Therapeutic combinations of anti-IGF-1R antibodies and other compounds
A technology of IGF-1R and antibodies, applied in the direction of antibody mimics/stents, antibodies, drug combinations, etc., can solve problems such as increased risk
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Embodiment 1
[1172] Selection of IGF-1R-specific Fab from phage library
[1173] Recombinant human IGF-1R ectodomain was used to screen for cells containing 3.5x10 10 A human native phagemid Fab library of unique clones (Hoet, R.M., et al. Nat Biotechnol. 23(3):344-8 (2005), ("Hoet et al.") is incorporated by reference in its entirety in this article). Biotinylated IGF1R-his and IGF1R-Fc proteins were used after two different panning arms. Proteins are captured on streptavidin-coated magnetic beads and incubated with phage. For IGF1R-Fc, biotinylated anti-Fc antibodies were captured on magnetic beads, which then captured the Fc fusion protein. Selections were made as described by Hoet et al. After 3 rounds of panning, the 479 bp gene III remnant was removed by MluI digestion, and the vector was religated for soluble Fab expression in TG1 cells. ELISA analysis of 920 clones with biotinylated IGF1R-His arms yielded 593 positive clones containing 33 unique sequences. ELISA analysis of 9...
Embodiment 2
[1175] Binding activity of Fab to IGF-1R expressed on tumor cells
[1176] The ability of Fabs to bind wild-type IGF-IR can be determined by flow cytometry using the MCF-7 tumor cell line.
[1177] MCF-7 cells (human breast cancer cells from NCI) were split 24 hours before setting up the assay to obtain a 70% confluent monolayer. The MCF-7 cell line is routinely maintained for less than 20 passages. Cells were dissociated with Cell Dissociation Buffer (Gibco Cat# 13151-014), counted, washed and adjusted to 1x10 6 cells / ml, then 1 ml of cells was added to each tube (12x75mm tube Falcon cat# 352054). Cells were pelleted by centrifugation at 1200 rpm for 5 minutes and the supernatant was removed, then 100 μl of diluted antibody was added to the cell pellet. The starting concentration of purified Fab tested can be 210 or 60 μg / ml diluted 1 :3 in FRCS buffer up to 0.001 μg / ml. The FACS buffer used throughout the assay was PBS (Ca++ / Mg++ free) with 1% BSA (Sigma Cat# A-7906) and...
Embodiment 3
[1182] Inhibition of ligand binding to IGF-1R by Fab
[1183] The ability of Fabs to block the binding of IGF-1 and IGF-2 ligands to IGF-1R can be tested by radioimmunoassay (RIA) assay.
[1184] Ligand Blocking Assay (RIA). Recombinant human IGF-1 (Cat# 291-G1), IGF-2 (Cat# 292-G2), insulin (Cat# Custom02) and human insulin receptor (Cat# 1544-1R) were purchased from R&D Systems, Inc., Minneapolis , MN. Insulin (Arg-Insulin, Cat#01-207) was purchased from Upstate Cell Signaling Solutions (Lake Placid, NY (now part of Millipore, Concord, MA (USA)). 125 I-rhIGF-1 (Cat# IM 172), 125 I-rhIGF-2 (Cat# IM238) and 125 I-rhInsulin (Cat# IM166) was purchased from Amersham Biosciences (Piscataway, NJ). AffiPure goat anti-human IgG, Fcγ fragment specific antibody (Cat # 109-005-098, Jackson IrnmunoResearch, West Grove, PA) was used to capture IGF-1R-Fc. Goat anti-mouse IgG HRP (Cat# 1030-05, Southern Biotech Birmingham, AL) was used as detection antibody.
[1185] IR3 (Ab-1, Cat. ...
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