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-11104744-B2
Publication Date
2021-08-31
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 locus-engineering framework for generating chimeric, including humanized, antibody-producing non-human mammals/cells using engineered immunoglobulin loci. It emphasizes the construction of transgenes that are capable of undergoing gene arrangement and, upon expression, produce immunoglobulin variable-region polypeptides.
A key aspect is avoiding detrimental conformation and effector changes described in prior antibody formats while still enabling production of chimeric antibodies. The engineered constructs combine non-endogenous immunoglobulin variable regions with chimeric constant regions, including combinations such as mouse CH2/CH3 with non-endogenous CH1/hinge, and maintain antibody function by the selected constant-region configuration.
The approach includes design and assembly of complex immunoglobulin transgenes using in silico DNA design and synthetic DNA assembly, along with BAC/YAC-based recombineering and homologous recombination to build large synthetic immunoglobulin loci. It also includes optional replacement of cis regulatory elements such as LCR/3 enhancers and recovery/restoration of functional regulation, including recovery of NF-baB binding in the Igbb 3 LCR, and inactivation of endogenous Ig loci via targeted deletions/mutations to drive exclusive transgene expression.
Claims Coverage
The provided material identifies two independent claims. Across these independent claims, the coverage centers on the architecture of an immunoglobulin light-chain variable region transgene that undergoes gene arrangement upon expression, with mouse-derived non-coding sequences between variable (V) exons and joining (J) coding sequences.
Canine light-chain variable transgene with mouse-derived V-noncoding and J-noncoding
A mouse whose genome comprises a transgene encoding a polypeptide comprising an immunoglobulin light chain variable region, where the transgene includes a plurality of immunoglobulin light chain variable (V) exons encoding canine immunoglobulin light chain variable (V) polypeptides, non-coding sequences between the V exons, a plurality of immunoglobulin light chain joining (J) coding sequences encoding canine immunoglobulin light chain joining (J) polypeptides, and non-coding sequences between the J coding sequences, with the non-coding sequences between the V exons and between the J coding sequences derived from mouse immunoglobulin light chain non-coding sequences, and the transgene is capable of undergoing gene arrangement such that upon expression it produces a polypeptide comprising the immunoglobulin light chain variable region.
Canine light-chain variable transgene in non-human mammalian cells with mouse-derived V-noncoding and J-noncoding
A non-human mammalian cell whose genome comprises a transgene encoding a polypeptide comprising a canine immunoglobulin light chain variable region, where the transgene includes a plurality of immunoglobulin light chain variable (V) exons encoding canine immunoglobulin light chain variable (V) polypeptides, non-coding sequences between the V exons, a plurality of immunoglobulin light chain joining (J) coding sequences encoding canine immunoglobulin light chain joining (J) polypeptides, and non-coding sequences between the J coding sequences, with the non-coding sequences between the V exons and between the J coding sequences derived from mouse immunoglobulin light chain non-coding sequences, and the transgene is capable of undergoing gene arrangement such that upon expression it produces a polypeptide comprising the immunoglobulin light chain variable region.
Both independent claims require a transgene that encodes a canine immunoglobulin light chain variable region with multiple V exons and multiple J coding sequences, separated by non-coding sequences derived from mouse immunoglobulin light chain non-coding sequences, and configured to undergo gene arrangement upon expression to produce the immunoglobulin light-chain variable-region polypeptide.
Stated Advantages
Avoiding detrimental conformation and effector changes described as occurring in prior antibody formats while enabling production of chimeric antibodies.
Maintaining antibody function by the selected constant-region configuration.
Recovery or restoration of functional regulation, including recovery of NF-baB binding in the Igbb 3 LCR.
Driving exclusive transgene expression through inactivation of endogenous Ig loci.
Assembling large synthetic Ig lambda and kappa light-chain transgenes with functional V-J diversity.
Documented Applications
Generating chimeric, including humanized, antibody-producing non-human mammals/cells using engineered immunoglobulin loci.
Building large synthetic immunoglobulin loci.
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