Intrabodies with defined framework that is stable in a reducing environment and applications thereof
AssigneeESBATech AG
InventorsAdrian Auf Der Maur, Alcide Barberis, Dominik Escher
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Abstract
A method for the isolation of CDRs in a defined framework that is stable and soluble in reducing environment is described as well as thus obtainable scFv. Starting from such scFv with defined framework a scFv library can be generated wherein the framework is conserved while at least one complementary determining region (CDR) is randomized. Such library, e.g. in yeast cells, is suitable for screening for antibody/CDR-interactions or for screening for antibodies.
Claims41
A method for the isolation of an scFv with defined framework that is stable and soluble in a reducing environment, wherein
- a) a scFv library with varied frameworks and constant CDRs is generated by mutation of at least one framework encoding region of DNA sequence of a scFv to a known antigen and by introduction of such mutations into suitable expression vectors,
- b) host cells able to express a specific known antigen and only surviving in the presence of antigen-scFv-interaction are transformed with said scFv library,
- c) the thus transformed host cells are cultivated under conditions suitable to express the antigen and the scFv and allowing cell survival only in the presence of antigen-scFv-interaction,
- d) the scFv expressed in surviving cells and having a defined framework that is stable and soluble in reducing environment is isolated.
A method for the generation of a scFv encoding DNA with a framework suitable for selective alterations in the CDR region, wherein specific restriction sites are introduced into the sequence of a defined, stable and soluble scFv encoding DNA by means of site directed mutagenesis.
A scFv comprising the framework defined in SEQ ID NO 1.
A method for the generation of a CDR library with a defined framework, that is stable and soluble in a reducing environment, wherein DNA sequences encoding a scFv of one of the previous claims are digested to replace at least one CDR per sequence by a modified CDR.
A library of intrabodies with at least one randomized CDR and defined framework that is stable and soluble under reductive conditions.
A method for screening for CDRs interacting with a specific antigen, wherein host cells transformed with a nucleic acid sequence encoding a known antigen are further transformed with a randomized CDR library with defined framework that is stable and soluble in a reducing environment, whereby the antigen and/or the scFv are linked to a marker system or part of a marker system thus that the cell cultured under selective conditions only survives in the presence of antigen/scFv-interaction, that thus transformed cells are cultivated under selective conditions, and that surviving cells are cultured and the intrabodies harvested.
A method for screening for an antigen interacting with an scFv, wherein host cells expressing at least one antigen of interest are transformed with at least one scFv with defined framework that is stable and soluble in reducing environment, or with a randomized CDR library with defined framework that is stable and soluble in reducing environment, whereby the antigens and/or the scFvs are linked to a marker system or part of a marker system thus that the cell cultured under selective conditions only survives in the presence of antigen/scFv-interaction, that thus transformed cells are cultivated under selective conditions, and that surviving cells are cultured and the scFvs harvested.
An scFv with defined framework as therapeutic or diagnostic or prophylactic agent.
Use of the scFv with defined framework for intracellular screenings.
A method for the identification of intrabody frameworks or intrabodies wherein suitable host cells are transformed with a library and a marker system, whereby said library is a fusion product of an intrabody library and at least part of said marker system, wherein said marker system is only activated in the presence of a fusion protein encoding a soluble and stable intrabody framework, and culturing said cells under conditions allowing the identification and selection of cells expressing a soluble and stable intrabody framework.
Dependencies are inferred from the claim text.