Anti-Fas antibodies

Inactive Publication Date: 2003-09-11
SANKYO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

0059] It is an object of the present invention to provide an anti-Fas antibody, or similar m

Problems solved by technology

Monoclonal antibodies that specifically bind to human Fas and which also have apoptosis-inducing activity are known, but none of them is capable of binding

Method used

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Examples

Experimental program
Comparison scheme
Effect test

reference example 2

Immunization of Mice and Preparation of Hybridoma

[0420] (2-1) Immunization

[0421] A sample of 1 ml of the crude human Fas fusion protein solution obtained in Reference Example 1 above (total protein: 100 .mu.g) was taken and, to this, were added 25 .mu.l of 2N HCl , 250 .mu.l of 9% w / v potash alum (final concentration: 1.1% w / v) and 25 .mu.l of 2N NaOH. The resulting mixture was adjusted to a pH of between about 6.5 and 7.0 by the addition of about 120 .mu.l of an aqueous solution of 10% (w / v) sodium hydrogencarbonate and left to stand at room temperature for about 30 minutes. After this time, 200 .mu.l of killed Bordetella pertussis (Wako Pure Chemical Industries, Ltd.; 1.2.times.10.sup.11 cells / ml) were added to the mixture in order to activate the T cells, and the mixture was administered intraperitoneally to a Fas knock-out mouse. The mouse used was prepared in accordance with the method described by Senju et al. [c.f. Senju, S. et al., (1996), International Immunology, 8, 423]. ...

reference example 3

Purification of HFE7A Monoclonal Antibody

[0437] The mouse-mouse hybridoma HFE7A obtained in Reference Example 2 (FERM BP-5828) was grown to a cell density of 1.times.10.sup.6 cells / ml by incubation in 1 l of ASF medium, containing 10% v / v FCS, at 37.degree. C. under 5% v / v CO.sub.2. The culture was then centrifuged (1,000 r.p.m., 2 minutes) and the supernatant discarded. The cell pellet was washed once with serum-free ASF medium, suspended in 1 l of serum-free ASF medium and incubated for 48 hours at 37.degree. C. under 5% v / v CO.sub.2. After this time, the culture was centrifuged (1,000 r.p.m. for 2 minutes) to recover the supernatant. This supernatant was then placed in a dialysis tube (exclusion m.w.: 12,000-14,000; Gibco BRL), and dialyzed against 10 volumes of 10 mM sodium phosphate buffer (pH 8.0). Partial purification of IgG from the inner solution was achieved using a high performance liquid chromatography apparatus (FPLC system; Pharmacia) under the following conditions:

[04...

reference example 4

cDNA Cloning

[0445] (4-1) Preparation of Poly(A).sup.+ RNA

[0446] Cells of the mouse-mouse hybridoma HFE7A (FERM BP-5828), obtained in Reference Example 2, were grown to a cell density of 1.times.10.sup.6 cells / ml in 1 l of ASF medium supplemented with 10% v / v FCS at 37.degree. C. under 5% v / v CO.sub.2. These cells were harvested by centrifugation and lyzed in the presence of guanidinium thiocyanate solution [4 M guanidinium thiocyanate, 1% v / v Sarcosyl, 20 mM EDTA, 25 mM sodium citrate (pH 7.0), 100 mM 2-mercaptoethanol, 0.1% v / v Antifoam A] and the lysate was recovered. Isolation of poly(A).sup.+ RNA was performed essentially as described in "Molecular Cloning A Laboratory Manual" [c.f. Maniatis, T., et al., (1982), pp. 196-198]. More specifically, the procedure was as follows.

[0447] The recovered cell lysate was sucked into and exhausted from a 10 ml-syringe equipped with a 21-gauge needle, several times. The cell lysate was layered over 3 ml of an aqueous solution of 5.7 M cesium ...

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Abstract

Anti-Fas antibodies which are cross-reactive with mouse and human Fas and are useful in the treatment of conditions attributable to abnormalities in the Fas/Fas ligand system.

Description

[0001] This application is a continuation-in-part application of U.S. application Ser. No. 09 / 053,583, filed Apr. 1, 1998 and U.S. application Ser. No. 09 / 408,646, filed Sep. 30, 1999; the entire contents of said applications are hereby incorporated by reference herein.[0002] The present invention relates to antibodies and fragments thereof, especially humanized antibodies, recognizing the Fas antigen, to DNA encoding all or part of such an antibody, and to agents, comprising such antibodies, for the prophylaxis and / or treatment of conditions arising from abnormalities in the Fas / Fas ligand system.[0003] The physiological death of cells in a living organism in the natural course of events is known as apoptosis, and is distinguished from the pathological death of cells, i.e. necrosis [c.f. Kerr et al., (1972), Br. J. Cancer, 26, 239 et seq.]. Apoptosis is an example of programmed cell death, which is where certain cells are programmed, in advance, to die in a living organism in the n...

Claims

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Application Information

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IPC IPC(8): A61K38/00C07K14/705C07K16/28
CPCA61K38/00A61K2039/505C07K14/70575C07K2317/34C07K2317/24C07K2319/00C07K16/2878
Inventor SERIZAWA, NOBUFUSAICHIKAWA, KIMIHISAOHSUMI, JUNOHTSUKI, MASAHIKOHARUYAMA, HIDEYUKITAKAHASHI, TOHRUYOSHIDA, HIROKOSHIRAISHI, AKIOYONEHARA, SHINNAKAHARA, KAORITAMAKI, IKUKO
Owner SANKYO CO LTD
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