Cysteine engineered fibronectin type iii domain binding molecules

A technology of cysteine ​​and fibronectin, applied in the direction of peptide/protein components, animal/human proteins, anti-animal/human immunoglobulins, etc.

Inactive Publication Date: 2019-04-26
JANSSEN BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, modification in cysteine ​​thiol groups obtained by mutation of various amino acid residues of the protein to cysteine ​​amino acids can be problematic, especially at unpaired (free Cys) residues or relative In the case of those residues susceptible to reaction or oxidation

Method used

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  • Cysteine engineered fibronectin type iii domain binding molecules
  • Cysteine engineered fibronectin type iii domain binding molecules
  • Cysteine engineered fibronectin type iii domain binding molecules

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0365] Example 1: Construction of Tencon Library

[0366] Tencon (SEQ ID NO:1) is an immunoglobulin-like scaffold type III fibronectin (FN3) domain designed from the consensus sequence of fifteen FN3 domains of human tenascin-C (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012; US Patent Publication 2010 / 0216708). The crystal structure of Tencon shows six surface exposed loops connecting seven β-strands. The selected residues within these loops or each loop can be randomized to construct a type III fibronectin (FN3) domain library that can be used to select novel molecules that bind to specific targets.

[0367] Tencon:

[0368] Lpapknlvvsevtedslrlswtapdaafdsfliqyqesekvgeainltvpgsersydltglkpgteytvsiygvkgghrsnplsaeftt (SEQ ID NO 1):

[0369] TCL1 library construction

[0370] The library TCL1 designed to randomize only the FG loop of Tencon (SEQ ID NO:1) was constructed for use with the cis-display system (Jacobs et al., Protein Engineering, Design, and Sele...

Embodiment 2

[0388] Example 2: Selection of type III fibronectin (FN3) domain that binds EGFR and inhibits EGF binding

[0389] Library screening

[0390] The cis display was used to select EGFR binding domains from TCL1 and TCL2 libraries. The recombinant human extracellular domain of EGFR fused to IgG1 Fc (R&D Systems) was biotinylated using standard methods and used for panning (residues 25-645 of the full-length EGFR of SEQ ID NO: 73). For in vitro transcription and translation (ITT), 2-6 µg of library DNA are incubated with 0.1 mM intact amino acids, 1X S30 premix fraction and 30 µL S30 extract (Promega) in a total volume of 100 µL and incubated at 30°C . After 1 hour, 450 μL of blocking solution (PBS pH 7.4, supplemented with 2% bovine serum albumin, 100 μg / mL herring sperm DNA, and 1 mg / mL heparin) was added, and the reaction was incubated on ice for 15 minutes. EGFR-Fc was assembled by mixing recombinant human EGF (R&D Systems) and biotinylated recombinant EGFR-Fc in blocking solutio...

Embodiment 3

[0408] Example 3: Characterization of EGFR-binding FN3 domain that inhibits EGF binding

[0409] Large-scale expression, purification and endotoxin removal

[0410] The 9 FN3 domains shown in Table 4 are enlarged to provide more materials for detailed characterization. Using an overnight culture containing each EGFR-binding FN3 domain variant, the overnight culture was diluted 1 / 80 in 0.8 L Terrific broth supplemented with 100 µg / mL ampicillin and inoculated into fresh medium, Incubate with shaking at 37°C. When the optical density at 600 nm reached about 1.2-1.5 by adding IPTG to a final concentration of 1 mM, the culture was induced and the temperature was lowered to 30°C. After 4 hours, the cells were collected by centrifugation, and the cell pellets were stored at -80°C until needed.

[0411] For cell lysis, the thawed pellets were thawed at 5mL BugBuster per gram of pellets ® Resuspended in supplement with 25U / mL Benzonase ® (Sigma-Aldrich) and 1kU / mL rLysozyme ™ (Novagen...

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Abstract

Cysteine engineered monospecific and bispecific EGFR and / or c-Met FN3 domain containing molecules comprising one or more free cysteine amino acids are prepared by mutagenizing a nucleic acid sequenceof a parent molecule and replacing one or more amino acid residues by cysteine to encode the cysteine engineered FN3 domain containing monospecific or bispecific molecules; expressing the cysteine engineered FN3 domain containing molecules; and recovering the cysteine engineered FN3 domain containing molecule, isolated cysteine engineered monospecific or bispecific FN3 domain containing moleculesmay be covalently attached to a detection label or a drug moiety and used therapeutically.

Description

Technical field [0001] The present invention relates to binding molecules engineered with cysteine ​​residues, and methods of making them and using them. More specifically, the present invention relates to type III fibronectin (FN3) domain molecules, which can bind to cysteine-engineered EGFR and / or c-Met. Background technique [0002] Epidermal growth factor receptor (EGFR or ErbBl or HER1) is a 170kDa transmembrane glycoprotein, which is encoded by the c-erbBl proto-oncogene. EGFR is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases (RTK), which includes HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). These RTKs share a homologous structure consisting of a ligand-binding extracellular domain (ECD), a single transmembrane domain, and an intracellular domain, which includes a catalytic kinase domain and a C-terminal tail. EGFR signal transduction is triggered by ligand binding and then induces conformational changes, dimerization and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/00A61K38/39C07K14/78C07K16/28C12N15/09
CPCC07K14/78C07K14/71C07K14/82C07K2319/31C07K2319/30C12N15/64C12N15/09C12N15/63C07K2319/74C07K2319/00C07K19/00A61K38/39A61K31/25C07K2318/20C07D207/448
Inventor M.安德森R.阿塔M.迪姆S.戈德伯格L.玄S.雅各布斯A.金D.克莱因S.摩尔斯K.奥奈尔K.皮查
Owner JANSSEN BIOTECH INC
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