BACKGROUND:Rheumatoid arthritis (RA) is characterized by synovial inflammation and hyperplasia, with fibroblast-like synoviocytes (FLS) playing a key pathogenic role. Cysteine metabolism is central to redox homeostasis and ferroptosis regulation, yet its mechanisms in RA-FLS remain poorly understood. This study investigates the role of AMD1, a polyamine metabolism enzyme, in regulating cysteine metabolism and ferroptosis sensitivity in RA-FLS. METHODS:Transcriptomic (GSE89408) and single-cell RNA-seq datasets (GSE200815, GSE246416) were analyzed using differential expression, WGCNA, and machine learning (LASSO, Random Forest, SVM). RA-FLS were subjected to AMD1 knockdown or overexpression, with functional assays for proliferation, migration, redox status, and ferroptosis markers. Rescue experiments used exogenous cysteine or Ferrostatin-1 (Fer-1). A collagen-induced arthritis (CIA) mouse model with local AAV-mediated AMD1 knockdown was used for in vivo validation. RESULTS:AMD1 was significantly upregulated in RA synovium and showed prominent expression in FLS. AMD1 knockdown reduced RA-FLS proliferation and migration, decreased polyamine and glutathione levels, increased ROS and lipid peroxidation, and promoted ferroptosis, as evidenced by reduced SLC7A11 and GPX4 expression, increased ACSL4 expression, and Fe2+ accumulation. Exogenous cysteine or Fer-1 partially reversed these effects. In CIA mice, local AMD1 knockdown alleviated joint swelling, cartilage destruction, and synovitis, while modulating ferroptosis-related protein expression. CONCLUSIONS:AMD1 regulates cysteine metabolism and ferroptosis susceptibility in RA-FLS, representing a promising therapeutic target for RA.

The development of anthelmintic resistance due to long-term and irregular use of drugs such as ivermectin (IVM) poses a major challenge to the prevention and control of haemonchosis caused by Haemonchus contortus. However, the mechanism of IVM resistance in H. contortus remains incompletely understood. Reactive oxygen species (ROS) generated under oxidative stress conditions serve as key regulators of autophagy. Reactive oxygen species induce autophagy, and autophagy mitigates oxidative stress-mediated damage, thereby enhancing cell survival. Although autophagy is known to contribute to H. contortus resistance, the involvement of ROS and autophagy in H. contortus resistance remains unclear. This study explores the regulatory role of ROS-mediated autophagy in H. contortus resistance to IVM, using sensitive and resistant strains as experimental subjects, with resistance assessed via larval migration inhibition tests and RT-qPCR, autophagy levels detected by RT-qPCR and transmission electron microscopy, and ROS levels determined by the DCFH-DA fluorescence probe assay. Results showed that baseline ROS levels were higher in the resistant strain than in the sensitive strain, and IVM treatment increased ROS levels in the sensitive strain (P < 0.05). After N-acetyl-L-cysteine (NAC)-mediated inhibition of ROS, autophagy levels and IVM resistance in the resistant strain were substantially reduced. Lipopolysaccharide (LPS)-induced upregulation of ROS led to substantial increases in autophagy levels and IVM resistance in the sensitive strain. The study demonstrates that ROS may enhance H. contortus resistance to IVM by inducing autophagy. This research shows a potential relationship between ROS, autophagy, and IVM resistance, providing insights into the mechanisms underlying resistance in H. contortus.

There is an urgent clinical need for safe and effective treatment agents and therapy targets for estrogen receptor negative (ER−) breast cancer. G protein-coupled receptor 30 (GPR30), which mediates non-genomic signaling of estrogen to regulate cell growth, is highly expressed in ER− breast cancer cells. We here showed that activation of GPR30 by the receptor-specific agonist G-1 inhibited the growth of ER− breast cancer cells in vitro . Treatment of ER− breast cancer cells with G-1 resulted in G2/M-phase arrest, downregulation of G2-checkpoint regulator cyclin B, and induction of mitochondrial-related apoptosis. The G-1 treatment increased expression of p53 and its phosphorylation levels at Serine 15, promoted its nuclear translocation, and inhibited its ubiquitylation, which mediated the growth arrest effects on cell proliferation. Further, the G-1 induced sustained activation and nuclear translocation of ERK1/2, which was mediated by GPR30/epidermal growth factor receptor (EGFR) signals, also mediated its inhibition effects of G-1. With extensive use of siRNA-knockdown experiments and inhibitors, we found that upregulation of p21 by the cross-talk of GPR30/EGFR and p53 was also involved in G-1-induced cell growth arrest. In vivo experiments showed that G-1 treatment significantly suppressed the growth of SkBr3 xenograft tumors and increased the survival rate, associated with proliferation suppression and upregulation of p53, p21 while downregulation of cyclin B. The discovery of multiple signal pathways mediated the suppression effects of G-1 makes it a promising candidate drug and lays the foundation for future development of GPR30-based therapies for ER− breast cancer treatment.

Proteins are the basic building blocks of life. Studying the protein expression mechanism is essential for understanding the cellular organization principles and the development of biotechnology. Protein expression, involving transcription, translation, folding, and post-translational modification, is a complicatedly regulated process affected by various cellular components and sequence features of the expressed protein. Establishing protein expression models based on expression data is of great significance for probing into the regulatory factors and mechanisms of protein expression. Here we review the recent research progress in the mechanism models for quantitatively simulating the protein expression process and the prediction algorithms based on artificial intelligence for analyzing the regulatory factors. Chemical reaction network models have been developed to mathematically describe the elementary processes in protein expression and simulate the influences of various cellular components such as RNA polymerase and tRNA. However, the experimental determination of the huge number of model parameters is a big challenge. The main objective of data-driven AI models is to study the effects of protein/DNA sequences of the target protein on its expression, and subsequently optimize the sequences to improve protein expression. Methods combining mechanism models and AI models have the potential to deepen our understanding of protein expression processes, providing theoretical and technical support for the efficient production of high-value proteins and coordinate the regulation of different proteins.

T cell receptors (TCRs), a promising therapeutic strategy for targeting intracellular antigens, can specifically recognize antigen peptide presented by human lymphocyte antigen (HLA) and mediate T cell responses to eliminate tumor cells. However, the low affinity of TCRs presents a great challenge to their broader clinical application. TCR-mimic (TCRm) antibodies, with similar antigen recognition capabilities as TCRs but higher affinity, offer a solution to these limitations, enabling the targeting of previously undruggable cancer antigens. Neverthelss, specificity remains a major challenge to ensure therapeutic accuracy and patient safety, as TCRm antibodies need to precisely target a single tumor-specific peptide antigen presented by HLA rather than other similar peptides. Using RenTCR-mimic transgenic mice, we describe the generation and preclinical evaluation of fully human TCRm antibodies by fusing them with a humanized anti-CD3 scFv to form T cell engager or assembled on T cell surface to build TCRm-T cells, further to specifically redirect T cells to lysis tumors by our proprietary RenTCR-mimic platform. We determine cross-reactive peptide sequences predicted by in silico or known off-target sequences. We further confirm the specificity and safety of TCRm antibodies. Despite low density antigen copies, TCRm antibodies elicited antibody-mediated T-cell recognition and cytotoxicity against antigen peptide-HLA complexes-restricted cell lines. Based on these methods, we perform screens to validate TCRm antibodies specificity. These experiments led us to discriminate between the target peptide and the potential off-target peptides presented by HLA and highlight the therapeutics potential of TCRm antibodies in tumor immunotherapy, offering a novel approach to expanding the repertoire of druggable cancer targets. Han Zhang, Wanbo Tang, Jun Du, Yue Zhang, Limin Zhao, Xin Jiao, Baihong Liu, Yi Yang. Development of fully human TCR-mimic antibodies targeting intracellular tumor antigens using humanized RenTCR-mimic transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4768.

This study delineates a hierarchical signaling axis driving gastric cancer (GC) progression through integrated transcriptomic and functional analyses. Single-cell sequencing and TCGA data identified CSF2 as a key oncogene, with elevated expression correlating with poor prognosis. Mechanistically, the transcription factor HES1 directly activates IGF2BP2 transcription, as confirmed by chromatin immunoprecipitation and dual-luciferase assays. IGF2BP2 subsequently stabilizes CSF2 mRNA via N6-methyladenosine (m6A) modification, validated through RNA immunoprecipitation and mRNA decay kinetics. Functional interrogation revealed that the HES1-IGF2BP2-CSF2 axis promotes GC cell growth, motility, and infiltrative capacity while inhibiting apoptosis. Critically, this axis orchestrates glycolytic reprogramming, evidenced by upregulated HK2/PKM2/LDHA expression, increased lactate/ATP production, and enhanced glycolytic flux. In vivo xenografts demonstrated accelerated tumor growth upon axis activation, with immunohistochemistry showing elevated Ki67 and reduced apoptosis. These results establish a novel signaling cascade wherein HES1 transcriptionally integrates IGF2BP2-mediated m6A epitranscriptomics and metabolic rewiring to fuel GC aggressiveness.

Therapeutic antibodies have become important drugs in the field of targeted therapy because of their high specificity to target antigens. Currently, over 100 antibody drugs have been approved for the treatment of various human diseases, yet the targets covered are indeed limited, with approximately half of these drugs targeting only 10 common targets. Approved drugs encompass not only monoclonal antibodies but also antibody-drug conjugates, bispecific antibodies, and antibody fragments. The scarcity of antibody drug targets and subsequent conjugation or engineering modifications pose challenges for the development of novel antibody drugs. In response to the growing demand for fully human antibody drugs, Biocytogen’s antibody discovery team has generated a library of antibodies targeting hundreds of antigens using fully human antibody transgenic mice (RenMice). Our RenMice undergo additional gene-editing procedures to knock out designated target genes. Subsequently, antibodies obtained by conventional immunization have a wide range of epitope-binding properties to homologous protein targets, but also exhibit cross-species reactivity. We have completed over 900 fully human antibody target projects and established a library containing over four hundred thousand off-the-shelf fully human antibody sequences. The antibodies generated from RenMice can be used in a variety of applications, and many of the TAA (tumor-associated antigen)-targeting antibodies have been screened for internalization activity to ensure suitability for ADC development. In addition to fully human monoclonal antibody derived from RenMab platform (fully human immunoglobulin variable domains replacing the mouse loci), RenLite-derived common light chain antibodies are easy to assemble into bispecific antibodies (BsAb) with low mismatch rate and ideal physiochemical properties, which are suitable for BsADC, biparatopic antibody (BpAb) and other modalities. Small and flexible fully human nanobodies based on RenNano platform (fully human heavy chain only) also provide solutions for specific antigen epitopes or multi-targets, multi-epitope antigens. Thus, the fully human antibody library can accelerate the development of antibody drugs. Ting Yi, Jun Du, Yue Zhang, Yuting Hu, Shujin Zhang, Xin Ji, Yi Yang. RenBiologics- a fully human antibody library generated by RenMice facilitates the development of antibody drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1576.

Aging is a highly complex process and one of the largest risk factors for many chronic diseases. Aronia melanocarpa (AM) is rich in bioactive phytochemicals with antioxidant, anti-inflammatory, and anticancer properties. However, little is known about its effects on aging. The objective of this study was to evaluate the effects of AM extract on lifespan and health-span using Caenorhabditis elegans as a representative model. The mechanisms of its effects were explored using transcriptomics and untargeted metabolomics. Results showed that the lifespan of C. elegans was significantly extended by 22.2% after high-dose AM treatment. AM improved the behavior and physiological functions of C. elegans by increasing the pharyngeal pumping rate, decreasing lipofuscin accumulation and the reactive oxygen species level, enhancing resistance to oxidative stress, and increasing the activities of superoxide dismutase and catalase. Transcriptome analysis showed that the pmk-1 gene (mitogen-activated protein kinase 1), which is involved in the MAPK signaling pathway, was the gene with the largest fold change after AM intervention. However, in the C. elegans pmk-1(km25) mutant, the beneficial effect of AM in improving nematode senescence disappeared. An untargeted metabolomics study showed that the levels of 4-hydroxyproline, rhamnose, and cysteine were increased after AM supplementation, and their extending effect on the lifespan and health-span of C. elegans were partly dependent on the pmk-1 gene. In conclusion, our results revealed that AM can promote the lifespan and health-span of C. elegans via the PMK-1 pathway, highlighting the potential of AM as a dietary supplement to delay aging.

Abstract Mutated KRAS proteins are ideal cancer targets, as they are expressed frequently and specifically in certain solid tumors. A large proportion of human colorectal cancer and pancreatic ductal adenocarcinoma express the tumor driver KRAS gene mutations G12V/G12D, but drugs targeting G12V/G12D are not available, revealing a huge unmet clinical need. While small molecules often fail to target the KRAS mutation G12V/G12D, T cell receptor-mimic (TCR-mimic) antibodies can specifically recognize KRAS mutations presented by human leukocyte antigen (HLA), opening up possibilities for targeting such intracellular antigens. Here, we discovered novel antibodies highly specific to G12V/HLA and G12D/HLA complexes by immunizing our proprietary RenTCR-mimicTM mice and using high-throughput Beacon-based screening. These TCR-mimic antibodies have higher affinities compared to endogenous TCRs, which may effectively reduce the possibility of tumor escape. Germline distribution analysis indicated their high sequence diversity, which suggests diverse epitope targeting. Although pancreatic cancer is extremely difficult to treat and has an extremely low KRAS mutant peptide-HLA complex density on the cell surface, our TCR-mimic antibodies exhibited potent in vitro tumor lysis activity when assembled into CD3 T cell engagers. Furthermore, these antibodies demonstrated convincing off-target safety. Together, our results indicate promising therapeutic potential of these KRAS mutation-targeted TCR-mimic antibodies for the treatment of solid tumors. Citation Format: Jun Du, Wanbo Tang, Xin Jiao, Limin Zhao, Pengfei Du, Yuqi Zhang, Jian Bao, Han Chen, Chaoshe Guo, W. Frank An. Targeting mutant KRAS proteins with novel TCR-mimic fully human antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6717.

Objective : To investigate the mechanism by which Astragalus mongholicus Bunge (AM), and Angelica sinensis Diels (AS) act in interstitial lung disease (ILD) based on computational prediction. Methods : We screened the ingredients of AM and AS in PubMed, the Web of Science, China National Knowledge Infrastructure (CNKI) Databases, etc. Then obtained the potential effective components. By sharing the same molecular with ILD, we got the possible target genes for ILD treatment and constructed components–targets–disease network with Cytoscape software. The CTD (Comparative Toxicogenomics Database) database was used for GO and KEGG enrichment analysis of these target genes. Results : 59 active ingredients that can be druggable were chosen from AM, 67 active ingredients were chosen from AS. 77 overlapping target genes for AM and ILD and 36 overlapping target genes for AS and ILD were acquired. The hub targets of AM were PTGS2, PTGS1,CDK2, MAOA, ESR1, TOP2A, GSK3B, ESR2, PPARG, NOS2, The hub targets of AS were PTGS2, GABRA1, PTGS1, CHRM1, SLC6A2, ADRA1B, ADRAIA, ADRB2, CHRM3, GABRA2, CHRM2. Quercetin, kaempferol, daidzein, pavilion, 7-Hydroxycoumarin, and 5-Hydroxycoumarin were the main active ingredients which have more effective targets. Prediction of the protein-protein interaction network showed PTGS2, GSK3B, PPARG, etc., were the important predicted targets. The enriched KEGG pathways, including the Immune System, Metabolism of lipids and lipoproteins, Cytokine Signaling in the Immune system, Generic Transcription Pathway, The interleukin pathway, Metabolism of proteins, PI3K-Akt signaling pathway, Metabolic pathways, Innate Immune System, Neuroactive ligand-receptor interaction, Metabolism, GPCR downstream signaling, Amine ligand-binding receptors, Class A/1, Calcium signaling pathway. Molecular docking showed that quercetin, kaempferol, daidzein, pavilion, 7-Hydroxycoumarin, 5-Hydroxycoumarin had good binding activities with PTGS2 and GSK3B, which mainly mediated PI3K/Akt and other important signaling pathways in the pathogenesis of ILD. Conclusion : The components in AS and AM share some common targets, such as PTGS2. AM and AS may ameliorate ILD through the PI3K-Akt signaling pathway which is mediated by GSK3B. PTGS2, PPARG may also be vital target genes in the treatment of ILD with AM and AS.

Background A popular target for tumor vaccines and TCR-T cell therapy is NY-ESO-1, a cancer testis antigen (CTA) family member. NY-ESO-1 is expressed at varying degrees in several cancers such as synovial sarcoma, neuroblastoma, melanoma, and breast cancer, but its expression in normal tissues is restricted (testis), which makes it an ideal target for limiting potential off-target toxicity. NY-ESO-1 is an intracellular antigen whose nine peptides (157-165) can be presented to the cell surface by HLA-A02 for T-cell recognition. Biocytogen's TCR-mimic (TCRm) platform is capable of developing TCR-like antibodies that target the NY-ESO-1/HLA-A02 complex to overcome limitations of low TCR affinity. Methods Biocytogen's unique RenMabTM mice were engineered to express HLA-A02 and immunized to obtain TCRm antibodies against NY-ESO-1/HLA-A02. To detect antibody binding to target or off-target cells, flow cytometry was performed. Surface plasmon resonance (SPR) measured the affinity between antibodies and the NY-ESO-1/HLA-A02 complex. Alanine scan substitution was performed to identify the residues critical for TCRm antibody recognition. Cytotoxicity and activation of TCRm-T cells were measured by LDH and an NFAT Jurkat reporter cell line, respectively. In vivo tumor suppression experiments tested the efficacy of NY-ESO-1 TCRm-T cell therapy in severely combined immunodeficient B-NDG mice inoculated with tumor cells. Results Through immuno-exclusive HLA-A02/RenMab mice, combined with high-throughput Beacon screening and sequence verification, fully humanized antibody sequences of NY-ESO-1/HLA-A02 with mature affinity in vivo were obtained. The obtained antibody affinity is at the nM level (TCR is generally at the μM level). We detected key antibody-antigen binding sites and speculated potential off-target peptides following an alanine scan, then using NFAT Jurkat reporter cells, we identified that our candidate antibodies exhibited low off-target risk. The optimal sequence was applied to cell therapy, and we observed that TCRm-CD8+ T cells demonstrated specific killing of target cells in vitro, and showed rapid and obvious clearance effect on NY-ESO-1+HLA-A02+ tumors with low abundance antigen in vivo. Conclusions Biocytogen's TCRm antibody discovery platform combines unique immune technology with a multiplex screening method to obtain a high-affinity NY-ESO-1/HLA-A02 antibody, which can bind and specifically kill HLA-A02-positive cell lines presenting NY-ESO-1. This TCRm platform overcomes the limitation of targeting extracellular antigens and displays a wider range of applications. Ethics Approval All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Biocytogen Beijing Co., Ltd.

Therapeutic antibodies have ushered in a new age of cancer immunotherapy. Historically, these therapies have targeted a limited subset of soluble and cell surface tumor-associated antigens (TAAs). T cell receptors (TCRs) on cytotoxic CD8+ T cells recognize peptide antigens bound to major histocompatibility class I (MHC-I) proteins, called HLA-A/B/C in humans. By this pathway, antigen is regularly sampled from the intracellular peptidome, processed, and presented to cytotoxic T cells. Expanding TCR-based recognition of soluble, intracellular TAAs presented on the surface of malignant cells by this mechanism is a propitious therapeutic strategy. Here we describe a novel platform for generating T cell receptor mimic (TCRm) antibodies using our humanized immunoglobulin (RenMabTM) mice engineered to express HLA. TCRm antibodies have the same binding properties as endogenous TCRs and recognize processed, HLA-bound peptides including intracellular tumor-associated antigens, viral oncoproteins, and cancer-testis antigen (CTA). TCRm antibodies bind peptide-HLA with high specificity and up to nanomolar affinity. Our optimized immunization protocols and high-throughput screening methods allow for one-step TCRm antibody generation. TCRm antibodies can also be used to assemble bispecific T cell engaging antibodies (BiTEs) to enhance tumor targeting of cytotoxic T cells. Biocytogen’s TCRm antibodies are a flexible and powerful tool for cancer immunotherapy. By enabling TCR-mediated recognition of an unrestricted repertoire of cancer neoantigens, TCRm antibodies may find broad clinical application. Citation Format: Jun Du, Taolin Liu, Wanbo Tang, Yue Zhang, Limin Zhao, Xin Jiao, Chao Sun, Pengfei Du, Yuqi Zhang, Baihong Liu, Qingcong Lin, Yi Yang. Targeting intracellular tumor antigens using fully human TCR mimic antibodies derived from HLA transgenic RenMiceTM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2752.

Background and aims: Aberrant long non-coding RNA (lncRNA) expression in cancer can be used as a potential diagnostic biomarker and therapeutic target. In the present study we determined the potential pathogenic mechanism underlying significant aberrant expression of lncRNAs in HepG2-ADR. Methods: First, we identified different levels of lncRNA expression in liver cancer and adjacent non-tumor tissues obtained from public data (GSE70880) in NCBI. Then, the results were verified in a sensitive liver cancer cell line (HepG2) and a adriamycin-resistant liver cancer cell line (HepG2-ADR). Then, the effects of lncRNAs on the phenotype and some biological characteristics were also determined in HepG2 and HepG2-ADR through overexpression and using siRNA interference methods. Results: We showed that lncRNA ENST00000425005 is highly expressed in a liver cancer-resistant cell line when compared with adjacent non-tumor tissues based on bioinformatics analysis and qPCR verification. Compared with the control group, overexpression of lncRNA ENST00000425005 significantly promoted proliferation and adhesiveness, but inhibited apoptosis in HepG2-ADR cells. In contrast, interference of lncRNA in HepG2-ADR cells suppressed proliferation and adhesiveness, and induced apoptosis. Conclusion: In conclusion, lncRNA ENST00000425005 promotes cell proliferation and invasion in drug-resistant liver cancer cells by regulating epithelial-mesenchymal transition-related gene expression and participating in the regulation of EGF and FGF7. (C) 2019 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

The mechanistic action of bromodomain-containing protein 4 (BRD4) in cancer motility, including epithelial-mesenchymal transition (EMT), remains largely undefined. We found that targeted inhibition of BRD4 reduces migration, invasion, in vivo growth of patient-derived xenograft (PDX), and lung colonization of breast cancer (BC) cells. Inhibition of BRD4 rapidly decreases the expression of Snail, a powerful EMT transcription factor (EMT-TF), via diminishing its protein stability and transcription. Protein kinase D1 (PRKD1) is responsible for BRD4-regulated Snail protein stability by triggering phosphorylation at Ser11 of Snail and then inducing proteasome-mediated degradation. BRD4 inhibition also suppresses the expression of Gli1, a key transductor of Hedgehog (Hh) required to activate the transcription of SNAI1, in BC cells. The GACCACC sequence (−341 to −333) in the SNAI1 promoter is responsible for Gli1-induced transcription of SNAI1. Clinically, BRD4 and Snail levels are increased in lung-metastasized, estrogen receptor-negative (ER-), and progesterone receptor-negative (PR-) breast cancers and correlate with the expression of mesenchymal markers. Collectively, BRD4 can regulate malignancy of breast cancer cells via both transcriptional and post-translational regulation of Snail.