Genetic engineering of non-human animals for the production of chimeric antibodies
Inventors
Green, Larry • Shizuya, Hiroaki
Assignees
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Publication Number
US-10836832-B2
Publication Date
2020-11-17
Expiration Date
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 heavy chain variable region (VH) polypeptide. A mouse is immunized with an antigen, and the mouse’s genome comprises a transgene that is capable of undergoing gene rearrangement to produce a polynucleotide sequence encoding the antibody or fragment comprising the human VH polypeptide. The transgene comprises a plurality of immunoglobulin heavy chain variable (V) exons encoding human immunoglobulin heavy chain V polypeptides, with non-coding sequences between the V exons derived from mouse immunoglobulin non-coding sequences.
The transgene further comprises a plurality of immunoglobulin heavy chain joining (J) coding sequences encoding human immunoglobulin heavy chain J polypeptides, with non-coding sequences between the J coding sequences derived from mouse immunoglobulin non-coding sequences. The method further includes recovering from the mouse a genomic DNA or cDNA comprising a nucleotide sequence encoding the human immunoglobulin VH polypeptide, and using the recovered nucleotide sequence for recombinantly producing the human immunoglobulin VH polypeptide.
The disclosed strategy also includes refinement of the transgene by specifying non-coding sequences derived from mouse immunoglobulin regulatory/termination elements and by incorporating additional immunoglobulin coding elements. The specification describes engineering approaches that use synthetic immunoglobulin light-chain transgenes and chimeric immunoglobulin heavy-chain constant-region components, together with inactivation of endogenous immunoglobulin loci, to support expression from engineered loci and generation of repertoires or display libraries.
Claims Coverage
The partial content provides one independent claim centered on a method of producing an antibody or antigen-binding fragment that comprises a human immunoglobulin heavy chain variable (VH) polypeptide. The claim is supported by three inventive features, with additional dependent claims refining the transgene architecture and downstream use of recovered sequences.
Rearrangeable transgene encoding human VH exons and J sequences
A transgene in a mouse genome that comprises a plurality of immunoglobulin heavy chain variable (V) exons encoding human immunoglobulin heavy chain V polypeptides, non-coding sequences between the V exons derived from mouse immunoglobulin non-coding sequences, a plurality of immunoglobulin heavy chain joining (J) coding sequences encoding human immunoglobulin heavy chain J polypeptides, and non-coding sequences between the J coding sequences derived from mouse immunoglobulin non-coding sequences, wherein the transgene is capable of undergoing gene rearrangement to produce a polynucleotide sequence encoding an antibody or fragment comprising the human immunoglobulin VH polypeptide.
Recover and recombinantly produce recovered human VH
Recovering from the mouse a genomic DNA or cDNA comprising a nucleotide sequence encoding the human immunoglobulin VH polypeptide, and recombinantly producing the human immunoglobulin VH polypeptide.
Immunize mouse to generate VH-encoding rearranged sequences
Immunizing a mouse with an antigen, wherein the mouse's genome comprises the rearrangeable transgene encoding human immunoglobulin VH components, to produce the antibody or fragment comprising the human immunoglobulin VH polypeptide via gene rearrangement.
The coverage centers on immunizing a transgenic mouse carrying a rearrangeable human VH transgene with mouse-derived non-coding sequences between V and J segments, then recovering genomic DNA or cDNA encoding the human VH for recombinantly producing the human immunoglobulin VH polypeptide. Dependent claims further specify mouse-derived non-coding elements, additional immunoglobulin coding components, and use of recovered VH sequences in an in vitro antibody display system.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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