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The decline of microglia in the dentate gyrus is a new phenomenon that may explain the pathogenesis of depression, and reversing this decline has an antidepressant effect. The development of strategies that restore the function of dentate gyrus microglia in under stressful conditions is becoming a new focus. Lymphocyte-activating gene-3 (LAG3) is an immune checkpoint expressed by immune cells including microglia. One of its functions is to suppress the expansion of immune cells. In a recent study, chronic systemic administration of a LAG3 antibody that readily penetrates the brain was reported to reverse chronic stress-induced hippocampal microglia decline and depression-like behaviors. We showed here that a single intranasal infusion of a LAG3 antibody (In-LAG3 Ab) reversed chronic unpredictable stress (CUS)-induced depression-like behaviors in a dose-dependent manner, which was accompanied by an increase in brain-derived neurotrophic factor (BDNF) in the dentate gyrus. Infusion of an anti-BDNF antibody into the dentate gyrus, construction of knock-in mice with the BDNF Val68Met allele, or treatment with the BDNF receptor antagonist K252a abolished the antidepressant effect of In-LAG3 Ab. Activation of extracellular signal-regulated kinase1/2 (ERK1/2) is required for the reversal effect of In-LAG3 Ab on CUS-induced depression-like behaviors and BDNF decrease in the dentate gyrus. Moreover, both inhibition and depletion of microglia prevented the reversal effect of In-LAG3 Ab on CUS-induced depression-like behaviors and impairment of ERK1/2-BDNF signaling in the dentate gyrus. These results suggest that In-LAG3 Ab exhibits an antidepressant effect through microglia-mediated activation of ERK1/2 and synthesis of BDNF in the dentate gyrus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The management of extensive bone loss remains a clinical challenge. Numerous studies are underway to develop a combination of biomaterials, biomolecules, and stem cells to address this challenge. In particular, the systemic administration of antibodies against sclerostin, a regulator of bone formation, was recently shown to enhance the bone repair efficiency of dense collagen hydrogels (DCHs) hosting murine dental pulp stem cells (mDPSCs). The aim of the present study was to assess whether these antibodies, encapsulated and released from DCHs, could promote craniofacial bone repair by the local inhibition of sclerostin. In vitro studies showed that antibody loading modified neither the hydrogel structure nor the viability of seeded mDPSCs. When implanted in a mouse calvaria critical-size bone defect, antibody-loaded DCHs showed repair capabilities similar to those of acellular unloaded DCHs combined with antibody injections. Importantly, the addition of mDPSCs provided no further benefit. Altogether, the local delivery of antisclerostin antibodies from acellular dense collagen scaffolds is highly effective for bone repair. The drastic reduction in the required amount of antibody compared to systemic injection should reduce the cost of the procedure, making the strategy proposed here a promising therapeutic approach for large bone defect repair.

Toll-like receptor (TLR) activation converts immunologically inactive tumors into immunologically active tumors by activating tumor residing antigen-presenting cells and recruitment of cytotoxic T lymphocytes. Targeted immune agonists (TIAs) are antibody drug conjugates with small-molecule TLR agonist payloads. The mechanism of action of TIAs involves tumor antigen recognition, Fcγ-receptor-dependent phagocytosis, and TLR-mediated activation to drive tumor killing by myeloid cells. Several new low DAR anti-HER2 TIAs conjugated with novel TLR7 or dual-TLR7/8 agonists with cleavable and noncleavable linkers were synthesized and profiled. In vitro studies demonstrated that these TIAs activate myeloid cells only in the presence of antigen-expressing cancer cells. Evaluation in ELISpot-based assays confirmed the low immunogenicity of these constructs. Systemic administration of the novel TIAs in tumor-bearing mice resulted in tumor reduction at low doses. These results provide a strong rationale for further development of the TIAs as a novel class of immunotherapeutics.

Myasthenia gravis (MG) is a chronic and severe disease of the skeletal neuromuscular junction (NMJ) in which the effects of neurotransmitters are attenuated, leading to muscle weakness. In the most common forms of autoimmune MG, antibodies attack components of the postsynaptic membrane, including the acetylcholine receptor (AChR) or muscle-specific kinase (MuSK). MuSK, a master regulator of NMJ development, associates with the low-density lipoprotein-related receptor 4 (Lrp4) to form the signaling receptor for neuronal Agrin, a nerve-derived synaptic organizer. Pathogenic antibodies to MuSK interfere with binding between MuSK and Lrp4, inhibiting the differentiation and maintenance of the NMJ. MuSK MG can be debilitating and refractory to treatments that are effective for AChR MG. We show here that recombinant antibodies, derived from MuSK MG patients, cause severe neuromuscular disease in mice. The disease can be prevented by a MuSK agonist antibody, presented either prophylactically or after disease onset. These findings suggest a therapeutic alternative to generalized immunosuppression for treating MuSK MG by selectively and directly targeting the disease mechanism., Competing Interests: Competing interests statement:C.S., B.V., K.S., and R.V. are employees of and have equity ownership in argenx BV. Issued patents: S.J.B., NYU Medical School, US9329182 S.J.B., Wei Zhang, Maartje Huijbers, Johannes J. Verschuuren, and Silvere M. van der Maarel; NYU Medical School and LUMC; US20150125442A1 S.J.B. et al., NYU Medical School and argenx; US11492401 Patent applications: M.G.H. et al., LUMC, WO2020/055241 M.G.H. et al., LUMC and argenx, WO2021/180676 R.V. et al.; argenx, Université de Montréal and NYU Medical School; WO2023/147489 R.V. et al.; argenx and NYU Medical School; WO2023/218099. S.J.B. is grateful for financial support for research from argenx.

The effective treatment of acute lung injury (ALI) remains a significant challenge. Patients with ALI demonstrate an abundance of proinflammatory mediators in both bronchoalveolar lavage fluid (BALF) and circulating plasma. Bardoxolone methyl (BM) is a semi-synthetic triterpenoid derived from oleanolic acid, a natural product known for its ability to inhibit proinflammatory signaling. GSDMD is a signaling protein involved in pyroptosis, a form of programmed cell death. It has been reported that its upstream proteins play a role in the pathogenesis of ALI. However, there is currently no research examining whether the effect of BM on the occurrence and development of ALI is associated with changes in GSDMD protein. In this study, we prepared nanostructured lipid carriers loaded with BM and conjugated with anti-PECAM-1 antibody (PECAM@BM NLCs). PECAM@BM NLCs were designed to specifically bind to pulmonary vascular endothelial cells that highly express the PECAM-1 receptors. We also aimed to investigate the protective effects of PECAM@BM NLCs on ALI and elucidate the underlying molecular mechanisms. The results demonstrated that PECAM@BM NLCs accumulated in the lung tissues and significantly alleviated the inflammatory injury of ALI. This was evidenced by the changes in the lung wet/dry ratio, the total protein concentration, proinflammatory cytokines in BALF, and the histopathological progress. Additionally, we elucidated that PECAM@BM NLCs had the ability to inhibit the assembly of NLRP3 inflammasome and pro-caspase-1 complex, thereby suppressing the induction of pyroptosis. This mechanism resulted in the inhibition of N-terminal GSDMD expression and effectively prevented the progression of ALI., Competing Interests: Declaration of Competing Interest We state explicitly that no potential competing interests exist., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background and Purpose: Chitinase-3-like 1 (CHI3L1) causes skin inflammation in the progression of atopic dermatitis. We investigated if anti-CHI3L1 antibody could prevent the development of atopic dermatitis and its mechanisms of action., Experimental Approach: The effect of CHI3L1 antibody on phthalic anhydride-induced atopic dermatitis animal model and in vitro reconstructed human skin (RHS) model were investigated. Expression and release of atopic dermatitis-related cytokines were determined using an enzyme-linked immunosorbent assay, and RT-qPCR, STAT3 and CXCL8 signalling were measured by western blotting., Key Results: Anti-CHI3L1 antibody suppressed phthalic anhydride-induced epidermal thickening, clinical score, IgE level and infiltration of inflammatory cells, and reduced phthalic anhydride-induced inflammatory cytokines concentration. In addition, CHI3L1 antibody treatment inhibited the expression of STAT3 activity in phthalic anhydride-treated skin. It was also confirmed that CHI3L1 antibody treatment alleviated atopic dermatitis-related inflammation in the RHS model. The inhibitory effects of CHI3L1 antibody was similar or more effective compared with that of the IL-4 antibody. We further found that CHI3L1 is associated with CXCL8 by protein-association network analysis. siRNA of CHI3L1 blocked the mRNA levels of CHI3L1, IL-1β, IL-4, CXCL8, TSLP, and the expression of CHI3L1 and p-STAT, and the level of CXCL8, whereas recombinant level of CXCL8 was elevated. Moreover, siRNA of STAT3 reduced the mRNA level of these cytokines. CHI3L1 and p-STAT3 expression correlated with the reduced CXCL8 level in the RHS in vitro model., Conclusion and Implications: Our data demonstrated that CHI3L1 antibody could be a promising effective therapeutic drug for atopic dermatitis., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

We evaluated the potential of sclerostin antibody (SclAb) therapy to enhance osseointegration of dental and orthopaedic implants in a mouse model (Brtl/+) mimicking moderate to severe Osteogenesis Imperfecta (OI). To address the challenges in achieving stable implant integration in compromised bone conditions, our aim was to determine the effectiveness of sclerostin antibody (SclAb) at improving bone-to-implant contact and implant fixation strength. Utilizing a combination of micro-computed tomography, mechanical push-in testing, immunohistochemistry, and Western blot analysis, we observed that SclAb treatment significantly enhances bone volume fraction (BV/TV) and bone-implant contact (BIC) in Brtl/+ mice, suggesting a normalization of bone structure toward WT levels. Despite variations in implant survival rates between the maxilla and tibia, SclAb treatment consistently improved implant stability and resistance to mechanical forces, highlighting its potential to overcome the inherent challenges of OI in dental and orthopaedic implant integration. These results suggest that SclAb could be a valuable therapeutic approach for enhancing implant success in compromised bone conditions., Competing Interests: Declaration of competing interest KMK is a consultant for NextCure, Inc. The remaining authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Marinobufagenin (MBG) is implicated in chronic kidney disease, where it removes Fli1-induced inhibition of the collagen-1. We hypothesized that (i) in nephrectomized rats, aortic fibrosis develops due to elevated plasma MBG and inhibited Fli1, and (ii) that the antibody to MBG reduces collagen-1 and improves vasodilatation. A partial nephrectomy was performed in male Sprague-Dawley rats. Sham-operated animals comprised the control group. At 5 weeks following nephrectomy, rats were administered the vehicle ( n = 8), or the anti-MBG antibody ( n = 8). Isolated aortic rings were tested for their responsiveness to sodium nitroprusside following endothelin-1-induced constriction. In nephrectomized rats, there was an increase in the intensity of collagen staining in the aortic wall vs. the controls. In antibody-treated rats, the structure of bundles of collagen fibers had ordered organization. Western blots of the aorta had lower levels of Fli1 (arbitrary units, 1 ± 0.05 vs. 0.2 ± 0.01; p < 0.001) and greater collagen-1 (arbitrary units, 1 ± 0.01 vs. 9 ± 0.4; p < 0.001) vs. the control group. Administration of the MBG antibody to rats reversed the effect of the nephrectomy on Fli1 and collagen-1 proteins. Aortic rings pretreated with endothelin-1 exhibited 50% relaxation following the addition of sodium nitroprusside (EC 50 = 0.28 μmol/L). The responsiveness of the aortic rings obtained from nephrectomized rats was markedly reduced (EC 50 = 3.5 mol/L) compared to the control rings. Treatment of rats with the antibody restored vasorelaxation. Thus, the anti-MBG antibody counteracts the Fli1-collagen-1 system and reduces aortic fibrosis.

Status epilepticus (SE) is a medical emergency associated with high mortality and morbidity. Na + , K + -ATPase, is a promising therapeutic target for SE, given its critical role in regulation of neuron excitability and cellular homeostasis. We investigated the effects of a Na + , K + -ATPase-activating antibody (DRRSAb) on short-term electrophysiological and behavioral consequences of pilocarpine-induced SE. Rats were submitted to pilocarpine-induced SE, followed by intranasal administration (2 μg/nostril). The antibody increased EEG activity following SE, namely, EEG power in theta, beta, and gamma frequency bands, assessed by quantitative analysis of EEG power spectra. One week later, DRRSAb-treated animals displayed less behavioral hyperreactivity in pick-up tests and better performance in novel object recognition tests, indicating that the intranasal administration of this Na + , K + -ATPase activator immediately after SE improves behavioral outcomes at a later time point. These results suggest that Na + , K + -ATPase activation warrants further investigation as an adjunctive therapeutic strategy for SE.

CD33 (Siglec-3) is a cell surface receptor expressed in approximately 90% of acute myeloid leukemia (AML) blasts, making it an attractive target for therapy of AML. Although previous CD33-targeting antibody-drug conjugates (ADC) like gemtuzumab ozogamicin (GO, Mylotarg) have shown efficacy in AML treatment, they have suffered from toxicity and narrow therapeutic window. This study aimed to develop a novelADCwith improved tolerability and a wider therapeutic window. GLK-33 consists of the anti-CD33 antibody lintuzumab and eight mavg-MMAU auristatin linkerpayloads per antibody. The experimental methods included testing in cell cultures, patient-derived samples, mouse xenograft models, and rat toxicology studies. GLK-33 exhibited remarkable efficacy in reducing cell viability within CD33-positive leukemia cell lines and primary AML samples. Notably, GLK-33 demonstrated antitumor activity at single dose as low as 300 mg/kg in mice, while maintaining tolerability at single dose of 20 to 30 mg/kg in rats. In contrast with both GO and lintuzumab vedotin, GLK-33 exhibited a wide therapeutic window and activity against multidrug-resistant cells. The development of GLK-33 addresses the limitations of previous ADCs, offering a wider therapeutic window, improved tolerability, and activity against drug-resistant leukemia cells. These findings encourage further exploration of GLK-33 in AML through clinical trials., (©2024 American Association for Cancer Research.)

Bone tissue represents the most frequent site of cancer metastasis. We developed a hemichannel-activating antibody, Cx43-M2. Cx43-M2, directly targeting osteocytes in situ, activates osteocytic hemichannels and elevates extracellular ATP, thereby inhibiting the growth and migration of cultured breast and osteosarcoma cancer cells. Cx43-M2 significantly decreases breast cancer metastasis, osteosarcoma growth, and osteolytic activity, while improving survival rates in mice. The antibody's inhibition of breast cancer and osteosarcoma is dose dependent in both mouse and human cancer metastatic models. Furthermore, Cx43-M2 enhances anti-tumor immunity by increasing the population and activation of tumor-infiltrating immune-promoting effector T lymphocytes, while reducing immune-suppressive regulatory T cells. Our results suggest that the Cx43-M2 antibody, by activating Cx43 hemichannels and facilitating ATP release and purinergic signaling, transforms the cancer microenvironment from a supportive to a suppressive state. Collectively, our study underscores the potential of Cx43-M2 as a therapeutic for treating breast cancer bone metastasis and osteosarcoma., Competing Interests: Declaration of interests J.X.J. is a co-founder, shareholder, and a member of the Scientific Advisory Board of AlaMab Therapeutics. J.X.J., M.A.R., S.G., W.X., N.Z., and Z.A. are listed as inventors on relevant patent applications that were exclusively licensed to AlaMab Therapeutics by the Board of Regents of the University of Texas system. The University of Texas has a financial interest in AlaMab in the form of licensing., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Human genetics and preclinical studies have identified key contributions of TREM2 to several neurodegenerative conditions, inspiring efforts to modulate TREM2 therapeutically. Here, we characterize the activities of three TREM2 agonist antibodies in multiple mixed-sex mouse models of Alzheimer's disease (AD) pathology and remyelination. Receptor activation and downstream signaling are explored in vitro, and active dose ranges are determined in vivo based on pharmacodynamic responses from microglia. For mice bearing amyloid-β (Aβ) pathology (PS2APP) or combined Aβ and tau pathology (TauPS2APP), chronic TREM2 agonist antibody treatment had limited impact on microglia engagement with pathology, overall pathology burden, or downstream neuronal damage. For mice with demyelinating injuries triggered acutely with lysolecithin, TREM2 agonist antibodies unexpectedly disrupted injury resolution. Likewise, TREM2 agonist antibodies limited myelin recovery for mice experiencing chronic demyelination from cuprizone. We highlight the contributions of dose timing and frequency across models. These results introduce important considerations for future TREM2-targeting approaches., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Targeted immunomodulation for reactivating innate cells, especially macrophages, holds great promise to complement current adaptive immunotherapy. Nevertheless, there is still a lack of high-performance therapeutics for blocking macrophage phagocytosis checkpoint inhibitors in solid tumors. Herein, a peptide-antibody combo-supramolecular in situ assembled CD47 and CD24 bi-target inhibitor (PAC-SABI) is described, which undergoes biomimetic surface propagation on cancer cell membranes through ligand-receptor binding and enzyme-triggered reactions. By simultaneously blocking CD47 and CD24 signaling, PAC-SABI enhances the phagocytic ability of macrophages in vitro and in vivo, promoting anti-tumor responses in breast and pancreatic cancer mouse models. Moreover, building on the foundation of PAC-SABI-induced macrophage repolarization and increased CD8 + T cell tumor infiltration, sequential anti-PD-1 therapy further suppresses 4T1 tumor progression, prolonging survival rate. The in vivo construction of PAC-SABI-based nano-architectonics provides an efficient platform for bridging innate and adaptive immunity to maximize therapeutic potency., (© 2024. The Author(s).)

The disassembly of the neuromuscular junction (NMJ) is an early event in amyotrophic lateral sclerosis (ALS), ultimately leading to motor dysfunction and lethal respiratory paralysis. The hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most common genetic mutation, and the dipeptide repeat (DPR) proteins have been shown to cause neurodegeneration. While no drugs can treat ALS patients efficiently, new treatment strategies are urgently needed. Here, we report that a MuSK agonist antibody alleviates poly-PR-induced NMJ deficits in C9orf72-ALS mice. The HB9-PR F/F mice, which express poly-PR proteins in motor neurons, exhibited impaired motor behavior and NMJ deficits. Mechanistically, poly-PR proteins interacted with Agrin to disrupt the interaction between Agrin and Lrp4, leading to attenuated activation of MuSK. Treatment with a MuSK agonist antibody rescued NMJ deficits, and extended the lifespan of C9orf72-ALS mice. Moreover, impaired NMJ transmission was observed in C9orf72-ALS patients. These findings identify the mechanism by which poly-PR proteins attenuate MuSK activation and NMJ transmission, highlighting the potential of promoting MuSK activation with an agonist antibody as a therapeutic strategy to protect NMJ function and prolong the lifespan of ALS patients., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The effective management of infectious diseases and the growing concern of antibiotic resistance necessitates accurate and targeted therapies, highlighting the importance of antibiotic susceptibility testing. This study aimed to develop a real-time impedimetric biosensor for identifying and monitoring bacterial growth and antibiotic susceptibility. The biosensor employed a gold 8-channel disk-shaped microelectrode array with specific antibodies as bio-recognition elements. This setup was allowed for the analysis of bacterial samples, including Staphylococcus aureus, Bacillus cereus, and Micrococcus luteus. These microorganisms were successfully cultured and detected within 1 h of incubation even with a minimal bacterial concentration of 10 CFU/ml. Overall, the developed biosensor array exhibits promising capabilities for monitoring S. aureus, B. cereus and M. luteus, showcasing an excellent linear response ranging from 10 to 10 4  CFU/ml with a detection limit of 0.95, 1.22 and 1.04 CFU/mL respectively. Moreover, real-time monitoring of antibiotic susceptibility was facilitated by changes in capacitance, which dropped when bacteria were exposed to antibiotic doses higher than their minimum inhibitory concentration (MIC), indicating suppressed bacterial growth. The capacitance measurements enabled determination of half-maximal cytotoxic concentrations (CC50) values for each bacteria-antibiotic pair. As a proof-of-concept application, the developed sensor array was employed as a sensing platform for the real time detection of bacteria in milk samples, which ensured the reliability of the sensor for in-field detection of foodborne pathogens and rapid antimicrobial susceptibility tests (ASTs)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Neutrophils play a pivotal role in innate immunity by releasing ROS through respiratory bursts to neutralize various pathogenic factors. However, excessive ROS release can cause tissue damage. Adenosine is an endogenous anti-inflammatory molecule inhibiting respiratory burst to protect the host. Adenosine aptamers with antibody-like properties and good stability are expected to act as adenosine antagonists with functional modulation capability. This study compares the effects of adenosine and its aptamer on the respiratory bursts of salivary polymorphonuclear leukocytes and circulating polymorphonuclear leukocytes using a programmable stopped-flow injection approach, ensuring rapid and efficient analysis while maintaining the neutrophils' viability. The results show that primed salivary polymorphonuclear leukocytes exhibit specificities that differ from circulating polymorphonuclear leukocytes. Adenosine aptamer can function as an inhibitory antagonist that distinguishes between physiologically controlled and excessive priming of neutrophils, showing potential application prospects in immunotherapy., Competing Interests: Declaration of competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cajaninstilbene acid (CSA), longistylin A (LLA), and longistylin C (LLC) are three characteristic stilbenes isolated from pigeon pea. The objective of this study was to evaluate the antibacterial activity of these stilbenes against Staphylococcus aureus and even methicillin-resistant Staphylococcus aureus (MRSA) and test the possibility of inhibiting biofilm formation. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of these stilbenes were evaluated. And the results showed that LLA was most effective against tested strains with MIC and MBC values of 1.56 μg/mL followed by LLC with MIC and MBC values of 3.12 μg/mL and 6.25 μg/mL as well as CSA with MIC and MBC values of 6.25 μg/mL and 6.25-12.5 μg/mL. Through growth curve and cytotoxicity analysis, the concentrations of these stilbenes were determined to be set at their respective 1/4 MIC in the follow-up research. In an anti-biofilm formation assay, these stilbenes were found to be effectively inhibited bacterial proliferation, biofilm formation, and key gene expressions related to the adhesion and virulence of MRSA. It is the first time that the anti-S. aureus and MRSA activities of the three stilbenes have been systematically reported. Conclusively, these findings provide insight into the anti-MRSA mechanism of stilbenes from pigeon pea, indicating these compounds may be used as antimicrobial agents or additives for food with health functions, and contribute to the development as well as application of pigeon pea in food science., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The incessant mutations of viruses, variable immune responses, and likely emergence of new viral threats necessitate multiple approaches to novel antiviral therapeutics. Furthermore, the new antiviral agents should have broad-spectrum activity and be environmentally stable. Here, we show that biocompatible tapered CuS nanoparticles (NPs) efficiently agglutinate coronaviruses with binding affinity dependent on the chirality of surface ligands and particle shape. L- penicillamine-stabilized NPs with left-handed curved apexes display half-maximal inhibitory concentrations (IC 50 ) as low as 0.66 pM (1.4 ng/mL) and 0.57 pM (1.2 ng/mL) for pseudo-type SARS-CoV-2 viruses and wild-type Wuhan-1 SARS-CoV-2 viruses, respectively, which are about 1,100 times lower than those for antibodies (0.73 nM). Benefiting from strong NPs-protein interactions, the same particles are also effective against other strains of coronaviruses, such as HCoV-HKU1, HCoV-OC43, HCoV-NL63, and SARS-CoV-2 Omicron variants with IC 50 values below 10 pM (21.8 ng/mL). Considering rapid response to outbreaks, exposure to elevated temperatures causes no change in the antiviral activity of NPs while antibodies are completely deactivated. Testing in mice indicates that the chirality-optimized NPs can serve as thermally stable analogs of antiviral biologics complementing the current spectrum of treatments., Competing Interests: Competing interests statement:The authors declare no competing interest.

The occurrence of many autoimmune diseases takes root on the disrupted balance among Treg cells, Teff cells, etc. Low-dose interleukin-2 (IL-2) cytokine demonstrates promising clinical efficacy in the expansion of Treg cells and the treatment of autoimmune diseases. However, its clinical application is hindered by the small therapeutic index and short half-life. Previous studies have shown that non-covalent complex of human IL-2 and anti-IL-2 antibody biases cytokine activity towards Treg cells and extends IL-2's half-life. The clinical translation of such complex is non-trivial. In this study, we discover an anti-human IL-2 antibody and engineer a covalently-linked single-agent fusion of human IL-2 and its antibody that selectively expands Treg cells and exhibits superior disease control activity in animal models of ulcerative colitis and systemic lupus erythematosus, with proper safety profile and good developability. These studies pave the road for its clinical development in diverse autoimmune diseases., (© 2024. The Author(s).)

The high burden of disease and the long-lasting sequelae following Streptococcus pyogenes (Strep A) infections make the development of an effective vaccine a global health priority. Streptolysin O (SLO), is a key toxin in the complex pathogenesis of Strep A infection. Antibodies are elicited against SLO after natural exposure and represent a key target for vaccine-induced immunity. Here we present the setup and characterization of a hemolysis assay to measure functionality of anti-SLO antibodies in human sera. Assay specificity, precision, linearity, reproducibility, and repeatability were determined. The assay was demonstrated to be highly sensitive, specific, reproducible, linear and performed well in assessing functionality of anti-SLO antibodies induced by exposed individuals. Moreover, different sources of critical reagents, in particular red- blood cells, have been compared and had minimal impact on assay performance. The assay presented here has throughput suitable for evaluating sera in vaccine clinical trials and sero-epidemiological studies to gain further insights into the functionality of infection- and vaccine-induced antibodies., Competing Interests: Declaration of competing interest This work was performed with funds from CARB-X, and with internal funding form GlaxoSmithKline Biologicals SA, who is the sponsor of the study.The work was also supported by funding from the New Zealand Ministry of Health (Te Whatu Ora) as part of an initiative to accelerate Strep A vaccine development for Aotearoa New Zealand. The external funders had no role in the design of the study, in the collection, analyses or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. GSK Vaccines Institute for Global Health Srl is an affiliate of GlaxoSmithKline Biologicals SA. M.C., L.R., L.M, M.I, F.B.S., D.G.M. and O.R. are employees of the GSK group of companies. FBS, MI, DGM, MC, and OR report ownership of GSK shares/share options., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Sclerostin is an extracellular inhibitor of canonical Wnt signaling that inhibits bone formation and stimulates bone resorption. Anti-sclerostin antibodies (Scl-Ab) have been developed as bone-building agents. DKK1, another extracellular inhibitor of the pathway, is upregulated in osteocytes in response to sclerostin inhibition. To further enhance bone-forming effects, a bispecific antibody inhibiting both sclerostin and DKK1 was created (AMG 147). In nonclinical safety studies, AMG 147 resulted in novel skull findings. In the rat, there was increased thickness of skull bones of neural crest origin due to increased subperiosteal compact lamellar and intramembranous woven bone. Externally, subperiosteal fibroblastic/osteoblastic stromal cell proliferation with woven bone and hemorrhage was also observed. Scl-Ab alone resulted in increased skull thickness in the rat, like AMG 147, but without the stromal cell proliferation/woven bone formation. In contrast to embryonic flat bone development, intramembranous bone formed similar to plexiform bone. In the monkey, AMG 147 resulted in macroscopic skull thickening due to a diffuse increase in appositional lamellar bone and increased intramembranous bone on both periosteal surfaces of all skull bones. These data demonstrate that dual inhibition of sclerostin and DDK1 results in unique effects on the skull not observed with sclerostin inhibition alone., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. J-GB, LC, and MF are employees of Charles River Laboratories. TM is an employee of Amgen Inc. and owns Amgen stock. RWB is a former employee of Amgen Inc., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Skin cancers, particularly keratinocyte cancers, are the most commonly diagnosed tumors. Although surgery is often effective in early-stage disease, skin tumors are not always easily accessible, can reoccur and have the ability to metastasize. More recently, immunotherapies, including intravenously administered checkpoint inhibitors, have been shown to control some skin cancers, but with off-target toxicities when used in combination. Our study investigated whether peritumoral administration of an antibody combination targeting PD-1, 4-1BB (CD137) and VISTA might control skin tumors and lead to circulating antitumor immunity without off-target toxicity., Methods: The efficacy of combination immunotherapy administered peritumorally or intravenously was tested using transplantable tumor models injected into mouse ears (primary tumors) or subcutaneously in flank skin (secondary tumors). Changes to the tumor microenvironment were tracked using flow cytometry while tumor-specific, CD8 T cells were identified through enzyme-linked immunospot (ELISPOT) assays. Off-target toxicity of the combination immunotherapy was assessed via serum alanine aminotransferase ELISA and histological analysis of liver sections., Results: The data showed that local administration of antibody therapy eliminated syngeneic murine tumors transplanted in the ear skin at a lower dose than required intravenously, and without measured hepatic toxicity. Tumor elimination was dependent on CD8 T cells and was associated with an increased percentage of CD8 T cells expressing granzyme B, KLRG1 and Eomes, and a decreased population of CD4 T cells including CD4 + FoxP3 + cells in the treated tumor microenvironment. Importantly, untreated, distal tumors regressed following antibody treatment of a primary tumor, and immune memory prevented growth of subcutaneous flank tumors administered 50 days after regression of a primary tumor., Conclusions: Together, these data suggest that peritumoral immunotherapy for skin tumors offers advantages over conventional intravenous delivery, allowing antibody dose sparing, improved safety and inducing long-term systemic memory. Future clinical trials of immunotherapy for primary skin cancer should focus on peritumoral delivery of combinations of immune checkpoint antibodies., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Conditionally activated molecules, such as Probody therapeutics (PbTx), have recently been investigated to improve antitumoral response while reducing systemic toxicity. PbTx are engineered to be proteolytically activated by proteases that are preferentially active locally in the tumor microenvironment (TME). Here, we perform an exploratory study using our recently published quantitative systems pharmacology model, previously validated for other drugs, to evaluate the effectiveness and targeting specificity of an anti-PD-L1 PbTx compared to the non-modified antibody. We have informed the model using the PbTx dynamics and pharmacokinetics published in the literature for anti-PD-L1 in patients with triple-negative breast cancer (TNBC). Our results suggest masking of the antibody slightly decreases its efficacy, while increasing the localization of active therapeutic component in the TME. We also perform a parameter optimization for the PbTx design and drug dosing regimens to maximize the response rate. Although our results are specific to the case of TNBC, our findings are generalizable to any conditionally activated PbTx molecule in solid tumors and suggest that design of a highly effective and selective PbTx is feasible., (© 2023 The Authors. CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Objective animal health evaluation is essential to determine welfare and discomfort in preclinical in vivo research. Body condition scores, body weight, and grimace scales are commonly used to evaluate well-being in murine rheumatoid arthritis (RA) and osteoarthritis experiments. However, nest-building, a natural behavior in mice, has not yet been evaluated in wild type (WT) or genetically modified rodents suffering from collagen antibody-induced arthritis (CAIA). To address this, we analyzed nesting behavior in WT mice, calcitonin gene-related peptide alpha-deficient (αCGRP -/- ) mice, and calcitonin receptor-deficient (Calcr -/- ) mice suffering from experimental RA compared to healthy control (CTRL) groups of the same genotypes. CAIA was induced in 10-12-week-old male mice, and clinical parameters (body weight, grip strength, clinical arthritis score, ankle size) as well as nesting behavior were assessed over 10 or 48 days. A slight positive association between the nest score and body weight and grip strength was found for animals suffering from CAIA. For the clinical arthritis score and ankle size, no significant associations were observed. Mixed model analyses confirmed these associations. This study demonstrates that clinical effects of RA, such as loss of body weight and grip strength, might negatively affect nesting behavior in mice. Assessing nesting behavior in mice with arthritis could be an additional, non-invasive and thus valuable health parameter in future experiments to monitor welfare and discomfort in mice. During severe disease stages, pre-formed nest-building material may be provided to animals suffering from arthritis., (© 2023. The Author(s).)

Hemopexin (HPX) is overexpressed in the retina of patients with diabetes and induces the breakdown of the blood-retinal barrier in vitro. The aim of this study was to evaluate whether HPX blockade by specific antibodies (aHPX) could avoid vascular leakage in vivo and microvascular angiogenesis in vitro and ex vivo. For this purpose, the effect of intravitreal (IVT) injections of aHPX on vascular leakage was evaluated in db/db mice and rats with streptozotocin-induced diabetes using the Evans Blue method. Retinal neurodegeneration and inflammation were also evaluated. The antiangiogenic effect of aHPX on human retinal endothelial cells (HRECs) was tested by scratch wound healing and tube formation using standardized methods, as well as by choroidal sprouting assays from retinal explants obtained in rats. We found that IVT injection of aHPX significantly reduced vascular leakage, retinal neurodegeneration, and inflammation. In addition, treatment with aHPX significantly reduced HREC migration and tube formation induced by high glucose concentration and suppressed choroidal sprouting even after vascular endothelial growth factor stimulation, with this effect being higher than obtained with bevacizumab. The antipermeability and antiangiogenic effects of IVT injection of aHPX suggest the blockade or inhibition of HPX as a new strategy for the treatment of advanced stages of diabetic retinopathy., Article Highlights: Hemopexin (HPX) is the best-characterized permeability factor in steroid-sensitive nephrotic syndrome. We have previously reported that HPX is overexpressed in the retina of patients with diabetes and induces the breakdown of the blood-retinal barrier in vitro. Here, we report that intravitreal injection of anti-HPX antibodies significantly reduces vascular leakage, retinal neurodegeneration, and inflammation in diabetic murine models and that the immunoneutralization of HPX exerts a significant antiangiogenic effect in vitro and in retinal explants. The blockade of HPX can be considered as a new therapy for advanced stages of diabetic retinopathy., (© 2023 by the American Diabetes Association.)

Despite the success of programmed cell death-1 (PD-1) and PD-1 ligand (PD-L1) inhibitors in treating solid tumors, only a proportion of patients respond. Here, we describe a first-in-class bifunctional therapeutic molecule, STAR0602, that comprises an antibody targeting germline Vβ6 and Vβ10 T cell receptors (TCRs) fused to human interleukin-2 (IL-2) and simultaneously engages a nonclonal mode of TCR activation with costimulation to promote activation and expansion of αβ T cell subsets expressing distinct variable β (Vβ) TCR chains. In solution, STAR0602 binds IL-2 receptors in cis with Vβ6/Vβ10 TCRs on the same T cell, promoting expansion of human Vβ6 and Vβ10 CD4 + and CD8 + T cells that acquire an atypical central memory phenotype. Monotherapy with a mouse surrogate molecule induced durable tumor regression across six murine solid tumor models, including several refractory to anti-PD-1. Analysis of murine tumor-infiltrating lymphocyte (TIL) transcriptomes revealed that expanded Vβ T cells acquired a distinct effector memory phenotype with suppression of genes associated with T cell exhaustion and TCR signaling repression. Sequencing of TIL TCRs also revealed an increased T cell repertoire diversity within targeted Vβ T cell subsets, suggesting clonal revival of tumor T cell responses. These immunological and antitumor effects in mice were recapitulated in studies of STAR0602 in nonhuman primates and human ex vivo models, wherein STAR0602 boosted human antigen-specific T cell responses and killing of tumor organoids. Thus, STAR0602 represents a distinct class of T cell-activating molecules with the potential to deliver enhanced antitumor activity in checkpoint inhibitor-refractory settings.

Receptor clustering is the most critical step to activate extrinsic apoptosis by death receptors belonging to the TNF superfamily. Although clinically unsuccessful, using agonist antibodies, the death receptors-5 remains extensively studied from a cancer therapeutics perspective. However, despite its regulatory role and elevated function in ovarian and other solid tumors, another tumor-enriched death receptor called Fas (CD95) remained undervalued in cancer immunotherapy until recently, when its role in off-target tumor killing by CAR-T therapies was imperative. By comprehensively analyzing structure studies in the context of the binding epitope of FasL and various preclinical Fas agonist antibodies, we characterize a highly significant patch of positively charged residue epitope (PPCR) in its cysteine-rich domain 2 of Fas. PPCR engagement is indispensable for superior Fas agonist signaling and CAR-T bystander function in ovarian tumor models. A single-point mutation in FasL or Fas that interferes with the PPCR engagement inhibited apoptotic signaling in tumor cells and T cells. Furthermore, considering that clinical and immunological features of the autoimmune lymphoproliferative syndrome (ALPS) are directly attributed to homozygous mutations in FasL, we reveal differential mechanistic details of FasL/Fas clustering at the PPCR interface compared to described ALPS mutations. As Fas-mediated bystander killing remains vital to the success of CAR-T therapies in tumors, our findings highlight the therapeutic analytical design for potentially effective Fas-targeting strategies using death agonism to improve cancer immunotherapy in ovarian and other solid tumors., (© 2023. The Author(s).)

Ndel1 oligopeptidase activity shows promise as a potential biomarker for diagnosing schizophrenia (SCZ) and monitoring early-stage pharmacotherapy. Ndel1 plays a pivotal role in critical aspects of brain development, such as neurite outgrowth, neuronal migration, and embryonic brain formation, making it particularly relevant to neurodevelopmental disorders like SCZ. Currently, the most specific inhibitor for Ndel1 is the polyclonal anti-Ndel1 antibody (NO Ab ), known for its high specificity and efficient anti-catalytic activity. NO Ab has been vital in measuring Ndel1 activity in humans and animal models, enabling the prediction of pharmacological responses to antipsychotics in studies with patients and animals. To advance our understanding of in vivo Ndel1 function and develop drugs for mental disorders, identifying small chemical compounds capable of specifically inhibiting Ndel1 oligopeptidase is crucial, including within living cells. Due to challenges in obtaining Ndel1's three-dimensional structure and its promiscuous substrate recognition, we conducted a high-throughput screening (HTS) of 2,400 small molecules. Nine compounds with IC 50 -values ranging from 7 to 56 μM were identified as potent Ndel1 inhibitors. Notably, one compound showed similar efficacy to NO Ab and inhibited Ndel1 within living cells, although its in vivo use may pose toxicity concerns. Despite this, all identified compounds hold promise as candidates for further refinement through rational drug design, aiming to enhance their inhibitory efficacy, specificity, stability, and biodistribution. Our ultimate goal is to develop druggable Ndel1 inhibitors that can improve the treatment and support the diagnosis of psychiatric disorders like SCZ., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The anti-inflammatory effect of technologically processed antibodies (TPA) to immune targets (MHC I and MHC II) was assessed in the carrageenan-induced rat paw edema model. The parameters "increase in edema" and "suppression of edema" significantly decreased (p<0.05) against the background of treatment with TPA and the reference drug indomethacin compared to the placebo group. The tested TPA produced an anti-inflammatory effect., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Delivering biologics to elicit a therapeutic response in the central nervous system (CNS) remains challenging due to the presence of the blood-brain barrier (BBB). Receptor-mediated transcytosis is a strategy to improve brain exposure after systemic drug administration. The availability of a clinically relevant in vitro BBB model is crucial to investigate transcytosis pathways and to predict the penetration of biologics into the CNS. We created a perfused human in vitro BBB model made of induced pluripotent stem cells (iPSC)-derived brain microvascular endothelial cells (BMEC) for studying transferrin receptor-mediated transcytosis. iPSC-derived BMEC were seeded in the top channel of a three-lane microfluidic device (OrganoPlate®). After 2 days in culture, the established cell model exhibited relevant BBB features, including physiological transendothelial electrical resistance in a transwell setting (1500 Ω*cm 2 ), reduced apparent permeability (Papp) to the fluorescence tracer Lucifer yellow (20-fold less than cell-free chips), expression of key BBB markers such as tight junctions proteins, transporters, receptors and functional P-gp efflux pump. Moreover, the model exhibited functional transferrin receptor-mediated uptake and transcytosis. To assess selective transferrin receptor-mediated transcytosis, a mixture of anti-human transferrin receptor (MEM-189) and control (sheep IgG anti-bovine serum albumin) antibodies was perfused in the top channel for 2 h. The Papp of MEM-189 was 11-fold higher than that of the control antibody, demonstrating facilitated receptor-mediated transcytosis. Compared to published work reporting a 2-fold ratio, this result is remarkable and establishes the suitability of our model for exploring receptor-mediated transcytosis and screening of antibodies for putative brain shuttle application. A perfused in vitro human model made of iPSC-derived BMEC with the chief characteristics (barrier tightness, functionality) of the human BBB can be applied to study transferrin receptor (TfR)-mediated transcytosis of therapeutic antibodies. This may bring critical advances in drug shuttle technology. Graphical abstract generated with biorender.com., (© 2023. The Author(s).)

The interactions between polymers and the immune system remains poorly controlled. In some instances, the immune system can produce antibodies specific to polymer constituents. Indeed, roughly half of pegloticase patients without immunomodulation develop high titers of anti-PEG antibodies (APA) to the PEG polymers on pegloticase, which then quickly clear the drug from circulation and render the gout treatment ineffective. Here, using pegloticase as a model drug, we show that addition of high molecular weight (MW) free (unconjugated) PEG to pegloticase allows us to control the immunogenicity and mitigates APA induction in mice. Compared to pegloticase mixed with saline, mice repeatedly dosed with pegloticase containing different MW or amount of free PEG possessed 4- to 12- fold lower anti-PEG IgG, and 6- to 10- fold lower anti-PEG IgM, after 3 rounds of pegloticase dosed every 2 weeks. The markedly reduced APA levels, together with competitive inhibition by free PEG, restored the prolonged circulation of pegloticase to levels observed in APA-naïve animals. In contrast, mice with pegloticase-induced APA eliminated nearly all pegloticase from the circulation within just four hours post-injection. These results support the growing literature demonstrating free PEG may effectively suppress drug-induced APA, which in turn may offer sustained therapeutic benefits without requiring broad immunomodulation. We also showed free PEG effectively blocked the PEGylated protein from binding with cells expressing PEG-specific B cell receptors. It provides a template of how we may be able to tune the interactions and immunogenicity of other polymer-modified therapeutics. STATEMENT OF SIGNIFICANCE: A major challenge with engineering materials for drug delivery is their interactions with the immune system. For instance, our body can produce high levels of anti-PEG antibodies (APA). Unfortunately, the field currently lack tools to limit immunostimulation or overcome pre-existing anti-PEG antibodies, without using broad immunosuppression. Here, we showed that simply introducing free PEG into a clinical formulation of PEG-uricase can effectively limit induction of anti-PEG antibodies, and restore their prolonged circulation upon repeated dosing. Our work offers a readily translatable method to safely and effectively restore the use PEG-drugs in patients with PEG-immunity, and provides a template to use unconjugated polymers with low immunogenicity to regulate interactions with the immune system for other polymer-modified therapeutics., Competing Interests: Declaration of Competing Interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: S.K.L. is an inventor of patents on the use of free PEG to block APA. The terms of these arrangements are managed by UNC-CH in accordance with its conflict-of-interest policies. A.C.N., M.S.B. and B.L. are employees of, and have stock in, Horizon Therapeutics., (Copyright © 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Intranasal administration of F(ab)2 fragments of anti-glutamate antibodies to 12-month-old C57BL/6 mice improves passive avoidance conditioning and have no effect on horizontal and vertical locomotor activity in the open-field test. In contrast to full-length antibodies to glutamate, their F(ab)2 fragments significantly increase the number of animals developed a conditioned passive avoidance reflex., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Background and Aims: Radiofrequency ablation (RFA) often results in incomplete ablation for medium-to-large and irregular tumors. To solve this clinical problem, we proposed a new treatment strategy of OK-432 in combination with an anti-programmed cell death protein 1 (αPD-1) antibody for residual tumors after incomplete RFA (iRFA) of hepatocellular carcinoma (HCC)., Approach and Results: The effect of OK-432 on immature dendritic cells (iDCs) was evaluated in vitro. A CCK-8 kit and ELISPOT were used to assess the killing effect of OK-432-induced CD8 + T cells in combination with an αPD-1 antibody on Hepa1-6 cells. We found that OK-432 significantly increased the maturation level of DCs, and OK-432-induced CD8 + T cells in combination with αPD-1 antibody significantly enhanced the function of CD8 + T cells. In the in vivo experiment, HCC model mice were treated with (1) pseudo iRFA + phosphate-buffered saline (PBS); (2) iRFA + PBS; (3) iRFA + OK-432; (4) iRFA + αPD-1; or (5) iRFA + OK-432 + αPD-1. We found that the combined therapy of OK-432 with αPD-1 antibody significantly increased the infiltration and function of CD8 + T cells and significantly decreased the number of FoxP3 + regulatory T cells in residual tumors after iRFA of HCC. Moreover, the smallest tumor volumes and the longest survival were observed in the triple combination treatment (iRFA+OK-432 +αPD-1 antibody) group compared with the other four groups., Conclusions: The combined therapy of OK-432 with αPD-1 antibody induced a strong antitumor immune response, which significantly inhibited the residual tumors after iRFA of HCC. This concept may provide a new treatment strategy to increase the curative efficacy of RFA for medium-to-large and irregular HCCs., Availability of Data and Material: The data of this study are available from the corresponding author on reasonable request., Competing Interests: Declaration of Competing Interest The authors declare no potential conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Non-small-cell lung cancer (NSCLC) and glioblastoma (GB) have poor prognoses. Discovery of new molecular targets is needed to improve therapy. Tax interacting protein 1 (TIP1), which plays a role in cancer progression, is overexpressed and radiation-inducible in NSCLC and GB. We evaluated the effect of an anti-TIP1 antibody alone and in combination with ionizing radiation (XRT) on NSCLC and GB in vitro and in vivo. NSCLC and GB cells were treated with anti-TIP1 antibodies and evaluated for proliferation, colony formation, endocytosis, and cell death. The efficacy of anti-TIP1 antibodies in combination with XRT on tumor growth was measured in mouse models of NSCLC and GB. mRNA sequencing was performed to understand the molecular mechanisms involved in the action of anti-TIP1 antibodies. We found that targeting the functional domain of TIP1 leads to endocytosis of the anti-TIP1 antibody followed by reduced proliferation and increased apoptosis-mediated cell death. Anti-TIP1 antibodies bound specifically (with high affinity) to cancer cells and synergized with XRT to significantly increase cytotoxicity in vitro and reduce tumor growth in mouse models of NSCLC and GB. Importantly, downregulation of cancer survival signaling pathways was found in vitro and in vivo following treatment with anti-TIP1 antibodies. TIP1 is a new therapeutic target for cancer treatment. Antibodies targeting the functional domain of TIP1 exhibited antitumor activity and enhanced the efficacy of radiation both in vitro and in vivo. Anti-TIP1 antibodies interrupt TIP1 function and are effective cancer therapy alone or in combination with XRT in mouse models of human cancer., Competing Interests: Declaration of Competing Interest Dr. Hallahan is a founder and shareholder of Medical Guidance Systems LLC, which is developing human anti-TIP1 antibodies. Vaishali Kapoor, and Abhay Kumar Singh are inventors on pending patents. Other authors state that there are no conflicts of interest. All authors agree to send an individual conflict of interest form if requested by the journal., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Drug‑resistance in hepatitis B virus (HBV), especially due to prolonged treatment with nucleoside analogs, such as lamivudine (LAM), remains a clinical challenge. Alternatively, several plant products and isolated phytochemicals have been used as promising anti‑HBV therapeutics with no sign of resistance. Among all known Rhus species, R. coriaria , R. succedanea and R. tripartite have been widely studied for their anti‑HBV efficacy, however, the effects of R. retinorrhoea have not been previously investigated. The current study reported the isolation of two flavonoids, namely sakuranetin (SEK) and velutin (VEL), from the dichloromethane fraction of R. retinorrhoea aerial parts using chromatography and spectral analyses. The two flavonoids (6.25‑50 µg/ml) were pre‑tested for non‑hepatocytotoxicity using an MTT assay and their dose‑ and time‑dependent inhibitory activities against HBV [hepatitis B surface antigen (HBsAg) and hepatitis B 'e' antigen (HBeAg)] in cultured HepG2.2.15 cells were assessed by ELISA. SEK and VEL at the selected doses (12.5 µg/ml) significantly inhibited HBsAg by ~58.8 and ~56.4%, respectively, and HBeAg by ~55.5 and ~52.4%, respectively, on day 5. The reference drugs LAM and quercetin (anti‑HBV flavonoids), suppressed the production of HBsAg/HBeAg by ~86.4/~64 and ~84.5/~62%, respectively. Furthermore, molecular docking of the flavonoids with HBV polymerase and capsid proteins revealed the formation of stable complexes with good docking energies, thus supporting their structure‑based antiviral mechanism. In conclusion, the present study was the first to demonstrate the anti‑HBV therapeutic activities of SEK and VEL isolated from R. retinorrhoea .

The use of treatments, such as programmed death protein 1 (PD1) or cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibodies, that loosen the natural checks upon immune cell activity to enhance cancer killing have shifted clinical practice and outcomes for the better. Accordingly, the number of antibodies and engineered proteins that interact with the ligand-receptor components of immune checkpoints continue to increase along with their use. It is tempting to view these molecular pathways simply from an immune inhibitory perspective. But this should be resisted. Checkpoint molecules can have other cardinal functions relevant to the development and use of blocking moieties. Cell receptor CD47 is an example of this. CD47 is found on the surface of all human cells. Within the checkpoint paradigm, non-immune cell CD47 signals through immune cell surface signal regulatory protein alpha (SIRPα) to limit the activity of the latter, the so-called trans signal. Even so, CD47 interacts with other cell surface and soluble molecules to regulate biogas and redox signaling, mitochondria and metabolism, self-renewal factors and multipotency, and blood flow. Further, the pedigree of checkpoint CD47 is more intricate than supposed. High-affinity interaction with soluble thrombospondin-1 (TSP1) and low-affinity interaction with same-cell SIRPα, the so-called cis signal, and non-SIRPα ectodomains on the cell membrane suggests that multiple immune checkpoints converge at and through CD47. Appreciation of this may provide latitude for pathway-specific targeting and intelligent therapeutic effect., (© 2023. The Author(s).)

For antibody-based drugs, it is important and relevant to study their toxic effects, which can often become limiting when prescribing this type of therapy. General toxicity of antiviral drug Ergoferon based on technologically processed antibodies was studied on sexually mature animals. Analysis of acute toxicity showed the absence of lethal outcomes when the drug was administered to adult rats at the maximum tolerated doses. In a study of repeated dose toxicity, no adverse effects of the drug were detected., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Heparin-induced thrombocytopenia (HIT) is a rare, iatrogenic condition, characterized by its potential severity and diagnostic difficulties. The diagnosis is based on a set of arguments allowing the calculation of a pre-test score pointing to HIT. There are rapid diagnostic tests for suspected HIT. Among these, the STic Expert® HIT has a good sensitivity to detect HIT. However, it must be performed within 2 hours after sampling. The aim of this study was to evaluate a delayed STic Expert® HIT test at 8 hours and in frozen plasma. Thirty-six patients were prospectively included for HIT testing between April 01, 2018, and July 1, 2022, at the University Rouen Hospital. For any request for HIT testing, an analysis by STic Expert® HIT was performed within 2 hours and 8 hours post-sampling. Any positive result was confirmed by a functional test, platelet aggregation with heparin, release of 14C-serotonin assay (SRA), and immunological assay by a research for anti-platelet factor 4 IgG antibodies. Twenty-three patients had a STic Expert® HIT. Sixteen presented platelet aggregations in the presence of heparin and had a positive anti-PF4 test, 17 had a positive SRA. Six patients had no HIT. For the test performed within 2 hours of collection, the Se = 100%, Sp = 68.42%, PPV = 73.91%, and NPV = 100%. The X2 = 18.21 with p < 0.001. For the test performed at 8 hours post sampling, the Se = 100%, Sp = 68.42%, PPV = 73.91% and NPV = 100%. The X2 = 18.21 with p < 0.001. In conclusion, we have demonstrated that the STic Expert® can be used to perform an HIT diagnostic test 8 hours after sampling and on thawed plasma. However, this study needs to be confirmed on a larger number of samples.

Background: Transthyretin amyloid (ATTR) cardiomyopathy is a progressive and fatal disease caused by misfolded transthyretin. Despite advances in slowing disease progression, there is no available treatment that depletes ATTR from the heart for the amelioration of cardiac dysfunction. NI006 is a recombinant human anti-ATTR antibody that was developed for the removal of ATTR by phagocytic immune cells., Methods: In this phase 1, double-blind trial, we randomly assigned (in a 2:1 ratio) 40 patients with wild-type or variant ATTR cardiomyopathy and chronic heart failure to receive intravenous infusions of either NI006 or placebo every 4 weeks for 4 months. Patients were sequentially enrolled in six cohorts that received ascending doses (ranging from 0.3 to 60 mg per kilogram of body weight). After four infusions, patients were enrolled in an open-label extension phase in which they received eight infusions of NI006 with stepwise increases in the dose. The safety and pharmacokinetic profiles of NI006 were assessed, and cardiac imaging studies were performed., Results: The use of NI006 was associated with no apparent drug-related serious adverse events. The pharmacokinetic profile of NI006 was consistent with that of an IgG antibody, and no antidrug antibodies were detected. At doses of at least 10 mg per kilogram, cardiac tracer uptake on scintigraphy and extracellular volume on cardiac magnetic resonance imaging, both of which are imaging-based surrogate markers of cardiac amyloid load, appeared to be reduced over a period of 12 months. The median N-terminal pro-B-type natriuretic peptide and troponin T levels also seemed to be reduced., Conclusions: In this phase 1 trial of the recombinant human antibody NI006 for the treatment of patients with ATTR cardiomyopathy and heart failure, the use of NI006 was associated with no apparent drug-related serious adverse events. (Funded by Neurimmune; NI006-101 ClinicalTrials.gov number, NCT04360434.)., (Copyright © 2023 Massachusetts Medical Society.)

Activated protein C (APC) is a pleiotropic coagulation protease with anticoagulant, anti-inflammatory, and cytoprotective activities. Selective modulation of these APC activities contributes to our understanding of the regulation of these physiological mechanisms and permits the development of therapeutics for the pathologies associated with these pathways. An antibody library targeting the nonactive site of APC was generated using llama antibodies (nanobodies). Twenty-one nanobodies were identified that selectively recognize APC compared with the protein C zymogen. Overall, 3 clusters of nanobodies were identified based on the competition for APC in biolayer interferometry studies. APC functional assays for anticoagulant activity, histone H3 cleavage, and protease-activated receptor 1 (PAR1) cleavage were used to understand their diversity. These functional assays revealed 13 novel nanobody-induced APC activity profiles via the selective modulation of APC pleiotropic activities, with the potential to regulate specific mechanisms for therapeutic purposes. Within these, 3 nanobodies (LP2, LP8, and LP17) inhibited all 3 APC functions. Four nanobodies (LP1, LP5, LP16, and LP20) inhibited only 2 of the 3 functions. Monofunction inhibition specific to APC anticoagulation activity was observed only by 2 nanobodies (LP9 and LP11). LP11 was also found to shift the ratio of APC cleavage of PAR1 at R46 relative to R41, which results in APC-mediated biased PAR1 signaling and APC cytoprotective effects. Thus, LP11 has an activity profile that could potentially promote hemostasis and cytoprotection in bleedings associated with hemophilia or coagulopathy by selectively modulating APC anticoagulation and PAR1 cleavage profile., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Significance Statement: Kidney-derived thrombopoietin (TPO) increases myeloid cell and platelet production during antibody-mediated chronic kidney disease (AMCKD) in a mouse model, exacerbating chronic thromobinflammation in microvessels. The effect is mirrored in patients with extracapillary glomerulonephritis associated with thromboinflammation, TGF β -dependent glomerulosclerosis, and increased bioavailability of TPO. Neutralization of TPO in mice normalized hematopoiesis, reduced chronic thromboinflammation, and ameliorated renal disease. The findings suggest that TPO is a relevant biomarker and a promising therapeutic target for patients with CKD and other chronic thromboinflammatory diseases.Neutralization of TPO in mice normalized hematopoiesis, reduced chronic thromboinflammation, and ameliorated renal disease. The findings suggest that TPO is a relevant biomarker and a promising therapeutic target for patients with CKD and other chronic thromboinflammatory diseases., Background: Chronic thromboinflammation provokes microvascular alterations and rarefaction, promoting organ dysfunction in individuals with various life-threatening diseases. Hematopoietic growth factors (HGFs) released by the affected organ may sustain emergency hematopoiesis and fuel the thromboinflammatory process., Methods: Using a murine model of antibody-mediated chronic kidney disease (AMCKD) and pharmacological interventions, we comprehensively monitored the response to injury in the circulating blood, urine, bone marrow, and kidney., Results: Experimental AMCKD was associated with chronic thromboinflammation and the production of HGFs, especially thrombopoietin (TPO), by the injured kidney, which stimulated and skewed hematopoiesis toward myelo-megakaryopoiesis. AMCKD was characterized by vascular and kidney dysfunction, TGF β -dependent glomerulosclerosis, and microvascular rarefaction. In humans, extracapillary glomerulonephritis is associated with thromboinflammation, TGF β -dependent glomerulosclerosis, and increased bioavailability of TPO. Analysis of albumin, HGF, and inflammatory cytokine levels in sera from patients with extracapillary glomerulonephritis allowed us to identify treatment responders. Strikingly, TPO neutralization in the experimental AMCKD model normalized hematopoiesis, reduced chronic thromboinflammation, and ameliorated renal disease., Conclusion: TPO-skewed hematopoiesis exacerbates chronic thromboinflammation in microvessels and worsens AMCKD. TPO is both a relevant biomarker and a promising therapeutic target in humans with CKD and other chronic thromboinflammatory diseases., (Copyright © 2023 by the American Society of Nephrology.)

Despite the benefit with cancer immunotherapies in clinical implication, immunotherapeutic resistance occurred in many patients and the mechanism remains unknown. Increasing evidence has revealed that cell-intrinsic programmed cell death ligand 1 (PD-L1) may play a non-negotiable part in immunotherapeutic resistance. Our present study aimed to elucidate the immune-independent acquired resistance mechanism to PD-L1 antibody. We found elevated PD-L1 expression induced by PD-L1 antibodies in cancer cell and vascular endothelial cells (VECs) with substantially acquired resistance to PD-L1 antibodies. Moreover, proliferation of resistant cells was accelerated and the apoptosis was reduced in the absence of immune compared with parental cells. Subsequently, we confirmed that the activation of the PI3K/AKT pathway is involved in the upregulation of PD-L1 expression. Finally, we found that low dose of anlotinib downregulated PD-L1 expression only in VECs via inhibiting the PI3K/AKT pathway; however, the same effect was not observed in cancer cells. To sum up, our findings revealed that upregulation of PD-L1 via activation of the PI3K/AKT signal pathway may promote acquired resistance to PD-L1 antibodies in an immune-independent manner. SIGNIFICANCE: These findings provide new mechanisms of immunotherapeutic resistance and effective evidence of anlotinib combined with immunotherapy., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Lassa virus (LASV) can cause severe acute systemic infection in humans. No approved antiviral drugs or vaccines are currently available. Antibody-based therapeutics are considered a promising treatment strategy in the management of LASV disease., Methods: We used chimeric Ifnar -/- C57BL/6 (Ifnar -/- Bl6 ) mice, a lethal LASV mouse model, to evaluate the protective efficacy of polyclonal antibodies purified from sera of rabbits hyperimmunized with virus-like particles displaying native-like LASV glycoprotein GP spikes., Results: Polyclonal anti-LASV GP antibodies provided 100% protection against lethal LASV infection in a pre- and post-exposure treatment setting and prevented LASV disease. Treatment also significantly lowered viremia level and virus load in organs. When treatment was initiated at the onset of symptoms, the hyperimmune antibodies provided partial protection and increased the survival rate by 80%., Conclusions: Our findings support the consideration of animal-derived hyperimmune antibodies targeting GP as an effective treatment option for highly pathogenic LASV.

Cytotoxic T lymphocytes (CTLs) are central effector cells in antitumor immunotherapy. However, the complexity of immunosuppressive factors in the immune system contributes to the low response rates of current CTL-based immunotherapies. Here, we propose a novel holistic strategy including a priming response, promoting activity, and relieving suppression of CTLs, aiming to strengthen the effect of personalized postoperative autologous nanovaccines. The nanovaccine (C/G-HL-Man) fused the autologous tumor cell membrane with dual adjuvants (CpG and cGAMP), and could effectively accumulate in lymph nodes and promote antigen cross presentation by dendritic cells to prime a sufficient specific-CTL response. The PPAR-α agonist fenofibrate was used to regulate T-cell metabolic reprogramming to promote antigen-specific CTLs activity in the harsh metabolic tumor microenvironment. Finally, the PD-1 antibody was used to relieve the suppression of specific-CTLs in the immunosuppressive tumor microenvironment. In vivo, the C/G-HL-Man exhibited strong antitumor effect in the B16F10 murine tumor prevention model and the B16F10 postoperative recurrence model. In particular, combination therapy with nanovaccines, fenofibrate, and PD-1 antibody effectively inhibited the progression of recurrent melanoma and prolonged the survival time. Our work highlights the critical role of the T-cell metabolic reprogramming and PD-1 blocking in autologous nanovaccines, offering a novel strategy for strengthening the function of CTLs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Inflammation plays an important role in the pathogenesis of diabetic retinopathy (DR). To precisely define the inflammatory mediators, we examined the transcriptomic profile of human retinal endothelial cells exposed to advanced glycation end products, which revealed the neutrophil chemoattractant chemokine CXCL1 as one of the top genes upregulated. The effect of neutrophils in the alteration of the blood-retinal barrier (BRB) was further assessed in wild-type C57BL/6J mice intravitreally injected with recombinant CXCL1 as well as in streptozotocin-induced diabetic mice. Both intravitreally CXCL1-injected and diabetic animals showed significantly increased retinal vascular permeability, with significant increase in infiltration of neutrophils and monocytes in retinas and increased expression of chemokines and their receptors, proteases, and adhesion molecules. Treatment with Ly6G antibody for neutrophil depletion in both diabetic mice as well as CXCL1-injected animals showed significantly decreased retinal vascular permeability accompanied by decreased infiltration of neutrophils and monocytes and decreased expression of cytokines and proteases. CXCL1 level was significantly increased in the serum samples of patients with DR compared with samples of those without diabetes. These data reveal a novel mechanism by which the chemokine CXCL1, through neutrophil recruitment, alters the BRB in DR and, thus, serves as a potential novel therapeutic target., Article Highlights: Intravitreal CXCL1 injection and diabetes result in increased retinal vascular permeability with neutrophil and monocyte recruitment. Ly6G antibody treatment for neutrophil depletion in both animal models showed decreased retinal permeability and decreased cytokine expression. CXCL1 is produced by retinal endothelial cells, pericytes, and astrocytes. CXCL1 level is significantly increased in serum samples of patients with diabetic retinopathy. CXCL1, through neutrophil recruitment, alters the blood-retinal barrier in diabetic retinopathy and, thus, may be used as a therapeutic target., (© 2023 by the American Diabetes Association.)

Background: Brain-directed immunotherapy is a promising strategy to target amyloid-β (Aβ) deposits in Alzheimer's disease (AD). In the present study, we compared the therapeutic efficacy of the Aβ protofibril targeting antibody RmAb158 with its bispecific variant RmAb158-scFv8D3, which enters the brain by transferrin receptor-mediated transcytosis., Methods: App NL-G-F knock-in mice received RmAb158, RmAb158-scFv8D3, or PBS in three treatment regimens. First, to assess the acute therapeutic effect, a single antibody dose was given to 5 months old App NL-G-F mice, with evaluation after 3 days. Second, to assess the antibodies' ability to halt the progression of Aβ pathology, 3 months old App NL-G-F mice received three doses during a week, with evaluation after 2 months. Reduction of RmAb158-scFv8D3 immunogenicity was explored by introducing mutations in the antibody or by depletion of CD4 + T cells. Third, to study the effects of chronic treatment, 7-month-old App NL-G-F mice were CD4 + T cell depleted and treated with weekly antibody injections for 8 weeks, including a final diagnostic dose of [ 125 I]RmAb158-scFv8D3, to determine its brain uptake ex vivo. Soluble Aβ aggregates and total Aβ42 were quantified with ELISA and immunostaining., Results: Neither RmAb158-scFv8D3 nor RmAb158 reduced soluble Aβ protofibrils or insoluble Aβ1-42 after a single injection treatment. After three successive injections, Aβ1-42 was reduced in mice treated with RmAb158, with a similar trend in RmAb158-scFv8D3-treated mice. Bispecific antibody immunogenicity was somewhat reduced by directed mutations, but CD4 + T cell depletion was used for long-term therapy. CD4 + T cell-depleted mice, chronically treated with RmAb158-scFv8D3, showed a dose-dependent increase in blood concentration of the diagnostic [ 125 I]RmAb158-scFv8D3, while concentration was low in plasma and brain. Chronic treatment did not affect soluble Aβ aggregates, but a reduction in total Aβ42 was seen in the cortex of mice treated with both antibodies., Conclusions: Both RmAb158 and its bispecific variant RmAb158-scFv8D3 achieved positive effects of long-term treatment. Despite its ability to efficiently enter the brain, the benefit of using the bispecific antibody in chronic treatment was limited by its reduced plasma exposure, which may be a result of interactions with TfR or the immune system. Future research will focus in new antibody formats to further improve Aβ immunotherapy., (© 2023. The Author(s).)

Non-small-cell lung cancer (NSCLC) is one of the deadliest cancers worldwide, and metastasis is considered one of the leading causes of treatment failure in NSCLC. Wnt/β-catenin signaling is crucially involved in epithelial-mesenchymal transition (EMT), a crucial factor in promoting metastasis, and also contributes to resistance developed by NSCLC to targeted agents. Frizzled-7 (Fzd7), a critical receptor of Wnt/β-catenin signaling, is aberrantly expressed in NSCLC and has been confirmed to be positively correlated with poor clinical outcomes. SHH002-hu1, a humanized antibody targeting Fzd7, was previously successfully generated by our group. Here, we studied the anti-tumor effects of SHH002-hu1 against NSCLC and revealed the underlying mechanism. First, immunofluorescence (IF) and near-infrared (NIR) imaging assays showed that SHH002-hu1 specifically binds Fzd7 + NSCLC cells and targets NSCLC tissues. Wound healing and transwell invasion assays indicated that SHH002-hu1 significantly inhibits the migration and invasion of NSCLC cells. Subsequently, TOP-FLASH/FOP-FLASH luciferase reporter, IF, and western blot assays validated that SHH002-hu1 effectively suppresses the activation of Wnt/β-catenin signaling, and further attenuates the EMT of NSCLC cells. Finally, the subcutaneous xenotransplanted tumor model of A549/H1975, as well as the popliteal lymph node (LN) metastasis model, was established, and SHH002-hu1 was demonstrated to inhibit the growth of NSCLC xenografts and suppress LN metastasis of NSCLC. Above all, SHH002-hu1 with selectivity toward Fzd7 + NSCLC and the potential of inhibiting invasion and metastasis of NSCLC via disrupting Wnt/β-catenin signaling, is indicated as a good candidate for the targeted therapy of NSCLC., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

G protein-coupled receptors (GPCRs) are important signal transducers that are phosphorylated upon activation at intracellular serine and threonine residues. Although antibodies that specifically recognize the phosphorylation state of GPCRs have been available for many years, efficient immunolocalization of phosphorylated receptors in their tissues of origin has not been possible. Here, we show that phosphorylation of receptors is highly unstable during routine immunohistochemical procedures, requiring the use of appropriate phosphatase inhibitors particular during tissue perfusion, post-fixation, and cryoprotection but not during immunostaining of tissue sections. We provide proof of concept using phosphorylation state-specific μ-opioid receptor (MOP) and cannabinoid receptor 1 (CB1) antibodies. Indeed, three of four well-characterized phosphosite-specific MOP antibodies, including pS375-MOP, pT376-MOP, and pT379-MOP, showed robust neuronal immunostaining in brain and spinal cord sections of opioid-treated mice only after inclusion of phosphatase inhibitors. We then extended this approach to the CB1 receptor and demonstrated that one of three newly-generated phosphosite-specific CB1 antibodies, namely pS425-CB1, showed striking staining of fibers and varicosities in brain slices from cannabinoid-treated mice. Although subsequent experiments showed that phospho-CB1 immunostaining was less sensitive to phosphatases, we conclude that the use of phosphatase inhibitors should always be considered in the development of immunohistochemical procedures for new phosphosite-specific GPCR antibodies. In summary, we anticipate that this improved protocol will facilitate the widespread use of phosphorylation state-specific antibodies to monitor the activation of endogenous GPCRs under physiological and pharmacological conditions. Our approach may also prove useful to confirm target engagement of GPCR drug candidates in native tissues., (© 2023. The Author(s).)