High throughput generation of human monoclonal antibody against peptide fragments derived from membrane proteins
OwnerShaobing Hua, Michelle H. Pauling +1inventors
InventorsShaobing Hua, Michelle H. Pauling, Li Zhu
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Abstract
Methods are provided for efficient, high throughput screening of antibody libraries against proteins targets, especially membrane proteins. In particular, methods are provided for screening a fully human antibody library against membrane proteins such as HIV coreceptors in yeast. More particularly, a library of human single chain antibodies is screened against peptide fragments derived from extracellular domains of human CCR5 and high affinity monoclonal antibodies against CCR5 are selected.
Claims29
A method for selecting a single chain antibody (scFv) against a peptide target in a yeast, comprising:
- expressing a library of scFv fusion proteins in yeast cells, each scFv fusion protein comprising either an activation domain or a DNA binding domain of a transcription activator and a scFv, the scFv comprising a VH of antibody whose sequence varies within the library, a VL of antibody whose sequence varies within the library independently of the VH, and a linker peptide which links the VH and VL;
- expressing a target fusion protein in the yeast cells expressing the scFv fusion proteins, the target fusion protein comprising either the DNA binding domain or the activation domain of the transcription activator which is not comprised in the scFv fusion proteins, and a target peptide;
- and
- selecting those yeast cells in which a reporter gene is expressed, the expression of the reporter gene being activated by a reconstituted transcriptional activator formed by binding of the scFv fusion protein to the target fusion protein.
Dependencies are inferred from the claim text.