Methods of selecting internalizing antibodies
AssigneeThe Regents of the University of California
InventorsJames D. Marks, Marie Alix Poul, Baltazar Becerril
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Abstract
This invention provides methods of selecting antibodies that are internalized into target cells. The methods generally involve contacting target cells with one or more members of an antibody phage display library. The members of the phage display library are also contacted with cells of a subtractive cell line. The target cells are then washed to remove the subtractive cell line cells and members of the phage display library that are non-specifically bound or weakly bound to the target cells. The target cells are cultured under conditions where members of the phage display library can be internalized if bound to an internalizing marker and internalized members of the phage display library are then identified.
Claims42
A method of selecting a polypeptide that is internalized into a target cell, said method comprising:
- i) contacting one or more target cells with one or more members of a phage display library displaying one or more polypeptides;
- ii) culturing the one or more target cells and enriching internalized library members under conditions where said internalized library members of said phage display library are enriched at least 30-fold as compared to on-internalized library members, wherein at least 30-fold enrichment is achieved in a single repetition of step I) and ii);
- and
- iii) identifying internalized library members of said phage display library, thereby selecting for a polypeptide that is internalized into the target cell.
A method of selecting a polypeptide that is internalized into a target cell, comprising:
- i) contacting one or more target cells with one or more members of a phage display library displaying one or more polypeptides;
- ii) culturing the one or more target cells under conditions wherein members of said phage display library bound to an internalizing marker become internalized;
- iii) reducing non-internalized members of said phage display library by removing phage trapped in an extracellular matrix;
- and
- iv) identifying members of said phage display library that are internalized into one or more of said target cells, where the internalized library members of said phage display library each display polypeptide that is internalized into a target cell.
Dependencies are inferred from the claim text.