A Human Monoclonal Antibody That Improves Glycemic Control
The extracellular domain of the human insulin receptor (hINSR; R&D Systems, Minneapolis, MN) was biotinylated with Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) according to the manufacturer's protocol. To obtain allosteric antibodies to the INSR, panning and subsequent screening were performed with the biotinylated extracellular domain of the hINSR maintained in the presence of saturating insulin (10 μmol/L; Sigma-Aldrich, St. Louis, MO) to block the orthosteric binding site. This biotinylated receptor:ligand complex was immobilized on streptavidin-coated magnetic Dynabeads M-280 (Invitrogen Dynal AS, Oslo, Norway) and panned against two scFv antibody phage display libraries (XOMA [US], LLC, Berkeley, CA; BioInvent, Lund, Sweden), using standard methods. After each round of panning, phage were deselected against streptavidin-coated magnetic Dynabeads M-280 to remove nonspecific phage antibodies. After three rounds of panning and deselection, bead-bound phage were eluted and used to infect TG1 bacterial cells (Stratagene, La Jolla, CA). Phage were then rescued with helper phage M13KO7 (New England Biolabs, Ipswich, MA). Individual colonies were picked and grown in 96-well plates used to generate bacterial periplasmic extracts according to standard methods. The lysate supernatants were assayed for INSR binding by flow cytometry (vide infra). For this purpose, CHO cells that were transfected with either the hINSR (CHO-hINSR) or mouse INSR (CHO-mINSR) were used. Both INSR transfected cell lines had ~250,000 receptors per cell compared with the untransfected cells, which had less than 5,000 INSR per cell as determined by flow cytrometry (similar numbers of IGF-IR were expressed on CHO-hIGF-IR cells; vide infra). The single chain fragment (scFv) with the highest affinity was reformatted to a fully human IgG2a monoclonal antibody, XMetA.
For flow cytometry, cells (2 × 10/mL) were washed and resuspended in PBS with 0.5% fatty acid-free bovine serum albumin and 0.1% sodium azide (Fluorescence-activated cell sorter buffer; Invitrogen, Carlsbad, CA). Either XMetA or an antikeyhole limpet hemocyanin IgG2a isotype control antibody (XOMA [US]) was diluted in FACS buffer and incubated with cells at 4°C for 60 min. Cells were then washed once and resuspended in Alexa Fluor 647–conjugated goat anti-human IgG (1:200; Invitrogen). The cells were incubated for 30 min at 4°C, washed twice, and analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
The effect of XMetA on the binding of insulin to the INSR was assessed in an equilibrium assay under conditions of a saturating antibody concentration (70 nmol/L). Human insulin (80 pmol/L; Sigma-Aldrich) and XMetA or isotype control antibody were incubated at 5°C with increasing concentrations of CHO-hINSR cells for 18 h. Cells were pelleted by centrifugation, and the amount of free insulin in solution was measured by immunofluorescence using a KinExA instrument. Insulin concentration data were curve-fit using KinExA software to yield the relative change in insulin binding affinity.
CHO cells at 37°C expressing mouse or human INSR or IGF-IR were incubated in serum-free culture medium with increasing concentrations of insulin, control antibody, or XMetA for 10 min. Total Akt and Akt phosphorylated at Ser473 and total Erk1/2 and Erk1/2 phosphorylated at Thr202/Tyr204; Thr185/Tyr187 were measured by enzyme-linked immunosorbent assay (ELISA; Meso Scale Discovery, Gaithersburg, MD). To evaluate the effect of XMetA on the hINSR and mINSR autophosphorylation, CHO cells were incubated with increasing concentrations of insulin, control antibody, or XMetA for 10 min and phosphotyrosine content of the INSR measured by ELISA (Millipore, Billerica, MA). To determine the effect of XMetA on autophosphorylation of the IGF-IR, CHO cells transfected with the human IGF-IR that had ~250,000 receptors per cell were used. Cells were preincubated at 37°C in serum-free medium with a saturating concentration of XMetA (33 nmol/L) for 15 min followed by a 10-min incubation at 37°C with IGF-I (100 nmol/L). Tyrosine-phosphorylated INSR or IGF-IR was determined by ELISA (Millipore).
To measure 2-deoxy-glucose uptake, 3T3 cells expressing hINSR (3T3-HIR cells; ~1 × 10 hINSR/cell) were incubated in serum-free medium for 1 h with increasing concentrations of XMetA, isotype control antibody, or insulin. [H]-2-deoxy-D-glucose was then added for 20 min, and its uptake was measured. MCF-7 cells (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium containing glucose at 4.5 g/L supplemented with 10% FBS and 2 mmol/L glutamine (Invitrogen) for normal maintenance. For the proliferation assay, cells (1 × 10) were seeded in 96-well white opaque microtiter plates and allowed to reattach for 24 h. After 24 h, cells were washed twice with prewarmed PBS and incubated in Dulbecco's modified Eagle's medium containing glucose at 1 g/L and no phenol red, supplemented with 0.1% FBS and 2 mmol/L glutamine (Invitrogen; "starvation media"). XMetA, isotype control antibody, or insulin (Sigma-Aldrich) was added to the cells for a further 24 h and cell proliferation was measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI).
Animal experiments were approved by the XOMA (US) Institutional Animal Care and Use Committee (IACUC) and performed in accordance with IACUC guidelines. All animals were maintained in a pathogen-free environment and allowed free access to food and water. Six- to eight-week-old male ICR mice (Harlan, Indianapolis, IN) were randomly divided into groups administered streptozotocin (STZ; Sigma) or treated with citrate buffer. Buffer-treated (control) mice were fed a standard chow diet (Harlan), and diabetic animals were fed a 40 kcal% fat, 35 kcal% sucrose diet (Research Diets, New Brunswick, NJ) on arrival. STZ was dissolved in citrate buffer and injected intraperitoneally (40 mg/kg) for 5 consecutive days during the third week after arrival. After an additional week, mice were divided into groups (n = 8) and treated twice weekly, intraperitoneally, with XMetA or an antikeyhole limpet hemocyanin isotype control antibody, as indicated. Blood glucose was measured using the α-TRAK Blood Glucose Monitoring System (Abbott, Chicago, IL), and hemoglobin A1c was evaluated using the A1CNow platform (Bayer HealthCare, Tarrytown, NY). After ~6 weeks of dosing, mice were killed after an overnight fast and terminal plasma was collected by cardiac puncture. Plasma insulin and C-peptide were measured by ELISA (Alpco Diagnostics, Salem, NH). XMetA was determined not to cross-react with capture or detection antibodies in these systems (data not shown). β-Hydroxybutyrate, cholesterol, triglycerides, and free fatty acids were determined by standard colorimetric methods (Wako Chemicals, Richmond, VA).
For glucose tolerance tests, mice were fasted overnight for 14 h followed by a glucose challenge (1 g/kg; Mediatech, Manassas, VA) by intraperitoneal injection or oral gavage, as indicated. Whole venous blood was obtained from the tail vein at 0, 15, 30, 60, and 120 min after challenge and evaluated for blood glucose. Insulin tolerance tests were carried out after a 4-h fast in animals by administering insulin (0.75 units/kg; Roche Diagnostics) intraperitoneally and measuring venous blood for glucose at 0, 30, 60, and 120 min after insulin challenge.
All values, unless otherwise indicated, are expressed as mean ± SEM. Statistical analyses were carried out using a two-tailed Student unpaired t test using Prism 5.0 graphing software (GraphPad, La Jolla, CA).
Research Design and Methods
XMetA Discovery
The extracellular domain of the human insulin receptor (hINSR; R&D Systems, Minneapolis, MN) was biotinylated with Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) according to the manufacturer's protocol. To obtain allosteric antibodies to the INSR, panning and subsequent screening were performed with the biotinylated extracellular domain of the hINSR maintained in the presence of saturating insulin (10 μmol/L; Sigma-Aldrich, St. Louis, MO) to block the orthosteric binding site. This biotinylated receptor:ligand complex was immobilized on streptavidin-coated magnetic Dynabeads M-280 (Invitrogen Dynal AS, Oslo, Norway) and panned against two scFv antibody phage display libraries (XOMA [US], LLC, Berkeley, CA; BioInvent, Lund, Sweden), using standard methods. After each round of panning, phage were deselected against streptavidin-coated magnetic Dynabeads M-280 to remove nonspecific phage antibodies. After three rounds of panning and deselection, bead-bound phage were eluted and used to infect TG1 bacterial cells (Stratagene, La Jolla, CA). Phage were then rescued with helper phage M13KO7 (New England Biolabs, Ipswich, MA). Individual colonies were picked and grown in 96-well plates used to generate bacterial periplasmic extracts according to standard methods. The lysate supernatants were assayed for INSR binding by flow cytometry (vide infra). For this purpose, CHO cells that were transfected with either the hINSR (CHO-hINSR) or mouse INSR (CHO-mINSR) were used. Both INSR transfected cell lines had ~250,000 receptors per cell compared with the untransfected cells, which had less than 5,000 INSR per cell as determined by flow cytrometry (similar numbers of IGF-IR were expressed on CHO-hIGF-IR cells; vide infra). The single chain fragment (scFv) with the highest affinity was reformatted to a fully human IgG2a monoclonal antibody, XMetA.
Binding of XMetA to INSR
For flow cytometry, cells (2 × 10/mL) were washed and resuspended in PBS with 0.5% fatty acid-free bovine serum albumin and 0.1% sodium azide (Fluorescence-activated cell sorter buffer; Invitrogen, Carlsbad, CA). Either XMetA or an antikeyhole limpet hemocyanin IgG2a isotype control antibody (XOMA [US]) was diluted in FACS buffer and incubated with cells at 4°C for 60 min. Cells were then washed once and resuspended in Alexa Fluor 647–conjugated goat anti-human IgG (1:200; Invitrogen). The cells were incubated for 30 min at 4°C, washed twice, and analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
Measurement of Insulin Binding Affinity
The effect of XMetA on the binding of insulin to the INSR was assessed in an equilibrium assay under conditions of a saturating antibody concentration (70 nmol/L). Human insulin (80 pmol/L; Sigma-Aldrich) and XMetA or isotype control antibody were incubated at 5°C with increasing concentrations of CHO-hINSR cells for 18 h. Cells were pelleted by centrifugation, and the amount of free insulin in solution was measured by immunofluorescence using a KinExA instrument. Insulin concentration data were curve-fit using KinExA software to yield the relative change in insulin binding affinity.
Insulin and IGF-I Receptor Signaling in Cultured Cells
CHO cells at 37°C expressing mouse or human INSR or IGF-IR were incubated in serum-free culture medium with increasing concentrations of insulin, control antibody, or XMetA for 10 min. Total Akt and Akt phosphorylated at Ser473 and total Erk1/2 and Erk1/2 phosphorylated at Thr202/Tyr204; Thr185/Tyr187 were measured by enzyme-linked immunosorbent assay (ELISA; Meso Scale Discovery, Gaithersburg, MD). To evaluate the effect of XMetA on the hINSR and mINSR autophosphorylation, CHO cells were incubated with increasing concentrations of insulin, control antibody, or XMetA for 10 min and phosphotyrosine content of the INSR measured by ELISA (Millipore, Billerica, MA). To determine the effect of XMetA on autophosphorylation of the IGF-IR, CHO cells transfected with the human IGF-IR that had ~250,000 receptors per cell were used. Cells were preincubated at 37°C in serum-free medium with a saturating concentration of XMetA (33 nmol/L) for 15 min followed by a 10-min incubation at 37°C with IGF-I (100 nmol/L). Tyrosine-phosphorylated INSR or IGF-IR was determined by ELISA (Millipore).
Glucose Transport and Proliferation Assays
To measure 2-deoxy-glucose uptake, 3T3 cells expressing hINSR (3T3-HIR cells; ~1 × 10 hINSR/cell) were incubated in serum-free medium for 1 h with increasing concentrations of XMetA, isotype control antibody, or insulin. [H]-2-deoxy-D-glucose was then added for 20 min, and its uptake was measured. MCF-7 cells (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium containing glucose at 4.5 g/L supplemented with 10% FBS and 2 mmol/L glutamine (Invitrogen) for normal maintenance. For the proliferation assay, cells (1 × 10) were seeded in 96-well white opaque microtiter plates and allowed to reattach for 24 h. After 24 h, cells were washed twice with prewarmed PBS and incubated in Dulbecco's modified Eagle's medium containing glucose at 1 g/L and no phenol red, supplemented with 0.1% FBS and 2 mmol/L glutamine (Invitrogen; "starvation media"). XMetA, isotype control antibody, or insulin (Sigma-Aldrich) was added to the cells for a further 24 h and cell proliferation was measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI).
Mouse Model of Diabetes
Animal experiments were approved by the XOMA (US) Institutional Animal Care and Use Committee (IACUC) and performed in accordance with IACUC guidelines. All animals were maintained in a pathogen-free environment and allowed free access to food and water. Six- to eight-week-old male ICR mice (Harlan, Indianapolis, IN) were randomly divided into groups administered streptozotocin (STZ; Sigma) or treated with citrate buffer. Buffer-treated (control) mice were fed a standard chow diet (Harlan), and diabetic animals were fed a 40 kcal% fat, 35 kcal% sucrose diet (Research Diets, New Brunswick, NJ) on arrival. STZ was dissolved in citrate buffer and injected intraperitoneally (40 mg/kg) for 5 consecutive days during the third week after arrival. After an additional week, mice were divided into groups (n = 8) and treated twice weekly, intraperitoneally, with XMetA or an antikeyhole limpet hemocyanin isotype control antibody, as indicated. Blood glucose was measured using the α-TRAK Blood Glucose Monitoring System (Abbott, Chicago, IL), and hemoglobin A1c was evaluated using the A1CNow platform (Bayer HealthCare, Tarrytown, NY). After ~6 weeks of dosing, mice were killed after an overnight fast and terminal plasma was collected by cardiac puncture. Plasma insulin and C-peptide were measured by ELISA (Alpco Diagnostics, Salem, NH). XMetA was determined not to cross-react with capture or detection antibodies in these systems (data not shown). β-Hydroxybutyrate, cholesterol, triglycerides, and free fatty acids were determined by standard colorimetric methods (Wako Chemicals, Richmond, VA).
Glucose and Insulin Tolerance Tests
For glucose tolerance tests, mice were fasted overnight for 14 h followed by a glucose challenge (1 g/kg; Mediatech, Manassas, VA) by intraperitoneal injection or oral gavage, as indicated. Whole venous blood was obtained from the tail vein at 0, 15, 30, 60, and 120 min after challenge and evaluated for blood glucose. Insulin tolerance tests were carried out after a 4-h fast in animals by administering insulin (0.75 units/kg; Roche Diagnostics) intraperitoneally and measuring venous blood for glucose at 0, 30, 60, and 120 min after insulin challenge.
Statistical Procedures
All values, unless otherwise indicated, are expressed as mean ± SEM. Statistical analyses were carried out using a two-tailed Student unpaired t test using Prism 5.0 graphing software (GraphPad, La Jolla, CA).
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