Genetic engineering of non-human animals for the production of chimeric antibodies

Inventors

Green, Larry • Shizuya, Hiroaki

Assignees

Ablexis LLC

Abstract

The invention provides non-human cells and mammals having a genome encoding chimeric antibodies and methods of producing transgenic cells and mammals. Certain aspects of the invention include chimeric antibodies, humanized antibodies, pharmaceutical compositions and kits. Certain aspects of the invention also relate to diagnostic and treatment methods using the antibodies of the invention.

Core Innovation

The invention provides a method of producing an antibody, or an antigen-binding fragment thereof, in which the antibody or fragment comprises a human immunoglobulin light chain variable (VL) polypeptide. A mouse is immunized with an antigen, where the mouse’s genome comprises a transgene that includes human immunoglobulin light chain V exons encoding human immunoglobulin light chain V polypeptides, non-coding sequences between the V exons, human immunoglobulin light chain J coding sequences encoding human immunoglobulin light chain J polypeptides, and non-coding sequences between the J coding sequences. The non-coding sequences between the V exons and between the J coding sequences are derived from mouse immunoglobulin non-coding sequences, and the transgene is capable of undergoing gene rearrangement to produce a polynucleotide sequence encoding the VL polypeptide.

The method recovers from the mouse genomic DNA or cDNA comprising a nucleotide sequence encoding the human immunoglobulin VL polypeptide. The method then recombinantly produces the human immunoglobulin VL polypeptide from the recovered nucleotide sequence. The disclosed concept is that the human VL polypeptide sequence is generated in the mouse via rearrangement of a transgene that combines human coding segments with mouse-derived non-coding sequences, followed by recovery and recombinant production.

In dependent implementations, the transgene architecture is further refined by providing specific combinations of human immunoglobulin light chain V and J segments, and by defining additional components that can contribute to an antibody or antigen-binding fragment comprising the human immunoglobulin VL polypeptide. These refinements include specifying cis-regulatory elements and selecting cis regulatory sequences from defined categories, such as promoters, enhancers, recombination signal sequences, splice acceptor sequences, and splice donor sequences. Additional refinements include specifying non-coding sequences upstream of the V exons as being selected from promoters and enhancers, and constraining the number of J-C coding sequence pairs for specific light-chain types.

Claims Coverage

The independent claim identified is the method of producing an antibody or antigen-binding fragment using a transgenic mouse with a specific human light chain VL transgene architecture that undergoes gene rearrangement. This claim is further supported by dependent claims that refine transgene composition and downstream recovery/production and optionally add screening and in vitro display implementations. The inventive coverage is centered on transgene-driven VL rearrangement in a mouse using human coding segments with mouse-derived non-coding sequences, followed by recovery of the VL-encoding nucleotide sequence and recombinant production of the VL polypeptide.

Overall, the claim coverage is directed to generating a human immunoglobulin light chain VL polypeptide by rearrangement of a human V/J transgene in an immunized mouse using mouse-derived non-coding sequences, then recovering the VL-encoding nucleotide sequence and recombinantly producing the human VL polypeptide. Dependent claims additionally refine the transgene’s cis-regulatory components and architectural constraints and may include B-lymphocyte screening and in vitro antibody display implementation.

Stated Advantages

Documented Applications