Transposition-mediated identification of specific binding or functional proteins

Inventors

Grawunder, Ulf

Assignees

NBE-Therapeutics AG

Abstract

The method disclosed herein describes a novel technology offering unparalleled efficiency, flexibility, utility and speed for the discovery and optimization of polypeptides having desired binding specificity and/or functionality, including antigen-binding molecules such as antibodies and fragments thereof, for desired functional and/or binding phenotypes. The novel method is based on transposable constructs and diverse DNA libraries cloned into transposable vectors and their transfection into host cells by concomitant transient expression of a functional transposase enzyme. This ensures an efficient, stable introduction of the transposon-based expression vectors into vertebrate host cells in one step, which can then be screened for a desired functional or binding phenotype of the expressed proteins, after which the relevant coding sequences for the expressed proteins, including antibodies and fragments thereof, can be identified by standard cloning and DNA sequencing techniques.

Core Innovation

The invention relates to transposition-mediated discovery and optimization of antibodies and antibody fragments. It generates a diverse collection of polynucleotides encoding at least 10^2 unique antibodies or fragments having different target or epitope binding specificities, with each polynucleotide disposed between inverted terminal repeat sequences that are recognized by and functional with at least one transposase enzyme.

A diverse collection of polynucleotides is introduced into vertebrate host cells, and at least one transposase enzyme functional with the inverted terminal repeat sequences is expressed in the vertebrate host cells so that the polynucleotides are integrated into the vertebrate host cell genomes. The integrated vertebrate host cell population expresses antibodies or fragments thereof with different target or epitope binding specificities, with the encoded sequences provided as either immunoglobulin V_H and V_L regions, or full-length immunoglobulin heavy and light chains.

The invention screens the vertebrate host cells to identify a vertebrate host cell expressing an antibody or fragment having a desired target or epitope binding specificity, followed by isolating the polynucleotide sequence encoding the identified antibody or fragment. The workflow supports screening based on binding phenotypes and recovery of encoding sequences for further characterization, including by genomic amplification, RT-PCR amplification, or next-generation deep sequencing.

Claims Coverage

The provided independent claims are clm-00001, clm-00006, and clm-00007. Across these independent claims, there are 6 main inventive features: generating an inverted-ITR flanked diverse polynucleotide library, introducing the library into vertebrate host cells, transposase-driven integration and expression in the host cells, screening host cells for desired binding specificity, isolating the encoding polynucleotide sequence, and constraining the ITRs to PiggyBac or Sleeping Beauty systems.

The independent claims collectively cover generating an antibody-encoding polynucleotide library flanked by transposase-functional inverted terminal repeats, introducing the library into vertebrate host cells, integrating and expressing the library via a compatible transposase, screening host cells for desired target or epitope binding specificity, and isolating the encoding polynucleotide sequence, with the library constrained to VH/VL or heavy/light formats and the ITRs restricted to PiggyBac and/or Sleeping Beauty systems.

Stated Advantages

Documented Applications