The fibroblasts were then co-cultured with cancer cell CM for 48 h. The fibroblasts were then stained for mIHC. The antibodies used in this section were: anti-COL1A1, anti-FAP, anti-α-SMA.\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\"\u003e\n \u003ch2\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells isolation\u003c/h2\u003e\n \u003cp\u003eMouse CD8⁺ T cells were isolated from C57BL/6 mouse using the EasySep™ mouse CD8⁺ T Cell Isolation Kit (STEMCELL Technologies). Spleens were harvested and mechanically dissociated in PBS containing 2% FBS, then passed through a 70 µm strainer. The resulting suspension was centrifuged at 1200 rpm for 10 minutes. After discarding the supernatant, the cell pellet was resuspended in 0.5-2 mL RoboSep™ Buffer (STEMCELL). Subsequently, 20 µL of mouse FcR Blocking Reagent and 50 µL of the Isolation Cocktail were added, followed by incubation at room temperature for 10 minutes. Then, 125 µL/mL of RapidSpheres™ were added and incubated for 5 minutes at room temperature. The cell suspension was diluted with 1 mL RoboSep™ Buffer, and CD8⁺ T cells were positively selected using a STEMCELL Separator. Finally, the isolated cells were activated for 2 hours with \u003cem\u003ein vivo\u003c/em\u003e anti-mouse CD3 Recombinant mAb (STARTER) and \u003cem\u003ein vivo\u003c/em\u003e anti-mouse CD28 mAb (STARTER) at appropriate concentrations.\u003c/p\u003e\n \u003cp\u003eTo prepare the magnetic bead buffer for isolating human CD8⁺ T cells, 2 mM EDTA and 2.5 g BSA standard protein were separately dissolved in 50 mL PBS, thoroughly mixed, filtered through a 0.22 µm filter, and then added to 400 mL PBS to obtain a final volume of 500 mL. Human Peripheral Blood Mononuclear Cells (PBMCs) were resuspended in 1 mL of the prepared bead buffer, and 1 × 10⁷ cells were transferred to a 1.5 mL microcentrifuge tube, followed by centrifugation at 300 × g for 5 minutes. After discarding the supernatant, the cell pellet was resuspended in 40 µL bead buffer. For CD8⁺ T cell isolation, 20 µL of CD8-Biotin antibody was added and incubated, followed by the addition of 20 µL anti-Biotin MicroBeads and thorough mixing. The mixtures were incubated for 15 minutes at 4°C protected from light, and magnetic separation was performed using the OctoMACS system (Miltenyi Biotec).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec27\"\u003e\n \u003ch2\u003eCD8\u003csup\u003e+\u003c/sup\u003e T cells infiltration assay\u003c/h2\u003e\n \u003cp\u003eAfter 48 hours of co-incubation with rhFzd7, the maturation of DCs (Isolated from human PBMC) was assessed by FCM based on the expression of CD80/CD86 markers. Mature DCs loaded with Fzd7 antigen were then co-cultured with CD8⁺ T cells (stained with Dil) for 48 hours at a DC:T cell ratio of 1:3 to generate adoptive T cells. Following the procedure of the aforementioned Transwell assay, 4 × 10⁴ MRC-5 cells were diluted in complete medium and mixed with an equal volume of Matrigel to form an ECM barrier. A total of 5 × 10⁴ A549 or H1975 cells (stained with DiO) were seeded into the upper chamber of an ultra-low attachment 24-well plate and treated with the respective antibodies according to the experimental groups. Then, 5 × 10⁴ adoptive T cells were added to the upper chamber. After 24 hours, the 24-well plate was examined under a fluorescence microscope to evaluate T cell infiltration.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\"\u003e\n \u003ch2\u003eSingle-cell RNA sequencing and analysis\u003c/h2\u003e\n \u003cp\u003eFollowing the induction of MRC5 into CAFs using A549-CM, the resulting CAFs were divided into three experimental groups (n = 3): Control, Atezolizumab (100 nM), and SHH-YN-Bi (100 nM). After a 24-hour incubation in 5% FBS DMEM, cells were collected \u003cem\u003evia\u003c/em\u003e PBS washing and centrifugation (1200 rpm, 5 min, twice). Single-cell suspensions meeting quality criteria (viability \u0026gt; 80%, \u0026gt; 1×10\u003csup\u003e6\u003c/sup\u003e cells/group) were used for library preparation. Single-cell isolation was achieved using Gel Bead-in-Emulsions (GEMs), followed by post-GEM-RT cleanup, cDNA amplification, and quality Control. Finally, sequencing was carried out on a DNBSEQ-T7 platform with a PE100/150 protocol. Data analysis was performed using the Majorbio Cloud (www.majorbio.com).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec29\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eAll results were presented as mean ± s.d., and all experiments were independently repeated at least three times with at least three independent biological samples. Student’s t-test and ANOVA were used to determine significant differences. GraphPad Prism (\u003cem\u003eV.10.0\u003c/em\u003e) was used for specific comparisons throughout the manuscript, with \u003cem\u003eP\u003c/em\u003e values indicated in figures. A \u003cem\u003eP\u003c/em\u003e value of \u0026lt; 0.05 was considered statistically significant. In the direct image presentation, the results from the same experiment were selected for presentation.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eShuyang Mao: Writing \u0026ndash; review \u0026amp; editing, Methodology, Conceptualization, Writing \u0026ndash; original draft, Investigation. Xiaofan Zhang: Funding acquisition, Software, Formal analysis, Data curation. Zitong Wang: Formal analysis, Data curation. Pan Zhou: Investigation, Data curation. Zixuan Wang: Investigation, Validation. Yijun Zhao: Investigation. Jiayi Ye: Investigation. Guoqing Wan: Formal analysis. Tong Wang: Data curation. Gangyi Hu: Data curation. Kexin Zhu: Data curation. Yuxia Liu: Investigation. Lin Yang: Funding acquisition, Conceptualization. Xiaofei Zhang: Funding acquisition, Supervision. Gang Huang: Funding acquisition, Conceptualization, Supervision. Qingqing Huang: Formal analysis, Conceptualization. Wei Xie: Writing \u0026ndash; review \u0026amp; editing, Supervision, Funding acquisition, Conceptualization. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThanks all support from Shanghai Key Laboratory of Molecular Imaging \u0026amp; Digital and Intelligent Empowerment Biomedical Innovation Center, Pudong Gongli Hospital, School of Pharmacy, Shanghai University of Medicine and Health Sciences. This article was supported by the Natural Science Foundation of Shanghai (23ZR1427400); Key Clinical Program of Shanghai Municipal Health Commission (20214Y0516); Construction project of Shanghai Key Laboratory of Molecular Imaging (18DZ2260400); National Natural Science Foundation of China (82203714, 32300295).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data reported in this article are available and will be shared by the lead contact upon request (Prof. Wei Xie, [email protected]).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhao S, Zhao H, Yang W, Zhang L. The next generation of immunotherapies for lung cancers. Nat Rev Clin Oncol. 2025;22(8):592\u0026ndash;616.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Chen Q, Shan Q, Liang T, Forde P, Zheng L. Clinical development of immuno-oncology therapeutics. 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Adv Mater. 2025;37(17):e2412974.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"npj-precision-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjprecisiononcology","sideBox":"Learn more about [npj Precision Oncology](http://www.nature.com/npjprecisiononcology/)","snPcode":"41698","submissionUrl":"https://submission.springernature.com/new-submission/41698/3","title":"npj Precision Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"NSCLC, Frizzled7, PD-L1, Wnt/β-Catenin signaling pathway, Tumor microenvironment, Immunotherapy, Cancer-associated fibroblast","lastPublishedDoi":"10.21203/rs.3.rs-9337927/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9337927/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImmunotherapy efficacy in non-small cell lung cancer (NSCLC) remains limited, largely due to aberrant activation of the Wnt/β-Catenin pathway. However, the precise role of its receptor Frizzled7 (Fzd7) in immune checkpoint inhibitor (ICI)-resistance remains unexplored. Here, we elucidate the role of Fzd7 in shaping the immunosuppressive tumor microenvironment (TME) and develop a novel bispecific antibody (named SHH-YN-Bi) that co-targets Fzd7 and programmed cell death ligand 1 (PD-L1) to overcome ICI-resistance. This study reveals that Fzd7 is remarkably enriched in human NSCLC tissues and correlates with PD-L1 expression and immune exclusion. Targeting Fzd7 sensitizes NSCLC to Atezolizumab in multiple orthotopic NSCLC models. SHH-YN-Bi was generated \u003cem\u003evia\u003c/em\u003e “Knob-into-Hole”, demonstrating effective dual binding to Fzd7 and PD-L1 and superior tumor targeting compared with Atezolizumab. We show that SHH-YN-Bi concurrently recruits and activates CD8\u003csup\u003e+\u003c/sup\u003e T cells in a basic leucine zipper transcriptional factor ATF-like 3 (Batf3)\u003csup\u003e+\u003c/sup\u003e conventional type 1 dendritic cells (cDC1s)-dependent manner to reprogram the TME, yielding robust antitumor activity in ICI-resistant NSCLC orthotopic models. Furthermore, scRNA-seq analysis uncovered potent correlation between Fzd7 and cancer-associated fibroblast (CAF)-mediated extracellular matrix (ECM) remodeling, and SHH-YN-Bi treatment induced a phenotypic switch of CAFs from a highly oncogenic to a normal state, associated with elevated CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration. SHH-YN-Bi overcomes ICI-resistance in NSCLC by simultaneously blocking the oncogenic Wnt3a/Fzd7/β-Catenin signaling and PD-1/PD-L1 axis. Our findings highlight the critical role of Fzd7 in immunosuppressive TME, particularly in CAF-mediated ECM remodeling, and offer a innovative approach for NSCLC treat.\u003c/p\u003e","manuscriptTitle":"A Novel Bispecific Antibody Targeting Frizzled7 and PD-L1 Reverses Immunotherapy Resistance in Non-Small Cell Lung Cancer by Reprogramming the Tumor Microenvironment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-29 09:13:39","doi":"10.21203/rs.3.rs-9337927/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-14T12:06:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36600134297902223512346770982687026076","date":"2026-04-24T06:22:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"193378223786652075473578039559802024200","date":"2026-04-23T23:41:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26620608280599295498192196325547513688","date":"2026-04-23T05:35:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-21T02:35:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-11T00:31:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-08T05:00:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Precision Oncology","date":"2026-04-07T00:52:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-precision-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjprecisiononcology","sideBox":"Learn more about [npj Precision Oncology](http://www.nature.com/npjprecisiononcology/)","snPcode":"41698","submissionUrl":"https://submission.springernature.com/new-submission/41698/3","title":"npj Precision Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1f8245df-6494-41c1-81c8-bd64a0832163","owner":[],"postedDate":"April 29th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-14T12:06:58+00:00","index":44,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67103626,"name":"Biological sciences/Cancer"},{"id":67103627,"name":"Biological sciences/Immunology"},{"id":67103628,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-04-29T09:13:40+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-29 09:13:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9337927","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9337927","identity":"rs-9337927","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}