Development and characterization of an antibody that recognizes influenza virus N1 neuraminidases.

Influenza A viruses (IAVs) continue to pose a huge threat to public health, and their prevention and treatment remain major international issues. Neuraminidase (NA) is the second most abundant surface glycoprotein on influenza viruses, and antibodies to NA have been shown to be effective against influenza infection. In this study, we generated a monoclonal antibody (mAb), named FNA1, directed toward N1 NAs. FNA1 reacted with H1N1 and H5N1 NA, but failed to react with the NA proteins of H3N2 and H7N9. In vitro, FNA1 displayed potent antiviral activity that mediated both NA inhibition (NI) and blocking of pseudovirus release. Moreover, residues 219, 254, 358, and 388 in the NA protein were critical for FNA1 binding to H1N1 NA. However, further validation is necessary to confirm whether FNA1 mAb is indeed a good inhibitor against NA for application against H1N1 and H5N1 viruses.

pHLIP-fused PD-L1 engages avelumab to elicit NK cytotoxicity under acidic conditions.

Natural killer (NK) cells represent key player in immune surveillance to eliminate transformed or malignant cells. One of mechanisms of action of NK cells is antibody-dependent cell-mediated cytotoxicity (ADCC) by recognizing tumor antigens on the surface of cancer cells. However, the heterogeneity of tumor antigens and the scarcity of membrane surface targets significantly restrict this strategy. Recently, we constructed a new cargo by tethering a low pH insertion peptide (pHLIP) to the C terminus of the ectodomain of programed death ligand-1 (PD-L1) and demonstrated its ability to modulate immune responses. Herein, the potential application of PD-L1-pHLIP in cancer therapy was determined. pHLIP tethering had no effect on the binding capacity of PD-L1 protein to an anti-PD-L1 antibody (i.e. avelumab). Association of pHLIP rendered PD-L1 segment display on the surface of cellular membrane in the acidic buffer instead of the neutral solution. Importantly, plate-coated or beads-coupled PD-L1-pHLIP enable robust activation and expression of cytotoxic mediators of NK cells via engaging avelumab. Overall, this work provides proof of concept that recombinant PD-L1 protein decorated on the cellular membrane driven by pHLIP in combination with appropriate monoclonal antibody has potentials to elicit NK cytotoxicity, which may represent a novel and promising therapeutic avenue in cancer.

A novel MARV glycoprotein-specific antibody with potentials of broad-spectrum neutralization to filovirus.

Marburg virus (MARV) is one of the filovirus species that cause deadly hemorrhagic fever in humans, with mortality rates up to 90%. Neutralizing antibodies represent ideal candidates to prevent or treat virus disease. However, no antibody has been approved for MARV treatment to date. In this study, we identified a novel human antibody named AF-03 that targeted MARV glycoprotein (GP). AF-03 possessed a high binding affinity to MARV GP and showed neutralizing and protective activities against the pseudotyped MARV in vitro and in vivo. Epitope identification, including molecular docking and experiment-based analysis of mutated species, revealed that AF-03 recognized the Niemann-Pick C1 (NPC1) binding domain within GP1. Interestingly, we found the neutralizing activity of AF-03 to pseudotyped Ebola viruses (EBOV, SUDV, and BDBV) harboring cleaved GP instead of full-length GP. Furthermore, NPC2-fused AF-03 exhibited neutralizing activity to several filovirus species and EBOV mutants via binding to CI-MPR. In conclusion, this work demonstrates that AF-03 represents a promising therapeutic cargo for filovirus-caused disease.

Dynamic aberrances of substantia nigra-relevant coactivation patterns in first-episode treatment-naïve patients with schizophrenia.

BACKGROUND: Although dopaminergic disturbances are well-known in schizophrenia, the understanding of dopamine-related brain dynamics remains limited. This study investigates the dynamic coactivation patterns (CAPs) associated with the substantia nigra (SN), a key dopaminergic nucleus, in first-episode treatment-naïve patients with schizophrenia (FES). METHODS: Resting-state fMRI data were collected from 84 FES and 94 healthy controls (HCs). Frame-wise clustering was implemented to generate CAPs related to SN activation or deactivation. Connectome features of each CAP were derived using an edge-centric method. The occurrence for each CAP and the balance ratio for antagonistic CAPs were calculated and compared between two groups, and correlations between temporal dynamic metrics and symptom burdens were explored. RESULTS: Functional reconfigurations in CAPs exhibited significant differences between the activation and deactivation states of SN. During SN activation, FES more frequently recruited a CAP characterized by activated default network, language network, control network, and the caudate, compared to HCs (F = 8.54, FDR-p = 0.030). Moreover, FES displayed a tilted balance towards a CAP featuring SN-coactivation with the control network, caudate, and thalamus, as opposed to its antagonistic CAP (F = 7.48, FDR-p = 0.030). During SN deactivation, FES exhibited increased recruitment of a CAP with activated visual and dorsal attention networks but decreased recruitment of its opposing CAP (F = 6.58, FDR-p = 0.034). CONCLUSION: Our results suggest that neuroregulatory dysfunction in dopaminergic pathways involving SN potentially mediates aberrant time-varying functional reorganizations in schizophrenia. This finding enriches the dopamine hypothesis of schizophrenia from the perspective of brain dynamics.

Functional characterization of AF-04, an afucosylated anti-MARV GP antibody.

Marburg virus (MARV), one member of the Filoviridae family, cause sporadic outbreaks of hemorrhagic fever with high mortality rates. No countermeasures are currently available for the prevention or treatment of MARV infection. Monoclonal antibodies (mAbs) are promising candidates to display high neutralizing activity against MARV infection in vitro and in vivo. Recently, growing evidence has shown that immune effector function including antibody-dependent cell-mediated cytotoxicity (ADCC) is also required for in vivo efficacy of a panel of antibodies. Glyco-engineered methods are widely utilized to augment ADCC function of mAbs. In this study, we generated a fucose-knockout MARV GP-specific mAb named AF-04 and showed that afucosylation dramatically increased its binding affinity to polymorphic FcγRIIIa (F176/V176) compared with the parental AF-03. Accordingly, AF-04-mediated NK cell activation and NFAT expression downstream of FcγRIIIa in effector cells were also augmented. In conclusion, this work demonstrates that AF-04 represents a novel avenue for the treatment of MARV-caused disease.

Functional epitopes and neutralizing antibodies of vaccinia virus.

Smallpox is an infectious disease caused by the variola virus, and it has a high mortality rate. Historically it has broken out in many countries and it was a great threat to human health. Smallpox was declared eradicated in 1980, and Many countries stopped nation-wide smallpox vaccinations at that time. In recent years the potential threat of bioterrorism using smallpox has led to resumed research on the treatment and prevention of smallpox. Effective ways of preventing and treating smallpox infection have been reported, including vaccination, chemical drugs, neutralizing antibodies, and clinical symptomatic therapies. Antibody treatments include anti-sera, murine monoclonal antibodies, and engineered humanized or human antibodies. Engineered antibodies are homologous, safe, and effective. The development of humanized and genetically engineered antibodies against variola virus via molecular biology and bioinformatics is therefore a potentially fruitful prospect with respect to field application. Natural smallpox virus is inaccessible, therefore most research about prevention and/or treatment of smallpox were done using vaccinia virus, which is much safer and highly homologous to smallpox. Herein we summarize vaccinia virus epitope information reported to date, and discuss neutralizing antibodies with potential value for field application.

Neural variability in three major psychiatric disorders.

Across the major psychiatric disorders (MPDs), a shared disruption in brain physiology is suspected. Here we investigate the neural variability at rest, a well-established behavior-relevant marker of brain function, and probe its basis in gene expression and neurotransmitter receptor profiles across the MPDs. We recruited 219 healthy controls and 279 patients with schizophrenia, major depressive disorder, or bipolar disorders (manic or depressive state). The standard deviation of blood oxygenation level-dependent signal (SDBOLD) obtained from resting-state fMRI was used to characterize neural variability. Transdiagnostic disruptions in SDBOLD patterns and their relationships with clinical symptoms and cognitive functions were tested by partial least-squares correlation. Moving beyond the clinical sample, spatial correlations between the observed patterns of SDBOLD disruption and postmortem gene expressions, Neurosynth meta-analytic cognitive functions, and neurotransmitter receptor profiles were estimated. Two transdiagnostic patterns of disrupted SDBOLD were discovered. Pattern 1 is exhibited in all diagnostic groups and is most pronounced in schizophrenia, characterized by higher SDBOLD in the language/auditory networks but lower SDBOLD in the default mode/sensorimotor networks. In comparison, pattern 2 is only exhibited in unipolar and bipolar depression, characterized by higher SDBOLD in the default mode/salience networks but lower SDBOLD in the sensorimotor network. The expression of pattern 1 related to the severity of clinical symptoms and cognitive deficits across MPDs. The two disrupted patterns had distinct spatial correlations with gene expressions (e.g., neuronal projections/cellular processes), meta-analytic cognitive functions (e.g., language/memory), and neurotransmitter receptor expression profiles (e.g., D2/serotonin/opioid receptors). In conclusion, neural variability is a potential transdiagnostic biomarker of MPDs with a substantial amount of its spatial distribution explained by gene expressions and neurotransmitter receptor profiles. The pathophysiology of MPDs can be traced through the measures of neural variability at rest, with varying clinical-cognitive profiles arising from differential spatial patterns of aberrant variability.

[Hippocampal Development Deviation and Its Relationship With Cognition in Major Psychiatric Disorders].

Objective: To investigate hippocampal development deviation and its association with cognition in patients with major psychiatric disorders (MPDs), including schizophrenia, bipolar disorder and major depressive disorder. Methods: The T1-weighted MRI data of 174 first-episode drug-naïve schizophrenia (FES) atients, 169 bipolar disorder (BD) patients, 184 major depressive disorder (MDD) patients, and 321 healthy controls were collected and their hippocampal volume was extracted after preprocessing with Freesurfer 5.3. A normative neurodevelopment model was applied to calculate the hippocampal deviation scores. Data on cognitive functions, including visual memory, attention, spatial working memory, were collected. Comparison by different sexes was made to identify difference between the hippocampal development deviation scores of the control group and those of the disease groups. We also investigated the moderating effect of age on the deviation score and explored the association between the deviation score and cognitive function. Results: The hippocampal development deviation scores of patients with MPDs were significantly lower than those of the healthy controls (false discovery rate [FDR]-P<0.05). Analysis of the moderating effect of age revealed lower deviation scores in young patients (<[25.83-28.56] yr.) and higher deviation scores in old patients (>[35.87-54.35] yr.) in comparison with those of the healthy controls. The right hippocampal deviation scores in male FES patients were positively correlated with the number of errors for tasks concerning spatial working memory ( r=0.32, FDR-P=0.04). Conclusion: Our findings suggest abnormal hippocampal development in MPDs patients and its different distribution in MPDs patients of different age groups. The hippocampal development deviation score may provide a new perspective for further understanding of etiology in MPDs.

[Frequency-Specific Alterations of Spontaneous Brain Activity in First-Episode Drug-Naïve Schizophrenia].

Objective: To investigate frequency-specific alterations of spontaneous brain activity in first-episode drug-naïve schizophrenia (SZ) patients and the associations with clinical symptoms. Methods: We collected the resting-state functional MRI (rs-fMRI) data from 84 first-episode drug-naïve SZ patients and 94 healthy controls (HCs) and calculated the amplitude of low-frequency fluctuations (ALFF) and regional homogeneity (ReHo) of four frequency bands, including slow-2, slow-3, slow-4, and slow-5. Two-sample t-tests were used to evaluate the intergroup differences in ALFF and ReHo, while partial correlation analyses were conducted to explore the associations between abnormal ALFF and ReHo and the severity of clinical symptoms in the SZ group. Results: Compared with HCs, the SZ group showed reduced ALFF in superior cerebellum and cerebellar vermis across slow-2, slow-3, and slow-4 bands, while increased ALFF was found in left superior temporal gyrus, middle temporal gyrus, and superior temporal pole at slow-4 band. Moreover, reduced ReHo was observed in the right precentral and postcentral gyri at slow-3 band in the SZ group. Additionally, the ALFF of left superior temporal gyrus, middle temporal gyrus, and superior temporal pole in slow-4 band showed a trend of positive correlation with the excited factor score of Positive and Negative Syndrome Scale (PANSS) in the SZ group. Conclusion: Our results suggest that local alterations of spontaneous brain activity were frequency-specific in first-episode drug-naïve SZ patients.

Identifying transdiagnostic biological subtypes across schizophrenia, bipolar disorder, and major depressive disorder based on lipidomics profiles.

Emerging evidence has demonstrated overlapping biological abnormalities underlying schizophrenia (SCZ), bipolar disorder (BP), and major depressive disorder (MDD); these overlapping abnormalities help explain the high heterogeneity and the similarity of patients within and among diagnostic categories. This study aimed to identify transdiagnostic subtypes of these psychiatric disorders based on lipidomics abnormalities. We performed discriminant analysis to identify lipids that classified patients (N = 349, 112 with SCZ, 132 with BP, and 105 with MDD) and healthy controls (N = 198). Ten lipids that mainly regulate energy metabolism, inflammation, oxidative stress, and fatty acylation of proteins were identified. We found two subtypes (named Cluster 1 and Cluster 2 subtypes) across patients with SCZ, BP, and MDD by consensus clustering analysis based on the above 10 lipids. The distribution of clinical diagnosis, functional impairment measured by Global Assessment of Functioning (GAF) scales, and brain white matter abnormalities measured by fractional anisotropy (FA) and radial diffusivity (RD) differed in the two subtypes. Patients within the Cluster 2 subtype were mainly SCZ and BP patients and featured significantly elevated RD along the genu of corpus callosum (GCC) region and lower GAF scores than patients within the Cluster 1 subtype. The SCZ and BP patients within the Cluster 2 subtype shared similar biological patterns; that is, these patients had comparable brain white matter abnormalities and functional impairment, which is consistent with previous studies. Our findings indicate that peripheral lipid abnormalities might help identify homogeneous transdiagnostic subtypes across psychiatric disorders.

Novel CRISPR/Cas9-mediated knockout of LIG4 increases efficiency of site-specific integration in Chinese hamster ovary cell line.

AIM: To investigate the impact of deficiency of LIG4 gene on site-specific integration in CHO cells. RESULTS: CHO cells are considered the most valuable mammalian cells in the manufacture of biological medicines, and genetic engineering of CHO cells can improve product yield and stability. The traditional method of inserting foreign genes by random integration (RI) requires multiple rounds of screening and selection, which may lead to location effects and gene silencing, making it difficult to obtain stable, high-yielding cell lines. Although site-specific integration (SSI) techniques may overcome the challenges with RI, its feasibility is limited by the very low efficiency of the technique. Recently, SSI efficiency has been enhanced in other mammalian cell types by inhibiting DNA ligase IV (Lig4) activity, which is indispensable in DNA double-strand break repair by NHEJ. However, this approach has not been evaluated in CHO cells. In this study, the LIG4 gene was knocked out of CHO cells using CRISPR/Cas9-mediated genome editing. Efficiency of gene targeting in LIG4-/--CHO cell lines was estimated by a green fluorescence protein promoterless reporter system. Notably, the RI efficiency, most likely mediated by NHEJ in CHO, was inhibited by LIG4 knockout, whereas SSI efficiency strongly increased 9.2-fold under the precise control of the promoter in the ROSA26 site in LIG4-/--CHO cells. Moreover, deletion of LIG4 had no obvious side effects on CHO cell proliferation. CONCLUSIONS: Deficiency of LIG4 represents a feasible strategy to improve SSI efficiency and suggests it can be applied to develop and engineer CHO cell lines in the future.

TIM-1 Augments Cellular Entry of Ebola Virus Species and Mutants, Which Is Blocked by Recombinant TIM-1 Protein.

Ebola virus, a member of the Filoviridae family, utilizes the attachment factors on host cells to support its entry and cause severe tissue damage. TIM-1 has been identified as a predominant attachment factor via interaction with phosphatidylserine (PS) localized on the viral envelope and glycoprotein (GP). In this study, we give the first demonstration that TIM-1 enhances the cellular entry of three species of Ebola virus, as well as those harboring GP mutations (A82V, T544I, and A82V T544I). Furthermore, two TIM-1 variants (i.e., TIM-1-359aa and TIM-1-364aa) had comparable effects on promoting Zaire Ebola virus (EBOV) attachment, internalization, and infection. Importantly, recombinant TIM-1 ectodomain (ECD) protein could decrease the infectivity of Ebola virus and display synergistic inhibitory effects with ADI-15946, a monoclonal antibody with broad neutralizing activity to Ebola virus. Of note, EBOV strains harboring GP mutations (K510E and D552N), which were refractory to antibody treatment, were still sensitive to TIM-1 protein-mediated impairment of infectivity, indicating that TIM-1 protein may represent an alternative therapeutic regimen when antibody evasion occurs. IMPORTANCE The viral genome has acquired numerous mutations with the potential to increase transmission during the 2013-to-2016 outbreak of Ebola virus. EBOV strains harboring GP mutations (A82V, T544I, and A82V T544I), which have been identified to increase viral infectivity in humans, have attracted our attention. Herein, we give the first report that polymorphic TIM-1 enhances the infectivity of three species of Ebola virus, as well as those harboring GP mutations (A82V, T544I, and A82V T544I). We show that recombinant TIM-1 ECD protein could decrease the infectivity of Ebola virus with or without a point mutation and displays synergistic inhibitory effects with ADI-15946. Furthermore, TIM-1 protein potently blocked cell entry of antibody-evading Ebola virus species. These findings highlight the role of TIM-1 in Ebola virus infection and indicate that TIM-1 protein represents a potential therapeutic avenue for Ebola virus and its mutated species.

A Novel Humanized Anti-Abrin A Chain Antibody Inhibits Abrin Toxicity In Vitro and In Vivo.

Abrin, a type-II ribosome inactivating protein from the seed of Abrus precatorius, is classified as a Category B bioterrorism warfare agent. Due to its high toxicity, ingestion by animals or humans will lead to death from multiple organ failure. Currently, no effective agents have been reported to treat abrin poisoning. In this study, a novel anti-abrin neutralizing antibody (S008) was humanized using computer-aided design, which possessed lower immunogenicity. Similar to the parent antibody, a mouse anti-abrin monoclonal antibody, S008 possessed high affinity and showed a protective effect against abrin both in vitro and in vivo, and protected mice that S008 was administered 6 hours after abrin. S008 was found that it did not inhibit entry of abrin into cells, suggesting an intracellular blockade capacity against the toxin. In conclusion, this work demonstrates that S008 is a high affinity anti-abrin antibody with both a neutralizing and protective effect and may be an excellent candidate for clinical treatment of abrin poisoning.

Selection and Characterization of FD164, a High-Affinity Signal Regulatory Protein α Variant with Balanced Safety and Effectiveness, from a Targeted Epitope Mammalian Cell-Displayed Antibody Library.

Phagocytic resistance plays a key role in tumor-mediated immune escape, so phagocytosis immune checkpoints are a potential target for cancer immunotherapy. CD47 is one of the important phagocytosis immune checkpoints; thus, blocking the interaction between CD47 and signal regulatory protein α (SIRPα) may provide new options for cancer treatment. Using computer-aided targeted epitope mammalian cell-displayed antibody library, we screened and obtained an engineered SIRPα variant fragment crystallizable fusion protein, FD164, with higher CD47-binding activity than wild-type SIRPα Compared with wild-type SIRPα, FD164 has approximately 3-fold higher affinity for binding to CD47, which further enhanced its phagocytic effect in vitro and tumor suppressor activity in vivo. FD164 maintains the similar antitumor activity of the clinical research drug Hu5F9 in the mouse xenograft model. Furthermore, FD164 combined with rituximab can significantly improve the effect of single-agent therapy. On the other hand, compared with Hu5F9, FD164 does not cause hemagglutination, and its ability to bind to red blood cells or white blood cells is weaker at the same concentration. Finally, it was confirmed by computer structure prediction and alanine scanning experiments that the N45, E47, 52TEVYVK58, K60, 115EVTELTRE122, and E124 residues of CD47 are important for SIRPα or FD164 recognition. Briefly, we obtained a high-affinity SIRPα variant FD164 with balanced safety and effectiveness. SIGNIFICANCE STATEMENT: Up to now, few clinically marketed drugs targeting CD47 have been determined to be effective and safe. FD164, a potential signal regulatory protein α variant fragment crystallizable protein with balanced safety and effectiveness, could provide a reference for the development of antitumor drugs.

Identification of a novel protective human monoclonal antibody, LXY8, that targets the key neutralizing epitopes of staphylococcal enterotoxin B.

Staphylococcal enterotoxin B (SEB), one of the exotoxins produced by Staphylococcus aureus, is the key toxin that causes poisoning reactions and toxic shock syndrome. In the current research work, a novel human antibody named LXY8 was screened from a human phage display antibody library, and LXY8 blocked the interaction between SEB and the T cell receptor (TCR). The binding activity between LXY8 and SEB was 0.525 nM. Furthermore, LXY8 could effectively inhibit the SEB-induced activation of peripheral blood mononuclear cells and release of cytokines. In the BALB/c mouse model, LXY8 effectively neutralized SEB toxicity in vivo. Finally, based on computer-guided molecular modeling, we designed a series of SEB mutation sites; these sites facilitated the determination of the key residues (i.e.176EFNN179) of SEB recognized by LXY8. The research revealed that the 176EFNN179 residues of SEB are important for specific antibody-antigen recognition. The results may be helpful for the development of antibody-based therapy for SEB-induced toxic shock syndrome.

pH Low Insertion Peptide-Modified Programmed Cell Death-Ligand 1 Potently Suppresses T-Cell Activation Under Acidic Condition.

Programmed cell death-ligand 1 (PD-L1)/PD-1 axis is critical for maintenance of immune homeostasis by limiting overactivation of effector T-cell responses. The impairment of PD-L1/PD-1 signals play an important role in the pathogenesis of inflammatory diseases, making this pathway an ideal target for novel therapeutics to induce immune tolerance. Given weakly acidic environment as a putative hallmark of inflammation, in this study we designed a new cargo by linking the ectodomain of murine PD-L1 to the N terminus of pHLIPs, a low pH-responding and membrane-insertion peptide, and demonstrated its potent immune-suppressive activity. Specifically, PD-L1-pHLIP spanned the cellular membrane and perfectly recognized its ligand PD-1 in acidic buffer. Immobile PD-L1-pHLIP actively inhibited T-cell proliferation and IFN-γ production. Importantly, soluble PD-L1-pHLIP retained its function to dampen T-cell responses under acidic condition instead of neutral aqueous solution. Overall, these data suggest that PD-L1-pHLIP has potentials to be a novel therapeutic avenue for T-cell-mediated inflammatory diseases.

Immunoregenerative effects of the bionically cultured Sanghuang mushrooms (Inonotus sanghuagn) on the immunodeficient mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Description of the pharmacological activities of Sanghuang mushrooms (Inonotus Sanghuang) can be traced back to Tang dynasty of China 1300 years ago. This mushroom has been widely accepted in China, Japan, Korea and certain regions of Europe as a nutraceutical medicine for enhancing immunity or an alternative medicine for prevention or inhibition of tumorigenesis. However, this mushroom is rarely available from the mulberry trees in the wild because of the rigorous conditions needed for formation of the Sanghuang mushrooms. AIM OF THE STUDY: This study aims to establish a practical protocol for culture, particularly for a bunch of production of Sanghuang mushrooms possibly to commercialize the cultured Sanghuang based on deep comparison of quality and pharmacological activities between the cultured and the wild Sanghuang. MATERIALS AND METHODS: A phylogenetic tree containing five strains of the wild Sanghuang was constructed using rDNA markers. Different temperatures and medium compositions were surveyed to develop a practical protocol for culture of the Sanghuang mushrooms. 5-fluorouracil was used to induce the immunodeficient mice. Chemotherapeutic components and pharmacological activities were deeply analyzed between a cultured strain (SG) and three strains of the wild Sanghuang. RESULTS: Maintenance of a temperature of 22-28 °C and a high relative humidity of 90-95%, and use of a high ratio (80%) of mulberry tree sticks in the medium were critical to successful culture of Sanghuang. The cultured mushrooms were yellow with a uniform shape, while the wild Sanghuang was dark brown with a smaller and irregular shape. The cultured mushrooms contained significantly higher levels of polysaccharides, amino acids, and water-soluble nutraceuticals, whereas flavones in the wild Sanghuang were significantly higher (P < 0.05). Use of a dose of 8 mg/kg or 16 mg/kg to immunoregenerate the immunodeficient mice was comparable between the cultured and wild Sanghang based on analysis of hematological parameters and histological examination of the thymus and spleen in the treated mice. CONCLUSIONS: This study highlights the potential of the immunoregenerative functions of the cultured Sanghuang for cancer chemotherapy and suggests that the cultured Sanghuang can be an alternative to wild Sanghuang used for nutraceutical medicine.

Engineered AXL-ECD-Fc variants that abolish the AXL/Gas6 interaction suppress tumor cell migration.

AXL receptor tyrosine kinase ligand (AXL), a tyrosine kinase receptor that is commonly overexpressed in numerous types of cancer, significantly promotes drug resistance and metastasis in tumor cells. Inhibition of the AXL/growth arrest-specific 6 (Gas6) signaling pathway is emerging as a potential anticancer therapeutic strategy. In the present study, on the basis of the three-dimensional complex structure of AXL/Gas6, the critical residues (E56, E59 and T77) in AXL binding to Gas6 were determined using computer graphics analysis and the distance geometry method. Subsequently, four-variant AXL-ECD-Fc-M1 (G32S, D87G, V92A and G127R) and AXL-ECD-Fc-M2 (G32A, D87A, V92A and G127A) were predicted as high-affinity mutants; AXL-ECD-Fc-M3 (E56R and T77R) and AXL-ECD-Fc-M4 (E59R and T77R) were predicted as low-affinity mutants. The results of the present study revealed that the half-maximal effect concentrations of AXL-ECD-Fc-M1 and AXL-ECD-Fc-M2 were ~0.141 and 0.375 µg/ml, respectively, whereas that of the wild-type protein (AXL-ECD-Fc-WT) was 0.514 µg/ml. Furthermore, adding the high-affinity mutants into culture medium to capture free Gas6 significantly inhibited AXL/Gas6 binding and thus blocked the downstream signaling pathway. In addition, the high-affinity mutants effectively suppressed the migration and metastasis of SKOV3 and A549 cells. Conversely, compared with AXL-ECD-Fc-WT, the low-affinity AXL mutants AXL-ECD-Fc-M3 and AXL-ECD-Fc-M4 lost all inhibitory activities. These findings highlight AXL as a potential therapeutic target and demonstrated that the key residues E56, E59 and T77 may be crucial sites for abolishing the activity of the AXL/Gas6 pathway in cancer therapy.

A novel human anti-AXL monoclonal antibody attenuates tumour cell migration.

TAM family members (TYRO3, AXL and MERTK) play essential roles in the resolution of inflammation and in infectious diseases and cancer. AXL, a tyrosine kinase receptor, is commonly overexpressed in several solid tumours and numerous hematopoietic malignancies including acute myeloid leukaemia, acute lymphocytic leukaemia, chronic myeloid leukaemia, chronic lymphocytic leukaemia and multiple myeloma. AXL significantly promotes tumour cell migration, invasion and metastasis, as well as angiogenesis. AXL also plays an important role in inflammation and macrophage ontogeny. Recent studies have revealed that AXL contributes to leukaemic phenotypes through activation of oncogenic signalling pathways that lead to increased cell migration and proliferation. To evaluate the mechanisms underlying the role of AXL signalling in tumour metastasis, we screened a phage display library to generate a novel human monoclonal antibody, named DAXL-88, that recognizes both human and murine AXL. The concentrations of DAXL-88 required for 50% maximal binding to human and murine AXL were 0.118 and 0.164 µg/mL, respectively. Furthermore, DAXL-88 bound to human AXL with high affinity (KD  ~ 370 pM). DAXL-88 blocked the interaction between AXL and its ligand, growth arrest-specific gene 6 (GAS6), with a half maximal inhibitory concentration of 2.16 µg/mL. Moreover, DAXL-88 inhibited AXL/GAS6-dependent cell signalling, which is implicated in cell migration and invasion. In conclusion, the novel anti-AXL DAXL-88 high-affinity antibody blocks the interaction between AXL and GAS6 and inhibits tumour cell migration and invasion induced by GAS6. Thus, DAXL-88 offers promise for the development of targeted therapeutic strategies in solid tumours, leukaemias and other lymphoid neoplasms.