An Fc-SPINK1 fusion protein inhibits pancreatic inflammation in a mouse model

An Fc-SPINK1 fusion protein inhibits pancreatic inflammation in a mouse model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article An Fc-SPINK1 fusion protein inhibits pancreatic inflammation in a mouse model Jeffrey C. Way, D. Heid, M. Theiss, S. F. Nørrelykke, L. Barck, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6658843/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Pancreatitis results from premature activation and impaired inactivation of pancreatic proteases, primarily trypsin, leading to self-digestion, tissue necrosis, fibrosis, and inflammation. SPINK1 is a pancreas-specific inhibitor of trypsin that prevents premature trypsin activation, and could be a candidate therapeutic. However, because of its small size, SPINK1 would be subject to rapid renal clearance, making it ineffective. To construct a long half-life therapeutic inhibitor of trypsin for pancreatitis treatment we fused this protein to the C-terminus of an IgG1 antibody Fc element, increasing the size to ~ 78 kDa, thereby exceeding the renal clearance threshold and providing for FcRn-mediated recycling out of cells. A non-glycosylated form of Fc-SPINK1 was expressed in the yeast Pichia pastoris . Fc-SPINK1 inhibits trypsin enzyme activity in vitro . The blood pharmacokinetics in mice are consistent with a three-compartment distribution model and a terminal half-life of ~ 3 days. In a cerulein-induced mouse model of pancreatitis, Fc-SPINK1 significantly ameliorated cell death and immune cell infiltration. We developed an automated image analysis technique to quantify pancreatitis-associated loss of tissue cohesion, and found that Fc-SPINK1 also reduced this effect. This study demonstrates the potential of Fc-SPINK1 as a rationally designed therapy for pancreatitis. Biological sciences/Biotechnology Biological sciences/Cell biology Biological sciences/Drug discovery Biological sciences/Physiology Pancreatitis ERCP Fc fusion trypsin Image analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Pancreatitis is a major unmet medical challenge, affecting nearly 9 million people annually and resulting in more than 100,000 deaths/year. [ 1 – 2 ] Acute pancreatitis has a high mortality, in the range of 3–16%. [ 3 – 6 ] Patients with chronic pancreatitis can have acute attacks, can also progress to pancreatic cancer, and may also have inflammatory diabetes that is often mistaken for Type II diabetes. The disease is influenced by genetic factors, alcohol consumption, trauma, gallstones and also occurs as a complication of endoretrocholiangopancreatographic surgery (ERCP). [ 7 ] Gallstone-driven pancreatitis is well-treated by surgery, and acute pancreatitis involves a SIRS-like cytokine storm that is challenging to treat in any case. In contrast, chronic and ERCP-induced pancreatitis have a predictable course that may allow for pharmacological intervention. Genetic studies have demonstrated the central role of trypsin in the development of pancreatitis, and point to a multicomponent regulatory system for controlling trypsin activity. [ 8 ] Trypsin cleaves the pro-form of other digestive enzymes including prochymotrypsin. Mature chymotrypsin can then cleave and inactivate trypsin. In addition, the small, pancreas-specific protein SPINK1 inhibits trypsin. Gain of function mutations in cationic trypsinogen (PRSS-1) and loss of function mutations in SPINK1 and chymotrypsin significantly increase the risk of pancreatitis. [ 9 – 12 ] In the pancreatic acinar cells, pro-trypsin, pro-chymotrypsin, other digestive enzymes, and SPINK1 are co-localized in vesicles that normally fuse with the apical surface to release their contents into a duct system that leads to the small intestine. In pancreatitis, these vesicles aberrantly fuse with lysosomes, and the lysosomal hydrolase cathepsin B activates trypsin, initiating a chain reaction and autolysis of acinar cells with the destruction of surrounding tissue. [ 13 – 15 ] Studies in mice associate the loss of cathepsin B with reduced trypsin activity and milder pancreatitis. [ 13 ][ 14 ] Various agents have been investigated for the treatment of pancreatitis, including antisecretory molecules, protease inhibitors, calcium modulators, immunomodulators, anti-inflammatories, and antioxidants. For trypsin inhibitors, the small protein Aprotinin/BPTI and small molecule gabexate mesylate were both extensively clinically tested against acute pancreatitis and generally failed, [ 16 – 18 ] most likely because both of these molecules have a very short plasma half-life (2 hours and < 1 minute, respectively), [ 17 – 19 ] and because in acute pancreatitis the risk of death is likely driven by inflammatory cytokines, and it is too late to address the initiating problem of trypsin overactivity. [ 20 ] However, clinical trials have shown that 13-hour or 6.5 hour infusions of gabexate mesylate prior to ERCP did significantly reduce the incidence of pain and acute pancreatitis after the procedure. [ 21 – 22 ] Unfortunately, the long infusion times before surgery make this specific treatment impractical. These trials demonstrate that trypsin inhibition could be of use in prophylaxis of pancreatitis before ERCP, but also underscore the importance of pharmacokinetics in driving adoption of a particular treatment. As the human body’s natural endogenous trypsin inhibitor, SPINK1 (serine protease inhibitor Kazal type 1) is a promising therapeutic element. Compared to BPTI, SPINK1 has the advantage that it is an endogenous human protein and less likely to induce an immune response after repeated administration in treatment of chronic disease. In this work, we demonstrate that an Fc-SPINK1 fusion protein can reduce the severity of pancreatitis in a mouse model. Results Design, production, and purification of Fc-SPINK1, and in vitro trypsin inhibition The trypsin inhibitors SPINK1 and BPTI (bovine pancreatic trypsin inhibitor; Aprotinin) both have a molecular weight of < 7,000 Daltons, which will result in rapid renal clearance and to make these proteins unsuitable as drugs. We fused SPINK1 and BPTI to the C-terminus of the Fc fragment of human IgG to create an Fc-SPINK1 and Fc-BPTI dimers with a molecular weight of about 78 kDa, which is well above the renal clearance threshold and should significantly improve pharmacokinetics [ 23 ] (Fig. 1 a-c). The Fc region contains three mutations relative to the natural human IgG1 sequence (Fig. 1 b). First, amino acid 5, normally cysteine, is mutated to serine because this cysteine normally forms a disulfide bond with the antibody light chain, which is not present in this construct. Second, the aspartic acid at position 82 is normally asparagine, but this is the site of N-linked glycosylation of the Fc region. Removing this glycosylation has the effect of significantly reducing binding to both Fc receptors and to the complement component C1q, [ 24 ] and also allows for safe, low-cost expression in the yeast Pichia pastoris instead of mammalian cells; specifically, Pichia generates a high-mannose-type N-linked oligosaccharide modification that would be recognized as foreign by mammals, [ 25 ] but our molecule has been engineered to not be glycosylated. Third, amino acid 232 is normally the C-terminal lysine of a secreted IgG1, but this amino acid can be cleaved in both natural antibodies and fusion proteins, so it is mutated to alanine to prevent potential cleavage. [ 26 ] The SPINK1 and BPTI modules were placed at the C-terminus of Fc instead of the N-terminus, because fusions in the Fc-X orientation are generally expressed at a higher level. [ 23 ] No linker was used because the C-terminus of Fc is somewhat unstructured and may form a natural linker, and because both fusion termini are far from known sites of protein interaction (Fig. 1 c). The Pichia pastoris expression system was used for the production procedures, and fusion proteins were purified to near homogeneity by protein A chromatography (Fig. 1 d-e; Methods). A trypsin inhibition assay (Fig. 1 f) demonstrated the efficacy of both constructs in inhibiting trypsin at molar ratios of 1:1 (0.429 nM each) and 1:0.5 Fc-SPINK1-trypsin. (We generally did not observe partial inhibition of trypsin by Fc-SPINK1 at low molar ratios of SPINK1:trypsin; we hypothesize that trypsin might inactivate Fc-SPINK1 under the conditions we used.) In vivo efficacy in cerulein-induced mouse model of acute pancreatitis Fc-SPINK1 and Fc-BPTI were evaluated as potential therapeutic agents in a mouse model of cerulein-induced pancreatitis. Cerulein is a frog-derived toxic peptide that stimulates a moderate, transient pancreatitis. [ 27 – 28 ] Pancreatitis was induced by seven injections of cerulein into C57Bl/6N mice, with a single injection of Fc-SPINK1, Fc-BPTI, or vehicle, followed by harvest of the pancreas and blood 18 hours after the first injection (Fig. 2 A). Pancreases were fixed, sectioned, and stained with hematoxylin/eosin (H&E) or with anti-CD11b (staining macrophages, neutrophils, and certain other immune cells [ 29 ] ) (Fig. 2 B-G). To date, necrosis and immune cell infiltration is quantified visually. This slow process requires subsampling of tissue. [ 41 ] To increase statistical power, we performed semi-automated analysis on all available image slides with respect to necrotic cells (Fig. 2 H-I), and immune cell infiltration (Fig. 2 J-K). In H&E-stained healthy pancreas tissue, the acinar cells are normally tightly associated with each other (Fig. 2 B). After cerulein treatment, the cells are dramatically separated in H&E-stained sections (Fig. 2 C). [ 30 ] To quantitate this effect in an unbiased way, we developed a new automated image analysis method (Fig. 2 L-M; see below). In one experiment, we treated mice with 5 mg of Fc fusion protein after the third cerulein injection. This high dose was based on the fact that mammals synthesize large amounts of trypsin, and only a small amount of the injected Fc-SPINK1 fusion protein will distribute into the pancreas (see Discussion). Fc-SPINK1 reduced markers of cerulein-induced pancreatitis (Fig. 2 ). Specifically, acinar cell necrosis and plasma amylase levels were reduced in cerulein + Fc-SPINK1-treated mice compared to mice treated with cerulein and vehicle or Fc-BPTI (Fig. 2 N-O). Quantitative semi-automated image analysis of necrotic cells showed a significant reduction in the Fc-SPINK1-treated group compared to the cerulein control (Fig. 2 , Supplementary 1). Fc-BPTI did not appear to reduce acinar cell necrosis or amylase levels. Pancreatic amylase, which is released during cerulein-induced inflammation, is a human biomarker for the detection of pancreatic injury. Blood plasma amylase activity (Fig. 2 D) was reduced in the Fc-SPINK1-treated group (*p < 0.05). There was no reduction in amylase activity in the plasma of Fc-BPTI-treated mice. Typically, in the cerulein-induced pancreatitis mouse model, plasma amylase levels peak after the last cerulein injection. [ 30 ] In this experiment, at the 18 hour time-point, the amylase levels are returning to baseline, but the effect of Fc-SPINK1 can still be observed. In a second experiment, the effects of different Fc-SPINK1 doses administered by intravenous (IV) and intraperitoneal (IP) injection were investigated (Fig. 3 and Supplementary Fig. 2), using doses of 1, 2.5, or 5 mg per 20-gram mouse. A dose response was observed with the IV-injected mice. Mice receiving 2.5 or 5 mg/mouse showed significantly less necrosis than mice treated with 0 or 1 mg, and the tissue showed progressively ameliorated junctional integrity with increasing IV dosing. In the intraperitoneally dosed animals, the separate dose groups did not show significantly less cell necrosis than the untreated group, but there was a trend towards a therapeutic effect (Supplementary Fig. 2). Taken together, the results indicate that pancreatitis symptoms decrease with high enough doses of Fc-SPINK1, and that a maximally effective dose may not have been reached. Mice of the C57Bl6J strain develop a more severe cerulein-induced pancreatitis than C57Bl6N mice. In a third experiment, when C57Bl6J mice were treated with cerulein and Fc-SPINK1 following the timeline in Fig. 2 A, a moderate reduction in disease severity was observed. However, when the C57Bl6J mice were given Fc-SPINK1 by IP injection prior to the cerulein injections, we observed a stronger effect, seeing both a statistically significant reduction in cell necrosis and cell separation in the pancreases of cerulein-treated mice (Fig. 4 ). Prophylactic treatment is particularly relevant to ERCP-induced pancreatitis. Image analysis We used automated and semi-automated image analysis to quantitate the extent of cell death, lymphocyte infiltration, and cell separation, which are visual markers of inflammation. Cell separation may be related to edematous fluid influx, and is henceforth also referred to as edema. Each analyzed image corresponds to a whole-pancreas cross-section. Images that showed staining-defects or were acquired with different microscope-settings than the rest of their batch, were excluded from analysis. First, exocrine tissue (acinar cells) was segmented using a random-forest-based pixel classifier in the open-source digital pathology software QuPath (v. 0.5.1.). [ 42 – 43 ] A pixel-classifier assigns a class to each pixel (such as background, exocrine tissue) based on manually annotated ground truth examples. To ensure pixel classifiers for Edema and Necrosis were trained on well-randomized samples, square regions at a random location within exocrine tissue were automatically selected and combined into a training image to which manual annotations were added. After pixel-classification, manual corrections were made by removing wrongly segmented (false positive) areas. The Edema, Necrosis, and CD11b + scoring functions were generated as follows (see Supplementary Information for details). Within exocrine segments, the space between cells was segmented using a QuPath pixel classifier. In inflamed tissue, acinar cells are rounded, possibly due to a loss of tight junctional integrity that may relate to edematous fluid influx into tissue or to separation of cells during fixation. Hence, the intercellular space forms a netlike topology. Branch points signify a loss of tight junction integrity at multicellular junctions. For the Edema function, the number of branch points was therefore used to quantify inflammation. For the Necrosis function, necrotic cells were segmented using a QuPath pixel classifier, and false positive segments were manually removed. The ratio of necrotic cells to exocrine tissue was used to quantify this aspect of pancreatitis. The CD11b + function is based on the fact that inflammation correlates with CD11B + immune cell invasion. In IHC images, CD11B + cells and tissue (exocrine and endocrine) were segmented using a QuPath pixel classifier, and the percentage of tissue area classed as CD11B + was used to quantify inflammation. The Shapiro-Wilk test of normality was conducted for each treatment condition to assess the normality of sample distributions, with a significance level of α = 0.05. If the null hypothesis of at least one condition within an experiment was rejected, the Mann-Whitney-U test [ 31 ] was used with the alternative hypothesis that sick samples show greater indicators of inflammation than healthy or treated samples. When the null hypothesis of the Shapiro-Wilk test [ 32 ] of normality was not rejected, Welch’s one-tailed t-test [ 33 ] was applied. Pharmacokinetics We investigated the dynamics of Fc-SPINK1 in the blood system using Tg276 mice. [ 34 ] Because Fc-SPINK1 is large enough to prevent renal clearance, pharmacokinetics is expected to be dominated by trafficking through cells with various Fc receptors. The Tg276 mice express human versions of FcRn and the various Fc-gamma receptors, so the pharmacokinetics of Fc-SPINK1 (in which the Fc and SPINK1 elements are both based on human proteins) will be more accurately represented. The concentration of Fc-SPINK1 in blood plasma was followed over time (Fig. 6). Analysis of the data using a two-compartment model indicates a beta-phase half-life of 21.51 hours, an area under the curve (AUC) of 3190 (µgཥh)/ml or 132.9 (µgཥdays)/ml for a 100 µg dose, and a clearance rate of 0.75 ml/day. Discussion Pancreatitis is an extremely painful and sometimes fatal inflammation of the pancreas in which excess trypsin activation plays a major role. To treat pancreatitis, we designed an Fc-SPINK1 fusion protein that would inhibit extracellular trypsin, and also have a long enough plasma half-life to be practical as a therapeutic protein. SPINK1 is attractive because it is a natural human protein, unlike bovine pancreatic trypsin inhibitor (BPTI/Aprotinin), which is only expressed in ruminants [ 35 ] and might therefore be immunogenic. In addition, SPINK1 has a high specificity for trypsin and a much lower affinity than BPTI for other serine proteases. We placed SPINK1 at the C-terminus of the Fc region because Fc fusion proteins are generally more highly expressed in this configuration. [ 23 ] We expressed this protein in the yeast Pichia pastoris , purified it, and showed that it inhibits trypsin in vitro (Fig. 1 ). Fc-SPINK1 reduced the indications of pancreatitis in the cerulein-induced mouse model of this disease. (Fig. 2 – 4 , Supplementary Fig. 1). Cerulein, a toxic peptide from the Australian green tree frog with a C-terminus similar to cholecystokinin, hyperstimulates the pancreas and, after 7 injections leads to a mild pancreatitis that resolves after about 24 hours. [ 27 ][ 30 ] When C57Bl6N mice were treated with cerulein, 18 hours after the first injection the pancreas showed signs of pancreatitis: dead acinar cells, infiltrating CD11b + leukocytes, and spaces between cells that may represent edema or loss of junctional integrity. When mice received 5 mg of Fc-SPINK1 after the third cerulein injection, cell death and microscopic edema was reduced (Figs. 2 A-N). Blood levels of pancreatic amylase were slightly increased in cerulein-treated mice but reduced back to baseline in mice receiving Fc-SPINK1 as well (Fig. 2 O). These effects were observed at the higher intravenous doses of a dose-escalation experiment in which we administered 0, 1, 2.5 or 5 mgs/mouse of Fc-SPINK1 to cerulein-treated C57Bl6N mice (Fig. 3 ). In addition, when mice of the C57Bl6J strain, which develop a more severe cerulein-induced pancreatitis than C57Bl6N mice, were treated with Fc-SPINK1 prior to the cerulein injections, a reduction in cell death and leukocyte infiltration was observed (Fig. 4 ). Taken together these results indicate that Fc-SPINK1 can reduce the intensity of pancreatitis in the cerulein-treated mouse model. One dramatic effect of pancreatitis in mouse models is the separation of cells in H&E-stained sections of the pancreas of cerulein-treated mice. Previously, researchers scored this by visual inspection (blinded to sample identity), which is tedious and may miss subtle effects. As part of this work, we developed an automated scoring system in which the spaces between cells in H&E stained sections are identified, abstracted as lines, and the branchpoints in the resulting pattern is quantitated (Fig. 5 ). This approach may work in part because non-pathological spaces between cells, corresponding to ducts and spaces between pancreatic lobes, do not have extensive branching patterns and will not contribute greatly to the score. The Fc-SPINK1 protein has a long plasma half-life (Fig. 6). When the data are interpreted with a two-compartment model, the elimination half-life is about 21 hours, while interpretation with a more physiological 3 compartment model indicates an elimination phase half-life of about 4 days (Supplementary Fig. 4). These results suggest that the Fc-SPINK1 molecule tested here would be adequate for prophylactic administration prior to the ERCP procedure, but improvement of the plasma half-life would be ideal for chronic pancreatitis patients who may need life-long treatment. The initiation of pancreatitis is thought to occur when overstimulation of exocrine secretion leads to fusion of acinar secretory vesicles with the wrong membrane, such as the basolateral cell surface or a lysosome or endosome. In particular, upon cerulein treatment, fusion of a trypsinogen-containing vesicle with a vesicle containing cathepsin B leads to trypsin activation within 30 minutes of treatment. [ 30 ] A cathepsin B knockout mouse does not show this early activation, but still shows pancreatitis phenotypes after multiple cerulein injections. Geisz et al. [ 30 ] hypothesize that trypsin autoactivation also occurs in the interstitial space and, through cleavage of protease-activated receptors, may initiate pancreatitis. The fact that Fc-SPINK1 reduces the severity of pancreatitis in the cerulein mouse model supports the idea that interstitial autoactivation plays a role in this model, and that cerulein-induced pancreatitis is not solely driven by intracellular trypsin activation. As an injected drug, Fc-SPINK1 has advantages compared to small molecule trypsin inhibitors. Camostat is a serine protease inhibitor that is approved in Japan for treatment of pancreatitis. [ 36 ] Layer et al. found that when healthy human volunteers were given oral camostat, trypsin activity was inhibited throughout the small intestine, but also increased levels of pancreatis amylase and lipase in the intestine. [ 37 ] These authors inferred that this occurred through increased pancreatic secretion, and the results were evidence for a feedback loop between protease levels in the intestine and pancreatic activity. These observations suggest that any orally delivered trypsin inhibitor might be problematic for treatment of pancreatitis, since there will always be more drug in the intestine than in the pancreas, and during periods between doses when drug levels fall, pancreatic secretion could be stimulated. A high dose of Fc-SPINK1 is required to successfully treat the cerulein-dosed mouse. This is likely because mammals produce a large amount of trypsin, and to be effective, Fc-SPINK1 needs to be in molar excess relative to active extracellular trypsin. As a point of comparison, the human body secretes between 10 and 100 mgs of trypsin per day into the small intestine. [ 38 – 39 ] The pancreas weighs about 80 grams – slightly more than 0.1% of body weight – so only a small fraction of systemically administered Fc-SPINK1 will distribute into the pancreas. The fraction of trypsin that is activated and redirected into the accessible extracellular space during pancreatitis is unknown but may be large. With allometric scaling, a dose of 5 milligrams in a 25-gram mouse corresponds to a dose of about 1–2 grams in a human. Administration of such a large amount of protein is less challenging in humans than in mice because humans are amenable to IV infusion. Patients undergoing an Endoscopic retrograde cholangiopancreatography (ERCP) procedure have about a 10% chance of experiencing post-procedure pancreatitis, which usually resolves but can be debilitating or fatal. Typically, before the procedure, a patient is given an IV infusion of 250 mls to 1 liter of fluid to make the ducts more accessible. Fc-SPINK1 could be administered prophylactically in this infusion to reduce the chances of developing pancreatitis. The infusion volume would allow for a large dose of Fc-SPINK1 without requiring an exotic formulation for a high protein concentration. [ 40 ] The plasma half-life of Fc-SPINK1 is in an appropriate range, since post-ERCP pancreatitis occurs within 24 hours of the procedure. Taken together, our results indicate that Fc-SPINK1 is a promising candidate for treatment and prevention of pancreatitis. Methods DNA construction . Geneious Prime version 2020.1.2 was used to organize sequence data, design, and validate cloning strategies, maps, and primers. Each construct made during this research project was sequence-validated by sanger sequencing provided by GENEWIZ (South Plainfield, NJ, USA). All plasmids used in this study are based on the expression vector pPICZαA (Thermo Fischer Scientific, V19520). This plasmid allows methanol-inducible, secreted expression in Pichia pastoris . Plasmids were constructed by standard techniques. Fc and SPINK1 sequences were codon-optimized for expression in Pichia pastoris . Protein expression in P. pastoris . Expression and purification of the Fc-SPINK1 and Fc-BPTI fusion proteins was carried out using standard techniques, which are detailed in Supplementary Information. In brief, the methanol-inducible P. pastoris system was used to express secreted Fc fusion proteins, which were concentrated from supernatant and purified using standard Protein A chromatography. The SDS-polyacrylamide gel depicted in Fig. 1 e, which illustrates the expression and purification of Fc-SPINK1, complies with digital image and integrity policies ( https://www.nature.com/srep/journal-policies/editorial-policies#digital-image ) as follows: (1) the gel depicted was a single SDS gel, and no lanes were removed in the image; (2) all of the molecular weight markers visible in the original gel are depicted; (3) the gel image was ‘cropped’ at the top of the gel, removing only the wells, at the bottom of the gel, and on the sides outside the area where samples were run; and (4) the gel image was recorded with normal contrast. No other images of this gel are available. Protein characterization: Trypsin inhibition activity . Trypsin-inhibitor fusion proteins were tested in vitro for trypsin binding capacity. Therefore, trypsin inhibitor proteins and trypsin were diluted to a concentration of 50 µM in PBS. Trypsin was mixed with the respective inhibitor protein in ratios from 1:1 to 1:0.0625, including a positive and negative control for trypsin activity, and incubated at room temperature for 5 min. Trypsin activity in each sample was measured using the Trypsin Activity Colorimetric Assay Kit (Abcam, ab102523). Here, trypsin activity was measured with a substrate that is cleaved by trypsin to generate p-nitroaniline ( p -NA) which is detected at λ = 405 nm. In brief, reactions were diluted 1:50 with PBS. Samples and controls were mixed with 50 µl reaction mix and incubated at 25°C for 60 min. Absorption at 405 nm was measured and normalized to the trypsin positive control without trypsin inhibitor Cerulein-induced acute pancreatitis model Pancreatitis was induced by intraperitoneal (IP) administration of the peptide cerulein according to Lampel and Kern. [ 28 ] Cerulein (50 µg/kg dissolved in 50 µl endotoxin-free PBS − 1 µg for a mouse with 20 g) was injected once per hour to a total of 7 injections. Mice were closely monitored throughout the injection period. Illness was observed within 1–2 hours after the first injections. Administration of trypsin inhibitor proteins . Trypsin inhibitor fusion proteins were administered in endotoxin-free PBS either intraperitoneal (IP) or intravenously (IV) by tail vein injection. Maximum volumes for IP and IV injections were 200 µl and 100 µl, respectively. Measurement of serum α-amylase activity. Serum activity of α -amylase, a digestive enzyme made by the pancreas and a clinical biomarker for pancreatitis was measured using the Amylase Activity Colorimetric Assay Kit (abcam, ab102523) according to manufacturer’s instructions. Histopathology: Hematoxylin and Eosin (H&E) staining and immunohistochemistry (IHC) . Immediately after necropsy, pancreas tissue was fixed in 10% Neutral Buffered Formalin for 24 hours before embedding, cutting, and staining. Slide scanning for all samples and IHC staining for CD11b was performed by Histowiz (Brooklyn, NY, USA). Declarations Data availability The datasets generated and/or analyzed during the current study are available in the Zenodo repository, with the following links: Experiment 1 (Figure 2): Batch 11571: https://zenodo.org/records/15586925 Batches 11564: https://zenodo.org/records/15587055 Dose-response experiments (Figure 3): Batch 14628: https://zenodo.org/records/15587055 Batch 14550: https://zenodo.org/records/15587142 Prophylaxis experiments (Figure 4) Batch 25868: https://zenodo.org/records/15587101 Raw IHC data: Batches 11564 and 14628: https://zenodo.org/records/15587055 These files can be opened with QuPath (https://qupath.github.io/) and other specialized image-viewing applications. Pharmacokinetics . Pharmacokinetics of trypsin inhibitor fusion proteins were studied by IV administration of the respective protein. Tail vein blood was collected 5 min, 1 h, 3 h, 6 h, 12 h, 24 h, 72 h and 96 h post-drug injection and stored as EDTA-plasma at -20°C until analysis. ELISAs based on human SPINK1 capture and human Fc detection were used to quantitate fusion protein in serum, using standard procedures described in Supplementary Information. Animal welfare. All mouse experiments were carried out under protocol IS00002593, which has been approved by the Harvard Medical Area Standing Committee on Animals (HMS-IACUC). All mouse experiments were carried out in accordance with relevant state and federal guidelines and regulations. Accordance with ARRIVE guidelines. Mouse experiments were carried out and reported in accordance with the “ARRIVE Essential 10” guidelines (https://arriveguidelines.org) as follows. (1) The ‘experimental unit’ was the individual mouse. Controls are indicated in the figure legends and text for each experiment. (2) The studies reported here are exploratory, so there was no specific expectation of effect sizes or numbers of animals that needed to be tested. The sample sizes were limited by availability of the Fc-SPINK1 test protein, which we manufactured ourselves. Generally, dose groups of 4 or 5 mice were tested. For some experiments smaller numbers of mice were tested or evaluated in an exploratory manner, and not all pancreases were imaged due to expense, especially for CD11b+ staining. For the sake of completeness, these data are reported in Supplementary Information, but are generally not statistically meaningful. (3) Individual mice from a dataset were excluded only if a mouse died prematurely. In one case, a set of images was excluded from analysis because the pixel size was different from that of the other images, and the machine learning-based analysis appeared to give spurious results. Scores for each mouse are individually reported for all of the experiments where Fc-SPINK1 was tested. (4). Randomization was not used because the mice in each experiment were all the same age. (5) Blinding was used in preliminary manual analysis of images but was not necessary for computer-evaluated images. (6) Outcomes are described in the text and figure legends. The overall hypothesis being tested was that Fc-SPINK1 treatment would reduce the severity of pancreatitis markers induced by cerulein, but we did not have a specific hypothesis about how strong the effect would be, what the optimal dose of Fc-SPINK1 would be, or when the maximal effect would be observed; hence this is an exploratory study. (7) Statistical methods are described above and in the Figure Legends. (8) All mice were purchased from either Charles River Labs or Jackson Labs, and were 8- to 12-week old females. The use of C57Bl/6N and C57Bl/6J substrains of mice, which have different pancreatitis phenotypes, is described in the text. Mice were naïve; they had not been used for other experiments. (9) Specifics of the pancreatitis induction and treatment protocol are described above and in the main text. All mice were acclimatized for at least 1 week after shipping before the experiment was performed. (10) The statistical analysis of each experiment is presented in the figures, main text and methods. Euthanasia and humane treatment of mice. As part of the pancreatitis induction and testing protocol, mice were sacrificed at the end of the experiment before the pancreases were harvested. Euthanasia was performed using standard carbon dioxide asphyxiation, followed by cervical dislocation, in accordance with the IACUC-approved mouse protocol cited above. Acknowledgments: We thank Harvard Medical School for a Q-FASTR grant and the Wyss Institute for Biologically Inspired Engineering for validation project support. We also thank Andrea Geisz for extensive discussions on pancreatitis mechanisms and techniques. Author contributions: JCW, DH, LBa and LBu were responsible for the analysis and interpretation of the data and for the preparation of this manuscript. DH, LBa, LBu, JAB, TD, PH, DN, RS, TS were responsible for protein expression, and/or performing and planning the experiments. MT and SFN were responsible for imaging algorithm development, analysis, interpretation, preparation of the manuscript KR conceptualized the Fc-SPINK1 fusion protein and constructed the first versions. ARG, AV and MSV were responsible for verifying the accuracy and performance of the animal studies. PAS was responsible for funding and study supervision, and assisted with preparation of the manuscript. Competing interests statement. Jeffrey Way, Katherine Redfield Chan, Daniel Heid, and Dominik Niopek are inventors on a patent application covering SPINK1 fusion proteins that has been assigned to Harvard University. References M. H. Forouzanfar et al., “Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990–2015: a systematic analysis for the Global Burden of Disease Study 2015,” Lancet, vol. 388, no. 10053, pp. 1659–1724, 2016, doi: 10.1016/S0140-6736(16)31679-8. D. Yadav and A. B. Lowenfels, “The epidemiology of pancreatitis and pancreatic cancer,” Gastroenterology, vol. 144, no. 6, pp. 1252–1261, 2013, doi: 10.1053/j.gastro.2013.01.068. O. J. Hines and S. J. Pandol, “Management of severe acute pancreatitis,” BMJ, vol. 367, pp. 1–12, 2019, doi: 10.1136/bmj.l6227. J. Norman, “The role of cytokines in the pathogenesis of shigellosis,” J. Pediatr. Gastroenterol. 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Silver","email":"","orcid":"","institution":"Harvard Medical School","correspondingAuthor":false,"prefix":"","firstName":"P.","middleName":"A.","lastName":"Silver","suffix":""}],"badges":[],"createdAt":"2025-05-13 22:08:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6658843/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6658843/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86667652,"identity":"905e84a8-79a1-4fc2-b41f-bf268ea149a2","added_by":"auto","created_at":"2025-07-14 11:15:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":135757,"visible":true,"origin":"","legend":"\u003cp\u003eThe fusion proteins Fc-SPINK1 and Fc-BPTI are constructed from the Fc region of a human IgG1 antibody and SPINK1 resp. BPTI. SPINK1 is a naturally derived human trypsin inhibitor, BPTI is found in bovine pancreas. (a) Schematic structure of Fc-SPINK1 or Fc-BPTI. (b) Sequence of the mature, secreted form of Fc-SPINK1. (c) The predicted 3D structure of Fc-SPINK1 and Fc-BPTI. (d) Overview of the production and purification procedure of Fc-SPINK1 and Fc-BPTI in the yeast \u003cem\u003ePichia pastoris\u003c/em\u003e. (e) The major production and purification steps are visualized using Coomassie stained SDS-PAGE. The data displays that Fc-SPINK1 (32 kDa) was effectively purified and concentrated. (f) The trypsin inhibition assay demonstrates the inhibition of trypsin by Fc-SPINK1 and Fc-BPTI in a para-nitrophenol-based assay. Both constructs successfully inhibit trypsin at a 1:1 and 1:0.5 ratio.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6658843/v1/e427a7d300461e3b42f65db6.png"},{"id":86667654,"identity":"9550be0f-e3ad-457b-acce-b4e1c7c7b311","added_by":"auto","created_at":"2025-07-14 11:15:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":503480,"visible":true,"origin":"","legend":"\u003cp\u003eTreatment of cerulein-induced pancreatitis with Fc-SPINK1 reduces symptom severity.\u003cstrong\u003e (A)\u003c/strong\u003e Workflow of the experiment. N=5 C57Bl6N mice per dose group. \u003cstrong\u003e(B-D)\u003c/strong\u003e Hematoxylin/Eosin staining of pancreas tissue sections from mice treated with \u003cstrong\u003e(B) \u003c/strong\u003eseven hourly injections of PBS, \u003cstrong\u003e(C)\u003c/strong\u003e seven hourly injections of cerulein, or \u003cstrong\u003e(D)\u003c/strong\u003eseven hourly injections of cerulein plus an injection of 5 mgs of Fc-SPINK1. The results indicate that in cerulein-treated mice, a number of cells become necrotic (light pink staining), and cells also suffer from a loss of junctional integrity, going from a cuboid shape to a rounded shape with fluid between cells. Treatment with Fc-SPINK1 may lessen these phenotypes. \u003cstrong\u003e(E-G)\u003c/strong\u003e Anti-CD11b staining of pancreas tissue sections from mice treated with PBS, cerulein, or cerulein plus Fc-SPINK1. The results indicate that in cerulein-treated mice, CD11b+ leukocytes (e.g., macrophages and NK cells) invade the pancreas. Treatment with Fc-SPINK1 may lessen this phenotype. \u003cstrong\u003e(H-M)\u003c/strong\u003e Image processing methods for automated, unbiased evaluation of stained tissue sections. See Supplementary Information for algorithmic details. Scale bars are 100 microns. \u003cstrong\u003e(H-I)\u003c/strong\u003eSemi-automated scoring of cell necrosis. Yellow false-colored areas are those scored as necrotic. Magenta false-colored area indicates exocrine tissue \u003cstrong\u003e(J-K)\u003c/strong\u003eAutomated scoring of CD11b+ infiltration. Yellow false-colored cells are CD11b+. Magenta false-colored cells indicate exocrine tissue. \u003cstrong\u003e(L-M) \u003c/strong\u003eAutomated scoring of loss of junctional integrity in the pancreas. This algorithm segments gaps between cells to generate a network pattern (green false-color) and then calculates the number of branch points in the network (normalized to the area of the tissue section; Figure 5). \u003cstrong\u003e(N)\u003c/strong\u003e Results of semi-automated necrotic cell staining. Pancreases of PBS-treated mice show no necrotic cells, while pancreases of cerulein treated mice showed a increased level of cell necrosis. Injection of Fc-SPINK1 appeared to lessen cell necrosis, while Fc-BPTI had no apparent effect on cell necrosis. To improve the statistical analysis, the scores of mice treated with cerulein alone and cerulein plus Fc-BPTI were combined and compared to scores from mice treated with cerulein plus Fc-SPINK1. Horizontal bars indicate mean ± standard error of mean (SEM). Red diamonds indicate mean weighted by tissue-area. P-values: one-tailed Mann-Whitney-U test. \u003cstrong\u003e(O)\u003c/strong\u003e Plasma amylase levels in mice treated with PBS, cerulein, and Fc-BPTI or FC-SPINK1, 18 hours after the first injection of cerulein. Levels of amylase in mice treated with cerulein and Fc-SPINK1 were lower than in mice treated with either cerulein or cerulein and Fc-BPTI (*p\u0026lt;0.05, one-tailed Student’s T-test).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6658843/v1/fc10204191eb444231f78657.png"},{"id":86667657,"identity":"6cdff424-5494-4fdd-992d-673da39ce1cb","added_by":"auto","created_at":"2025-07-14 11:15:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40942,"visible":true,"origin":"","legend":"\u003cp\u003eDose-responsive reduction in \u003cstrong\u003e(A)\u003c/strong\u003e cerulein-induced pancreatitic necrosis and \u003cstrong\u003e(B)\u003c/strong\u003e loss of tissue integrity. C57Bl6N mice were treated with seven injections of cerulein and one injection of Fc-SPINK1 as in Figure 2A. Horizontal bars indicate mean ± SEM. Red diamonds indicate mean weighted by tissue-area. P-values: One-tailed Welch’s t-test.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6658843/v1/1afa5c3d4a261d72e2e2e4d8.png"},{"id":86667655,"identity":"2d90fbc4-4e08-4f30-940f-48e7712e274e","added_by":"auto","created_at":"2025-07-14 11:15:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33613,"visible":true,"origin":"","legend":"\u003cp\u003eProphylactic treatment with Fc-SPINK1 reduces effects of cerulein-induced pancreatitis. For both panels,* is p\u0026lt;0.05; ** is p\u0026lt;0.01; ** is p\u0026lt;0.001. \u0026nbsp;\u003cstrong\u003e(A)\u003c/strong\u003e Necrotic cells were quantitated in H\u0026amp;E-stained pancreas cross-sections, in which mice were treated with Fc-SPINK1 either during the series of cerulein injections as in Figure 2A, or before the first cerulein injection (“prophylaxis”). Black dots represent necrosis scores, and red diamonds represent the mean score, weighted by sample area. P-values: One-tailed Mann-Whitney-U test. \u003cstrong\u003e(B)\u003c/strong\u003e Branchpoint analysis was applied to the same images. Black dots represent branchpoint scores, and red diamonds represent the mean score weighted by the area of each cross-section. The results suggest that prophylactic treatment with Fc-SPINK1 has a stronger effect on inhibition of cerulein-induced pancreatitis than treatment after initial cerulein injections. P-values: One-tailed Welch’s t-test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6658843/v1/bcb38ff52bf2bdfddae8cc55.png"},{"id":86667664,"identity":"175daa65-8fea-4e81-9c60-8458cb6baa5e","added_by":"auto","created_at":"2025-07-14 11:15:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":436118,"visible":true,"origin":"","legend":"\u003cp\u003eBranchpoint analysis to quantitate reduction of tissue integrity. \u003cstrong\u003e(A)\u003c/strong\u003e Segmented gaps between cells \u003cstrong\u003e(B) \u003c/strong\u003eSkeletonized gaps: the network of gaps is turned into a graph. \u003cstrong\u003e(C)\u003c/strong\u003e Counting the branch point of this graph serves as a proxy for how separated the cells are. \u003cstrong\u003e(B)\u003c/strong\u003eand \u003cstrong\u003e(C)\u003c/strong\u003e: Multicolored dots indicate individual branch points.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6658843/v1/e2cc15e9715d2af60e9d7cb3.png"},{"id":86667661,"identity":"35ac0563-aff5-4650-b110-16c26e0d7f68","added_by":"auto","created_at":"2025-07-14 11:15:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59247,"visible":true,"origin":"","legend":"\u003cp\u003ePharmacokinetics (and distribution) of Fc-SPINK1. (A) Compartment model for estimating PK parameters. (B) Concentration of Fc-SPINK1 in the blood after IV injection of 100 mcg/mouse (N=4). The inset shows estimated pharmacokinetic parameters. The black curve is a two-parameter fit of the data. The brown lines are point-by-point straight line connections between averages of each time point.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6658843/v1/9b92adcc1db2bdb02f281c3d.png"},{"id":91865658,"identity":"81d7c4c6-bc8f-480e-96d8-27cddc2fe449","added_by":"auto","created_at":"2025-09-22 13:10:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1865159,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6658843/v1/e465d4c9-653f-4918-b904-eba057437fde.pdf"},{"id":86667653,"identity":"a90493c5-ccbf-49f0-9df1-bc95c5191dfa","added_by":"auto","created_at":"2025-07-14 11:15:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":920134,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6658843/v1/2590448fbe712a8edb18e4a9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"An Fc-SPINK1 fusion protein inhibits pancreatic inflammation in a mouse model","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatitis is a major unmet medical challenge, affecting nearly 9\u0026nbsp;million people annually and resulting in more than 100,000 deaths/year.\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e Acute pancreatitis has a high mortality, in the range of 3\u0026ndash;16%.\u003csup\u003e[\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e Patients with chronic pancreatitis can have acute attacks, can also progress to pancreatic cancer, and may also have inflammatory diabetes that is often mistaken for Type II diabetes. The disease is influenced by genetic factors, alcohol consumption, trauma, gallstones and also occurs as a complication of endoretrocholiangopancreatographic surgery (ERCP).\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e Gallstone-driven pancreatitis is well-treated by surgery, and acute pancreatitis involves a SIRS-like cytokine storm that is challenging to treat in any case. In contrast, chronic and ERCP-induced pancreatitis have a predictable course that may allow for pharmacological intervention.\u003c/p\u003e\u003cp\u003eGenetic studies have demonstrated the central role of trypsin in the development of pancreatitis, and point to a multicomponent regulatory system for controlling trypsin activity.\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e Trypsin cleaves the pro-form of other digestive enzymes including prochymotrypsin. Mature chymotrypsin can then cleave and inactivate trypsin. In addition, the small, pancreas-specific protein SPINK1 inhibits trypsin. Gain of function mutations in cationic trypsinogen (PRSS-1) and loss of function mutations in SPINK1 and chymotrypsin significantly increase the risk of pancreatitis. \u003csup\u003e[\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e In the pancreatic acinar cells, pro-trypsin, pro-chymotrypsin, other digestive enzymes, and SPINK1 are co-localized in vesicles that normally fuse with the apical surface to release their contents into a duct system that leads to the small intestine. In pancreatitis, these vesicles aberrantly fuse with lysosomes, and the lysosomal hydrolase cathepsin B activates trypsin, initiating a chain reaction and autolysis of acinar cells with the destruction of surrounding tissue. \u003csup\u003e[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e Studies in mice associate the loss of cathepsin B with reduced trypsin activity and milder pancreatitis.\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e][\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eVarious agents have been investigated for the treatment of pancreatitis, including antisecretory molecules, protease inhibitors, calcium modulators, immunomodulators, anti-inflammatories, and antioxidants. For trypsin inhibitors, the small protein Aprotinin/BPTI and small molecule gabexate mesylate were both extensively clinically tested against acute pancreatitis and generally failed,\u003csup\u003e[\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e most likely because both of these molecules have a very short plasma half-life (2 hours and \u0026lt;\u0026thinsp;1 minute, respectively),\u003csup\u003e[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e and because in acute pancreatitis the risk of death is likely driven by inflammatory cytokines, and it is too late to address the initiating problem of trypsin overactivity.\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e However, clinical trials have shown that 13-hour or 6.5 hour infusions of gabexate mesylate prior to ERCP did significantly reduce the incidence of pain and acute pancreatitis after the procedure.\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e Unfortunately, the long infusion times before surgery make this specific treatment impractical. These trials demonstrate that trypsin inhibition could be of use in prophylaxis of pancreatitis before ERCP, but also underscore the importance of pharmacokinetics in driving adoption of a particular treatment.\u003c/p\u003e\u003cp\u003eAs the human body\u0026rsquo;s natural endogenous trypsin inhibitor, SPINK1 (serine protease inhibitor Kazal type 1) is a promising therapeutic element. Compared to BPTI, SPINK1 has the advantage that it is an endogenous human protein and less likely to induce an immune response after repeated administration in treatment of chronic disease. In this work, we demonstrate that an Fc-SPINK1 fusion protein can reduce the severity of pancreatitis in a mouse model.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eDesign, production, and purification of Fc-SPINK1, and\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003etrypsin inhibition\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe trypsin inhibitors SPINK1 and BPTI (bovine pancreatic trypsin inhibitor; Aprotinin) both have a molecular weight of \u0026lt;\u0026thinsp;7,000 Daltons, which will result in rapid renal clearance and to make these proteins unsuitable as drugs. We fused SPINK1 and BPTI to the C-terminus of the Fc fragment of human IgG to create an Fc-SPINK1 and Fc-BPTI dimers with a molecular weight of about 78 kDa, which is well above the renal clearance threshold and should significantly improve pharmacokinetics\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-c).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe Fc region contains three mutations relative to the natural human IgG1 sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). First, amino acid 5, normally cysteine, is mutated to serine because this cysteine normally forms a disulfide bond with the antibody light chain, which is not present in this construct. Second, the aspartic acid at position 82 is normally asparagine, but this is the site of N-linked glycosylation of the Fc region. Removing this glycosylation has the effect of significantly reducing binding to both Fc receptors and to the complement component C1q,\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e and also allows for safe, low-cost expression in the yeast \u003cem\u003ePichia pastoris\u003c/em\u003e instead of mammalian cells; specifically, \u003cem\u003ePichia\u003c/em\u003e generates a high-mannose-type N-linked oligosaccharide modification that would be recognized as foreign by mammals,\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e but our molecule has been engineered to not be glycosylated. Third, amino acid 232 is normally the C-terminal lysine of a secreted IgG1, but this amino acid can be cleaved in both natural antibodies and fusion proteins, so it is mutated to alanine to prevent potential cleavage.\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e The SPINK1 and BPTI modules were placed at the C-terminus of Fc instead of the N-terminus, because fusions in the Fc-X orientation are generally expressed at a higher level.\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e No linker was used because the C-terminus of Fc is somewhat unstructured and may form a natural linker, and because both fusion termini are far from known sites of protein interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ePichia pastoris\u003c/em\u003e expression system was used for the production procedures, and fusion proteins were purified to near homogeneity by protein A chromatography (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed-e; Methods). A trypsin inhibition assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef) demonstrated the efficacy of both constructs in inhibiting trypsin at molar ratios of 1:1 (0.429 nM each) and 1:0.5 Fc-SPINK1-trypsin. (We generally did not observe partial inhibition of trypsin by Fc-SPINK1 at low molar ratios of SPINK1:trypsin; we hypothesize that trypsin might inactivate Fc-SPINK1 under the conditions we used.)\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eefficacy in cerulein-induced mouse model of acute pancreatitis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFc-SPINK1 and Fc-BPTI were evaluated as potential therapeutic agents in a mouse model of cerulein-induced pancreatitis. Cerulein is a frog-derived toxic peptide that stimulates a moderate, transient pancreatitis.\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e Pancreatitis was induced by seven injections of cerulein into C57Bl/6N mice, with a single injection of Fc-SPINK1, Fc-BPTI, or vehicle, followed by harvest of the pancreas and blood 18 hours after the first injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Pancreases were fixed, sectioned, and stained with hematoxylin/eosin (H\u0026amp;E) or with anti-CD11b (staining macrophages, neutrophils, and certain other immune cells\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-G). To date, necrosis and immune cell infiltration is quantified visually. This slow process requires subsampling of tissue.\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e To increase statistical power, we performed semi-automated analysis on all available image slides with respect to necrotic cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-I), and immune cell infiltration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ-K). In H\u0026amp;E-stained healthy pancreas tissue, the acinar cells are normally tightly associated with each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). After cerulein treatment, the cells are dramatically separated in H\u0026amp;E-stained sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e To quantitate this effect in an unbiased way, we developed a new automated image analysis method (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL-M; see below).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn one experiment, we treated mice with 5 mg of Fc fusion protein after the third cerulein injection. This high dose was based on the fact that mammals synthesize large amounts of trypsin, and only a small amount of the injected Fc-SPINK1 fusion protein will distribute into the pancreas (see Discussion). Fc-SPINK1 reduced markers of cerulein-induced pancreatitis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Specifically, acinar cell necrosis and plasma amylase levels were reduced in cerulein\u0026thinsp;+\u0026thinsp;Fc-SPINK1-treated mice compared to mice treated with cerulein and vehicle or Fc-BPTI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN-O). Quantitative semi-automated image analysis of necrotic cells showed a significant reduction in the Fc-SPINK1-treated group compared to the cerulein control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Supplementary 1). Fc-BPTI did not appear to reduce acinar cell necrosis or amylase levels.\u003c/p\u003e\u003cp\u003ePancreatic amylase, which is released during cerulein-induced inflammation, is a human biomarker for the detection of pancreatic injury. Blood plasma amylase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) was reduced in the Fc-SPINK1-treated group (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was no reduction in amylase activity in the plasma of Fc-BPTI-treated mice. Typically, in the cerulein-induced pancreatitis mouse model, plasma amylase levels peak after the last cerulein injection.\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e In this experiment, at the 18 hour time-point, the amylase levels are returning to baseline, but the effect of Fc-SPINK1 can still be observed.\u003c/p\u003e\u003cp\u003eIn a second experiment, the effects of different Fc-SPINK1 doses administered by intravenous (IV) and intraperitoneal (IP) injection were investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Supplementary Fig.\u0026nbsp;2), using doses of 1, 2.5, or 5 mg per 20-gram mouse. A dose response was observed with the IV-injected mice. Mice receiving 2.5 or 5 mg/mouse showed significantly less necrosis than mice treated with 0 or 1 mg, and the tissue showed progressively ameliorated junctional integrity with increasing IV dosing. In the intraperitoneally dosed animals, the separate dose groups did not show significantly less cell necrosis than the untreated group, but there was a trend towards a therapeutic effect (Supplementary Fig.\u0026nbsp;2). Taken together, the results indicate that pancreatitis symptoms decrease with high enough doses of Fc-SPINK1, and that a maximally effective dose may not have been reached.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMice of the C57Bl6J strain develop a more severe cerulein-induced pancreatitis than C57Bl6N mice. In a third experiment, when C57Bl6J mice were treated with cerulein and Fc-SPINK1 following the timeline in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, a moderate reduction in disease severity was observed. However, when the C57Bl6J mice were given Fc-SPINK1 by IP injection prior to the cerulein injections, we observed a stronger effect, seeing both a statistically significant reduction in cell necrosis and cell separation in the pancreases of cerulein-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Prophylactic treatment is particularly relevant to ERCP-induced pancreatitis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eImage analysis\u003c/h2\u003e\u003cp\u003eWe used automated and semi-automated image analysis to quantitate the extent of cell death, lymphocyte infiltration, and cell separation, which are visual markers of inflammation. Cell separation may be related to edematous fluid influx, and is henceforth also referred to as edema.\u003c/p\u003e\u003cp\u003eEach analyzed image corresponds to a whole-pancreas cross-section. Images that showed staining-defects or were acquired with different microscope-settings than the rest of their batch, were excluded from analysis. First, exocrine tissue (acinar cells) was segmented using a random-forest-based pixel classifier in the open-source digital pathology software QuPath (v. 0.5.1.).\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e A pixel-classifier assigns a class to each pixel (such as background, exocrine tissue) based on manually annotated ground truth examples. To ensure pixel classifiers for Edema and Necrosis were trained on well-randomized samples, square regions at a random location within exocrine tissue were automatically selected and combined into a training image to which manual annotations were added. After pixel-classification, manual corrections were made by removing wrongly segmented (false positive) areas.\u003c/p\u003e\u003cp\u003eThe Edema, Necrosis, and CD11b\u0026thinsp;+\u0026thinsp;scoring functions were generated as follows (see Supplementary Information for details). Within exocrine segments, the space between cells was segmented using a QuPath pixel classifier. In inflamed tissue, acinar cells are rounded, possibly due to a loss of tight junctional integrity that may relate to edematous fluid influx into tissue or to separation of cells during fixation. Hence, the intercellular space forms a netlike topology. Branch points signify a loss of tight junction integrity at multicellular junctions. For the Edema function, the number of branch points was therefore used to quantify inflammation. For the Necrosis function, necrotic cells were segmented using a QuPath pixel classifier, and false positive segments were manually removed. The ratio of necrotic cells to exocrine tissue was used to quantify this aspect of pancreatitis. The CD11b\u0026thinsp;+\u0026thinsp;function is based on the fact that inflammation correlates with CD11B\u0026thinsp;+\u0026thinsp;immune cell invasion. In IHC images, CD11B\u0026thinsp;+\u0026thinsp;cells and tissue (exocrine and endocrine) were segmented using a QuPath pixel classifier, and the percentage of tissue area classed as CD11B\u0026thinsp;+\u0026thinsp;was used to quantify inflammation.\u003c/p\u003e\u003cp\u003eThe Shapiro-Wilk test of normality was conducted for each treatment condition to assess the normality of sample distributions, with a significance level of α\u0026thinsp;=\u0026thinsp;0.05. If the null hypothesis of at least one condition within an experiment was rejected, the Mann-Whitney-U test\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e was used with the alternative hypothesis that sick samples show greater indicators of inflammation than healthy or treated samples. When the null hypothesis of the Shapiro-Wilk test\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e of normality was not rejected, Welch\u0026rsquo;s one-tailed t-test\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e was applied.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePharmacokinetics\u003c/h3\u003e\n\u003cp\u003eWe investigated the dynamics of Fc-SPINK1 in the blood system using Tg276 mice.\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e Because Fc-SPINK1 is large enough to prevent renal clearance, pharmacokinetics is expected to be dominated by trafficking through cells with various Fc receptors. The Tg276 mice express human versions of FcRn and the various Fc-gamma receptors, so the pharmacokinetics of Fc-SPINK1 (in which the Fc and SPINK1 elements are both based on human proteins) will be more accurately represented. The concentration of Fc-SPINK1 in blood plasma was followed over time (Fig.\u0026nbsp;6). Analysis of the data using a two-compartment model indicates a beta-phase half-life of 21.51 hours, an area under the curve (AUC) of 3190 (\u0026micro;gཥh)/ml or 132.9 (\u0026micro;gཥdays)/ml for a 100 \u0026micro;g dose, and a clearance rate of 0.75 ml/day.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePancreatitis is an extremely painful and sometimes fatal inflammation of the pancreas in which excess trypsin activation plays a major role. To treat pancreatitis, we designed an Fc-SPINK1 fusion protein that would inhibit extracellular trypsin, and also have a long enough plasma half-life to be practical as a therapeutic protein. SPINK1 is attractive because it is a natural human protein, unlike bovine pancreatic trypsin inhibitor (BPTI/Aprotinin), which is only expressed in ruminants\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e and might therefore be immunogenic. In addition, SPINK1 has a high specificity for trypsin and a much lower affinity than BPTI for other serine proteases. We placed SPINK1 at the C-terminus of the Fc region because Fc fusion proteins are generally more highly expressed in this configuration.\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e We expressed this protein in the yeast \u003cem\u003ePichia pastoris\u003c/em\u003e, purified it, and showed that it inhibits trypsin \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFc-SPINK1 reduced the indications of pancreatitis in the cerulein-induced mouse model of this disease. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Fig.\u0026nbsp;1). Cerulein, a toxic peptide from the Australian green tree frog with a C-terminus similar to cholecystokinin, hyperstimulates the pancreas and, after 7 injections leads to a mild pancreatitis that resolves after about 24 hours.\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e][\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e When C57Bl6N mice were treated with cerulein, 18 hours after the first injection the pancreas showed signs of pancreatitis: dead acinar cells, infiltrating CD11b\u003csup\u003e+\u003c/sup\u003e leukocytes, and spaces between cells that may represent edema or loss of junctional integrity. When mice received 5 mg of Fc-SPINK1 after the third cerulein injection, cell death and microscopic edema was reduced (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-N). Blood levels of pancreatic amylase were slightly increased in cerulein-treated mice but reduced back to baseline in mice receiving Fc-SPINK1 as well (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO). These effects were observed at the higher intravenous doses of a dose-escalation experiment in which we administered 0, 1, 2.5 or 5 mgs/mouse of Fc-SPINK1 to cerulein-treated C57Bl6N mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In addition, when mice of the C57Bl6J strain, which develop a more severe cerulein-induced pancreatitis than C57Bl6N mice, were treated with Fc-SPINK1 prior to the cerulein injections, a reduction in cell death and leukocyte infiltration was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Taken together these results indicate that Fc-SPINK1 can reduce the intensity of pancreatitis in the cerulein-treated mouse model.\u003c/p\u003e\u003cp\u003eOne dramatic effect of pancreatitis in mouse models is the separation of cells in H\u0026amp;E-stained sections of the pancreas of cerulein-treated mice. Previously, researchers scored this by visual inspection (blinded to sample identity), which is tedious and may miss subtle effects. As part of this work, we developed an automated scoring system in which the spaces between cells in H\u0026amp;E stained sections are identified, abstracted as lines, and the branchpoints in the resulting pattern is quantitated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This approach may work in part because non-pathological spaces between cells, corresponding to ducts and spaces between pancreatic lobes, do not have extensive branching patterns and will not contribute greatly to the score.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe Fc-SPINK1 protein has a long plasma half-life (Fig.\u0026nbsp;6). When the data are interpreted with a two-compartment model, the elimination half-life is about 21 hours, while interpretation with a more physiological 3 compartment model indicates an elimination phase half-life of about 4 days (Supplementary Fig.\u0026nbsp;4). These results suggest that the Fc-SPINK1 molecule tested here would be adequate for prophylactic administration prior to the ERCP procedure, but improvement of the plasma half-life would be ideal for chronic pancreatitis patients who may need life-long treatment.\u003c/p\u003e\u003cp\u003eThe initiation of pancreatitis is thought to occur when overstimulation of exocrine secretion leads to fusion of acinar secretory vesicles with the wrong membrane, such as the basolateral cell surface or a lysosome or endosome. In particular, upon cerulein treatment, fusion of a trypsinogen-containing vesicle with a vesicle containing cathepsin B leads to trypsin activation within 30 minutes of treatment.\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e A cathepsin B knockout mouse does not show this early activation, but still shows pancreatitis phenotypes after multiple cerulein injections. Geisz et al.\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e hypothesize that trypsin autoactivation also occurs in the interstitial space and, through cleavage of protease-activated receptors, may initiate pancreatitis. The fact that Fc-SPINK1 reduces the severity of pancreatitis in the cerulein mouse model supports the idea that interstitial autoactivation plays a role in this model, and that cerulein-induced pancreatitis is not solely driven by intracellular trypsin activation.\u003c/p\u003e\u003cp\u003eAs an injected drug, Fc-SPINK1 has advantages compared to small molecule trypsin inhibitors. Camostat is a serine protease inhibitor that is approved in Japan for treatment of pancreatitis.\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e Layer et al. found that when healthy human volunteers were given oral camostat, trypsin activity was inhibited throughout the small intestine, but also increased levels of pancreatis amylase and lipase in the intestine.\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e These authors inferred that this occurred through increased pancreatic secretion, and the results were evidence for a feedback loop between protease levels in the intestine and pancreatic activity. These observations suggest that any orally delivered trypsin inhibitor might be problematic for treatment of pancreatitis, since there will always be more drug in the intestine than in the pancreas, and during periods between doses when drug levels fall, pancreatic secretion could be stimulated.\u003c/p\u003e\u003cp\u003eA high dose of Fc-SPINK1 is required to successfully treat the cerulein-dosed mouse. This is likely because mammals produce a large amount of trypsin, and to be effective, Fc-SPINK1 needs to be in molar excess relative to active extracellular trypsin. As a point of comparison, the human body secretes between 10 and 100 mgs of trypsin per day into the small intestine.\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e The pancreas weighs about 80 grams \u0026ndash; slightly more than 0.1% of body weight \u0026ndash; so only a small fraction of systemically administered Fc-SPINK1 will distribute into the pancreas. The fraction of trypsin that is activated and redirected into the accessible extracellular space during pancreatitis is unknown but may be large. With allometric scaling, a dose of 5 milligrams in a 25-gram mouse corresponds to a dose of about 1\u0026ndash;2 grams in a human. Administration of such a large amount of protein is less challenging in humans than in mice because humans are amenable to IV infusion.\u003c/p\u003e\u003cp\u003ePatients undergoing an Endoscopic retrograde cholangiopancreatography (ERCP) procedure have about a 10% chance of experiencing post-procedure pancreatitis, which usually resolves but can be debilitating or fatal. Typically, before the procedure, a patient is given an IV infusion of 250 mls to 1 liter of fluid to make the ducts more accessible. Fc-SPINK1 could be administered prophylactically in this infusion to reduce the chances of developing pancreatitis. The infusion volume would allow for a large dose of Fc-SPINK1 without requiring an exotic formulation for a high protein concentration.\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e The plasma half-life of Fc-SPINK1 is in an appropriate range, since post-ERCP pancreatitis occurs within 24 hours of the procedure. Taken together, our results indicate that Fc-SPINK1 is a promising candidate for treatment and prevention of pancreatitis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eDNA construction\u003c/b\u003e. Geneious Prime version 2020.1.2 was used to organize sequence data, design, and validate cloning strategies, maps, and primers. Each construct made during this research project was sequence-validated by sanger sequencing provided by GENEWIZ (South Plainfield, NJ, USA). All plasmids used in this study are based on the expression vector pPICZαA (Thermo Fischer Scientific, V19520). This plasmid allows methanol-inducible, secreted expression in \u003cem\u003ePichia pastoris\u003c/em\u003e. Plasmids were constructed by standard techniques. Fc and SPINK1 sequences were codon-optimized for expression in \u003cem\u003ePichia pastoris\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eProtein expression in\u003c/b\u003e \u003cb\u003eP. pastoris\u003c/b\u003e. Expression and purification of the Fc-SPINK1 and Fc-BPTI fusion proteins was carried out using standard techniques, which are detailed in Supplementary Information. In brief, the methanol-inducible \u003cem\u003eP. pastoris\u003c/em\u003e system was used to express secreted Fc fusion proteins, which were concentrated from supernatant and purified using standard Protein A chromatography.\u003c/p\u003e\u003cp\u003eThe SDS-polyacrylamide gel depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, which illustrates the expression and purification of Fc-SPINK1, complies with digital image and integrity policies (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.nature.com/srep/journal-policies/editorial-policies#digital-image\u003c/span\u003e\u003cspan address=\"https://www.nature.com/srep/journal-policies/editorial-policies#digital-image\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) as follows: (1) the gel depicted was a single SDS gel, and no lanes were removed in the image; (2) all of the molecular weight markers visible in the original gel are depicted; (3) the gel image was \u0026lsquo;cropped\u0026rsquo; at the top of the gel, removing only the wells, at the bottom of the gel, and on the sides outside the area where samples were run; and (4) the gel image was recorded with normal contrast. No other images of this gel are available.\u003c/p\u003e\u003cp\u003e\u003cb\u003eProtein characterization: Trypsin inhibition activity\u003c/b\u003e. Trypsin-inhibitor fusion proteins were tested in vitro for trypsin binding capacity. Therefore, trypsin inhibitor proteins and trypsin were diluted to a concentration of 50 \u0026micro;M in PBS. Trypsin was mixed with the respective inhibitor protein in ratios from 1:1 to 1:0.0625, including a positive and negative control for trypsin activity, and incubated at room temperature for 5 min.\u003c/p\u003e\u003cp\u003eTrypsin activity in each sample was measured using the Trypsin Activity Colorimetric Assay Kit (Abcam, ab102523). Here, trypsin activity was measured with a substrate that is cleaved by trypsin to generate p-nitroaniline (\u003cem\u003ep\u003c/em\u003e-NA) which is detected at λ\u0026thinsp;=\u0026thinsp;405 nm.\u003c/p\u003e\u003cp\u003eIn brief, reactions were diluted 1:50 with PBS. Samples and controls were mixed with 50 \u0026micro;l reaction mix and incubated at 25\u0026deg;C for 60 min. Absorption at 405 nm was measured and normalized to the trypsin positive control without trypsin inhibitor\u003c/p\u003e\u003cp\u003e\u003cb\u003eCerulein-induced acute pancreatitis model\u003c/b\u003e Pancreatitis was induced by intraperitoneal (IP) administration of the peptide cerulein according to Lampel and Kern.\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e Cerulein (50 \u0026micro;g/kg dissolved in 50 \u0026micro;l endotoxin-free PBS \u0026minus;\u0026thinsp;1 \u0026micro;g for a mouse with 20 g) was injected once per hour to a total of 7 injections. Mice were closely monitored throughout the injection period. Illness was observed within 1\u0026ndash;2 hours after the first injections.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAdministration of trypsin inhibitor proteins\u003c/b\u003e. Trypsin inhibitor fusion proteins were administered in endotoxin-free PBS either intraperitoneal (IP) or intravenously (IV) by tail vein injection. Maximum volumes for IP and IV injections were 200 \u0026micro;l and 100 \u0026micro;l, respectively.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMeasurement of serum α-amylase activity.\u003c/b\u003e Serum activity of α -amylase, a digestive enzyme made by the pancreas and a clinical biomarker for pancreatitis was measured using the Amylase Activity Colorimetric Assay Kit (abcam, ab102523) according to manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHistopathology: Hematoxylin and Eosin (H\u0026amp;E) staining and immunohistochemistry (IHC)\u003c/b\u003e. Immediately after necropsy, pancreas tissue was fixed in 10% Neutral Buffered Formalin for 24 hours before embedding, cutting, and staining. Slide scanning for all samples and IHC staining for CD11b was performed by Histowiz (Brooklyn, NY, USA).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available in the Zenodo repository, with the following links:\u003c/p\u003e\n\u003cp\u003eExperiment 1 (Figure 2):\u003c/p\u003e\n\u003cp\u003eBatch 11571: https://zenodo.org/records/15586925 \u003c/p\u003e\n\u003cp\u003eBatches 11564: https://zenodo.org/records/15587055 \u003c/p\u003e\n\u003cp\u003eDose-response experiments (Figure 3):\u003c/p\u003e\n\u003cp\u003eBatch 14628: https://zenodo.org/records/15587055 \u003c/p\u003e\n\u003cp\u003eBatch 14550: https://zenodo.org/records/15587142 \u003c/p\u003e\n\u003cp\u003eProphylaxis experiments (Figure 4)\u003c/p\u003e\n\u003cp\u003eBatch 25868: https://zenodo.org/records/15587101 \u003c/p\u003e\n\u003cp\u003eRaw IHC data:\u003c/p\u003e\n\u003cp\u003eBatches 11564 and 14628: https://zenodo.org/records/15587055 \u003c/p\u003e\n\u003cp\u003eThese files can be opened with QuPath (https://qupath.github.io/) and other specialized image-viewing applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePharmacokinetics\u003c/strong\u003e. Pharmacokinetics of trypsin inhibitor fusion proteins were studied by IV administration of the respective protein. Tail vein blood was collected 5 min, 1 h, 3 h, 6 h, 12 h, 24 h, 72 h and 96 h post-drug injection and stored as EDTA-plasma at -20°C until analysis. ELISAs based on human SPINK1 capture and human Fc detection were used to quantitate fusion protein in serum, using standard procedures described in Supplementary Information. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal welfare.\u003c/strong\u003e All mouse experiments were carried out under protocol IS00002593, which has been approved by the Harvard Medical Area Standing Committee on Animals (HMS-IACUC). All mouse experiments were carried out in accordance with relevant state and federal guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccordance with ARRIVE guidelines.\u003c/strong\u003e Mouse experiments were carried out and reported in accordance with the “ARRIVE Essential 10” guidelines (https://arriveguidelines.org) as follows. (1) The ‘experimental unit’ was the individual mouse. Controls are indicated in the figure legends and text for each experiment. (2) The studies reported here are exploratory, so there was no specific expectation of effect sizes or numbers of animals that needed to be tested. The sample sizes were limited by availability of the Fc-SPINK1 test protein, which we manufactured ourselves. Generally, dose groups of 4 or 5 mice were tested. For some experiments smaller numbers of mice were tested or evaluated in an exploratory manner, and not all pancreases were imaged due to expense, especially for CD11b+ staining. For the sake of completeness, these data are reported in Supplementary Information, but are generally not statistically meaningful. (3) Individual mice from a dataset were excluded only if a mouse died prematurely. In one case, a set of images was excluded from analysis because the pixel size was different from that of the other images, and the machine learning-based analysis appeared to give spurious results. Scores for each mouse are individually reported for all of the experiments where Fc-SPINK1 was tested. (4). Randomization was not used because the mice in each experiment were all the same age. (5) Blinding was used in preliminary manual analysis of images but was not necessary for computer-evaluated images. (6) Outcomes are described in the text and figure legends. The overall hypothesis being tested was that Fc-SPINK1 treatment would reduce the severity of pancreatitis markers induced by cerulein, but we did not have a specific hypothesis about how strong the effect would be, what the optimal dose of Fc-SPINK1 would be, or when the maximal effect would be observed; hence this is an exploratory study. (7) Statistical methods are described above and in the Figure Legends. (8) All mice were purchased from either Charles River Labs or Jackson Labs, and were 8- to 12-week old females. The use of C57Bl/6N and C57Bl/6J substrains of mice, which have different pancreatitis phenotypes, is described in the text. Mice were naïve; they had not been used for other experiments. (9) Specifics of the pancreatitis induction and treatment protocol are described above and in the main text. All mice were acclimatized for at least 1 week after shipping before the experiment was performed. (10) The statistical analysis of each experiment is presented in the figures, main text and methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEuthanasia and humane treatment of mice. \u003c/strong\u003eAs part of the pancreatitis induction and testing protocol, mice were sacrificed at the end of the experiment before the pancreases were harvested. Euthanasia was performed using standard carbon dioxide asphyxiation, followed by cervical dislocation, in accordance with the IACUC-approved mouse protocol cited above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank Harvard Medical School for a Q-FASTR grant and the Wyss Institute for Biologically Inspired Engineering for validation project support. We also thank Andrea Geisz for extensive discussions on pancreatitis mechanisms and techniques. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e JCW, DH, LBa and LBu were responsible for the analysis and interpretation of the data and for the preparation of this manuscript. DH, LBa, LBu, JAB, TD, PH, DN, RS, TS were responsible for protein expression, and/or performing and planning the experiments. MT and SFN were responsible for imaging algorithm development, analysis, interpretation, preparation of the manuscript KR conceptualized the Fc-SPINK1 fusion protein and constructed the first versions. ARG, AV and MSV were responsible for verifying the accuracy and performance of the animal studies. PAS was responsible for funding and study supervision, and assisted with preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests statement.\u003c/strong\u003e Jeffrey Way, Katherine Redfield Chan, Daniel Heid, and Dominik Niopek are inventors on a patent application covering SPINK1 fusion proteins that has been assigned to Harvard University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eM. H. Forouzanfar et al., \u0026ldquo;Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990\u0026ndash;2015: a systematic analysis for the Global Burden of Disease Study 2015,\u0026rdquo; Lancet, vol. 388, no. 10053, pp. 1659\u0026ndash;1724, 2016, doi: 10.1016/S0140-6736(16)31679-8.\u003c/li\u003e\n \u003cli\u003eD. Yadav and A. B. 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Studies of aglycosylated chimeric mouse-human IgG: Role of Carbohydrate in the Structure and Effector Functions Mediated by the Human IgG Constant Region. J. Immunol. 143, 2595-2601 (1989).\u003c/li\u003e\n \u003cli\u003eLove, K.R., Dalvie, N.C., \u0026amp; Love, J.C. The yeast stands alone: the future of protein biologic production. Current Opinion in Biotechnology, 53:50\u0026ndash;58, 2018.\u003c/li\u003e\n \u003cli\u003eGillies, S.D., Burger, C., Lo, K.-M. enhancing the circulating half-life of antibody-based fusion proteins. US Patent 7,790,415. 2010.\u003c/li\u003e\n \u003cli\u003eMay, R.J., Conlon, T.P. Erspamer, V., Gardner, J.D. Actions of peptides isolated from amphibian skin on pancreatic acinar cells. Am. J. Physiol. ;235(2):E112-8, (1978).\u003c/li\u003e\n \u003cli\u003eM. Lampel and H. F. Kern, \u0026ldquo;Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue,\u0026rdquo; Virchows Arch. A Pathol. Anat. Histol., vol. 373, no. 2, pp. 97\u0026ndash;117, 1977, doi: 10.1007/BF00432156.\u003c/li\u003e\n \u003cli\u003eSolovjov DA, Pluskota E, Plow EF (January 2005). \u0026quot;Distinct roles for the alpha and beta subunits in the functions of integrin alphaMbeta2\u0026quot;. The Journal of Biological Chemistry. 280 (2): 1336\u0026ndash;1345. doi:10.1074/jbc.M406968200. PMID 15485828.\u003c/li\u003e\n \u003cli\u003eGeisz, A., Tran, T., Orekhova, A., \u0026amp; Sahin-Toth, M. Trypsin Activity in Secretagogue-induced Murine Pancreatitis Is Solely Elicited by Cathepsin B and Does Not Mediate Key Pathologic Responses. Gastroenterology 164:684\u0026ndash;687, (2023).\u003c/li\u003e\n \u003cli\u003eMann, H. B. \u0026amp; Whitney, D. R. On a Test of Whether one of Two Random Variables is Stochastically Larger than the Other. Annals of Mathematical Statistics 18,50\u0026ndash;60 (1947).\u003c/li\u003e\n \u003cli\u003eShapiro, S.S. \u0026amp; Wilk, M.B. 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Feedback regulation of human pancreatic secretion. Gastroenterology 98,1311-1319 (1990).\u003c/li\u003e\n \u003cli\u003eFedail, S.S., Harvey, R.F., Salmon, P.R., Brown, P. \u0026amp; Read, A.D. Trypsin and lactoferrin levels in pure pancreatic juice in patients with pancreatic disease. Gut 20, 983-986 (1979).\u003c/li\u003e\n \u003cli\u003eIshiguro, H. et al. Physiology and pathophysiology of bicarbonate secretion by pancreatic duct epithelium. Nagoya J. Med. Sci. 74, 1-18 (2012).\u003c/li\u003e\n \u003cli\u003eGaridel, P., Kuhn, A.B., Schaefer, L.V., Karow-Zwick, A.R. \u0026amp; Blech, M. High-concentration protein formulations: How high is high? Eur. J. Pharmaceutics and Biopharmaceutics 119, 353\u0026ndash;360 (2017).\u003c/li\u003e\n \u003cli\u003eGeisz, A., Jansco, Z., Nemeth, B.C., Hegyi, E., Sahin-Toth, M. Natural single-nucleotide deletion in chymotrypsinogen C gene increases severity of secretagogue-induced pancreatitis in C57BL/6 mice. JCI Insight. 2019;4(14):e129717. https://doi. org/10.1172/jci.insight.129717.\u003c/li\u003e\n \u003cli\u003eBankhead, P., Loughrey, M.B., Fern\u0026aacute;ndez, J.A. et al. QuPath: Open source software for digital pathology image analysis. Sci Rep 7, 16878 (2017). https://doi.org/10.1038/s41598-017-17204-5.\u003c/li\u003e\n \u003cli\u003eMiura, K., Noerrelykke, S. Reproducible image handling and analysis. EMBO J. 40(3), e105889. (2021) doi: 10.15252/embj.2020105889.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pancreatitis, ERCP, Fc fusion, trypsin, Image analysis","lastPublishedDoi":"10.21203/rs.3.rs-6658843/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6658843/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePancreatitis results from premature activation and impaired inactivation of pancreatic proteases, primarily trypsin, leading to self-digestion, tissue necrosis, fibrosis, and inflammation. SPINK1 is a pancreas-specific inhibitor of trypsin that prevents premature trypsin activation, and could be a candidate therapeutic. However, because of its small size, SPINK1 would be subject to rapid renal clearance, making it ineffective. To construct a long half-life therapeutic inhibitor of trypsin for pancreatitis treatment we fused this protein to the C-terminus of an IgG1 antibody Fc element, increasing the size to ~\u0026thinsp;78 kDa, thereby exceeding the renal clearance threshold and providing for FcRn-mediated recycling out of cells. A non-glycosylated form of Fc-SPINK1 was expressed in the yeast \u003cem\u003ePichia pastoris\u003c/em\u003e. Fc-SPINK1 inhibits trypsin enzyme activity \u003cem\u003ein vitro\u003c/em\u003e. The blood pharmacokinetics in mice are consistent with a three-compartment distribution model and a terminal half-life of ~\u0026thinsp;3 days. In a cerulein-induced mouse model of pancreatitis, Fc-SPINK1 significantly ameliorated cell death and immune cell infiltration. We developed an automated image analysis technique to quantify pancreatitis-associated loss of tissue cohesion, and found that Fc-SPINK1 also reduced this effect. This study demonstrates the potential of Fc-SPINK1 as a rationally designed therapy for pancreatitis.\u003c/p\u003e","manuscriptTitle":"An Fc-SPINK1 fusion protein inhibits pancreatic inflammation in a mouse model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 11:15:52","doi":"10.21203/rs.3.rs-6658843/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6db5f82b-a737-4ff2-b3b7-4e4d4d0f6459","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51207556,"name":"Biological sciences/Biotechnology"},{"id":51207557,"name":"Biological sciences/Cell biology"},{"id":51207558,"name":"Biological sciences/Drug discovery"},{"id":51207559,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2025-09-22T13:10:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-14 11:15:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6658843","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6658843","identity":"rs-6658843","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}