Fc Protein Research Articles

Crystal structure and mutational analysis of the human TRIM7 B30.2 domain provide insights into the molecular basis of its binding to glycogenin-1

Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel β-sheets of seven and six strands, arranged as a distorted β-sandwich. Furthermore, two long loops partially cover the concave face of the β-sandwich defined by the β-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.

Lack of evidence for expression and function of IL-39 in human immune cells.

Members of the IL-6/IL-12 cytokine family are critical regulators of innate and adaptive immunity and have emerged as key players controlling inflammatory and autoimmune disorders. This cytokine family comprises of IL-12, IL-23, IL-27, and IL-35, each consisting of distinct α- and β-cytokine subunits that form heterodimers. A new member of this family, IL-39, was identified in the murine species and was shown to consist of the IL-23p19 and Epstein-Barr Virus-induced 3 (EBI3) subunits. Subsequently, it was shown that IL-39 was implicated in the immunopathogenesis of murine experimental lupus erythematosus. The existence of IL-39 in the human system has yet to be confirmed. Based on the clinical success of IL-23p19 neutralizing approaches in moderate-to-severe psoriasis, anti-IL-23p19 antibodies in the clinic may not only neutralize IL-23, but additionally IL-39, implying that IL-39 might also contribute to the pathogenesis of psoriasis. It is therefore pivotal to demonstrate IL-39 expression and to characterize its function in the human system. In this study, we provided evidence for the existence of secreted heterodimeric p19 and EBI3 complexes in supernatants originating from p19 and EBI3 transfected HEK293FT cells. We attempted to detect IL-39 expression from stimulated human primary B cells, human keratinocytes and in vitro polarized human macrophages. Whereas, the expression of p19 and EBI3 mRNA was elevated, we failed to detect p19 and EBI3 heterodimers. Functional assays were conducted with conditioned media containing human IL-39 or with a human recombinant IL-39 Fc protein. Immune cells targeted by IL-39 in mouse, such as neutrophils and PBMCs, did not respond to human IL-39 stimulation and IL-39 failed to activate STAT3 in a reporter cell line. These results suggest that, while the secretion of p19/EBI3 complexes can be forced in human cells, it is secreted below the lower quantity of detection or it has no functional role.

The effects of ephrinB2 signaling on proliferation and invasion in glioblastoma multiforme.

The aggressive nature of glioblastoma multiforme (GBM) may be attributed to the dysregulation of pathways driving both proliferation and invasion. EphrinB2, a membrane-bound ligand for some of the Eph receptors, has emerged as a critical target regulating these pathways. In this study, we investigated the role of ephrinB2 in regulating proliferation and invasion in GBM using intracranial and subcutaneous xenograft models. The Cancer Genome Atlas analysis suggested high transcript and low methylation levels of ephrinB2 as poor prognostic indicators in GBM, consistent with its role as an oncogene. EphrinB2 knockdown, however, increased tumor growth, an effect that was reversed by ephrinB2 Fc protein. This was associated with EphB4 receptor activation, consistent with the data showing a significant decrease in tumor growth with ephrinB2 overexpression. Mechanistic analyses showed that ephrinB2 knockdown has anti-invasive but pro-proliferative effects in GBM. EphB4 stimulation following ephrinB2 Fc treatment in ephrinB2 knockdown tumors was shown to impart strong anti-proliferative and anti-invasive effects, which correlated with decrease in PCNA, p-ERK, vimentin, Snail, Fak, and increase in the E-cadherin levels. Overall, our study suggests that ephrinB2 cannot be used as a sole therapeutic target. Concomitant inhibition of ephrinB2 signaling with EphB4 activation is required to achieve maximal therapeutic benefit in GBM.

P235 Serological biomarkers of interstitial matrix and basement membrane remodelling correlate the Modified Mayo Endoscopic Score (mMES) for ulcerative colitis

Abstract Background Endoscopy is a mainstay in ulcerative colitis (UC) for disease activity monitoring. The modified Mayo Endoscopic Score (mMES) for UC takes into account disease extent and hereby additional objectivity can be achieved for endoscopic assessment. However, endoscopic evaluation of the intestinal mucosa is time-consuming and inconvenient for the patients. Therefore, surrogate biomarkers for endoscopic examinations are warranted. The aim of this study was to evaluate serological neo-epitope biomarkers of type IV collagen degradation (C4M) and formation (PRO-C4), type III collagen degradation (C3M), type V collagen formation (PRO-C5) and macrophage activity (VICM) and their association with the mMES for UC. Methods Endoscopic disease activity was scored using mMES for UC patients (n = 63). Patients were stratified according to the disease activity severity based on mMES (remission = 0–1, mild = 2–4, moderate = 5–6, severe >6). ELISA was applied for biomarker measurements (C4M, PRO-C4, C3M, PRO-C5 and VICM) in serum. Pearson correlations and one-way-ANOVA with FDR correction were applied for statistical analysis. A multivariate regression model was developed in order to combine the ECM biomarkers as a composite index. This model was correlated with the mMES and compared with FC and C-reactive protein (CRP) levels. Results All the biomarkers correlated to mMES (VICM: r = 0.35, p = 0.0063; PRO-C5: r = 0.33, p = 0.009; C3M: r = 0.32, p = 0.011; C4M: r = 0.28, p = 0.0255; PRO-C4: r = 0.25, p = 0.044). Stratification of the patients revealed that VICM, C3M, and C4M serum levels were significantly elevated in UC patients with severe mMES compared with patients in remission (VICM: p = 0.0158, C3M: p = 0.04), mild disease activity (VICM: p = 0.0002, C3M: p = 0.0049, C4M: p = 0.0084) and moderate disease activity (VICM: p = 0.0166). Biomarkers that correlated significantly with the mMES were included in the multivariate regression model as a composite biomarker index. The biomarker model correlated significantly with mMES (r = 0.45, p = 0.0002) (Figure 1A) and the model was also able to discriminate between the different stages of disease activity (p = 0.0007) (Figure 1D). FC correlated with mMES (r = 0.48, p = 0.0001) (Figure 1B and E), however CRP did not correlate with mMES (Figure 1C and F). Conclusion The combined tissue-remodelling biomarkers correlated significantly with mMES for UC. The ECM serological biomarkers performed better than CRP, and equally good as FC. Thus, serological biomarkers of ECM remodelling, especially basement membrane degradation, interstitial matrix degradation and macrophage activity biomarkers may be used as surrogate markers for endoscopic disease activity assessment for UC.

Rational design of transient gene expression process with lipoplexes for high-level therapeutic protein production in HEK293 cells

Abstract Transient gene expression, an attractive technology that enables the rapid production of recombinant proteins for the development of biopharmaceutical candidates at an early stage, offers an interesting alternative to the time-consuming generation of stable recombinant cell lines. In this study, a highly efficient transient gene expression system was established by optimizing significant factors; namely, the host cells, lipid-based transfection reagent, enhancer, and expression vector. HEK293E and Expi293 F cells were evaluated to determine the best host cells to use, and the cell passage number and initial cell density were optimized for high transient gene expression. Cationic liposomes were selected as the vehicle for plasmid DNA delivery and optimized with respect to the co-lipid complex and DNA-to-lipid ratio. Among the cationic lipids, DC-Chol/DOPE gave a more than 2-fold increase in production yield. Following gene delivery into cells, butyric acid (a histone deacetylase inhibitor) supplementation increased protein production by a maximum of 4-folds through transcriptional regulation. Additionally, an expression vector bearing the E77 element at the 3′ end of the polyadenylation signal sequence further enhanced the human-Fc and bevacizumab yields by 7% and 15%, respectively. Finally, our novel combination method was successfully applied to human IgG and Fc protein production using Expi293 F cells.

Effect of allotypic variation of human IgG1 on the thermal stability of disulfide-linked knobs-into-holes mutants of the Fc for stable bispecific antibody design.

BackgroundDisulfide-linked knobs-into-holes (dKiH) mutation is a well-validated antibody engineering technique to force heterodimer formation of different Fcs for efficient production of bispecific antibodies. An artificial disulfide bond is created between mutated cysteine residues in CH3 domain of human IgG1 Fc whose positions are 354 of the “knob” and 349 of the “hole” heavy chains. The disulfide bond is located adjacent to the exposed loop with allotypic variations at positions 356 and 358. Effects of the variation on the biophysical property of the Fc protein with dKiH mutations have not been reported.MethodsWe produced dKiH Fc proteins of high purity by affinity-tag fusion to the hole chain and IdeS treatment, which enabled removal of mispaired side products. Thermal stability was analyzed in a differential scanning calorimetry instrument.ResultsWe firstly analyzed the effect of the difference in allotypes of the Fcs on the thermal stability of the heterodimeric Fc. We observed different melting profiles of the two allotypes (G1m1 and nG1m1) showing slightly higher melting temperature of G1m1 than nG1m1. Additionally, we showed different characteristics among heterodimers with different combinations of the allotypes in knob and hole chains.ConclusionAllotypic variations affected melting profiles of dKiH Fc proteins possibly with larger contribution of variations adjacent to the disulfide linkage.

Abstract 2248: Generation and formatting of a bispecific Affimer® biotherapeutic for the inhibition of the LAG-3 and PD-L1 pathway

Abstract Programmed Cell Death-Ligand 1 (PD-L1) binds to its receptor, Programmed Cell Death protein (PD-1) and is expressed on both a range of activated immune cells and tumor cells. Lymphocyte Activation Gene-3 (LAG-3) is a member of the Ig superfamily and is expressed on activated T cells, NK cells, DCs and B cells. Both the PD-1/PD-L1 and the LAG-3 pathway are involved in immune suppression, leading to the down-regulation of T cell activity and the generation of exhausted T cells, allowing tumor escape and metastasis. Despite the impressive success of PD-L1 targeted immunotherapy, there remains a large proportion of patients with many tumor types that fail to benefit or acquire resistance during therapy. Consequently, it is necessary to consider future combinations of immunotherapies to enhance the clinical outcomes and reduce tumor escape. LAG-3 is upregulated on T-cells following PD-1/PD-L1 pathway blockade, indicating that targeting of both pathways as a combination may lead to more durable antitumor responses. To reach the full potential of LAG-3/PD-L1 dual blockade the development of a bispecific immunotherapy may further augment T-cell activation. Affimer biotherapeutics are a new protein scaffold with great potential for the generation of biotherapeutics. Based on the human protease inhibitor Stefin A, the scaffold is small (14kDa), lacks any post-translational modifications such as disulfide bonds and expresses at high levels. Affimers against a target are generated through the use of proprietary, highly diverse phage display libraries that have been created by engineering in two nine amino acid peptide loops into the scaffold backbone. We have identified a range of potent Affimer biotherapeutic antagonists to both human PD-L1 and LAG-3. When formatted as a bispecific Fc fusion to both the N and C-terminal of a human IgG1 Fc protein the Affimers demonstrate pM to nM binding affinities and binding to both target antigens simultaneously without compromising the affinities. The bispecific Fc fusion has been shown to block the interaction of LAG-3 with its ligand, MHC class II and PD-L1 with both PD-1 and CD80 by competitive ELISA. Functionally, the LAG-3/PD-L1 bispecific enhanced T-cell activation in vitro in both SEB-driven and mixed lymphocyte reaction assays. The Fc fusion format expresses over 500 mg/L from Expi293F mammalian transient transfections and can be purified to over 95% purity. The pharmacokinetic (PK) properties showed half-life extension of the Fc fusion format in mouse by using the IgG Fc-FcRn recycling pathway, dosing at 10 mg/kg resulted in a half-life extension of over 90 hours. We have demonstrated that the Affimer scaffold protein has all the necessary attributes to be developed as a bispecific LAG-3/PD-L1 immunotherapy. The Affimer scaffold was formatted as a bispecific Fc fusion molecule with ease, had good biophysical properties and shown to be well tolerated in vivo. Citation Format: Amrik Basran, Emma Jenkins, Estelle Adam, Michele Writer, Floriane Laurent, Assa Oumie, Jyrki Sivula, Jennifer Hillman, Lisa Strong, Maureen West, Emma Stanley. Generation and formatting of a bispecific Affimer® biotherapeutic for the inhibition of the LAG-3 and PD-L1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2248.

Characterization of intact glycopeptides reveals the impact of culture media on site-specific glycosylation of EPO-Fc fusion protein generated by CHO-GS cells.

With the increasing demand to provide more detailed quality attributes, more sophisticated glycan analysis tools are highly desirable for biopharmaceutical manufacturing. Here, we performed an intact glycopeptide analysis method to simultaneously analyze the site-specific N- and O-glycan profiles of the recombinant erythropoietin Fc (EPO-Fc) protein secreted from a Chinese hamster ovary glutamine synthetase stable cell line and compared the effects of two commercial culture media, EX-CELL (EX) and immediate advantage (IA) media, on the glycosylation profile of the target protein. EPO-Fc, containing the Fc region of immunoglobulin G1 (IgG1) fused to EPO, was harvested at Day 5 and 8 of a batch cell culture process followed by purification and N- and O-glycopeptide profiling. A mixed anion exchange chromatographic column was implemented to capture and enrich N-linked glycopeptides. Using intact glycopeptide characterization, the EPO-Fc was observed to maintain their individual EPO and Fc N-glycan characteristics in which the EPO region presented bi-, tri-, and tetra-branched N-glycan structures, while the Fc N-glycan displayed mostly biantennary glycans. EPO-Fc protein generated in EX medium produced more complex tetra-antennary N-glycans at each of the three EPO N-sites while IA medium resulted in a greater fraction of bi- and tri-antennary N-glycans at these same sites. Interestingly, the sialylation content decreased from sites 1-4 in both media while the fucosylation progressively increased with a maximum at the final IgG Fc site. Moreover, we observed that low amounts of Neu5Gc were detected and the content increased at the later sampling time in both EX and IA media. For O-glycopeptides, both media produced predominantly three structures, N1F1F0SOG0, N1H1F0S1G0, and N1H1F0S2G0, with lesser amounts of other structures. This intact glycopeptide method can decipher site-specific glycosylation profile and provide a more detailed characterization of N- and O-glycans present for enhanced understanding of the key product quality attributes such as media on recombinant proteins of biotechnology interest.

Facile and Efficient Chemoenzymatic Semisynthesis of Fc-Fusion Compounds for Half-Life Extension of Pharmaceutical Components.

The formation of Fc-fusions, in which biologically active molecules and the Fc fragment of antibodies are linked to each other, is one of the most efficient and successful half-life extension technologies to be developed and applied to peptide and protein pharmaceuticals thus far. Fc-fusion compounds are generally produced by recombinant methods. However, these cannot be applied to artificial middle molecules, such as peptides with non-natural amino acids, unnatural cyclic peptides, or pharmaceutical oligonucleotides. Here, we developed a simple, efficient, semisynthetic method for Fc-fusion production involving our previously developed enzymatic N-terminal extension reaction (i.e., NEXT-A reaction) and strain-promoted azide-alkyne cycloaddition, achieving quantitative conversion and high selectivity for the N-terminus of the Fc protein. An Fc-fusion compound prepared by this method showed comparable biological activity to that of the original peptide and a long-circulating plasma half-life. Thus, the proposed method is potentially applicable for the conjugation of a wide range of pharmaceutical components.

Transcriptomic Analysis Reveals Erythroferrone Modulates BMP6 Signaling Pathways Involved in Iron Homeostasis and Metabolism

Introduction: Erythroferrone (ERFE), the recently identified erythroid regulator of iron absorption, is a member of the C1q/TNF-related protein (CTRP) family. It is produced in erythroblasts in response to an increased erythropoietic drive, downregulates hepcidin expression and thereby promotes intestinal iron absorption and mobilization. The levels of Erfe are inappropriately high under conditions of ineffective erythropoiesis in inherited anemias such as thalassemia and congenital dyserythropoietic anemias. However, the mechanism of Erfe mediated Hepcidin inhibition remains unknown. Here, we use transcriptomic analysis of the human hepatoma cell line, Huh7 to identify genes and pathways involved in hepatocyte response to Erfe.Methods: Huh7 cells were seeded in a 6-well cell culture plate. Twenty four hours later, the cells were washed with PBS, and treated with a recombinant murine ERFE monomeric Fc (mEFRE-FC, 10µg/ml) protein or a control IgG (10 ug/ml) for 1h, 6h or 24h in growth media. RNA was isolated, followed by RNA quantification and quality assessment using a 2100 Agilent Bioanalyzer. ERFE is known to regulate Hepcidin antimicrobial peptide (HAMP) transcription and a treatment effect on HAMP expression was demonstrated by qPCR prior to sequencing. A total of 27 mRNA sequencing libraries were constructed from 1ug of human total RNA with the Illumina TruSeq Stranded mRNA Sample Prep protocol and single-end 75 bp reads were generated on an Illumina NextSeq 500. DESeq2 statistical package was used for differential expression testing. Multiple comparisons were adjusted for using a false discovery rate of 5%. Additionally, results were filtered to consider only those genes that demonstrated a fold-change point estimate >2 in either direction. Pathway analysis was performed using the Biological Process gene sets from the Gene Ontology annotation.Results: Compared to control treated Huh7 cells, 3 transcripts were differentially regulated following a one hour treatment with mERFE-Fc, ID1 (Log2 fold-change -1.55, q-value 4.99E-09), BPIFB2 (Log2 fold-change -1.88, q-value 0.036) and ANO1 (Log2 fold-change -1.43, q-value 0.019). A larger number of genes were differentially regulated with longer treatments, 32 genes at 6h and 828 genes at 24h. Among selected genes differentially expressed at 24h, between control treated and Erfe treated cells, we observed a significant reduction in expression of genes known to be directly upregulated by bone morphogenetic proteins (BMPs), including DNA binding protein inhibitor ID1(ID1), DNA binding protein inhibitor ID2 (ID2), DNA binding protein inhibitor ID3 (ID3) and HAMP. BMP6 has been demonstrated to regulate several biological processes such as iron metabolism in the liver, adipogenesis, and insulin sensitization. Interestingly, we also observed upregulation of SLC27A1 (or FATP1, fatty acid transporter, Log2 fold-change 2.27, q-value 0.003), GDNF (Log2 fold-change 2.57, q-value 2.4E-05), GIPR (Log2 fold-change 2.53, q-value 7.9E-07) upon treatment with Erfe at 24h. Previous work has demonstrated that all of these genes are downregulated by BMP6 treatment 1-3. Pathway analysis indicated a number of genes differentially regulated in the GOBP (gene ontology biological processes) iron ion homeostasis pathway, BMP signaling or TGF-β receptor signaling pathway. This assessment further indicated downregulation of cellular hormone levels and metabolic processes and ion homeostasis as biological processes impacted by erythroferrone.Conclusions: Significant differences in gene expression occur in hepatocytes upon interaction with erythroferrone. Many genes in the iron homeostasis, BMP6 signaling metabolic processes were also differentially regulated. This further supports the mechanism of ERFE on iron homeostasis. DisclosuresJasuja:Pfizer: Employment. Sawant:Pfizer: Employment. Pittman:Pfizer: Employment. Quan:Pfizer: Employment.

Development of a novel affinity chromatography resin for platform purification of bispecific antibodies with modified protein a binding avidity.

There is strong interest in the production of bispecific monoclonal antibodies that can simultaneously bind two distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Regeneron's bispecific technology, based upon a standard IgG, consists of a heterodimer of two different heavy chains, and a common light chain. Coexpression of two heavy chains leads to the formation of two parental IgG impurities, the removal of which is facilitated by a dipeptide substitution in the Fc portion of one of the heavy chains that ablates Fc Protein A binding. Therefore, the affinity capture (Protein A) step of the purification process must perform both bulk capture and high resolution of these mAb impurities, a task current commercially available resins are not designed for. Resolution can be further impaired by the ability of Protein A to bind some antibodies in the variable region of the heavy chain (VH). This article details development of a novel Protein A resin. This resin combines an alkali stable ligand with a base matrix exhibiting excellent mass transfer properties to allow high capacity single step capture and resolution of bispecific antibodies (bsAbs) with high yields. The developed resin, named MabSelect SuRe™ pcc, is implemented in GMP production processes for several bsAbs. © 2018 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:650–658, 2018

Fused IgY Fc and Polysaccharide Adjuvant Enhanced the Immune Effect of the Recombinant VP2 and VP5 Subunits-A Prospect for Improvement of Infectious Bursal Disease Virus Subunit Vaccine.

Infectious bursal disease virus (IBDV) is a highly contagious pathogen that causes damage in lymphoid organs and remains a threat to the poultry industry worldwide. Currently, subunit vaccines based on VP2 antigen expressed in prokaryotic systems are widely used in clinical settings. However, the immunogenicity of VP2 vaccines is limited because of their inherent defect that the structure of the antigen expressed in Escherichia coli (E. coli) may be different from its natural conformation. In this study, we fused VP2 and VP5 protective antigen genes and linked the chicken IgY Fc gene onto it. The eukaryotic expression plasmid carrying the fusion gene was transformed into Pichia pastoris (P. pastoris) to express the recombinant VP2–VP5–Fc protein. The recombinant protein was used as immunogen for evaluating immune response, and the recombinant VP2–Fc and VP2 proteins expressed in P. pastoris and the commercial VP2 subunit vaccines were used as controls. Moreover, Taishan Pinus massoniana pollen polysaccharide (TPPPS), an immunomodulator found by our laboratory, was used as adjuvant to investigate its immune modulatory effects on immunogens. Chickens were divided into six groups and inoculated with VP2–VP5–Fc+TPPPS, VP2–VP5–Fc, VP2–Fc, VP2 vaccine, commercial VP2 subunit vaccine, and phosphate buffered saline (PBS). The recombinant VP2 subunit vaccine expressed in P. pastoris exhibited higher immunogenicity than the commercial VP2 subunit vaccine. The VP2–Fc protein showed a better effect than the VP2 protein, and the VP2–VP5–Fc subunit further improved the immune effects. In addition, TPPPS was proved to be a good immunopotentiator for the VP2–VP5–Fc subunit vaccine. Hence, the recombinant VP2–VP5–Fc subunit combined with TPPPS adjuvant exhibits potential as efficient IBDV vaccine to prevent infectious bursal disease.

Engineered cross-reacting nanobodies simplify comparative oncology between humans and dogs.

Antibodies cross-reacting with homologue antigens in different species would be essential reagents for the development of comparative oncology studies. In comparison with conventional immunoglobulin Gs, recombinant nanobodies (single-domain variable regions of heavy-chain only antibodies of Camelidae origin) can be easily isolated in vitro and engineered into a variety of reagents with optimized characteristics for different research and clinical applications. We recovered an anti-human epidermal growth factor receptor 2 (anti-HER2) nanobody from a naïve llama library by direct panning on whole cells and expressed it fused to Fc and green fluorescent protein. These immunoreagents were assessed by flow cytometry and immunofluorescence with both human and canine cells overexpressing HER2 and its canine homologue dog epidermal growth factor receptor 2. The reported data illustrate the potential of using this class of antibodies in comparative oncology and suggest some development perspectives enabled by in vitro panning of pre-immune nanobody libraries.

Interaction between Galectin-9/TIM-3 pathway and follicular helper CD4+ T cells contributes to viral persistence in chronic hepatitis C

Both Galectin 9 (Gal-9)/T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) pathway and follicular helper CD4+ T (Tfh) cells play important roles in persistent hepatitis C virus (HCV) infection. Thus, we aimed to investigate the regulatory role of interaction between Gal-9/TIM-3 pathway and Tfh cells in chronic hepatitis C. A total of 44 chronic hepatitis C patients and 19 normal controls (NCs) were enrolled in this study. Purified CD4+ T cells were cultured by TIM-3 Fc protein, recombinant Gal-9, or IL-21 for 48h. TIM-3 expression, Tfh proportion, and IL-21 production was measured, respectively. The immunomodulatory role of Gal-9/TIM-3 and IL-21 was also investigated in HCV cell culture system in vitro. We found that the percentage corresponding to both TIM-3-positive and CXCR5+ICOS+ Tfh cells within CD4+ T cells, which correlated with HCV RNA replication, was significantly elevated in patients with chronic hepatitis C in comparison with those in NCs. Moreover, blockade of Gal-9/TIM-3 pathway by TIM-3 Fc protein increased Tfh cells proportion, IL-21 mRNA and protein expression within purified CD4+ T cells, while activation of Gal-9/TIM-3 signaling by Gal-9 stimulation decreased IL-21 production in both patients with chronic HCV infection and healthy individuals. Meanwhile, high concentrations (100 and 200ng/mL) of IL-21 stimulation also elevated TIM-3 expression on CD4+ T cells in chronic hepatitis C. Furthermore, TIM-3 blockage and IL-21 stimulation suppressed mRNA expressions of HCV-induced antiviral proteins (myxovirus resistance A and oligoadenylate synthetase) in Huh7.5 cells without affecting viral replication in HCV cell culture system. The interaction between Gal-9/TIM-3 pathway and Tfh cells contributed to viral persistent in chronic HCV infection, which might be pivotal for development of new therapeutic approaches for chronic hepatitis C.

Improving Antibody-Based Cancer Therapeutics Through Glycan Engineering.

Antibody-based therapeutics has emerged as a major tool in cancer treatment. Guided by the superb specificity of the antibody variable domain, it allows the precise targeting of tumour markers. Recently, eliciting cellular effector functions, mediated by the Fc domain, has gained traction as a means by which to generate more potent antibody therapeutics. Extensive mutagenesis studies of the Fc protein backbone has enabled the generation of Fc variants that more optimally engage the Fcγ receptors known to mediate cellular effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and cellular phagocytosis. In addition to the protein backbone, the homodimeric Fc domain contains two opposing N-linked glycans, which represent a further point of potential immunomodulation, independent of the Fc protein backbone. For example, a lack of core fucose usually attached to the IgG Fc glycan leads to enhanced ADCC activity, whereas a high level of terminal sialylation is associated with reduced inflammation. Significant growth in knowledge of Fc glycosylation over the last decade, combined with advancement in genetic engineering, has empowered glyco-engineering to fine-tune antibody therapeutics. This has culminated in the approval of two glyco-engineered antibodies for cancer therapy: the anti-CCR4 mogamulizumab approved in 2012 and the anti-CD20 obinutuzumab in 2013. We discuss here the technological platforms for antibody glyco-engineering and review the current clinical landscape of glyco-engineered antibodies.

The role of interferon lambda during influenza, Staphylococcus aureus super-infection.

Abstract While pandemic influenza A H1N1 infection causes significant morbidity and mortality on its own, it also renders an individual more susceptible to secondary infection with bacterial pathogens such as Staphylococcus aureus. We have previously shown that type I interferons produced by influenza impair bacterial defense six to seven days post-influenza infection. However, the role of the predominant type III interferons (interferon lambda/IFNL, IL-28A/B) in bacterial super-infection is less clear. Thus, we explored the role of IFNL in influenza, S. aureus super-infection. First, we examined the impact of attenuating IFNL signaling directly before bacterial challenge. We found that treatment with the IFNLR1 Fc protein significantly increased bacterial burden in the lung compared with controls. However, when we challenged mice with super-infection that have constitutively attenuated IFNL, we found reduced bacterial burden compared to controls, which was significantly correlated with the degree of IFNL expression. While cellular inflammation was not altered, preliminary results suggest that higher bacterial burden is significantly correlated with higher weight loss, suggesting that mice with constitutively attenuated IFNL are protected from both influenza infection and bacterial super-infection. It has been published that mice lacking IFNL receptor 1 have lower bacterial burden during super-infection, which is corroborated by data from our constitutively attenuated IFNL mice. However, attenuation of IFNL signaling directly prior to bacterial challenge increases bacterial burden, suggesting that the timing of IFNL production regulates susceptibility to secondary bacterial infection.

Soluble vimentin binds to P‐selectin and attenuates leukocytic infiltration into the lungs in murine endotoxin induced acute lung injury

BackgroundAcute lung injury (ALI) is a devastating condition that culminates as inflammation of the lungs mediated by leukocytic infiltration. In animal models, platelets and platelet P‐selectin are well described mediators of ALI and other forms of inflammation through enhancing capture and transmigration of neutrophils across vascular endothelium. Vimentin is an intermediate filament protein mainly thought to be involved in maintaining the cytoskeleton. Recently, there has been increasing evidence of vimentin's role during inflammation, although its precise role is unclear. We had recently observed that recombinant soluble vimentin (sVim) blocks neutrophil adhesion to platelets, endothelial cells, and P‐selectin monolayers; whether these observations translate into attenuation of ALI is unknown. Therefore, we hypothesize that soluble vimentin sVim blocks P‐selectin‐PSGL‐1 interactions and will decrease leukocytic infiltration into the lungs of endotoxin‐exposed mice.MethodsAll experiments were approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine and the Michael E. DeBakey Veterans Affairs Medical Center. Recombinant sVim was expressed in E. coli and His‐purified using affinity chromatography. Binding kinetics of sVim to P‐selectin was determined using both enzyme‐linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). ELISA was performed by detecting sVim binding to immobilized P‐selectin/Fc or PSGL‐1/Fc protein. In SPR (BIACore), sVim was perfused over immobilized P‐selectin/Fc or IgG protein to determine the equilibrium dissociation constant KD. To determine whether sVim inhibits leukocytic infiltration into lungs after endotoxin exposure, C57BL/6 mice were treated with intraperitoneal injections of sVim (3 mg/kg) or vehicle control followed by endotoxin (O111:B4; 15 mg/kg) 1 hour later and then euthanized at 96 hours. Mice were evaluated by blinded investigators for both physical activity and degree of lung inflammation. Activity was determined using the mouse clinical assessment score for sepsis (M‐CASS) and lung inflammation was determined by alveolar septal thickening and leukocytic infiltration on hematoxylin and eosin stained sections.ResultssVim preferentially binds to P‐selectin/Fc and but not PSGL‐1/Fc in ELISA. Using SPR, sVim binds to P‐selectin/Fc with a KD of 125 ± 1.2 nM and not to IgG control. Endotoxin‐exposed mice treated with sVim were more active as determined by the M‐CASS (1.9 ± 0.2 vs 2.5 ± 0.2; 4‐point scale where lower is better; n=10/grp, p<0.05). Mice receiving sVim also had less alveolar septal thickening and leukocytic infiltration into the lungs after exposure to endotoxin.ConclusionSoluble vimentin binds to P‐selectin and not P‐selectin glycoprotein ligand‐1 and decreases leukocytic infiltration into the lungs of endotoxin‐exposed mice. Further studies need to take place to determine whether vimentin also improves gas exchange in the lungs. Finally, dose‐response studies need to be performed to determine the optimal dose and timing of sVim to improve outcomes in acute lung injury.Support or Funding InformationNIH/NHLBI HL‐116524, NIH/NIGMS GM‐112806

Decoy receptor 3 analogous supplement protects steatotic rat liver from ischemia–reperfusion injury

BackgroundFor steatotic livers, pharmacological approaches to minimize the hepatic neutrophil and macrophage infiltration, and cytokine and chemokine release in ischemia–reperfusion (IR) injury are still limited. Tumor necrosis factor (TNF)-α superfamily-stimulated pathogenic cascades and M1 macrophage/Kupffer cells (KC) polarization from Th1 cytokines are important in the pathogenesis of IR liver injury with hepatic steatosis (HS). Conversely, anti-inflammatory M2 macrophages produce Th2 cytokine (interleukin-4), which reciprocally enhances M2 polarization. Toll-like receptor 4-activated KCs can release proinflammatory mediators, skew M1 polarization and escalate liver IR injury. Decoy receptor 3 (DcR3) could be potential agents simultaneously blocking the IR liver injury-related pathogenic changes and extend the survival of steatotic graft. MethodsRats were fed with methionine and choline-deficient high-fat diet (MCD HFD) for 6 weeks to induce HS. Preliminary experiments with HS group and IR group were conducted, and either immunoglobulin G Fc protein or DcR3 analogue was treated for 14 days in all groups to evaluate the severity. In the Zucker rat-focused experiments, various serum and hepatic substances, M1 polarization, and hepatic microcirculation were assessed. ResultsWe found that serum/hepatic DcR3 levels were lower in nonalcoholic fatty liver disease patients with HS. DcR3a protected Zucker rats with HS from IR liver injury. The beneficial effects of DcR3a supplement were mediated by inhibiting hepatic M1 polarization of KCs, decreasing serum/hepatic TNFα, nitric oxide, nitrotyrosine, soluble TNF-like cytokine 1A, Fas ligand, and interferon-γ levels, neutrophil infiltration, and improving hepatic microcirculatory failure among rats with IR-injured steatotic livers. Additionally, downregulated hepatic TNF-like cytokine 1A/Fas-ligand and toll-like receptor 4/nuclear factor-κB signals were found to mediate the DcR3a-related protective effects of steatotic livers from IR injury. ConclusionUsing multimodal in vivo and in vitro approaches, we found that DcR3a analogue was a potential agent to protect steatotic liver against IR injury by simultaneous blockade of the multiple IR injury-related pathogenic changes.

Expression of a Human Prostatic Acid Phosphatase (PAP)-IgM Fc Fusion Protein in Plants Using In vitro Tissue Subculture.

In this study, prostatic acid phosphatase (PAP), which is overexpressed in human prostate cancer cells, was cloned to be fused to the IgM constant fragment (Fc) for enhancing immunogenicity and expressed in transgenic tobacco plants. Then, the transgenic plants were propagated by in vitro tissue subculture. Gene insertion and expression of the recombinant PAP-IgM Fc fusion protein were confirmed in each tested the first, second, and third subculture generations (SG1, SG2, and SG3, respectively). Transcription levels were constantly maintained in the SG1, SG2, and SG3 leaf section (top, middle, and base). The presence of the PAP-IgM Fc gene was also confirmed in each leaf section in all tested subculture generations. RNA expression was confirmed in all subculture generations using real-time PCR and quantitative real-time PCR. PAP-IgM Fc protein expression was confirmed in all leaves of the SG1, SG2, and SG3 recombinant transgenic plants by using quantitative western blotting and chemiluminescence immunoassays. These results demonstrate that the recombinant protein was stably expressed for several generations of in vitro subculture. Therefore, transgenic plants can be propagated using in vitro tissue subculture for the production of recombinant proteins.

CD80 Expressed by CD8+ T Cells Contributes to PD-L1-Induced Apoptosis of Activated CD8+ T Cells.

Tumor cells are capable of limiting antitumor CD8+ T cell responses through their cell surface expression of PD-L1. In addition to PD-1 expressed by CD8+ T cells, PD-L1 also binds to CD80 expressed by CD8+ T cells. The influence of the PD-L1/CD80 interaction on CD8+ T cell function has not been fully characterized, so we sought to investigate the impact of the PD-L1/CD80 interaction on PD-L1-induced apoptosis of activated CD8+ T cells. We found that CD8+ T cells that lacked CD80 expression got activated to the same extent as wild-type CD8+ T cells, but when cultured with anti-CD3 and PD-L1/Fc protein, activated CD8+ T cells that lacked CD80 expression survived better than activated wild-type CD8+ T cells. These findings indicate that PD-L1 induces apoptosis in activated CD8+ T cells in part by signaling through CD80. Thus, in the design and implementation of checkpoint blockade therapies that target PD-L1, it is essential that both binding partners for PD-L1, PD-1, and CD80 are considered.