Prolonging the circulatory half-life of C1 esterase inhibitor via albumin fusion

Sangavi Sivananthan; Varsha Bhakta; Negin Chaechi Tehrani; William P Sheffield

doi:10.1371/journal.pone.0305719

. 2024 Oct 23;19(10):e0305719. doi: 10.1371/journal.pone.0305719

Prolonging the circulatory half-life of C1 esterase inhibitor via albumin fusion

Sangavi Sivananthan ¹, Varsha Bhakta ^1,², Negin Chaechi Tehrani ¹, William P Sheffield ^1,^2,^*

Editor: Yash Gupta³

PMCID: PMC11498661 PMID: 39441778

Abstract

Hereditary Angioedema (HAE) is an autosomal dominant disease characterized by episodic swelling, arising from genetic deficiency in C1-esterase inhibitor (C1INH), a regulator of several proteases including activated Plasma kallikrein (Pka). Many existing C1INH treatments exhibit short circulatory half-lives, precluding prophylactic use. Hexahistidine-tagged truncated C1INH (trC1INH lacking residues 1–97) with Mutated N-linked Glycosylation Sites N216Q/N231Q/N330Q (H₆-trC1INH(MGS)), its murine serum albumin (MSA) fusion variant (H₆-trC1INH(MGS)-MSA), and H₆-MSA were expressed in Pichia pastoris and purified via nickel-chelate chromatography. Following intravenous injection in mice, the mean terminal half-life of H₆-trC1INH(MGS)-MSA was significantly increased versus that of H₆-trC1INH(MGS), by 3-fold, while remaining ~35% less than that of H₆-MSA. The extended half-life was achieved with minimal, but significant, reduction in the mean second order rate constant of Pka inhibition of H₆-trC1INH(MGS)-MSA by 33% relative to that of H₆-trC1INH(MGS). Our results validate albumin fusion as a viable strategy for half-life extension of a natural inhibitor and suggest that H₆-trC1INH(MGS)-MSA is worthy of investigation in a murine model of HAE.

Introduction

Hereditary Angioedema (HAE) is an autosomal dominant disease characterized by an acute episodic swelling in the stomach, face, limbs, and throat; if the airways are involved HAE attacks can be life-threatening [1, 2]. There are two subtypes of HAE. Type 1 is characterized by both reduced C1 inhibitor (C1INH) antigen and activity levels, while type 2 is marked by normal antigenic but reduced activity C1INH levels. Both stem from mutations in SERPING1 [3]. C1INH is a glycoprotein serine protease inhibitor belonging to the serpin superfamily [4, 5]. The multifaceted activities of C1INH include roles in vascular permeability, anti-inflammatory function, coagulation, and fibrinolysis [6–9].

C1INH is the largest serpin (110kDa) and is hyperglycosylated with 10 sites of N-glycosylation and 24 sites of O-glycosylation [10, 11]. Most of these sites lie in a 100 amino acid N-terminal extension not found in other members of the serpin superfamily [5], with the exception of 3 N-linked glycosylation sites, N216, N231, and N330 [11] (numbered according to mature protein sequence). Several research groups have demonstrated that recombinant C1INH retains wild-type-like inhibitory activity when its N-terminal 97 residues were deleted [12–14], but, at least in the baculovirus/insect cell expression system, combining this truncation with the triple mutation N216Q/N231Q/N330Q to eliminate glycosylation abrogated detectable expression [13].

C1INH plays a pivotal role in inhibiting various proteases, including C1s in the complement system and plasma kallikrein (Pka) [15]. Following cleavage, Pka catalyzes the cleavage of high molecular weight kininogen, liberating bradykinin. Bradykinin controls fluid balance between the circulation and tissues via the B2 receptor, whose binding results in endothelial cell contraction, vasodilation, and nitric oxide production [16]. Bradykinin levels correlate with HAE attack severity and are higher in affected limbs than unaffected limbs in the same HAE patients during attacks [17, 18]. These observations, combined with the clinical efficacy of replacement therapy with either C1INH concentrates or drugs inhibiting Pka, support a causative role for unregulated bradykinin activity in HAE [19–22].

Over the last decade, the clinical management of HAE has expanded from treating attacks acutely to preventing attacks via prophylactic treatment [23]. Fewer drugs are approved for HAE prophylaxis than for acute treatment; for example, the only existing recombinant C1INH in clinical use, conestat alfa, is unsuitable for prophylactic use due to its short half-life [24]. Theoretically, prophylactic coverage for HAE patients would be easier to achieve with extended half-life (EHL) products, as has been demonstrated with protein drugs used to treat patients with hemophilia [25]. One approach to an EHL for HAE treatment would be a C1INH-albumin fusion protein. Fusing a therapeutic protein to albumin typically extends its plasma residency time, becoming more like that of albumin; plasma-derived human serum albumin (HSA) exhibits a plasma half-life of 19 days [26]. We hypothesized that albumin fusion would not impair C1INH-mediated inhibition of Pka and would extend its circulatory half-life in vivo. To facilitate an EHL strategy for C1INH, we simplified C1INH by removal of its N-terminal 97 amino acids, mutation of its serpin domain N-linked glycosylation sites and addition of an N-terminal hexahistidine tag to facilitate purification. We report that the inhibitory properties of simplified, truncated, non-glycosylated C1INH were largely unaffected by genetic fusion to mouse serum albumin (MSA) and that this fusion protein acquired an extended plasma residency time in mice in vivo approaching that of hexahistidine-tagged recombinant MSA.

Methods

Expression and purification of H₆-trC1INH(MGS), H₆-MSA, and H₆-trC1INH(MGS)-MSA fusion protein in P. pastoris

Clone Manager 10 (Sci Ed Software LLC) was used for design and oligonucleotides, or other synthetic DNA sequences (gBlock^TM) were purchased from Integrated DNA Technologies (IDT). All restriction and DNA modification enzymes were bought from Thermo Fisher Scientific (Waltham, Massachusetts, United States) (XhoI, catalogue number (cat. #) FD0694, EcoRI, cat. # FD0274, AccIII, cat. # FD0534). A hexahistidine-tagged truncated C1INH_98-478 with Mutated Glycosylation Sites (MGS; N216Q/N231Q/N330Q) (H₆-trC1INH(MGS)) was designed and the resulting 1192 base pair (bp) H₆-C1INH gBlock^TM product was restricted with XhoI and EcoRI and inserted between these sites in pPICZ9ssamp [27, 28] to yield pPICZ9ssH₆-trC1INH(MGS). An MSA cDNA was modified for expression via PCR, as directed by sense oligonucleotide 5’-TCTCTCGAGA AAAGACATCA CCATCACCAT CACGAAGCAC ACAAGAGTGA GATCGCC-3’ and antisense oligonucleotide 5’-CCTAGGGAAT TCCTAGGCTA AGGCGTCTTT GCATCTAGTG AC-3’ and catalyzed by heat-stable polymerase Phusion under conditions directed by its manufacturer (Thermo Fisher Scientific, cat. # XXXXXX). The resulting amplicon was restricted with XhoI and EcoRI and inserted between these sites in pPICZ9ssamp to form pPICZ9ssH₆-MSA. Fusion construct H₆-trC1INH(MGS)-MSA, was assembled by ligating a 1206 bp H₆-trC1INH(MGS)-(G₄S)₃ gBlock^TM product restricted with XhoI and AccIII, and a 1797 bp (G₄S)₃-MSA gBlock^TM product restricted with AccIII and EcoRI. These fragments were then inserted between the Xho1 and EcoRI sites of pPICZ9ssamp vector to yield pPICZ9ssH₆-trC1INH(MGS)-MSA. All plasmid candidates were verified by Sanger dideoxy sequencing of the relevant open reading frames by the McMaster University Genomics Facility. Purified plasmid DNA was linearized with appropriate restriction enzymes and were used to transform Pichia pastoris (P. pastoris) strain X-33 to Zeocin resistance. The transformed cell lines were cultured, induced with methanol, and the secreted proteins H₆-trC1INH(MGS), H₆-trC1INH(MGS)-MSA, and H₆-MSA were purified from conditioned media using nickel-chelate affinity chromatography, as previously described [29].

Protease inhibition assays with H₆-trC1INH(MGS) and H₆-trC1INH(MGS)-MSA

Second-order rate constants (k₂) and stoichiometries of inhibition (SI) were determined using chromogenic assays as previously described [30]. Pka was purchased from Enzyme Research Laboratories (South Bend, IN, USA) (cat. # 1303) and complement component 1, s subcomponent (C1s), activated, two chain form, was bought from Sigma Aldrich (Oakville, ON, Canada) (cat. # 204879-250UG). The velocity of amidolysis of chromogenic substrate S2302 (DiaPharma, West Chester, OH, USA) (cat.# 82034039) by Pka or of chromogenic substrate Pefachrome™-C1E (CH₃CO- benzyloxycarbonyl Lys-Gly-Arg-para-nitroanilide monoacetate) (DiaPharma) (cat. # DPG08703) by C1s was assessed in 96-well flat-bottom microtiter plates (Immulon IV, Thermo Fisher Scientific) (cat. # 1424563) at 37°C in PPNE buffer (20 mM sodium phosphate pH 7.4, 0.1% (w/vol) polyethylene glycol 8000, 100 mM sodium chloride, and 0.1 mM disodium ethylenediaminetetraacetic acid). The measurements were performed at a wavelength of 405 nm using an Elx808 Absorbance Microplate Reader (Biotek, Winooski, VT, USA). For the determination of k₂, reactions included 10 nM Pka (Enzyme Research, South Bend, USA) or C1s (Millipore Sigma, Massachusetts, USA) and 200 nM pdC1INH (Athens Research and Technology, Athens, Georgia, USA) (cat. # 16-16-031509) or C1INH recombinant variants, incubated at 30-second intervals and halted with 100 μM S2302 or 400 μM Pefachrome™-C1E chromogenic substrate. To ascertain the SI, reactions included 10 nM Pka and 10–50 nM pdC1INH/C1INH recombinant variants, incubated for 2-hours and stopped with 100 μM S2302.

Electrophoretic analysis of H₆-trC1INH(MGS) and Pka interaction

The electrophoretic profile of the interaction between pdC1INH/H₆-trC1INH(MGS) with Pka was visualized on 10% (w/vol) sodium dodecyl sulfate (SDS)-polyacrylamide gels (SDS-PAGE, under reducing conditions. Reactions involved a final concentration of 5 μM pdC1INH/H₆-trC1INH(MGS) and 1 μM Pka in PPNE buffer and were conducted at 37°C for 5-minutes. Reactions were then terminated by adding 1/3 the reaction volume of concentrated 4x SDS-PAGE sample buffer, and samples containing the entire reaction volume were subjected to electrophoresis. Gels were stained with Coomassie Brilliant Blue as described previously [31]. Polyacrylamide gels were imaged by scanning using a model XR GelDoc system (Bio-Rad Laboratories, Mississauga, ON, Canada) and nitrocellulose immunoblots prepared as described [30] were imaged by scanning using a model Azure 280 system (Azure Biosystems, CA, USA), generating Tagged Image File (TIF) format outputs.

Detection of C1INH binding to kallikrein by modified ELISA

An enzyme-linked immunosorbent assay (ELISA) was employed to detect H₆-trC1INH(MGS)-MSA: Pka complexes, as described, with modifications [31]. In 96-well flat-bottom microtiter plates, 250 ng of Pka (in 0.1 ml of PBS) was coated overnight. Subsequently, unbound Pka was removed, and the wells were washed with PBS-T (136 mM sodium chloride, 2.7 mM potassium chloride, 10 mM disodium phosphate, 1.8 mM monopotassium phosphate, and 0.1% Tween-20). H₆-trC1INH(MGS)-MSA (1 μg or 5 μg) or pdC1INH (500 ng or 1 μg) were diluted in 1% bovine serum albumin (BSA) in PBS-T and incubated in the Pka-coated 96-well flat bottom microtiter plates for 1-hour. Wells were subsequently washed with modified PBS-T buffers containing 1 M sodium chloride and 1% Tween-20 for the remaining washing steps. C1INH-related protein binding to immobilized Pka was detected with 100 ng per well of goat anti-human C1INH antibody conjugated to horseradish peroxidase (HRP) (Affinity Biologics, Ancaster, Canada) (cat. # GACINH-HRP); colour generation was monitored using 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate solution (Thermo Fisher Scientific) (cat. # 34021), 0.1 ml/well, and was stopped by adding an equal volume of 2M sulphuric acid after 5 minutes, prior to quantification using an Elx808 Absorbance Microplate Reader.

In vivo clearance and protein detection in mice

All in vivo experiments in this study used CD1 mice (Charles River, Wilmington, MA, USA) and were approved by the Animal Research Ethics Board of the Faculty of Health Sciences, McMaster University, via Animal Utilization Protocol 20-02-06. The ARRIVE guidelines (https://arriveguidelines.org/arrive-guidelines) were followed (specifically the ARRIVE Essential 10). Mice were typically 1–2 months old. All mice were acclimatized to their surroundings in the Central Animal Facility of the Faculty of Health Sciences of McMaster University for at least one week after arrival. Mice were always maintained under anesthesia during the procedure, using 3% isoflurane. For clearance studies, mice were intravenously injected with either 50 μg H₆-trC1INH(MGS), 50 μg H₆-MSA, or 100 μg H₆-trC1INH(MGS)-MSA, each in 0.2 ml of saline solution. The dose was selected in order to administer equimolar doses of the three proteins to be compared, allowing for the greater molecular weight of H₆-trC1INH(MGS)-MSA than the other two proteins, to ensure that 1% of the initial dose could be detected in plasma samples using ELISA, and to not exceed ~20% of the plasma concentration of C1INH in wild-type mice, expected to be ~0.25 mg/ml [6]. Serial blood samples were collected from the tail over time, and recombinant proteins remaining in plasma derived from these samples were quantified by ELISA with minor modifications [31]. Groups of six mice comprising three male and three female mice were used in all cases, with sample size selection being based on results of previous studies [29, 32]. Three groups were initially compared, those receiving H₆-trC1INH(MGS) or H₆-trC1INH(MGS)-MSA or H₆-MSA, the latter serving as a control. Subsequently two additional groups of six mice each receiving H₆-trC1INH(MGS) or H₆-trC1INH(MGS)-MSA were compared. A total of 30 mice were used in this study, weighing 31 ± 4 g (mean ± SD). Neither randomization nor blinding were employed. All animals were included in the analysis; there were no exclusions. After the final blood sample was drawn, mice were euthanized by cervical dislocation under anesthetic cover. To capture H₆-trC1INH(MGS) and H₆-trC1INH(MGS)-MSA, 0.1 ml of 1 μg/ml goat anti-human C1INH (Affinity Biologics, Ancaster, ON, Canada) (cat. # GACINH) was coated, per well, on a 96-well flat-bottom microtiter plate, overnight. Bound C1INH proteins were detected using 0.3 μg/ml goat anti-human C1INH HRP-conjugated antibody in 1% BSA in PBS, with colour detection as described above. Plasma-derived C1INH was used to construct a standard curve linear between 0.5 and 20 ng/ml. For detecting H₆-MSA, plasma samples diluted 1:1 with PBS were incubated with 500ng of Biotin-SP-conjugated affinity-purified Rabbit Anti-His tag antibody (Jackson ImmunoResearch Laboratories, PA, USA) (cat. # 300-065-240) for 1-hour before transfer to streptavidin-coated plates (Thermo Fisher Scientific) (cat. # 15120). Bound antibody-MSA complexes were detected with 0.25 μg/ml HRP-conjugated goat polyclonal anti-MSA antibodies (Affinity Biologicals) (cat. # AB19195) in 1% BSA in PBS, with colour generation as described above. Purified H₆-trC1INH(MGS)-MSA was used to construct a standard curve linear between 0.5 ng/ml and 20 ng/ml. Terminal half-lives were determined using a two-compartment model and curve-stripping as previously described [33]. The area under the curve was calculated via GraphPad Prism version 9.0 (Insight Partners, New York, NY, USA).

Statistical analysis and significance testing

Data are presented as the mean ± the standard deviation (SD). Statistical analysis was conducted using GraphPad Prism version 9.0. A p-value of < 0.05 was indicative of statistical significance. For multiple comparisons, data were assessed using One-way ANOVA with post-tests.

Results

Novel recombinant proteins

In this study, three recombinant proteins were designed, expressed, and purified, using a P. pastoris expression system, as previously described. In each case, recombinant minigenes were under the control of the methanol-inducible alcohol oxidase 1 (AOX1) promoter, and recombinant proteins were secreted into the conditioned media by the Saccharomyces cerevisiae yeast prepro-α factor 80 amino acid cleavable prepro signal sequence, terminating in the Kex2/Ste13 protease cleavage site (SLEKR↓EA) [34]. Each protein thus shared the Glu-Ala dipeptide followed by a hexahistidine tag on its N-terminus; their predicted primary sequences are shown in S1 Appendix. Fig 1A shows the three recombinant proteins H₆-trC1INH(MGS), H₆-trC1INH(MGS)-MSA, and H₆-MSA, with reference to full-length plasma-derived C1INH (pdC1INH).

The electrophoretic profile of the recombinant proteins is shown in Fig 1B, following nickel chelate affinity chromatography. H₆-trC1INH(MGS) was purified to homogeneity and contained a single polypeptide of 43 kDa, matching its predicted molecular weight of 43,697 Da. Similarly, H₆-trC1INH(MGS)-MSA exhibited a single polypeptide band of approximately 110 kDa, consistent with its predicted molecular weight of 110, 315 Da. Both C1INH variant proteins reacted with antibodies specific to human C1INH and hexahistidine; in addition, H₆-trC1INH(MGS)-MSA acquired immunoreactivity with antibodies specific for MSA. H₆-MSA preparations comprised a major polypeptide of 67 kDa consistent with the predicted molecular weight of 66,713 Da, as well as minor polypeptide comprising approximately 10% of the total protein of approximately 55 kDa. Both the major and minor species reacted not only with anti-MSA antibodies on immunoblots but also with anti-hexahistidine, indicating that the latter was intact on its N-terminus but was missing C-terminal residues.

Characterization of fused and unfused truncated, non-glycosylated C1INH proteins

The kinetics of protease inhibition by H₆-trC1INH(MGS) and H₆-trC1INH(MGS)-MSA were compared to that mediated by pdC1INIH, as shown in Table 1. Neither truncation, prevention of N-linked glycosylation, nor hexahistidine tagging appeared to impact inhibition of C1s, as the k₂ for C1s inhibition did not differ from pdC1INH. Adding albumin to the list of changes similarly did not impact the inhibition of C1s by H₆-trC1INH(MGS)-MSA. In contrast, small but significant reductions in mean k₂ for Pka inhibition were noted for both unfused and fused trC1INH(MGS) proteins, of 13.4% for H₆-trC1INH(MGS) and 45.2% for H₆-trC1INH(MGS)-MSA versus pdC1INH. Mean SI values were significantly increased for H₆-trC1INH(MGS) and H₆-trC1INH(MGS)-MSA compared to pdC1INH, by factors of 1.6 and 2.1, respectively, but did not differ significantly between each other. The results indicate small but significant reductions in both the rate and efficiency of inhibition for the variant recombinant C1INH proteins versus their pdC1INH counterpart.

Table 1. Pharmacokinetic characterization of activity of C1INH recombinant proteins.

Protein name	k₂ versus Pka (X 10⁴ M^-1s^-1)	k₂ versus C1s (X 10⁴ M^-1s^-1)	SI versus Pka
pdC1INH	2.3 ± 0.2^***	5.7 ± 0.5	3.3 ± 0.06^***^{^^^}
H₆-trC1INH(MGS)	2.01 ± 0.07^***	5 ± 0.5	5.3 ± 0.3
H₆-trC1INH(MGS)-MSA	1.3 ± 0.08	5.1 ± 0.4	6.9 ± 0.1

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Results are the mean of 5 determinations (SI or k₂ for kallikrein), ± SD, or 3 determinations (k₂ for C1s).

***, p < 0.001 versus H₆-trC1INH(MGS)-MSA.

^^^, p < 0.001 versus H₆-trC1INH(MGS).

Inhibitors of the serpin class of protease inhibitors typically form denaturation-resistant, SDS-stable complexes [35]. Inhibitory complexes between Pka and plasma-derived inhibitors were first examined by SDS-PAGE. As shown in Fig 2Ai, commercial purified Pka was comprised of multiple polypeptide chains when electrophoresed under reducing conditions, including a catalytic light chain of ~37 kDa. This polypeptide chain was almost fully converted into novel, C1INH-dependent high molecular weight complexes when Pka was reacted with an excess of either pdC1INH or H₆-trC1INH(MGS), of 147 kDa or 80 kDa, respectively. In contrast, when the same reaction was carried out with Pka and excess H₆-trC1INH(MGS)-MSA (Fig 2Aii), no higher molecular weight serpin-enzyme complex was formed, although the Pka catalytic light chain appeared to be consumed to a similar extent as in the reaction with H₆-trC1INH(MGS). Two new polypeptide chains were generated from H₆-trC1INH(MGS)-MSA on exposure to Pka, of ~70 kDa and ~40 kDa.

Fig 2 — A) i. Gel-based assays of complex formation. 10% SDS-PAGE gels are shown electrophoresed under reducing conditions and stained with Coomassie Brilliant Blue. Serpins (pdC1INH, H₆-trC1INH(MGS), or H₆-trC1INH(MGS)-MSA) were reacted with Pka at a 5:1 molar ratio for 5-minutes at 37°C. B) Graph of binding assay between pdC1INH:Pka and H₆-trC1INH(MGS)-MSA:Pka at varying ratios (500 ng-1 μg) with PBS-T washing condition of 1M NaCl and 1% Tween-20. The mean and SD of 3 determinations is shown.

To reconcile the apparent discrepancy of a substantial k₂ for Pka inhibition with the inability to detect SDS-stable complexes between H₆-trC1INH(MGS)-MSA and Pka by SDS-PAGE, we sought independent verification of complex formation. Active Pka was immobilized on microtiter plate wells, incubated with pdC1INH or H₆-trC1INH(MGS)-MSA, washed under stringent high-salt, high detergent conditions, and captured C1INH antigenic material was detected with C1INH-specific antibodies. As shown in Fig 2B, bound C1INH was detected when either pdC1INH or H₆-trC1INH(MGS)-MSA was reacted with immobilized Pka, in a dose-dependent manner, although the signal was higher with pdC1INH than with H₆-trC1INH(MGS)-MSA. These results, like the k₂ findings, are suggestive of tightly bound inhibitory complex formation between Pka and H₆-trC1INH(MGS)-MSA even if the complexes are not SDS-stable.

In vivo clearance of recombinant proteins

H₆-trC1INH(MGS), H₆-trC1INH(MGS)-MSA, and H₆-MSA proteins were separately injected into groups of mice. The average relative recovery in serial plasma samples is shown in Fig 3A (log-linear plot) and Fig 3B (linear-linear plot), where the profile of H₆-trC1INH(MGS) in mice exhibited a distinct and more rapidly cleared trajectory from that of H₆-trC1INH(MGS)-MSA or H₆-MSA, within minutes of injection. After 1 hour, only 27 ± 10% of the initial dose of H₆-trC1INH(MGS) remained in the circulation, which was significantly lower than the residual levels of H₆-trC1INH(MGS)-MSA or H₆-MSA (90 ± 4% and 95 ± 3%, respectively) (Fig 3C). By 4 hours post-injection, only 2.2 ± 0.5% of the initial dose of H₆-trC1INH(MGS) remained in the circulation, significantly less than H₆-trC1INH(MGS)-MSA or H₆-MSA (48 ± 10% and 60 ± 8%, respectively) (Fig 3D). While residual levels of H₆-trC1INH(MGS)-MSA and H₆-MSA did not exhibit a significant difference at 1 hour, the difference became significant by 4 hours post-injection.

Fig 3 — A) Logarithmic-linear plot of H₆-trC1INH(MGS), H₆-trC1INH(MGS)-MSA, and H₆-MSA percentage residual protein in plasma versus time in hours post-injection. B) Linear plot of H₆-trC1INH(MGS), H₆-trC1INH(MGS)-MSA, and H₆-MSA percentage residual protein in plasma versus time in hours post-injection. C) Percentage residual protein in plasma at 1-hour post-injection. D) Percentage residual protein in plasma at 4-hours post-injection. E) Percentage residual protein in plasma at the times, in hours, specified on the x axis, following injection of H₆-trC1INH(MGS)-MSA (light grey bars) or H₆-MSA (dark grey bars). The mean of 6 determinations ± SD is shown in all cases. *, p < 0.05, ****, p<0.0001 for statistical comparisons indicated by horizontal bars (ns denotes non-significant).

Our initial design of the in vivo clearance experiment was limited by local animal research ethics requirements which restricted the number of blood samples that could be taken in a 24-hour period. For H₆-trC1INH(MGS) that design covered >95% of the total clearance curve, but <50% of that of the less rapidly cleared MSA-containing proteins (see Fig 3A–3D). More extrapolation was therefore required to estimate the half-life of those proteins than for H₆-trC1INH(MGS). Accordingly, an additional experiment was performed in which identical doses of H₆-trC1INH(MGS)-MSA and H₆- MSA as previously employed were administered to additional groups of 6 mice each, and blood samples were obtained 16–28 hours after injection. As shown in Fig 3E, these results supported the initial, shorter experiment, and showed that substantial quantities of H₆-trC1INH(MGS)-MSA (19 ± 1%) and H₆- MSA (22 ± 1%) 18 and 28 hours after injection, respectively.

Pharmacokinetic analysis

The use of a two-compartment model to resolve clearance curves allowed the calculation of terminal half-lives, alongside the area under the curve (AUC) for the three proteins, are presented in Table 2. Fusion to albumin significantly extended the mean terminal half-life of C1INH by 3.0-fold. However, H₆-trC1INH(MGS)-MSA still exhibited a 1.5-fold more rapid clearance compared to H₆-MSA. Furthermore, there was a significant 3.8-fold increase in the observed AUC for H₆-trC1INH(MGS)-MSA versus H₆-trC1INH(MGS), while remaining 1.1-fold less than the AUC of H₆-MSA. The results suggest that albumin fusion significantly extended the plasma residency time of H₆-trC1INH(MGS)-MSA compared to H₆-trC1INH(MGS), as well as the terminal half-life, supporting our hypothesis, although the extension fell short of achieving the longer values observed for H₆-MSA. No effect of sex on terminal half-life was noted for any of the three proteins; when half-life values were grouped by sex for each protein, no significant differences were noted (as indicated by p values of 0.1 to 0.7 by Mann-Whitney non-parametric test). Similarly, no effect of body weight was noted for any of the three proteins; when half-life values were divided into heavier half versus lighter half per protein per group, no significant differences were noted (as indicated by p values of 0.4 to 0.99 by Mann-Whitney non-parametric test).

Table 2. H₆-trC1INH(MGS), H₆-trC1INH(MGS)-MSA, H₆-MSA pharmacokinetic analysis of clearance.

Protein name	Terminal half-life (hours)	Area Under the observed Curve (AUC; %-hours)
H₆-trC1INH(MGS)	5 ± 2	80 ± 20
H₆-trC1INH(MGS)-MSA	14 ± 3^*	310 ± 20^***
H₆-MSA	21 ± 8^***	340 ± 8^***

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Results are the mean of 6 determinations, ± SD.

*, p < 0.05

***, p < 0.001 versus H₆-trC1INH(MGS).

Discussion

The chief findings of this study were that the inhibitory properties of C1INH were maintained in a truncated, non-glycosylated variant, that these properties were largely maintained when this variant was fused to serum albumin, and that the fusion protein remained in the circulation of mice after intravenous injection for considerably longer than its unfused equivalent. Although the results supported our initial hypothesis, the extension in plasma residency fell short of that exhibited by recombinant albumin alone.

Unfused H₆-trC1INH(MGS) left the circulation rapidly after injection, as expected for a protein of 43 kDa, below the glomerular filtration limit. Such proteins are expected to be filtered in the glomerular structures of the kidneys, and not re-adsorbed in the renal tubules [36]. Consistent with this expectation, the clearance curve for H₆-trC1INH(MGS) diverged from those of H₆-trC1INH(MGS)-MSA or H₆-MSA at the earliest sampled time point. Both plasma residency time, as quantified by the observed AUC, and terminal catabolism, as quantified by the terminal half-life, increased 3- to 4-fold when MSA was fused to H₆-trC1INH(MGS). H₆-trC1INH(MGS)-MSA approached, but did not reach, the long plasma residency and slow rate of terminal clearance of H₆-MSA, achieving mean levels of 92% of the AUC and 66% of the terminal half life of that hexahistidine-tagged albumin. The mean terminal half-life of H₆-MSA, 21.4 hours, was less than that reported for plasma-derived MSA in previous studies (27.7 hours [37]– 35 hours [38]), a difference potentially arising either from hexahistidine tagging, recombinant production, or methodological differences such as protein radioiodination in previous studies. In this work we elected to follow the fusion proteins without modification via ELISA of plasma samples, in part due to initial observations that pdC1NH lost the ability to form SDS-stable complexes with Pka after iodination. Our results were nonetheless internally consistent and showed a significant extension of half-life and reduction in clearance rate for H₆-trC1INH(MGS)-MSA versus H₆-trC1INH(MGS). The improved pharmacokinetic profile was likely obtained by the combined mechanisms of avoidance of renal filtration and the conferring of recycling via murine FcRn [39].

Fusion to albumin generally increases the plasma half-life of fused proteins, but it can interfere with the biological function of the attached protein, to varying extents. Miyakawa et al. reported that fusion of interferon γ to MSA increased the observed clearance AUC of the fusion protein 10-fold versus unfused interferon γ, but at the cost of the loss of ~99% of its biological activity [40]. Less dramatic losses in activity were reported by our laboratory in fusing the Kunitz Protease Inhibitor (KPI) domain of Protease Nexin 2 to HSA, where the affinity of KPI for its target protease FXIa significantly declined 2.1-fold with fusion [41]. Similarly, fusing FIX to HSA resulted in substantial losses in specific activity compared to unfused recombinant FIX of up to 100-fold, unless a cleavable spacer was used that could liberate FIX from HSA [42]. In this study the rate of C1s inhibition by H₆-trC1INH(MGS) was unaffected by MSA fusion and the rate of Pka inhibition was reduced 1.8-fold. These outcomes likely arose from favourable interactions between the H₆-trC1INH(MGS) moiety of the fusion protein and the target proteases, unimpeded by MSA, which was designed to be distanced from that moiety by a substantive triplicated Gly₄Ser spacer.

Previous investigators have established that truncated, glycosylated recombinant C1 inhibitor retained the inhibitory capacity of full-length C1INH against several protease targets. Coutinho et al. deleted the N-terminal 97 residues of C1INH and showed, qualitatively, similar inhibitory activity of the deletion mutant as wild-type C1INH produced in cultured COS-1 cells [12]. Bos et al. expressed the same truncation mutant (Δ97) in P. pastoris and reported comparable association rate constants differing by less than two-fold for wild-type and truncated C1INH for C1s, Pka, and FXIIa [14]. Rossi et al. made the Δ97 truncation using baculovirus expression vectors expressed in cultured Sf21 insect cells, finding equivalent complex formation with C1s on gel analysis both for truncated and hexahistidine-tagged C1inhΔ97-ht and for untagged C1inhΔ97, and for truncated variants with mutated N216 and/or N231 sites; truncated variants with mutated N216, N231, and N330 sites failed to be expressed [13]. Our results contrasted with those of Rossi et al. in that we were able to obtain enough hexahistidine-tagged, truncated, non-glycosylated H₆-trC1INH(MGS) to determine quantitatively its identical kinetics of C1s inhibition and its similar (85%) kinetics of Pka inhibition to its plasma-derived counterpart. The discordance suggests that glycosylation on N330 is obligatory only for expression in Sf21 insect cells [13] and not for eukaryotic cells in general.

While many studies of albumin fusion proteins employ HSA as a fusion partner rather than a species-matched albumin, it is known that the serum half-life of HSA fusion proteins can be underestimated in mice. This underestimation arises because the binding affinity of HSA to murine FcRn is about 10-fold weaker than its binding to human FcRn [43]. Moreover, HSA-conjugated drugs persist longer in the circulation of transgenic mice expressing human FcRn than in wild-type mice [44]. Site-specific conjugation of superfolder green fluorescent protein (sfGFP) to MSA or HSA resulted in a significantly longer mean half-life for the sfGFP-MSA conjugate (27.7 hours) than for sfGFP-HSA (16.3 hours) [45]. In addition, use of MSA as the fusion partner could make it easier to determine proof-of-concept for the utility of H₆-trC1INH(MGS)-MSA in knockout mice genetically deficient in C1INH [6].

Purified plasma-derived C1INH concentrates have been reported to have mean half-lives in HAE patients ranging from 32.7 [46] to 74.1 hours [47]. These values might seem longer than those reported in this study for both H₆-trC1INH(MGS) and H₆-trC1INH(MGS)MSA were it not for the principle of allometric scaling, which holds that drug or protein clearance is proportional to the body weight of an animal or human[48]. The mean terminal catabolic half-life of HSA in humans is 19 days [26]. Extrapolating from our results in mice, we speculate that the half-life of a H₆-trC1INH(MGS)-HSA fusion protein would be significantly longer than that of plasma-derived C1INH concentrates, although it is unlikely that the full extension of half-life to 19 days would be achieved.

Although H₆-trC1INH(MGS)-MSA inhibited Pka with similar kinetics to either unfused H₆-trC1INH(MGS) or pdC1INH, in contrast to those proteins, the fusion protein did not form detectable SDS-stable complexes with Pka. Our failure to detect covalent serpin-protease complexes with this combination of serpin and protease may have arisen for several reasons. Firstly, our choice of reaction conditions could have been responsible; greater excesses of H₆-trC1INH(MGS)-MSA over Pka might have been required because of its mean SI of 6.8, versus 5.3 for H₆-trC1INH(MGS) (although these values did not differ statistically) and 3.2 for pdC1INH; others have estimated the latter value, for pdC1INH, at approximately 2 [14]. Secondly, it is possible that the fusion protein formed a strongly stabilized non-covalent inhibitory complex. There are precedents for such unusual variant serpins. Ciaccia et al. did not detect a covalent complex between the L444R variant of heparin cofactor II and α-thrombin in the presence of heparin, despite strong inhibition of activity [49]. Similarly, Rossi et al. described the lack of covalent complex formation between Complement Component 1, r subcomponent-like protein (C1rLP) and C1inhΔ97, despite strong inhibition of C1rLP activity [13]. Finally, relatively few fusion proteins involving serpins have been described in the literature. Alpha-1 antitrypsin (AAT), the most abundant serpin in human plasma, loses the ability to inhibit its natural target, neutrophil elastase, when fused to the N-terminus of IgG1 Fc [50]. In contrast, AAT M358R variant retains function when fused, via its N-terminus, to the C-terminus of bacteriophage T7 coat protein 10, as evidenced by its formation of SDS-stable complexes with thrombin [51]. Thus, H₆-trC1INH(MGS) retains potent anti-Pka activity but may inhibit its target via an atypical mechanism.

The rate of inhibition of Pka by C1INH is surprisingly low compared to those achieved by other serpins in regulating other proteases [35]. Nevertheless, the susceptibility of C1INH-deficient individuals to HAE and their successful treatment with pdC1INH concentrates show that it is high enough to maintain control of bradykinin generation. In this study we showed that fusion protein H₆-trC1INH(MGS)-MSA gains extended half-life and maintains most of rate of Pka inhibition exhibited by pdC1INH. Future studies in C1INH knockout mice will be required to determine if this profile is sufficiently favourable to warrant continued development via substitution of the MSA moiety by HSA and investigation in mice with human FcRn, or if protein engineering to re-direct a different serpin towards Pka inhibition at higher rates, like the approach taken by de Maat et al. [52] would be a superior strategy.

Supporting information

S1 Appendix

(1) Protein sequences and lengths. The primary sequence of all recombinant proteins employed in this study are shown, along with the length in amino acids, the molecular weight in Daltons, and the calculated isoelectric point. (2) Raw data shown in Table 1. (3) Raw data shown in Fig 2B. (3) Raw data shown in Fig 3A–3D. (4) Raw data shown in Fig 3E. (5) Raw data shown in Table 2.

(DOCX)

pone.0305719.s001.docx^{(41.1KB, docx)}

S2 Appendix. This file contains uncropped original images of all gels and blots shown in this study.

(PDF)

pone.0305719.s002.pdf^{(477.4KB, pdf)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This study was made possible through a Canadian Blood Services (CBS) Graduate Fellowship Program award to SS, and via a CBS Discovery Research Grant DRG2023-WS to WS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0305719.r001

Decision Letter 0

Yash Gupta

10 Apr 2024

PONE-D-24-10397Prolonging the circulatory half-life of C1 esterase inhibitor via albumin fusionPLOS ONE

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Authors need to update their literature survey with more pertinent studies and paralells are needed to be drawn comparing the merits of the findings.
Authors also need to revise methods while furnishing details of reagent / biologicals sources.
There is a missing information regarding sex and weight having effect on outcome and dosing flexiblity according to weight.

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Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Journal: PLOS ONE

Manuscript Number: PONE-D-24-10397

Article Type: Research Article

Title: Prolonging the circulatory half-life of C1 esterase inhibitor via albumin fusion

Recommendation: Publish after minor revisions are noted.

Comments:

The manuscript by Sivananthan and colleagues described the Hereditary Angioedema (HAE) an autosomal dominant episodic swelling disease occur in various body parts due to deficiency in C1-esterase inhibitor (C1INH), a regulator of several proteases eg. Plasma Kallikrein (Pka). Till date, many C1INH treatments have been employed to prevent this disease yet exhibit short circulatory half-life. In this study, they designed, expressed and purified recombinant proteins H6-trC1INH(MGS), H6-trC1INH(MGS)-MSA, and H6-MSA using Pichia pastoris. They found the significant extended circulatory half-life of recombinant H6-trC1INH(MGS)-MSA compared to H6-trC1INH(MGS).

Author also tried to perform vivo study using the CDI mice in which they injected the N-glycosylated C1INH recombinant protein with /or without albumin fusion, separately. They found the mean terminal half-life of H6-trC1INH(MGS)-MSA was 3-fold higher with reduction in Pka inhibition than that of wildtype H6-trC1INH(MGS). Suggesting that H6-trC1INH(MGS)-MSA discovery is advantage in mice model of HRE.

Overall, this an interesting manuscript showing extended circulatory half-life of N-glycosylated C1INH recombinant protein via albumin fusion.

Comment 1: Please add the catalog number for all purchased reagents. For examples: Antibodies, Pka, Complement component.

Comment 2: What is the rationale for dose determination of N-glycosylated C1INH recombinant proteins injected to mice model?

Comment 3: Please see the page 15, line 287,H6-trC1INH(MGS)-MSA or H6-MSA (90±4% and 95%±3%, respectively. please delete % from 95%.

Comment 4: Please tale a look on Y-axis scale of figure 3A. It looks to weird. There should be 50% residual protein in the middle of y axis scale instead of 10%. Please correct it accordingly.

Reviewer #2: In this work, Sivanathan et al. suggest that albusion prolongs the circulatory half-life of C1 esterase inhibitor. The title is suitable and precise, and the abstract adequately summarises work presented. The work is clear and straightforward and presented well. The data presented, structured, and the manuscript is well written with targeted experiment.

Points

1. Authors showed that albumin fusion extends the half-life of the inhibitor, but studies have already shown the half-life has extended up to several days.

The recent manuscript by Martinez-Saguer et al. suggests that the residual half-life of C1-esterase inhibitor (C1INH) treatment is up to 144 hours. (doi.org/10.1016/j.jacig.2023.100178.)

2. The authors should provide further valid points, such as why albumin fusion has better therapeutic potential than already available options, which have a shelf life for several days.

3. Do authors measure the protein level beyond the 4-hour time point?

4. Do authors observe any significant differences in experimental outcomes between male and female mice?

5. The authors mentioned that mice weighed between 25 and 35 g, the range of weight difference seems broad. Does the weight of mice have any difference in clearance of the protein?

6. In the methods section “In vivo clearance and protein detection in mice” did the authors used separate standards for H6-MSA and 6-trC1INH(MGS) or used the same standards (Purified H6-trC1INH(MGS)-MSA) for all the residual protein quantification.

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Reviewer #1: Yes: Brijesh Singh Chauhan

Reviewer #2: No

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Attachment

Submitted filename: Comments_PLOS ONE.docx

pone.0305719.s003.docx^{(15.4KB, docx)}

PLoS One. 2024 Oct 23;19(10):e0305719. doi: 10.1371/journal.pone.0305719.r002

Author response to Decision Letter 0

28 May 2024

We have attached a Response to Reviewers file and also reproduce the document below:

Response to Reviewers of PONE-D-24-10397

Our submission was invited for revision 2024-04-10 if we addressed all points raised during the review process. We have done so. Our responses are organized under “Journal Requirements”, “Reviewer 1”, and “Reviewer 2”.

Journal Requirements

1. The editor directed us to ensure that the manuscript meets PLOS ONE’s style requirements. We have verified that we have done so in our revised submission.

2. The editor asked us to enlarge our financial disclosure. We have done so in our cover letter, which now states the financial disclosure as “This study was made possible through a Canadian Blood Services (CBS) Graduate Fellowship Program award to SS, and via a CBS Discovery Research Grant DRG2023-WS to WS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

3. The Editor pointed out that we had repeated funding information in the Acknowledgements Section of the manuscript and that this error needed to be corrected. We have deleted the Acknowledgements section from the revised manuscript. In addition, we have further enlarged the Funding Statement to contain the previously misplaced text, “Due to the funding of the discovery research program at CBS by a contribution agreement with Health Canada, a department of the federal government of Canada, this publication must contain, as a condition of funding, the statement, “The views herein do not necessarily represent the views of the federal government of Canada.””

4. The editor pointed out that we needed to provide all uncropped and unadjusted images underlying all gel and blot results. These have now been provided in accordance with journal policies as S2 Appendix in Supporting Information.

5. The editor asked that our full ethics statement be provided. We have verified that our Animal Utilization Protocol details are provided in the Methods, including the full name of the issuing body.

6. The editor reminded us to include captions for Supporting Information files at the end of the manuscript and to update in-text citations where appropriate. We have added captions for the two Supporting Information files (S1 Appendix and S2 Appendix). In-text citations have been doublechecked for updating.

7. The editor concluded by asking that we review the reference list and to note any changes to the reference list. We have carried out this review. We report that we have added 3 references to address reviewers’ concerns, as described in detail in the response to reviewers’ comments below (see Reviewer 2, Point 1). We added an additional citation of a pre-existing reference in providing additional information in the text concerning dose selection (see Reviewer 1, Point 2).

Reviewer 1

1. The reviewer requested that catalog numbers be provided for all purchased reagents. These numbers have now been provided. All purchased reagents, a total of 16 items, are now identified by catalogue number.

2. The reviewer asked for the rationale for the dose determination of the C1INH recombinant proteins that we injected into mice. This information has now been provided in the Methods section, where we now state, “The dose was selected in order to give equimolar doses of the three proteins to be compared, allowing for the greater molecular weight of H6-trC1INH(MGS)-MSA than the other two proteins, to ensure that 1% of the initial dose could be detected in plasma samples using ELISA, and to not exceed ~20% of the plasma concentration of C1INH in wild-type mice, expected to be ~0.25 mg/ml [6].”

3. The reviewer found a typographical error and requested its correction. We have corrected “95%±3%” to read “95±3%”.

4. The reviewer commented that the Y-axis scale of Figure 3A looked odd, and that there should be 50% residual protein in the middle of the Y axis rather than 10%. The reason for this feature of Figure 3A is that it is a log-linear plot, with the Y axis being logarithmic in scale. Because protein clearance is typically described by a biphasic natural decay function, we thought this was the best way to present the data. However, to respect the reviewer’s perspective, we have added a linear-linear plot of the same data as a new panel in the revised figure, Figure 3B (rev).

Reviewer 2

1. The reviewer commented that our data supported an extension of C1INH half-life associated with albumin fusion but pointed out that a recent study by Martinez-Saguer et al. reported a half-life of several days for clinical grade C1INH concentrates in HAE patients. We have added a section to the Discussion referencing the study and addressing this point. Briefly, therapeutic protein clearance follows the principle of allometric scaling, in which animals with low body weights (or total body surface area) exhibit faster protein clearance than humans. Martinez-Saguer reported a half-life of 74.1 ± 19 hours (mean ± SD) for purified human plasma-derived C1INH in 20 HAE patients treated with a dose of 20 IU/kg. The half-life of human albumin in humans is 19 days. We therefore hypothesize that a C1INH-albumin fusion protein would persist in the circulation for much longer than current products like that employed by Martinez-Saguer. Moreover, due to allometry, the half-life of albumin in mice is only one day. Our data are predictive of a half-life in humans of a C1INH-albumin fusion protein intermediate between that of current products and albumin. The Discussion has been modified to make these points clearer. In so doing we added 3 references: to the Martinez-Saguer et al. study, to a similar study examining the pharmacokinetics of another plasma-derived C1INH concentrate in HAE patients, and to an article about allometry, i.e. dose extrapolations between animals and humans.

2. The reviewer’s second point was a direction to explain why albumin fusion has good therapeutic potential. We have done so, as explained in Point 1.

3. The reviewer asked if we measured the protein level beyond the 4-hour time point. In our original submission, we did not. However, we have added additional data to the revised manuscript. For H6-MSA and H6-trC1INH-MSA, we verified that substantial quantities of the proteins remained in the murine circulation at longer time points of 16 – 28 hours post-injection, recognizing that the original experiment required much more extrapolation to characterize the half-lives of these longer-lived proteins than H6-trC1INH(MGS), which was >95% removed from the circulation within the original four-hour period. These additional data are now shown in new panel E of revised Figure 3.

4. The reviewer asked if we observed any differences in experimental outcomes between male and female mice. To answer this question, we analyzed half-lives by sex. We did not note any effect of sex, at n=3 per sex per protein, no significant difference between male and female mouse terminal half-lives was detected. This analysis is now reported in the Results section, specifically in the “Pharmacokinetic analysis subsection”.

5. The reviewer commented on the weight range of our mice, initially described as “between 25g and 35 g”. We have provided more detailed information, calculating a mean weight of 31 ± 4 g (mean ± SD, n=30) for all mice employed in this study, in the revised Methods section. We also stratified protein half-lives into two categories, heavier and lighter, and compared protein half-lives. At n=3 per weight class per protein, no significant difference between heavier and lighter mouse terminal half-lives was detected. This analysis is now reported in the Results section, specifically in the “Pharmacokinetic analysis subsection”, after the new statements concerning sex and half-life.

6. The reviewer asked if we used the same or different standards to quantify H6-MSA and H6-trC1INH(MGS). As stated in the Methods section, we used purified plasma-derived C1INH as a standard for the ELISA-based determination of H6-trC1INH(MGS) and H6-trC1INH(MGS)-MSA and we used purified recombinant H6-trC1INH(MGS)-MSA for the determination of H6-MSA. While it would arguably have been better to use the same standard for all 3 proteins, it should be noted that both ELISA protocols yielded identical sensitivities.

My co-authors and I are appreciative of the careful work of the editor and peer reviewers and have attempted to answer all queries and make all requested modifications to our work. We acknowledge that this revision has improved the scientific quality of our manuscript and hope that it is now acceptable for publication in PLOS ONE.

Sincerely,

William P. Sheffield, PhD

Professor, Pathology and Molecular Medicine, McMaster University

Hamilton, Ontario, Canada

Attachment

Submitted filename: Response to Reviewers PONE-D-24-10397.docx

pone.0305719.s004.docx^{(19.1KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0305719.r003

Decision Letter 1

Yash Gupta

5 Jun 2024

Prolonging the circulatory half-life of C1 esterase inhibitor via albumin fusion

PONE-D-24-10397R1

Dear Dr. Sheffield,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Yash Gupta, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0305719.r004

Acceptance letter

Yash Gupta

13 Aug 2024

PONE-D-24-10397R1

PLOS ONE

Dear Dr. Sheffield,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Appendix

(DOCX)

pone.0305719.s001.docx^{(41.1KB, docx)}

S2 Appendix. This file contains uncropped original images of all gels and blots shown in this study.

(PDF)

pone.0305719.s002.pdf^{(477.4KB, pdf)}

Attachment

Submitted filename: Comments_PLOS ONE.docx

pone.0305719.s003.docx^{(15.4KB, docx)}

Attachment

Submitted filename: Response to Reviewers PONE-D-24-10397.docx

pone.0305719.s004.docx^{(19.1KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

Prolonging the circulatory half-life of C1 esterase inhibitor via albumin fusion

Sangavi Sivananthan

Varsha Bhakta

Negin Chaechi Tehrani

William P Sheffield

Roles

Abstract

Introduction

Methods

Expression and purification of H6-trC1INH(MGS), H6-MSA, and H6-trC1INH(MGS)-MSA fusion protein in P. pastoris

Protease inhibition assays with H6-trC1INH(MGS) and H6-trC1INH(MGS)-MSA

Electrophoretic analysis of H6-trC1INH(MGS) and Pka interaction

Detection of C1INH binding to kallikrein by modified ELISA

In vivo clearance and protein detection in mice

Statistical analysis and significance testing

Results

Novel recombinant proteins

Fig 1. C1-esterase inhibitor (C1INH) recombinant proteins.

Characterization of fused and unfused truncated, non-glycosylated C1INH proteins

Table 1. Pharmacokinetic characterization of activity of C1INH recombinant proteins.

Fig 2. Complex characterization of C1INH recombinant proteins.

In vivo clearance of recombinant proteins

Fig 3. Protein clearance of H6-trC1INH(MGS), H6-trC1INH(MGS)-MSA, and H6-MSA.

Pharmacokinetic analysis

Table 2. H6-trC1INH(MGS), H6-trC1INH(MGS)-MSA, H6-MSA pharmacokinetic analysis of clearance.

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Yash Gupta

Roles

Author response to Decision Letter 0

Decision Letter 1

Yash Gupta

Roles

Acceptance letter

Yash Gupta

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Expression and purification of H₆-trC1INH(MGS), H₆-MSA, and H₆-trC1INH(MGS)-MSA fusion protein in P. pastoris

Protease inhibition assays with H₆-trC1INH(MGS) and H₆-trC1INH(MGS)-MSA

Electrophoretic analysis of H₆-trC1INH(MGS) and Pka interaction

Fig 3. Protein clearance of H₆-trC1INH(MGS), H₆-trC1INH(MGS)-MSA, and H₆-MSA.

Table 2. H₆-trC1INH(MGS), H₆-trC1INH(MGS)-MSA, H₆-MSA pharmacokinetic analysis of clearance.