Background: Coagulation factor VIII represents one of the oldest protein-based therapeutics, serving as an effective hemophilia A treatment for half a century. Optimal treatment consists of repeated intravenous infusions of blood coagulation factor VIII (FVIII) per week for life. Despite overall treatment success, significant limitations remain, including treatment invasiveness, duration, immunogenicity, and cost. These issues have inspired research into the development of bioengineered FVIII products and gene therapies. Objectives: To structurally characterize a bioengineered construct of FVIII, termed ET3i, which is a human/porcine chimeric B domain-deleted heterodimer with improved expression and slower A2 domain dissociation following proteolytic activation by thrombin. Methods: The structure of ET3i was characterized with X-ray crystallography and tandem mass spectrometry-based glycoproteomics. Results: Here, we report the 3.2 Å crystal structure of ET3i and characterize the distribution of N-linked glycans with LC-MS/MS glycoproteomics. This structure shows remarkable conservation with the human FVIII protein and provides a detailed view of the interface between the A2 domain and the remaining FVIII structure. With two FVIII molecules in the crystal, we observe two conformations of the C2 domain relative to the remaining FVIII structure. The improved model and stereochemistry of ET3i served as a scaffold to generate an improved, refined structure of human FVIII. With the original datasets at 3.7 Å and 4.0 Å resolution, this new structure resulted in improved refinement statistics. Conclusions: These improved structures yield a more confident model for next-generation engineering efforts to develop FVIII therapeutics with longer half-lives, higher expression levels, and lower immunogenicity.

Patients with hemophilia A present with spontaneous and sometimes life-threatening bleeding episodes that are treated using blood coagulation factor VIII (fVIII) replacement products. Although effective, these products have limited availability worldwide due to supply limitations and product costs, which stem largely from manufacturing complexity. Current mammalian cell culture manufacturing systems yield around 100 µg/l of recombinant fVIII, with a per cell production rate of 0.05 pg/cell/day, representing 10,000-fold lesser production than is achieved for other similar-sized recombinant proteins (e.g. monoclonal antibodies). Expression of human fVIII is rate limited by inefficient transport through the cellular secretory pathway. Recently, we discovered that the orthologous porcine fVIII possesses two distinct sequence elements that enhance secretory transport efficiency. Herein, we describe the development of a bioengineered fVIII product using a novel lentiviral-driven recombinant protein manufacturing platform. The combined implementation of these technologies yielded production cell lines that biosynthesize in excess of 2.5 mg/l of recombinant fVIII at the rate of 9 pg/cell/day, which is the highest level of recombinant fVIII production reported to date, thereby validating the utility of both technologies.

Essentials Factor VIII inhibitors are the most serious complication in patients with hemophilia A. Aggregates in biopharmaceutical products are an immunogenic risk factor. Aggregates were identified in recombinant full-length factor VIII products. Aggregates in recombinant factor VIII products are identified by analytical ultracentrifugation. Summary: Background The development of inhibitory anti-factor VIII antibodies is the most serious complication in the management of patients with hemophilia A. Studies have suggested that recombinant full-length FVIII is more immunogenic than plasma-derived FVIII, and that, among recombinant FVIII products, Kogenate is more immunogenic than Advate. Aggregates in biopharmaceutical products are considered a risk factor for the development of anti-drug antibodies. Objective To evaluate recombinant full-length FVIII products for the presence of aggregates. Methods Advate, Helixate and Kogenate were reconstituted to their therapeutic formulations, and subjected to sedimentation velocity (SV) analytical ultracentrifugation (AUC). Additionally, Advate and Kogenate were concentrated and subjected to buffer exchange by ultrafiltration to remove viscous cosolvents for the purpose of measuring s 20,w values and molecular weights. Results The major component of all three products was a population of ~7.5 S heterodimers with a weight-average molecular weight of ~230 kDa. Helixate and Kogenate contained aggregates ranging from 12 S to at least 100 S, representing ≈ 20% of the protein mass. Aggregates greater than 12 S represented < 3% of the protein mass in Advate. An approximately 10.5 S aggregate, possibly representing a dimer of heterodimers, was identified in buffer-exchanged Advat e and Kogenate. SV AUC analysis of a plasma-derived FVIII product was confounded by the presence of von Willebrand factor in molar excess over FVIII. Conclusions Aggregate formation has been identified in recombinant full-length FVIII products, and is more extensive in Helixate and Kogenate than in Advate. SV AUC is an important method for characterizing FVIII products.

The objective of this investigation is to engender greater confidence in the validity of binding equations derived for multivalent ligands on the basis of reacted-site probability theory. To that end, a demonstration of the theoretical interconnection between expressions derived by the classical stepwise equilibria and reacted-site probability approaches for univalent ligands is followed by the use of the traditional stepwise procedure to derive binding equations for bivalent and trivalent ligands. As well as demonstrating the unwieldy nature of the classical binding equation for multivalent ligand systems, that exercise has allowed numerical simulation to be used to illustrate the equivalence of binding curves generated by the two approaches. The advantages of employing a redefined binding function for multivalent ligands are also confirmed by subjecting the simulated results to a published analytical procedure that has long been overlooked.

Background The most important complication in hemophilia A treatment is the development of inhibitory anti-Factor VIII (FVIII) antibodies in patients after FVIII therapy. Patients with severe hemophilia who express no endogenous FVIII, i.e. cross-reacting material (CRM), have the greatest incidence of inhibitor formation. However, current mouse models of severe hemophilia A produce low levels of truncated FVIII. The lack of a corresponding mouse model hampers the study of inhibitor formation in the complete absence of FVIII protein. Objectives We aimed to generate and characterize a novel mouse model of severe hemophilia A (designated the F8TKO strain) lacking the complete coding sequence of F8 and any FVIII CRM. Methods Mice were created on a C57BL/6 background using Cre-Lox recombination and characterized using in vivo bleeding assays, measurement of FVIII activity by coagulation and chromogenic assays, and anti-FVIII antibody production using ELISA. Results All F8 exonic coding regions were deleted from the genome and no F8 mRNA was detected in F8TKO mice. The bleeding phenotype of F8TKO mice was comparable to E16 mice by measurements of factor activity and tail snip assay. Similar levels of anti-FVIII antibody titers after recombinant FVIII injections were observed between F8TKO and E16 mice. Conclusions We describe a new C57BL/6 mouse model for severe hemophilia A patients lacking CRM. These mice can be directly bred to the many C57BL/6 strains of genetically engineered mice making it valuable for studying the impact of a wide variety of genes on FVIII inhibitor formation on a defined genetic background.

Anti-drug antibodies to coagulation factor VIII (fVIII), often termed inhibitors, present the greatest economical and treatment related obstacle in the management of hemophilia A. Although several genetic and environmental risk factors associated with inhibitor development have been identified, the precise mechanisms responsible for the immune response to exogenous fVIII therapies remain undefined. Clinical trials suggest there is an increased immunogenic potential of recombinant fVIII compared to plasma-derived products. Additional biochemical and immunological studies have demonstrated that changes in recombinant fVIII production and formulation can alter fVIII structure and immunogenicity. Recently, one study demonstrated increased immunogenicity of the recombinant fVIII product Helixate in hemophilia A mice following oxidation with hypochlorite (ClO−). It is widely reported that protein aggregates within drug products can induce adverse immune reactions in patients. Several studies have therefore investigated the prevalence of molecular aggregates in commercial recombinant products with and without use-relevant stress and agitation. To investigate the potential link between oxidation-induced immunogenicity and molecular aggregation, we analyzed the recombinant fVIII product, Helixate, via sedimentation velocity analytical ultracentrifugation following oxidation with ClO−. At 80 μM ClO−, a concentration that reduced the specific-activity by 67%, no detectable increase in large molecular aggregates (s > 12 S) was observed when compared to non-oxidized fVIII. This lack of aggregates was demonstrated both in commercial excipient as well as a HEPES buffered saline formulation. These data suggest that oxidation induced immunogenicity is independent of aggregate-mediated immune response. Therefore, our data support multiple, independent mechanisms underlying fVIII immunogenicity.

Several candidate HIV subunit vaccines based on recombinant envelope (Env) glycoproteins have been advanced into human clinical trials. To facilitate biopharmaceutical production, it is necessary to produce these in CHO (Chinese Hamster Ovary) cells, the cellular substrate used for the manufacturing of most recombinant protein therapeutics. However, previous studies have shown that when recombinant Env proteins from clade B viruses, the major subtype represented in North America, Europe, and other parts of the world, are expressed in CHO cells, they are proteolyzed and lack important glycan-dependent epitopes present on virions. Previously, we identified C1s, a serine protease in the complement pathway, as the endogenous CHO protease responsible for the cleavage of clade B laboratory isolates of-recombinant gp120s (rgp120s) expressed in stable CHO-S cell lines. In this paper, we describe the development of two novel CHOK1 cell lines with the C1s gene inactivated by gene editing, that are suitable for the production of any protein susceptible to C1s proteolysis. One cell line, C1s-/-CHOK1 2.E7, contains a deletion in the C1s gene. The other cell line, C1s-/-MGAT1-CHOK1 1.A1, contains a deletion in both the C1s gene and the MGAT1 gene, which limits glycosylation to mannose-5 or earlier intermediates in the Nlinked glycosylation pathway. In addition, we compare the substrate specificity of C1s with thrombin on the cleavage of both rgp120 and human Factor VIII, two recombinant proteins known to undergo unintended proteolysis (clipping) when expressed in CHO cells. Finally, we demonstrate the utility and practicality of the C1s-/-MGAT1-CHOK1 1.A1 cell line for the expression of clinical isolates of clade B Envs from rare individuals that possess broadly neutralizing antibodies and are able to control virus replication without anti-retroviral drugs (elite neutralizer/controller phenotypes). The Envs represent unique HIV vaccine immunogens suitable for further immunogenicity and efficacy studies.

Bacterial toxin–antitoxin systems are important factors implicated in growth inhibition and plasmid maintenance. Type II toxin–antitoxin pairs are regulated at the transcriptional level by the antitoxin itself. Here, we examined how the HigA antitoxin regulates the expression of the Proteus vulgaris higBA toxin–antitoxin operon from the Rts1 plasmid. The HigBA complex adopts a unique architecture suggesting differences in its regulation as compared to classical type II toxin–antitoxin systems. We find that the C-terminus of the HigA antitoxin is required for dimerization and transcriptional repression. Further, the HigA structure reveals that the C terminus is ordered and does not transition between disorder-to-order states upon toxin binding. HigA residue Arg40 recognizes a TpG dinucleotide in higO2, an evolutionary conserved mode of recognition among prokaryotic and eukaryotic transcription factors. Comparison of the HigBA and HigA-higO2 structures reveals the distance between helix-turn-helix motifs of each HigA monomer increases by ~4 Å in order to bind to higO2. Consistent with these data, HigBA binding to each operator is twofold less tight than HigA alone. Together, these data show the HigB toxin does not act as a co-repressor suggesting potential novel regulation in this toxin–antitoxin system.

The most significant complication for patients with severe cases of congenital or acquired hemophiliaAis the development of inhibitor antibodies against coagulation factor VIII (fVIII). The C2 domain of fVIII is a significant antigenic target of antifVIII antibodies. Here, we have utilized small angle x-ray scattering (SAXS) and biochemical techniques to characterize interactions between two different classes of anti-C2 domain inhibitor antibodies and the isolated C2 domain. Multiple assays indicated that antibodies 3E6 and G99 bind independently to the fVIII C2 domain and can form a stable ternary complex. SAXS-derived numerical estimates of dimensional parameters for all studied complexes agree with the proportions of the constituent proteins. Ab initio modeling of the SAXS data results in a long kinked structure of the ternary complex, showing an angle centered at the C2 domain of ∼130°. Guided by biochemical data, rigid body modeling of subunits into the molecular envelope of the ternary complex suggests that antibody 3E6 recognizes a C2 domain epitope consisting of the Arg2209-Ser2216 and Leu 2178-Asp2187 loops. In contrast, antibody G99 recognizes the C2 domain primarily through the Pro2221-Trp2229 loop. These two epitopes are on opposing sides of the fVIII C2 domain, are consistent with the solvent accessibility in the context of the entire fVIII molecule, and provide further structural detail regarding the pathogenic immune response to fVIII.

Background: The hierarchical hemostasis response involves a self-inhibitory feature of von Willebrand factor (VWF) that has not been fully characterized. The residues flanking the A1 domain of VWF are important in this self-inhibition by forming an autoinhibitory module (AIM) that masks the A1 domain. Objectives: To delimit the AIM sequence and to evaluate the cooperative interplay between the discontinuous AIM regions. Methods: ELISA, flow cytometry, a thermal stability assay and hydrogen–deuterium exchange (HDX) mass spectrometry were used to characterize recombinant VWF A1 fragments varying in length. Results: The longest A1 fragment (rVWF1238–1493) showed higher inactivity in binding the platelet receptor glycoprotein (GP) Ibα and greater thermostability than its shorter counterparts. The HDX results showed that most of the N-terminal residues and residues 1459–1478 at the C-terminus of rVWF1238–1493 have slower deuterium uptake than the residues in its denatured counterpart, implying that these residues may interact with the A1 domain. In contrast, residues 1479–1493 showed less difference from the denatured form, indicating that these residues are unlikely to be involved in binding the A1 domain. The A1 fragment that lacks either the entire C-terminal flanking region of the AIM (C-AIM), i.e. rVWF1238–1461, or the entire N-terminal flanking region of the AIM (N-AIM), i.e. rVWF1271–1493, showed high GPIbα-binding affinity and low thermostability, suggesting that removal of either N-terminal or C-terminal residues resulted in loss of AIM inhibition of the A1 domain. Conclusion: The AIM is probably composed of residues 1238–1271 (N-AIM) and 1459–1478 (C-AIM). Neither the N-AIM nor the C-AIM alone could fully inhibit binding of the A1 domain to GPIbα.