Site-specific PEGylation Research Articles

Site-specific PEGylation of an anti-CEA/CD3 bispecific antibody improves its antitumor efficacy.

IntroductionBispecific antibodies that engage immune cells to kill cancer cells are actively pursued in cancer immunotherapy. Different types of bispecific antibodies, including single-chain fragments, Fab fragments, nanobodies, and immunoglobulin Gs (IgGs), have been studied. However, the low molecular weight of bispecific antibodies with single-chain or Fab fragments generally leads to their rapid clearance in vivo, which limits the therapeutic potential of these bispecific antibodies.Materials and methodsIn this study, we used a site-specific PEGylation strategy to modify the bispecific single-domain antibody-linked Fab (S-Fab), which was designed by linking an anticarcinoembryonic antigen (anti-CEA) nanobody with an anti-CD3 Fab.ResultsThe half-life (t1/2) of PEGylated S-Fab (polyethylene glycol-S-Fab) was increased 12-fold in vivo with a slightly decreased tumor cell cytotoxicity in vitro as well as more potent tumor growth inhibition in vivo compared to S-Fab.ConclusionThis study demonstrated that PEGylation is an effective approach to enhance the antitumor efficacy of bispecific antibodies.

The Locational Impact of Site-Specific PEGylation: Streamlined Screening with Cell-Free Protein Expression and Coarse-Grain Simulation.

Although polyethylene glycol (PEG) is commonly used to improve protein stability and therapeutic efficacy, the optimal location for attaching PEG onto proteins is not well understood. Here, we present a cell-free protein synthesis-based screening platform that facilitates site-specific PEGylation and efficient evaluation of PEG attachment efficiency, thermal stability, and activity for different variants of PEGylated T4 lysozyme, including a di-PEGylated variant. We also report developing a computationally efficient coarse-grain simulation model as a potential tool to narrow experimental screening candidates. We use this simulation method as a novel tool to evaluate the locational impact of PEGylation. Using this screen, we also evaluated the predictive impact of PEGylation site solvent accessibility, conjugation site structure, PEG size, and double PEGylation. Our findings indicate that PEGylation efficiency, protein stability, and protein activity varied considerably with PEGylation site, variations that were not well predicted by common PEGylation guidelines. Overall our results suggest current guidelines are insufficiently predictive, highlighting the need for experimental and simulation screening systems such as the one presented here.

Characterisation of the site-specific monoPEGylated rhG-CSF analogue pegteograstim

We describe the characterisation of a novel monoPEGylated recombinant human granulocyte colony-stimulating factor analogue, pegteograstim (Neulapeg), prepared by site-specific 20 kDa maleimide-PEG conjugation. An additional cysteine was inserted between Gly136 and Ala137 of filgrastim (methionyl human granulocyte colony-stimulating factor) for site-specific PEGylation, and Cys18 of filgrastim was replaced with Ser18 to prevent unwanted PEGylation. Pegteograstim was produced by Escherichia coli and purified by cation exchange chromatography, and its structural, physicochemical, biological and immunological properties were investigated. Male Sprague-Dawley rats were administered pegteograstim (100 μg/kg) and the pharmacokinetics and pharmacodynamics compared with those of filgrastim.The results of long-term stability testing of pegteograstim revealed no significant change in its quality attributes at 2–8 °C for 36 months. In addition, pegteograstim was stable under the accelerated conditions (25 ± 2 °C, RH of 60 ± 5%) for 6 months. The site-specific monoPEGylated pegteograstim is a highly pure, stable and novel drug for long-lasting treatment of chemotherapy-induced neutropenia.

Polyethylene Glycol Based Changes to β-Sheet Protein Conformational and Proteolytic Stability Depend on Conjugation Strategy and Location.

The development of chemical strategies for site-specific protein modification now enables researchers to attach polyethylene glycol (PEG) to a protein drug at one or more specific locations (i.e., protein PEGylation). However, aside from avoiding enzyme active sites or protein-binding interfaces, distinguishing the optimal PEGylation site from the available alternatives has conventionally been a matter of trial and error. As part of a continuing effort to develop guidelines for identifying optimal PEGylation sites within proteins, we show here that the impact of PEGylation at various sites within the β-sheet model protein WW depends strongly on the identity of the PEG-protein linker. The PEGylation of Gln or of azidohomoalanine has a similar impact on WW conformational stability as does Asn-PEGylation, whereas the PEGylation of propargyloxyphenylalanine is substantially stabilizing at locations where Asn-PEGylation was destabilizing. Importantly, we find that at least one of these three site-specific PEGylation strategies leads to substantial PEG-based stabilization at each of the positions investigated, highlighting the importance of considering conjugation strategy as an important variable in selecting optimal PEGylation sites. We further demonstrate that using a branched PEG oligomer intensifies the impact of PEGylation on WW conformational stability and also show that PEG-based increases to conformational stability are strongly associated with corresponding increases in proteolytic stability.

Site-Specific PEGylated Adeno-Associated Viruses with Increased Serum Stability and Reduced Immunogenicity

Adeno-associated virus (AAV) is one of the most extensively studied and utilized viral vectors in clinical gene transfer research. However, the serum instability and immunogenicity of AAV vectors significantly limit their application. Here, we endeavored to overcome these limitations by developing a straightforward approach for site-specific PEGylation of AAV via genetic code expansion. This technique includes incorporation of the azide moiety into the AAV capsid protein followed by orthogonal and stoichiometric conjugation of a variety of polyethylene glycols (PEGs) through click chemistry. Using this approach, only the chosen site(s) was consistently PEGylated under mild conditions, preventing nonselective conjugation. Upon a series of in vitro examinations, AAVs conjugated with 20-kD PEG at sites Q325+1, S452+1, and R585+1 showed a 1.7- to 2.4-fold stability improvement in pooled human serum and a nearly twofold reduction in antibody recognition. Subsequent animal research on Sprague Dawley rats displayed a promising 20% reduction in antibody inducement and a higher virus titer in the blood. Together, our data demonstrate successful protection of an AAV vector from antibody neutralization and blood clearance, thereby increasing the efficiency of therapeutic gene delivery.

Oxidized catechol-derived poly (ethylene glycol) for thiol-specific conjugation

Site-specific PEGylation of clinically used protein via free cysteine residue has been widely applied for a homogeneous product. Herein, we described a new thiol-specific PEGylation strategy based on catechol-derived reactive quinone species. Catechol-derived polyethylene glycol (PEG) was synthesized by coupling linear PEG N-hydroxysuccinimide to dopamine and then oxidized to quinone. PEG-dopaquinone (PEG-DAQ) mostly modifying the free cysteines of two model proteins of truncated flagellin (CBLB502) and recombined human ciliary neurotrophic factor (rhCNTF) evidenced its thiol-specificity. The yield of PEG-DAQ-rhCNTF under neutral pH value was 87.5%, compared with PEG-maleimide of 92.3% and PEG-vinylsulfone of 17.6%, respectively. About 95% reactive capacity for PEG-DAQ was maintained after incubation in aqueous solvent for 96h, indicating that the reagent was superiorly hydro-stable. Moreover, it was found that quenching the PEGylation reaction by excess cysteine was necessary to obtain more stable conjugate. The resulted PEG-DAQ-rhCNTF remained about 93.5% of the initial products after storage for 14days. As a comparison, PEG-MAL-rhCNTF was remained to 71.2%. Our results demonstrated that PEG-dopaquinone had properties of high selectivity and reactivity with cysteine thiol, and superior stability for both the reagent itself and its conjugate, showing great promise for developing as an alternative reagent for thiol-selective modification.

Production and characterization of a PEGylated derivative of recombinant human deoxyribonuclease I for cystic fibrosis therapy

Recombinant human deoxyribonuclease I (rhDNase) is the mucolytic agent most widely used for the treatment of respiratory disease in cystic fibrosis. However, rhDNase is rapidly cleared from the lungs which implies a high dosing frequency and limited patient adherence. The aim of this study was to produce a long-acting PEGylated derivative of rhDNase presenting a preserved enzymatic activity. Site-specific PEGylation on the N-terminal (N-ter) leucine residue of rhDNase was achieved by reductive alkylation at acidic pH using linear 20kDa, linear 30kDa or two-arm 40kDa polyethylene glycol (PEG) propionaldehydes. Yields of mono-PEGylated products ranged between 45% and 61%. Conjugation to PEG fully preserved the secondary structure and the in vitro enzymatic activity of the native protein. These properties offer interesting perspectives for in vivo inhalation studies of the PEGylated enzyme.

In silico designing of a new cysteine analogue of hirudin variant 3 for site specific PEGylation.

Hirudin is an anticoagulant agent of the salivary glands of the medicinal leech. Recombinant hirudin (r-Hir) displays certain drawbacks including bleeding and immunogenicity. To solve these problems, cysteine-specific PEGylation has been proposed as a successful technique. However, proper selection of the appropriate cysteine residue for substitution is a critical step. This study has, for the first time, used a computational approach aimed at identifying a single potential PEGylation site for replacement by cysteine residue in the hirudin variant 3 (HV3). Homology modeling (HM) was performed using MODELLER. All non-cysteine residues of the HV3 were replaced with the cysteine. The best model was selected based on the results of discrete optimized protein energy score, PROCHECK software, and Verify3D. The receptor binding was investigated using protein-protein docking by ClusPro web tool which was then visualized using LigPlot+ software and PyMOL. Finally, multiple sequence alignment (MSA) using ClustalW software and disulfide bond prediction were performed. According to the results of HM and docking, Q33C, which was located on the surface of the protein, was the best site for PEGylation. Furthermore, MSA showed that Q33 was not a conserved residue and LigPlot+ software showed that it is not involved in the hirudin-thrombin binding pocket. Moreover, prediction softwares established that it is not involved in disulfide bond formation. In this study, for the first time, the utility of the in silico approach for creating a cysteine analogue of HV3 was introduced. Our study demonstrated that the substitution of Q33 by cysteine probably has no effect on the biological activity of the HV3. However, experimental analyses are required to confirm the results.

Solid Phase PEGylation of Uricase

Uricase is used as a therapeutic enzyme to treat gout and hyperuricemia which also finds application in cancer chemotherapy. Site-specific PEGylation of uricase using methoxy polyethylene glycol maleimide (mPEG-mal) through thiol PEGylation reaction is an effective way to overcome the demerit of its short plasma half-life in blood as well as for the enhancement of its therapeutic potential. However, conventional solution-phase PEGylation for the synthesis of conjugates leads to lower yields of the desired PEGylated product and thus falls short of commercial attraction. In order to preserve the bioactivity of PEGylated product, selectivity, and extent of covalent conjugation, a methodology for on-column/solid phase PEGylation of uricase enzyme using size-exclusion reaction chromatography (SERC) has been attempted. Sephadex G-100 was used as a chromatographic solid media, wherein synthesis of mono and di-PEGylated uricase molecules was observed which were efficiently separated from their non-PEGylated counterparts.

A Therapeutic Uricase with Reduced Immunogenicity Risk and Improved Development Properties.

Humans and higher primates are unique in that they lack uricase, the enzyme capable of oxidizing uric acid. As a consequence of this enzyme deficiency, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit resulting in crystallization in joints, acute inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Krystexxa® is a hyper-PEGylated pig-baboon chimeric uricase indicated for chronic refractory gout that induces an immunogenic response in 91% of treated patients, including infusion reactions (26%) and anaphylaxis (6.5%). These properties limit its use and effectiveness. An innovative approach has been used to develop a therapeutic uricase with improved properties such as: soluble expression, neutral pH solubility, high E. coli expression level, thermal stability, and excellent activity. More than 200 diverse uricase sequences were aligned to guide protein engineering and reduce putative sequence liabilities. A single uricase lead candidate was identified, which showed low potential for immunogenicity in >200 human donor samples selected to represent diverse HLA haplotypes. Cysteines were engineered into the lead sequence for site specific PEGylation and studies demonstrated >95% PEGylation efficiency. PEGylated uricase retains enzymatic activity in vitro at neutral pH, in human serum and in vivo (rats and canines) and has an extended half-life. In canines, an 85% reduction in serum uric acid levels was observed with a single subcutaneous injection. This PEGylated, non-immunogenic uricase has the potential to provide meaningful benefits to patients with gout.

Site-Specific Thiol-mediated PEGylation of Streptokinase Leads to Improved Properties with Clinical Potential.

Streptokinase (SK) is an efficient thrombolytic agent that dissolves fibrin blood clots with clinical efficiency comparable to the high priced drug, tissue plasminogen activator (tPA). However, being of bacterial origin, its major drawbacks are its potentially high antigenicity, and relatively short circulating half-life (approximately 10-15 min). In the present investigation, an attempt has been made to address both these shortcomings by site-specific pegylation, and to obtain longer lasting thrombolytics, which are consistent with clinical requirements. Therefore, we employed available three-dimensional structural information on SK to carry out site-specific cysteine incorporation at 'optimal' surfaceexposed sites within all the three domains in streptokinase followed by pegylation with 20KDa PEG groups, and screening for biologically active variants. Interestingly, some of these SK PEG-conjugates exhibited considerably subdued immunereactivity along with enhanced in-vitro proteolytic stability profiles and extended circulating in-vivo half-lives (2 to 20-fold compared to that of native unconjugated SK) depending upon location and number of PEG-groups per molecule obtained in homogeneous form. The obtained results are a promising approach for favorably modulating immune-reactivity and half-life by cysteine- specific PEGylation of SK to achieve therapeutic attributes desirable for the treatment of different circulatory disorders, such as ischemic stroke, myocardial infarction and pulmonary embolism.

Intein-mediated site-specific synthesis of tumor-targeting protein delivery system: Turning PEG dilemma into prodrug-like feature.

Poor tumor-targeted and cytoplasmic delivery is a bottleneck for protein toxin-based cancer therapy. Ideally, a protein toxin drug should remain stealthy in circulation for prolonged half-life and reduced side toxicity, but turn activated at tumor. PEGylation is a solution to achieve the first goal, but creates a hurdle for the second because PEG rejects interaction between the drugs and tumor cells therein. Such PEG dilemma is an unsolved problem in protein delivery. Herein proposed is a concept of turning PEG dilemma into prodrug-like feature. A site-selectively PEGylated, gelatinase-triggered cell-penetrating trichosanthin protein delivery system is developed with three specific aims. The first is to develop an intein-based ligation method for achieving site-specific modification of protein toxins. The second is to develop a prodrug feature that renders protein toxins remaining stealthy in blood for reduced side toxicity and improved EPR effect. The third is to develop a gelatinase activatable cell-penetration strategy for enhanced tumor targeting and cytoplasmic delivery. Of note, site-specific modification is a big challenge in protein drug research, especially for such a complicated, multifunctional protein delivery system. We successfully develop a protocol for constructing a macromolecular prodrug system with intein-mediated ligation synthesis. With an on-column process of purification and intein-mediated cleavage, the site-specific PEGylation then can be readily achieved by conjugation with the activated C-terminus, thus constructing a PEG-capped, cell-penetrating trichosanthin system with a gelatinase-cleavable linker that enables tumor-specific activation of cytoplasmic delivery. It provides a promising method to address the PEG dilemma for enhanced protein drug delivery, and importantly, a facile protocol for site-specific modification of such a class of protein drugs for improving their druggability and industrial translation.

Improved variants of SrtA for site-specific conjugation on antibodies and proteins with high efficiency.

Sortase mediated ligation is a highly specific platform for conjugation that relies on the specificity of the transpeptidase Sortase A (SrtA) for short peptide sequences (LPXTG and GGG). SrtA retains its specificity while accepting a wide range of potential substrates, but its broad use is limited by the wild-type enzyme’s poor kinetics, which require large amounts of SrtA and extended reaction times for efficient conjugation. Prior explorations have aimed to improve the kinetics of SrtA with limited success. Herein we describe the discovery of further improved SrtA variants with increased efficiency for the conjugation reaction, and demonstrate their robustness in labelling proteins and antibodies in a site-specific manner. Our variants require significantly lower amounts of enzyme than WT SrtA and can be used to attach small molecules to the N or C-terminus of the heavy or light chain in antibodies with excellent yields. These improved variants can also be used for highly efficient site-specific PEGylation.

PEGylation and HAylation via catechol: α-Amine-specific reaction at N-terminus of peptides and proteins.

The development of chemoselective, site-specific chemistries for proteins/peptides is essential for biochemistry, pharmaceutical chemistry, and other fields. In this work, we found that catechol, which has been extensively utilized as an adhesive molecule for material-independent surface chemistry and as a crosslinker in hydrogel preparation, specifically reacts with N-terminal α-amines, avoiding the ε-amine group in lysine. A conjugate of methoxy-poly(ethylene glycol)-catechol called mPEG-cat chemoselectively reacts with N-terminal amine groups at neutral pH resulting in site-specific PEGylation. To demonstrate the versatility of this catechol chemoselective reaction, we used four proteins (lysozyme, basic-fibroblast growth factor (bFGF), granulocyte-colony stimulating factor (G-CSF), insulin, and erythropoietin (EPO)) as well as two peptides (hinge-3 and laminin-derived peptide (LDP)). All the tested macromolecules showed N-terminal site-specific modifications. Furthermore, we prepared another catechol grafted conjugate called hyaluronic acid-catechol (HA-cat) to demonstrate that this catechol-involved chemoselective chemistry is not specific for PEG conjugates. This new catechol chemoselective chemistry could be a new platform for the functionalization of proteins and peptides for a variety of purposes. Considering the fact that biological activities of proteins or peptides depend largely on their 3-dimensional conformation, the orientation-controllable reaction is very important for preserving the intrinsic functionality of them. In addition to PEG, many other bio-polymers such as oligonucleotides, antibodies, and oligosaccharides have been conjugated with proteins or peptides for various biomedical applications. Although several chemoselective conjugation chemistries have been reported, conjugation efficiencies are different depending on types of proteins or polymers, and thus there've been strong needs for the development of alternative strategy of chemoselective conjugation that can be applied for a variety of therapeutic proteins towards high biological activities. We are certain this new catechol chemoselective chemistry could be a new platform for the functionalization of proteins and peptides for various purposes.

OPTIMIZATION OF THE PHARMACOKINETIC (PK) AND PHARMACODYNAMIC (PD) PROPERTIES OF AN ANTI-ABETA ANTIBODY M266 FAB FRAGMENT VARIANT BY SITE-SPECIFIC PEGYLATION

OPTIMIZATION OF THE PHARMACOKINETIC (PK) AND PHARMACODYNAMIC (PD) PROPERTIES OF AN ANTI-ABETA ANTIBODY M266 FAB FRAGMENT VARIANT BY SITE-SPECIFIC PEGYLATION

Conjugation Strategy Strongly Impacts the Conformational Stability of a PEG-Protein Conjugate

Site-specific PEGylation is an important strategy for enhancing the pharmacokinetic properties of protein drugs, and has been enabled by the recent development of many chemoselective reactions for protein side-chain modification. However, the impact of these different conjugation strategies on the properties of PEG-protein conjugates is poorly understood. Here we show that the ability of PEG to enhance protein conformational stability depends strongly on the identity of the PEG-protein linker, with the most stabilizing linkers involving conjugation of PEG to planar polar groups near the peptide backbone. We also find that branched PEGs provide superior stabilization relative to their linear counterparts, suggesting additional applications for branched PEGs in protein stabilization.

Pharmacological studies of pegylated anti-TNF-α antibodies

Objective Rheumatoid arthritis (RA) is a systemic disease with chronic inflammation of the joints, and is caused by increased inflammatory cytokines such as TNF-α. The inhibition of TNF-α was found to effectively control inflammation, and block the progress of the disease. This study designed a new and efficient strategy to develop TNF-α antibodies. Methods Prokaryotic engineered cells were growing in a small scale bioreactor, and Fab' products (TMP5) were purified by affinity chromatography. Then TMP5 was modified by 20 kDa site-specific pegylation, and purified by SP sepharose column. Activities and affinities of TMP5 and PEG-TMP5 in vitro were studied. Results TMP5 was effectively produced after fermentation, periplasmic extraction and purification. The purity of TMP5 was more than 96%. After pegylation by 20 kDa PEG, the affinity and activity of PEG-TMP5 were (6.48±0.86)×10-9 M and (7.58±0.61)×10-11 M respectively, and were in the same range as positive controls. Conclusion PEG-TMP5 is a promising drug candidate for treating rheumatoid arthritis. Key words: Antibodies; Tumor necrosis factor α; Pegylation; pharmacology in vitro

New developments in the management of moderate-to-severe hemophilia B.

Hemophilia B is an X-linked genetic deficiency of coagulation factor IX (FIX) activity associated with recurrent deep tissue and joint bleeding that may lead to long-term disability. FIX replacement therapy using plasma-derived protein or recombinant protein has significantly reduced bleeding and disability from hemophilia B, particularly when used in a prophylactic fashion. Although modern factor replacement has excellent efficacy and safety, barriers to the broader use of prophylaxis remain, including the need for intravenous (IV) access, frequent dosing, variability in individual pharmacokinetics, and cost. To overcome the requirement for frequent factor dosing, novel forms of recombinant FIX have been developed that possess extended terminal half-lives. Two of these products (FIXFc and rIX-FP) represent fusion proteins with the immunoglobulin G1 (IgG1) Fc domain and albumin, respectively, resulting in proteins that are recycled in vivo by the neonatal Fc receptor. The third product has undergone site-specific PEGylation on the activation peptide of FIX, similarly resulting in a long-lived FIX form. Clinical trials in previously treated hemophilia B patients have demonstrated excellent efficacy and confirmed less-frequent dosing requirements for the extended half-life forms. However, gaps in knowledge remain with regard to the risk of inhibitor formation and allergic reactions in previously untreated patient populations, safety in elderly patients with hemophilia, effects on in vivo FIX distribution, and cost-effectiveness. Additional strategies designed to rebalance hemostasis in hemophilia patients include monoclonal-antibody-mediated inhibition of tissue factor pathway inhibitor activity and siRNA-mediated reduction in antithrombin expression by the liver. Both of these approaches are long acting and potentially involve subcutaneous administration of the drug. In this review, we will discuss the biology of FIX, the evolution of FIX replacement therapy, the emerging FIX products possessing extended half-lives, and novel “rebalancing” approaches to hemophilia therapy.

Studies on the Site-specific PEGylation Induced Interferences Instigated in Uricase Quantification Using the Bradford Method

Uricase from Bacillus fastidiosus was site-specifically PEGylated using methoxypolyethyleneglycol-maleimide (mPEG-mal) of different molecular weights (750 Da, 5 kDa, 10 kDa) via Thiol PEGylation strategy. The obtained monoPEGylated uricase conjugates were characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the molecular weight of single subunit of the conjugate was found to be 42.6, 48.1 and 56.3 kDa with respect to different molecular weights of m-PEG-mal. The influence of PEGylation on the quantification of uricase using protein quantification techniques like Bradford assay, RP-HPLC detection and Dumbroff method has been evaluated. A gradual decline in the absorbance value was observed with the advancement of the PEGylation reaction, indicating an interferences in the protein quantification due to PEGylation. The extent of interference highly dependence on mPEG-mal concentration and its chain length. The present study indicates that the quantification of PEGylation induced interferences caused in protein measured ought to be prudently measured at every discrete step of the PEGylation process.

A Factor VIII with Site-Specific Pegylation Attenuates the Binding with Inhibitors Results in the Retention of Hemostatic Effect

Factor (F) VIII inhibitors develop as alloantibodies in mainly severe hemophilia A (HA) patients after frequent infusion of FVIII concentrates and also as autoantibodies in previously normal individuals resulting in acquired HA. The appearance of autoantibodies usually results in severe bleeding and complicates the hemostatic treatment. The epitopes recognized by FVIII inhibitors are commonly located in the A2 and/or C2 domain of the FVIII molecule. BAY 94-9027(BAY 94) is a B-domain deleted (BDD) recombinant (r)FVIII with site-specific attachment of polyethylene glycol (PEG) that has shown an extended half-life in HA patients as well as animal models of HA (Coyle et al. JTH 2014). The hemostatic effect of BAY 94 in the presence of the inhibitors remains unknown. In this study, we investigated the effects of BAY 94 on the inhibitory activity of antibodies with various epitopes developed in acquired HA as well as congenital HA. Firstly, we assessed the binding of BAY 94 or BDD to the three monoclonal antibodies (mAbs) recognizing the different epitopes with the different inhibitory kinetics pattern (type1 or 2) (monoA; A1 type1, ESH4;C2 type1 and ESH8;C2 type2), and the seven polyclonal inhibitor IgGs obtained from three congenital HA (Case1(recognizing epitope(s) in A2 and C2 type1), 2(C2 type1), 3(C2 type2)) and four acquired HA (Case 4(A2 and C2 type1), 5(A2), 6(C2 type2), 7(C2 type1)) patients plasmas purified with the protein G column. Antibodies were covalently coupled (~5 ng/mm2) to a sensor chip surface, utilizing surface plasmon resonance (SPR) assay to assess binding. BAY 94 bound to the immobilized anti-A1 mAb, recognizing the acidic region, with modest lower binding affinity (Kd ~4-fold higher) than BDD. An anti-C2 mAb ESH8 also bound to BAY 94 with an ~8-fold higher Kd compared to BDD. Another anti-C2 ESH4 bound to BDD with similar binding affinity to anti-C2 ESH8, however, of interest, this mAb failed to bind to BAY 94. Case 1-IgG, recognizing both A2 and C2 domains, bound to BAY 94 with an ~30-fold lower binding affinity (Kd ~ 1.5nM) compared to BDD (Kd ~0.05nM). Similarly, the binding potentials of BAY 94 to Case 2-IgG (Kd ~ 42nM) was markedly decreased compared to BDD (Kd ~ 4.7nM). In addition, Case 3-IgG bound to BDD (Kd ~ 3.1nM) but little to BAY 94. Four anti-FVIII autoAbs (Case 4,5,6 and 7) bound to BDD with weaker affinities compared to anti-FVIII alloAbs and mAbs. However, all autoAbs almost failed to bind to BAY 94. Further, to confirm the hemostatic function of BAY 94 with inhibitors, we evaluated the FVIII:C with BAY 94 or BDD in the presence of anti-FVIII Abs by a one-stage clotting assay. Anti-A1 mAb, monoA as well as anti-C2 ESH8 decreased the FVIII:C levels in BAY 94 and BDD similarly,in a dose-dependent manner. The inhibition of FVIII:C by anti-C2 mAb ESH4, however, had little effect on BAY 94 activity up to the max IgG concentration of 10 nM employed. Similarly, in anti-C2 alloAb Case 2, possessing the similar characteristic of ESH4, the inhibitory effects of this IgG was weaker for BAY 94 compared to BDD. The four anti-FVIII autoAbs decreased the FVIII:C levels of BAY 94 more modestly than BDD. Particularly the inhibitory effect of anti-C2 autoAb Case 6, as well as mAb ESH4, were quite different between BDD and BAY 94. Taken together, BAY 94 bound to anti-FVIII autoAbs as well as alloAbs with weaker binding affinity compared to BDD, independent of its epitope and kinetic mode, resulting in higher residual FVIII:C levels of BAY 94 relative to BDD. BAY 94-9027 is a site-specific pegylated rFVIII with extended half-life in vivo and the data presented here suggests the attached PEG could facilitate its hemostatic function even in the presence of inhibitors, by attenuating the binding with inhibitors. DisclosuresYada:Chugai Pharmaceutical Co., Ltd: Research Funding. Nogami:F.Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai Paharmaceutical Co., Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Patel:Bayer HealtheCare LLC: Employment. Sim:Bayer HealtheCare LLC: Employment. Kauser:Bayer HealtheCare LLC: Employment. Shima:Bayer: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Baxalta: Honoraria, Research Funding; Kaketsuken: Honoraria; Chugai Pharmaceutical Co., Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Honoraria, Research Funding; Biogen: Honoraria, Research Funding.