Fc Fusion Research Articles

Antibody and Antibody Mimetic Immunotherapeutics

Antibody and Antibody Mimetic Immunotherapeutics

High-Affinity Internalizing Human scFv-Fc Antibody for Targeting FGFR1-Overexpressing Lung Cancer.

Targeted delivery of anticancer drugs using antibodies specific for tumor-associated antigens represents one of the most important approaches in current immuno-oncology research. Fibroblast growth factor receptor 1 (FGFR1) has been demonstrated to be a high-frequency targetable oncogene specific for smoking-associated lung cancers, present in over 20% of lung squamous cell carcinoma cases. This report describes the generation of a potent, fully human antibody fragment in scFv-Fc format efficiently targeting FGFR1. Antibody phage display was used to select high-affinity scFv antibody fragments against the extracellular domain of FGFR1(IIIc). Enzyme immunoassay (ELISA) and surface plasmon resonance (SPR) analysis were used for antibody screening and characterization. The best binder (named D2) was cloned to diabody and Fc fusion formats. All D2 antibodies demonstrated high affinity for FGFR1 with dissociation constants of 18 nmol/L (scFvD2), 0.82 nmol/L (scFvD2 diabody), and 0.59 nmol/L (scFvD2-Fc). scFvD2 was found to be exquisitely selective for FGFR1 versus other FGFR family members and bound FGFR1 even in the presence of its natural ligand FGF2, as shown by competitive analysis. Confocal microscopy revealed that scFvD2-Fc was specifically and rapidly internalized by a panel of cell lines overexpressing FGFR1. Finally, it was demonstrated that scFvD2-Fc mediated specific delivery of a cytotoxic payload into lung cancer cells harboring oncogenic FGFR1 gene amplifications.Implications: This study reports a highly specific internalizing antibody fragment that can serve as a therapeutic targeting agent for efficient delivery of cytotoxic drugs into FGFR1-positive lung cancer cells. Mol Cancer Res; 15(8); 1040-50. ©2017 AACR.

Abstract 2646: Intratumoral delivery of TTI-621 (SIRPαFc), a CD47-blocking immunotherapeutic, inhibits tumor growth and prolongs animal survival in a subcutaneous B cell lymphoma model

Abstract Tumor cells often evade macrophage-mediated destruction by increasing cell surface expression of CD47, which delivers an anti-phagocytic (“do-not-eat”) signal by binding the inhibitory signal-regulatory protein α (SIRPα) receptor on macrophages. We have previously shown that blockade of the CD47-SIRPα pathway using TTI-621, a soluble SIRPα-IgG1 Fc fusion protein, triggers macrophage phagocytosis of tumor cells in vitro as well as inhibits tumor growth in vivo when delivered systemically. In the current study, the efficacy of intratumoral delivery of TTI-621 was evaluated in a subcutaneous diffuse large B-cell lymphoma (Toledo) xenograft model. Tumor bearing mice were randomized into treatment groups when tumor volumes reached approximately 120 mm3. Weekly intratumoral administration of TTI-621 at 10, 1 and 0.1 mg/kg dose levels resulted in statistically significant decreases in tumor growth and improved survival relative to vehicle control treatment. Notably, at day 50 post-tumor implantation 100% survival was achieved at the highest dose level (vs. 0% survival with vehicle control treatment). Moreover, weekly intratumoral administration of TTI-621 was efficacious even at a high tumor load setting in which the pre-dose volumes were approximately 300 mm3. Flow cytometry analysis of the dissociated tumor samples demonstrated no significant change in the numbers of M1 and M2 tumor-associated macrophages (TAMs) following intratumoral administration of TTI-621. Nevertheless, TTI-621 dramatically increased the phagocytosis of Toledo cells by both M1 and M2 TAMs to a similar extent ex vivo, suggesting that TTI-621 is efficacious in increasing the phagocytosis of tumor cells by a heterogeneous population of TAMs. Collectively, these results demonstrate that TTI-621 is efficacious when delivered intratumorally and can increase the phagocytosis of tumor cells by both M1 and M2 TAM populations. These data support the evaluation of intratumoral administration of TTI-621 in cancer patients, and a Phase I study of intratumorally delivered TTI-621 in patients with percutaneously accessible solid tumors and mycosis fungoides is ongoing (NCT02890368). Citation Format: Gloria H. Lin, Marilyse Charbonneau, Vien Chai, Alison M. O'Connor, Bolette Bossen, Hui Chen, Mark Wong, Natasja N. Viller, Emma Linderoth, Lisa D. Johnson, Xinli Pang, Jeffery Winston, Penka S. Petrova, Robert A. Uger. Intratumoral delivery of TTI-621 (SIRPαFc), a CD47-blocking immunotherapeutic, inhibits tumor growth and prolongs animal survival in a subcutaneous B cell lymphoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2646. doi:10.1158/1538-7445.AM2017-2646

The structure-function relationship of disulfide bonds in etanercept

Etanercept is a TNFα receptor Fc fusion protein used for the treatment of rheumatic disease and psoriasis. Physicochemical and functional investigation of process fractions during development of the etanercept biosimilar GP2015 (Erelzi®) revealed a correlation between reduced potency and incorrect disulfide bridging between specific cysteines in the receptor domain. This novel structure-function relationship was found to be the molecular basis for reduced potency in recent Enbrel® batches, which exhibit higher levels of incorrect disulfide bridging. Interestingly, incorrect disulfide bridging was found to be reversible under serum-like redox conditions, restoring potency to normal levels. This redox dependent reversibility suggests that these variants are likely not relevant for clinical efficacy once the drug enters the bloodstream. Nonetheless, incorrect disulfide bridging in etanercept represents a new quality attribute that is critical for biopharmaceutical functionality and should thus be carefully monitored and controlled to guarantee patient safety.

Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch

To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHOBRI/rcTA) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHOBRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900mg/L were reproducibly achieved for a Fc fusion protein and up to 350mg/L for an antibody after 14days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1α-HTLV constitutive promoters. These results suggest that the cumate-inducible CHOBRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins.

The structural basis for the functional comparability of factor VIII and the long‐acting variant recombinant factor VIII Fc fusion protein

Background Fusion of the human IgG1 Fc domain to the C-terminal C2 domain of B-domain-deleted (BDD) factor VIII (FVIII) results in the recombinant FVIII Fc (rFVIIIFc) fusion protein, which has a 1.5-fold longer half-life in humans. Objective To assess the structural properties of rFVIIIFc by comparing its constituent FVIII and Fc elements with their respective isolated components, and evaluating their structural independence within rFVIIIFc. Methods rFVIIIFc and its isolated FVIII and Fc components were compared by the use of hydrogen-deuterium exchange mass spectrometry (HDX-MS). The structure of rFVIIIFc was also evaluated by the use of X-ray crystallography, small-angle X-ray scattering (SAXS), and electron microscopy (EM). The degree of steric interference by the appended Fc domain was assessed by EM and surface plasmon resonance (SPR). Results HDX-MS analysis of rFVIIIFc revealed that fusion caused no structural perturbations in FVIII or Fc. The rFVIIIFc crystal structure showed that the FVIII component is indistinguishable from published BDD FVIII structures. The Fc domain was not observed, indicating high mobility. SAXS analysis was consistent with an ensemble of rigid-body models in which the Fc domain exists in a largely extended orientation relative to FVIII. Binding of Fab fragments of anti-C2 domain antibodies to BDD FVIII was visualized by EM, and the affinities of the corresponding intact antibodies for BDD FVIII and rFVIIIFc were comparable by SPR analysis. Conclusions The FVIII and Fc components of rFVIIIFc are structurally indistinguishable from their isolated constituents, and show a high degree of structural independence, consistent with the functional comparability of rFVIIIFc and unmodified FVIII.

Protein Adsorption to In-Line Filters of Intravenous Administration Sets

Ensuring compatibility of administered therapeutic proteins with intravenous administration sets is an important regulatory requirement. A low-dose recovery during administration of low protein concentrations is among the commonly observed incompatibilities, and it is mainly due to adsorption to in-line filters. To better understand this phenomenon, we studied the adsorption of 4 different therapeutic proteins (2 IgG1s, 1 IgG4, and 1 Fc fusion protein) diluted to 0.01 mg/mL in 5% glucose (B. Braun EcoFlac; B. Braun Melsungen AG, Melsungen, Germany) or 0.9% sodium chloride (NaCl; Freeflex; Fresenius Kabi, Friedberg, Germany) solutions to 8 in-line filters (5 positively charged and 3 neutral filters made of different polymers and by different suppliers). The results show certain patterns of protein adsorption, which depend to a large extent on the dilution solution and filter material, and to a much lower extent on the proteins' biophysical properties. Investigation of the filter membranes' zeta potential showed a correlation between the observed adsorption pattern in 5% glucose solution and the filter's surface charge, with higher protein adsorption for the strongly negatively charged membranes. In 0.9% NaCl solution, the surface charges are masked, leading to different adsorption patterns. These results contribute to the general understanding of the protein adsorption to IV infusion filters and allow the design of more efficient compatibility studies.

Optimization-based framework for resin selection strategies in biopharmaceutical purification process development.

This work addresses rapid resin selection for integrated chromatographic separations when conducted as part of a high‐throughput screening exercise during the early stages of purification process development. An optimization‐based decision support framework is proposed to process the data generated from microscale experiments to identify the best resins to maximize key performance metrics for a biopharmaceutical manufacturing process, such as yield and purity. A multiobjective mixed integer nonlinear programming model is developed and solved using the ε‐constraint method. Dinkelbach's algorithm is used to solve the resulting mixed integer linear fractional programming model. The proposed framework is successfully applied to an industrial case study of a process to purify recombinant Fc Fusion protein from low molecular weight and high molecular weight product related impurities, involving two chromatographic steps with eight and three candidate resins for each step, respectively. The computational results show the advantage of the proposed framework in terms of computational efficiency and flexibility. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:1116–1126, 2017

New interleukin-15 superagonist (IL-15SA) significantly enhances graft-versus-tumor activity

Interleukin-15 (IL-15) is a potent cytokine that increases CD8+ T and NK cell numbers and function in experimental models. However, obstacles remain in using IL-15 therapeutically, specifically its low potency and short in vivo half-life. To help overcome this, a new IL-15 superagonist complex comprised of an IL-15N72D mutation and IL-15RαSu/Fc fusion (IL-15SA, also known as ALT-803) was developed. IL-15SA exhibits a significantly longer serum half-life and increased in vivo activity against various tumors. Herein, we evaluated the effects of IL-15SA in recipients of allogeneic hematopoietic stem cell transplantation. Weekly administration of IL-15SA to transplant recipients significantly increased the number of CD8+ T cells (specifically CD44+ memory/activated phenotype) and NK cells. Intracellular IFN-γ and TNF-α secretion by CD8+ T cells increased in the IL-15SA-treated group. IL-15SA also upregulated NKG2D expression on CD8+ T cells. Moreover, IL-15SA enhanced proliferation and cytokine secretion of adoptively transferred CFSE-labeled T cells in syngeneic and allogeneic models by specifically stimulating the slowly proliferative and nonproliferative cells into actively proliferating cells.We then evaluated IL-15SA's effects on anti-tumor activity against murine mastocytoma (P815) and murine B cell lymphoma (A20). IL-15SA enhanced graft-versus-tumor (GVT) activity in these tumors following T cell infusion. Interestingly, IL-15 SA administration provided GVT activity against A20 lymphoma cells in the murine donor leukocyte infusion (DLI) model without increasing graft versus host disease. In conclusion, IL-15SA could be a highly potent T- cell lymphoid growth factor and novel immunotherapeutic agent to complement stem cell transplantation and adoptive immunotherapy.

Simultaneous Catabolite Identification and Quantitation of Large Therapeutic Protein at the Intact Level by Immunoaffinity Capture Liquid Chromatography-High-Resolution Mass Spectrometry.

As therapeutic recombinant fusion proteins become more widely applicable for the treatment of various types of diseases, there is an increased demand for universal methods such as liquid chromatography (LC)-mass spectrometry (MS) for the determination of their pharmacokinetic properties, particularly their catabolism. The most common approach of analyzing proteins by LC-MS is to digest them into peptides, which can serve as surrogates of the protein. Alternatively, we have developed a novel high-resolution mass spectrometry (HRMS) based approach for analyzing large-molecule proteins at the intact level in biological samples without digestion. We established an immunoaffinity capture LC-HRMS method to quantify the intact parent molecule while simultaneously identifying catabolites for recombinant fusion proteins. We describe this method using dulaglutide, a glucagon-like peptide 1 (GLP1)-Fc fusion protein. Two proteolytic sites within the GLP1 peptide sequence of dulaglutide were identified using this novel LC-HRMS analysis in vivo in mice. These proteolytic sites were identified with the intact molecule being quantified simultaneously. Together with the trypsin digestion based LC-MS/MS analysis using surrogate peptides from different domains of the analyte, an insightful understanding of the pharmacokinetics and in vivo biotransformation of dulaglutide was obtained. Thus, this method enables simultaneous acquisition of both intact drug concentration and important catabolite information for this recombinant fusion protein, providing valuable insight into the integrity of the molecule and its catabolism in vivo. This is critical for designing and screening novel protein therapeutics and for understanding their pharmacokinetics and pharmacodynamics. With continuing advancement of LC-HRMS and software, this method can be very beneficial in drug discovery and development.

Integrin-targeted cancer immunotherapy elicits protective adaptive immune responses.

Certain RGD-binding integrins are required for cell adhesion, migration, and proliferation and are overexpressed in most tumors, making them attractive therapeutic targets. However, multiple integrin antagonist drug candidates have failed to show efficacy in cancer clinical trials. In this work, we instead exploit these integrins as a target for antibody Fc effector functions in the context of cancer immunotherapy. By combining administration of an engineered mouse serum albumin/IL-2 fusion with an Fc fusion to an integrin-binding peptide (2.5F-Fc), significant survival improvements are achieved in three syngeneic mouse tumor models, including complete responses with protective immunity. Functional integrin antagonism does not contribute significantly to efficacy; rather, this therapy recruits both an innate and adaptive immune response, as deficiencies in either arm result in reduced tumor control. Administration of this integrin-targeted immunotherapy together with an anti-PD-1 antibody further improves responses and predominantly results in cures. Overall, this well-tolerated therapy achieves tumor specificity by redirecting inflammation to a functional target fundamental to tumorigenic processes but expressed at significantly lower levels in healthy tissues, and it shows promise for translation.

Fusion of Fc domain to Factor IX (FIX) modulates the Th2 biased response to FIX and triggers Th1 immunity in hemophilia B treated mice.

Abstract Fusion of the IgG1 constant region domain (Fc) to therapeutic proteins increases the plasma half-life of drugs via binding to the neonatal Fc receptor (FcRn). However, potential interactions with Fc gamma-receptors (FcγR) expressed by immune cells may effect the immunogenicity of these drugs. The coagulation factors VIII (FVIII) and IX (FIX) for the treatment of hemophilia A & B (HA & HB) are the only drugs available with and without Fc fusion and offer an opportunity to experimentally study the effect of Fc fusion. Anti FVIII & FIX antibodies (ADAs) and rare cases of anaphylaxis in HB patients, are serious safety concerns. Using a mouse model for HB, we compared the immune responses to infusions of recombinant human FIX (hFIX) and hFIX fused to mouse IgG2a-Fc (hFIX-mFc). The mFc allowed species-specific Fc-FcγR interactions and prevented immune response to the foreign hFc. Treatment with hFIX-mFc caused earlier onset of anti-FIX IgG and elicited higher FIX-neutralizing antibody levels. Treatment with hFIX triggered Th2 biased immunity i.e., elevated plasma IgE titers, and Th2 cytokine profile of FIX-specific CD4 T cells and a few anaphylactic events. The fusion of Fc altered the response to a Th1 type with increased plasma IFN-γ levels, and IFN-γ secretion by effector CD4 T cells. hFIX-mFc binds to soluble FcγRs and FcγRs on natural killer cells (which also secrete IFNγ) and antigen presenting cells (dendritic cells, and macrophages source of IL-12). We show that Fc fusion enhances the presentation of hFIX to CD4 T cells. We propose that high plasma availability and targeting of APC provide plausible mechanisms by which the Fc fusion may modulate the CD4 T cell response to hFIX towards a Th1 like response, thus avoiding allergic responses.

CGEN-15001: a Novel B7-like regulator of immune homeostasis and inducer of Ag-specific tolerance

Abstract B7 proteins play critical immunomodulatory roles and provide attractive targets for development of novel therapies for cancer and autoimmunity, both of which involve improper immune tolerance. A major medical need in autoimmunity is restoration of immune tolerance to self-antigens. CGEN-15001, an Fc fusion protein of a novel B7-like protein that we identified, regulates immune homeostasis by inhibiting Th1/Th17 responses while enhancing Th2 and anti-inflammatory cytokines such as IL-10, and promoting iTreg differentiation. This was observed in vitro using murine cells and in PBMCs of healthy donors and MS patients, as well as in vivo in R-EAE model and in HY transplantation model. Short-term administration of CGEN-15001 in R-EAE model of MS and in NOD model of T1D resulted in durable therapeutic effects which lasted long after cessation of treatment, suggestive of restoration of immune tolerance. Treg blockade with anti-IL-10 or anti-TGFβ, and transient Treg neutralization with anti-CD25 abolished these therapeutic effects. Importantly, transferring CD4+ T cells from CGEN-15001-treated mice to naïve recipients protected them from R-EAE induction in Ag-specific manner, demonstrating induction of Ag-specific tolerance. In contrast, tolerance transfer was not achieved following CTLA4-Ig treatment in spite of the remission it induced in the donor mice. In summary, CGEN-15001 has a unique mode of action, combining immunomodulation and regulation of immune homeostasis, as well as re-establishment of Ag-specific tolerance via enhancing Treg differentiation, leading to durable disease amelioration. These findings support the potential of CGEN-15001 to provide a promising therapeutic approach across autoimmune diseases.

Novel immunomodulatory proteins generated via directed evolution of variant IgSF domains

Abstract The immunoglobulin superfamily (IgSF) is a large, diverse family of proteins expressed on immune cells that have been extensively targeted for treatment of cancers and autoimmune diseases. Most of the therapeutic strategies targeting this family have focused on high affinity antibodies that bind a single receptor. Moreover, wild-type IgSF receptors typically exhibit low affinities for their counter-structures, limiting their utility in therapeutic modulation of immune responses. We have developed a novel variant Ig domain (vIgD™) platform using directed evolution and yeast display to affinity mature human IgSF extracellular domains. In this platform, libraries of mutagenized IgSF domains were selected for enhanced or altered affinity to specific recombinant proteins. Fc fusion proteins incorporating these evolved immunomodulatory IgSF domains were then tested in vitro for their ability to either agonize or antagonize T cell responses. Multiple novel vIgD™ fusion proteins have been generated that significantly attenuate T cell activation in vitro as assessed by proliferation and cytokine production. Lead molecules also exhibited in vivo efficacy in the human PBMC-NSG™ GVHD mouse model. Efficacy in vitro and in vivo was superior to wild-type IgSF domains due to the induced alterations in affinity for cognate ligand and through specifically directed changes in their ability to bind additional counter-structures. Our results demonstrate that vIgDs™ can be developed to acquire unique biochemical properties that significantly enhance therapeutic utility as immunomodulatory agents. This vIgD™ therapeutic platform has broad potential to enhance the activity of biologics in treatment of both autoimmune disease and cancer.

Biological Activities of IL-15 superagonist - IL-15 Mutein:IL-15RaFc complex following Intravenous or Subcutaneous Administration

Abstract ALT-803 is a fusion protein complex consisting of IL-15N72D superagonist and a dimeric IL-15 receptor alpha (IL-15Rα) sushi domain IgG1 Fc fusion protein. When administered to mice, ALT-803 is capable of inducing NK and CD8+ T cell proliferation and activation, as well as potent antitumor responses. ALT-803 is currently in clinical studies using an intravenous (iv) route of administration. We were interested in exploring treatment regimens using subcutaneous (sc) administration of ALT-803 as an alternative to iv administration in order to lessen the adverse side effects that were observed. In this study, we compared the pharmacokinetics, immunostimulation, and anti-tumor efficacy of iv and sc administration of ALT-803 in C57BL/6 mice. We found the half-life of ALT-803 to be 7.5 hrs for iv administration vs. 7.7 hrs for sc. The maximal detected serum concentration of ALT-803 was 495 ng/ml at 16 hr time point following sc administration or 3926 ng/ml at 0.5 hr time point following iv administration. Similar bio-distribution of radio-labeled ALT-803 in mice was observed using quantitative PET-scan studies. We also demonstrated that ALT-803 administered iv or sc induced comparable proliferation of CD8+ T cells and NK cells and similarly activated immune cells which resulted in the reduction of tumor burden. A toxicity study of mice receiving multiple injections of ALT-803 for 4 weeks by iv or sc administration revealed that comparable immune system-related changes were observed and ALT-803 was well tolerated. The gradual absorbance into blood stream and lower maximal blood level of ALT-803 in sc injected mice, along with similar antitumor efficacy supports the use of ALT-803 by sc administration in patients with metastatic malignancies.

Ebola virus: Development of Filovirus glycoprotein Fc fusion proteins as a vaccine candidate

Abstract Krishnamurthy Konduru and Gerardo Kaplan Laboratory of Emerging Pathogens, Division ofEmerging and Transfusion Transmitted Diseases, Center for Biologics Evaluationand Research, Food and Drug Administration, Silver Spring, MD 20993. Recent Ebola virus(EBOV) epidemic in West Africa underscores the urgent need of vaccines ortherapeutics. Here we describe the development of EBOV and Marburg virus (MARV) subunit vaccines based on the extracellular domain of the viral glycoprotein (GP) fused to the Fc fragment of human IgG (EBOV gp-Fc or MARV gp-Fc) and expressed in mammalian cells. These GP fusion proteins undergo the complex post-translational processing observed in the membrane-bound GP. In vaccinated mice, EBOV gp-Fc induced high levels of total and neutralizing anti-GP antibodies and elicited T-cell immunity characterized by the production of IFNγ (Konduru et al. 2011). Vaccinated guinea pigs developed a strong humoral response as measured by total and neutralizing antibodies to GP, and were completely protected against EBOV lethal challenge (Konduru et al. 2016). We are currently extending our studies to the filo virus lethal challenge model in monkeys. Our preliminary data indicates that EBOV gp-Fc and MARV gp-Fc induces total and neutralizing antibodies as well as recall CD8+ T-cell immunity as assessed by the production of IFNγ and TNFα in PBMC. Further experiments, including protection against lethal challenge in monkeys, will be required to determine whether Filovirus GP-Fc fusion proteins could be developed as a candidate vaccine.

Recombinant human tumor necrosis factor receptor type Ⅱ-IgG Fc fusion protein for treatment of occupational medicamentosa-like dermatitis induced by trichloroethylene

Objective: To investigate the efficacy and safety of the recombinant human tumor necrosis factor receptor Ⅱ-IgG Fc fusion protein (rhTNFR: Fc, etanercept) for the treatment of occupational medicamentosa-like dermatitis induced by trichloroethylene (OMLDT) . Methods: In September 2011 to February 2016, 12 patients with OMLDT were treated with etanercept 25 mg, subcutaneous injection, twice per week, doubling of first dose. The course of treatment was 6 weeks. The drug eruption area and severity index (DASI) score, the proportion of patients achieving a 50%, 75% and 90% reduction in DASI (DASI50, DASI75, DASI90) and the serum level of TNF-α were used to assess the efficacy at different times. Adverse reactions were also recorded and evaluated. The results were statistically analyzed by nonparametric Friedman test and repetitive measurement ANOVA using the software SPSS19.0. Results: After 4 weeks treatment, the DASI score decreased form 56.33±7.02 to 0.50±0.91 (P<0.01) . The DASI50, DASI75 and DASI90 were all increased to 12 (100%) . The serum level of TNF-α decreased form (43.74±41.62) pg/ml to (3.03±0.47) pg/ml (P<0.01) . Statistically significant difference was observed from the above indexes. There were no adverse reactions in clinical application. Conclusion: Recombinant human tumor necrosis factor receptor Ⅱ-IgG Fc fusion protein may be a safe and effective drug in the treatment of OMLDT.

Lysibodies are IgG Fc fusions with lysin binding domains targeting Staphylococcus aureus wall carbohydrates for effective phagocytosis

The cell wall of Gram-positive bacteria contains abundant surface-exposed carbohydrate molecules that are highly conserved within and often across species. The potential therapeutic usefulness of high-affinity antibodies to cell wall carbohydrates is unquestioned, however obtaining such antibodies is challenging due to the poor overall immunogenicity of these bacterial targets. Autolysins and phage lysins are peptidoglycan hydrolases, enzymes that have evolved over a billion years to degrade bacterial cell wall. Such wall hydrolases are modular enzymes, composed of discrete domains for high-affinity binding to cell wall carbohydrates and cleavage activity. In this study, we demonstrate that binding domains from autolysins and lysins can be fused to the Fc region of human IgG, creating a fully functional homodimer (or "lysibody") with high-affinity binding and specificity for carbohydrate determinants on the bacterial surface. Furthermore, we demonstrate that this process is reproducible with three different binding domains specific to methicillin-resistant Staphylococcus aureus (MRSA). Cell-bound lysibodies induced the fixation of complement on the bacterial surface, promoted phagocytosis by macrophages and neutrophils, and protected mice from MRSA infection in two model systems. The lysibody approach could be used to target a range of difficult-to-treat pathogenic bacteria, given that cell wall hydrolases are ubiquitous in nature.

Modulating immunogenicity of factor IX by fusion to an immunoglobulin Fc domain: a study using a hemophilia B mouse model

Background Fc fusion is a platform technology used to increase the circulating half-life of protein and peptide therapeutics. However, there are potential immunological consequences with this approach, such as changes in the molecule's immunogenicity as well as possible interactions with a repertoire of Fc receptors (FcR) that can modulate immune responses. Objectives/Methods Using a mouse hemophilia B (HB) model, we compared the immune responses to infusions of recombinant human factor IX (hFIX) and hFIX fused to mouse IgG2a-Fc (hFIX-mFc). The mFc was employed to allow species-specific Fc-FcγR interactions. Results Although treatment with hFIX-mFc altered the early development of anti-FIX IgG, no significant differences in anti-FIX antibody titers were observed at the end of the treatment regimen (5 weeks) or upon anamnestic response (5 months). However, treatment with hFIX-mFc elicited higher FIX-neutralizing antibody levels and resulted in reduced IgE titers compared with the hFIX-treated group. Additionally, differences in plasma cytokine levels and in vitro CD4+ T-cell responses suggest that whereas hFIX treatment triggered a Th2-biased immune response, hFIX-mFc treatment induced Th1-biased CD4+ T cells. We also show that hFIX-mFc bound to soluble FcγRs and engaged with FcγRs on different cell types, which may impact antigen presentation. Conclusions These studies provide a model system to study how Fc-fusion proteins may affect immune mechanisms. We used this model to demonstrate a plausible mechanism by which Fc fusion may modulate the IgE response to hFIX. This model may be appropriate for investigating the rare but severe IgE-mediated anaphylaxis reaction to hFIX infusions in HB patients.

Establishment of the first WHO International Standard for etanercept, a TNF receptor II Fc fusion protein: Report of an international collaborative study

Etanercept, a recombinant human tumor necrosis factor (TNF) receptor Fc fusion protein is an effective treatment option in adults with rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis or plaque psoriasis and paediatrics with juvenile idiotypic arthritis and plaque psoriasis.Patent expiration in Europe and intense development of various etanercept products worldwide triggered a need for an international reference standard to facilitate determination of biological activity. Therefore, three candidate preparations of etanercept were lyophilized and evaluated in a multi-centre collaborative study comprising twenty eight laboratories from 15 countries for their suitability to serve as an international standard for the bioactivity of TNF receptor II Fc fusion proteins (international nonproprietary name, Etanercept). The preparations were tested for neutralization activity against the third TNF-α international standard (IS) in different in vitro cell-based assays, e.g., cytotoxicity, apoptosis and reporter gene methods. Regardless of the assay and the amount of TNF-α IS used, potency estimates for the different preparations were very similar. An indication of the inhibitory activity of etanercept in terms of the biological activity of the TNF-α IS based on ED50 data derived from a limited number of laboratories using a cytotoxicity assay was also derived. Results indicated that the candidate preparation coded 13/204 was stable and suitable to serve as an international standard for the biological activity of etanercept. Therefore, the preparation coded 13/204 was established by the WHO Expert Committee on Biological Standardization (ECBS) in 2015 as the WHO first International Standard for TNF receptor II Fc fusion protein (INN, etanercept) with an assigned in vitro bioactivity of 10,000IU per ampoule.It should be noted that this first-in-class international standard for a Fc fusion protein, available from the National Institute for Biological Standards and Control and also as a biological reference preparation (BRP) from the European Directorate for the Quality of Medicines and Healthcare, is intended for controlling the performance of biological assays for etanercept and to support the establishment of in-house bioassay standards. This standard is not intended for describing the labelling or dosage of etanercept therapeutic products or for use as a comparator (reference product) for biosimilarity determination.