Her2 binding proteins based on di-ubiquitin muteins

A protein-binding and diubiquitin-binding technology, applied in immunoglobulins, fusion polypeptides, drug combinations, etc., can solve problems such as complex molecular structures of monoclonal antibodies

Active Publication Date: 2018-03-27
NAVIGO PROTEINS GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, monoclonal antibodies have major disadvantages such as complex molecular structure, large size and challenging production methods
Additionally, treating the disease with currently available Her2-binding molecules is not effective in all patients and can have serious side effects

Method used

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  • Her2 binding proteins based on di-ubiquitin muteins
  • Her2 binding proteins based on di-ubiquitin muteins
  • Her2 binding proteins based on di-ubiquitin muteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Example 1. Identification of binding proteins

[0127] Library construction and cloning Two ubiquitin moieties (each containing seven randomized amino acid positions) were synthesized by triplet technology (MorphoSys Slonomics, Germany) to obtain a well-balanced amino acid distribution. A mixture of 19 amino acids encoding pre-made double-stranded triplets excluding cysteine ​​was used for the synthesis. The two ubiquitin moieties were directly linked in a head-to-tail direction (without a linker between the two ubiquitin moieties) to generate a protein of 152 amino acids with 14 randomized amino acid positions. The sequence of diubiquitin with 14 randomized positions is shown in SEQ ID NO: 3:

[0128]MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQXLXFAGKQLEDGRTLSDYNIQKESTLXLXLXXXAAMQIFVXTXTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDYNIXXXXXLHLVLRLRAA.

[0129] The 14 randomized amino acids correspond to positions 42, 44, 68, 70, 72, 73, 74, 82, 84, 138, 139, 140, 141...

Embodiment 2

[0137] Example 2. Expression and purification of Her2-binding proteins

[0138] Affilin molecules were cloned into expression vectors using standard methods known to those skilled in the art, purified and analyzed as described below. All Affilin proteins are expressed and highly purified by affinity chromatography and gel filtration. After affinity chromatography purification, the System and Superdex TM Size exclusion chromatography (SE HPLC or SEC) was performed on a 200 HiLoad 16 / 600 column (GE Healthcare). The column has a volume of 120ml and is equilibrated with 2CV. Samples were applied with purification buffer B at a flow rate of 1 ml / min. Fraction collection was started when the signal strength reached 10 mAU. After SDS-PAGE analysis, positive fractions were pooled and their protein concentration was quantified.

[0139] Further analysis included SDS-PAGE, SE-HPLC and RP-HPLC. Protein concentration was determined by absorbance measurement at 280 nm using the mo...

Embodiment 3

[0140] Example 3. Solubility Analysis of Her2 Binding Proteins

[0141] Supernatants and resuspended pellets were analyzed by NuPage Novex 4-12% Bis-Tris SDS gels and stained using Coomassie. Protein was recovered from the pellet by adding 8M urea. Her2 binding protein exhibits at least 80% soluble (SEQ ID NO:5,27,30,37,38), at least 90% soluble (SEQ ID NOs:6,20,23,28,34), at least 95% soluble expression (SEQ ID NO:7,9,10,11,22,29), 100% soluble (SEQ ID NO:8,12,13,14,15,16,17,18,19,21,25,26, 33,35,36) high solubility.

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Abstract

The present invention relates to new Her2 binding molecules based on di-ubiquitin muteins. The invention further refers to Her2 binding proteins optionally fused or conjugated to a moiety modulating pharmacokinetics or to a therapeutically or diagnostically active component. The invention further relates to the use of these Her2 binding proteins in medicine, preferably for use in the diagnosis ortreatment of cancer.

Description

technical field [0001] The present invention relates to novel Her2 binding molecules based on di-ubiquitin muteins. The present invention also relates to Her2 binding proteins, optionally fused or conjugated to moieties that regulate pharmacokinetics or to therapeutically or diagnostically active components. The invention also relates to the use of these Her2-binding proteins in medicine, preferably in the diagnosis or treatment of cancer. Background technique [0002] Increased expression of the membrane-bound receptor tyrosine kinase Her2 plays an important role in the development and progression of many breast cancers, but also in ovarian, gastric and uterine cancers, especially invasive cancers. Overexpression of this oncogene has been reported for malignancies, mainly those of epithelial origin, and is associated with cancer recurrence and poor prognosis. Three domain proteins (extracellular, transmembrane, intracellular tyrosine kinase domains) mediate cell prolifera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/32C07K14/47C12N15/62
CPCC07K14/47C07K2318/20C07K16/32A61P35/00C07K16/2863C07K2317/31C07K2317/92C07K2317/94C07K2319/00
Inventor 弗洛里安·塞泰莱玛德勒恩·茨瓦格玛加·格洛泽伊瓦·博塞-德内克埃里克·菲德勒乌尔里希·豪普茨
Owner NAVIGO PROTEINS GMBH
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