Mathieu Ing

Brian E Hew

David C Fritzinger

Sandrine Delignat

Sébastien Lacroix-Desmazes

Carl-Wilhelm Vogel
email: cvogel@cc.hawaii.edu

Julie Rayes

Absence of a neutralizing antibody response to humanized cobra venom factor in mice

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Cobra venom factor (CVF) is the complement-activating protein in cobra venom. It is a structural and functional analog of complement component C3. When CVF is added to serum, it binds Factor B and leads to the formation of an alternative pathway C3/C5 convertase. The convertase (CVF,Bb) is physico-chemically stable, and resistant to inactivation by complement regulatory proteins Factors H and I, causing rapid cleavage of C3 and C5, and resulting in the depletion of the serum complement (reviews: [START_REF] Vogel | Cobra venom factor: Structure, function, and humanization for therapeutic complement depletion[END_REF][START_REF] Vogel | Cobra Venom Factor: The Unique Component of Cobra Venom that Activates the Complement System[END_REF]. The functional similarity of CVF to C3 is corroborated by its structure. Both proteins are synthesized as single-chain pro-proteins which are subsequently processed into the mature proteins with corresponding chain structures (Fig. 1). The sequence identity between CVF and mammalian C3 is about 50%, with a similarity of about 70% [START_REF] Fritzinger | Molecular cloning and derived primary structure of cobra venom factor[END_REF]. Both proteins exhibit a highly similar crystal structure with identical domains [START_REF] Janssen | Structure of C3b reveals conformational changes that underlie complement activity[END_REF][START_REF] Janssen | Insights into complement convertase formation based on the structure of the factor Bcobra venom factor complex[END_REF][START_REF] Krishnan | The crystal structure of cobra venom factor, a cofactor for C3-and C5-convertase CVF,Bb[END_REF].

Ever since it had been demonstrated that CVF can be safely administered to laboratory animals [START_REF] Cochrane | Depletion of plasma complement in vivo by a protein of cobra venom: Its effect on various immunologic reactions[END_REF], the ability of CVF to exhaustively activate, and thereby deplete, complement has been exploited in innumerable studies as a tool to study the role of complement in host defense as well as pathogenesis of disease. As a matter of fact, our knowledge of the role of complement in the pathogenesis of many diseases was established by comparing normal animals with complementdepleted animals. As complement depletion by CVF eliminates the pathogenic contribution of complement to the disease process, complement depletion has more recently been recognized as a potentially valuable therapeutic approach in diseases with complement pathogenesis (Vogel andFritzinger, 2007, Fritzinger et al., 2008;[START_REF] Vogel | Humanized cobra venom factor: Structure, activity, and therapeutic efficacy in preclinical disease models[END_REF]. However, CVF is immunogenic, generating an antibody response that limits its usefulness for complement depletion to a single injection (Cochrane et al., 1970, Pryjma and[START_REF] Pryjma | Prolonged C3 depletion by cobra venom factor in thymus-deprived mice and its implication for the role of C3 as an essential second signal for B-cell triggering[END_REF]. After identifying that the very Cterminal part of CVF harbors the important structures for forming a stable convertase [START_REF] Grunwald | Cobra venom factor: An intact β-Chain is not required for activity[END_REF][START_REF] Hew | Hybrid proteins of cobra venom factor and cobra C3: Tools to identify functionally important regions in cobra venom factor[END_REF], we attempted to overcome the limitation of immunogenicity by creating chimeric proteins in which C-terminal amino acid residues of human C3 were replaced by the homologous sequence from CVF. These chimeric proteins are human C3 derivatives, termed "humanized CVF" (hCVF), which exhibit rapid complement depletion in vitro and in vivo just like natural CVF [START_REF] Fritzinger | Functional characterization of human C3/cobra venom factor hybrid proteins for therapeutic complement depletion[END_REF][START_REF] Vogel | Cobra venom factor: Structure, function, and humanization for therapeutic complement depletion[END_REF][START_REF] Vogel | Humanized cobra venom factor: Structure, activity, and therapeutic efficacy in preclinical disease models[END_REF]. Our lead hCVF protein (termed HC3-1496) consists of human C3 in which the 168 C-terminal amino acid residues are homologous to CVF. HC3-1496 exhibits an overall protein sequence identity with human C3 of 94.3%; and even within the 168 C-terminal residues from CVF, 44.05% are identical to human C3 (Fig. 1). hCVF has been shown in multiple preclinical disease models with complement pathogenesis to be a promising therapeutic agent. These include age-related macular degeneration (AMD) [START_REF] Fritzinger | Complement depletion with humanized cobra venom factor in a mouse model of agerelated macular degeneration[END_REF], myocardial and gastrointestinal ischemia reperfusion injury [START_REF] Gorsuch | Humanized cobra venom factor decreases myocardial ischemia-reperfusion injury[END_REF][START_REF] Vogel | Humanized cobra venom factor: Structure, activity, and therapeutic efficacy in preclinical disease models[END_REF], ventilator-induced lung injury [START_REF] Takahashi | Complement 3 is involved with ventilator-induced lung injury[END_REF], collagen-induced arthritis [START_REF] Fritzinger | Derivatives of human complement component C3 for therapeutic complement depletion: A novel class of therapeutic agents[END_REF], paroxysmal nocturnal hemoglobinuria (PNH) [START_REF] Vogel | Cobra venom factor: Structure, function, and humanization for therapeutic complement depletion[END_REF], myasthenia gravis [START_REF] Vogel | Humanized cobra venom factor: Structure, activity, and therapeutic efficacy in preclinical disease models[END_REF], and monoclonal antibody therapy of lymphoma [START_REF] Wang | Depletion of the C3 component of complement enhances the ability of rituximabcoated target cells to activate human NK cells and improves the efficacy of monoclonal antibody therapy in an in vivo model[END_REF]. In three disease models (AMD, arthritis, and myasthenia gravis), multiple injections of hCVF resulted in depletion for up to 30 days, suggesting the absence of a neutralizing antibody response to hCVF. This observation was recently extended in a mouse model of hemophilia A, aimed at reducing the anti-Factor VIII immune response to treatment with recombinant Factor VIII in Factor VIII knockout mice. A significant reduction in the levels of induced anti-Factor VIII antibodies was achieved by four cycles of complement depletion with hCVF in weekly intervals (Rayes et al., 2018). Here we report that repeated injections of hCVF into Factor VIII knock-out mice, as well as wild-type mice, lead to repeated complement depletion; and although antibodies are generated against hCVF, crossreacting with natural CVF, recombinant CVF, and human C3, they do not neutralize the complementdepleting activity of hCVF.

Material and methods

Materials.

Restriction enzymes and calf intestinal phosphatase were from New England Biolabs (Beverly, MA). T4 DNA ligase was either from New England Biolabs, or from Invitrogen (Carlsbad, CA). Oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA). Fastart TAQ DNA polymerase and Fastart High Fidelity DNA polymerase were obtained from Roche Applied Sciences, Inc (Indianapolis, IN). The D. melanogaster S2 expression plasmid, pMT/Bip-V5-HisA , all Drosophila S2 media, and fetal calf serum were from Invitrogen. Human complement protein C3 was purchased from Merck Millipore (Merck Chemicals Ltd, Nottingham, United Kingdom). Natural CVF was purified from lyophilized Naja kaouthia venom (Miami Venom Laboratories, Punta Gorda, Florida, USA) as described (Vogel and Müller-Eberhard, 1984). Recombinant CVF (rCVF) was produced in D. melanogaster S2 cells as described [START_REF] Kock | Structure and function of recombinant cobra venom factor[END_REF]. rCVF is a mixture of C3-like and C3b-like proteins [START_REF] Kock | Structure and function of recombinant cobra venom factor[END_REF]) (Fig. 1). Polyclonal goat antimouse C3 antibodies were purchased from MP Biomedicals (Illkirch, France). Biotinylated polyclonal goat anti-mouse C3 antibodies and streptavidin-conjugated horseradish peroxidase (HRP) were from Expression and purification of HC3-1496. pOptiVEC-1496(sig)-3# and pOptiVEC-1496-3# were transfected into CHO cells by electroporation. Following transfection, stably transfected cells were selected using either 200 nM or 500 nM methotrexate in liquid media. High-producing clones were then selected by plating between 2,500 and 10,000 cells onto semisolid media containing a fluorescent anti-CVF antibody in petri dishes. Colonies were allowed to grow for 10 days, and colonies producing HC3-1496 were detected, either by detecting fluorescent colonies, or colonies with a ring of immunoprecipitates. Colonies producing high yields of HC3-1496 were selected, and plaque purified. The HC3-1496 protein was purified by a combination of Capto-Q, Butyl FF HiTrap, and Q-HP HiTrap chromatography. Purified HC3-1496 is a mixture of C3-like and C3b-like proteins [START_REF] Fritzinger | Functional characterization of human C3/cobra venom factor hybrid proteins for therapeutic complement depletion[END_REF].

Animals and in vivo complement depletion. Wild-type C57Bl/6 mice (Janvier, Saint-Berthevin, France) and Factor VIII exon 16-invalidated C57Bl/6 mice (gift from Prof. H.H. Kazazian, University of Pennsylvania School of Medicine, Philadelphia, PA, USA) [START_REF] Bi | Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A[END_REF] were handled at 8-12 weeks-old, in agreement with French ethical authorities (authorization #23BA53). Complement depletion was performed by intraperitoneal injections of nCVF or hCVF in mice (1 mg/kg, diluted in PBS), once a week for 4 consecutive weeks. Retro-orbital sampling was performed to collect blood once a week for four weeks three hours after each hCVF injection, and EDTA (50 mM) was added to the blood samples to prevent ex vivo complement activation. Plasma was isolated from the blood and kept at -80°C until use.

Measurement of circulating C3 in mouse plasma.

Ninety-six-well flat-bottomed ELISA plates (Nunc Maxisorp ® , Roskilde, Denmark) were coated with a polyclonal goat anti-mouse C3 antibody at 3.3 µg/ml in bicarbonate buffer, pH 9.5, overnight at 4°C. Wells were then blocked in PBS, 0.1% Tween-20 and 3% bovine serum albumin (BSA), pH 7.4. Plasma were serially diluted in blocking buffer and added to anti-C3 coated wells for 1 hour at 37°C. Bound C3 was revealed using a biotinylated polyclonal goat anti-mouse C3 antibody (1.6 µg/ml), followed by streptavidin conjugated to horseradish peroxidase using o-phenylenediamine dihydrochloride (OPD) as substrate. Absorbance was measured at 492 nm. Serially diluted normal plasma was used as standard and considered at 100% C3 level. The level of residual C3 in mice was expressed as a percentage of the value in normal plasma. Although cross-reactivity between CVF and antisera to mammalian C3 is very weak (Vogel et al., 1984), we ensured that the measurements of plasma C3 levels after injection with nCVF or hCVF were not altered by any potential cross-reaction (see Supplementary Fig. 1).

Titration of anti-human C3

, anti-rCVF, anti-hCVF, and anti-nCVF IgG. ELISA plates were coated with human C3 (hC3), hCVF, nCVF, and rCVF (10 nM) in bicarbonate buffer, pH 9.5, overnight at 4°C. After blocking with PBS, 0.1% Tween-20 and 3% BSA, pH 7.4, serially diluted plasma was incubated for 1 hour at 37°C. Bound IgG were revealed using a HRP-coupled polyclonal goat anti-mouse IgG antibody and the OPD substrate. Absorbance was then measured at 492 nm.

Statistical analysis. Statistical significance was assessed using the double-sided nonparametric Mann-Whitney test.

Results

Repeated administration of hCVF causes repeated depletion of complement in vivo.

In order to assess the efficiency of hCVF for repeated complement depletion in mice, we compared complement C3 depletion following weekly injections of humanized (hCVF) or natural CVF (nCVF) in mice. This experiment was performed in Factor VIII knock-out mice, a preclinical model of hemophilia A, a pathology in which we have observed an important role of complement in the elicitation of anti-Factor VIII antibodies (Rayes et al., 2018). The first injection of either hCVF or nCVF resulted in rapid and drastic complement depletion in all animals as measured by the residual mouse C3 (muC3) levels in plasma. Indeed, circulating muC3 levels decreased from 100% to 6.4% [4.5-11.4%] (median with [interquartile range]) for nCVF (Fig. 2A) and to 19.5% [8.3-27.8%] for hCVF (Fig. 2B), with significant mouse-to-mouse variation. Figure 3 shows a time course of C3 depletion after a single injection of hCVF in Factor VIII knock-out mice, demonstrating that the absence of Factor VIII does neither affect the rapid depletion of C3 nor its return to normal levels after one to two days as shown previously for wild-type mice and other species [START_REF] Vogel | Cobra venom factor: Structure, function, and humanization for therapeutic complement depletion[END_REF]. Subsequent weekly injections of hCVF consistently caused efficient complement depletion to residual levels of C3 of about 20% [14-27%], no different from what was seen after the first injection, even at week four (Fig. 2B). In contrast, second and third weekly administrations of nCVF failed to deplete complement in a consistent manner, with resulting C3 levels varying greatly between normal and essentially fully depleted (Fig. 2A); and only a fourth weekly administration rendered nCVF essentially inactive (Fig. 2A). These results strongly suggest that repeated administration of hCVF, in contrast to nCVF, does not elicit a neutralizing antibody response.

Repeated administration of hCVF induces a non-neutralizing humoral immune response in mice.

To exclude the remote possibility that the absence of Factor VIII affected the antibody response to hCVF, we repeated the four weekly injection schedule with hCVF in C57Bl/6 wild-type mice. As previously observed in Factor VIII-deficient mice, injections of hCVF in wild-type mice resulted in a significant reduction of circulating C3 level: 43.9% [30.0-53.4] (Fig. 4A), again with significant mouse-to-mouse variation. After four weeks, we also determined the presence of anti-hCVF IgG antibodies. Mice receiving hCVF treatment developed anti-hCVF antibodies, as compared to the PBS control group (0.236 [0.115-0.507] AU vs 0.052 [0.049-0.056] AU, respectively) (Fig. 4B). However, there was significant variation from mouse to mouse, with approximately one quarter of the animals exhibiting essentially non-detectable antibody levels, and another quarter very low levels (Fig. 4B).

Moreover, the anti-hCVF IgG levels in individual mice did not correlate with the levels of remaining muC3 (r²=0.0440, p=0.3750) (Fig. 4C). These results suggest that hCVF exhibits an overall low degree of immunogenicity in mice, and that anti-hCVF IgG produced in mice lack neutralizing activity toward hCVF.

Anti-hCVF antibodies recognize antigenic epitopes on both CVF and human C3. hCVF protein HC3-1496 is a human C3 derivative with nCVF-like functions, obtained by replacing a short region of the C-terminus of the human C3 α-chain with the homologous sequence from nCVF (Fig. 1). The protein sequence of the chimeric single-chain pro-protein HC3-1496 is 94.3% identical to human C3; this includes the 168 residues long CVF portion of the protein where 44.05% of the residues are identical to human C3. hCVF protein HC3-1496, like other hCVF proteins [START_REF] Fritzinger | Functional characterization of human C3/cobra venom factor hybrid proteins for therapeutic complement depletion[END_REF] and recombinant CVF (rCVF) [START_REF] Kock | Structure and function of recombinant cobra venom factor[END_REF], is usually expressed as a mixture of a C3-like and C3b-like form (Fig. 1). In order to identify the immunogenic regions of hCVF in mice, we assessed the cross-reactivity of the anti-hCVF antibodies with nCVF, rCVF, and human C3 (huC3). The anti-hCVF antibodies recognized rCVF and nCVF similarly to hCVF (Fig. 5A,B). Anti-hCVF antibodies also bound to huC3, although many mice had essentially non-detectable levels of anti-hCVF antibodies cross-reacting with huC3 (Fig. 5C). The mean level of anti-hCVF antibodies cross-reacting with huC3 was significantly lower compared to hCVF, albeit only at a p-value of 0.02.

There was a highly significant correlation when the levels of anti-hCVF were plotted against the levels of anti-nCVF, anti-rCVF, and anti-huC3 antibodies (p<0.0001 in each case; Fig. 5D,E,F), indicating that mice with higher overall levels of anti-hCVF antibodies also showed increased binding to both CVF and huC3 epitopes, excluding the possibility that the higher antibody levels reflect preferential immunogenicity of CVF-specific or huC3-specific epitopes.

As shown above for anti-hCVF (Fig 4C ), the levels of anti-nCVF, anti-rCVF, and anti-huC3 did not display any significant correlation with the levels of remaining circulating C3 in individual mice after the fourth injection with hCVF (Fig. 5G,H,I). This observation is consistent with the fact that the antibodies elicited against hCVF do not interfere with its C3-depleting activity, and are devoid of a neutralizing function toward hCVF.

Discussion

Humanized CVF (hCVF) is a term coined for human C3 derivatives where the C-terminal portion of the C3 α-chain has been exchanged with the homologous sequence from CVF, thereby introducing the CVF-specific functions of forming a physico-chemically stable C3 convertase, and exhibiting resistance to inactivation by the regulatory proteins Factors H and I. These two properties of hCVF, like natural CVF from cobra venom, are a prerequisite for continuous C3 activation, leading to complement depletion both in vitro and in vivo [START_REF] Fritzinger | Functional characterization of human C3/cobra venom factor hybrid proteins for therapeutic complement depletion[END_REF][START_REF] Vogel | Humanized cobra venom factor: Structure, activity, and therapeutic efficacy in preclinical disease models[END_REF][START_REF] Vogel | Cobra Venom Factor: The Unique Component of Cobra Venom that Activates the Complement System[END_REF]. hCVF was developed as a biopharmaceutic for therapeutic complement depletion in diseases where the complement system is involved in the disease pathogenicity. The efficacy of complement depletion with hCVF was subsequently demonstrated in multiple animal models of disease [START_REF] Vogel | Humanized cobra venom factor: Structure, activity, and therapeutic efficacy in preclinical disease models[END_REF]Rayes et al., 2018).

CVF has been used for well over four and a half decades for safe complement depletion in laboratory animals, from mice to primates. The sole adverse effect known is an acute but fleeting pulmonitis [START_REF] Till | Activation of C5 by cobra venom factor is required in neutrophil-mediated lung injury in the rat[END_REF][START_REF] Mulligan | Requirement and role of C5a in acute lung inflammatory injury in rats[END_REF], observed only after massive complement activation in plasma by nCVF. The nCVF-dependent convertase not only cleaves C3 but also C5. The resulting massive generation of C5a overwhelms the body's protective mechanisms, leading to C5a-mediated neutrophil activation with subsequent sequestration to the lungs and causing pulmonitis. In contrast to nCVF, the convertase formed with hCVF does not cleave C5 [START_REF] Vogel | Cobra venom factor: Structure, function, and humanization for therapeutic complement depletion[END_REF][START_REF] Vogel | Humanized cobra venom factor: Structure, activity, and therapeutic efficacy in preclinical disease models[END_REF]. Accordingly, complement depletion with hCVF does not generate C5a; and even intra-arterial injection into the pulmonary artery of cynomolgus monkeys had no effect on pulmonary function. Consistent with the decades-long experience of safe complement depletion with nCVF, and the absence of C5a generation by hCVF, no adverse effects of complement depletion with hCVF were seen in any of the animal models of disease [START_REF] Vogel | Humanized cobra venom factor: Structure, activity, and therapeutic efficacy in preclinical disease models[END_REF].

In addition to efficacy and safety, immunogenicity is an important property of any biotherapeutic. We found that hCVF efficiently depletes complement in mice after each of four weekly administrations. In contrast, nCVF resulted in consistent and efficient complement depletion only after the first administration. Subsequent injections over two weeks resulted in inconsistent complement depletion, with significant variation from mouse to mouse. A fourth injection rendered nCVF essentially inactive. These results confirm earlier studies that demonstrated that CVF is a potent immunogen, eliciting neutralizing antibodies [START_REF] Cochrane | Depletion of plasma complement in vivo by a protein of cobra venom: Its effect on various immunologic reactions[END_REF]Pryjma and Humphrey, 1974), and that nCVF is a reliable reagent for complement depletion in nCVF naïve animals only. Our finding that hCVF exhibits effective complement depletion after repeated administration indicates that hCVF does not induce a neutralizing antibody response, at least after multiple injections over a four-week period.

There is always a significant variation from animal to animal, both with regard to the initial complement levels (Fig. 4A) and the extent of complement depletion (Figs. 2B,3,4A). We have no experimental evidence, or any explanation, why complement depletion with nCVF would differ from hCVF, and that the absence of Factor VIII would affect the complement depletion by nCVF or hCVF. Although means and interquartile ranges were somewhat different, 25% of the remaining C3 levels in wild-type mice after depletion with hCVF (Fig. 4A) were at the same level of Factor VIII knock-out mice (Fig. 2B), and that many Factor VIII knock-out mice after depletion with hCVF exhibited similarly low remaining C3 levels as seen after depletion with nCVF (compare Figs. 2B and3 with Fig. 2A).

We analyzed the antibody response to hCVF after four weekly administrations. We detected anti-hCVF IgG antibodies in the murine plasma. However, the overall antigenicity of hCVF appears to be low. There was significant variation in the antibody levels of individual mice, with approximately one quarter of the animals exhibiting essentially non-detectable antibody levels, and another quarter very low levels. The anti-hCVF antibodies cross-reacted with nCVF and rCVF as well as huC3, indicating that both CVF-specific epitopes and huC3-specific epitopes of hCVF contribute to the immunogenicity of hCVF in mice. Our data do not allow to locate antigenic epitopes on the hCVF protein. Based on primary protein sequence differences to murine C3, there are CVF-specific residues in the human C3 portion of hCVF, and huC3-specific residues in the nCVF-portion of hCVF. However, the number of mouse plasma with very low to non-detectable antibodies cross-reacting with human C3 was much lower compared to plasma cross-reacting with rCVF and nCVF, suggesting that CVF-specific sequence differences are more immunogenic despite the fact that there are significantly more huC3-specific sequence differences. On the other hand, we found that mice with higher overall levels of anti-hCVF antibodies also showed increased binding to both CVF and huC3 epitopes, thereby excluding the possibility that the higher antibody levels reflect preferential immunogenicity of CVF-specific or huC3-specific epitopes.

Although mice mount an antibody response to hCVF, our data demonstrate that these antibodies are not neutralizing the complement-depleting activity of hCVF. This is further supported by the observation that there was no correlation between anti-hCVF antibody levels and remaining C3 levels in complement-depleted mice. There are likely several explanations for the low immunogenicity of hCVF and the lack of neutralizing antibodies. For one, the protein sequence of HC3-1496 is highly homologous to murine C3 (approximately 78% identity in the large human C3 portion, and approximately 43% identity within the C-terminal 168 residues). Moreover, hCVF, just like nCVF and the C3 proteins of all mammals, must contain the identical protein structures required for binding to Factor B and forming a convertase. These include the C345C domain at the C-terminus of C3 which binds the Bb portion of Factor B, and other domains of C3, in particular the the CUB g domain, which is required for the initial contact with the Ba portion of Factor B, first described by us [START_REF] O'keefe | A novel cleavage product of human complement component C3 with structural and functional properties of cobra venom factor[END_REF], and subsequently confirmed by crystallography of the CVF,B complex [START_REF] Janssen | Insights into complement convertase formation based on the structure of the factor Bcobra venom factor complex[END_REF]. These structures of C3 are therefore highly conserved throughout the mammals (and likely all vertebrates), and consequently not immunogenic. Lastly, just like complement depletion with hCVF reduces the antibody response to Factor VIII (Rayes et al., 2018), the hCVF-induced complement depletion may aid in lowering its own immunogenicity.

Our results were obtained by injecting hCVF into mice. It may therefore not be unreasonable to speculate that hCVF might be even less immunogenic in humans as the total number of different amino acids between HC3-1496 and murine C3 will be reduced from approximately 400 to less than 100 residues between HC3-1496 and human C3, all within the 168 amino acid long C-terminal sequence only. Moreover, the three-dimensional structure of the C-terminal sequence has been predicted by in silico analysis to be essentially identical between human C3 and hCVF [START_REF] Fritzinger | Derivatives of human complement component C3 for therapeutic complement depletion: A novel class of therapeutic agents[END_REF][START_REF] Vogel | Humanized cobra venom factor: Structure, activity, and therapeutic efficacy in preclinical disease models[END_REF]. However, immunogenicity is not only determined by primary structure differences, and complement depletion by hCVF in itself may affect an antibody response differently in humans than in mice. Ultimately, the degree of immunogenicity of hCVF in humans is unpredictable, and will have to await clinical trials.

In conclusion, hCVF is a promising therapeutic agent for the many diseases with complement pathogenesis. It has been shown to be efficacious in multiple animal disease models, it has no known adverse effects, and as shown here, it does not induce neutralizing antibodies in mice, at least not after multiple injections over a one-month period. The effect of longer-term complement depletion with hCVF on the risk of developing infections is unknown. However, complement depletion with hCVF in never complete. There is always a significant amount of C3 remaining, along with normal levels of C5 and the other terminal complement components, ensuring some protection against infections. This is convincingly demonstrated by CVF-transgenic mice which exhibit a normal life span and, in contrast to C3 knock-out mice, no tendency to develop infections [START_REF] Andrä | Generation and characterization of transgenic mice expressing cobra venom factor[END_REF][START_REF] Fritzinger | Complement depletion with humanized cobra venom factor in a mouse model of agerelated macular degeneration[END_REF].

Figure Legends

Figure 1: Schematic representation of the chain structures of human C3, hCVF (HC3-1496), and CVF. Shown are the single-chain structures of pro-C3, pro-HC3-1496, and pro-CVF. The Ntermini are to the left. The chain homologies are indicated. Please note that the nomenclature of the three chains of nCVF (α-chain, β-chain, γ-chain) was coined before their homology to C3 chains was established by N-terminal sequencing [START_REF] Eggertsen | Molecular Characterization of the complement activating protein in the venom of the Indian cobra (Naja n. siamensis)[END_REF], immunologic cross-reaction [START_REF] Eggertsen | Antigenic relationships between human and cobra complement factors C3 and cobra venom factor (CVF) from the Indian cobra (Naja naja)[END_REF], and molecular cloning [START_REF] Fritzinger | Molecular cloning and derived primary structure of cobra venom factor[END_REF]. Both hCVF and rCVF are usually expressed as a mixture of C3-like and C3b-like proteins [START_REF] Fritzinger | Functional characterization of human C3/cobra venom factor hybrid proteins for therapeutic complement depletion[END_REF][START_REF] Kock | Structure and function of recombinant cobra venom factor[END_REF].

Please note that rCVF is fully active without processing into the three-chain structure of nCVF [START_REF] Kock | Structure and function of recombinant cobra venom factor[END_REF].

Figure 2 .

 2 Figure 2. Repeated injection of hCVF, but not natural CVF, causes repeated complement depletion in mice.Factor VIII-deficient mice were injected intraperitoneally with (Panel A) nCVF (n=8) or (Panel B) hCVF (HC3-1496, n=24) at a dose of 1 mg/kg, once a week for 4 consecutive weeks. Blood was collected before (Week 0) and weekly 3 hours post-CVF injection (Weeks 1-4). Residual circulating mouse C3 (muC3) level was measured by sandwich ELISA. Results are expressed as a percentage of circulating C3 in normal plasma, measured in mice before depletion and considered at 100%. Horizontal bars represent the median of each time point.

Figure 3 .

 3 Figure 3. Time course of C3 depletion with hCVF in Factor VIII knock-out mice. Factor VIII knock-out mice were injected intraperitoneally with 20 μg hCVF. Blood was collected at different time points as indicated, and C3 levels were measured in plasma by sandwich ELISA.

Figure 4 .

 4 Figure 4. Repeated administration of hCVF is associated with a non-neutralizing humoral immune response. Wild-type mice were injected intraperitoneally with PBS (n=20) or HC3-1496 (hCVF, n=20) at a dose of 1 mg/kg, once a week for 4 consecutive weeks. Four weeks following the first injection, blood was collected and residual mouse C3 (muC3) and anti-hCVF antibody levels were measured in the plasma. Panel A. Residual muC3 levels in plasma was measured by ELISA. Results are expressed as a percentage compared to a normal C3 plasma level, considered at 100%. Horizontal bars represent the median for each group. Panel B. Anti-hCVF IgG levels in plasma were assessed by ELISA. Results are expressed in optical densities measured at 492 nm (arbitrary units, AU). Horizontal bars represent the median for each group. Panel C. Correlation between the level of anti-hCVF IgG and the residual percentage of circulating muC3 in mice treated with hCVF. The linear regression curve is represented as a full line curve, and the p and r² values are indicated.

Figure 5 .

 5 Figure 5. Antibodies against hCVF recognize both CVF-and human C3-specific epitopes. Wildtype mice were injected intraperitoneally with PBS (n=20) or HC3-1496 (hCVF, n=20) once a week for four consecutive weeks, and blood was collected four weeks following the first injection. Panels A, B, C. Levels of anti-natural CVF (nCVF, Panel A), anti-recombinant CVF (rCVF, Panel B), and anti-human C3 (huC3, Panel C) IgG in the plasma were assessed by ELISA. Results are expressed as optical densities measured at 492 nm (arbitrary units, AU). Horizontal bars represent the median for each group. Panels D, E, F. Correlations between the levels of anti-nCVF (Panel D), anti-rCVF (Panel E), and anti-huC3 (Panel F) IgG compared to the level of anti-hCVF IgG are shown. The linear regression curves are represented as full line curves, and the corresponding p and r² values are indicated. Panels G, H, I. Correlations between the levels of anti-nCVF (Panel G), anti-rCVF (Panel H), and anti-huC3 (Panel I) IgG and the residual circulating C3 levels in hCVF-treated mice are shown. The linear regression curves are represented as full line curves, and the corresponding p and r² values are indicated.

Acknowledgments

This study was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie (UPMC) Paris 6, by a grant from Pfizer (Aspire Haemophilia research award 2016 WI212828), by a grant from Incode Corporation, San Diego, CA, and by funds from the University of Hawaii Cancer Center. MI was the recipient of a fellowship from Ministère de l'enseignement supérieur et de la recherche.

Authors' contributions

Designed research: MI, SLD, CWV, JR Performed experiments: MI, BEH, DCF, SD, JR Analyzed data: MI, SLD, CWV, JR Wrote the paper: MI, SLD, CWV, JR

Supplementary Material

To exclude the possibility that the measurements of C3 levels in mice after injection with nCVF or hCVF by the sandwich ELISA assay were falsely overestimated by any cross-reaction of nCVF or hCVF with the anti-muC3antibodies used in the assay, we assessed the possible cross-reaction. Supplemental Fig. 1 shows that neither nCVF, rCVF, nor hCVF of up to 150 μg/ml cross-reacts with the anti-muC3 antibodies employed in the assay. Given that mice (of approximately 20 g body weight) were injected with 20 μg hCVF or nCVF, 150 μg/ml is at least one order of magnitude above the potential peak plasma concentration. We had previously shown that the peak plasma concentration in mice after injection of 10 μg hCVF was 1.5 μg/ml, two orders of magnitude below 150 μg/ml.

Figure Legend

Supplemental Figure 1: Lack of cross-reactivity of nCVF, rCVF, and hCVF with anti-muC3. The sandwich ELISA assay for murine C3 was performed with serial 1:10 dilutions of nCVF (*), rCVF (▼), and hCVF (Δ), starting at 150 μg/ml. Mouse plasma from Factor VIII knock-out mice at a 1:10 dilution (with an estimated concentration of C3 of 150 μg/ml) and serial 1:10 dilutions served as positive control (•). Results are expressed in optical densities measured (arbitrary units, AU).


Sandrine Delignat

Ivan Peyron

Maria El Ghazaly

Srinivas V Kaveri

Jan Rohde

Frank Mueller

Sebastien Lacroix-Desmazes
email: sebastien.lacroix-desmazes@crc.jussieu.fr

Biochemical characterization and immunogenicity of Neureight, a recombinant full-length factor VIII produced by fed-batch process in disposable bioreactors

Keywords:

Hemophilia A is a X-linked recessive bleeding disorder consecutive to the lack of circulating pro-coagulant factor VIII (FVIII). The most efficient strategy to treat or prevent bleeding in patients with hemophilia A relies on replacement therapy using exogenous FVIII. Commercially available recombinant FVIII are produced using an expensive perfusion technology in stainless steel fermenters. A fed-batch fermentation technology was recently developed to produce 'Neureight', a full-length recombinant human FVIII, in Chinese hamster ovary (CHO) cells. Here, we investigated the structural and functional integrity and lack of increased immunogenicity of Neureight, as compared to two commercially available full-length FVIII products, Helixate and Advate, produced in baby hamster kidney or CHO cells, respectively. Our results demonstrate the purity, stability and functional integrity of Neureight with a standard specific activity of 4235 ± 556 IU/mg. The glycosylation and sulfation profiles of Neureight were similar to that of Advate, with the absence of the antigenic carbohydrate epitopes α-Gal and Neu5Gc, and with sulfation of Y1680, that is critical for FVIII binding to von Willebrand factor (VWF). The endocytosis of Neureight by human immature dendritic cells was inhibited by VWF, and its half-life in FVIIIdeficient mice was similar to that of Advate, confirming unaltered binding to VWF. In vitro and in vivo assays indicated a similar immunogenicity for Neureight, Advate and Helixate. In conclusion, the production of fulllength FVIII in a fed-batch fermentation mode generates a product that presents similar biochemical, functional and immunogenic properties as products developed using the classical perfusion technology.

Introduction

Hemophilia A is a X-linked recessive bleeding disorder consecutive to the lack of circulating pro-coagulant factor VIII (FVIII) [START_REF] Mannucci | The hemophilias-from royal genes to gene therapy[END_REF]. Treatment and prevention of bleedings in patients with hemophilia A rely on replacement therapy using either plasma-derived or recombinant FVIII [START_REF] Peyvandi | A randomized trial of factor VIII and neutralizing antibodies in hemophilia A[END_REF]. FVIII is among the most expensive protein therapeutics on the market owing to the low yields of production of the glycoprotein in the case of recombinant products and to the different steps required to eliminate potential viral agents [START_REF] Coppola | Potter Study, Cost-effectiveness analysis of late prophylaxis vs. ondemand treatment for severe haemophilia A in Italy[END_REF]. The poor stability of FVIII [START_REF] Spencer | Lentiviral vector platform for production of bioengineered recombinant coagulation factor VIII[END_REF][START_REF] Fantacini | The F309S mutation increases factor VIII secretion in human cell line[END_REF] is an additional constraint that participates in increase of production costs. Production processes for recombinant FVIII (both full-length or B domain-deleted) are all based on expensive perfusion technology in stainless steel fermenters [START_REF] Boedeker | Recombinant factor VIII (Kogenate®) for the treatment of hemophilia A: the first and only world-wide licensed recombinant protein produced in high-throughput perfusion culture[END_REF][START_REF] Boedeker | Production processes of licensed recombinant factor VIII preparations[END_REF][START_REF] Riedel | A new technology standard for safety and efficacy in factor VIII replacement therapy: designing an advanced category rFVIII concentrate[END_REF]. The use of continuous perfusion technology allows the growth of FVIII-producing cells to high concentrations while ensuring a relatively short protein residence time [START_REF] Pollock | Fed-batch and perfusion culture processes: economic, environmental, and operational feasibility under uncertainty[END_REF][START_REF] Kim | Effect of transmembrane pressure on Factor VIII yield in ATF perfusion culture for the production of recombinant human Factor VIII co-expressed with von Willebrand factor[END_REF]. Shorter residence times were proposed to allow efficient production and less disruption of the recombinant FVIII (rFVIII) protein [START_REF] Wurm | Production of recombinant protein therapeutics in cultivated mammalian cells[END_REF], thus minimizing the generation of potentially immunogenic FVIII degradation products. To address the need for better supply of affordable rFVIII at global level, efforts have focused on cell line development, process improvements and optimization of expression rates in commercial mammalian cell culture processes. Nevertheless, expression does typically not exceed 0.1 mg FVIII/L cell culture supernatant for a full-length FVIII [START_REF] Soukharev | Expression of factor VIII in recombinant and transgenic systems[END_REF].

We recently developed a fed-batch fermentation technology based on disposable fermentation technology to produce a full-length recombinant human FVIII. Compared to perfusion technology, fed-batch processes have significant advantages, such as ease of process validation and characterization, better lot definition and consistency, ease of downstream processing, overall reduced time to product approval, reduced medium consumption, reduced waste generation, less manpower requirements [START_REF] Pollock | Fed-batch and perfusion culture processes: economic, environmental, and operational feasibility under uncertainty[END_REF][START_REF] Whitford | Fed-batch mammalian cell culture[END_REF], easier technology transferability and less automation requirements. Current state-of-the-art single-use disposable technologies have been recognized as an increasing trend in biopharmaceutical manufacturing due to several advantages like reduction of the risk for cross-contamination, elimination of cleaning steps and an increase in flexibility, ease for handling and an estimated reduction in costs between 20 and 40% [START_REF] Shukla | Single-use disposable technologies for biopharmaceutical manufacturing[END_REF][START_REF] Levine | Single-Use Technology and Modular Construction[END_REF][START_REF] Noack | Single-Use Stirred Tank Reactor BIOSTAT CultiBag STR: Characterization and Applications, Single-Use Technology in Biopharmaceutical Manufacture[END_REF]. In the present work, the new fulllength FVIII, referred to as Neureight, was compared to available fulllength recombinant products, and the immunogenicity was assessed in in vitro and in vivo assays.

Material and methods

Sources of FVIII and VWF

Neureight is a recombinant full-length FVIII produced in eukaryotic CHO cells using a fed-batch process in a disposable setting in the absence of any stabilizing agents such as of VWF or human serum albumin. The purification of Neureight involves a step of affinity chromatography using the VIIIselect matrix (GE Healthcare, Piscataway, NI, USA) followed by traditional ion exchange, a hydrophobic interaction chromatography steps and virus reduction steps such as detergent treatment and nanofiltration. Helixate and Advate were purchased from CSL-Behring (Marburg, Germany) and Shire (Vienna, Austria), respectively. For in vitro studies, the three full-length FVIII were reconstituted in their respective excipients and dialyzed against DMEM-F12 for 2 h at 4 °C. FVIII were then aliquoted and stored at -80 °C until use. FVIII:Ag and FVIII:C in aliquots were assessed by Asserachrom (Stago, Asnières sur Seine, France) and by chromogenic assay (Dade-Behring, Marburg, Germany), respectively. Total protein content was measured by Bradford using bovine serum albumin as a standard. FVIII levels are indicated as International Units (IU) per ml based on the FVIII:Ag levels. Wilfactin (LFB, Les Ulis, France) was used a source of VWF. It was dialyzed in DMEM-F12 during 2 h at 4 °C, and aliquoted and stored at -80 °C.

Electrophoretic and Western blot analyses

SDS-PAGE was performed using 4-12% Bis-TrisNuPage gels (Life Technologies, Carlsbad, California, USA) followed by a staining with a Silver Express Stain Kit (Life Technologies). Western blotting was carried out using an anti-FVIII antibody GMA-012 (Green Mountain Antibodies, Burlington, VT, USA) which recognizes the A2 domain epitopes at residues 497-510 and 584-593, followed by an alkaline phosphatase conjugated anti-mouse antibody (Sigma-Aldrich Chemie GmbH, Munich, Germany).

Glycan profiling

MALDI-TOF MS analysis of N-glycans was performed after deglycosylation and derivatization of the separated and purified N-glycans. The permethylated glycans were further purified by a C18 cartridge and lyophilized, solubilized with 50% v/v methanol:water and mixed with MALDI mass spectrometry matrix before analysis. Positive ion reflectron MALDI mass spectra were acquired on a VOYAGER DE PRO (AB Sciex). Relative intensities (%) of N-glycans were determined based on the peak heights of the corresponding deisotoped monoisotopic mass ions. Interpretation of glycan structures corresponding to monoisotopic masses was performed using ExPASyGlycoMod tool and GlycoWork Bench.

Identification and assignment of sulfation sites, glycosylation sites and glycopeptides

Tryptic digestion and detection of the sulfo-and glycopeptides by MS and tandem MS (MS/MS) was performed to elucidate the sulfation and glycosylation sites and the corresponding glycans. The yielded tryptic glycopeptides were purified and enriched for the respective analysis by MALDI-TOF MS where positive ion linear MALDI mass spectra were acquired in different mass ranges and with multiple acquisition conditions on a MALDI-TOF/TOF Autoflex III spectrometer (Bruker Daltonics). Further, the separated glycopeptide-enriched fractions were deglycosylated, and the yielded peptides were also analyzed by MALDI-TOF MS where positive ion reflectron and linear MALDI mass spectra were acquired in different mass ranges and with multiple acquisition conditions on a MALDI-TOF/TOF Autoflex III spectrometer (Bruker Daltonics). This was done for determining which of the theoretical N-glycosylation sites are actually occupied with N-glycans.

For identification of sulfation sites, the obtained deglycosylated peptides were also injected on a nano-HPLC system coupled to an ion trap nano-electrospray mass spectrometer (LTQvelos, ThermoScientific). Acquisition of mass data was performed in a mass range of 300 to 2000 Da excluding monocharged ions. Proteome Discoverer ThermoElectron (v: 1.4) was used to analyze acquired LC-MS/MS data files.

Preparation of monocyte-derived dendritic cells

Blood from healthy donors was anonymously obtained from Etablissement Français du Sang Ile-de-France (Ivry-sur-Seine, France). Monocytes from peripheral blood mononuclear cells were isolated using anti-CD14 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) from heparinized buffy coats of healthy donors. Purified monocytes were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, L-glutamine and antibiotics in the presence of 500 IU/10 6 cells of IL-4 (R&D Systems, Lille, France) and 1000 IU/ 10 6 cells GM-CSF (ImmunoTools, Friesoythe, Germany). After 5 days, the non-adherent immature dendritic cells (DCs)-enriched fraction was harvested and the immature status was confirmed by the expression of surface phenotypic markers (data not shown).

Incubation of FVIII with MO-DCs

For maturation experiments, immature 5-day old MO-DCs (2.5x10 5 cells/well) were incubated in 500 µl of X-VIVO 15 (Lonza) with the different FVIII preparations at 1 or 10 IU/ml for 48 h. Surface expression of CD80, CD83, CD86, CD40 and HLA-DR was investigated by flow cytometry on 10,000 acquired cells per condition. As a positive control for induction of maturation, we used LPS at 100 ng/ml. Cells incubated alone were used as a negative control.

For FVIII endocytosis experiments, immature MO-DCs (4 × 10 5 cells/well) were incubated for 120 min with the FVIII at 50 to 400 IU/ml in 200 µl of X-VIVO 15 at 4 °C or 37 °C. Intracellular FVIII was recognized after permeabilization with 0.5% saponin, using the FITClabeled monoclonal antibody (anti-A2 FVIII domain mAb77IP52H7, 10 µg/ml, a kind gift from LFB, Les Ulis, France). 10,000 cells were acquired for each condition for flow cytometry analysis. Percentages of cells positive for FVIII were then calculated. Uptake was quantified as the difference in the % cells at 37 °C and 4 °C. When indicated, FVIII (400 IU/ml, 215 nM) was pre-incubated in X-VIVO 15 with VWF (87.5 IU/ml, 3.6 µM) for 20 min at room temperature, prior to incubation with MO-DCs. mAb77IP52H7 does not interfere with the interaction of FVIII with VWF (not shown).

FVIII-specific T-cell activation assay

Five-day old immature MO-DCs derived from monocyte of a healthy donor with the HLA class II DRB1*0101/0301 haplotype, were cultured in RPMI-1640 medium supplemented with 10% FCS, 2-ME, L-glutamine and antibiotics in 96-round bottom plates, with the 1G8-A2 FVIII-specific HLA-DRB1*0101-restricted mouse T cell hybridoma [START_REF] Delignat | Predictive immunogenicity of Refacto AF[END_REF] (10 4 DCs:10 5 T cells) in the presence of the different FVIII preparations at 3, 10 and 30 IU/ml. Twenty four hours later, supernatants were collected and the Il-2 secreted by activated T cells was measured by ELISA.

FVIII-deficient mice

FVIII-deficient mice were 8 to 10-week old males on a C57BL6 background (a gift from Prof Kazazian, University of Pennsylvania School of Medicine, Philadelphia) and were generated by in-house breeding. Our animal facility uses conventional SPF housing conditions where a maximum of 5 mice are kept in ventilated type IIL cages containing normal bedding, vegetal nests and cardboard tunnels at a temperature of 22 °C and 12-hour cycles of light/darkness. Mice were maintained on ad libidum normal diet A03 (SAFE, Augy, France) as well as water by automatic distribution. Mice were checked every day. Mice were sacrificed at the end of each experiment following decerebration under anesthesia (2-4% isofluorane). Mice were handled in agreement with French ethical authorities (Comité National de Réflexion Ethique sur l'Expérimentation Animale, Groupe hospitalier Pitié-Salpêtrière, Paris, France, authorization # 02058.04).

Half-life of FVIII

FVIII (1 IU) in 100 µl was administered intravenously to anesthetized (2-4% isofluorane) FVIII-deficient mice. The residual FVIII:Ag levels were measured at different time points (n ≥ 6 mice per time point and per FVIII product) using a FVIII-specific ELISA assay (Asserachrom, Stago). The data are expressed as the percentage of initial FVIII:Ag level (measured 5 min after administration) versus time. The half-lives were calculated using Graphpad prism by fitting the data to the two-phase decay model with the plateau constraint set to 0.

Anti-FVIII immune response in FVIII-deficient mice

Anesthetized FVIII-deficient mice were injected retro-orbital with 1 IU of each FVIII in its respective excipient at day 0, 7, 14, and 21. Blood was drawn under anesthesia by retro-orbital bleeding 5 days after the 4th administration of FVIII. Decomplemented plasma (56 °C for 30 min) was kept at -20 °C until use. For titration of anti-FVIII IgG, ELISA plates (Nunc) were coated with FVIII (2 μg/mL, Recombinate®) for 1 h at 37 °C, and blocked with PBS-1% BSA. Plasma diluted in PBS-1% BSA was then incubated for 1 h at 37 °C. Bound IgG was revealed using a HRP-coupled monoclonal anti-mouse IgG and substrate. The mouse monoclonal anti-FVIII IgG mAb6 (a gift from Prof. J.M. Saint-Remy, KUL, Belgium) was used as standard. Results are shown in µg/ml mAb6-equivalent. For measurement of FVIII inhibitory titers, mouse plasma samples diluted in veronal buffer were incubated with a standard human plasma (Dade-Behring) for 2 h at 37 °C. The residual procoagulant FVIII activity was measured using a chromogenic assay following the manufacturer's recommendations (Dade-Behring). Bethesda titers, expressed in Bethesda units (BU)/mL, are defined as the reciprocal of the dilution of plasma that produces 50% residual FVIII activity.

Results

Production of Neureight

Neureight was produced using a novel fed-batch disposable fermentation concept in a 400 L scale using a high producer CHO cell line. Expression of recombinant full-length FVIII reached up to 30 IU/mL cell culture supernatant. The process was completely serum-free and did not contain animal-derived stabilizer. Purification of Neureight was carried out by applying several chromatographic steps including affinity chromatography. The purity and structural integrity of Neureight were compared to that of commercially available recombinant FVIII products, Helixate and Advate upon separation by SDS-PAGE with or without digestion with thrombin (Fig. 1A). The three recombinant FVIII demonstrated protein bands between 80 and 200 kDa, that correspond to the FVIII precursor, as well as the separated light chains (LC) at 80 kDa, and the separated heavy (HC), characterized by different molecular weight bands ranging from 90 kDa and higher that correlated to the variable lengths of the B domain, with the A1-A2 domains lacking the B domain as the smallest HC species at 90 kDa. Minor species were also detected between 40 and 50 kDa for Neureight, and to a lesser extent in Helixate and Advate, which can be correlated to the A1 and A2 domains as inferred from the thrombin digested profiles. Digestion of FVIII by thrombin generated the expected 73, 50, and 43 kDa polypeptide fragments that correspond to the activated LC, and to the A1 and A2 domains of the HC, respectively. Thrombin digestion of Neureight confirmed the absence of contaminating protein in the preparation. Indeed, all the protein bands on the gel were sensitive to thrombin cleavage and demonstrated changes in migration profile, thus showing that they belong to FVIII.

The identity of the three products was further confirmed by Western blotting using monoclonal anti-A2 and anti-LC IgG (Fig. 1B and1C, respectively). After dialysis against DMEM-F12, specific activities were 4235 ± 556, 3923 ± 1180 and 6171 ± 824 IU/mg for Neureight, Helixate and Advate, respectively (Table 1, means ± SD of 3 different lots), further substantiating the proper structure and function of Neureight.

Glycan profiling

N-glycan profiles of Neureight revealed both high mannose type glycan forms (Man)5(GlcNAc)2 to (Man)9(GlcNAc)2 and complex type glycans (Table 2). The majority of complex structures were fucosylated and sialylated. The level of sialylated N-glycans was determined with a percentage relative abundance of 59% for Neureight, as compared to 86% for Advate. High mannose type glycans were determined with a percentage relative abundance of 11% in the case of Neureight, compared to 4% in that of Advate. Importantly, the N-glycan profiles did not show the presence of the antigenic epitope Neu5Gc or of the nonhuman antigenic epitope galactose-α1,3-galactose (Gal-alpha-Gal) on N-glycans of the proteins. Neu5Gc was also undetectable in O-glycan profiling.

Identification and assignment of sulfation sites, glycosylation sites and glycopeptides

The occupation sites and the type of glycan structures found for each of the 16 identified N-glycosylation sites were similar between Neureight and Advate (Table 2). For both molecules, the N41 glycosylation site was mainly occupied with complex bi-antennary glycans, mostly core-fucosylated and asialylated structures, the N943 and N2118 glycosylation sites both contained only high mannose type glycans, the N239 and N1066 sites presented with high mannose types and non-sialylated complex glycans, and the N1412 site was occupied by sialylated bi-antennary structures. For both Neureight and Advate, the N1255 glycosylation site was occupied only by high mannose-type glycans, while the N1259 glycosylation site was occupied by a variety of bi-and tri-antennary complex glycans with and without sialic acid (not shown). Tyrosine (Y) sulfation was identical between Advate and Neureight in terms of determined sites of tyrosine sulfation: the 714-733 peptide harboring the three tyrosine sulfation sites Y718, 719 and 723 was sulfated (Table 3). Both molecules showed sulfation of Y1680 (see Table 3), which is critical for the FVIII-VWF interaction.

Induction of maturation of monocyte-derived dendritic cells (MO-DCs)

In order to assess the potential immunogenicity of Neureight, we compared the capacity of the three FVIII products to induce the maturation of human immature MO-DCs. Incubation of 5-day old MO-DCs with LPS, used as a positive control, resulted in the overexpression of CD80, CD83, CD40 and HLA-DR (Fig. 2). In contrast, and as described previously [START_REF] Pfistershammer | Recombinant factor VIII and factor VIII-von Willebrand factor complex do not present danger signals for human dendritic cells[END_REF], Advate and Helixate failed to induce the maturation of MO-DCs. Likewise, Neureight did not alter the immature profile of MO-DCs, irrespective of the FVIII concentration used in the assay. The results also reflect the lack of contamination of Neureight with endotoxins. Mean ± SD of 3 different lots for each product after dialysis against DMEM-F12. °FVIII:C was measured using a chromogenic assay. * FVIII:Ag was measured using Asserachrom.

† Protein concentration was measured in a Bradford assay using OVA as a standard.

‡ Specific activity reflects the IU of FVIII:C per mg of proteins (Bradford assay). * The tri-sulfated peptide was not detected as it is too unstable to be seen in the negative MALDI mode, but is likely to be present. The fact that mono-and di-sulfated peptides are also visible in the linear positive mode is a good indication that these ions are generated by spontaneous fragmentation of the trisulfated molecule.

Endocytosis of Neureight by MO-DCs

The endocytosis of Neureight, Advate and Helixate by 5-day old immature MO-DCs was measured as a function of the concentration of FVIII. The 3 FVIII products were endocytosed in a dose-dependent manner (Fig. 3A). VWF has been reported to inhibit the endocytosis of FVIII by dendritic cells [START_REF] Dasgupta | VWF protects FVIII from endocytosis by dendritic cells and subsequent presentation to immune effectors[END_REF][START_REF] Herczenik | Uptake of blood coagulation factor VIII by dendritic cells is mediated via its C1 domain[END_REF]. We thus compared the protective effect of VWF on FVIII endocytosis in our assay. The three FVIII products (400 IU/ml) were pre-incubated alone or with a 30-fold molar excess of VWF. FVIII was endocytosed by 37 ± 10, 40 ± 4 and 27 ± 4% of the cells in the case of Advate, Helixate, and Neureight, respectively (Fig. 3B). When the FVIII products were pre-incubated with VWF, percentages of FVIII-positive cells were reduced to 8 ± 2, 12 ± 2 and 14 ± 4%, respectively. VWF thus reduced the endocytosis of FVIII by 70 ± 15, 68 ± 9 or 49 ± 15% in the case of Advate, Helixate, Neureight, respectively (Fig. 3C). Differences between products were not statistically significant. Normal binding of Neureight to VWF was further substantiated by the observation of unaltered circulating halflife in FVIII-deficient mice, as compared to Advate (Fig. 4A).

Activation of FVIII-specific CD4+ T cells

To test whether the endocytosis of Neureight by MO-DCs leads to the activation of T cells in vitro, we took advantage of a FVIII-specific mouse T-cell hybridoma that is restricted to the human HLA-DRB1*0101 allele, and can be activated by human MO-DCs with the HLA-DRB1*0101 haplotype or by splenocytes from HLA-DRB1*0101transgenic SureL1 mice [START_REF] Delignat | Predictive immunogenicity of Refacto AF[END_REF]. The three different FVIII products were presented by human MO-DCs to T cells in a dose-dependent manner and to similar levels (Fig. 3D), suggesting that Neureight is endocytosed to a similar extent as Helixate and Advate by antigen-presenting cells, at FVIII concentrations below 100 IU/ml.

Immunogenicity of Neureight in FVIII-deficient mice

The three recombinant FVIII products were administered intravenously once a week for 4 weeks to FVIII-deficient mice at a quantity close to replacement doses in the human (50 IU/kg). After 4 administrations of FVIII, there was no statistical difference between the levels of anti-FVIII IgG induced by the three recombinant FVIII products (1816.0 ± 519.9, 1896.0 ± 478.9 and 680.7 ± 329.9 µg/ml for Advate, Helixate and Neureight, respectively, Fig. 4B). Levels of FVIII inhibitors, measured by the Bethesda assay (Fig. 4C), were statistically different between mice treated with Advate (123.3 ± 50.4 BU/ml, mean ± SEM) and mice treated with Neureight (15.7 ± 10.2 BU/ml, P < .01). There was however no significant difference between Helixate (120.2 ± 43.6 BU/ml) and Neureight.

Discussion

The objective of the present work was to validate the structural and functional integrity and lack of increased immunogenicity of Neureight, a recombinant full-length FVIII produced in CHO cells using a novel fed-batch fermentation mode based on a disposable concept. Neureight demonstrated a migration profile and sensitivity to digestion by thrombin, similar to that of Advate and Kogenate, two commercially available full-length FVIII produced using the classical perfusion technology. Importantly, thrombin digestion revealed the absence of contaminating non-cleavable protein bands in the three preparations, indicating similar degrees of purity of the end products. Importantly, the cell culture supernatant was harvested at high cell viabilities (> 90%) which may contribute to the quality of Neureight by limiting the accumulation of harmful/toxic metabolic by-products and keeping low levels of apoptotic cells. The purity and functional integrity of Neureight was further confirmed by the determination of the specific Fig. 2. Induction of maturation of MO-DCs by different recombinant FVIII products. Neureight, Advate and Helixate were incubated at 1 or 10 IU/ml (FVIII:Ag) with 5day old immature human MO-DCs for 48 h. The expression of CD80, CD83, CD86, CD40 and HLA-DR was measured by flow cytometry and is expressed as percent of positive cells (CD80, CD83) and mean fluorescence intensity (MFI, CD40, HLA-DR). LPS was used as a positive control for the induction of MO-DC maturation. The results represent means and SEM of 3 independent experiments. Differences were not statistically significant (ns) as assessed using a twotailed non-parametric Mann-Whitney test. activities that were in the range reported for commercially available products, i.e., 4000-10,000 IU/mg for Advate and 2600-6800 IU/mg for Helixate [START_REF] Brooker | Registry of Clotting Factor Concentrates, Facts and Figures[END_REF].

Glycosylation is the most common post-translational modification observed in extracellular and integral membrane proteins. It impacts proper folding and efficient secretion of many extracellular proteins. Glycosylation also plays a significant role in determining the plasma half-life and biologic activity of several proteins. FVIII is extensively Nglycosylated, especially within the B-domain, with a variety of complex-, hybrid-and high mannose-type glycan structures [START_REF] Hansson | Post-translational modifications in proteins involved in blood coagulation[END_REF]. As anticipated, the comparison of Neureight and Advate, that are both produced in CHO cells, by mass-spectrometry confirmed the heavily glycosylated profile of the two proteins with complex, hybrid-and high mannose-type glycosylations. The identical glycan structures were present at the same sites on both molecules, and there was a complete absence of the antigenic carbohydrate epitopes α-Gal and Neu5Gc [START_REF] Kannicht | Characterisation of the post-translational modifications of a novel, human cell line-derived recombinant human factor VIII[END_REF]. Tyrosine sulfation is another post-translational modification that takes place in the trans-Golgi apparatus. Tyrosine sulfation of FVIII is required for full pro-coagulant activity as well as for optimal binding to VWF. Our study confirms the mono and di-sulfation of Neureight and Advate at the 714-733 peptide, while the tri-sulfated peptide was too unstable to be seen in the negative MALDI mode for both products. More importantly for proper binding to VWF, our study also confirms sulfation at Y1680.

The immunogenicity of a protein may result from different properties [START_REF] Lacroix-Desmazes | Dynamics of factor VIII interactions determine its immunologic fate in hemophilia A[END_REF], including the capacity to be endocytosed by antigen-presenting cells, the capacity to provide maturation signals that trigger the maturation of the antigen-presenting cells, the capacity to intracellularly bind to MHC class II and be presented to T cells, as well as the presence in the organism of CD4+ T cells that are specific for the therapeutic protein-derived peptides and that are able to be activated (i.e., absence of active tolerance). Both full-length and B domain-deleted therapeutic FVIII products have been shown to be endocytosed by human MO-DCs. Although controversial, the endocytosis of FVIII implicates mannose-ending sugars and charged amino-acid residues in the C1 and C2 domains of FVIII [START_REF] Dasgupta | A role for exposed mannosylations in presentation of human therapeutic self-proteins to CD4+ T lymphocytes[END_REF][START_REF] Wroblewska | Modification of an exposed loop in the C1 domain reduces immune responses to factor VIII in hemophilia A mice[END_REF][START_REF] Gangadharan | The C1 and C2 domains of blood coagulation factor VIII mediate its endocytosis by dendritic cells[END_REF]. Studies have ruled out a role for LRP and related receptors in the endocytic process by MO-DCs and implicated mannose-sensitive receptors such as CD206 [START_REF] Dasgupta | Factor VIII bypasses CD91/LRP for endocytosis by dendritic cells leading to T-cell activation[END_REF][START_REF] Navarrete | Endocytic receptor for pro-coagulant factor VIII: relevance to inhibitor formation[END_REF]. In our hands, Neureight was endocytosed with a dose-dependency identical to that of Advate and Helixate, suggesting that the moieties implicated in FVIII internalization are identical irrespective of the method used to produce FVIII (fed-batch or perfusion approaches). Besides, the Fig. 3. Endocytosis and presentation of recombinant FVIII. Panel A. Immature five-day-old MO-DCs were incubated in X-VIVO 15 medium containing 50 to 400 IU/ml FVIII (FVIII:Ag) for 120 min at 37 °C or at 4 °C. The percent of cells having endocytosed FVIII was measured by subtracting the fluorescence measured at 4 °C from that measured at 37 °C minus. Results depict percent of positive cells. Error bars indicate SEM for 4 independent experiments with MO-DCs from 4 different healthy donors. ns: not significant as assessed using a two-way ANOVA. Panels B and C. FVIII (400 IU/ml FVIII:Ag) was pre-incubated for 20 min alone or in the presence of 87.5 IU/ml plasma-derived VWF at room temperature. Mixtures were then incubated at 4 °C or 37 °C in X-VIVO 15 medium containing 400 IU/ml FVIII:Ag. ns: not significant as assessed by the non-parametric Kruskal-Wallis test. In panel C, percent inhibition in the presence of VWF was calculated as the % FVIII-positive cells measured in the presence of VWF versus that measured in the absence of VWF. Results are mean ± SEM of 3 independent experiments. Panel D. Five-day old immature MO-DCs from healthy donors with the HLA class II DRB1*0101/0301 haplotypes, were cultured with the 1G8-A2 FVIII specific HLA-DRB1*0101restricted mouse T-cell hybridoma in the presence of FVIII (1, 3, 10 and 30 IU/ml FVIII:Ag). Supernatant was collected after 24 h and Il-2 was measured in supernatant. Representative of three experiments. Data were normalized according to the IL-2 production measured for Helixate at 30 IU/ml FVIII:Ag and are depicted as % activation of the T-cell hybridoma. ns: not significant as assessed using a two-way ANOVA.

internalized Neureight was presented to a CD4+ T-cell hybridoma to a similar level as the two therapeutic proteins and both by mouse (not shown) and human antigen-presenting cells. The latter set of data indicates that the intracellular processing of Neureight does not differ from that of Advate and Helixate.

Previous work had documented that FVIII does not alter the maturation state of MO-DCs [START_REF] Pfistershammer | Recombinant factor VIII and factor VIII-von Willebrand factor complex do not present danger signals for human dendritic cells[END_REF]. Indeed, incubation of Advate and Helixate with MO-DCs had no effect on the maturation state of the cells as assessed by the expression levels of CD80, CD83, CD40 and HLA-DR. Likewise, Neureight did not impart on the maturation profile of FVIII, suggesting that its binding to the surface of MO-DCs is identical to that of the other recombinant FVIII, and that it does not contain significant levels of endotoxins.

Interestingly, the levels of anti-FVIII IgG induced by Neureight in FVIII-deficient mice were identical to those induced by Advate and Helixate. In contrast, the inhibitory titers developed by the mice following administration of Neureight were significantly lower than that generated by the other FVIII proteins. An important difference between the in vivo model of severe hemophilia A and the in vitro predictive tests for FVIII immunogenicity used in the present work, is the presence or absence of VWF, respectively. VWF is a chaperon molecule that protects FVIII in the circulation from degradation by circulating enzymes, and transports FVIII to the site of bleeding. Controversial results suggest that VWF-containing FVIII may be less immunogenic in hemophilia A patients [START_REF] Peyvandi | A randomized trial of factor VIII and neutralizing antibodies in hemophilia A[END_REF][START_REF] Calvez | Inhibitor incidence with recombinant vs. plasma-derived FVIII in previously untreated patients with severe hemophilia A: homogeneous results from four published observational studies[END_REF][START_REF] Gouw | Factor VIII products and inhibitor development in severe hemophilia A[END_REF] and that VWF reduces the immunogenicity of FVIII in FVIII-deficient mice [START_REF] Behrmann | Von Willebrand factor modulates factor VIII immunogenicity: comparative study of different factor VIII concentrates in a haemophilia A mouse model[END_REF][START_REF] Delignat | Comparison of the immunogenicity of different therapeutic preparations of human factor VIII in the murine model of hemophilia A[END_REF][START_REF] Qadura | Recombinant and plasma-derived factor VIII products induce distinct splenic cytokine microenvironments in hemophilia A mice[END_REF]. Here, we confirmed that VWF reduces the internalization of Helixate and Advate by MO-DCs in vitro [START_REF] Dasgupta | VWF protects FVIII from endocytosis by dendritic cells and subsequent presentation to immune effectors[END_REF]. Similarly, the endocytosis of Neureight was reduced, although without reaching statistical significance, in the presence of a physiological molar excess of VWF. The lack of statistical significance may be due to the somewhat lower amount of Neureight that is endocytosed, as compared to Advate and Helixate, when FVIII concentrations > 100 IU/ml are used in the assay. In addition, the half-life of Advate and Neureight were identical in FVIII-deficient mice, thus confirming the structural integrity of Neureight as well as its conserved ability to bind VWF. Taken together, our data predict that the immunogenicity of Neureight, at least as assessed using in vitro immunological assays or using the experimental model of severe hemophilia A, is similar to that of Advate and Helixate.

In conclusion, the production of full-length FVIII in a fed-batch fermentation mode generates a product that presents similar biochemical and functional properties as products developed using the classical perfusion technology. Further, the immunogenicity profile of the fed-batch fermentation product is not different from that of commercially available full-length products. Adoption of the fed-batch fermentation strategy should allow a substantial decrease in production costs in the range of 20-40% and permit the delivery of an efficient and safe recombinant FVIII product to developing countries.

Fig. 1 .

 1 Fig. 1. Purity of Neureight and sensitivity to thrombin cleavage. Molecular weight markers (lane 1), Neureight (lanes 2-3), Helixate (lanes 4-5) and Advate (lanes 6-7) were separated by 4-12% SDS-PAGE under non-denaturing conditions (200 ng/lane) directly (lanes 2, 4 and 6) or after incubation with thrombin (2 IU) for 30 min at 37 °C (lanes 3, 5 and 7). Gels were either silver stained (Panel A) or transferred onto nitrocellulose membranes (Panels B and C). Blotted proteins were recognized using an anti-A2 domain monoclonal IgG (Panel B, GMA-012) or an anti-LC monoclonal IgG (Panel C, GMA-8018) and a labelled secondary anti-mouse IgG. Identity of the bands is indicated in the right of each panel (HC: heavy chain; LC: light chain; A1 and A2: A1 and A2 domains of the heavy chain; A3C1C2: thrombin-cleaved light chain).

Fig. 4 .

 4 Fig. 4. Half-life and immunogenicity of FVIII in FVIII-deficient mice. Panel A. FVIII-deficient mice were injected with 1 IU FVIII:Ag (Advate: 6 mice; Neureight: 13 mice). Blood was collected 5, 15, 30, 60, 120, 240, 360 and 480 min after FVIII injection. FVIII in plasma was quantified by Asserachrom. Results are expressed in % of FVIII considering t = 5 min as 100%. Half-lives were fitted to two-phase decay non-linear regression curves using Graphpad Prism. Data depict means and SEM. Inset table: values for slow and fast half-lives expressed in min and area under the curves expressed in %*min. Panels B and C. FVIII-deficient mice (7-8 mice per group) received Advate, Helixate or Neureight (1 IU FVIII:Ag) intravenously once a week for 4 weeks. Mice were bled 5 days after the 4th FVIII administration. Anti-FVIII IgG and inhibitory antibodies were measured by ELISA (Panel B) and Bethesda assay (Panel C). Data depict means and SEM. Statistical significances were assessed with the non-parametric double-sided Mann-Whitney test (ns: not significant).

Table 1

 1 FVIII:C and FVIII:Ag levels.

		FVIII:Ag * (IU/ml) FVIII:C °(IU/ml) Proteins †	Specific
				(µg/ml)	activity ‡ (IU/
					mg)
	Neureight 4032 ± 2530	1971 ± 424	498 ± 133	4235 ± 556
	Helixate	3451 ± 411	1629 ± 891	449 ± 290	3923 ± 1180
	Advate	4271 ± 480	1717 ± 747	310 ± 77	6171 ± 824

Table 2

 2 Identification and assignment of glycosylation sites and glycopeptides.

			Neureight		Advate
	Site (N) Isolated peptide Mass range of detected	Description	Mass range of detected	Description
			glycopeptides (Da)		glycopeptides (Da)
	N41	37-47	2588.79-3349.53	complex bi-antennary glycans mostly	2589.49-3349.63	complex bi-antennary glycans mostly
				core-fucosylated and asialylated		core-fucosylated and asialylated
				structures		structures
	N943	926-946	3851.20-4015.48	high mannose type structures (Man	3850.76-4013.48	high mannose type structures (Man 8-9)
				8-9)
	N239	231-240	2408.80-3058.59	high mannose type and non-sialylated	2409.13-3058.97	high mannose type and non-sialylated
	N1066	1061-1067	2289.88-2815.15	complex structures	2288.14-2815.46	complex structures
	N1412	1407-1422	3867.55	Sialylated biantennary structure	3866.54	Sialylated biantennary structure
	N2118	2117-2136	3411.34-3897.38	high mannose type structures (Man	3412.72-3898.33	high mannose type structures (Man 6-9)
				6-9)

Table 3

 3 Identification and assignment of sulfation sites.

	Sulfation	Peptide	Number of	Theoretical	Neureight	Advate
	(Y)	position	sulfates	average mass
				(Da)	Measured average mass
					(Da)
	Y718,	714-733 *	1	2425.6	2424.9	2425.2
	Y719,		2	2505.6	2505.7	2505.8
	Y723
	Y1680	1674-1689	1	2079.1	2079.1	2079.1

Acknowledgements

This work was supported by INSERM, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie Paris 6, and by a grant from Minapharm Pharmaceuticals S.A.E (Cairo, Egypt). Glycosylation and sulfation studies were performed by Proteodynamics SARL, France.