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Detection of a posttranslationally modified polypeptide by a bi-valent binding agent

a technology of bivalent binding agent and polypeptide, which is applied in the field of detection of posttranslationally modified polypeptides by bivalent binding agents, can solve the problems detection and quantification of secondarily modified polypeptides, and the discovery of complex modifications in one protein is recen

Inactive Publication Date: 2013-10-31
ROCHE DIAGNOSTICS OPERATIONS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text reveals that the method used in this invention allows for flexibility in the length of the linker, which is beneficial.

Problems solved by technology

However, very complicated modifications in one protein are lately discovered in many processes.
Detection and quantitation of a secondarily modified polypeptide, however, requires sophisticated tools and techniques.
The immunological detection of a posttranslationally modified polypeptide has consistently turned out to be rather difficult.
Various types of problems may be encountered.
It may be difficult to obtain a required immunogen in sufficient purity and quantity.
Especially when there is a need for an antibody of highly reproducible, consistent quality, e.g. a monoclonal antibody, it may turn out very demanding to obtain such an antibody.
However, many binding agents generated by routine procedures show cross-reactions to other polypeptides with the same kind of posttranslational modification, do not exhibit the required affinity to the epitope recognized and / or show cross-reactivity to the non-modified polypeptide.

Method used

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  • Detection of a posttranslationally modified polypeptide by a bi-valent binding agent
  • Detection of a posttranslationally modified polypeptide by a bi-valent binding agent
  • Detection of a posttranslationally modified polypeptide by a bi-valent binding agent

Examples

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Effect test

example 1

Bi-Valent Binding Agent to Troponin T

[0283]1.1 Monoclonal Antibodies and Fab′-Fragments

[0284]Two monoclonal antibodies binding to human cardiac Troponin T at different, non-overlapping epitopes, epitope A′ and epitope B′, respectively, were used. Both these antibodies are used in the current Roche Elecsys™ Troponin T assay, wherein Troponin T is detected in a sandwich immuno assay format.

[0285]Purification of the monoclonal antibodies from culture supernatant was carried out using state of the art methods of protein chemistry.

[0286]The purified monoclonal antibodies are protease digested with either pre-activated papain (anti-epitope A′ MAb) or pepsin (anti-epitope B′ MAb) yielding F(ab′)2 fragments that are subsequently reduced to Fab′-fragments with a low concentration of cysteamin at 37° C., i.e. A and B, respectively, in Formula I (A-a′:a-S-b:b′-B). The reaction is stopped by separating the cysteamin on a Sephadex G-25 column (GE Healthcare) from the polypeptide-containing part ...

example 2

Bi-Valent Binding Agent to Phosphorylated IGF-1R

[0325]2.1 Monoclonal Antibody Development (mAb 8.1.2, mAb 1.4.168 and mAB 30.4.33)

[0326]a) Immunization of Mice

[0327]BALB / C mice are immunized at week 0, 3, 6 and 9, respectively. Per immunization 100 μg of the conjugate comprising the phosphorylated peptide pIGF-1R (1340-1366) (SEQ ID NO:11) is used. This peptide had been phosphorylated at tyrosine 1346 (=1346-pTyr) and coupled to KLH via the C-terminal cysteine (=Aoc-Cys-MP-KLH-1340) to yield the conjugate used for immunization. At weeks 0 and 6, respectively, the immunization is carried out intraperitoneally and at weeks 3 and 9, respectively, subcutanuosly at various parts of the mouse body.

[0328]b) Fusion and Cloning

[0329]Spleen cells of immunized mice are fused with myeloma cells according to Galfre G., and Milstein C., Methods in Enzymology 73 (1981) 3-46. In this process ca 1×108 spleen cells of an immunized mouse are mixed with 2×107 myeloma cells a(P3×63-Ag8653, ATCC CRL1580)...

example 3

Bi-Valent Binding Agent to Phosphorylated HER3

[0421]The receptor tyrosine kinase family of HER proteins consists of four members: HER1, HER2, HER3 and HER4. Upon ligand binding, the receptors dimerize as homo- or heterodimers in various ways to trigger different signal transduction pathways, depending on the ligand and the expression levels of each of the four family members. For example, HER3 undergoes a conformational shift when it is bound to its ligands Neuregulin1 (NRG1) or Neuregulin2 (NRG2) and the HER3 dimerization domain is exposed and it can interact with other HER receptors. Upon dimerization, HER3 becomes phosphorylated. In this example, we developed a dual binder to detect the phosphorylated form of HER3.

[0422]3.1 Monoclonal Antibody Development (mAb 7.2.32 and mAb 4.1.15)

[0423]a) Immunization of Mice

[0424]Balb / c and NMRI mice are immunized with HER3(1243-1267)[KLH-MP-Cys-UZU-1243]amide or pHER3(1283-1295)[pTyr1289; KLH-MP-Cys-UZU-1283]amide. The initial immunization do...

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Abstract

A bi-valent binding agent having a first monovalent binder that binds to a polypeptide epitope of a target polypeptide, a second monovalent binder that binds to a posttranslational polypeptide modification on the target polypeptide and a linker. Further disclosed are methods for the detection of a posttranslationally modified target polypeptide, for making the disclosed bi-valent binding agent, and for use of the disclosed bi-valent binding agent in histological staining procedures.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of International Application No. PCT / EP2011 / 073560, filed Dec. 21, 2011, which claims the benefit of European Patent Application No. 10196687.7, filed Dec. 23, 2010, and European Patent Application No. 11173832.4, filed Jul. 13, 2011, the disclosures of which are hereby incorporated by reference in their entirety.SEQUENCE LISTING[0002]The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 19, 2013, is named SEQUENCE_LISTING—27204US.txt, and is fifteen thousand eight hundred and thirteen bytes in size.BACKGROUND OF THE DISCLOSURE[0003]The primary structure of a polypeptide, i.e. its sequence, is determined by the nucleic acid coding for it. However, knowing the primary structure of a polypeptide is only part of the story. Many polypeptides—estimates range from 50 to 9...

Claims

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Application Information

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IPC IPC(8): C07K16/46
CPCC07K16/468G01N33/531
Inventor GERG, MICHAELHEINDL, DIETERKLEIN, CHRISTIANMERTENS, ALFREDSCHMID, VOLKERSCHRAEML, MICHAELSOUKUPOVA, MONIKATACKE, MICHAEL
Owner ROCHE DIAGNOSTICS OPERATIONS INC
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