GLP2-2G Gene Construction and Expression
GLP2-2G-XTEN gene construction was performed as previously described 
. The final construct comprised the gene encoding the cellulose binding domain (CBD) from Clostridium thermocellum
(accession #ABN54273), a tobacco etch virus (TEV) protease recognition site (ENLYFQ), the GLP2-2G sequence, and an 864 amino acid XTEN sequence under control of a T7 promoter. The removal of CBD from GLP2-2G-XTEN is mediated by the TEV protease, which is co-expressed on the same plasmid but from a separate operon using the constitutive GroE promoter. The host strain for expression, AmE025, was derived from W3110 (Yale CGSC #4474) in which the fhuA
gene was deleted by P1 transduction from strain JW0146-2 (Yale CGSC #8416) and the lambda DE3 prophage was integrated onto the chromosome using a λDE3 lysogenization kit (EMD Chemicals USA #69734). GLP2-2G-XTEN was expressed in a 5L glass jacketed fermentation vessel with a B. Braun Biostat B controller, with an initial growth temperature of 37°C, followed by a reduction to 26°C upon addition of IPTG at 20 hours run time. After a total fermentation run time of 45 hours the culture was harvested by centrifugation, yielding cell pellets ~1 kg in wet weight. The pellets were stored frozen at −80°C until purification was initiated.
The E. coli cell pellet was re-suspended in 20 mM Tri-HCl pH 7.5, 50 mM NaCl and lysed by homogenization. The resulting cell lysate was heat coagulated at 85°C for 10 minutes, rapidly cooled to 10°C and clarified by centrifugation. The supernatant was collected and stored at 4°C for purification. GLP2-2G-XTEN protein was purified to homogeneity out of the clarified, heat-treated lysate using three bind and elute chromatography steps, two anion exchange and one hydrophobic interaction, run on an AKTA FPLC system. The final material was formulated using diafiltration in 20 mM Tris-HCl pH 7.5, 135 mM NaCl, sterile filtered using a 0.22 micron filter, and frozen at −80°C until further use. Overall purification yield was approximately 30%.
Analytical Size Exclusion Chromatography
Analytical size exclusion chromatography (SEC) was performed using a BioSep-SEC-s4000, 7.8 × 600 mm HPLC column (Phenomenex) connected to an LC2010 integrated HPLC system equipped with an autosampler and a UV/VIS detector (Shimadzu). The system and the column were equilibrated in 50 mM NaPO4 pH 6.5, 300 mM NaCl at a flow rate of 0.5 mL/min at ambient temperature. For column performance, a SEC column check standard (Phenomenex, AL0-3042) was used. For sample analysis, 20 µL of 1 mg/mL purified GLP2-2G-XTEN was injected and the absorbance at 214 nm was monitored for 75 min.
200 µg of purified GLP2-2G-XTEN protein was desalted by solid phase extraction using Extract-Clean C18 column (Discovery Sciences). Desalted protein solution in 0.1% formic acid, 50% acetonitrile was infused at 4 µl/min into a QSTAR XL mass spectrometer (AB Sciex). Multi-charge TOF spectrum was acquired in 800–1400 amu range. Zero-charge spectrum was obtained by Bayesian reconstruction in 10–100 kDa range.
Human GLP2-2G peptide (HGDGSFSDEMNTILDNLAARDFINWLIQTKITD) was purchased from American Peptide (catalog # - 304076). The purity was greater than 95% as confirmed by HPLC and MS.
Potency Assay – GPCR
Sample analysis was performed by Millipore’s GPCRProfiler® service using Millipore’s cloned human GLP-2 Receptor -expressing cell line made in the Chem-11 host (Millipore catalog #HTS164C) and their standard assay conditions. Calcium flux was monitored in real-time by FLIPR analysis after addition of serial dilutions of GLP2-2G-XTEN and GLP2-2G. Calcium flux was monitored in real-time by FLIPR analysis after addition of serial dilutions of GLP2-2G-XTEN and GLP2-2G. Eight and four replicates of the assay were performed on GLP2-2G-XTEN and GLP2-2G, respectively. After baseline corrections were applied, percentage activation relative to the Millpore reference agonist Emax were calculated. Then, dose response curves and EC50 values were generated and calculated using GraphPad Prism (Sigmoidal Dose Response).
Animal Ethics Statement
Animal welfare for these studies was in compliance with the U.S. Department of Agriculture’s (USDA) Animal Welfare Act (9 CFR Parts 1, 2 and 3). The Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Academy Press, Washington, D.C., was followed. The contract facilities performing this work maintain an Animal Welfare Assurance statement with the National Institutes of Health, Office of Laboratory Animal Welfare. In order to ensure compliance, all protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of each contract facility before the initiation of treatment. No procedures or test articles were used that would cause more than momentary pain or distress to the animals. Detailed protocols, in-life summaries, and study reports are on file at Amunix, Inc.
Monkeys completed a quarantine and acclimation period and only healthy animals were selected for study. All monkeys were housed individually in stainless steel cages and provided environmental enrichment during the study. Fluorescent lighting was provided via an automatic timer for approximately 12 hours per day. Food and water was available ad libitum. Temperature and humidity was monitored and recorded daily and maintained to the maximum extent possible between 64 to 84°F and 30 to 70%, respectively.
Pharmacokinetic studies in mice were performed using female C57BL/6 mice dosed subcutaneously (3 mice per time point) with 25 nmol/kg (2 mg/kg) GLP2-2G-XTEN. Plasma samples were collected at pre-dose and at selected time points over 120 hours in heparinized collection tubes. Pharmacokinetic studies in rats were done using catheterized female Wistar rats (3 rats per group) dosed subcutaneously with 25 nmol/kg and 200 nmol/kg GLP2-2G-XTEN. Plasma samples were collected at pre-dose and at selected time points over 168 hours in heparinized collection tubes.
The pharmacokinetics of GLP2-2G-XTEN after intravenous and subcutaneous injection was determined in male cynomolgus monkeys. Monkeys (6 per group) were dosed with 25 nmol/kg GLP2-2G-XTEN by intravenous or subcutaneous administration using a cross-over study design. Plasma samples were taken pre-dose and at selected time points post-dose up to 21 days (504 hours) following injection in heparinized collection tubes.
ELISA for Pharmacokinetic Experiments
A quantitative sandwich enzyme-linked immunosorption assay (ELISA) technique was developed to measure GLP2-2G-XTEN in mouse, rat and cynomolgus monkey plasma. In the assay, standards, controls and test samples were incubated with mouse anti-XTEN monoclonal antibody (ProSci, 4D9G3) which was immobilized on a microtiter plate at a concentration of 8 µg/mL. After incubation, unbound material was washed away and GLP2-2G-XTEN was detected using biotinylated rabbit polyclonal to human GLP2 antibody (Phoenix Pharmaceuticals, B-G-028) at a 1
20,000 dilution, followed by streptavidin-HRP (Pierce, 21130) at 0.1 µg/mL, and visualized with a TMB peroxidase substrate (Immunochemistry Technologies, SUB1). The assay has a range of 480–0.47 ng/mL in 3% plasma.
Analysis of Pharmacokinetic Experiments
Pharmacokinetic curves were analyzed by fitting a non-compartmental model to the profile from each animal. The resulting pharmacokinetic parameters were averaged for the final results. Bioavailability was determined by dividing the area under the curve (AUC∞) from the subcutaneous administration and the AUC∞ from the intravenous administration on a per animal basis. The results were averaged for the final data. Analysis was performed using Phoenix WinNonLin software (Pharsight, Cary NC).
Rat Intestinotrophic Studies
Small intestine growth in rats was measured as a primary pharmacodynamic endpoint. GLP2-2G peptide, GLP2-2G-XTEN or vehicle was administered via subcutaneous injection into male Sprague-Dawley rats weighing 200–220 grams (10–12 rats per group). GLP2-2G peptide was dosed using the previously published regimen of 12.5 nmol/kg (0.05 mg/kg) twice daily for 12 days. GLP2-2G-XTEN was dosed at 25 nmol/kg once daily for 12 days. After sacrifice, a midline incision was made, the small intestines were removed, stretched to their maximum length and the length recorded. The fecal material was flushed from the lumen and the small intestinal wet weight recorded.
Male Wistar rats (180–220 grams) were weighed and randomized into treatment groups of ten rats each to ensure balance for average body weight across the treatment groups. Using a prophylactic study design, rats were injected subcutaneously with vehicle, GLP2-2G peptide (12.5 nmol/kg twice per day) or GLP2-2G-XTEN (25 nmol/kg once daily, 25 nmol/kg every other day for a total of three doses, or 75 nmol/kg once, as indicated), starting three days prior to indomethacin administration on day −3. Rats were fasted for 12 hours before receiving the first subcutaneous injection of freshly prepared indomethacin (7 mg/kg) on day 0; a second injection of indomethacin was administered 24 hours later on day 1. For the remainder of the experiment rats had unlimited access to food and water. Rats were euthanized 24 hours after receiving the second dose of indomethacin on day 2.
Disease readouts for the indomethacin model were performed as follows: 24 hours after the last indomethacin injection, rats were injected intravenously with 1 mL 1% Evan’s Blue dye. Thirty minutes later rats were anesthetized and exsanguinated. A midline incision was made in the abdomen and the rats were scored for the presence of adhesions. The small intestine was removed and the length recorded. The intestinal lumen was flushed with sterile 0.9% sodium chloride for injection and the length of the ulcerated area recorded. The intestines were gently blotted to remove excess fluid and weighed. The small intestine was scored for adhesions and trans-ulcerations using the following scoring system: Adhesions: none
3; transmural ulceration: none
Rat small intestine samples consisted of a 3 cm section of proximal jejunum and a 3 cm section of mid-jejunum collected 15 cm and 30 cm from the pylorus, respectively. Samples were fixed in 10% neutral buffered formalin. Samples were trimmed into multiple sections without bias toward lesion presence or absence. These sections were placed in cassettes, embedded in paraffin, microtomed at approximately 4 microns thickness, and stained with hematoxylin and eosin (H&E). The slides were evaluated microscopically by a board certified veterinary pathologist and scored for villous height as well as infiltration/inflammation, mucosal atrophy, villi/crypt appearance, abscesses/ulceration. A 1 to 4 severity grading scale was used, where 1
marked/severe, reflecting the combination of the cellular reactions seen histopathologically.
Parametric variables (small intestine length, small intestine weight, TNFα levels) were compared using an ANOVA with a Tukey-Kramer post hoc test for individual pairwise comparisons using an overall alpha of 0.05. Non-parametric variables (adhesion score, ulceration score) were compared with the vehicle control using a Mann Whitney U test with a Bonferroni correction for the p-value to create an overall alpha of 0.05. Calculations were performed using JMP or Excel as appropriate.