Colorectal cancer (CRC) is a common malignant tumor with high incidence and mortality worldwide. Conventional therapeutic strategies, such as surgical resection and chemotherapy, which result in improved outcomes for some patients, are greatly hindered by drug resistance and off-target toxicity. Small interfering RNA (siRNA)-mediated RNA interference has provided a breakthrough for precision CRC therapy. However, efficient delivery of siRNA to the tumor site with its complex tumor microenvironment remains challenging. Here, an ionizable mesoporous silica nanoparticle (MSN)-based nanocarrier with high siRNA loading capacity and enhanced cell membrane penetration capability is developed for effective CRC therapy. By modifying diethylamino groups onto the surface of MSNs featuring maze-like pores, a new siRNA delivery carrier, denoted as MPMNC, is successfully prepared. MPMNC exhibits improved cellular uptake efficiency in CT26 cells and a significant fluorescence silencing effect in EGFP-293T cells. Furthermore, aurora-B siRNA (siAURKB), which downregulates the expression of AURKB in tumor cells to induce apoptosis, is loaded onto MPMNC to afford the nanomedicine siAURKB@MPMNC, which delivers siAURKB to tumor cells and inhibits tumor growth effectively (tumor growth inhibition rate of 64.9%) through intratumor injection in CRC model mice. Additionally, it exhibits excellent biocompatibility in vitro and in vivo. Overall, this work presents a novel nanomedicine, siAURKB-loaded MSN, highlighting its surface modification with ionizable amino groups, which offers a promising therapeutic paradigm for CRC.

Combined blockade of PD-L1 and VEGF represents a promising therapeutic strategy for cancer, yet enhancing anti-VEGF efficacy remains challenging. Here, we engineered a novel bispecific antibody, PLVBP, by fusing a novel high-affinity anti-PD-L1 antibody (T0004) with four VEGF binding domains derived from VEGFR1 D2 (domain II). PLVBP was efficiently expressed in mammalian cells and purified via a three-step chromatography process, achieving over 98% purity. Comprehensive structural characterization confirmed its homogeneity, stability, and preserved secondary structure. Surface plasmon resonance (SPR) analysis revealed that PLVBP binds to PD-L1 and VEGF165 with sub-nanomolar affinities. Functional assays demonstrated that PLVBP maintained potent PD-L1 binding while exhibiting significantly enhanced VEGF binding affinity and capacity compared to bevacizumab. Furthermore, in cell-based assays, PLVBP effectively suppressed VEGF-induced HUVEC proliferation and migration, confirming its VEGF-neutralizing activity. Furthermore, PLVBP simultaneously engaged both PD-L1 and VEGF, validating its dual-targeting capability. Preliminary stability assessments indicated that PLVBP largely maintained its physicochemical and functional integrity during long-term storage in non-optimized buffer. These findings highlight PLVBP as a promising bispecific therapeutic candidate with enhanced avidity-driven VEGF capture, providing a strong foundation for further preclinical development.

Background Insights into basic bone biology and genetic disorders have led to advances in the treatment of osteoporosis. However, new drugs for the treatment of osteoporosis in men remain insufficient. We investigated the efficacy of a novel anti-Dkk1 monoclonal antibody (Dkk1-mAb) and its sequential alendronate treatment in a mouse model of osteoporosis induced by orchiectomy. Methods A specific Dkk1-mAb was generated using the hybridoma technology. A total of 40 male C57BL/6 mice aged 12 weeks underwent orchidectomy or sham surgery. At 8 weeks postoperatively, the orchiectomized mice were randomly divided into 4 groups (8 in each group) to receive treatment of Dkk1-mAb, alendronate, Dkk1-mAb followed by alendronate, or placebo. After 8 weeks of treatment, the mice were euthanized, and bone mineral density (BMD), skeletal mechanical properties, bone histology, bone turnover biomarkers, serum levels of Dkk1 and sclerostin, muscle properties, and activity of the Wnt signaling were evaluated. Results After 8 weeks of treatment, serum Dkk1 levels were lower in Dkk1-mAb and sequential groups than placebo group (P < 0.001, P < 0.05). L1-5 BMD was higher in Dkk1-mAb, alendronate, and sequential groups by 11.9 %, 22.8 %, and 23.1 % than placebo group (P < 0.05 or P < 0.001), with BMD at left femur increased by 12.8 % and 21.0 % in alendronate and sequential groups than placebo group (P < 0.05 or P < 0.001). The sequential group exhibited higher vertebral trabecular volume/total volume (39.9 %), bone surface/total volume (25.8 %), trabecular BMD (4.0 %), and trabecular thickness (13.1 %) than Dkk1-mAb group (P < 0.01 or P < 0.001), and higher cortical BMD, thickness than Dkk1-mAb (2.9 %, P < 0.01; 6.6 %, P < 0.05) and alendronate (2.2 %, 5.7 %, all P < 0.05) groups. All treatment groups showed greater bone strength, cross-sectional area of muscle, and grip strength than placebo group. The expression of Lef1 was significantly increased in bones of Dkk1-mAb and sequential groups. No pathological abnormalities in vital organs were found in all groups, suggesting that Dkk1-mAb had a good safety profile. Conclusions The novel Dkk1-mAb has therapeutic potential in osteoporosis, which can increase BMD and bone strength, improve bone microarchitecture and muscle performance of orchiectomy-induced osteoporotic mice through activating the WNT pathway, and its sequential treatment with alendronate achieves additive benefits. The Translational Potential of this Article This study found that the novel Dkk1-mAb can increase BMD and bone strength, improve bone microarchitecture and muscle performance of orchiectomy-induced osteoporosis mice, and sequential treatment with Dkk1-mAb and alendronate achieves additive benefits. Dkk1-mAb has translational potential for the treatment of osteoporosis.

Colorectal cancer (CRC) ranks as one of the leading causes of cancer-related mortality globally. NPDC1 is a novel regulator involved in cell proliferation and is upregulated in CRC. However, the biological function and mechanism of NPDC1 driving CRC progression have not been investigated. We integrated single-cell RNA-seq data and bulk RNA-seq cohorts to identify prognostic epithelial gene clusters. The R package “ClusterGVis” was employed to categorize six distinct gene clusters within epithelial cells, following Cox regression identifying poor prognosis genes (HR > 1) in the C1 cluster showing progressive upregulation across the four stages. NPDC1 expression was validated by quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC) and immunofluorescence (IF). Functional impacts on proliferation, metastasis, and immune microenvironment were assessed using CCK8 assays, EdU staining, colony formation, transwell assays and flow cytometry. Additionally, gene set enrichment analysis (GSEA) based on KEGG terms was performed to investigate the potential signaling pathways and biological functions associated with NPDC1 in CRC. The regulatory role of NPDC1 in tumor progression was assessed establishing subcutaneous xenograft tumor model and lung metastasis model of mouse CRC. NPDC1 is significantly upregulated in KRAS mutant CRC and correlates with poor prognosis. Functional experiments demonstrated that NPDC1 drives CRC proliferation in vitro and in vivo but does not affect apoptosis, migration, or invasion. Mechanistically, KRAS mutation-induced glutamine metabolism elevates NPDC1 expression via JUND, activating the PI3K-AKT pathway to promote tumor growth independently of immune modulation. Collectively, our results reveal NPDC1 as a KRAS-glutamine axis effector that specifically regulates CRC proliferation via PI3K/AKT signaling, suggesting that NPDC1 could serve as a potential therapeutic target for CRC treatment, particularly in KRAS mutant CRC.

Penetrating orocutaneous and oropharyngeal fistulas (POFs) are difficult to treat due to continuous oral muscle movement and saliva leakage, and persistent bacterial infection and inflammation in the oral environment. However, healing biomaterials that can simultaneously block the penetrating wound, exert antibacterial and anti-inflammatory effects and degrade timely to accommodate fistula closure are rarely reported. Here, we report an organic-inorganic hybrid hydrogel termed MPMQb@gel, which exhibits robust mechanical strength, flexibility, and adhesion ability, enabling effective POF blockage. It consists of hemiaminal structure-containing polymeric hydrogel and Mn-polydopamine nanoparticles cross-linked via aldehyde-amine condensation. Triggered by excessive reactive oxygen species (ROS) in the POF microenvironment, MPMQb@gel gradually degrades and releases Mn2+, quercetin (Que), and basic fibroblast growth factor (bFGF) to achieve synergistic antibacterial, anti-inflammatory and pro-healing effects. In a New Zealand rabbit model of severe POF infection, this hybrid hydrogel realized complete POF healing within 12 days with administration every 4 days, markedly faster than commercial iodoform gauze-treated group. RNA sequencing and mechanistic analysis reveal that MPMQb@gel suppresses Th1 and Th17 cell differentiation, inhibit the Th17/IL-17-associated signaling axis and modulate the TLR2/STAT3 pathway to promote a stable immune microenvironment, thereby attenuating the inflammatory response and ultimately promoting POF repair. Collectively, by addressing the critical challenge of reconciling wound contraction with the requirement for durable occlusion through a controllably biodegradable organic-inorganic hybrid hydrogel, this work offers a transformative therapeutic strategy for POFs and penetrating wounds.

Succinimide (Asu), a critical intermediate in asparagine deamidation and aspartic acid isomerization, represents a potential critical quality attribute (CQA) of therapeutic antibodies but remains challenging to accurately quantify due to its pH-dependent instability during conventional peptide mapping (PM)-based analysis. To address this limitation, we developed a novel rapid subunit-based multi-attribute method (MAM) that combines IdeS-mediated antibody cleavage with liquid chromatography-mass spectrometry (LC-MS) for rapid, simultaneous monitoring of Asu and other post-translational modifications (PTMs), including oxidation and glycosylation. This approach significantly reduces sample processing time and minimizes Asu degradation by operating under optimized pH conditions (pH 6.5). The method was rigorously validated, demonstrating excellent specificity, precision and accuracy across multiple antibody isotypes. Forced oxidation studies further confirmed strong correlation with traditional PM results, underscoring the method's reliability for comprehensive PTM profiling. As the first reported rapid subunit-based MAM specifically optimized for Asu analysis, this strategy provides a rapid, accurate, and comprehensive (RAC) tool for enhanced quality control and stability monitoring of therapeutic antibodies, thereby supporting improved biopharmaceutical production efficiency and product safety.

Fc-containing GLP-1 therapeutics exhibit complex post-translational modification (PTM) heterogeneity, necessitating advanced analytical methods for quality control (QC) and process analytical technology (PAT). We developed a reverse-phase liquid chromatography (RP-LC) method for the PTM-specific profiling of these biologics. Using dulaglutide (IgG4-Fc) as a model, critical parameters─including mobile-phase additives, acid concentration, and shallow gradients─were optimized to resolve PTM variants (e.g., hydroxylation, N-terminal truncation, disulfide reduction, glycosylation) within 40 min. Mass spectrometry (MS) compatibility was enabled by adopting difluoroacetic acid (DFA) as an alternative ion-pairing reagent to support intact-mass characterization of variants. This enabled the identification of additional PTMs not readily resolved under the initial RP-LC conditions, including site-specific HyK-Gal-Glc O-glycosylation and process-dependent truncations. The method also allowed for the direct quantification of critical impurities and the detection of process-induced variants across biosimilar clones. The method was further demonstrated on the IgG2-subtype GLP-1-Fc-fusion (supaglutide), showing applicability across the two Fc subtypes examined without additional optimization. This robust, MS-compatible RP-LC platform provides a rapid, accurate, and comprehensive (RAC) means of conducting PTM-specific QC for Fc-GLP-1 therapeutics, supporting PAT implementation and accelerating biosimilar and next-generation drug development.

Macrophage anti-tumor efficacy requires coordinated inflammatory activation and phagocytic function, whether a tumor-intrinsic metabolic regulator simultaneously determines both macrophage anti-tumor mechanisms remains unclear. Here, we screened and identified that farnesyl-diphosphate farnesyltransferase 1 (FDFT1) drives the dual inhibition of macrophage activation and phagocytic function and promotes tumor progression. Mechanistically, tumor-intrinsic FDFT1 directly binds to STAT3 and facilitates the later phosphorylation, which induces PD-L1-dependent suppression of macrophage phagocytosis. Concurrently, FDFT1 binds to and stabilizes cholesterol 25-hydroxylase (CH25H) to promote the secretion of 25-hydroxycholesterol (25HC), suppressing proinflammatory activation of macrophages. Furthermore, FDFT1-mediated dual anti-tumor pathways were validated in mouse tumor models and correlated with clinical pathophysiology. Notably, a small-molecule drug FDFT1-I (2123) targeting FDFT1 inhibits both STAT3-PD-L1 and CH25H/25HC pathways and improves anti-tumor immunity. Collectively, our findings highlight FDFT1 as a tumor-intrinsic metabolic factor promoting tumor development via dual macrophage-dependent mechanisms, suggesting FDFT1 as a promising target for tumor therapy.

IntroductionOsteoporosis remains a major global health challenge, and current single-pathway therapies often fail to achieve coordinated bone remodeling. Dickkopf-1 (DKK-1) is a potent inhibitor of the Wnt/β-catenin signaling pathway and plays a critical role in osteoporotic bone loss. Neutralization of DKK-1 represents a promising therapeutic strategy to promote bone formation while simultaneously inhibiting bone resorption.MethodsA humanized anti-DKK-1 monoclonal antibody (IgG4) was generated via CDR grafting combined with structure-guided back-mutations. Its binding affinity and specificity were assessed by ELISA and surface plasmon resonance. The antibody's ability to restore Wnt/β-catenin signaling was evaluated in human bone marrow mesenchymal stem cells (hMSCs) by Western blotting. Osteogenic differentiation and mineralization were assessed by ALP activity assay and Alizarin Red S staining, respectively. An osteoblast-osteoclast Transwell co-culture model was established to evaluate the antibody's effects on osteoclastogenesis via the OPG/RANKL axis, with osteoclast differentiation assessed by TRAP staining and qPCR. Developability properties, including stability and immunogenicity, were also characterized.ResultsThe humanized antibody bound to DKK-1 with high affinity (EC₅₀ = 32.8 ng/mL, KD = 2.760 × 10⁻¹⁰ mol/L) and showed no cross-reactivity to DKK-2, DKK-3, or DKK-4. In hMSCs, the antibody restored Wnt/β-catenin signaling, as evidenced by nuclear β-catenin accumulation, GSK-3β phosphorylation, and c-Myc upregulation, leading to increased ALP activity, OPG secretion, and mineralization. In the Transwell co-culture model, the antibody restored the OPG/sRANKL molar ratio and significantly suppressed osteoclast marker gene expression (ACP5, CALCR, CTSK, ITGB3, MMP9, and NFATC1) as well as TRAP-positive multinucleated cell formation (from 29 ± 5 per field in the undifferentiated control to 2 ± 1 per field in the antibody-treated group). The antibody also exhibited favorable developability properties, including long-term stability at 2 -8°C for over two years and low cellular immunogenicity.DiscussionThese findings demonstrate that the humanized anti-DKK-1 antibody exerts dual anabolic and anti-resorptive effects through the Wnt/β-catenin/OPG/RANKL axis. This dual mechanism of action positions the antibody as a promising therapeutic candidate for osteoporosis, warranting further preclinical and clinical evaluation.

Nucleic acid therapeutics are transforming modern medicine to a new era, yet their intrinsic instability, immunogenicity, and poor intracellular transport continue to limit clinical translation. Although lipid-based nanoparticles have driven recent successes, formulation constraints such as cold-chain dependence and particle instability motivate complementary solutions. Silica-based nanomaterials provide a robust alternative due to their structurally programmable architectures, large accessible surface area, and chemically accessible silanol network, enabling rational tuning of nucleic acid loading, stabilization, and biodistribution. However, existing relevant studies remain fragmented and lack clear summary to provide generalizable design rules. This review integrates current progress and establishes a coherent conceptual basis connecting foundational considerations of carrier systems, NA loading strategies, stimuli-responsive release mechanisms, and therapeutic functions. By emphasizing physicochemical compatibility between silica matrices and NA cargos, we outline actionable design principles to guide the development of next-generation bio-modulatory silica platforms—especially for emerging RNA therapeutics.

Background: The potent topoisomerase I inhibitor SN-38, the active metabolite of irinotecan, is limited in clinical application due to severe systemic toxicity. Prodrug strategies enabling selective activation in the tumor microenvironment offer a promising approach to improve its therapeutic index. This study aims to rationally design, synthesize, and systematically evaluate novel disulfide-based SN-38 prodrugs engineered for redox-responsive activation in hypoxic tumors. Methods: Two novel disulfide-based SN-38 prodrugs (SN-38-CSS and SN-38-LSS) were designed and synthesized; SN-38-CSS incorporates a constrained cis-piperazine-fused six-membered cyclic disulfide linker, while SN-38-LSS contains a linear disulfide tether, to differentially exploit the upregulated thioredoxin (Trx/TrxR) system in hypoxic tumor microenvironments. Results: Both prodrugs demonstrated high stability under physiological pH conditions and in human plasma, minimizing premature release. Crucially, they exhibited selective, rapid degradation in the presence of dithiol reductants (TCEP and DTT), mimicking Trx system activity, while remaining stable towards monothiols (GSH, L-Cys). In vitro cytotoxicity assays revealed that the prodrugs exhibited significantly reduced toxicity compared to SN-38 under normoxic conditions across most tested cell lines. However, under hypoxic conditions, their activity was significantly restored. Specifically, SN-38-CSS exhibited cytotoxicity comparable to SN-38 against MCF-7 and NCI-N87 cells, whereas SN-38-LSS showed lower activation efficiency. Conclusions: SN-38-CSS is identified as a promising redox and hypoxia dual-responsive prodrug candidate, highlighting the strategic use of cyclic disulfide linkers for achieving high selectivity and controlled drug release within the tumor microenvironment.

Mycoplasma species are prevalent microbial contaminants in the production of biological products, such as monoclonal antibodies, posing significant threats to the safety and efficacy of these products. Current regulatory guidelines as well as pharmacopoeias mandate the demonstration of the absence of Mycoplasma in the cell culture and further downstream processing to ensure product safety. Despite recent advancements in sensitive detection techniques for Mycoplasma in eucaryotic expression systems, these methods remain complex and time-consuming. There is a pressing need for a rapid, simple, and sensitive process analytical technology (PAT) for Mycoplasma detection. Here, we report the first development and application of a recombinase polymerase amplification (RPA)-assisted CRISPR-Cas12a (RPA-CRISPR/Cas12a) system spcifically tailored for Mycoplasma detection in biopharmaceutical production. This system combines the high-sensitivity isothermal nucleic acid amplification capabilities of RPA with the trans-cleavage activity of CRISPR-Cas12a reporter probes, enabling the rapid and accurate detection of Mycoplasma, accommodating various experimental requirements and application scenarios. By designing RPA universal primers and crRNA targeting the highly conserved sequences of Mycoplasma 16S rRNA and optimizing reaction conditions, we achieved dual-specific recognition with unprecedented efficiency in bioprocessing samples. All tested Mycoplasma specimens were detectable with limits between 10 and 0.1 copies/μL, with the whole process taking less than 1 hour. We further evaluated the feasibility of this method in detecting Mycoplasma in the cell culture of antibody products and further downstream processing samples. This method reduces the risk of false-positive signals due to non-specific amplification, enhancing detection sensitivity and specificity while significantly reducing analysis, representing the first PAT-compatible method for rapid Mycoplasma monitoring in antibody manufacturing, thereby providing robust assurance for the quality and safety of biological products.

The identification of predictive markers to determine the premetastatic phase before metastasis is critical for developing effective strategies for early detection and prevention. By applying the dynamic network biomarker (DNB) approach to analyze time‐series transcriptomic data from a pulmonary metastasis HCC mouse model, it is revealed that the premetastatic phase occurred during the fourth week after implantation. A total of 142 DNB genes are identified as functionally important biomarkers for HCC metastasis, among which 60S ribosomal protein L6 (RPL6) is a core DNB member. RPL6 is significantly upregulated in HCC tissues with extrahepatic metastasis and is strongly correlated with poor prognosis in HCC patients. RPL6 promotes the invasion and metastasis of HCC cells, both in vitro and in vivo. Mechanistically, RPL6 directly binds to the HMGCS1 mRNA 3′UTR, a rate‐limiting enzyme in cholesterol biosynthesis, thus increasing HMGCS1 mRNA stability and protein expression and subsequently elevating intracellular cholesterol level. Elevated cholesterol inhibits the ubiquitin‐dependent degradation of HIF‐1α, which further results in activation of HIF‐1α signaling pathway. Together, this study provides new insights into the dynamic transcriptome profiles of HCC pulmonary metastasis and establishes an important role for the RPL6‐HMGCS1‐HIF‐1α axis in HCC metastasis, suggesting potential prognostic biomarkers and therapeutic targets in HCC.

Immune checkpoint blockade (ICB) therapy offers hope for improved outcomes in lung cancer treatment, but its effectiveness is restricted by the presence of an immunosuppressive tumor microenvironment (TME), resulting in a limited response rate (< 20%). Here this study reports a tumor‐site glutathione (GSH)/glutathione peroxidase (GPX4) dual‐depletion strategy to induce tumor ferroptosis and amplify cuproptosis via a GSH‐responsive polydopamine‐based hybrid nanoparticle (termed CACuPDA). This approach triggers cellular lysis to reverse immunosuppressive TME and further enhance the therapeutic efficacy of lung tumors combined with anti‐PD‐L1‐based ICB therapy. The released cinnamaldehyde (CA) can stimulate reactive oxygen species production, while Cu 2+ can directly deplete GSH and suppress GPX4. Interestingly, Cu 2+ induces cuproptosis by downregulating ferredoxin (FDX1) expression, whereas reduced Cu + can catalyze hydroxyl radicals (·OH) generation from overexpressed H 2 O 2 at the tumor site. The redox imbalance amplifies ferroptosis and cuproptosis in lung tumor cells, releasing substantial amounts of cellular contents into the immunosuppressive TME, as evidenced by an increased amount of cytotoxic T cells and a decreased amount of immunosuppressive Treg cells. In addition, in vivo experimental results revealed that CACuPDA enhanced the therapeutic effect of anti‐PD‐L1 by about fivefold for lung tumor treatment, providing a promising strategy to improve ICB therapy for lung tumors.

To survive nutrient stress caused by rapid proliferation and dysfunctional vasculature, tumor cells extensively reprogram their metabolic pathways, including the tricarboxylic acid (TCA) cycle representing a critical remodeling node. Functioning as a key TCA cycle intermediate, malate bridges fumarate and oxaloacetate, both of which are metabolites known to play significant roles in tumorigenesis. However, whether malate itself regulates tumor progression and the specific mechanism remain unclear. In this study, we demonstrate that oral administration of malate significantly inhibits the growth of colorectal cancer (CRC) xenografts in both nude mice and immunocompetent models, suggesting its antitumor effects are immunity-independent. Mechanistically, we found that malate acts as an allosteric regulator of pyruvate kinase M2 (PKM2), binding to it and initiating a cascade that promotes the ubiquitin-mediated proteasomal degradation of cell division cycle 25 A (CDC25A). This reduction in CDC25A enhances the inhibitory phosphorylation of CDK1 at Tyr15, leading to cell cycle arrest and suppression of proliferation. Clinical analyses further support these findings, showing decreased malate levels in human CRC tissues. Moreover, the expression of malate-metabolizing enzymes, MDH1 and FH, is significantly correlated with activity of the CDC25A/p-CDK1 signaling axis. Collectively, our results identify malate as a non-metabolic regulator of the cell cycle, operating through the PKM2-CDC25A-CDK1 pathway, and propose a novel therapeutic strategy targeting metabolic mediators of cell proliferation in cancer.

Ensuring the quality of pharmaceutical products, particularly for complex recombinant protein drugs such as TNF receptor 2-Fc fusion proteins (TNFR2-Fc, Etanercept), poses significant public health challenges. These products, including biosimilars and follow-on versions, exhibit intricate glycosylation patterns and heterogeneous post-translational modifications, complicating their analytical assessment. The Chinese market, hosting four different TNFR2-Fc products, presents a unique regulatory challenge for rapid differentiation and quality control. This study developed a novel mass spectrometry-based multiattribute method (MAM) to address this challenge, enabling simultaneous monitoring of multiple quality attributes and effective differentiation among products from various manufacturers. Conventional techniques initially indicated high purity across all products, but these methods provided limited capabilities for differentiation. The improved MAM approach, involving desialylation, partial deglycosylation, and digestion steps, minimizes heterogeneity and simplifies analysis. This method successfully indicates differences in primary amino acid sequences and specific quality attributes, allowing for a clear differentiation among manufacturers. Notably, products from manufacturers A and B, as well as C and D, despite their high similarity, could be differentiated by their O-glycan profiles. Further activity evaluations revealed that the products from manufacturers C and D exhibited lower binding and biological activity, potentially due to differences in primary amino acid sequences or disulfide bond mismatches. Additionally, all products demonstrated similar Fc-effector functions. In conclusion, this study underscores the variability among TNFR2-Fc products in the Chinese market and the necessity for robust regulatory oversight. The MAM method developed herein serves as a rapid, accurate, and technologically advanced platform for quality control with significant implications for regulatory authorities, healthcare providers, and patients in ensuring access to safe and effective TNFR2-Fc products.

The development of high-concentration monoclonal antibody (mAb) formulations for subcutaneous administration is faces critical stability challenges, particularly oxidation and aggregation, which compromise efficacy and safety. While antioxidants are commonly employed, existing studies predominantly focus on low concentrations, leaving the potential of high-concentration antioxidants underexplored. Here, we present the first systematic evaluation of high-concentration L-methionine (L-Met,>20 mM) as a novel antioxidant to address these limitations. Through accelerated stability testing coupled with multi-dimensional analytical techniques, we demonstrated that L-Met at concentrations exceeding 20 mM surpasses conventional antioxidants in mitigating oxidation and aggregation. Synergy with 200 mM trehalose further enhanced stability by reducing oxidative degradation and inhibiting protein aggregation. Comprehensive biophysical analyses confirmed no adverse effects, with some aspects showing improved outcomes in structural integrity, colloidal stability, and thermal behavior. The optimized formulation (25 mM L-Met +200 mM trehalose) also exhibited robust protection against light-induced degradation and broad applicability across therapeutic antibodies. This work pioneers a high-concentration antioxidant strategy, addressing a critical gap in mAb formulation science and offering a translatable solution for stabilizing next-generation high-concentration biologics.

Glioblastoma (GBM) remains a therapeutically intractable central nervous system malignancy with limited treatment options. Apatinib, a selective vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitor, shows emerging potential in recurrent or refractory glioblastoma, yet its impact on the cerebrospinal fluid (CSF) glycome remains unexplored. In this pioneering longitudinal study, we characterize the N-glycome dynamics in paired CSF specimens from GBM patients pre- and post-apatinib treatment using hydrophilic interaction-based ultra performance liquid chromatography (HILIC-UPLC)-fluorescence mass spectrometry-based glycan profiling. Our results highlight potential alterations in specific glycoforms, particularly an increase in GalNAcβ1-4GlcNAc (LacdiNAc or LDN), core fucosylation(a modification catalyzed by α1,6-fucosyltransferase (Fut8), which transfers L-fucose from GDP-fucose to the innermost GlcNAc residue), and bisecting glycans(a β1,4-linked GlcNAc attached to the core β-mannose residue), a decrease in sialylation and biantennary glycans among treatment responders. These shifts correlate with attenuated immunosuppressive signatures, suggesting enhanced immunomodulation that may contribute to apatinib's therapeutic efficacy. This work uncovers the remodeling of the CSF glycome driven by apatinib, providing a novel molecular lens for understanding its anti-angiogenic/immune-modulating mechanisms and proposing CSF N-glycans as dynamic biomarkers for treatment efficacy in GBM.

The role of galectin-4 (LGALS4) in colorectal cancer (CRC) progression and the tumor immune microenvironment (TIME) is poorly understood. Through analysis of single-cell profiling data from various colon tissues, this work identifies tumor suppressor genes critically involved in CRC development. The impact of LGALS4 on CT26 cell growth was evaluated through a combination of in vitro and in vivo experiments. The potential mechanisms regulating LGALS4 expression and LGALS4 regulatory biological functions were investigated using different analytical and detection assays. In vivo experiments evaluated the synergistic anti-tumor effect of LGALS4 overexpression and PDL1 neutralizing antibody. This study revealed that the expression of LGALS4 was significantly reduced in clinical CRC tissue samples. Multimodal analysis combined with experimental verification suggested that TP53 mutations might mediate the downregulation of LGALS4 expression. Functional studies indicated that overexpression of LGALS4 did not affect the biological functions of CT26 cells, such as proliferation, apoptosis, and migration, but significantly inhibited the growth of subcutaneous transplanted tumor. Mechanistically, LGALS4 exerts its effect by improving the TIME. Specifically, it activates the CCL4/CCR5 axis through the NF-κB signaling pathway, thereby promoting the recruitment of various immune cells, including macrophages. Moreover, overexpression of LGALS4 can synergistically enhance anti-tumor effects with PD-L1 neutralizing antibodies. Collectively, these findings demonstrate a novel role for TP53-mediated LGALS4 in regulating tumor progression. This work identifies LGALS4 as a promising therapeutic target and provides a foundation for the development of combined immunotherapy based on galectin for CRC.