Methods for producing antigen-binding proteins against foreign antigens
Inventors
VORONINA, Vera • MacDonald, Lynn • Prissette, Marine • LAI, Ka-Man Venus • Badithe, Ashok • Murphy, Andrew J. • Droguett, Gustavo • Frendewey, David • Zambrowicz, Brian
Assignees
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Publication Number
US-12342801-B2
Publication Date
2025-07-01
Expiration Date
Abstract
Methods and compositions are provided for generating antigen-binding proteins against a foreign antigen of interest.
Core Innovation
The invention relates to a method of generating antigen-binding proteins against a human foreign antigen of interest by creating a transgenic F0 generation mouse with reduced tolerance to the human foreign antigen of interest. A self-antigen orthologous to the human foreign antigen of interest is targeted within a target genomic locus by introducing Cas9 protein or a nucleic acid encoding Cas9 protein together with a first guide RNA and a second guide RNA to enable genome modification.
The target genomic locus comprises all or part of a gene encoding a self-antigen orthologous to the human foreign antigen of interest, and the genomic locus is modified in a pair of corresponding chromosomes to produce a biallelic modification including a biallelic deletion such that expression of the self-antigen is eliminated. After producing the transgenic F0 generation mouse with the self-antigen expression eliminated, the mouse is immunized with the human foreign antigen of interest to initiate an immune response and produce antigen-binding proteins against the human foreign antigen of interest.
The transgenic F0 generation mouse comprises, in its germline, ectopic nucleic acid sequence encoding a mouse ADAM6 protein, and human immunoglobulin heavy chain V, D, and J gene segments operably linked to a mouse heavy chain constant region gene at an endogenous mouse immunoglobulin locus, together with human immunoglobulin light chain V and J gene segments operably linked to a mouse light chain constant region gene sequence. The mouse is incapable of forming an antibody that comprises a human variable region and a human constant region.
Claims Coverage
The provided excerpt includes one independent claim. It contains a multi-part inventive scheme with genome engineering to eliminate self-antigen expression, followed by immunization to generate antigen-binding proteins, and includes specific transgenic germline components with ectopic mouse ADAM6 and human immunoglobulin variable region gene segments linked to mouse constant regions.
Biallelic deletion of a self-antigen orthologous gene to eliminate expression
Introducing Cas9 protein or a nucleic acid encoding Cas9 protein, a first guide RNA and a second guide RNA into mouse one-cell stage embryos or mouse ES cells, where the guide RNAs hybridize to recognition sequences within a target genomic locus comprising all or part of a gene encoding a self-antigen orthologous to the human foreign antigen of interest, and producing a transgenic F0 generation mouse with a biallelic deletion such that expression of the self-antigen is eliminated.
Immunization of a reduced-tolerance transgenic F0 mouse to generate antigen-binding proteins
Immunizing the transgenic F0 generation mouse with the human foreign antigen of interest and maintaining the mouse under conditions sufficient to initiate an immune response such that the transgenic F0 generation mouse produces antigen-binding proteins against the human foreign antigen of interest.
Transgenic germline configuration with ectopic ADAM6 and human Ig variable segments linked to mouse constant regions
Providing the transgenic F0 generation mouse comprising in its germline an ectopic nucleic acid sequence encoding a mouse ADAM6 protein functional in a male mouse, a heavy chain locus comprising human immunoglobulin heavy chain V, D, and J gene segments operably linked to a mouse immunoglobulin heavy chain constant region gene at an endogenous mouse immunoglobulin locus, and a light chain locus comprising human immunoglobulin light chain V and J gene segments operably linked to a mouse immunoglobulin light chain constant region gene sequence, wherein rearrangement forms human heavy chain and light chain variable regions operably linked to mouse constant regions, and wherein the mouse is incapable of forming an antibody that comprises a human variable region and a human constant region.
Across the independent claim, the inventive core is a workflow that engineers a transgenic mouse with reduced tolerance by biallelic deletion of a self-antigen orthologous gene to eliminate its expression, then immunizes the resulting transgenic F0 generation mouse with the human foreign antigen to elicit antigen-binding proteins, using a defined germline configuration that includes ectopic mouse ADAM6 and human immunoglobulin V(D)J segments linked to mouse constant regions.
Stated Advantages
Reduced tolerance to the human foreign antigen of interest, enabling production of antigen-binding proteins against the human foreign antigen of interest.
Elimination of expression of the self-antigen by biallelic deletion of all or part of the gene encoding the self-antigen orthologous to the human foreign antigen.
Faster production and improved antibody repertoire or epitope diversity after immunization.
Documented Applications
Generating antigen-binding proteins against a human foreign antigen of interest using a reduced-tolerance transgenic mouse produced by biallelic deletion of a self-antigen orthologous gene, followed by immunization of the transgenic F0 generation mouse.
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