Antigen derived from extracellular domain of multi-transmembrane protein and uses thereof
Inventors
Shin, Young Kee • Kim, Young-Deug • Choi, Jun Young • Lee, Myung Seok
Assignees
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Publication Number
US-9751941-B2
Publication Date
2017-09-05
Expiration Date
Abstract
A multi-transmembrane protein antigen includes a polypeptide corresponding to an extracellular loop of the multi-transmembrane protein, the N-terminal and C-terminal of the polypeptide being fixed on a solid substrate or the N-terminal and C-terminal being attached to both ends of a linker to form a cyclic structure, an antibody specifically binding to the antigen or an antigen-binding fragment thereof, and a method for screening an antibody specifically binding to the antigen. The present invention may be usefully employed for effective production of antibodies for multi-transmembrane proteins that play important roles in disease-related phenomena such as cell signaling.
Core Innovation
The invention relates to a multi-transmembrane protein antigen comprising a polypeptide corresponding to an extracellular loop of a multi-transmembrane protein. The polypeptide has an N-terminal and a C-terminal, and these termini are fixed on a solid substrate, or the N-terminal and C-terminal are attached to both ends of a linker to form a cyclic structure. The extracellular loop polypeptide is exemplified by SEQ ID NO: 3 or 4, including claudin 3 and claudin 4.
Cyclic presentation is described using linkers and linker contexts, including aminohexanoic acid, polyglycine, and polyalanine, with an option in which the linker is attached to a water-soluble resin and further enclosed by polyethylene glycol (PEG). The document also describes comparative preparation and antigen formats, including loop-type versus linear antigen forms.
The invention further covers antibodies and antigen-binding fragments specific to the multi-transmembrane protein antigen, including Fab, F(ab')2, scFv, and dAb, with CDR-defined components. An antibody screening approach using a humanized antibody library and a phage display library is described, including screening comparing loop-type versus linear antigens.
Claims Coverage
The document includes one independent claim directed to a multi-transmembrane protein extracellular-loop antigen presented with immobilized termini or a cyclic structure, with the extracellular-loop sequence specified as SEQ ID NO: 3 or 4. Dependent claims refine the termini presentation and specify linker and linker-resin contexts, including cyclic-linker details and PEG-enclosed resin.
Extracellular loop antigen with fixed termini or cyclic structure
A multi-transmembrane protein antigen comprising a polypeptide corresponding to an extracellular loop of a multi-transmembrane protein, wherein an N-terminal and a C-terminal of the polypeptide are fixed on a solid substrate, or an N-terminal and a C-terminal of the polypeptide are attached to both ends of a linker to form a cyclic structure.
Extracellular-loop polypeptide defined by SEQ ID NO: 3 or 4
The polypeptide corresponding to the extracellular loop of the multi-transmembrane protein is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 or 4.
Overall claim coverage centers on presenting an extracellular loop of a multi-transmembrane protein as an antigen with either fixed N- and C-termini on a solid substrate or a cyclic structure formed by attachment to a linker, with the extracellular-loop sequence specified as SEQ ID NO: 3 or 4.
Stated Advantages
Substantially improved antibody reactivity for loop-type antigens versus linear antigens.
Improved antibody binding affinity for loop-type antigens versus linear antigens, described as notably higher by SPR.
Documented Applications
Use of a humanized antibody library and phage library display to screen for antibodies specific to the multi-transmembrane protein antigen, including comparing loop-type versus linear antigen formats.
Assessment of antigen-antibody binding using ELISA, FACS, and SPR, including evaluation of antibody reactivity and binding affinity for loop-type versus linear antigens.
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