Intrabodies with defined framework that is stable in a reducing environment and applications thereof
View Patent ↗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.
1. A method for screening for intrabodies with CDRs interacting with a specific antigen comprising:
(a) identification of intrabody framework which is soluble and stable in a reducing environment by a method which comprises the following steps:
i) transformation of suitable host cells with a nucleic acid library, said library encoding a fusion protein comprising an intrabody and a transcriptional activation domain wherein said transcriptional activation domain is only active as part of a fusion protein comprising an intrabody moiety which is soluble and stable in a reducing environment, and
ii) culturing said cells under conditions allowing the identification and selection of cells expressing a fusion protein comprising an intrabody moiety which is soluble and stable in a reducing environment by detection of a reporter gene that is expressed by the interaction of the transcriptional activation domain with a DNA binding domain in the host cell wherein the interaction of the transcriptional activation domain with the DNA binding domain results in the growth or identification of cells indicating that the intrabody frameworks or intrabodies are stable and soluble under reducing conditions and is not dependent upon the presence of the antigen for which the intrabody is specific and identifying intrabody frameworks that are soluble and stable in a reducing environment;
(b) preparation of a randomized intrabody CDR library by exchanging at least one nucleic acid sequence encoding a CDR in an intrabody with a framework which has been identified by the method of step a);
(c) transformation of host cells with a randomized intrabody CDR library of step (b) and with a nucleic acid sequence encoding a known antigen, whereby the antigen and/or the intrabody of the CDR library are linked to a marker system or part of a marker system such that the cells cultured under selective conditions only survive in the presence of antigen/intrabody-interaction which activates the marker system;
(d) cultivation of the cells under selective conditions allowing cell survival only in the presence of antigen/intrabody-interaction; and
(e) harvesting the intrabodies interacting with the known antigen from surviving cells.
2. The method of claim 1 , wherein said marker protein of step a) has a selectable activity, which is an enzymatic activity or a fluorescence activity.
3. The method of claim 1 , wherein the cell is an eukaryotic cell.
4. The method of claim 1 wherein the DNA sequence encoding the antigen and the DNA sequence encoding the scFv both encode chimeric molecules with the antigen or scFv, respectively, both linked to part of a transcription activating system linked to a survival allowing marker.
5. The method of claim 4 , wherein the antigen is fused to a DNA binding domain and the scFv is fused to a transcriptional activator domain or the antigen is fused to a transcriptional activator domain and the scFv is fused to a DNA binding domain.
6. The method of claim 3 wherein the cell is a yeast cell.