Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease with an urgent need for novel therapeutic strategies. M2 macrophage-derived TGF-β1 promotes fibroblast myogenesis, contributing to IPF pathogenesis. Targeting macrophage polarization and fibroblast function thus represents an effective therapeutic approach for treating IPF. Here, we identify B-cell lymphoma 9 (BCL9) as a key upstream regulator implicated in IPF pathogenesis. We demonstrate that BCL9 drives the macrophage M2 program through the MerTK-ERK-SPP1 axis. Notably, pharmacological inhibition of BCL9 with our novel peptide, hsBCL9Z96, effectively attenuates pulmonary fibrosis by reprogramming macrophage-fibroblast crosstalk. Specifically, BCL9 inhibition promotes fibroblast lipogenesis via TGF-β1 signaling, which in turn supports alveolar type 2 (AT2) cell expansion. This macrophage-orchestrated fibroblast phenotypic switch from myogenic to lipogenic is visually corroborated by spatial transcriptomic analyses and immunofluorescence staining of human lung tissues. Functionally, the pathological role of BCL9 and efficacy of hsBCL9Z96 are validated in human cellular models, including IPF patient-derived cells, confirming its translational significance. Collectively, our findings not only elucidate a novel BCL9-driven macrophage-fibroblast-AT2 cell axis in IPF but also establish hsBCL9Z96 as a promising first-in-class therapeutic candidate, providing a strong rationale for targeting BCL9-mediated Wnt signaling in clinical IPF treatment.

Peroxisome proliferator-activated receptor α (PPARα) represents a critical therapeutic target for dyslipidemia. To discover novel scaffold agonists, we used Pemafibrate as a template to construct a focused chemical library with the infiniSee software. An integrated virtual screening approach, including molecular docking, MM-GBSA binding free energy calculations, ADMET profiling, and visual inspection, was applied to identify a hit compound. Subsequent structural optimization led to the synthesis of 40 derivatives. Among the tested compounds, XYQ3-B11 was identified as the most potent PPARα activator. In the luciferase reporter assay, XYQ3-B11 exhibited an EC50 value of 8.33 μM, better than the reference agonist WY14643 (EC50 = 16.10 μM). Notably, XYQ3-B11 possesses a distinctive hybrid architecture, comprising a central pyridine ring, a 2-fluorobenzoate moiety, and an indole-derived side chain. These findings highlight XYQ3-B11 as a promising novel chemotype for the start of development of PPARα agonists.

Background and Purpose The CKLF-like MARVEL transmembrane domain containing superfamily (CMTM) has an important role in tumour immunity. The role of CMTM8 in tumour immune microenvironment (TIME) remains unknown. The aim was to investigate the relationship between CMTM8 and antitumour immunity.Experimental Approach Lentiviral system was established to achieve functional edition of CMTM8. CMTM8 in tumour progression and TIME was investigated by using subcutaneous xenograft mice tumour models. CMTM8 targeting was achieved using an adeno-associated virus (AAV)-based shRNA vector.Key Results Clinical data showed that high expression of CMTM8 in melanoma and colorectal cancer is associated with lower patient survival rates and poorer intra-tumoural immune cell infiltration. Mouse melanoma and colon cancer models, CMTM8 expression markedly accelerated tumour growth by regulating intra-tumoural infiltration of CD8+ T cells and NK cells. RNA sequencing of CMTM8-overexpressed B16 cells showed that Notch pathway is significantly up-regulated, and blockade of Notch pathway in vivo resulted in lower intra-tumoural infiltration of CD8+ T cells and NK cells, and confined tumour progression. CMTM8 could down-regulate the EGFR pathway by promoting endocytosis of EGFR, possible leading to higher expression of Notch1. Similar this observed in human colon cancer in a PBMC-engrafted NCG model. Besides, we developed the CMTM8-targeting adeno-associated virus, which effectively up-regulated intra-tumoural infiltration of CD8+ T cells and NK cells and inhibited tumour growth.Conclusion and Implications Tumour-intrinsic CMTM8 could possibly regulate TIME via EGFR-Notch axis and that CMTM8 is a promising target for tumour immunotherapy, especially for treatment of colorectal cancer or melanoma patients.

The application of immunocapture LC-MS/MS in drug testing has expanded considerably over the past decade, offering significant advantages in analysis, development, and identification. In this study, we developed and validated a universal immunocapture LC-MS/MS assay for the detection of humanized IgG1-based drugs in cynomolgus monkey sera. The method selected a 12-amino acid characteristic peptide from the CH1 region of IgG1 as a quantification surrogate and used the heavy stable isotope-labeled same peptide as an internal standard. Furthermore, humanized IgG1-based drugs were immunocaptured using magnetic streptavidin beads pre-bound to biotinylated anti-human IgG1 Fc antibodies, followed by trypsin digestion. The related liquid chromatography was conducted on a Shiseido C18 column (2.0 mm × 100 mm, 3 μm) equipped on a Shimadzu Nexera X2 UHPLC system. Quantification of the humanized IgG1 -based drugs was performed on a LCMS-8050 triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) mode with positive electrospray ionization. The developed assay was validated for parameters including calibration curve, precision, accuracy, selectivity, recovery, and stability. The obtained results all met the acceptance criteria valid for ligand-binding assays (LBAs). Besides, involving humanized IgG1 -based drugs exhibited good stability in cynomolgus monkey sera under different tested conditions. After validation, the assay was successfully applied to the pharmacokinetic studies of two humanized IgG1-based antibody-drug conjugates (ADCs), respectively, generating comparable pharmacokinetic profiles to related LBAs. Together, the results confirm the assay's reliability, universality, and feasibility for quantifying the target drugs.

Immune checkpoint inhibitors (ICIs), especially CTLA-4 inhibitors (αCTLA-4), exhibit a high incidence of colitis as an immune-related adverse event (irAE) during cancer treatment, severely limiting patient benefit. Clinically, both treatment interruption and existing intervention drugs for ICI-mediated colitis may compromise antitumor efficacy. However, there is inadequate research on the pathogenesis of ICI-mediated colitis, with findings often conflicting. Here, we first established multiple clinically relevant animal models, including an immuno-humanized ICI-mediated colitis model. Through time-series transcriptomics, we discovered that αCTLA-4-induced colonic toxicity exhibits characteristics ranging from early metabolic reprogramming represented by glycolysis to later immune disorders represented by Th17 responses. By targeting colonic CTLA-4+ T cells, αCTLA-4 blocked CD80/CD86-CTLA-4 interaction, thereby activating the PI3K-AKT-mTOR pathway. Subsequently, mTOR mediated metabolic reprogramming in T cells, shifting them from Treg-biased oxidative phosphorylation to Th17-biased glycolysis. The colonic toxicity of αCTLA-4 has also been demonstrated to depend on the PI3K-AKT-mTOR pathway, glycolysis, and Th17 responses. Notably, metformin significantly relieved ICI-mediated colitis by inhibiting mTOR without impeding antitumor efficacy. Collectively, these findings highlighted the metabolic-immune axis in the colonic toxicity of ICI and provided a clinically superior intervention strategy. ### Competing Interest Statement The authors have declared no competing interest.

It has been a consensus that hepatic microenvironment composed by the non-parenchymal cells networks play a critical role during liver fibrogenesis, with the crosstalk between hepatic stellate cells (HSCs) and macrophages being of paramount importance. Interleukin 11(IL-11) has been implicated as a pro-fibrogenic cytokine, where its function in/between hepatocytes and HSCs has been revealed. But whether IL-11 participates in the interaction of HSCs and macrophages and related mechanism remains obscure. Our research demonstrates that HSC-derived IL-11 operates through a dual mechanism of autocrine activation and paracrine reprogramming to drive the fibrosis. AAV6-mediated IL-11 overexpression in the HSCs aggravated hepatic fibrosis induced by CCl4 in C57/B6 mice, accompanied by a marked increase of M2 macrophages. Mechanistically, the autocrine signaling of IL-11 activated HSCs directly, potently enhancing the contractility, migration, and collagen production of HSCs through GP130-SFK-YAP pathway. Furthermore, IL-11 also functioned as a paracrine signal of HSCs activation that synergized with IL-4 to polarize macrophages into a profibrotic M2-like phenotype. This reprogramming was achieved through the coordinated activation of PI3K-mTOR signaling to promote TGF-β synthesis and STAT3 pathway to elevate chemokine levels. The necessity of macrophages in this process was proven when their depletion blunted the pro-fibrogenic effects of IL-11 overexpression. Consequently, therapeutic inhibition of IL-11 with a nanobody alleviated fibrosis and reversed macrophage polarization. Our findings proposed a self-amplifying loop where HSC-derived IL-11 directly activates fibrogenesis and simultaneously reprograms macrophages to create a feed-forward cycle that relentlessly drives disease progression.

BACKGROUND:Tumour-associated macrophages are an indispensable part of the tumour immune microenvironment, exhibiting phenotypic and functional plasticity that enables them to play an important role in modulating the immune response. A large number of RNA editing events have been identified during macrophage functional remodelling. APOBEC3 is a well-known family of base-editing enzymes, but whether and how it regulates macrophage function is unclear. METHODS:In this study, we mainly used genetically engineered mice to demonstrate how host APOBEC3 deficiency affects tumour progression. RESULTS:We found that the polarisation state of macrophages in the tumour microenvironment was altered by host APOBEC3 deficiency and that knockout of host APOBEC3 significantly inhibited tumour growth and metastasis in a macrophage-dependent manner. Moreover, APOBEC3 deficiency remodelled macrophage function, improved their polarisation state, and enhanced their phagocytic capacity. Mechanistically, APOBEC3 knockout attenuated lipid uptake by macrophages in a PPARγ pathway-dependent way, leading to an increase of M1/M2 ratio in macrophages. CONCLUSIONS:These findings reveal that APOBEC3 deficiency drives lipid metabolism reprogramming in macrophages and remodels their functions, thereby inducing a strong anti-tumour immune response, providing a proof-of-concept and new insights for the development of the APOBEC3 family for novel tumour therapies. Host APOBEC3 deficiency regulates macrophage function through lipid metabolic remodelling, ultimately leading to tumour suppression (Created with BioRender.com).

The essential role of B cells and B cell intrinsic molecules in tumor immunity is beginning to be recognized. Tumor cell CKLF-like MARVEL transmembrane domain-containing protein 6 (CMTM6) is a novel tumor immunoregulator involved in maintaining membrane levels of several important molecules, such as programmed cell death ligand 1 (PD-L1) and CD58. Host CMTM6 may also play a function in the tumor microenvironment. Here, we found that CMTM6 was highly expressed in splenic B cells and tumor-infiltrating B cells. CMTM6 deficiency resulted in impaired splenic development, germinal center B cell differentiation, memory B cell differentiation, T/B cell interaction and B cell anti-tumor immune responses. Through multi-omics data mining and B-cell agonist screening, we identified that CMTM6 interacted with CD40 and maintained CD40 membrane levels in B cells. CMTM6 cis-interacts with CD40 and inhibits ubiquitin/proteasome-mediated CD40 degradation. CMTM6 deficiency led to impaired CD40 signaling-mediated B cell activation, survival, proliferation, differentiation and T/B cell interaction. In vivo, CMTM6 deficiency leads to a significant decrease in the anti-tumor activity of immune checkpoint blockade (ICB) therapy and B cell-dependent CD40 agonists. Collectively, B-cell intrinsic CMTM6 maintains B cell CD40 levels and signaling to promote B cell function and anti-tumor immunity.

GL-2401 is a novel antibody-drug conjugate (ADC) made by site-specific enzymatic conjugation of monomethyl auristatin E (MMAE) to trastuzumab at the N297 glycosylation site. We aimed to develop two reliable bioanalytical methods for quantifying both total antibody (TAb) and conjugated antibody of GL-2401 in rat serum for exploration of its pharmacokinetic (PK) profiles. In this study, we developed two affinity-capture LC-MS/MS methods, of which one for the conjugated antibody and the other for the TAb in rat serum. In both methods, the target analytes in rat serum were firstly enriched using a related biotinylated capture reagent immobilized on streptavidin magnetic beads, followed by washes and on-bead trypsin digestion. Thereafter, the characteristic peptide IYPTNGYTR, derived from the complementarity determining region 2 (CDR2) of trastuzumab, served as the single surrogate analyte for quantification of both conjugated antibody and TAb. The methods were subsequently validated, showing good accuracy and precision for quantifying the conjugated antibody and the TAb within the ranges of 0.50-100.00 μg/mL and 0.50-40.00 μg/mL, respectively. Finally, the validated methods were applied to a rat PK study. GL-2401 demonstrated higher serum and linker stability compared to conventionally conjugated ADCs, providing a basis for further application of GL-2401 in other animal species.

Peroxisome proliferator-activated receptor α (PPARα), an important member of the nuclear receptor superfamily, plays a crucial role in regulating lipid metabolism, glucose homeostasis, and inflammation. Its dysfunction is associated with metabolic diseases such as hypertriglyceridemia and non-alcoholic fatty liver disease, making PPARα a significant therapeutic target. In this study, a potential novel PPARα agonist lead compound, LY-23 (EC₅₀ = 11.91 μM), was identified through virtual screening of the ChemDiv database followed by biological validation. Based on preliminary structure-activity relationship (SAR) analysis, structural modification and optimization of three key regions of LY-23 were carried out, resulting in fifteen structurally novel derivatives, whose synthetic routes incorporated photocatalytic steps as an advanced synthetic strategy. Among these derivatives, compound GJX-230 (EC₅₀ = 10.42 μM) exhibited the highest agonist activity in a luciferase reporter gene assay. Further studies demonstrated that GJX-230 selectively upregulated the expression of HMGCS2. Molecular docking studies elucidated the binding modes of the active compounds within the PPARα ligand-binding domain. This work not only provides a series of new chemical foundations, but also establishes a clear SAR framework and preliminary biological insights, which may facilitate the future development of more selective and safer PPARα agonists.

Peptide vaccines offer a flexible, rapidly updatable platform for responding to SARS-CoV-2 antigenic drift. However, many candidates target a single epitope class, predominantly CD4 +/CD8 + T-cell epitopes or short linear B-cell epitopes, limiting their capacity to induce both high-titer neutralizing antibodies and robust T-cell immunity. Here, to elicit potent humoral and cellular responses, we used AI-aided epitope prediction tools to analyze 18 Omicron subvariants and designed two candidate peptides from the RBD of XBB.1.5, designated LY54-XBB (L455-Y508) and P67-XBB (Y351-K378). In silico TCR-pMHC binding analysis and structural modeling showed that both peptides contained T-cell epitopes. The immunogenicity of the two peptide nanoemulsions was validated in murine and non-human primate (NHP) models. Mixed vaccination elicited RBD-binding, ACE2-blocking, and pseudovirus-neutralizing antibody responses, together with a Th1-biased cross-reactive cellular immune response, with no observable adverse reactions. Importantly, mixed vaccination protected the lungs of HLA-A2/DR1-hACE2 transgenic mice from the SARS-CoV-2 BA.5 variant challenge. In addition, ex vivo stimulation of hPBMCs from COVID-19 convalescent plasma donors confirmed that both peptides elicited antigen-specific CD4 + and CD8 + T-cell responses, validating the inclusion of effective T-cell epitopes. These complementary peptides, supported by both experimental and computational validation, represent promising rapidly updatable booster candidates. Our epitope-based pipeline provides a generalizable framework for vaccine design that may help sustain population immunity against SARS-CoV-2 and other antigenically diverse pathogens.

Interleukin-11 (IL-11), a member of the IL-6 cytokine family, is well-recognized for its role in driving fibrosis and stromal remodeling. Extensive research on this fibroblast-associated cytokine have focused on its roles in tissue scarring and extracellular matrix deposition. However, emerging evidence has unveiled its sophisticated role in immunomodulation, extending far beyond its conventional pro-fibrotic functions. This review demonstrates how IL-11 influences phenotypic shifts of immune cell plasticity within both innate and adaptive compartments. In the myeloid lineage, IL-11 orchestrates macrophage polarization and macrophage-to-mesenchymal transition (MMT), regulates neutrophil extracellular traps (NETs) formation, and modulates the plasticity of NK cells, while in the lymphoid compartment, it influences T helper cell differentiation, regulatory T cell stability, and B cell responses. Of note, the effect of IL-11 on immune cells may be exerted either directly through engagement with the target cells or indirectly via intercellular crosstalk. Furthermore, we also highlight the therapeutic potential of modulating the IL-11 signaling axis through monoclonal antibodies, siRNAs, peptides, recombinant proteins, and small molecules to restore immune homeostasis across multiple disease states.

Accurate measurement of interleukin-8 (IL-8) in non-human primates is crucial for research into inflammation and tumors; however, sensitive enzyme-linked immunosorbent assays (ELISAs) for non-clinical studies remain limited. We aimed to develop and validate a sensitive method for IL-8 quantification in cynomolgus monkey serum. Using two recombinant IL-8-specific antibodies and a surrogate matrix, a sandwich-type ELISA was developed, involving sequential incubations with samples, biotinylated antibody, and horseradish peroxidase-labeled Streptavidin (SA-HRP). The assay was validated against regulatory guidelines, achieving a lower limit of quantification of 4.69 pg/mL as well as good accuracy and precision. The assay was neither affected by serum matrix nor hemolysis, nor did it cross-react with other cytokines. Furthermore, the assay showed dilution linearity, no hook effect, and good recovery and parallelism. Finally, IL-8 in monkey serum exhibited sufficient stability for analysis. After validation, the assay was applied to 19 healthy cynomolgus monkey serum samples, and the results correlated well with those from a commercial assay. In conclusion, the developed ELISA is a sensitive, accurate, and reliable method for IL-8 quantification in cynomolgus monkey serum, suitable for non-clinical studies in cynomolgus monkeys.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, interstitial lung disease lacking an efficient drug to reverse it. Thus, there is an urgent need to elucidate the complex pathogenesis of IPF and identify new therapeutic targets. It has been revealed that the pathophysiology of IPF is a highly orchestrated process that includes multiple cell types in which the contribution of endothelial cells (ECs) has attracted researchers' attention. However, although the involvement of ECs in fibrosis has been recognized, the underlying key molecules driving these changes are not well defined. In this study, we revealed that von Willebrand factor (VWF), a marker of damaged ECs, and endothelial dysfunction are positively correlated with IPF progression on the basis of reanalysis of gene expression profiles of patients with IPF. Next, we discovered that VWF deficiency attenuated fibrosis in experimental models, including human cell lines (in vitro) and mice (in vivo). Mechanistically, VWF deficiency inhibited endothelial-to-mesenchymal transition, regulated vascular abnormalities, and limited M2 macrophage infiltration, which were achieved, at least in part, by the inhibition of Wnt signaling. Our findings provided evidence for the pivotal role of ECs in IPF and revealed that VWF might be a driving factor of endothelial-to-mesenchymal transition, suggesting that VWF can be developed as a potential therapeutic target against IPF.

ETHNOPHARMACOLOGICAL RELEVANCE:Qingfeiyin decoction (QFY), a well-documented traditional Chinese medicine formula, is used to treat a variety of lung diseases. However, the effect of QFY on idiopathic pulmonary fibrosis (IPF) is still unclear. AIM OF THE STUDY:This study aimed to investigate the effect of QFY on IPF, identify its bioactive components, and reveal the possible mechanism. MATERIALS AND METHODS:A bleomycin-induced pulmonary fibrosis mouse model was established to evaluate the anti-IPF effects of QFY. The bioactive ingredients in QFY were identified by HPLC-MS, following which their in vitro effects were examined by the cell models of epithelial-to-mesenchymal transition, fibroblast-to-myofibroblast transition and macrophage polarization. Network pharmacology and clinical dataset (GSE110147) were integrated to predict the possible mechanism of the bioactive ingredients, which were verified by in vitro experiments, molecular docking and molecular dynamics simulation. RESULTS:QFY demonstrated dose-dependent amelioration of IPF pathological phenotypes. Oroxylin A (OA), Geniposide (GDS), Baicalin (BCL), and Genipin-1-gentiobioside (GG) were identified with significant lung tissue enrichment, where OA and BCL exhibited obvious improvement in EMT, FMT, and macrophage polarization experiments. Network pharmacology and bioinformatics analyses revealed the association of OA/GDS/BCL/GG with the PI3K-AKT signaling pathway, which were confirmed by their effects on the migration and apoptosis of A549 and HFL1 cells. Concurrently, molecular docking and molecular dynamics simulations demonstrated strong binding affinities of BCL and OA for p110α and p110γ subunits of PI3K. CONCLUSIONS:Our work demonstrates the potential of QFY in the treatment of IPF, which might due to the bioactive ingredients in the lung affecting the PI3K signaling pathway.

Supplementary Table and Figures S1-S21

Pancreatic ductal adenocarcinoma (PDAC) remains refractory to current immune checkpoint blockade (ICB) therapies, necessitating innovative therapeutic strategies. Emerging evidence implicates aberrant sialoglycan upregulation as a key mediator of immune evasion in PDAC. Herein, we report Y-320, a highly potent oral sialylation inhibitor discovered through high-throughput screening. Y-320 suppresses α-2,3/2,6-sialylation in PDAC cells (IC50 ≈ 200 nM) with >300-fold higher activity than the known pan-inhibitor P-3Fax-Neu5Ac. Structural analyses reveal competitive occupation of multiple sialyltransferases' substrate-binding pockets as Y-320's action mechanism. In vivo, Y-320 significantly inhibits tumor growth and remodels the tumor immune microenvironment. Mechanistic studies establish that the therapeutic efficacy of Y-320 depends on the coordinated engagement between CD8+ T cell and macrophage. Importantly, Y-320 synergizes with anti-PD-1 therapy to overcome ICB resistance in PDAC, demonstrating superior tumor suppression compared to monotherapies. Our findings demonstrate that Y-320 shows promise for use as a therapeutic agent for cancer and validates sialylation inhibition as a novel glycoimmune checkpoint strategy for PDAC and other immunotherapy-resistant malignancies.

Aberrant sialylated glycosylation in the tumor microenvironment is a novel immune suppression pathway, which has garnered significant attention as a targetable glycoimmune checkpoint for cancer immunotherapy to address the dilemma of existing therapies. However, rational drug design and in-depth mechanistic studies are urgently required for tumor sialic acid to become valuable glycoimmune targets. In this study, we explored the positive correlation of PD-L1 and sialyltransferase expression in clinical colorectal cancer tissues and identified their mutual regulation effects in macrophages. Subsequently, we characterized a new sialidase with excellent properties from human oral symbiotic bacteria and then developed a novel nanobody-enzyme fusion protein, designated as Nb16-Sia, to concurrently target the PD-L1 and sialic acid. Results from syngeneic colon tumor models reveal superior efficacy of Nb16-Sia over monotherapy and combinations, which could remodel the tumor immune microenvironment. Mechanistically, Nb16-Sia, which could repolarize macrophages from the tumor-promoting M2 to anti-tumor M1 phenotype via the C-type lectin pathway, exerted its antitumor efficacy mainly by regulating tumor-associated macrophages. Our strategy of nanobody-enzyme fusion protein effectively enables the delivery of sialidase, allows the collaboration between anti-PD-L1 nanobody and sialidase in combating tumors, and holds considerable promise for further development.