Polypeptides having binding affinity for HER2

A technology of affinity and structural domain, applied in the field of new peptides, can solve problems such as bone marrow suppression

Active Publication Date: 2006-08-09
阿菲博迪生物技术公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Radioimmunotherapy with isotope-conjugated antibodies also leads to myelosuppression

Method used

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  • Polypeptides having binding affinity for HER2
  • Polypeptides having binding affinity for HER2
  • Polypeptides having binding affinity for HER2

Examples

Experimental program
Comparison scheme
Effect test

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. Library panning and clone selection

[0111] 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 A Z protein variant (Affibody  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 Selection of Affibodies that bind antigens  molecular. After these four rounds of selection rounds, a total of 91 clones were selected for ph...

Embodiment 2

[0124] Z HER2A Binding to HER2 expressing cells

[0125] cell culture

[0126] 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.

[0127] Radiolabeled

[0128] 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 re...

Embodiment 3

[0133] Expression and Characteristic Analysis of HER2 Binding Polypeptide Dimer

[0134] DNA vector construction and protein production

[0135] 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. The DNA Sequencer ABI Prism  The introduced Z was confirmed by DNA sequencing on the 3700 Analyzer (Applied Biosystems, Foster City, CA). HER2A fragment. Escherichia coli strain RR1ΔM15 (Rther, Nucleic Acids Res 10: 5765-5772 (1982)) was used as the bacterial host during the cloning process. 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 chro...

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Abstract

A polypeptide is provided, which has a binding affinity for HER2 and which is related to a domain of staphylococcal protein A (SPA) in that the sequence of the polypeptide corresponds to the sequence of the SPA domain having from 1 to about 20 substitution mutations. Nucleic acid encoding the polypeptide, as well as expression vector and host cell for expressing the nucleic acid, are also provided. Also provided is the use of such a polypeptide as a medicament, and as a targeting agent for directing substances conjugated thereto to cells overexpressing HER2. Methods, and kits for performing the methods, are also provided, which methods and kits rely on the binding of the polypeptide to HER2.

Description

technical field [0001] The present invention relates to a novel polypeptide binding to human epidermal growth receptor factor 2 (hereinafter referred to as HER2). The polypeptide is related to a domain of staphylococcal protein A (SPA), wherein the sequence of the polypeptide corresponds to the sequence of the SPA domain, with at least one substitution mutation. The present invention also relates to the use of the HER2-binding polypeptide as a medicine, more particularly to its use in the preparation of a medicine for treating cancer characterized by overexpression of HER2. Background technique [0002] Affibody  molecular [0003] This molecule is related to protein Z, derived from the B domain of staphylococcal protein A (SPA) (Nilsson B et al (1987) Protein Engineering 1, 107-133), and was derived from this class of molecules by using different interacting target molecules (See WO95 / 19374; WO00 / 63243; Nord K et al (1995) Prot Eng 8:601-608; Nord K et al (1997) Nature ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31A61P35/00C07K
Inventor 约根·卡尔松斯特凡·斯托尔托弗·埃里克松埃琳·贡内里乌松弗雷德里克·尼尔松
Owner 阿菲博迪生物技术公司
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