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Anti-IgE antibodies

a technology of anti-ige antibodies and antibodies, which is applied in the field of anti-ige antibodies, can solve the problems of conformational changes, reduction of binding affinity, and anaphylaxis of dangerous life-threatening conditions

Inactive Publication Date: 2008-01-03
GENENTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention is about improving a polypeptide that has a specific affinity to a target molecule. The method involves identifying aspartyl residues that are prone to isomerization and substituting them with alternative residues to screen for improved affinity. The invention also includes a method of improving the affinity of an anti-IgE antibody by replacing a specific residue with another residue and modifying other residues through phage display. The invention also includes a composition of improved affinity anti-IgE antibodies and a method of reducing or inhibiting the production of histamine. Finally, the invention also includes a method of treating IgE-mediated disorders by administering the improved antibodies or functional fragments thereof.

Problems solved by technology

However anaphylactic shock, a dangerous life-threatening condition anaphylaxis is usually provoked by insect stings or parental medication.
The end result is the insertion of an additional methyl group into the polypeptide backbone of the antibody, which can result in conformational changes and reduction in binding affinity.

Method used

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Examples

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example i

Preparation of Monoclonal Antibodies to IgE

[0382] Eight monoclonal antibodies (MAE10-MAE17) with the ability to block the binding of IgE to FcεR1 were prepared. Monoclonal antibodies to IgE were prepared from the supernatants of U266B1 cells (ATCC TIB 196) using affinity chromatography on an isolated anti-IgE antibody (Genentech MAE1). For MAE12, five BALB / c female mice, age six weeks, were immunized in their foot pads with 10 μg of purified IgE in Ribi's adjuvant. Subsequent injections were done in the same manner at one and three weeks after the initial immunizations. Three days after the final injection, the inguinal and popliteal lymph nodes were removed and pooled, and a single cell suspension was made by passing the tissue through steel gauze. The cells were fused at a 4:1 ratio with mouse myeloma P3X63-Ag8.653 (ATCC CRL 1580) in high glucose (DMEM) containing 50% w / v polyethylene glycol 4000. For MAE14, MAE15, and MAE13 the immunizations were done in a similar manner except...

example 2

Preparation of Humanized MaE11

Introduction:

[0421] The following example describes various preparations of humanized MaE11 wherein residues were modified via site-directed mutagenesis to arrive at 12 anti-IgE MaE11 variants [F(ab)1-12]. The residues of F(ab)12 were used as the template to create rhuMaE25 or E25, a highly potent anti-IgE antibody described in Application PCT / US92 / 06860, filed Aug. 14, 1992.

Methods:

[0422] The murine anti-human IgE mAb MaE11, shown in FIG. 1, was modified by site-directed mutagenesis (Kunkel, T. A. (1985), Proc. Natl. Acad. Sci. USA 82: 488) from a deoxyuridine-containing template containing a human k-subgroup I light chain and human subgroup III heavy chain (VH-CHI) in a pUC119-based plasmid, pAK2 (Carter et al. (1992), Proc. Natl. Aca. Sci. USA 89: 4285) to obtain the variant F(ab)-1. F(ab)-2 was constructed from the F(ab)-1 template and all other humanized F(ab) variants were constructed from a template of F(ab)-2. The plasmids were transformed...

example 3

Histamine Release Assay

[0452] Introduction:

[0453] This is a rat mast cell histamine assay (RMCHA) which measures quantitatively the biological activity of a recombinant humanized, monoclonal anti-IgE antibody based on the ability of the antibody to block histamine release from allergen-sensitized RBL 48 cells. Furthermore, this determination is made under physiological conditions similar to those of the human body. The RBL 48 cell line was derived from the parental rat mast cell line RBL 2H3 which has been subsequently transfected with the α-subunit of the high affinity human IgE receptor (FcεRI). Gilfillan A. M. et al., J. Immunol. 149(7): 2445-2451 (1992).

[0454] Methods:

[0455] RBL 48 cells (Gilfillan et al., supra) are grown in sIMDM, Iscove's modified Dulbecco's media supplemented with 10% fetal calf serum, 2 mM glutamine, and 500μg / ml of active geneticin (Gibco, #860-1811) in a T175 tissue culture flask (Falcon #3028) at 37° C. in a humidified 5% CO2 incubator (Fischer, mode...

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Abstract

The present invention relates to a method for adjusting the affinity of a polypeptide to a target molecule by a combination of steps, including: (1) the identification of aspartyl residues which are prone to isomerization; (2) the substitution of alternative residues and screening the resulting mutants for affinity against the target molecule. In a preferred embodiment, the method of subtituting residues is affinity maturation with phage display (AMPD). In a further preferred embodiment the polypeptide is an antibody and the target molecule is an antigen. In a further preferred embodiment, the antibody is anti-IgE and the target molecule is IgE. In another embodiment, the invention relates to an anti-IgE antibody having improved affinity to IgE.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation application of U.S. Ser. No. 10 / 791,619, filed Mar. 2, 2004, which is a continuation of U.S. Ser. No. 09 / 716,028, filed Nov. 17, 2000, now U.S. Pat. No. 6,723,833, which is a division of U.S. Ser. No. 09 / 109,207, filed Jun. 30, 1998, now U.S. Pat. No. 6,172,213, which claims the benefit under 35 U.S.C. §119(e) of U.S. Ser. No. 60 / 051,554, filed Jul. 2, 1997, all of which are incorporated herein by reference.BACKGROUND OF THE INVENTION [0002] The present invention relates to immunoglobulin E (IgE), IgE antagonists, anti-IgE antibodies capable of binding to human IgE, and to a method of improving polypeptides, including anti-IgE antibodies. [0003] IgE is a member of the immunoglobulin family that mediates allergic responses such as asthma, food allergies, type 1 hypersensitivity and the familiar sinus inflammation suffered on a widespread basis. IgE is secreted by, and expressed on the surface of B-cells...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395A61P11/06A61P37/00C07K16/00C12N15/13C12N5/10C12P21/00C07K16/42C12P21/08
CPCA61K39/39566C07K16/4291C07K2316/96C07K2317/55C07K2317/565C07K2317/56A61K2300/00A61P11/06A61P37/00C07K2317/76
Inventor LOWMAN, HENRY B.PRESTA, LEONARD G.JARDIEU, PAULA M.LOWE, JOHN
Owner GENENTECH INC
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