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Tnf-alpha antigen-binding proteins

A protein and antigen binding technology, applied in the direction of anti-animal/human immunoglobulin, immunoglobulin, anti-cytokine/lymphokine/interferon immunoglobulin, etc.

Inactive Publication Date: 2015-11-11
GLAXOSMITHKLINE INTPROP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many of these modifications have been shown to vary and sometimes conversely result in different antibodies

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0161] Example 1: Cloning of antibody expression vector

[0162] DNA expression constructs encoding the variable heavy (VH) and variable light (VL) domains of the anti-TNFα antibody were previously prepared de novo and included restriction sites for cloning into mammalian expression vectors. Both the heavy and light chain variable region sequences were sequence optimized for expression in mammalian cells (for methodology see WO2009024567 and Kotsopoulou et al. J Biotechnol (2010) 146:186-193). Information describing the heavy and light chain variable region sequences can be found in US Pat. No. 6,090,382. To generate the constructs used in this study, variable heavy domain (VH) sequences were amplified using PCR. PCR primers contain HindIII and A Spel restriction site to form the VH domain framework containing the signal sequence for cloning into the pTT mammalian expression vector containing the human γ1 constant region. Similarly, the VL domain sequence was amplified b...

Embodiment 2

[0164] Example 2: Engineering Transformation of the Fc Region

[0165] Forward and reverse priming primers were used to introduce modifications (M252Y / S254T / T256E and T250Q / M428L) in the human γ1 constant region of the plasmid encoding the heavy chain of pascolizumab (anti-IL-4 antibody) using the Quikchange protocol (Promega).

[0166] A PCR fragment encoding the VH domain of an anti-TNFα antibody was generated using a previously constructed codon-optimized vector as a template as described in Example 1 above. The fragment obtained using HindIII and SpeICloned into the pTT expression vector containing the modified human gamma 1 constant region as described in the paragraph above. The plasmid encoding the heavy chain of anti-TNFα antibody with M252Y / S254T / T256E modification was named SJC324. The plasmid encoding the heavy chain with T250Q / M428L modification was named SJC323.

[0167] Forward and reverse priming primers were used to introduce modifications in the human γ1 c...

Embodiment 3

[0168] Example 3: Antibody expression in HEK2936 cells using pTT episomal vector

[0169] Expression plasmids encoding the above heavy and light chains were transiently co-transfected into HEK2936E cells. Expressed antibodies were purified from supernatants by affinity chromatography using 1 mL HiTrap protein A columns (GE Healthcare). Table 1 below shows a list of the antibodies produced.

[0170] Some antibodies are also expressed in CHO cells using a different set of expression vectors. See Examples 13, 14 and 15 for descriptions of use in molecular biology, expression and purification.

[0171] Table 1: List of expressed antibodies

[0172]

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Abstract

The present invention provides liquid formulations comprising antigen binding proteins which bind specifically to TNF-alpha and histidine buffer. For example novel variants of anti-TNF antibodies such as adalimumab which show increased binding to the FcRn receptor or increased half life compared to adalimumab. Also provided are compositions comprising the antigen binding proteins and uses of such compositions in treatment of disorders and disease.

Description

technical field [0001] The present invention relates to novel variants of anti-TNF antibodies and formulations of these antigen binding proteins. Background of the invention [0002] FcRn, also known as the neonatal Fc receptor in adult mammals, plays an important role in maintaining serum antibody levels by acting as a protective receptor that binds and rescues IgG isotype antibodies from degradation. IgG molecules are endocytosed by endothelial cells, and if they bind FcRn, are recycled out into circulation. In contrast, IgG molecules that do not bind FcRn enter cells and are targeted to the lysosomal pathway, where they are degraded. [0003] The nascent FcRn receptor is thought to be involved in antibody clearance and transcytosis across tissues (see, Junghans R.P (1997) Immunol. Res 16.29-57 and Ghetie et al. (2000) Annu. Rev. Immunol. 18, 739-766). [0004] WO9734631 discloses compositions comprising mutant IgG molecules having increased serum half-life and having at...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24A61K39/395
CPCC07K16/241A61K39/39591A61K31/52A61P1/04A61P17/06A61P19/02A61P29/00A61P43/00A61K39/39566A61K2039/55511C07K2317/92
Inventor G.科罗茨S.莫拉尔-米特里卡
Owner GLAXOSMITHKLINE INTPROP
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