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Binding proteins specific for insulin-like growth factors and uses thereof

A technology that binds proteins and growth factors, applied in the direction of anti-growth factor immunoglobulin, anti-animal/human immunoglobulin, recombinant DNA technology, etc., can solve the problems of breast tumor tumor suppression in pancreas

Inactive Publication Date: 2009-07-29
ASTRAZENECA AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Additionally, in xenograft tumor models, IGF-IR blockade resulted in significantly inhibited growth of breast, kidney, and pancreas tumors in vivo

Method used

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  • Binding proteins specific for insulin-like growth factors and uses thereof
  • Binding proteins specific for insulin-like growth factors and uses thereof
  • Binding proteins specific for insulin-like growth factors and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

Immunization and Titration

immunity

[0146] Recombinant human IGF-I and IGF-II (available from R&D Systems, Inc. (Minneapolis, MN, catalog numbers 291-G1 and 292-G2, respectively)) were used as antigens. through sequential immunization Mice (XenoMouse strains XMG2 and XMG4 (3C-1 strain), Abgenix, Inc. Fremont, CA) produce monoclonal antibodies against IGF-I / II. XenoMouse animals were immunized via the pawpad route for all injections. The total volume of each injection was 50 μl / mouse, 25 μl / paw pad. A total of 10 mice were immunized in each group. 10 μg of IGF-I or IGF-II alone or with keyhole as vehicle per injection per mouse Hemocyanin (KLH) antigen-conjugated IGF-I or IGF-II, see Table 2 for details. The first injection was prepared in Dulbecco's PBS (DPBS) and mixed 1:1 v / v with Titermax Gold adjuvant (SIGMA, cat# T2684, lot# K1599). Each mouse was then administered with 25 μg Adju-Phos (aluminum phosphate gel, catalog number 1452-250, lot number 8937, HCI Bio...

Embodiment 2

Lymphocyte recovery, B cell isolation, fusion and generation of hybridomas

[0147] The immunized mice were killed by cervical dislocation, and the draining lymph nodes were collected from each group and combined. Lymphocytes were dissociated by trituration in DMEM to release the cells from the tissue and suspended in DMEM. To count the number of cells, add 0.9ml DMEM / 1×10 8 Lymphocytes were added to the cell pellet to gently but completely resuspend the cells. Use 100 μl CD90+ magnetic beads / 1×10 8 cells, label the cells by incubating the cells with magnetic beads for 15 minutes at 4°C. will contain up to 10 8 positive cells (or a total of 2×10 9 cells) of the magnetically labeled cell suspension was applied to the LS+ column, and the column was washed with DKEM. The entire effluent was collected as a CD90 negative fraction (the majority of these cells are expected to be B cells).

[0148] Fusion was carried out by mixing the above-washed enriched B cells with non-secre...

Embodiment 3

Selection of Candidate Antibodies by ELISA

[0154] After 14 days of culture, hybridoma supernatants were screened for IGF-I / II-specific monoclonal antibodies. ELISA plates (Fisher, Cat. No. 12-565-136) were used in coating buffer (0.1M carbonate buffer, pH 9.6, NaHCO 3 8.4 g / L) in 50 μl / well human IGF-I or IGF-II (2 μg / ml) and then incubated overnight at 4°C. After incubation, the plate was washed 3 times with wash buffer (0.05% Tween 20 in PBS). 200 μl / well of blocking buffer (0.5% BSA, 0.1% Tween 20, 0.01% Thimerosal in Ix PBS) was added and the plate was incubated for 1 hour at room temperature. After incubation, wash the plate 3 times with wash buffer. 50 μl / well of hybridoma supernatant and positive and negative controls were added and the plate was incubated for 2 hours at room temperature.

[0155]After incubation, wash the plate 3 times with wash buffer. 100 μl / well of the detection antibody goat anti-huIgGFc-HRP (Catalog No. H10507) was added and the plate was i...

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Abstract

Binding proteins, such as antibodies directed to IGF-II with cross-reactivity to IGF-I and uses of such antibodies are described. In particular, fully human monoclonal antibodies directed to the IGF-II with cross-reactivity to IGF-I are disclosed. Also discussed are nucleotide sequences encoding, and amino acid sequences comprising, heavy and light chain immunoglobulin molecules, particularly sequences corresponding to contiguous heavy and light chain sequences spanning the framework regions and / or complementarity determining regions (CDR's), specifically from FR1 through FR4 or CDR1 through CDR3.

Description

[0001] Cross References to Related Applications [0001] Pursuant to Title 35, United States Code, Section 119, this application claims U.S. Provisional Application Serial No. 60 / 750,085 filed December 13, 2005, U.S. Provisional Application Serial No. 60 / 750,772 filed December 14, 2005, and February 17, 2005 US Provisional Application Serial No. 60 / 774,747, filed on May 24, 2006, and US Provisional Application Serial No. 60 / 808,183, filed May 24, 2006, each of which is hereby incorporated by reference in its entirety. field of invention Background of the invention [0002] The present invention relates to binding proteins capable of binding insulin-like growth factor-2 (IGF-II) but cross-reactive with insulin-like growth factor-1 (IGF-I), and uses of these binding proteins. More specifically, the present invention relates to monoclonal antibodies directed against IGF-II but cross-reactive with IGF-I and uses of these antibodies. Aspects of the invention also relate to hybri...

Claims

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Application Information

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IPC IPC(8): A61K39/395C07K16/22C12N15/09
Inventor O·雷伯G·加兹特-博恩斯坦X·杨S·A·卡特利奇D·W·唐格G·加兹特－博恩斯坦
Owner ASTRAZENECA AB
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