Immunoglobulins

An antibody, therapeutic antibody technology, applied in the field of immunoglobulin, which can solve the problems of low potency, slow onset, and poor tolerance

Inactive Publication Date: 2007-07-11
GLAXO GROUP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Low potency levels, slow onset, toxicity, poor tolerability, and increased resistance over time hamper the use of these treatments, including methotrexate (MTX), sulfasalazine, gold, and leflunomide

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0332] Examples 1-6 relate to the production and engineering of antibody 15E10. Example 7 relates to the production and engineering of antibody 10D3.

[0333] 1. Production of monoclonal antibodies

[0334] Monoclonal antibodies are generally produced from hybridomas as described in E Harlow and D Lane, Antibodies a Laboratory Manual, Cold Spring Harbor Laboratory, 1988 . Fusion of mouse myeloma cells with B lymphocytes from mice immunized with the target antigen. The myeloma fusion partner immortalizes the hybridoma cells while providing antibody production capacity by the B lymphocytes.

[0335] Four SJL mice were immunized by intraperitoneal injection of a suspension of glycosylated human OSM (hOSM) produced in CHO cells in RIBI adjuvant (Sigma). After 2 weeks, mice were boosted with hOSM alone, followed, after a further 2 weeks, with hOSM neutralized with an anti-site III monoclonal antibody (OM4 / 11.17; OSM:Mab 1:1.5 wt:wt). Mice were boosted with hOSM to direct the ...

Embodiment 7

[0693] Example 7 - Antibody 10D3

[0694] 7.1. Production of monoclonal antibodies

[0695] Hybridoma 10D3 was generated as detailed in Example 1 above.

[0696] 7.2. Cloning of the 10D3 variable region

[0697] Total RNA was extracted from clone 10D3 hybridoma cells, and cDNAs for the variable domains of the heavy and light chains were generated by reverse transcription using murine leader-specific primers and antibody constant regions of the predetermined isotype (IgG1 / κ). The cDNAs of the heavy and light chain variable domains were then cloned into vector pCR2.1 for sequencing.

[0698] 7.2.1 RNA extraction

[0699] 10 of 10D3 were cloned from hybridomas using Promega's SV Total RNA Isolation System following the manufacturer's instructions. 6 Total RNA was extracted from the cell pellet.

[0700] 7.2.2 Reverse transcription

[0701] RNA was reverse transcribed using primers specific for the murine leader sequence and murine IgG gamma 2a / kappa constant regio...

Embodiment 8

[0885] Example 8. gp130 inhibition ELISA

[0886] OSM sequentially binds gp130 and either the OSM receptor or the LIF receptor. The experiments described here allow the measurement of OSM (eg hOSM) bound to gp130 on ELISA plates. In addition, this experiment allows measuring the inhibition of OSM binding to the gp130 receptor by antibodies directed against OSM site II.

[0887] 8.1 Materials

[0888] 1. Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A)

[0889] 2. Human gp130-Fc 100μg / ml (R&D Systems, 671-GP-100)

[0890] 3. PBS

[0891] 4.BSA (Sigma A7030)

[0892] 5. Human recombinant OSM 10 μg / ml (R&D Systems, non-glycosylated)

[0893] 6. Biotinylated anti-human OSM 50 μg / ml (R&D Systems, BAF295)

[0894] 7. Streptavidin HRP (Amersham RPN4401)

[0895] 8. 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma)

[0896] 9. Sulfuric acid

[0897] 10. Tween 20 (Sigma P7949)

[0898] 8.2 Preparation of reagents

[0899] 1. Plate preparation: Human gp130-Fc w...

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PUM

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Abstract

The present invention concerns immunoglobulins, such as antibodies, which specifically bind Oncostatin M (OSM), particularly human OSM (hOSM) and modulate the interaction between OSM and gp130. In typical embodiments, OSM is glycosylated. The invention also concerns antibodies that modulate the interaction between both Site II and Site III of OSM and their respective interacting partners. Further disclosed are pharmaceutical compositions, screening and medical treatment methods.

Description

field of invention [0001] The present invention relates to immunoglobulins which specifically bind Oncostatin M (OSM), in particular human OSM (hOSM). More specifically, the invention relates to antibodies that specifically bind hOSM. The invention also relates to methods of treating diseases or disorders using said immunoglobulins, pharmaceutical compositions containing said immunoglobulins, and methods of production. Other aspects of the invention will be apparent from the description below. Background of the invention [0002] Oncostatin M is a 28KDa glycoprotein belonging to the interleukin 6 (IL-6) family of cytokines, which includes IL-6, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophic Protein-1 (CT-1) and cardiotrophin-1-like cytokines (see Kishimoto T et al. (1995) Blood 86:1243-1254), which share the gp130 transmembrane signaling receptor (see Taga T and Kishimoto T (1997) Annu. Rev. Immunol. 15:797-819). OSM was originally di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24G01N33/53A61K39/395
Inventor J·H·埃利斯A·埃安－杜瓦尔V·杰马彻维斯基C·普鲁普顿N·T·拉普森M·R·韦斯特
Owner GLAXO GROUP LTD
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