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The cytokine interleukin-6 (IL-6) plays a crucial role in autoimmune and inflammatory diseases. Understanding the precise mechanism of IL-6 interaction at the amino acid level is essential to develop IL-6-inhibiting compounds. In this study, we employed computer-guided drug design tools to predict the key residues that are involved in the interaction between IL-6 and its receptor IL-6R. Subsequently, we generated IL-6 mutants and evaluated their binding affinity to IL-6R and the IL-6R - gp130 complex, as well as monitoring their biological activities. Our findings revealed that the R167A mutant exhibited increased affinity for IL-6R, leading to enhanced binding to IL-6R - gp130 complex and subsequently elevated intracellular phosphorylation of STAT3 in effector cells. On the other hand, although E171A reduced its affinity for IL-6R, it displayed stronger binding to the IL-6R - gp130 complex, thereby enhancing its biological activity. Furthermore, we identified the importance of R178 and R181 for the precise recognition of IL-6 by IL-6R. Mutants R181A/V failed to bind to IL-6R, while maintaining an affinity for the IL-6 - gp130 complex. Additionally, deletion of the D helix resulted in complete loss of IL-6 binding affinity for IL-6R. Overall, this study provides valuable insights into the binding mechanism of IL-6 and establishes a solid foundation for future design of novel IL-6 inhibitors.
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Interleucina-6 , Simulación del Acoplamiento Molecular , Unión Proteica , Receptores de Interleucina-6 , Interleucina-6/metabolismo , Interleucina-6/genética , Humanos , Receptores de Interleucina-6/metabolismo , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/química , Receptor gp130 de Citocinas/metabolismo , Receptor gp130 de Citocinas/genética , Receptor gp130 de Citocinas/química , Mutagénesis Sitio-Dirigida , Sitios de Unión , Factor de Transcripción STAT3/metabolismo , Fosforilación , MutaciónRESUMEN
MIL77-3 is one component of antibody cocktail that is produced in our lab and represents an effective regimen for animals suffering from Zaire Ebolavirus (EBOV) infection. MIL77-3 is engineered to increase its affinity for the FcγRIIIa (CD16a) by deleting the fucose in the framework region. The potential effects of this modification on host immune responses, however, remain largely unknown. Herein, we demonstrated that MIL77-3 recognized secreted glycoproptein (sGP), produced by EBOV, and formed the immunocomplex to potently augment antibody-dependent cytotoxicity of human peripheral blood-derived natural killer cells (pNKs), including CD56dim and CD56bright subpopulations, in contrast to the counterparts (Mab114, rEBOV548, fucosylated MIL77-3). Intriguingly, this effect was not observed when NK92-CD16a cell line was utilized and restored by the addition of beads-coupled or membrane-anchored sGP in combination with MIL77-3. Furthermore, sGP bound to unrecognized receptors on T cells contaminated in pNKs rather than NK92-CD16a cells. Administration of beads-coupled sGP/MIL77-3 complex in mice elicited NK activation. Overall, this work reveals an immune-stimulating function of sGP/MIL77-3 complex by triggering cytotoxic activity of NK cells, highlighting the necessity to evaluate the potential impact of MIL77-3 on host immune reaction in clinical trials. IMPORTANCE: Zaire Ebolavirus (EBOV) is highly lethal and causes sporadic outbreaks. The passive administration of monoclonal antibodies (mAbs) represents a promising treatment regimen against EBOV. Mounting evidence has shown that the efficacy of a subset of therapeutic mAbs in vivo is intimately associated with its capacity to trigger NK activity, supporting glycomodification of Fc region of anti-EBOV mAbs as a putative strategy to enhance Fc-mediated immune effector function as well as protection in vivo. Our work here uncovers the potential harmful influence of this modification on host immune responses, especially for mAbs with cross-reactivity to secreted glycoproptein (sGP) (e.g., MIL77-3), and highlights it is necessary to evaluate the NK-stimulating activity of a fucosylated mAb engaged with sGP when a new candidate is developed.
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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.
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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.
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Ebolavirus , Fiebre Hemorrágica Ebola , Marburgvirus , Humanos , Anticuerpos Antivirales , Simulación del Acoplamiento Molecular , Glicoproteínas , Fiebre Hemorrágica Ebola/prevención & control , Ebolavirus/químicaRESUMEN
Induced pluripotent stem cells (iPSCs) can be personalized and differentiated into neural stem cells (NSCs), thereby effectively providing a source of transplanted cells for spinal cord injury (SCI). To further improve the repair efficiency of SCI, we designed a functional neural network tissue based on TrkC-modified iPSC-derived NSCs and a CBD-NT3-modified linear-ordered collagen scaffold (LOCS). We confirmed that transplantation of this tissue regenerated neurons and synapses, improved the microenvironment of the injured area, enhanced remodeling of the extracellular matrix, and promoted functional recovery of the hind limbs in a rat SCI model with complete transection. RNA sequencing and metabolomic analyses also confirmed the repair effect of this tissue from multiple perspectives and revealed its potential mechanism for treating SCI. Together, we constructed a functional neural network tissue using human iPSCs-derived NSCs as seed cells based on the interaction of receptors and ligands for the first time. This tissue can effectively improve the therapeutic effect of SCI, thus confirming the feasibility of human iPSCs-derived NSCs and LOCS for SCI repair and providing a valuable direction for SCI research.
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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.
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Marburgvirus , Anticuerpos Monoclonales/uso terapéuticoRESUMEN
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.
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CD47, as an innate immune checkpoint molecule, is an important target of cancer immunotherapy. We previously reported that a high-affinity SIRPα variant FD164 fused with IgG1 subtype Fc showed a better antitumor effect than wild-type SIRPα in an immunodeficient tumor-bearing model. However, CD47 is widely expressed in blood cells, and the drugs targeting CD47 may cause potential hematological toxicity. Herein, we modified the FD164 molecule by Fc mutation (N297A) to inactivate the Fc-related effector function and named it nFD164. Moreover, we further studied the potential of nFD164 as a candidate drug targeting CD47, including the stability, in vitro activity, antitumor activity of single or combined drugs in vivo, and hematological toxicity in humanized CD47/SIRPα transgenic mouse model. The results show that nFD164 maintains strong binding activity to CD47 on tumor cells, but has weak binding activity with red blood cells or white blood cells, and nFD164 has good drug stability under accelerated conditions (high temperature, bright light and freeze-thaw cycles). More importantly, in the immunodeficient or humanized CD47/SIRPα transgenic mice bearing tumor model, the combination of nFD164 and anti-CD20 antibody or anti-mPD-1 antibody had a synergistic antitumor effect. Especially in transgenic mouse models, nFD164 combined with anti-mPD-1 significantly enhanced tumor suppressive activity compared with anti-mPD-1 (P < 0.01) or nFD164 (P < 0.01) as a single drug and had fewer hematology-related side effects than FD164 or Hu5F9-G4. When these factors are taken together, nFD164 is a promising high-affinity CD47-targeting drug candidate with better stability, potential antitumor activity, and improved safety profile.
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Antígeno CD47 , Neoplasias , Ratones , Animales , Antígeno CD47/metabolismo , Inmunoterapia/métodos , Modelos Animales de Enfermedad , Ratones Transgénicos , Sistemas de Liberación de Medicamentos , Neoplasias/tratamiento farmacológico , FagocitosisRESUMEN
The Interleukin-2 Family contains six kinds of cytokines, namely IL-2, IL-15, IL-4, IL-7, IL-9, and IL-21, all of which share a common γ chain. Many cytokines of the IL-2 family have been reported to be a driving force in immune cells activation. Therefore, researchers have tried various methods to study the anti-tumor effect of cytokines for a long time. However, due to the short half-life, poor stability, easy to lead to inflammatory storms and narrow safety treatment window of cytokines, this field has been tepid. In recent years, with the rapid development of protein engineering technology, some engineered cytokines have a significant effect in tumor immunotherapy, showing an irresistible trend of development. In this review, we will discuss the current researches of the IL-2 family and mainly focus on the application and achievements of engineered cytokines in tumor immunotherapy.
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Citocinas , Neoplasias , Humanos , Citocinas/metabolismo , Interleucina-2/uso terapéutico , Inmunoterapia/métodos , Neoplasias/terapiaRESUMEN
The mechanisms of self-renewal and pluripotency maintenance of human pluripotent stem cells (hPSCs) have not been fully elucidated, especially for the role of those poorly characterized long noncoding RNAs (lncRNAs). ESRG is a lncRNA highly expressed in hPSCs, and its functional roles are being extensively explored in the field. Here, we identified that the transcription of ESRG can be directly regulated by OCT4, a key self-renewal factor in hPSCs. Knockdown of ESRG induces hPSC differentiation, cell cycle arrest, and apoptosis. ESRG binds to MCM2, a replication-licensing factor, to sustain its steady-state level and nuclear location, safeguarding error-free DNA replication. Further study showed that ESRG knockdown leads to MCM2 abnormalities, resulting in DNA damage and activation of the p53 pathway, ultimately impairs hPSC self-renewal and pluripotency, and induces cell apoptosis. In summary, our study suggests that ESRG, as a novel target of OCT4, plays an essential role in maintaining the cell survival and self-renewal/pluripotency of hPSCs in collaboration with MCM2 to suppress p53 signaling. These findings provide critical insights into the mechanisms underlying the maintenance of self-renewal and pluripotency in hPSCs by lncRNAs.
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Componente 2 del Complejo de Mantenimiento de Minicromosoma , Células Madre Pluripotentes , ARN Largo no Codificante , Proteína p53 Supresora de Tumor , Humanos , Diferenciación Celular/genética , Supervivencia Celular/genética , Componente 2 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 2 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Células Madre Pluripotentes/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Psoriasis is a common, immune-mediated skin disease characterized by epidermal hyperproliferation and chronic skin inflammation. Long noncoding RNAs are >200 nucleotide-long transcripts that possess important regulatory functions. To date, little is known about the contribution of long noncoding RNAs to psoriasis. In this study, we identify LINC00958 as a long noncoding RNA overexpressed in keratinocytes (KCs) from psoriasis skin lesions, in a transcriptomic screen performed on KCs sorted from psoriasis and healthy skin. Increased levels of LINC00958 in psoriasis KCs were confirmed by RT-qPCR and single-molecule in situ hybridization. Confocal microscopy and analysis of subcellular fractions showed that LINC00958 is mainly localized in the cytoplasm of KCs. IL-17A, a key psoriasis cytokine, induced LINC00958 in KCs through C/EBP-ß and the p38 pathway. The inhibition of LINC00958 led to decreased proliferation as measured by Ki-67 expression, live cell analysis imaging, and 5-ethynyl-2-deoxyuridine assays. Transcriptomic analysis of LINC00958-depleted KCs revealed enrichment of proliferation- and cell cycleârelated genes among differentially expressed transcripts. Moreover, LINC00958 depletion led to decreased basal and IL-17Aâinduced phosphorylation of p38. Furthermore, IL-17Aâinduced KC proliferation was counteracted by the inhibition of LINC00958. In summary, our data support a role for the IL-17Aâinduced long noncoding RNA, LINC00958, in the pathological circuits of psoriasis by reinforcing IL-17Aâinduced epidermal hyperproliferation.
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Psoriasis , ARN Largo no Codificante , Humanos , Interleucina-17/genética , Interleucina-17/metabolismo , ARN Largo no Codificante/genética , Epidermis/metabolismo , Psoriasis/genética , Psoriasis/metabolismo , Queratinocitos/metabolismo , Proliferación Celular/genéticaRESUMEN
Antibody or antibody-like protein drugs related to tumor immunotherapy are now widely used. Here, we describe an antibody-fusion protein drug IMAB362-mIL-21 with mouse IL-21 (mIL-21) fused into the C-terminal domain of IMAB362 (a clinical antibody drug against Claudin18.2), that we expect can achieve tumor targeting and activate local anti-tumor immune response more effectively, while reducing the systemic side effects of individual cytokines. In vitro assays comparing the fusion protein IMAB362-mIL-21 to IMAB362 and mIL-21, IMAB362-mIL-21 was able to recognize its cognate antigen Claudin18.2 and natural receptor mIL-21R with similar binding affinities, mediate equivalent ADCC activity and activate IL-21R-mediated downstream signal pathway. In in vivo assays, IMAB362-mIL-21 produced stronger anti-tumor effects compared with IMAB362 or mIL-21 or their combination at equimolar concentrations. Moreover, according to routine blood indicators, mIL-21-Fc and the combined treatment group had significant decreases (P < 0.01) in red blood cells (RBC), hemoglobin (HGB) and hematocrit (HCT), while the IMAB362-mIL-21 group did not. The above results have shown that IMAB362-mIL-21 can produce better anti-tumor effects without obvious hematological toxicity, which is sufficient to show that this kind of antibody-cytokine protein has better application value than IMAB362 or IL-21 as single drugs or in combination. Therefore, this bifunctional molecule combined tumor-targeting and immune activation effectively and has good application prospects.
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Neoplasias , Ratones , Animales , Neoplasias/tratamiento farmacológico , Interleucinas , Inmunoterapia , Transducción de Señal , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
Several clinical studies demonstrate that there exist other immune checkpoints overexpressed in some PD-1 inhibitor-resistant tumor patients. Among them, Lymphocyte-activation gene 3 (LAG-3) is one of the important immune checkpoint molecules and has been clinically demonstrated to have synergistic anti-tumor effects in combination with PD-1 antibody. In this study, we designed a novel 'knob-in-hole' PD-1/LAG-3 bispecific antibody (BsAb) YG-003D3. In conclusion, the BsAb maintained the similar affinity and thermal stability to the parental antibody, and the BsAb structure can be independent of each other in the process of double-target recognition, and the recognition activity will not be affected. Moreover, the BsAb can not only target PD-1 and LAG-3 on single cell simultaneously, but also bridge the two kinds of cells expressing PD-1 and LAG-3, so as to release the 'brake system of immune checkpoints' and activate immune cells to exert anti-tumor effects more effectively. Especially in the PBMCs activation assay, YG-003D3 induced stronger IFN-γ, IL-6, and TNF-α secretion compared to anti-PD-1 or anti-LAG-3 single drug group or even combined drug group. In the tumor killing experiment of PBMC in vitro, YG-003D3 has a better ability to activate PBMC to kill tumor cells than anti-PD-1 or anti-LAG-3 single drug group or even combined drug group, and the killing rate is as high as 20%. In a humanized PD-1/LAG-3 transgenic mouse subcutaneous tumor-bearing model, YG-003D3 showed good anti-tumor activity, even better than that of the combination group at the same molar concentration. Further studies have shown that YG-003D3 could significantly alter the proportion of immune cells in the tumor microenvironment. In particular, the proportion of CD45+, CD3+ T, CD8+ T cells in tumor tissue and the proportion of CD3+ T, CD8+ T, CD4+ T cells in peripheral blood were significantly increased. These results suggest that YG-003D3 exerts a potent antitumor effect by activating the body 's immune system. In summary, the BsAb YG-003D3 has good anti-tumor activity, which is expected to become a novel drug candidate for cancer immunotherapy.
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Anticuerpos Biespecíficos , Neoplasias , Ratones , Animales , Linfocitos T CD8-positivos , Leucocitos Mononucleares , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Activación de Linfocitos , Inmunoterapia , Neoplasias/tratamiento farmacológico , Microambiente TumoralRESUMEN
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.
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Sistemas CRISPR-Cas , Edición Génica , Animales , Células CHO , Sistemas CRISPR-Cas/genética , Cricetinae , Cricetulus , Reparación del ADN por Unión de Extremidades/genética , ADN Ligasa (ATP)/genéticaRESUMEN
Sialic acid-binding Ig-like lectin-15 is an important immunosuppressive molecule considered to be a key target in next-generation tumor immunotherapy. In this study, we screened 22 high-affinity antibodies that specifically recognize human Siglec-15 by using a large human phage antibody library, and five representative sequences were selected for further study. The results showed the binding activity of five antibodies to Siglec-15 (EC50 ranged from 0.02368 µg/mL to 0.07949 µg/mL), and in two Siglec-15-overexpressed cell lines, three antibodies had the strongest binding activity, so the two clones were discarded for further study. Subsequently, the affinity of three antibodies were measured by bio-layer interferometry technology (5-9 × 10E-09M). As the reported ligands of Siglec-15, the binding activity of Siglec-15 and sialyl-Tn, cluster of differentiation 44, myelin-associated glycoprotein, and leucine-rich repeat-containing protein 4C can be blocked by three of the antibodies. Among these, 3F1 had a competitive advantage. Then, the antibody 3F1 showed an obvious antibody-dependent cell-mediated cytotoxicity effect (EC50 was 0.85 µg/mL). Further, antibody 3F1 can reverse the inhibitory effect of Siglec-15 on lymphocyte proliferation (especially CD4+T and CD8+T) and cytokine release Interferon-γ. Given the above results, 3F1 was selected as a candidate for the in vivo pharmacodynamics study. In the tumor model of Balb/c Nude mice, 3F1 (10 mg/kg) showed certain antitumor effects [tumor growth inhibition (TGI) was 31.5%], while the combination of 3F1 (5 mg/kg) and Erbitux (5 mg/kg) showed significant antitumor effects (TGI was 48.7%) compared with the PBS group. In conclusion, novel human antibody 3F1 has antitumor activity and is expected to be an innovative candidate drug targeting Siglec-15 for tumor immunotherapy. SIGNIFICANCE STATEMENT: Siglec-15 is considered as an important target in the next generation of tumor immunotherapy. 3F1 is expected to be the most promising potential candidate for targeting Siglec-15 for cancer treatment and could provide a reference for the development of antitumor drugs.
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Antígenos CD , Neoplasias , Animales , Antígenos CD/metabolismo , Humanos , Inmunoglobulinas , Lectinas/química , Lectinas/metabolismo , Ligandos , Proteínas de la Membrana , Ratones , Ratones Desnudos , Neoplasias/tratamiento farmacológicoRESUMEN
The continuous mutation of SARS-CoV-2 has presented enormous challenges to global pandemic prevention and control. Recent studies have shown evidence that the genome sequence of SARS-CoV-2 nucleocapsid proteins is relatively conserved, and their biological functions are being confirmed. There is increasing evidence that the N protein will not only provide a specific diagnostic marker but also become an effective treatment target. In this study, 2G4, which specifically recognizes the N protein, was identified by screening a human phage display library. Based on the computer-guided homology modelling and molecular docking method used, the 3-D structures for the 2G4 scFv fragment (VH-linker-VL structure, with (G4S)3 as the linker peptide in the model), SARS-CoV-2 N protein and its complex were modelled and optimized with a suitable force field. The binding mode and key residues of the 2G4 and N protein interaction were predicted, and three mutant antibodies (named 2G4-M1, 2G4-M2 and 2G4-M3) with higher affinity were designed theoretically. Using directed point mutant technology, the three mutant antibodies were prepared, and their affinity was tested. Their affinity constants of approximately 0.19 nM (2G4-M1), 0.019 nM (2G4-M2) and 0.075 nM (2G4-M3) were at least one order of magnitude lower than that of the parent antibody (3 nM; 2G4, parent antibody), as determined using a biolayer interferometry (BLI) assay. It is expected that high-affinity candidates will be used for diagnosis and even as potential therapeutic drugs for the SARS-CoV-2 pandemic.
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COVID-19 , SARS-CoV-2 , Anticuerpos Monoclonales , Afinidad de Anticuerpos , Técnicas de Visualización de Superficie Celular , Humanos , Simulación del Acoplamiento Molecular , SARS-CoV-2/genéticaRESUMEN
Rationale: White matter repair is critical for the cognitive and neurological functional recovery after ischemic stroke. M2 microglia are well-documented to enhance remyelination and their extracellular vesicles (EVs) mediate cellular function after brain injury. However, whether M2 microglia-derived EVs could promote white matter repair after cerebral ischemia and its underlying mechanism are largely unknown. Methods: EVs were isolated from IL-4 treated microglia (M2-EVs) and untreated microglia (M0-EVs). Adult ICR mice subjected to 90-minute transient middle cerebral artery occlusion received intravenous EVs treatment for seven consecutive days. Brain atrophy volume, neurobehavioral tests were examined within 28 days following ischemia. Immunohistochemistry, myelin transmission electron microscope and compound action potential measurement were performed to assess white matter structural remodeling, functional repair and oligodendrogenesis. The effects of M2-EVs on oligodendrocyte precursor cells (OPCs) were also examined in vitro. EVs' miRNA sequencing, specific miR-23a-5p knockdown in M2-EVs and luciferase reporter assay were used to explore the underlying mechanism. Results: M2-EVs reduced brain atrophy volume, promoted functional recovery, oligodendrogenesis and white matter repair in vivo, increased OPC proliferation, survival and differentiation in vitro. miR-23a-5p was enriched in M2-EVs and could promote OPC proliferation, survival and maturation, while knocking down miR-23a-5p in M2-EVs reversed the beneficial effects of M2-EVs both in vitro and in vivo. Luciferase reporter assay showed that miR-23a-5p directly targeted Olig3. Conclusion: Our results demonstrated that M2 microglia could communicate to OPCs through M2-EVs and promote white matter repair via miR-23a-5p possibly by directly targeting Olig3 after ischemic stroke, suggesting M2-EVs is a novel and promising therapeutic strategy for white matter repair in stroke and demyelinating disease.
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Isquemia Encefálica , Vesículas Extracelulares , Accidente Cerebrovascular Isquémico , MicroARNs , Sustancia Blanca , Animales , Atrofia/patología , Isquemia Encefálica/patología , Vesículas Extracelulares/patología , Ratones , Ratones Endogámicos ICR , MicroARNs/farmacología , Microglía , Sustancia Blanca/patologíaRESUMEN
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.