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Agents that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represent promising approaches for HIV cure. Here, we develop and evaluate a trispecific antibody (Ab), N6/αCD3-αCD28, that targets three independent proteins: (1) the HIV envelope via the broadly reactive CD4-binding site Ab, N6; (2) the T cell antigen CD3; and (3) the co-stimulatory molecule CD28. We find that the trispecific significantly increases antigen-specific T-cell activation and cytokine release in both CD4 + and CD8 + T cells. Co-culturing CD4 + with autologous CD8 + T cells from ART-suppressed HIV + donors with N6/αCD3-αCD28, results in activation of latently-infected cells and their elimination by activated CD8 + T cells. This trispecific antibody mediates CD4 + and CD8 + T-cell activation in non-human primates and is well tolerated in vivo. This HIV-directed antibody therefore merits further development as a potential intervention for the eradication of latent HIV infection. One of the main hurdles to curing HIV infection are viral reservoirs. Here, the authors develop a trispecific antibody and demonstrate its ability to simultaneously activate and target latently HIV−1 infected cells for elimination by T cells as an alternative strategy for HIV cure.

BackgroundWe previously developed a saline-formulated mixture of 4 synthetic messenger ribonucleic acids (mRNAs) encoding the cytokines IL-12, IFN-α, GM-CSF, and IL-15 sushi (IL-15-IL-15Rα receptor fusion) that promoted systemic immunity and tumor eradication upon intratumoral administration in multiple mouse tumor models.Here, we explored whether stabilization of the mRNAs through a lipoplex (LPX) formulation could confer increased expression of the encoded cytokines to result in improved antitumor efficacy. To this end, we developed a pH-responsive amphoteric LPX by mixing preformed liposomes with mRNA in aqueous/aqueous solutions. The resulting complex was designed to destabilize under neutral pH in circulation, leading to a limited exposure in non-target organs following intratumoral administration.MethodsBiological activity of LPX-formulated mRNA-encoded cytokines or reporter genes were compared to that of naked mRNA in vivo using C57BL/6 and BALB/c mice harboring TC-1, B16F10 and CT26 tumors, respectively. Following intratumoral administration, cytokines were analyzed in serum and organs by ELISA and luciferase expression was followed by bioluminescent imaging. Anti-tumor efficacy was assessed by tumor growth inhibition and survival of CT26-tumor bearing mice and expansion of tumor-reactive T cells were analyzed by flow cytometry. Abscopal effects were assessed in C57BL/6 mice bearing B16F10 subcutaneous tumors on both flanks.ResultsIn vivo studies demonstrated a favorable tumor-to-liver translation ratio of luciferase reporter mRNA formulated with the LPX, supporting the hypothesis that the LPX would destabilize in circulation. The LPX formulation further increased target expression of each of the four encoded cytokines in tumors ranging from 7 to 47-fold compared to saline solution in B16F10-tumor bearing animals. Intratumoral administration of LPX-mRNA to mice with subcutaneous CT26 colorectal tumors significantly inhibited tumor growth, with stronger tumor growth inhibition when compared to treatment with saline-formulated mRNA. This increased antitumor activity was correlated with stronger pharmacodynamic responses, including expansion of T cells reactive against the tumor antigen gp70. C57BL/6 mice bearing B16F10 subcutaneous tumors on both flanks received an intratumoral injection of LPX-mRNA on one side. Treatment resulted in significant inhibition of tumor growth and prolonged survival. Antitumor efficacy was further enhanced when combined with anti-PD-1 antibody, resulting in complete tumor regression in a subset of mice, consistent with an abscopal response.ConclusionsTogether, these results demonstrate the potent anticancer activity of a novel LPX-formulated mRNA mixture.Ethics ApprovalAnimal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) and in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

Background One hypothesis of lack of effectiveness in most current clinical trials for the AD (Alzheimer’s disease) is the advanced stages of the disease at the time of pharmacological intervention. To aim a robust effect from disease‐modifying therapies, there is an urgent need for biomarkers that can identify patients with MCI and early stages of AD. In this study, we combined in‐house large‐scale LC‐MS and new emerging highly sensitive NULISA proteomics analysis on a medium sized AD cohort sample, aiming an early‐stage protein biomarker discovery in MCI and AD. Method 59 AD set plasma and 65 AD set CSF were purchased from the vendor. Donor classification strictly followed the disease inclusion/exclusion criteria. One aliquot from each sample were sent to Alamar Bioscience for NULISAseq analysis on ARGOTM system using 120‐plex CNS disease panel. In house LC‐MS was performed on the same set of samples. Result Of 59 AD set plasma samples (19 HC, 22 MCI AND 18 AD), 3 significantly up‐regulated protein in MCI and 16 significantly down‐regulated protein in AD and MCI were identified by NULISAseq 120plex CNS panel. One significantly up‐regulated protein in MCI and 3 significantly down‐regulated proteins in AD were also confirmed by in house LC‐MS. Of 65 AD set CSF samples (19 HC, 22 MCI and 24 AD), 12 significantly up‐regulated proteins and 11 significant down‐regulated protein in AD and MCI were identified by NULISAseq 120plex CNS panel. CSF protein profiling by in house LC‐MS is still pending. 5 (3 from plasma, and 2 from CSF) significantly up‐regulated novel proteins in MCI and CD have been reported to be associated with neuronal connectivity, neuro inflammation or acting as a neuromodulator. Sample correlation in CSF between NULISAseq and Simoa (Single Molecule Array) was also verified by NFL and pTau181. Both proteins displayed excellent correlation (R2 was 0.88 and 0.92). Conclusion 5 novel significantly upregulated protein biomarkers in MCI and AD were identified by combined in house MS and new emerging NULISAseq technologies. The integrated proteomic method could be a powerful tool to identify the molecular signature and pathological pathways associated with AD and MCI.

Effective antitumour immunity depends on the orchestration of potent T cell responses against malignancies 1 . Regression of human cancers has been induced by immune checkpoint inhibitors, T cell engagers or chimeric antigen receptor T cell therapies 2 – 4 . Although CD8 T cells function as key effectors of these responses, the role of CD4 T cells beyond their helper function has not been defined. Here we demonstrate that a trispecific antibody to HER2, CD3 and CD28 stimulates regression of breast cancers in a humanized mouse model through a mechanism involving CD4-dependent inhibition of tumour cell cycle progression. Although CD8 T cells directly mediated tumour lysis in vitro, CD4 T cells exerted antiproliferative effects by blocking cancer cell cycle progression at G1/S. Furthermore, when T cell subsets were adoptively transferred into a humanized breast cancer tumour mouse model, CD4 T cells alone inhibited HER2 + breast cancer growth in vivo. RNA microarray analysis revealed that CD4 T cells markedly decreased tumour cell cycle progression and proliferation, and also increased pro-inflammatory signalling pathways. Collectively, the trispecific antibody to HER2 induced T cell-dependent tumour regression through direct antitumour and indirect pro-inflammatory/immune effects driven by CD4 T cells. A trispecific antibody targeting HER2 and T cells stimulates CD4 T cell-dependent inhibition of of breast cancer cell proliferation in vitro and tumor regression in a humanized mouse model.

The choroid plexus (ChP) plays a role in cerebrospinal fluid (CSF) production, protein transport and clearance, and central nervous system homeostasis. Emerging evidence suggests that ChP dysfunction is implicated in the pathogenesis of neurodegenerative diseases. We aim to investigate ChP involvement in the pathophysiology of Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy bodies (DLB) using CSF proteomics. We included individuals with AD (n = 150), PD (n = 75), DLB (n = 53), and controls (n = 47) from 6 centers of the European Platform for Neurodegenerative Diseases (EPND) project. AD was defined as abnormal CSF Aβ42/40 (Roche NeuroToolKit) without meeting clinical criteria of PD or DLB. Controls had normal cognition and CSF Aβ42/40. Using the Olink Explore 3072 assay, 2902 CSF proteins were quantified. Proteins were classified as highly expressed in the ChP using the Allen Brain Atlas. Pairwise comparisons of protein concentrations between disease and controls were conducted, adjusting for age and sex. Weighted Gene Co-expression Network Analysis (WGCNA) was performed on ChP-expressed proteins to identify co-expression modules, followed by Wilcoxon tests to compare module expression profiles across groups. Pathway enrichment analysis was conducted using Gene Ontology. Of the 2902 proteins quantified, 799 were highly expressed by the ChP. In PD versus controls, 37% of the dysregulated proteins were highly expressed in the ChP, compared to 30% in AD versus controls and 28% in DLB versus controls (Figure 1). The dysregulated ChP-expressed proteins in AD, PD and DLB were predominantly distinct and associated with different underlying pathways (Figure 2). WGCNA identified four co-expression modules related to the proteins with high ChP expression. Module 1, related to innate immunity (complement), was decreased in AD versus all other groups. Module 2, linked to transcriptional and translational processes, was increased in PD versus controls and AD. Module 3, related to the extracellular matrix, was increased in DLB versus AD. No significant differences were observed for module 4 (Figure 3). Our findings highlight the distinct involvement of ChP-expressed proteins in the pathophysiology of AD, PD, and DLB, revealing disease-specific patterns. Further research is essential to elucidate mechanistic involvement of the ChP in disease progression across these disorders.

NM23/NDP kinases play an important role in development and cancer but their biological function is unknown, despite an intriguing collection of biochemical properties including nucleoside-diphosphate kinase (NDP kinase), DNA binding and transcription, a mutator function, and cleavage of unusually structured DNA by means of a covalent enzyme-DNA complex. To assess the role of the nuclease in human NM23-H2, we sought to identify the amino acid responsible for covalent catalysis. By sequencing a DNA-linked peptide and by site-directed mutagenesis, we identified lysine-12, a phylogenetically conserved residue, as the amino acid forming the covalent complex with DNA. In particular, the ε-amino group acts as the critical nucleophile, because substitution with glutamine but not arginine completely abrogated covalent adduct formation and DNA cleavage, whereas the DNA-binding properties remained intact. These findings and chemical modification data suggest that phosphodiester-bond cleavage occurs by a DNA glycosylase/lyase-like mechanism known as the signature of base excision DNA repair nucleases. Involvement of NM23/NDP kinase in a DNA repair pathway would be consistent with its role in normal and tumor cell development. Additionally, lysine-12, which is known in the x-ray crystallographic structure to lie in the catalytic pocket involved in the NDP kinase phosphorylation reaction, was found essential also for the NDP kinase activity of NM23-H2, suggesting that the two catalytic activities of NM23-H2 are fundamentally connected.

Background Aggregation of amyloid beta (Aβ) and tau proteins is a hallmark of Alzheimer's disease (AD) and tauopathies. In AD, extracellular deposition of Aβ peptide precedes tau aggregation and the onset of neuroinflammatory processes. Both neuroinflammation and tau deposition are associated with synaptic loss, neurodegeneration, and cognitive decline. However, the precise sequence of events from amyloid deposition to tau aggregation, neuroinflammation, and neurodegeneration remains unclear. A cross‐sectional analysis of tau and other proteins in the cerebrospinal fluid (CSF) of AD patients can provide insight into the inflammatory and degenerative processes. Tau levels may be used as a proxy for disease progression, and we hypothesize that the expression of neuroinflammatory markers will be modulated in response to increasing tau aggregation. Method To investigate this, we collaborated with the EPND‐biomarker consortium to analyze over 150 human CSF samples from AD and control patients. We measured levels of Aβ1‐40, Aβ1‐42, pTau181, and total tau, and conducted proteomic profiling using the O‐link and SomaScan platforms. Next, AD patients were stratified into Aβ+Tau+ and Aβ+Tau‐ groups to identify biomarkers associated with Aβ and/or tau aggregation. Result As expected, inflammation markers were elevated in AD CSF samples compared to controls. Interestingly, we observed distinct correlations between these markers and amyloid versus tau pathology biomarkers. Approximately 10% of the measured CSF proteins (∼250 proteins) showed significant differences (p < 0.05) between AD and control samples. CSF samples with elevated tau levels exhibited altered levels of proteins involved in the complement cascade, cytokine‐cytokine receptor interactions, cell adhesion, and vesicular transport. Conclusion Increased inflammation markers were observed in AD. Proteins linked to innate immune response and synaptic loss were significantly altered in relation to tau aggregation. These findings are crucial for guiding therapeutic strategies for neurodegenerative diseases and assessing their efficacy.

Background The choroid plexus (ChP) plays a role in cerebrospinal fluid (CSF) production, protein transport and clearance, and central nervous system homeostasis. Emerging evidence suggests that ChP dysfunction is implicated in the pathogenesis of neurodegenerative diseases. We aim to investigate ChP involvement in the pathophysiology of Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy bodies (DLB) using CSF proteomics. Method We included individuals with AD (n = 150), PD (n = 75), DLB (n = 53), and controls (n = 47) from 6 centers of the European Platform for Neurodegenerative Diseases (EPND) project. AD was defined as abnormal CSF Aβ42/40 (Roche NeuroToolKit) without meeting clinical criteria of PD or DLB. Controls had normal cognition and CSF Aβ42/40. Using the Olink Explore 3072 assay, 2902 CSF proteins were quantified. Proteins were classified as highly expressed in the ChP using the Allen Brain Atlas. Pairwise comparisons of protein concentrations between disease and controls were conducted, adjusting for age and sex. Weighted Gene Co‐expression Network Analysis (WGCNA) was performed on ChP‐expressed proteins to identify co‐expression modules, followed by Wilcoxon tests to compare module expression profiles across groups. Pathway enrichment analysis was conducted using Gene Ontology. Result Of the 2902 proteins quantified, 799 were highly expressed by the ChP. In PD versus controls, 37% of the dysregulated proteins were highly expressed in the ChP, compared to 30% in AD versus controls and 28% in DLB versus controls (Figure 1). The dysregulated ChP‐expressed proteins in AD, PD and DLB were predominantly distinct and associated with different underlying pathways (Figure 2). WGCNA identified four co‐expression modules related to the proteins with high ChP expression. Module 1, related to innate immunity (complement), was decreased in AD versus all other groups. Module 2, linked to transcriptional and translational processes, was increased in PD versus controls and AD. Module 3, related to the extracellular matrix, was increased in DLB versus AD. No significant differences were observed for module 4 (Figure 3). Conclusion Our findings highlight the distinct involvement of ChP‐expressed proteins in the pathophysiology of AD, PD, and DLB, revealing disease‐specific patterns. Further research is essential to elucidate mechanistic involvement of the ChP in disease progression across these disorders.

Technical lignins are by-products formed in the amount of about 70 million tons annually during the chemical and biochemical processing of plant biomass in the world. Most technical lignins are burned for energy. No more than 2% of technical lignins are supplied to the market mainly in the form of lignosulfonates due to their good solubility and surface activity. Modification of lignosulfonates can improve their properties and expand the field of practical use. In this study, we carried out a modification of sodium lignosulfonates by nitrosation under the conditions of electrochemical treatment in the presence of sodium nitrite. Chemical nitrosation with a mixture of sodium nitrite and hydrochloric or acetic acid was also performed for comparison. Electrochemical processing was performed in a diaphragmless cell using stainless steel electrodes. The process was controlled by spectrophotometry of the resulting solutions with a change in the voltage (3-20 V) and sodium nitrite consumption (0-30%) for 15-40 min. The final solutions of nitrosated lignosulfonates were subjected to ultrafiltration. The molecular weights of the products in concentrates were determined using high performance liquid chromatography. The elemental composition of nitrosated lignosulfonates in the concentrate was determined after drying. The nitrogen content in the product reaches 1.8%. The number-average molecular weight changed from 6.3 kDa (initial lignosulfonates) to 7.3-20.7 kDa depending on the processing conditions. Electrochemical nitrosation eliminates the use of acid and achieves a similar degree of nitrosation in less time.