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Antagonistic anti-hFAS ligand human antibodies and fragments thereof

An antibody and human technology, applied in the direction of anti-animal/human immunoglobulins, antibodies, fungi, etc., can solve the problem of slow removal of human antibodies

Inactive Publication Date: 2006-11-08
ELI LILLY & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This causes human antibodies to be cleared from the body more slowly than non-human or partially human antibodies

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Functional activity assay with soluble hFasL using Jurkat assay

[0087] FasL / enhancer matrices were prepared at 4x concentrations. 1× Matrix contains 50ng / ml recombinant human soluble FasL (Alexis  Biochemicals, Cat #522-001) and 1 μg / ml anti-FLAG M2 mouse monoclonal antibody (Boost; Sigma Chemical Co., Cat # F-3165). 1X matrix was used as a "100% apoptosis" reference. Jurkat cell culture medium without FasL or booster was used as "0% apoptosis" reference.

[0088] The matrix was incubated at room temperature for 1 hour. For each assay, 25 [mu]l of 4X Boost Fas Ligand Matrix or reference sample was added to each well of a 96-well plate. Subsequently, 25 μl of inhibitor sample (3E1 or 4G11 anti-hFasL antibody) or reference sample was added to each well. This addition dilutes all samples and matrix to half their original concentration. Incubate the samples at room temperature for 45 to 60 minutes. Subsequently, at 10 6 50 μl of Jurkat cells were added...

Embodiment 2

[0089] Example 2: CHO-K1 / Jurkat assay using membrane-bound FasL

[0090] The CHO-K1 cell line stably expressing Del.huFasL, the non-cleavable version of hFasL, was processed to analyze the ability of antibodies 3E1 and 4G11 to block membrane-bound FasL activity. This cell line expresses surface layer FasL, which induces Jurkat apoptosis when co-cultured with Jurkat cells.

[0091] Adherent CHO-1 cells were performed using DMEM:F-12 (3:1), 5% FBS, 40 μg / ml L-proline (Sigma), 50 μg / ml gentamicin (Sigma), and 600 μg / ml G418 Preparation of medium. For each assay, approximately 104 CHO-K1 cells (Del. huFasL or parental CHO-K1 ) were added to each well of a 96-well plate. The cells were incubated overnight at 37°C under 5% carbon dioxide. The medium was removed and 100 μl of each inhibitor sample (3E1 or 4G11 antibody; serial dilutions covering a concentration range) or reference (medium) was added to each well. The cells were incubated for 1 hour at 37°C under 5% carbon dioxide...

Embodiment 3

[0092] Example 3: Affinity Measurement of Monoclonal Antibodies

[0093] The response of various anti-hFasL antibodies to recombinant human soluble FasL (Alexis  Biochemicals, catalog #522-001). The BIAcore(R) exploits the optical signature of surface plasmon resonance to detect changes in the protein concentration of interacting molecules in a dextran biosensor matrix. All reagents and materials were purchased from BIAcore(R) AB (Upsala, Sweden) unless otherwise noted. All measurements were performed at room temperature. Samples were dissolved in HBS-EP buffer (150 mM NaCl, 3 mM EDTA, 0.005% (w / v) surfactant P-20 and 10 mM HEPES, pH 7.4). Goat anti-human Fc antibody at a level of 500 response units (RU) was immobilized in chambers 1 and 2 of the B1 sensor chip by using an amino-binding kit.

[0094] Binding of rhsFasL was assessed using multiple assay cycles. Each cycle was performed at a flow rate of 50 μl / min and consisted of the following steps: injection of 10 μL of...

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PUM

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Abstract

Human antibodies, preferably recombinant human antibodies that specifically bind to human Fas Ligand (hFasL) are disclosed. These antibodies have high affinity for hFasL, a slow off rate for hFasL dissociation and neutralize a Fas Ligand activity in vitro and in vivo. An antibody of the invention can be a full-length antibody or an antigen-binding portion thereof. The antibodies, or antigen-binding portions, of the invention are useful for neutralizing Fas Ligand activity, e.g., in a human subject suffering from a disorder in which hFas Ligand activity is detrimental. Nucleic acids, vectors and host cells for expressing the recombinant anti-hFasL human antibodies, and the methods for synthesizing the recombinant human antibodies are also encompassed by the invention.

Description

Background technique [0001] Fas ligand ("FasL") is a protein that has the activity of inducing apoptosis in cells expressing the Fas antigen ("Fas"). Apoptosis of cells expressing the Fas antigen is believed to be induced by the binding of Fas to FasL on the cell surface, resulting in the transmission of an apoptotic signal to the cell via the Fas antigen. The nucleic acid and protein sequences of FasL of human, mouse and rat origin are disclosed in US Patent 6,348,334 (incorporated herein by reference). [0002] Human Fas ligand ("hFasL") is a 40 kDa amino acid type II membrane-bound protein that belongs to the TNF family. Membrane-bound FasL can be cleaved by metalloproteases to produce soluble FasL, a predominantly non-covalently linked homotrimer (Mariani, et al., Eur. J. Immunol. 25:2303-7 (1995); Kayagaki, et al., J. Exp. Med. 182: 1777-83 (1995); Tanaka, et al., EMBO 14(6): 1129-35 (1995)). Soluble FasL exhibits less cytotoxicity than membrane-bound FasL (Nagata, Ann...

Claims

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Application Information

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IPC IPC(8): C07K16/00C12N15/13C12N15/63A61K39/395C12N15/09A61P1/00A61P1/04A61P1/16A61P3/10A61P9/00A61P9/10A61P11/00A61P13/12A61P17/02A61P19/02A61P21/04A61P25/00A61P29/00A61P31/04A61P31/12A61P31/14A61P31/16A61P31/18A61P31/20A61P35/00A61P37/02A61P37/06A61P43/00C07K16/28C12N1/15C12N1/19C12N1/21C12N5/10C12N15/12C12P21/08
CPCA61K2039/505C07K16/2875C07K2317/21C07K2317/56C07K2317/565C07K2317/76A61P1/00A61P1/04A61P1/16A61P11/00A61P13/12A61P17/02A61P19/02A61P21/04A61P25/00A61P29/00A61P31/04A61P31/12A61P31/14A61P31/16A61P31/18A61P31/20A61P35/00A61P37/02A61P37/06A61P43/00A61P9/00A61P9/10A61P3/10
Inventor J·S·兰卡斯特
Owner ELI LILLY & CO
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