INHBA promotes tumor growth and induces resistance to PD-L1 blockade by suppressing IFN-γ signaling.

Inhibin beta A (INHBA) and its homodimer activin A have pleiotropic effects on modulation of immune responses and tumor progression, but it remains uncertain whether tumors may release activin A to regulate anti-tumor immunity. In this study we investigated the effects and mechanisms of tumor intrinsic INHBA on carcinogenesis, tumor immunity and PD-L1 blockade. Bioinformatic analysis on the TCGA database revealed that INHBA expression levels were elevated in 33 cancer types, including breast cancer (BRCA) and colon adenocarcinoma (COAD). In addition, survival analysis also corroborated that INHBA expression was negatively correlated with the prognosis of many types of cancer patients. We demonstrated that gain or loss function of Inhba did not alter in vitro growth of colorectal cancer CT26 cells, but had striking impact on mouse tumor models including CT26, MC38, B16 and 4T1 models. By using the TIMER 2.0 tool, we figured out that in most cancer types, Inhba expression in tumors was inversely associated with the infiltration of CD4+ T and CD8+ T cells. In CT26 tumor-bearing mice, overexpression of tumor INHBA eliminated the anti-tumor effect of the PD-L1 antibody atezolizumab, whereas INHBA deficiency enhanced the efficacy of atezolizumab. We revealed that tumor INHBA significantly downregulated the interferon-γ (IFN-γ) signaling pathway. Tumor INHBA overexpression led to lower expression of PD-L1 induced by IFN-γ, resulting in poor responsiveness to anti-PD-L1 treatment. On the other hand, decreased secretion of IFN-γ-stimulated chemokines, including C-X-C motif chemokine 9 (CXCL9) and 10 (CXCL10), impaired the infiltration of effector T cells into the tumor microenvironment (TME). Furthermore, the activin A-specific antibody garetosmab improved anti-tumor immunity and its combination with the anti-PD-L1 antibody atezolizumab showed a superior therapeutic effect to monotherapy with garetosmab or atezolizumab. We demonstrate that INHBA and activin A are involved in anti-tumor immunity by inhibiting the IFN-γ signaling pathway, which can be considered as potential targets to improve the responsive rate of PD-1/PD-L1 blockade.

Identification and characterization of an α-1,3 mannosidase from Elizabethkingia meningoseptica and its potential attenuation impact on allergy associated with cross-reactive carbohydrate determinant.

Core α-1,3 mannose is structurally near the core xylose and core fucose on core pentasaccharide from plant and insect glycoproteins. Mannosidase is a useful tool for characterization the role of core α-1,3 mannose in the composition of glycan related epitope, especially for those epitopes in which core xylose and core fucose are involved. Through functional genomic analysis, we identified a glycoprotein α-1,3 mannosidase and named it MA3. We used MA3 to treat allergen horseradish peroxidase (HRP) and phospholipase A2 (PLA2) separately. The results showed that after MA3 removed α-1,3 mannose on HRP, the reactivity of HRP with anti-core xylose polyclonal antibody almost disappeared. And the reactivity of MA3-treated PLA2 with anti-core fucose polyclonal antibody decreased partially. In addition, when PLA2 was conducted enzyme digestion by MA3, the reactivity between PLA2 and allergic patients' sera diminished. These results demonstrated that α-1,3 mannose was an critical component of glycan related epitope.

A Natural Compound Containing a Disaccharide Structure of Glucose and Rhamnose Identified as Potential N-Glycanase 1 (NGLY1) Inhibitors.

N-glycanase 1 (NGLY1) is an essential enzyme involved in the deglycosylation of misfolded glycoproteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway, which could hydrolyze N-glycan from N-glycoprotein or N-glycopeptide in the cytosol. Recent studies indicated that NGLY1 inhibition is a potential novel drug target for antiviral therapy. In this study, structure-based virtual analysis was applied to screen candidate NGLY1 inhibitors from 2960 natural compounds. Three natural compounds, Poliumoside, Soyasaponin Bb, and Saikosaponin B2 showed significantly inhibitory activity of NGLY1, isolated from traditional heat-clearing and detoxifying Chinese herbs. Furthermore, the core structural motif of the three NGLY1 inhibitors was a disaccharide structure with glucose and rhamnose, which might exert its action by binding to important active sites of NGLY1, such as Lys238 and Trp244. In traditional Chinese medicine, many compounds containing this disaccharide structure probably targeted NGLY1. This study unveiled the leading compound of NGLY1 inhibitors with its core structure, which could guide future drug development.

New laboratory evidence for the association between endothelial dysfunction and COVID-19 disease progression.

There is growing evidence that angiotensin-converting enzyme 2 is highly expressed on endothelial cells, endothelial dysfunction plays a critical role in coronavirus disease 2019 (COVID-19) progression, but laboratory evidence is still lacking. This study established a multicenter retrospective cohort of 966 COVID-19 patients from three hospitals in Wuhan, China. We found that male (62.8% vs. 46.5%), old age [72 (17) vs. 60.5 (21)], and coexisting chronic diseases (88.5% vs. 60.0%) were associated with poor clinical prognosis in COVID-19. Furthermore, the deteriorated patients exhibited more severe multiorgan damage, coagulation dysfunction, and extensive inflammation. Additionally, a cross-sectional study including 41 non-COVID-19 controls and 39 COVID-19 patients assayed endothelial function parameters in plasma and showed that COVID-19 patients exhibited elevated vascular cell adhesion molecule-1 (VCAM-1) (median [IQR]: 0.32 [0.27] vs. 0.17 [0.11] µg/ml, p < 0.001), E-selectin (21.06 [12.60] vs. 11.01 [4.63] ng/ml, p < 0.001), tissue-type plasminogen activator (tPA) (0.22 [0.12] vs. 0.09 [0.04] ng/ml, p < 0.001), and decreased plasminogen activator inhibitor-1 (0.75 [1.31] vs 6.20 [5.34] ng/ml, p < 0.001), as compared to normal controls. Moreover, VCAM-1 was positively correlated with d-dimer (R = 0.544, p < 0.001); tPA was positively correlated with d-dimer (R = 0.800, p < 0.001) and blood urea nitrogen (R = 0.638, p < 0.001). Our findings further confirm the strong association between endothelial dysfunction and poor prognosis of COVID-19, which offers a rationale for targeting endothelial dysfunction as a therapeutic strategy for COVID-19.

In vivo-induced argininosuccinate lyase plays a role in the replication of Brucella abortus in RAW264.7 cells.

Brucellosis caused by Brucella species is a zoonotic disease with a serious impact on public health and the livestock industry. To better understand the pathogenesis of the disease, in vivo-induced antigen technology (IVIAT) was used to investigate the in vivo-induced antigens of Brucella abortus in this study. A genomic expression library of B. abortus was constructed and screened using pooled bovine B. abortus-positive sera by IVIAT. In total, 33 antigens were identified. Five antigens were further expressed and tested for their seroreactivity against 33 individual bovine B. abortus-positive sera by Western blot analysis. The results showed a highest positive rate of 32/33 for argininosuccinate lyase (ASL), indicating that ASL may be used as a candidate marker for serodiagnosis of brucellosis. Furthermore, an asl gene-deleted mutant strain S2308ΔASL was constructed, and the intracellular survival and replication of the mutant strain in RAW264.7 cells were investigated. Interestingly, the numbers of bacteria recovered from cells infected with mutant strain S2308ΔASL were similar at all time points observed from 0 h to 96 h post-infection, suggesting the asl gene plays an important role in the bacterial replication in RAW264.7 cells. Real-time quantitative PCR (qPCR) analysis showed that the mRNA levels in S2308ΔASL were decreased for BvrR, BvrS and virB5 when compared with those in S2308 (P<0.05). Our results not only expand the knowledge of Brucella intracellular replication but also expand the list of candidates for serodiagnostic markers of brucellosis.

Enzymatic activity analysis and catalytic essential residues identification of Brucella abortus malate dehydrogenase.

Malate dehydrogenase (MDH) plays important metabolic roles in bacteria. In this study, the recombinant MDH protein (His-MDH) of Brucella abortus was purified and its ability to catalyze the conversion of oxaloacetate (OAA) to L-malate (hereon referred to as MDH activity) was analyzed. Michaelis Constant (Km) and Maximum Reaction Velocity (Vmax) of the reaction were determined to be 6.45 × 10(-3) M and 0.87 mM L(-1)min(-1), respectively. In vitro studies showed that His-MDH exhibited maximal MDH activity in pH 6.0 reaction buffer at 40°C. The enzymatic activity was 100%, 60%, and 40% inhibited by Cu(2+), Zn(2+), and Pb(2+), respectively. In addition, six amino acids in the MDH were mutated to investigate their roles in the enzymatic activity. The results showed that the substitutions of amino acids Arg 89, Asp 149, Arg 152, His 176, or Thr 231 almost abolished the activity of His-MDH. The present study will help to understand MDH's roles in B. abortus metabolism.

Characterization of the immunogenicity and pathogenicity of malate dehydrogenase in Brucella abortus.

Brucella abortus is a gram-negative, facultative intracellular pathogen that causes brucellosis, a chronic zoonotic disease resulting in abortion in pregnant cattle and undulant fever in humans. Malate dehydrogenase (MDH), a key enzyme in the tricarboxylic acid cycle, plays important metabolic roles in aerobic energy producing pathways and in malate shuttle. In this study, the MDH-encoding gene for malate dehydrogenase mdh of B. abortus S2308 was cloned, sequenced and expressed. Western blot analysis demonstrated that MDH is an immunogenic membrane-associated protein. In addition, recombinant MDH showed sero-reactivity with 30 individual bovine B. abortus-positive sera by enzyme-linked immunosorbent assay, indicates that MDH may be used as a candidate marker for sero-diagnosis of brucellosis. Furthermore, MDH exhibits fibronectin and plasminogen-binding ability in immunoblotting assay. Inhibition assays on HeLa cells demonstrated that rabbit anti-serum against MDH significantly reduced both bacterial adherence and invasion abilities (p < 0.05), suggesting that MDH play a role in B. abortus colonization. Our results indicated that MDH is not only an immunogenic protein, but is also related to bacterial pathogenesis and may act as a new virulent factor, which will benefit for further understanding the MDH's roles in B. abortus metabolism, pathogenesis and immunity.

Identification and characterization of emGalaseE, a ß-1,4 galactosidase from Elizabethkingia meningoseptica, and its application on living cell surface.

The biological function of terminal galactose on glycoprotein is an open field of research. Although progress had being made on enzymes that can remove the terminal galactose on glycoproteins, there is a lack of report on galactosidases that can work directly on living cells. In this study, a unique beta 1,4 galactosidase was isolated from Elizabethkingia meningoseptica (Em). It exhibited favorable stability at various temperatures (4-37 °C) and pH (5-8) levels and can remove ß-1, 4 linked galactoses directly from glycoproteins. Using Alanine scanning, we found that two acidic residues (Glu-468, and Glu-531) in the predicted active pocket are critical for galactosidase activity. In addition, we also demonstrated that it could cleave galactose residues present on living cell surface. As this enzyme has a potential application for living cell glycan editing, we named it emGalaseE or glycan-editing galactosidase I (csgeGalaseI). In summary, our findings lay the groundwork for further investigation by presenting a simple and effective approach for the removal of galactose moieties from cell surface.

Suppression of Tumor or Host Intrinsic CMTM6 Drives Antitumor Cytotoxicity in a PD-L1-Independent Manner.

CKLF-like MARVEL transmembrane domain-containing protein 6 (CMTM6) is known to be a regulator of membranal programmed death ligand 1 (PD-L1) stability and a factor associated with malignancy progression, but the effects and mechanisms of CMTM6 on tumor growth, as well as its potential as a target for therapy, are still largely unknown. Here, we show that CMTM6 expression increased with tumor progression in both patients and mice. Ablation of CMTM6 significantly reduced human and murine tumor growth in a manner dependent on T-cell immunity. Tumor CMTM6 suppression broke resistance to immune-checkpoint inhibitors and remodeled the tumor immune microenvironment, as specific antitumor cytotoxicity was enhanced and contributed primarily to tumor inhibition. Without the PD-1/PD-L1 axis, CMTM6 suppression still significantly dampened tumor growth dependent on cytotoxic cells. Furthermore, we identified that CMTM6 was widely expressed on immune cells. T-cell CMTM6 levels increased with sustained immune activation and intratumoral immune exhaustion and affected T cell-intrinsic PD-L1 levels. Host CMTM6 knockout significantly restrained tumor growth in a manner dependent on CD8+ T cells and not entirely dependent on PD-L1. Thus, we developed and evaluated the antitumor efficacy of CMTM6-targeting adeno-associated virus (AAV), which effectively mobilized antitumor immunity and could be combined with various antitumor drugs. Our findings reveal that both tumor and host CMTM6 are involved in antitumor immunity with or without the PD-1/PD-L1 axis and that gene therapy targeting CMTM6 is a promising strategy for cancer immunotherapy.

Characterization and Molecular Mechanism of Aminoglycoside-6-Adenyl Transferase Associated with Aminoglycoside Resistance from Elizabethkingia meningoseptica.

Purpose: Elizabethkingia meningoseptica (EM) is a multi-drug-resistant bacterium of global concern for its role in nosocomial infection and is generally resistant to aminoglycoside antibiotics. In the whole genome of an EM strain (FMS-007), an aminoglycoside-6-adenyl transferase gene (ant(6)FMS-007) was predicted. This study aimed to characterize the biochemical function of ANT(6)FMS-007 and analyze the relationship between genotype and phenotype of ant(6) in clinical EM isolates, so as to provide evidence for clinical precision drug use. This study could establish a method for the verification of known or unknown functionally resistant genes. Methods: A total of 42 EM clinical isolates were collected from clinical departments during 2015-2023. The phenotype of aminoglycoside antibiotics was analyzed by broth microdilution (BMD) and Kirby-Bauer (K-B) methods. The whole-length ant(6) from EM clinical isolates was analyzed by polymerase chain reaction (PCR) and sequencing. The biochemical function of predictive ANT(6)FMS-007 from the FMS-007 whole genome was identified by 3D plate experiment and mass spectrometry analysis. Candidate active sites were predicted by multi-species sequence alignment and molecular docking, and other important sites were identified in the comparison of ant(6) genotypes and phenotypes of EM clinical isolates. Drug susceptibility test was used to verify the function of these sites. Results: The predictive ANT(6)FMS-007 protein could inactivate STR by modifying STR with ATP to form STR-AMP. Four active sites (Asp-38, Asp-42, Lys-95, and Lys-213) of ANT(6)FMS-007 were identified. Thirty-one EM clinical isolates (74%) carried the ant(6) gene. Eight EM clinical isolates containing the ant(6) gene had MIC values (<=32µg/mL) lower by at least 16-fold than FMS-007 (512µg/mL) for STR, and N59H and K204Q were the common mutations in the ant(6) gene. Conclusion: This assay verified the biochemical function of the predictive gene ant(6)FMS-007 and could provide an alternative method to study resistant gene function in multi-drug-resistant bacteria. The inconsistency between genotype and phenotype of resistant genes indicated that the combination of resistance gene detection and functional analysis could better provide precision medicine for clinical use.

Carbohydrate Strengthens the Immunotherapeutic Effect of Small-Molecule PD-L1 Inhibitors.

PD-1/PD-L1 checkpoint blockade has demonstrated great success in cancer immunotherapy. Small-molecule PD-L1 inhibitors also attract significant research interests but remain challenging in the efficacy and safety. Carbohydrate moiety and carbohydrate-binding proteins (lectins) play important roles in immune modulation including antigen recognition and presenting. Herein, we reported a novel strategy to strengthen the immunotherapeutic effect of small-molecule PD-L1 inhibitors by introducing sugar motifs, which may utilize the carbohydrate-mediated immune enhancement for cancer treatment. The data revealed that glycoside compounds containing mannose or N-acetylglucosamine exhibited the best results in IFN-γ secretion. Moreover, compared to the nonglycosylated compounds, glycosides C3 and C15 demonstrated significant lower cytotoxicity and effective in vivo antitumor potency in the CT26 and melanoma B16-F10 tumor models with good tolerance. Notably, tumor-infiltrating lymphocyte (TIL) analysis validated increased CD3+, CD4+, CD8+, and granzyme B+ T cells after glycoside treatments. This work presents a new concept to improve the immunotherapy.

Development of a bridging ELISA for detection of antibodies against ZV0203 in cynomolgus monkey serum.

Immunogenicity has been a major concern in the safety evaluation of therapeutic proteins. The assessment of the unwanted immunogenicity of the therapeutic proteins performed in animals prior to clinical trials has been a regulatory requirement. In preclinical studies of therapeutic proteins, cynomolgus monkeys are usually the most relevant animal species. ZV0203, a recombinant humanized anti-human epidermal growth factor receptor 2 monoclonal antibody covalently bound to a cytotoxic drug (Duo-5), possesses a novel format of antibody drug conjugates. In this study, we reported the development, validation, and application of a bridging enzyme-linked immunosorbent assay for the detection of antibodies against ZV0203 in cynomolgus monkey serum. Drug interference at low positive control (18.0 ng/mL) and high positive control (130 ng/mL) of anti-ZV0203 antibodies was not observed when ZV0203 concentration is below 1.74 µg/mL and 1.49 µg/mL, respectively. In addition, no interference was found from mouse IgG1, but interference was observed with human IgG1. No effect of hemolysis was found on the analysis results of the testing samples present in 100% pooled rabbit serum containing 2% (V/V) erythrocyte hemolysates. Besides, spiked anti-ZV0203 antibody in rabbit serum was stable after 5 freeze/thaw cycles. The results showed that the method is suitable for the detection of anti-ZV0203 antibodies in cynomolgus monkey serum. The assay was also successfully applied in the repeated dose study of ZV0203.

Noncanonical PD-1/PD-L1 Axis in Relation to the Efficacy of Anti-PD Therapy.

With programmed death 1/ligand 1 (PD-1/PD-L1) as the cornerstone, anti-PD antibodies have pioneered revolutionary immunotherapies for malignancies. But most patients struggled to respond to anti-PD owing to primary or acquired resistance or even hyperprogression, pointing to more efforts needed to explore this axis. PD-1 constrains T-cell immunoreactivity via engaging with PD-L1 of tumor/myeloid cells is the canonical PD-1/PD-L1 axis function mode. Studies are increasingly aware of the impact of noncanonical PD-1/PD-L1 expression in various cancers. PD-L1 induced on activated T-cells ligates to PD-1 to mediate self-tolerance or acts on intratumoral myeloid cells and other T-cells, affecting their survival, differentiation and immunophenotyping, leading to tumor immunosuppression. Myeloid PD-1 interferes with their proliferation, differentiation, cytokine secretion and phagocytosis, mediating remarkable pro-tumor effects. Tumor cell intrinsic PD-1 signaling has diverse functions in different tumors, resulting in pro-proliferation or proliferation inhibition. These nonclassical PD-1/PD-L1 functions may be novel anti-PD mechanisms or causes of treatment resistance. This review highlights the nonnegligible role of T-cell-intrinsic PD-L1 and tumor/myeloid PD-1 in the cell interplay network and the complex impact on the efficacy of anti-PD antibodies. Reconsidering and rational utilization of the comprehensive PD-1/PD-L1 axis could cumulate breakthroughs in precision treatment and combination for anti-PD therapies.

Different Immunoregulation Roles of Activin A Compared With TGF-ß.

Activin A, a critical member of the transforming growth factor-ß (TGF-ß) superfamily, is a pluripotent factor involved in allergies, autoimmune diseases, cancers and other diseases with immune disorder. Similar to its family member, TGF-ß, activin A also transmits signals through SMAD2/SMAD3, however, they bind to distinct receptors. Recent studies have uncovered that activin A plays a pivotal role in both innate and adaptive immune systems. Here we mainly focus its effects on activation, differentiation, proliferation and function of cells which are indispensable in the immune system and meanwhile make some comparisons with those of TGF-ß.

PD-L1/TLR7 dual-targeting nanobody-drug conjugate mediates potent tumor regression via elevating tumor immunogenicity in a host-expressed PD-L1 bias-dependent way.

BACKGROUND: Various tumors are insensitive to immune checkpoint blockade (ICB) therapy. Toll-like receptors (TLRs) establish the link between innate and adaptive immunity, which can assist T-cell activation and serve as promising targets for combination to enhance ICB therapy. Here, we aimed to improve efficacy for anti-programmed death ligand 1 (PD-L1) therapy by developing a PD-L1/TLR7 dual-targeting nanobody-drug conjugate (NDC), based on the PD-L1 nanobodies and TLR7 agonist we developed. METHODS: PD-L1 nanobodies were obtained by phage display screening and identified through T-cell activation bioassay, in vivo imaging and quantitative biodistribution study. Immune activation and PD-L1-inducing of TLR7 agonists were evaluated in diverse innate cell models. We constructed PD-L1/TLR7 dual-targeting NDCs by chemically coupling PD-L1 nanobodies and TLR7 agonists. The antitumor effect was evaluated via several murine or humanized solid tumor models. Immunophenotyping, immune cell depletion, tumor rechallenge, RNA sequencing and PD-L1-deficient models were combined to determine the mechanism for NDCs function. The dynamics of the in vivo behaviors of NDCs were assessed based on multiorgan changes in PD-L1 levels. RESULTS: The screened PD-L1 nanobodies were characterized as tumor-targeting and alleviated T-cell immunosuppression. The TLR7 agonists induced broad innate immune responses and intratumoral PD-L1 expression on antigen-presenting cells (APCs), and its antitumor effect was dependent on intratumoral delivery. The combination of TLR7 agonists and PD-L1 nanobodies activated both innate and adaptive immunity and upregulated PD-L1-related signaling pathways. After coupling to form dual-targeting NDCs, TLR7 agonists and PD-L1 nanobodies exerted synergistic antitumor effects and safety in either 'hot' or 'cold' tumor and early or advanced tumor models, reshaped the tumor immune microenvironment and induced antitumor immune memory. CD8+ T cells and natural killer cells were the main effector cells for NDCs to function. NDCs can promote PD-L1 expression on intratumoral APCs and tumor cells, and subsequently achieve targeted enrichment in tumors. Moreover, the efficacy of NDCs is biased toward dependence on host expression of PD-L1. CONCLUSIONS: The novel PD-L1/TLR7 dual-targeting NDC exhibited potent efficacy against heterogeneous tumors through orchestrating innate and adaptive immunity, which could act as a promising strategy to improve ICB therapy and shows prospects for clinical development.