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Antibodies to erbb2

A technology of antibody and monoclonal antibody, applied in the field of preparation and use of anti-ErbB2 antibody such as to treat cancer, human antibody

Inactive Publication Date: 2009-09-02
ASTRAZENECA AB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] Although EGF and TGFα do not bind ErbB2, EGF stimulates EGFR and ErbB2 to form a heterodimer, which activates EGFR and results in transphosphorylation of ErbB2 in the heterodimer

Method used

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  • Antibodies to erbb2
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  • Antibodies to erbb2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0394] Immunization and Titration

[0395] Immunization

[0396] A recombinant human ErbB2-ECD / Fcγ1 fusion protein comprising the extracellular domain of human ErbB2 and the Fc region of human IgG1 was obtained from R&D Systems, Inc. (Minneapolis, MN catalog #1129-ER / CF) for use as an immunogen. Monoclonal antibodies against ErbB2 were sequentially immunized by injection via the footpad route Mice (XenoMouse strainXM3B-3, Abgenix, Inc. Fremont, CA) were developed. The first injection was with 10 μg of recombinant human ErbB2-ECD / Fcγ1 per mouse in Titermax Gold (Sigma, catalog #T2684, lot #K1599). Subsequent 10 boosts were performed with 10 μg recombinant human ErbB2-ECD / Fcγ1 per mouse in 15 μl qCpG (ImmunEasy Mouse Adjuvant, Cat #303101; Qiagen), mixed with 5 μl Adju-Phos (Aluminophosphate Gel, Cat #1452- 250, HCI Biosector) mixed. The total volume per injection was 50 μl / mouse, 25 μl / footpad. Mice were immunized twice a week for 5 weeks and fusions were performed on...

Embodiment 2

[0404] Lymphocyte recovery, B cell isolation, fusion and hybridoma generation

[0405]Lymph nodes (LN) were harvested from immunized mice and processed into 3 ml sterile FASC buffer (PBS, 2% FBS). LN cells were filtered through a 40 μm cell strainer, spun down at 400 g for 3 minutes and resuspended in 3 ml fresh FACS buffer. Cells were counted and biotinylated antibodies against CD90 (Pharmingen, Cat #553002), CD4 (Pharmingen, Cat #553728), CD8 (Pharmingen, Cat #553029) and IgM (Pharmingen, Cat #555781 ) were subsequently added. Cells and the above antibodies were gently mixed and incubated on ice for 10 minutes. Cells were spun down again and washed 1x with FACS buffer. SA Dynal beads (M-280) were added to the cells at a 4:1 bead to target cell ratio and incubated for 12 minutes at room temperature with rotation. Place the cells / beads in the 15ml tube in the magnetic field of the Dynal magnet for 2 minutes. The supernatant containing the IgM-fraction was transferred to ...

Embodiment 3

[0409] Antibody screening by FMAT / FACS

[0410] After 14 days of culture, hybridoma supernatants were screened for ErbB2-specific antibodies by FMAT (Fluorometric Microvolume Assay Technology). Briefly, 4275 B300.19 / hErbB2 (positive cells) or B300.19 (ErbB2-negative cells) cells were incubated with 400 ng / ml Cy5 goat anti-human IgG (JIR, cat #109) in 15 μl FACS buffer. -176-098) were mixed and then incubated with 15 μl hybridoma supernatant for 3 hours at room temperature. Positive control antibodies were anti-hErbB2(2C4) with Cy5 goat anti-mouse IgGγ (JIR, catalog #115-176-071 ) combined with SA-Cy5 (JIR catalog #016-170-084, 350 ng / ml) or Goat anti-mouse IgG-Biot (Southern Biotechnology / SB Cat #1030-08, 400 ng / ml) was used for detection. Anti-KLH G1 antibody (Gmix, in-house) was used as isotype control. Plates were read on a FMAT 8100 HTS system from Applied Biosystems. Fluorescent signal and counts were determined after data analysis, and 362 positive hybridomas showi...

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Abstract

The present invention relates to antibodies including human antibodies and antigen-binding portions thereof that specifically bind to ErbB2, preferably human ErbB2. In another embodiment, the antibodies or antigen-binding portions thereof inhibit ErbB2. The invention also relates to antibodies that are chimeric, bispecific, derivatized, single chain antibodies or portions of fusion proteins. The invention also relates to isolated heavy and light chain immunoglobulins or portions thereof derived from human anti-ErbB2 antibodies and nucleic acid molecules encoding such immunoglobulins. The present invention also relates to methods of using the antibodies and compositions for diagnosis and treatment. The invention also provides gene therapy methods using nucleic acid molecules encoding the heavy and / or light immunoglobulin molecules that comprise the human anti-ErbB2 antibodies. The invention also relates to transgenic animals or plants comprising nucleic acid molecules of the present invention.

Description

[0001] Cross references to related applications [0002] This application claims priority under 35 U.S.C. §119 to U.S. Provisional Application Serial No. 60 / 835,514, filed August 4, 2006, which is hereby incorporated by reference in its entirety. field of invention [0003] The present invention relates to anti-ErbB2 antibodies, particularly human antibodies, and methods for making and using anti-ErbB2 antibodies, eg, to treat cancer. Background of the invention [0004] The ErbB family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival. The receptor family includes 4 distinct members including epidermal growth factor receptor (EGFR or ErbB1), HER2 (ErbB2 or p185neu), HER3 (ErbB3) and HER4 (ErbB4). [0005] EGFR, encoded by the erbB1 gene, has been causally implicated in human malignancies. In particular, increased expression of EGFR has been observed in breast, bladder, lung, head, neck and gastric cancers, as well as gliobl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/32A61K39/395A61P35/00C12N5/10C12N15/13C12N15/63A01K67/027C12N5/07C12N5/071C12N5/09C12N15/113
CPCC07K16/40A61K39/39558C07K16/32C07K2317/76C07K2317/21C07K2317/73A61K45/06A61K2039/505C07K2317/33C07K2317/92A61P35/00C12N15/11A61K39/395A01K67/027
Inventor S·A·卡蒂拉奇J·董M·希金森I·富尔茨J·S·康
Owner ASTRAZENECA AB
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