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Abstract Certain immune-checkpoint inhibitors have a narrow therapeutic window (TW) as cancer therapeutics, and engineered dual-/multi-targeting agents could potentially widen the TW to bring true clinical benefits. We report a new rationally-designed bispecific-antibody (BsAb), HX009, simultaneously targeting PD1 and CD47 to improve both the efficacy and safety over the respective single-targeting agents by grafting the extracellular domain of SIRPα onto the parental anti-PD1-monoclonal antibody, HX008. This resulted in an IgG4-based “2 × 2” symmetric structure but with an intentionally-reduced CD47-binding affinity, suggesting a novel candidate cancer immunotherapy. Specifically, HX009 has binding affinity constant of 8.951 × 10–9 M for human PD1 and 2.557 × 10–8 M for human CD47, respectively, where the CD47 binding is significantly weaker as compared to the binding affinity of HX008 to PD1 as well as the binding affinity of SIRPα-Fc to CD47, leading to little binding to RBCs and platelets and is contrasting to many CD47-agents in development. However, HX009 effectively and simultaneously binds to the PD1 and CD47 on PD1+CD47+ T-cells via cis-binding and elicits enhanced T cell activation compared to the parental HX008. HX009 caused little cytokine-release in human peripheral blood mononuclear cells. HX009 cross-species binds to cynomolgus monkey PD1/CD47 but not to rodents, making cynomolgus monkeys the choice of species to investigate the pharmacokinetics (PK) and toxicology of HX009. HX009’s anti-tumor activities were confirmed in several humanized preclinical mouse models by determining either its anti-PD1 (humanized hu-CD47-MC38 models) or anti-CD47 (HuT-102 lymphoma CDX and three PDX-AML models) functions, although limited available humanized models have hindered broadly demonstration of enhanced anti-tumor activities contributed from the dual targeting of the BsAb. The expanded DLBCL-PDX trial data suggested that both EBV-status and OX40 expression could potentially be two positive predictors for response to HX009. An intravenous (IV) infusion PK study in cynomolgus monkey revealed its largely vasculature distribution, terminal half-life (T1/2) of ~ 50 h, and dose-proportional exposure without accumulation. The anti-drug antibody (ADA) was observed in all monkeys as expected, affecting the PK parameters of repeated administration. The IV single-dose toxicology study with a 14-day observation revealed a maximum tolerated dose of 150 mg/kg, while the repeated-dose (once weekly for 4 weeks, 5 doses in total) study showed a highest non-severely toxic dose (HNSTD) of 15 mg/kg. The desired preclinical PK and safety profiles, along with its antitumor activity, support HX009’s candidacy for its clinical development.

Abstract Retinol is widely used to treat skin ageing because of its effect on cell differentiation, proliferation and apoptosis. However, its potential benefits appear to be limited by its skin permeability. Herein, we investigated the transcutaneous behavior of retinol in semisolid cosmetics, in both in vitro and in vivo experiments. In vitro experiments used the modified Franz diffusion cell combined with Raman spectroscopy. In in vivo experiments, the content of retinol in rat skin and plasma was detected with HPLC. Retinol in semisolid cosmetics was mainly concentrated in the stratum corneum in the skin of the three animal models tested, and in any case did not cross the skin barrier after a 24 h dermatologic topical treatment in Franz diffusion cells tests. Similar results were obtained in live mice and rats, where retinol did not cross the skin barrier and did not enter the blood circulation. Raman spectroscopy was used to test the penetration depth of retinol in skin, which reached 16 μm out of 34 μm in pig skin, whereas the skin of mouse and rat showed too strong bakground interference. To explore epidermal transport mechanism and intradermal residence, skin transcriptomics was performed in rats, which identified 126 genes upregulated related to retinol transport and metabolism, relevant to the search terms “retinoid metabolic process” and “transporter activity”. The identity of these upregulated genes suggests that the mechanism of retinol action is linked to epidermis, skin, tissue and epithelium development.

Abstract CSF1R is a receptor tyrosine kinase responsible for the growth/survival/polarization of macrophages and overexpressed in some AML patients. We hypothesized that a novel multi-kinase inhibitor (TKi), narazaciclib (HX301/ON123300), with high potency against CSF1R (IC50 ~ 0.285 nM), would have anti-AML effects. We tested this by confirming HX301’s high potency against CSF1R (IC50 ~ 0.285 nM), as well as other kinases, e.g. FLT3 (IC50 of ~ 19.77 nM) and CDK6 (0.53 nM). An in vitro proliferation assay showed that narazaciclib has a high growth inhibitory effect in cell cultures where CSF1R or mutant FLT3-ITD variants that may be proliferation drivers, including primary macrophages (IC50 of 72.5 nM) and a subset of AML lines (IC50

Abstract Both PD1/PD-L1 and CD47 blockades have demonstrated limited activity in most subtypes of NHL save NK/T-cell lymphoma. The hemotoxicity with anti-CD47 agents in the clinic has been speculated to account for their limitations. Herein we describe a first-in-class and rationally designed bispecific antibody (BsAb), HX009, targeting PD1 and CD47 but with weakened CD47 binding, which selectively hones the BsAb for tumor microenvironment through PD1 interaction, potentially reducing toxicity. In vitro characterization confirmed: (1) Both receptor binding/ligand blockade, with lowered CD47 affinity; (2) functional PD1/CD47 blockades by reporter assays; (3) T-cell activation in Staphylococcal-enterotoxin-B-pretreated PBMC and mixed-lymphocyte-reaction. In vivo modeling demonstrated antitumor activity in Raji-B and Karpass-229-T xenograft lymphomas. In the humanized mouse syngeneic A20 B-lymphoma (huCD47-A20) HuGEMM model, which has quadruple knocked-in hPD1xhPD-L1xhCD47xhSIRPα genes and an intact autologous immune-system, a contribution of effect is demonstrated for each targeted biologic (HX008 targeting PD1 and SIRPα-Fc targeting CD47), which is clearly augmented by the dual targeting with HX009. Lastly, the expression of the immune-checkpoints PD-L1/L2 and CD47 seemed co-regulated among a panel of lymphoma-derived-xenografts, where HX009 maybe more effective in those with upregulated CD47. Our data warrants HX009’s further clinical development for treating NHLs.

Abstract Cancers are immunologically heterogeneous. A range of immunotherapies target abnormal tumor immunity via different mechanisms of actions (MOAs), particularly various tumor-infiltrate leukocytes (TILs). We modeled loss of function (LOF) in four common anti-PD-1 antibody-responsive syngeneic tumors, MC38, Hepa1-6, CT-26 and EMT-6, by systematical depleting a series of TIL lineages to explore the mechanisms of tumor immunity and treatment. CD8+-T-cells, CD4+-T-cells, Treg, NK cells and macrophages were individually depleted through either direct administration of anti-marker antibodies/reagents or using DTR (diphtheria toxin receptor) knock-in mice, for some syngeneic tumors, where specific subsets were depleted following diphtheria toxin (DT) administration. These LOF experiments revealed distinctive intrinsic tumor immunity and thus different MOAs in their responses to anti-PD-1 antibody among different syngeneic tumors. Specifically, the intrinsic tumor immunity and the associated anti-PD-1 MOA were predominately driven by CD8+ cytotoxic TILs (CTL) in all syngeneic tumors, excluding Hepa1-6 where CD4+ Teff TILs played a key role. TIL-Treg also played a critical role in supporting tumor growth in all four syngeneic models as well as M2-macrophages. Pathway analysis using pharmacodynamic readouts of immuno-genomics and proteomics on MC38 and Hepa1-6 also revealed defined, but distinctive, immune pathways of activation and suppression between the two, closely associated with the efficacy and consistent with TIL-pharmacodynamic readouts. Understanding tumor immune-pathogenesis and treatment MOAs in the different syngeneic animal models, not only assists the selection of the right model for evaluating new immunotherapy of a given MOA, but also can potentially help to understand the potential disease mechanisms and strategize optimal immune-therapies in patients.

Due to their relatively high compatibility with specific photonic structures, strong light-matter interactions and unique nonlinear optical response, two-dimensional (2D) materials, such as graphene and transition metal dichalcogenides, are attractive for ultrafast photonics applications. Here, we fabricate MoS2/graphene nanocomposites by a typical hydrothermal method. In addition, we systematically investigate their nonlinear optical responses. Our experiments indicate that the combined advantages of ultrafast relaxation, a broadband response from graphene, and the strong light-matter interaction from MoS2, can be integrated together by composition. The optical properties in terms of carrier relaxation dynamics, saturation intensity and modulation depth suggest great potential for the MoS2/graphene nanocomposites in photonics applications. We have further fabricated 2D nanocomposites based optical saturable absorbers and integrated them into a 1.5 μm Erbium-doped fiber laser to demonstrate Q-switched and mode-locked pulse generation. The fabrication of 2D nanocomposites assembled from different types of 2D materials, via this simple and scalable growth approach, paves the way for the formation and tuning of new 2D materials with desirable photonic properties and applications. [ABSTRACT FROM AUTHOR]

To explore the role of YAP, a key effector of the Hippo pathway, in temporomandibular joint (TMJ) ankylosis. The temporal and spatial expression of YAP was detected via immunohistochemistry and multiplex immunohistochemistry on postoperative Days 1, 4, 7, 9, 11, 14 and 28 in a sheep model. Isolated mesenchymal stem cells (MSCs) from samples of the Day 14. The relative mRNA expression of YAP was examined before and after the osteogenic induction of MSCs. A YAP-silenced MSC model was constructed, and the effect of YAP knockdown on MSC function was examined. YAP is expressed in the nucleus of the key sites that determine the ankylosis formation, indicating that YAP is activated in a physiological state. The expression of YAP increased gradually over time. Moreover, the number of cells coexpressing of RUNX2 and YAP-with the osteogenic active zone labelled by RUNX2-tended to increase after Day 9. After the osteogenic induction of MSCs, the expression of YAP increased. After silencing YAP, the osteogenic, proliferative and migratory abilities of the MSCs were inhibited. YAP is involved in the early development of TMJ bony ankylosis. Inhibition of YAP using shRNA might be a promising way to prevent or treat TMJ ankylosis., (© 2024. The Author(s).)

Plasma trimethylamine N-oxide (TMAO) is associated with coronary atherosclerotic plaque and cardiovascular disease risk, but associations between gut microbes in acute coronary syndrome (ACS) and post-ST-segment elevation myocardial infarction (post-STEMI) events are unknown. We investigated associations between gut microbial taxa and systemic TMAO levels and the possible TMAO contribution to incident post-STEMI cardiovascular events., Patients and Methods: A total of 60 patients, including 30 with unstable angina pectoris (UAP), 30 post-STEMI and 30 healthy controls, were enrolled from June to November 2017. Metagenomic sequencing was performed and TMAO and IL-6 were detected., Results: Minimal discriminators of gut microbial taxa (top 40) distinguished ACS patients from controls. Serum TMAO levels were positively associated with increased abundance of Aerococcaceae, Ruminococcaceae&#95;UCG.005, Ruminococcaceae&#95;UCC.014 and X. Eubacterium&#95;fissicatena, and decreased abundance of Lachnospiraceae&#95;FCS020 (P < 0.05). Elevated serum TMAO levels correlated independently with ACS (P < 0.05). Risk stratification for incident major adverse cardiovascular events (MACE) improved at one year in patients with serum TMAO levels ≦2.19 µM. Serum interleukin-6 levels were not significantly increased in patients with ACS and post-STEMI MACE., Conclusions: ACS and incident post-STEMI MACE may be associated with the gut bacteria choline metabolite TMAO. The specific gut microbial taxa identified in association with serum TMAO levels may be potential predictive biomarkers for accurate diagnosis of ACS onset.

Increasing evidence has shown that Chinese herbal medicine (CHM) has promising therapeutic effects in colorectal cancer (CRC); however, the active ingredients and potential targets remain unclear. In this study, we aimed to investigate the relative molecular targets of the Chinese herbs that have been found effective in treating metastatic CRC (mCRC) based on clinical data and network pharmacology. In multivariate analysis CHM resulted an independent prognostic factor. The hazard ratio was 0.103 (95% confidence interval = 0.064-0.164; P < 0.001). Compared with the non-CHM group, the median survival time of the CHM group was also improved (40 versus 12 months; P < 0.001). Eighteen out of 295 herbs showed significant correlation with survival results (P < 0.05). Bioinformatics analysis indicated that the 18 herbs realize anti-CRC activity mainly through suppressing the proliferative activity of ERBB2, peroxisome proliferator-activated receptor gamma, and retinoid X receptor, suppressing angiogenesis via inhibition of VEGFR and VEGFA expression, inhibiting the phosphatidylinositol-3-kinase/AKT1 signaling pathway directly through SRC and AKT1, and reducing tumor necrosis factor-induced inflammation.

Hepes simplex Virus type 1 (HSV-1) is an enveloped DNA virus that can cause lytic and latent infection. miRNAs post-transcriptionally regulate gene expression, and our previous work has indicated that HSV-1 infection induces miR-101 expression in HeLa cells. The present study demonstrates that HSV-1-induced miR-101 is mainly derived from its precursor hsa-mir-101-2, and the HSV-1 immediate early gene ICP4 (infected-cell polypeptide 4) directly binds to the hsa-mir-101-2 promoter to activate its expression. RNA-binding protein G-rich sequence factor 1 (GRSF1) was identified as a new target of miR-101; GRSF1 binds to HSV-1 p40 mRNA and enhances its expression, facilitating viral proliferation. Together, ICP4 induces miR-101 expression, which downregulates GRSF1 expression and attenuates the replication of HSV-1. This allows host cells to maintain a permissive environment for viral replication by preventing lytic cell death. These findings indicate that HSV-1 early gene expression modulates host miRNAs to regulate molecular defense mechanisms. This study provides novel insight into host-virus interactions in HSV-1 infection and may contribute to the development of antiviral therapeutics.