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US-20260123608-A1 - NON-HUMAN ANIMALS HAVING MODIFIED IMMUNOGLOBULIN HEAVY CHAIN CONSTANT REGION LOCUS AND USES THEREOF

US20260123608A1US 20260123608 A1US20260123608 A1US 20260123608A1US-20260123608-A1

Abstract

Non-human animals (and/or non-human cells) and methods of using the same are provided, which non-human animals (and/or non-human cells) have a genome comprising human antibody-encoding sequences (i.e., immunoglobulin genes). Non-human animals described herein express antibodies that are of IgA, IgD, or IgM isotypes. Non-human animals provided herein are, in some embodiments, characterized by expression of IgA antibodies that contain human heavy chain and light chain variable domains and rodent constant domains. Methods for producing antibodies from non-human animals are also provided, which antibodies contain human variable regions and rodent constant regions.

Inventors

  • John McWhirter
  • Chunguang Guo
  • Lynn MacDonald
  • Andrew J. Murphy

Assignees

  • REGENERON PHARMACEUTICALS, INC.

Dates

Publication Date
20260507
Application Date
20250806

Claims (20)

  1. 1 . A genetically modified rodent whose germline genome comprises an engineered endogenous immunoglobulin heavy chain locus that comprises an immunoglobulin heavy chain constant region comprising a rodent immunoglobulin heavy chain alpha (Igha) constant region gene, wherein the genetically modified rodent only produces immunoglobulin heavy chains that comprise a rodent Igha constant domain encoded by the rodent Igha constant region gene.
  2. 2 . The genetically modified rodent of claim 1 , wherein the rodent's germline genome further comprises one or more genetic alterations at the endogenous immunoglobulin heavy chain locus, wherein the one or more genetic alterations disrupt expression from rodent immunoglobulin heavy chain constant region genes: mu (Ighm), delta (Ighd), gamma (Ighg), and epsilon (Ighe) constant region genes.
  3. 3 - 5 . (canceled)
  4. 6 . The genetically modified rodent of claim 2 , wherein the one or more genetic alterations comprises one or more deletions.
  5. 7 . The genetically modified rodent 6 , wherein the one or more deletions remove all or part of each of the rodent immunoglobulin heavy chain constant region genes: Ighm, Ighd, Ighg, and Ighe constant region genes.
  6. 8 . (canceled)
  7. 9 . The genetically modified rodent of claim 1 , wherein the engineered endogenous immunoglobulin heavy chain locus comprises in 5′ to 3′ order: (i) a rodent intronic enhancer (Eμ); (ii) a rodent switch mu region (Sμ); and (iii) a rodent Igha constant region gene.
  8. 10 - 13 . (canceled)
  9. 14 . The genetically modified rodent of claim 1 , wherein the engineered endogenous immunoglobulin heavy chain locus comprises in 5′ to 3′ order: (i) one or more human V H gene segments, one or more human D H gene segments, and one or more human J H gene segments; (ii) a rodent intronic enhancer (Eμ); (iii) a rodent switch mu region (Sμ); (iv) a rodent Igha constant region gene; and optionally (v) a rodent 3′ IgH regulatory region 3′RR.
  10. 15 - 27 . (canceled)
  11. 28 . The genetically modified rodent of claim 1 , wherein the germline genome of the rodent comprises an engineered endogenous immunoglobulin light chain locus comprising: (a) one or more non-rodent mammal Vκ gene segments, and (b) one or more non-rodent mammal Jκ gene segments, wherein the one or more non-rodent mammal Vκ gene segments and the one or more non-rodent mammal Jκ gene segments are operably linked to a Cκ gene.
  12. 29 - 37 . (canceled)
  13. 38 . The genetically modified rodent of claim 1 , wherein the germline genome of the rodent comprises an engineered endogenous immunoglobulin light chain locus comprising: (a) one or more non-rodent mammal Vλ gene segments, and (b) one or more non-rodent mammal Jλ gene segments, wherein the one or more non-rodent mammal Vλ gene segments and one or more non-rodent mammal Jλ gene segments are operably linked to a rodent immunoglobulin light chain constant region gene.
  14. 39 - 46 . (canceled)
  15. 47 . A genetically modified rodent whose germline genome comprises an engineered endogenous immunoglobulin heavy chain locus that comprises an immunoglobulin heavy chain constant region comprising rodent immunoglobulin heavy chain constant region genes Ighm, Ighd, and Igha, wherein the genetically modified rodent only produces immunoglobulin heavy chains that comprise a rodent Igha, a rodent Ighm, or a rodent Ighd constant domain encoded by the rodent Igha constant region gene, the rodent Ighm constant region gene, or the rodent Ighd constant region gene, respectively.
  16. 48 - 52 . (canceled)
  17. 53 . A method of making a genetically modified rodent, the method comprising genetically modifying the germline genome of a rodent so that the rodent comprises an immunoglobulin heavy chain constant region comprising a rodent Igha constant region gene and only produces immunoglobulin heavy chains that comprise a rodent Igha constant domain encoded by the rodent Igha constant region gene.
  18. 54 . A method of making a genetically modified rodent, the method comprising genetically modifying the germline genome of a rodent so that the rodent comprises an immunoglobulin heavy chain constant region comprising rodent immunoglobulin heavy chain constant region genes Ighm, Ighd, and Igha, and only produces immunoglobulin heavy chains that comprise a rodent Igha, a rodent Ighm, or a rodent Ighd constant domain encoded by the Igha constant region gene, the rodent Ighm constant region gene, or the rodent Ighd constant region gene, respectively.
  19. 55 . A method of producing an antibody, the method comprising the steps of: (a) immunizing a genetically modified rodent according to claim 1 with an antigen of interest; (b) maintaining the genetically modified rodent under conditions sufficient for the genetically modified rodent to produce an immune response to the antigen of interest; and (c) recovering from the genetically modified rodent: (i) an antibody that binds the antigen of interest, (ii) a nucleotide that encodes a human light or heavy chain variable domain, a light chain, or a heavy chain of an antibody that binds the antigen of interest, or (iii) a cell that expresses an antibody that binds the antigen of interest.
  20. 56 . A method of making an antibody, comprising: (a) expressing a first nucleotide sequence that encodes an immunoglobulin heavy chain in a host cell, wherein the first nucleotide sequence includes a human heavy chain variable region sequence that was identified from a genetically modified rodent according to claim 1 ; (b) expressing a second nucleotide sequence that encodes an immunoglobulin light chain in a host cell; (c) culturing the host cell so that immunoglobulin light chains and immunoglobulin heavy chains are expressed and form an antibody; and (d) obtaining the antibody from the host cell or host cell culture.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63/679,947, filed Aug. 6, 2024, the contents of which is hereby incorporated by reference in its entirety. SEQUENCE LISTING The instant application contains a Sequence Listing in electronic format entitled “2010794-3024_SL.xml” which was created on Jul. 22, 2025, and which has a size of 22,284 bytes. The contents of the file “2010794-3024_SL.xml” are incorporated by reference herein. BACKGROUND Human antibodies are the most rapidly growing class of therapeutics. Of the technologies that are currently used for their production, the development of genetically modified non-human animals (e.g., rodents) engineered with genetic material encoding human antibodies, in whole or in part, has revolutionized the field of human therapeutic monoclonal antibodies for the treatment of various diseases. Still, development of improved in vivo systems for generating human monoclonal antibodies that maximize human antibody repertoires in genetically modified non-human animals is needed. SUMMARY The present disclosure provides, among other things, a non-human animal (e.g., a rodent, e.g., a mouse or rat) whose germline genome has been modified such that the animal produces antibodies of only particular isotypes (e.g., IgA isotype, IgM isotype, and/or IgD isotype). For example, in some embodiments, a non-human animal (e.g., a rodent, e.g., a mouse or rat) described herein comprises a germline genome that comprises an engineered endogenous immunoglobulin heavy chain locus that comprises an immunoglobulin heavy chain constant region comprising a non-human animal (e.g., a rodent, e.g., a mouse or rat) immunoglobulin heavy chain alpha (Igha) constant region, wherein the genetically modified non-human animal produces only immunoglobulin chains that comprise IgA constant domain encoded by a Igha constant region gene. In another embodiment, a non-human animal (e.g., a rodent, e.g., a mouse or rat) described herein comprises a germline genome that comprises an engineered endogenous immunoglobulin heavy chain locus that comprises immunoglobulin heavy chain constant region genes Ighm, Ighd, and Igha, wherein the animal only produces immunoglobulin heavy chains that comprise an IgA, an IgM, or an IgD constant domain encoded by an Igha constant region gene, an Ighm constant region gene, or an Ighd constant region gene, respectively. Animals (e.g., rodents, e.g., mice or rats) described herein, when immunized with an antigen of interest, produce specific populations of antibodies. A genetically modified non-human animal (e.g., a rodent, e.g., a mouse or rat) that produces only, primarily, or an increased titer of IgA antibodies provides a new tool for antibody discovery, particularly taking advantage of the unique properties of IgA antibodies, which have traditionally proven difficult to isolate, purify and recombinantly produce. Additionally, IgA antibodies may provide new beneficial properties for therapeutic antibodies, particularly for difficult targets that traditional IgG antibodies have been unsuccessful at targeting. Antibodies with IgG isotype are most prevalent among circulating antibodies, accounting for approximately 80%. The second most prevalent isotype is monomeric IgA, which accounts for about 15% of circulating antibodies. However, IgA antibodies are the main class of antibodies found in the mucosa of the intestinal lining and is produced at a higher rate than any of the other isotypes (about 40-60 mg/kg per day) (see Bohlander, Fabian. Frontiers in Immunology 14 (2023): 1127339, which is herein incorporated by reference). IgA antibodies have unique structure and function within the immune system, including preventing infection (particularly from invading pathogens at mucosal surfaces) and controlling inflammation, that make them an ideal class of antibody for therapeutic antibody discovery. IgA antibodies have a strong effector function through the IgA-specific receptor FcαRI and are efficacious at killing tumor cells (see e.g., van Tetering, Geert, et al. Antibodies 9.4 (2020): 70, which is herein incorporated by reference). As such, efforts have been made to generate therapeutic IgA. However, challenges exist with developing therapeutic IgA antibodies, which include, among other things, lack of animal models due to differences in IgA antibodies in humans and rodents, difficulty in producing recombinant IgA antibodies due to its complex structure and heavy glycosylation, and its short half-life (see Bohlander, 2023). Consequently, most therapeutic antibodies to date have an IgG isotype. The prevalence of IgG antibodies as therapeutics is in part because of IgG's abundance in serum, long half-life, and body of literature related to IgG antibodies. The present disclosure addresses the difficulties in identifying, isolating and studying IgA antibodies (e.g., short half-life and lower quantity in serum), and is a pr