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GITR binding molecules and uses therefor

A technique of combining molecules and compositions, which is applied in the field of GITR binding molecules and their uses, and can solve problems such as inability to protect cells from apoptosis

Inactive Publication Date: 2008-07-09
GITR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

GITR expression protects T cells from apoptosis induced by anti-CD3 antibody treatment, but not by other apoptotic agents

Method used

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  • GITR binding molecules and uses therefor
  • GITR binding molecules and uses therefor
  • GITR binding molecules and uses therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0386] Example 1: Isolation and purification of 6C8

[0387] The 6C8 antibody is IgG2b, kappa type. Purification of this antibody revealed the presence of two heavy chains ( figure 1 ). This can be due to taking one of the two glycosylations or contamination with another antibody (Ab). Size exclusion chromatography showed only one peak ( figure 2 ).

[0388] The 6C8 antibody was purified as follows:

[0389] 1. Wash 20ml protein G (Pharmacia HR10 / 30) with 5 column volumes of dPBS

[0390] 2. Load 1L (1st round) or 2L (2nd round) hGITR (6C8) supernatant

[0391] 3. Wash with 10 column volumes of dPBS

[0392] 4. Elute directly into 1M Tris (20-25% v; v) with 100 mM citrate, pH 2.8

[0393] 5. Strip with 100mM citrate (pH 2.8), 0.3M NaCl

Embodiment 2

[0394] Example 2: Identification of 6C8

[0395] 6C8 antibody and cells transfected with GITR-L-M ( image 3 ) and activated PBL ( Figure 4 ) combined. Saturation curves of biotin-labeled anti-GITR on activated lymphocytes suggest good relative affinity ( Figure 5 ).

[0396] The 6C8 antibody has co-stimulatory activity on T lymphocytes activated with suboptimal anti-CD3 ( Figure 6 ). Costimulation of this antibody did not reach the same level as CD28, but was comparable to the anti-GITR commercial product (R&D).

[0397] The 6C8 antibody did not induce apoptosis in activated lymphocytes (Figure 7). Lymphocytes were activated with PHA, and antibodies were added 3 days later. Compared to YTH 655, an anti-human CD2 known to induce apoptosis in activated lymphocytes, 6C8 did not increase apoptosis in activated T lymphocytes.

[0398] 6C8 antibody did not block the primary mixed lymphocyte reaction (MLR) ( Figure 8 ). TRX1 (anti-human CD4) was used as a positive cont...

Embodiment 3

[0399] Example 3: 6C8 antibody counteracts the inhibitory effect of regulatory T cells on T effector cells

[0400] Antibody 6C8 was able to block the suppressive effect induced by regulatory T cells ( Figure 9 ). CD4+ / CD25+ cells were added to CD4+ / CD25- cells at different ratios. The cells were stimulated with anti-CD3 and anti-CD28 bound to microwell plates. When the ratio is 1:1, CD4+ / CD25+ cells are able to eliminate the proliferation of CD4+ / CD25- cells. Addition of 6C8 to the cultures blocked this inhibitory effect in a dose-dependent manner.

[0401] When T cells were stimulated by anti-CD3 alone (not co-stimulation with anti-CD28), CD4+ / CD25+ cells were added to CD4+ / CD25- cells, no inhibitory effect was observed, and indeed the anti-GITR antibody was has a weak co-stimulatory effect ( Figure 10 ).

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Abstract

The present invention provides binding molecules that specifically bind to GITR, e.g., human GITR (hGITR), on T cells and dendritic cells. Binding molecules of the invention are characterized by binding to hGITR with high affinity, in the presence of a stimulating agent, e.g., CD3, are agonistic, and abrogate the suppression of Teff cells by Treg cells. Various aspects of the invention relate to binding molecules, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such binding molecules. Methods of using a binding molecule of the invention to detect human GITR or to modulate human GITR activity, either in vitro or in vivo, are also encompassed by the invention.

Description

[0001] related application [0002] This application is entitled to U.S. Provisional Patent Application 60 / 665,322, filed March 25, 2005, entitled "GITR Binding Molecules and Uses Thereof," and to U.S. Provisional Patent Application No. 60 / 665,322, filed June 3, 2005, entitled "GITR Binding Molecules and Uses Thereof." Priority to US Provisional Patent Application 60 / 687,265, both applications are hereby incorporated by reference in their entirety. Background technique [0003] Members of the tumor necrosis factor and TNF receptor (TNFR) superfamily regulate a variety of biological functions, including cell proliferation, differentiation and survival. Using differential display to identify T cell mRNAs induced by the synthetic glucocorticoid dexamethasone, Nocentini et al. ((1997) Proc. Member mouse cDNA. Its corresponding gene is named GITR, which stands for Glucocorticoid-Inducible TNFR Family-Related Gene (also known as TNFRSF18). Like other TNFRs, the extracellular doma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00A61P31/00
CPCY02A50/30
Inventor L·M·史密斯格雷齐纳·齐曼斯卡保罗·波纳思迈克尔·罗森韦格乔斯·F·庞特
Owner GITR
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