Polypeptides having binding affinity for HER2
An affinity, functional peptide technology, applied in the field of new peptides, can solve problems such as myelosuppression
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Embodiment 1
[0109] Selection and Research of HER2 Binding Polypeptides
[0110] In this experiment, multiple HER2-binding polypeptides of the invention were selected from a library containing many different SPA domain-related polypeptides and subsequently characterized.
[0111] Library panning and clone selection
[0112] A combinatorial phage display library has been substantially prepared by Nord K et al (1995, supra). The library pool (pool) used in this study contains 8.7×10 8 Z protein variants ( molecule) having random amino acid residues at positions 9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 28, 32, and 35. Using biotinylated human HER2 extracellular domain (HER2-ECD) as target (recombinant human HER2 extracellular domain, amino acids 238-2109, provided by Fox Chase Cancer Center, Philadelphia, USA) in four panning cycles Antigen-binding molecular. After these four rounds of selection rounds, a total of 91 clones were selected for phage ELISA to analyze their HER2 binding act...
Embodiment 2
[0126] Z HER2A Binding to HER2 expressing cells
[0127] cell culture
[0128] The human breast cancer cell line SKBR-3 was purchased from ATCC (ATCC#HTB-30), and it is known that each cell of this cell line expresses about 2×10 6 HER2 molecules. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and PEST (100 IU / ml penicillin and 100 μg / ml streptomycin), all purchased from Biochrom KG (Berlin, Germany). Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2 and seeded in 3 cm petri dishes three days before the start of the experiment.
[0129] Radiolabeled
[0130] Labeling precursor, N-succinimidyl p-(trimethyl-stannyl)benzoate (SPMB) according to Orlova et al, Nucl Med Biol 27 : 827-835 (2000) method is prepared, the SPMB of 5 μ g is added to containing 5MBq 125 I in 5% acetic acid solution. 40 μg of an aqueous solution of Chloramine-T (Sigma, St. Louis, MO) was added to initiate the reaction. The rea...
Embodiment 3
[0135] Expression and Characteristic Analysis of HER2 Binding Polypeptide Dimer
[0136] DNA vector construction and protein production
[0137] Select a new affibody ligand as above, His 6 -Z HER2A , which has an affinity for the HER2 receptor. by coding Z HER2A The gene fragment of the polypeptide is subcloned into His 6 -Z HER2A to construct the dimer Z in the expression vector of HER2 Variants. DNA sequencer The introduced Z was confirmed by DNA sequencing on the 3700 Analyzer (Applied Biosystems, Foster City, CA). HER2A fragment. The Escherichia coli strain RR1ΔM15 ( Nucleic Acids Res 10:5765-5772 (1982)) as a bacterial host. The resulting vector encodes a dimeric Z under the control of the T7 promoter (Studier et al, Methods Enzymol 185:60-89 (1990)). HER2 Variant, (Z HER2A ) 2 , with N-terminal hexahistidyl (His 6 ) tag fusions that allow purification by immobilized metal ion affinity chromatography (IMAC).
[0138] Dimer Z HER2 The variant is express...
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