Genetically modified non-human animals and methods for producing heavy chain antibodies

Abstract

The present disclosure relates to genetically modified animals and methods for producing heavy chain antibodies. In one aspect, the genetically modified non-human animal comprises a modified immunoglobulin heavy chain locus, the modified immunoglobulin heavy chain locus comprises an IgG constant region gene, the IgG constant region gene encodes an IgG heavy chain constant region lacking a CH1 domain, and the genetically modified non-human animal expresses a heavy chain antibody. Also disclosed herein are anti-TFR1 antibodies, antigen-binding fragments, and uses thereof.

Description

[Technical Field]

[0001] Priority claims This application claims priority to PCT / CN2022 / 119188 filed on September 16, 2022 and PCT / CN2022 / 136246 filed on December 2, 2022. The entire contents of the foregoing are incorporated herein by reference.

[0002] Technical Field The present disclosure relates to genetically modified animals and methods for producing heavy chain antibodies.The present disclosure relates to anti-TFR1 antibodies, antigen-binding fragments, and uses thereof. [Background technology]

[0003] Therapeutic antibodies are one of the fastest-growing classes of therapeutic compounds, rapidly outpacing the growth of small molecule drugs. For example, monoclonal antibodies have revolutionized cancer treatment. However, the large size of traditional antibodies hinders their delivery to tumor cells in vivo. The minimal target recognition module of a traditional antibody consists of two non-covalently linked variable domains (VH and VL). The inherent hydrophobic interactions between the VH and VL domains limit the stability and solubility of engineered antibodies and often result in V domain aggregation and / or mispairing.

[0004] The discovery of heavy-chain antibodies has provided unprecedented opportunities to impact cancer therapy. These unique forms of camelid-derived antibodies lack the entire light chain and CH1 domain and consist of only a single variable domain, termed VHH. Recombinant VHHs are small (15–20 kDa) and strictly monomeric. They bind to their targets with nM affinity and are stable over a wide pH and temperature range. Molecular engineering is also easier with VHHs, facilitating the production of multivalent monoclonal antibodies compared to traditional recombinant antibodies and their fragments, which are subject to aggregation and reduced affinity. Furthermore, VHHs often bind epitopes that are less immunogenic than traditional antibodies.

[0005] Typically, therapeutic antibodies are human or humanized. Human or humanized antibodies can be generated by humanizing rodent antibodies (e.g., murine antibodies) or by using phage libraries. However, these animals and phage libraries typically cannot produce heavy chain antibodies. Instead, heavy chain antibodies are often derived from camelid heavy chain antibodies. These camelid heavy chain antibodies must be humanized. The humanization process can adversely affect binding affinity and introduce immunogenic epitopes into the antibody. Repetitive and time-consuming experiments are often required to improve the properties of these antibodies. In addition, in some cases, these antibodies can be immunogenic in patients, leading to a decline in their efficacy over time. Therefore, an efficient and reliable platform for producing human or humanized heavy chain antibodies and nanobodies is needed. Summary of the Invention

[0006] The present disclosure relates to genetically modified animals and cells with humanized immunoglobulin heavy chain variable region loci and truncated immunoglobulin heavy chain constant region loci. For example, the CH1 coding region within the IGHG1 gene can be knocked out, such that expressed IgG lacks the CH1 domain. In some embodiments, immunoglobulin light chain (e.g., kappa and lambda) loci are also knocked out. Upon immunization, the animals are capable of producing heavy chain antibodies with high affinity / diversity. In some embodiments, the heavy chain antibodies can be further engineered to generate nanobodies.

[0007] In one aspect, the present disclosure relates to a genetically modified non-human animal comprising an engineered immunoglobulin heavy chain locus, wherein in some embodiments the engineered immunoglobulin heavy chain locus comprises an IgG constant region gene, wherein in some embodiments the IgG constant region gene encodes an IgG heavy chain constant region lacking a CH1 domain, and wherein in some embodiments the genetically modified non-human animal expresses a heavy chain antibody. In some embodiments, the animal comprises exactly one IgG constant region gene. In some embodiments, the IgG heavy chain constant region gene is IGHG1. In some embodiments, the IgG heavy chain constant region comprises or consists of a CH2 domain and a CH3 domain, and optionally a hinge region.

[0008] In one aspect, the present disclosure relates to a genetically modified non-human animal whose genome comprises a germline genetic modification comprising a deletion of the IGHG3, IGHG2b, and IGHG2c genes and a deletion of the CH1 exon of the IGHG1 gene at the endogenous immunoglobulin heavy chain locus. In some embodiments, the germline genetic modification further comprises a deletion of the endogenous IGHE gene at the endogenous immunoglobulin heavy chain locus. In some embodiments, the genetic modification further comprises a deletion of endogenous Sγ2b, Sγ2c, and Sε switch regions at the endogenous immunoglobulin heavy chain locus. In some embodiments, the modified immunoglobulin heavy chain locus comprises a modified IGHG1 gene lacking a sequence encoding a CH1 domain, and in some embodiments, the modified IGHG1 gene comprises a sequence at least 80%, 90%, 95%, or 99% identical to SEQ ID NO: 1. In some embodiments, the genetic modification further comprises a deletion of an endogenous Sγ3 switch region at the endogenous immunoglobulin heavy chain locus. In some embodiments, the genome of the animal comprises a modified IGH1 gene lacking sequences encoding the endogenous Sμ, Sγ1, and Sα switch regions, and the CH1 domain, and endogenous IGHM, IGHδ, and IGHA genes. In some embodiments, the genetic modification further comprises a deletion of the endogenous IGHM and IGHδ genes at the endogenous immunoglobulin heavy chain locus. In some embodiments, the animal genome comprises a modified IGH1 gene lacking sequences encoding the endogenous Sμ, Sγ1, and Sα switch regions, and the CH1 domain, and an endogenous IGHA gene. In some embodiments, the Sμ and Sγ1 switch regions are linked to a sequence that is at least 80%, 90%, 95%, or 99% identical to SEQ ID NO:8. In some embodiments, the genetic modification further comprises a deletion of the CH1-encoding sequence of the IGHM gene at the endogenous immunoglobulin heavy chain locus. In some embodiments, the genome of the animal comprises endogenous Sμ, Sγ1, Sα switch regions, a modified IGHM gene lacking sequences encoding the CH1 domain, a modified IGHG1 gene lacking sequences encoding the CH1 domain, and endogenous IGHδ, IGHA genes.In some embodiments, the Sμ switch region and the modified IGHM gene are linked to a sequence at least 80%, 90%, 95%, or 99% identical to SEQ ID NO: 10. In some embodiments, the genetic modification further comprises a deletion of the CH1 exon of the IGHM gene and a deletion of the IGHδ gene at the endogenous immunoglobulin heavy chain locus. In some embodiments, the genome of the animal comprises endogenous Sμ, Sγ1, and Sα switch regions, a modified IGHM gene lacking a sequence encoding a CH1 domain, a modified IGHG1 gene lacking a sequence encoding a CH1 domain, and an endogenous IGHA gene. In some embodiments, the genetic modification further comprises a deletion of the CH1 exon of the IGHM gene and a deletion of the CH1 coding sequence of the IGHδ gene at the endogenous immunoglobulin heavy chain locus. In some embodiments, the genome of the animal comprises endogenous Sμ, Sγ1, Sα switch regions, a modified IGHM gene lacking sequences encoding a CH1 domain, a modified IGHδ gene lacking sequences encoding a CH1 domain, a modified IGHG1 gene lacking sequences encoding a CH1 domain, and an endogenous IGHA gene. In some embodiments, the modified IGHM gene is linked to a sequence at least 80%, 90%, 95%, or 99% identical to SEQ ID NO: 10, and the modified IGHδ gene comprises a sequence at least 80%, 90%, 95%, or 99% identical to SEQ ID NO: 41. In some embodiments, the modified IGHM gene comprises a sequence at least 80%, 90%, 95%, or 99% identical to SEQ ID NO: 13. In some embodiments, the genetic modification further comprises a deletion of an endogenous Sγ1 switch region at the endogenous immunoglobulin heavy chain locus. In some embodiments, the genome of the animal comprises a modified IGH1 gene lacking sequences encoding endogenous Sμ, Sγ3, Sα switch regions, and CH1 domains, and endogenous IGHM, IGHδ, and IGHA genes. In some embodiments, the genetic modification further comprises a deletion of the endogenous Sγ3 switch region at the endogenous immunoglobulin heavy chain locus.In some embodiments, the genetic modification further comprises a deletion of the endogenous IGHM gene and IGHδ gene at the endogenous immunoglobulin heavy chain locus. In some embodiments, the genome of the animal comprises an endogenous Sμ, Sα switch region, a modified IGH1 gene lacking sequences encoding a CH1 domain, and the endogenous IGHA gene. In some embodiments, the Sμ switch region and the modified IGH1 gene are linked to a sequence that is at least 80%, 90%, 95%, or 99% identical to SEQ ID NO:9. In some embodiments, the modified genome comprises a functional IGHM gene.

[0009] In some embodiments, if a sequence in the genome is replaced with the same sequence or a sequence from the same animal, the animal can still have the endogenous sequence.

[0010] In one aspect, the disclosure relates to a genetically modified non-human animal whose genome comprises, in 5' to 3' order, the following elements at an endogenous immunoglobulin heavy chain locus: an Sμ switch region, an IGHM gene, an IGHδ gene, an Sγ1 switch region, an IGH1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene. In some embodiments, the elements are operably linked. In some embodiments, the endogenous immunoglobulin heavy chain constant region locus consists of the following functional genes and switch regions: an Sμ switch region, an IGHM gene, an IGHδ gene, an Sγ1 switch region, an IGH1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene.

[0011] In one aspect, the disclosure relates to a genetically modified non-human animal whose genome comprises, in 5' to 3' order, the following elements at an endogenous immunoglobulin heavy chain locus: an Sμ switch region, an IGHM gene, an IGHδ gene, an Sγ3 switch region, an IGH1 gene lacking sequences encoding a CH1 domain, an Sα switch region, and an IGH1 gene. In some embodiments, the elements are operably linked. In some embodiments, the endogenous immunoglobulin heavy chain constant region locus consists of the following functional genes and switch regions: an Sμ switch region, an IGHM gene, an IGHδ gene, an Sγ3 switch region, an IGH1 gene lacking sequences encoding a CH1 domain, an Sα switch region, and an IGH1 gene.

[0012] In one aspect, the disclosure relates to a genetically modified non-human animal whose genome comprises, in 5' to 3' order, the following elements at an endogenous immunoglobulin heavy chain locus: an Sμ switch region, an Sγ1 switch region, an IGHG1 gene lacking sequences encoding a CH1 domain, an Sα switch region, and an IGHA gene. In some embodiments, the elements are operably linked. In some embodiments, the endogenous immunoglobulin heavy chain constant region locus consists of the following functional genes and switch regions: an Sμ switch region, an Sγ1 switch region, an IGHG1 gene lacking sequences encoding a CH1 domain, an Sα switch region, and an IGHA gene.

[0013] In one aspect, the disclosure relates to a genetically modified non-human animal whose genome comprises, in 5' to 3' order, the following elements at an endogenous immunoglobulin heavy chain locus: an Sμ switch region, an IGHG1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene. In some embodiments, the elements are operably linked. In some embodiments, the endogenous immunoglobulin heavy chain constant region locus consists of the following functional genes and switch regions: an Sμ switch region, an IGHG1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene.

[0014] In one aspect, the disclosure relates to a genetically modified non-human animal whose genome comprises, in 5' to 3' order, the following elements at an endogenous immunoglobulin heavy chain locus: an Sμ switch region, an IGHM gene lacking a sequence encoding a CH1 domain, an IGHδ gene, an Sγ1 switch region, an IGH1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene. In some embodiments, the elements are operably linked. In some embodiments, the endogenous immunoglobulin heavy chain constant region locus consists of the following functional genes and switch regions: an Sμ switch region, an IGHM gene lacking a sequence encoding a CH1 domain, an IGHδ gene, an Sγ1 switch region, an IGH1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene.

[0015] In one aspect, the disclosure relates to a genetically modified non-human animal whose genome comprises, in 5' to 3' order, the following elements at an endogenous immunoglobulin heavy chain locus: an Sμ switch region, an IGHM gene lacking a sequence encoding a CH1 domain, an Sγ1 switch region, an IGH1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene. In some embodiments, the elements are operably linked. In some embodiments, the endogenous immunoglobulin heavy chain constant region locus consists of the following functional genes and switch regions: an Sμ switch region, an IGHM gene lacking a sequence encoding a CH1 domain, an Sγ1 switch region, an IGH1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene.

[0016] In one aspect, the disclosure relates to a genetically modified non-human animal whose genome comprises, in 5' to 3' order, the following elements at an endogenous immunoglobulin heavy chain locus: an Sμ switch region, an IGHM gene lacking a sequence encoding a CH1 domain, an IGHδ gene lacking a sequence encoding a CH1 domain, an Sγ1 switch region, an IGH1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene. In some embodiments, the elements are operably linked. In some embodiments, the endogenous immunoglobulin heavy chain constant region locus consists of the following functional genes and switch regions: an Sμ switch region, an IGHM gene lacking a sequence encoding a CH1 domain, an IGHδ gene lacking a sequence encoding a CH1 domain, an Sγ1 switch region, an IGH1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene.

[0017] In some embodiments, the animal expresses a heavy chain antibody comprising an IgG heavy chain constant region lacking a CH1 domain. -7 Under M, 10 -8 Less than M or 10 -9 In some embodiments, the heavy chain antibody binds to a target antigen with a KD of less than M. In some embodiments, the heavy chain antibody comprises or consists of a variable region, a CH2 domain, and a CH3 domain. In some embodiments, the heavy chain antibody further comprises a transmembrane domain and / or a cytoplasmic domain. In some embodiments, the genetically modified non-human animal does not express an IgG antibody comprising a light chain. In some embodiments, the animal expresses IgM, IgD, and / or IgA (e.g., functional IgM, IgD, and / or IgA).

[0018] In some embodiments, the animal comprises one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes at an endogenous immunoglobulin heavy chain locus, and in some embodiments, the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operably linked and capable of undergoing VDJ rearrangement. In some embodiments, the animal comprises at least 150 human IGHV genes selected from Table 1, at least 20 human IGHD genes selected from Table 2, and at least 5 human IGHJ genes selected from Table 3. In some embodiments, the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at an endogenous immunoglobulin heavy chain locus on human chromosome 14 in a human subject. In some embodiments, the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at an endogenous immunoglobulin heavy chain locus on human chromosome 14 in a human cell. In some embodiments, the animal is a mouse, and the genetic modification at the animal's endogenous immunoglobulin heavy chain locus comprises a deletion of one or more mouse IGHV genes in Table 4, one or more mouse IGHD genes in Table 5, and / or one or more mouse IGHJ genes in Table 6. In some embodiments, the animal is a mouse, and the genetic modification at the animal's endogenous heavy chain immunoglobulin locus comprises a deletion of the contiguous sequence from the mouse IGHV1-85 gene to the mouse IGHJ4 gene. In some embodiments, the animal comprises unmodified human sequences from a human heavy chain immunoglobulin locus, and in some embodiments, the unmodified human sequences are at least 800 kb. In some embodiments, the animal comprises unmodified human sequences from a human heavy chain immunoglobulin locus from human IGHV(III)-82 to human IGHV1-2. In some embodiments, the animal comprises unmodified human sequences from a human heavy chain immunoglobulin locus from human IGHV(III)-82 to human IGHV6-1.In some embodiments, the animal comprises unmodified human sequences derived from a human heavy chain immunoglobulin locus from human IGHD1-1 to human IGHJ6, hi some embodiments, the animal comprises unmodified human sequences derived from a human heavy chain immunoglobulin locus from human IGHV(III)-82 to human IGHJ6.

[0019] In some embodiments, the genetically modified non-human animals described herein have a genome comprising a replacement of one or more endogenous IGHV, IGHD, and IGHJ genes with one or more human IGHV, IGHD, and IGHJ genes at an endogenous immunoglobulin heavy chain locus, wherein in some embodiments, the human IGHV, IGHD, and IGHJ genes are operably linked to one or more of endogenous IGHM, IGHδ, IGHG1 lacking a sequence encoding a CH1 domain, and IGHA genes. In some embodiments, one or more endogenous IGHV, IGHD, and IGHJ genes are replaced with at least 150 human IGHV genes from Table 1, at least 20 human IGHD genes from Table 2, and at least 5 human IGHJ genes from Table 3. In some embodiments, the animal is a mouse, and at least 180 mouse IGHV genes from Table 4, all of the mouse IGHD genes from Table 5, and all of the mouse IGHJ genes from Table 6 are replaced. In some embodiments, the animal is homozygous for the immunoglobulin heavy chain locus. In some embodiments, the animal is heterozygous for the immunoglobulin heavy chain locus. In some embodiments, the animal comprises an endogenous light chain immunoglobulin locus. In some embodiments, the animal comprises a disruption in an endogenous immunoglobulin light chain locus. In some embodiments, the animal lacks an endogenous immunoglobulin heavy chain variable region locus capable of rearranging and forming nucleic acid sequences encoding endogenous heavy chain variable domains. In some embodiments, the animal is capable of producing humanized antibodies. In some embodiments, the animal is a mammal. In some embodiments, the animal is a rodent. In some embodiments, the animal is a mouse. In some embodiments, the animal has substantially normal B-cell development and maturation.

[0020] In one aspect, the disclosure relates to a cell obtained from a genetically modified non-human animal described herein. In some embodiments, the cell is a B cell that expresses a chimeric immunoglobulin heavy chain comprising an immunoglobulin heavy chain variable domain derived from a rearrangement of one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, and in some embodiments, the immunoglobulin heavy chain variable domain is operably linked to a non-human heavy chain constant region. In some embodiments, the cell is an embryonic stem (ES) cell.

[0021] In one aspect, the present disclosure relates to a method of producing an antibody that specifically binds to an antigen, the method comprising: a) exposing a genetically modified non-human animal described herein to the antigen; b) producing a hybridoma from cells harvested from the animal; and c) harvesting a heavy chain antibody produced by the hybridoma. In some embodiments, the method further comprises sequencing the genome of the hybridoma.

[0022] In one aspect, the present disclosure relates to a method of making an antibody that specifically binds to an antigen, the method comprising: a) exposing a genetically modified non-human animal described herein to the antigen; b) sequencing a nucleic acid encoding a human immunoglobulin heavy chain variable region in a cell that expresses a heavy chain antibody that specifically binds to the antigen; and c) operably linking the nucleic acid encoding the human immunoglobulin heavy chain variable region to a nucleic acid encoding a human immunoglobulin heavy chain constant region in the cell.

[0023] In one aspect, the disclosure relates to a method of making an antibody that specifically binds to an antigen, the method comprising: a) obtaining a nucleic acid sequence encoding a human immunoglobulin heavy chain variable region in a cell that expresses a heavy chain antibody that specifically binds to the antigen, wherein in some embodiments the cell was obtained by exposing a genetically modified non-human animal as described herein to the antigen; b) operably linking the nucleic acid encoding the human immunoglobulin heavy chain variable region to a nucleic acid encoding a human immunoglobulin heavy chain constant region; and c) expressing the nucleic acid in the cell, thereby obtaining the antibody.

[0024] In one aspect, the present disclosure relates to a method of obtaining nucleic acids encoding an antibody binding domain that specifically binds to an antigen, the method comprising: a) exposing a genetically modified non-human animal described herein to the antigen; and b) sequencing nucleic acids encoding a human immunoglobulin heavy chain variable region in a cell that expresses a heavy chain antibody that specifically binds to the antigen.

[0025] In one aspect, the present disclosure relates to a method for generating an antibody that specifically binds to an antigen, the method comprising: a) exposing a genetically modified non-human animal described herein to the antigen; b) constructing a phage plasmid library using RNA prepared from immune cells (e.g., spleen cells) of the animal; c) screening the phage plasmid library; and d) sequencing nucleic acids encoding human immunoglobulin heavy chain variable regions from phage plasmids encoding heavy chain antibodies that specifically bind to the antigen. In some embodiments, screening comprises isolating phage that express immunoglobulin heavy chain variable regions based on binding affinity to the antigen.

[0026] In one aspect, the present disclosure relates to a method of obtaining a sample, the method comprising: a) exposing a genetically modified non-human animal described herein to the antigen; and b) obtaining the sample from the animal. In some embodiments, the sample is an immune cell, lymphoid tissue, spleen tissue, spleen cells, or B cells.

[0027] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to transferrin receptor 1 (TFR1), comprising a heavy chain single variable domain (VHH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein in some embodiments, the VHH CDR1 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a selected VHH CDR1 amino acid sequence, the VHH CDR2 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a selected VHH CDR2 amino acid sequence, and the VHH CDR3 region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a selected VHH CDR2 amino acid sequence. and in some embodiments, the selected VHH CDR1, 2, and 3 amino acid sequences are one of the following: (1) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 42, 43, and 44, respectively; (2) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 45, 46, and 47, respectively; (3) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 48, 49, and 50, respectively; (4) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 51, 52, and 53, respectively; (5) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 54, 55, and 56, respectively; (6) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 57, 58, and 59, respectively; (7) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 60, 61, and 62, respectively; and (8) The selected VHH CDR1, 2, and 3 amino acid sequences are shown in SEQ ID NOs: 63, 64, and 65, respectively.

[0028] In some embodiments, the VHH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 42, 43, and 44, respectively. In some embodiments, the VHH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively. In some embodiments, the VHH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 48, 49, and 50, respectively. In some embodiments, the VHH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 51, 52, and 53, respectively.

[0029] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that binds to TFR1, comprising a heavy chain single variable region (VHH) comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a selected VHH sequence; in some embodiments, the selected VHH sequence is selected from the group consisting of SEQ ID NOs: 66, 67, 68, and 69. In some embodiments, the VHH comprises the sequence of SEQ ID NO: 66. In some embodiments, the VHH comprises the sequence of SEQ ID NO: 67. In some embodiments, the VHH comprises the sequence of SEQ ID NO: 68. In some embodiments, the VHH comprises the sequence of SEQ ID NO: 69. In some embodiments, the antibody or antigen-binding fragment specifically binds to human TFR1, monkey TFR1, mouse TFR1, or chimeric TFR1. In some embodiments, the antibody or antigen-binding fragment is a human or humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a multispecific antibody (e.g., a bispecific antibody).

[0030] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof comprising VHH CDR1, 2, 3 of an antibody or antigen-binding fragment thereof described herein.

[0031] In some embodiments, the antibody or antigen-binding fragment comprises a human IgG Fc (e.g., a human IgG1 Fc). In some embodiments, the human IgG Fc comprises a non-asparagine residue (e.g., an alanine) at position 297 according to EU numbering. In some embodiments, the antibody or antigen-binding fragment comprises two or more heavy chain single variable domains.

[0032] In one aspect, the disclosure relates to a nucleic acid comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein. In some embodiments, the nucleic acid is a cDNA.

[0033] In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids described herein.

[0034] In one aspect, the present disclosure relates to a cell comprising the vector described herein. In some embodiments, the cell is a CHO cell. In one aspect, the present disclosure relates to a cell comprising one or more of the nucleic acids described herein.

[0035] In one aspect, the present disclosure relates to a method for producing an antibody or antigen-binding fragment thereof, the method comprising: (a) culturing a cell described herein under conditions sufficient for the cell to produce the antibody or antigen-binding fragment thereof; and (b) collecting the antibody or antigen-binding fragment thereof produced by the cell.

[0036] In one aspect, the present disclosure relates to an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof described herein covalently attached to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0037] In one aspect, the present disclosure relates to a method of treating a subject having a brain disease (e.g., brain cancer), the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof, or an antibody-drug conjugate described herein. In some embodiments, the antibody or antigen-binding fragment thereof, or antibody-drug conjugate is capable of crossing the blood-brain barrier (BBB) ​​of the subject.

[0038] In one aspect, the present disclosure relates to a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, described herein. In some embodiments, the cancer is brain cancer, lung cancer, gastric cancer, colorectal cancer, liver cancer, ovarian cancer, prostate cancer, leukemia, or breast cancer. In one aspect, the present disclosure relates to a method of identifying a subject as having a brain disease (e.g., brain cancer), the method comprising detecting a sample obtained from the subject with an antibody or antigen-binding fragment thereof described herein, thereby identifying the subject as having the brain disease. In some embodiments, the sample is a brain parenchyma sample from the subject. In some embodiments, the subject described herein is a human subject.

[0039] In one aspect, the present disclosure relates to a method of delivering an agent across the blood-brain barrier, the method comprising administering to a subject an agent covalently linked to an antibody or antigen-binding fragment thereof described herein. In some embodiments, the agent is an antibody or antibody-drug conjugate. In some embodiments, the agent is an anti-amyloid antibody.

[0040] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier. In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody drug conjugate described herein and a pharmaceutically acceptable carrier.

[0041] In one aspect, the present disclosure relates to antibodies, or antigen-binding fragments thereof, that cross-compete with the antibodies, or antigen-binding fragments thereof, described herein.

[0042] In one aspect, the disclosure provides a method of making an antibody that specifically binds to an antigen, the method comprising exposing an animal described herein to the antigen, obtaining (e.g., by sequencing) the sequence of a nucleic acid encoding a human heavy chain immunoglobulin variable region in a cell that expresses a chimeric heavy chain antibody that specifically binds to the antigen, and operably linking the nucleic acid encoding the human heavy chain immunoglobulin variable region to a nucleic acid encoding a human heavy chain immunoglobulin constant region in the cell.

[0043] The present disclosure also relates to progeny of the non-human mammal. In some embodiments, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

[0044] The present disclosure also provides cells comprising the targeting vectors described herein. The present disclosure also relates to cells (e.g., stem cells, embryonic stem cells, immune cells, B cells, T cells, or hybridomas) or cell lines derived from non-human mammals or their descendants, or primary cell cultures thereof. The present disclosure further relates to tissues, organs, or cultures thereof derived from non-human mammals or their descendants.

[0045] The present disclosure further relates to the use of non-human mammals or their progeny, animal models produced by the methods described herein, in the development of products related to the immunization process, the production of human antibodies, or in model systems for the study of pharmacology, immunology, microbiology, and medicine.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials well known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0047] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0048] [Figure 1] Schematic diagram of a heavy chain antibody. [Figure 2] Schematic structures of IgM, IgD, IgG, IgE, and IgA immunoglobulin isotypes are shown. Mouse and human immunoglobulin heavy chain locus genes are aligned and labeled with corresponding names. [Figure 3] 1 is a schematic diagram showing the constant region genes of the mouse immunoglobulin heavy chain locus, showing the Cγ1 gene structure, including exons CH1, H, CH2, CH3, M1, and M2. [Figure 4] 1 shows mutant alleles after genetic modification at the mouse immunoglobulin heavy chain constant region locus. [Figure 5] The gene structure of Cγ1ΔCH1 is shown, where the CH1 coding region is either deleted (left) or replaced with a Neo cassette (right). [Figure 6] Example 3 shows the workflow of gene modification using targeting vector V1. The 100,883-bp sequence from Sγ3 to Cε in the mouse immunoglobulin heavy chain constant region locus was replaced in a single step with a 16,076-bp sequence containing mouse Sγ1 and Cγ1ΔCH1 knock-in sequences to generate mutant allele 1. [Figure 7] Example 3 shows the workflow of gene modification using targeting vector V2. The 92,859-bp sequence from Cγ3 to Cε in the mouse immunoglobulin heavy chain constant region locus was replaced in a single step with a sequence containing the Cγ1ΔCH1 knock-in sequence to generate mutant allele 1′. [Figure 8] The workflow for gene modification using targeting vector V3 is shown in Example 3. A 16,434-bp sequence containing Cμ and Cδ was knocked out from mutant allele 1 to generate mutant allele 2. [Figure 9] The workflow for gene modification using targeting vector V4 is shown in Example 3. The sequence containing Cμ, Cδ, and Sγ1 was knocked out from mutant allele 1 to generate mutant allele 2′, so that Sμ and Cγ1ΔCH1′ were directly connected. [Figure 10]

[0039] Figure 3 shows the workflow of gene modification using targeting vector V5 (Example 3). The CH1 coding sequence in Cμ was knocked out from mutant allele 1 to generate mutant allele 3. [Figure 11] The workflow of gene modification using targeting vector V6 is shown in Example 3. The CH1 coding sequence in Cμ and the entire Cδ were knocked out from mutant allele 1 to generate mutant allele 4. [Figure 12] The workflow of gene modification using targeting vector V7 is shown in Example 3. The CH1 coding sequence in Cμ and the CH1 coding sequence in Cδ were knocked out from mutant allele 1 to generate mutant allele 5. [Figure 13] Figure 1 shows the results of a PCR assay using primer pairs L-GT-F1 / L-GT-R1 and R-GT-F2 / R-GT-R2, respectively, to verify the genotype of mutant allele 1. WT is the wild-type control. HO is the blank control. M is the marker. [Figure 14](A) Southern blot results of mutant allele 1-positive clones digested with BclI and hybridized with the LR probe. M is a marker. WT is wild-type. (B) Southern blot results of mutant allele 1-positive clones digested with ScaI and hybridized with the 3' probe. M is a marker. WT is wild-type. (C) Southern blot results of mutant allele 1-positive clones digested with XmnI and hybridized with the A probe. M is a marker. WT is wild-type. (D) Southern blot results of mutant allele 1-positive clones digested with BglII and hybridized with the 5' probe. M is a marker. WT is wild-type. [Figure 15] 1 shows the results of a PCR assay using primers DE-F1 and DE-R1 to verify knockout of the Cμ to Cδ sequence in mutant allele 2. [Figure 16] Shown are the results of a PCR assay using primers GT-Mut-F, GT-Mut-R, and GT-WT-R to verify knockout of the sequence from Cμ to Sγ1 in the mutant allele 2′. [Figure 17] 1 shows the results of a PCR assay using primers GT-3F and GT-3R to verify knockout of the CH1 coding sequence of Cμ in mutant allele 3. [Figure 18A] Figure 1 shows the results of a PCR assay using primers Mut-F and Mut-R to verify the sequence of CμΔCH1 in mutant allele 4. WT is the wild-type control. HO is the blank control. [Figure 18B] 1 shows the results of a PCR assay using primers F4 and R4 to verify the absence of Cδ in mutant allele 4. [Figure 19] Figure 1 shows the results of a PCR assay using primer pairs Mut-F / Mut-R and F3 / R3 to verify the sequences of CμΔCH1 and CδΔCH1 in mutant allele 5. WT is the wild-type control. HO is the blank control. [Figure 20]1 is an exemplary flow chart of a method for introducing human immunoglobulin genes into the mouse genome. [Figure 21] 1 is an overview of replacing mouse immunoglobulin heavy chain variable region locus sequences with human immunoglobulin heavy chain variable region locus sequences. [Figure 22] 1 shows the length distribution of CDR3 in the heavy chain variable region of antibodies produced by immunizing heterozygous Mut3 mice with antigen A. [Figure 23] 1 shows germline gene usage of variable region genes in mice heterozygous for the heavy chain mutant allele 3 genotype. [Figure 24] 1 shows the distribution of KD values ​​of antibodies raised against antigen A in heterozygous Mut3 mice (H / -). [Figure 25] 1 shows the distribution of KD values ​​of antibodies produced in homozygous Mut2 mice against antigen A. [Figure 26] 1 shows germline gene usage of variable region genes in homozygous Mut2 mice. [Figure 27] FIG. 1 is a schematic diagram showing the human immunoglobulin heavy chain (IGH) locus on chromosome 14 (14q32.33). [Figure 28-1] FIG. 1 is a schematic diagram showing the IGH locus on chromosome 12 (12F2) of the mouse (Mus musculus) (strain C57BL / 6). [Figure 28-2] Same as above [Figure 29-1] The IMGT repertoire of the human heavy chain immunoglobulin locus (IGH) is listed. [Figure 29-2] Same as above [Figure 29-3] Same as above [Figure 29-4] Same as above [Figure 29-5] Same as above [Figure 30-1] List the IMGT repertoire of mouse IGH. [Figure 30-2] Same as above [Figure 30-3] Same as above [Figure 30-4] Same as above [Figure 30-5] Same as above [Figure 30-6] Same as above [Figure 30-7] Same as above [Figure 30-8] Same as above [Figure 31-1] The sequences described in this disclosure are listed below. [Figure 31-2] Same as above [Figure 31-3] Same as above [Figure 31-4] Same as above [Figure 31-5] Same as above [Figure 31-6] Same as above [Figure 32] Figure 1 shows Western blot results of serum IgG levels in mice with mutant allele 2', mutant allele 3, and mutant allele 4 genotypes, respectively. Biot.Ladder is a protein marker. WT represents wild-type mice. [Figure 33] 1 shows the length distribution of CDR3 in the heavy chain variable region of antibodies produced by immunizing heterozygous Mut3 mice with human 4-1BB. [Figure 34] 1 shows germline gene usage of variable region genes in mice homozygous for the heavy chain mutant allele 3 genotype. [Figure 35] 1 shows the length distribution of CDR3 in the heavy chain variable region of antibodies produced by immunizing heterozygous Mut3 mice with human CD3ED and cynomolgus CD3ED. [Figure 36] 1 shows germline gene usage of variable region genes in mice homozygous for the heavy chain mutant allele 3 genotype. [Figure 37] The CDR sequences of the heavy chain variable regions of anti-TFR1 antibodies are listed according to Kabat numbering. [Figure 38] The CDR sequences of the heavy chain variable regions of anti-TFR1 antibodies are listed according to IMGT numbering. [Figure 39] The amino acid sequences of the heavy chain variable regions of anti-TFR1 antibodies are listed below. [Figure 40A]1 shows antibody concentrations in total brain protein of hTFR1 mice within 72 hours after intravenous (iv) administration of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6). [Figure 40B] This shows the ratio of antibody concentration in total brain protein to serum antibody concentration in hTFR1 mice within 72 hours after intravenous (iv) administration of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6). [Figure 40C] 1 shows antibody concentrations in the brain parenchyma of hTFR1 mice within 72 hours after intravenous (iv) administration of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6). [Figure 40D] 1 shows the ratio of antibody concentration in the brain parenchyma to serum antibody concentration in hTFR1 mice within 72 hours after intravenous (iv) administration of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6). [Figure 41] (A) shows the results of antibody concentration tests in the brain parenchyma 24 hours after intravenous (iv) administration of hIgG1(G1), JR141-N(G2), 23B8-N(G3), 24A1-N(G4), or 24G5-N(G5). (B) shows the results of antibody concentration tests in total brain protein (whole brain) 24 hours after intravenous (iv) administration of hIgG1(G1), JR141-N(G2), 23B8-N(G3), 24A1-N(G4), or 24G5-N(G5). [Figure 42] The results of antibody concentrations 6 hours or 24 hours after intravenous (iv) administration of JR141-N (G2 to G4) or 24G5-N (G5 to G7) are shown. hIgG1 was used as a negative control. [Figure 43] 1 shows germline gene usage of variable region genes in mice homozygous for the heavy chain mutant allele 3 genotype. [Figure 44]1 shows germline gene usage of variable region genes in heterozygous Mut2' mice (H / -). [Figure 45] 1 shows the distribution of KD values ​​of antibodies produced in heterozygous Mut2' mice (H / -) against human serum albumin. [Figure 46] 1 shows germline gene usage of variable region genes in mice homozygous for the heavy chain mutant allele 4 genotype. [Figure 47] 1 shows germline gene usage of variable region genes in mice homozygous for the heavy chain mutant allele 5 genotype. DETAILED DESCRIPTION OF THE INVENTION

[0049] The present disclosure relates to genetically modified animals and methods for producing heavy chain antibodies.

[0050] Heavy-chain antibodies (or heavy-chain-only antibodies) are antibodies that have only heavy chains (typically two heavy chains) and lack the two light chains typically found in antibodies. Naturally occurring heavy-chain antibodies have been found in cartilaginous fish (e.g., sharks) and camelids (e.g., llamas). For example, in cartilaginous fish, the immunoglobulin new antigen receptor (IgNAR) is a heavy-chain antibody. IgNARs have significant structural differences from other antibodies: they have five constant domains (CH) per chain instead of the usual three, several disulfide bonds in unusual positions, and the complementarity-determining region 3 (CDR3) forms an extended loop that covers the binding site for the light chain of other antibodies. These differences, combined with the phylogenetic age of cartilaginous fish, have led to the hypothesis that IgNARs may be more closely related to primitive antigen-binding proteins than mammalian immunoglobulins.

[0051] The only mammals with heavy-chain (IgG-like) antibodies are camelids, such as dromedaries, camels, llamas, and alpacas. Like all mammals, camelids (such as llamas) can produce conventional antibodies (e.g., IgG1) consisting of two heavy chains and two light chains linked in a Y-shape by disulfide bonds. However, they also produce two unique subclasses of IgG (also called heavy-chain IgGs): IgG2 and IgG3. These antibodies lack the CH1 region but still consist of only two heavy chains with antigen-binding domains (e.g., VHH) at their N-termini. Conventional IgGs require the combination of both heavy and light chain variable regions to enable a high diversity of antigen-antibody interactions. While single heavy and light chains still exhibit this ability, they exhibit significantly lower affinity compared to paired heavy and light chains. A unique feature of heavy-chain IgGs is the ability of their monomeric antigen-binding regions to bind antigens with specificity, affinity, and especially diversity comparable to conventional antibodies, without the need for pairing. This feature is mainly due to several key mutations in the amino acid sequences of the variable regions of the two heavy chains, which cause major structural changes compared to conventional Igs. The key substitutions in the variable regions not only prevent the light chains from binding to the heavy chains, but also prevent the unbound heavy chains from being recycled by immunoglobulin-binding proteins.

[0052] These heavy-chain antibody single variable domains (designated VHHs, sdAbs, or nanobodies) are the smallest antigen-binding domains generated by the adaptive immune system. It is well known that the complementarity-determining region 3 (CDR3) of the variable region of these antibodies is twice as long as that of conventional antibodies. This results in an expanded interaction surface with antigens, increasing the diversity of antigen-antibody interactions and compensating for the lack of light chains. The long CDR3 allows VHHs to reach crevices on proteins inaccessible to conventional antibodies, including functionally interesting sites such as the active site of an enzyme or the receptor-binding canyon on the surface of a virus. Furthermore, the addition of cysteine ​​residues makes the structure more stable, thus enhancing the strength of the interaction.

[0053] Compared to conventional antibodies with conventional antibody variable domains (VH and VL), VHHs offer numerous other advantages, including higher stability, solubility, expression yield, and refolding ability, as well as better in vivo tissue penetration. Furthermore, in contrast to the VH domain of conventional antibodies, VHHs exhibit no intrinsic tendency to bind to light chains. This facilitates the induction of heavy-chain antibodies in the presence of a functional light chain locus. Furthermore, because VHHs do not bind to VL domains, it is much easier to reformat VHHs into bispecific antibody constructs than constructs containing conventional VH-VL pairs or single domains based on VH domains.

[0054] A notable difference between camelid VHH and human VH domains is the length and orientation of the CDR3 loop. CDR3 corresponds to a unique region of an antibody molecule encoded by newly generated DNA elements during B cell development. Genetic recombination results in the fusion of the D element with adjacent V and J elements. During recombination, additional genetic diversity is generated by the addition and / or deletion of nucleotides at the junctions. Thus, the CDR3 loop provides a major contribution to antibody diversity and specificity. Some early transgenic heavy chain antibody animals have a limited number of variable region genes (IGHV, IGHD, and IGHJ), resulting in the inability to recognize some antigens despite the robust antigen response of wild-type animals (Janssens, Rick, et al. "Generation of heavy-chain-only antibodies in mice." Proceedings of the National Academy of Sciences 103.41 (2006):15130-15135). The present disclosure provides genetically modified animals with a fully human heavy chain antibody repertoire. Therefore, the variable domains produced by these animals can have the greatest possible diversity relative to human heavy chain variable domains, thereby maximizing the chances of obtaining fully humanized heavy chain antibodies.

[0055] Furthermore, because the entire sequence of the human immunoglobulin locus is introduced into the animal genome (without or only limited modifications), these genes can undergo V(D)J recombination in a manner very similar to that occurring in humans, reducing the risk of generating new immunogenic epitopes that can be recognized by the human immune system and thereby reducing immunogenicity. Immunogenicity can lead to the generation of anti-drug antibodies, potentially including efficacy. Here, the endogenous IGHV, IGHD, and IGHJ genes are effectively deleted. Antibodies generated by the antibody repertoire are unlikely to be immunogenic in humans. Furthermore, antibody production can be highly efficient, and due to efficient V(D)J recombination, production rates are similar to those of normal antibodies. Therefore, these antibodies are more suitable as therapeutic agents for humans. Therefore, genetically modified animals provide an advantageous platform for producing humanized heavy chain antibodies.

[0056] Furthermore, IgG1 is the most abundant antibody subtype in serum, has a long serum half-life, strong FcγR affinity, and possesses antibody-dependent cellular cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and other activities. IgG1 has unique advantages in the field of antibody drug development. Therefore, in one aspect, the present disclosure particularly relates to the preparation of humanized mice capable of producing heavy chain antibodies of the IgG1 subtype. Meanwhile, the coding sequences of all other IgG subtypes can be deleted. This creates an efficient and reliable platform for producing heavy chain antibodies in animals.

[0057] As used herein, the term "antibody" refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementarity-determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, heavy-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain the Fc region of a human antibody. The term antibody also includes derivatives, e.g., bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0058] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody, which portion is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0059] As used herein, the term "human antibody" means an antibody encoded by nucleic acid present in a human (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, a human antibody is recovered from a human or produced in human cell culture (e.g., in human hybridoma cells). In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line). In some embodiments, a human antibody is produced in a bacterial cell or yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a mouse) containing unrearranged or rearranged human immunoglobulin loci (e.g., a heavy or light chain human immunoglobulin locus).

[0060] As used herein, the term "chimeric antibody" refers to an antibody that contains sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human and murine antibodies). Non-limiting examples of chimeric antibodies include antibodies that contain variable domain sequences (e.g., all or part of the light and / or heavy chain variable domain sequences) of a human antibody and the constant domain of a non-human antibody. Further examples of chimeric antibodies are described herein and are well known in the art.

[0061] As used herein, the term "humanized antibody" means a non-human antibody that contains sequences derived from non-human (e.g., murine) immunoglobulin and that contains sequences derived from human immunoglobulin.

[0062] As used herein, the term "single-chain antibody" means a single polypeptide containing at least two immunoglobulin variable domains (e.g., the variable domains of a mammalian immunoglobulin heavy or light chain) that are capable of specifically binding to an antigen.

[0063] As used herein, the term "heavy chain antibody" refers to an antibody molecule that is composed only of heavy chains (usually two) and does not have any light chains.

[0064] As used herein, the term "VHH" refers to a variable domain derived from a heavy chain antibody. A VHH can specifically recognize an antigen without necessarily being paired with a VL. In some embodiments, a VHH (also referred to as an sdAb or nanobody) described herein is derived from any of the humanized heavy chain antibodies described herein. In some embodiments, a VHH, sdAb, or nanobody described herein is derived from a heavy chain antibody produced by any of the genetically modified non-human animals described herein.

[0065] As used herein, the terms "subject" and "patient" are used interchangeably throughout and refer to an animal, human, or non-human. Veterinary and non-veterinary applications are contemplated by the present disclosure. A human patient can be an adult human or a juvenile human (e.g., a human under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, patients include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pigs, minipigs), equines, canines, felines, bovines, and other domestic, livestock, and zoo animals.

[0066] As used herein, the phrases "specifically bind" and "specifically binds" when referring to an antibody mean that the antibody preferably interacts with the target molecule in preference to other molecules because the interaction is dependent on the presence of a particular structure (i.e., an antigenic determinant or epitope) on the target molecule; in other words, the reagent generally recognizes and binds to molecules containing a particular structure rather than whole molecules. An antibody that specifically binds to a target molecule can also be referred to as a target-specific antibody.

[0067] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers of amino acids of any length, of at least two amino acids.

[0068] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein and refer to polymers of nucleotides of any length, of at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0069] As used herein, the term "unmodified human sequence" means a sequence derived from a human subject, human cell, cultured human cell, or human cell line, which sequence is identical to a gene sequence in the human subject, human cell, cultured human cell, or human cell line.

[0070] Immunoglobulin heavy chain constant region locus The heavy chain immunoglobulin locus (also called IGH or immunoglobulin heavy chain locus) is a region on a chromosome (e.g., mouse chromosome 12) that contains the genes for the heavy chain of an antibody (or immunoglobulin). It includes sequences encoding the heavy chain variable region and the heavy chain constant region.

[0071] 2, mouse immunoglobulin heavy chain constant region genes include (in the following order): immunoglobulin heavy constant μ (IGHM, or Cμ), immunoglobulin heavy constant δ (IGHδ, or Cδ), immunoglobulin heavy constant gamma 3 (IGHG3, or Cγ3), immunoglobulin heavy constant gamma 1 (IGHG1, or Cγ1), immunoglobulin heavy constant gamma 2b (IGHG2b, or Cγ2b), immunoglobulin heavy constant gamma 2c (IGHG2c, or Cγ2c), immunoglobulin heavy constant epsilon (IGHE, or Cε), and immunoglobulin heavy constant alpha (IGHA, or Cα) genes. In some embodiments, immunoglobulin heavy constant gamma 2a (IGHG2a) is in place of IGHG2c. In contrast, human immunoglobulin constant region genes include (shown in the following order): immunoglobulin heavy constant μ (IGHM, or Cμ), immunoglobulin heavy constant delta (IGHδ, Cδ), immunoglobulin heavy constant gamma 3 (IGHG3, or Cγ3), immunoglobulin heavy constant gamma 1 (IGHG1, or Cγ1), immunoglobulin heavy constant epsilon P1 (pseudogene) (IGHEP1, or ψCε), immunoglobulin heavy constant alpha 1 (IGHA1, or Cα1), immunoglobulin heavy constant gamma P (non-functional) (IGHGP, or CγP, not shown), immunoglobulin heavy constant gamma 2 (IGHG2, or Cγ2), immunoglobulin heavy constant gamma 4 (IGHG4, Cγ4), immunoglobulin heavy constant epsilon (IGHE, or Cε), and immunoglobulin heavy constant alpha 2 (IGHA2, or Cα2) genes.

[0072] Immunoglobulin class switching (or isotype switching, or isotype exchange, or class switch recombination (CSR)) is a biological mechanism that changes the antibody production of B cells from one class to another (e.g., from isotype IgM to isotype IgG). During this process, the constant region portion of the antibody heavy chain changes, but the variable region of the heavy chain remains the same. Because the variable region does not change, class switching does not affect antigen specificity. Instead, the antibody can interact with different effector molecules while retaining affinity for the same antigen. This allows different daughter cells from the same activated B cell to produce antibodies of different isotypes or subtypes (e.g., IgG1, IgG2, etc.). Class switching occurs through a mechanism called class switch recombination (CSR). Class switch recombination is a biological mechanism that allows activated B cells to change the class of antibody produced during a process called isotype switching or class switching. In CSR, a portion of the antibody heavy chain locus is removed from the chromosome, and the gene segments surrounding the deleted portion are recombined, retaining functional antibody genes that can produce antibodies of different isotypes. Double-stranded breaks are generated in DNA at conserved nucleotide motifs called switch (S) regions, located upstream of gene segments encoding antibody heavy chain constant regions. These occur adjacent to all heavy chain constant region genes except Cδ. The DNA is nicked and destroyed at two selected switch regions by the activity of a series of enzymes, including activation-induced (cytidine) deaminase (AID), uracil DNA glycosylase, and apyrimidinic / purinic (AP) endonuclease. The intervening DNA between the switch regions is then excised from the chromosome, removing, for example, unwanted Cμ or Cδ heavy chain constant region sequences and allowing replacement with Cγ, Cα, or Cε constant region gene segments. The free ends of the DNA are rejoined by a process called nonhomologous end joining (NHEJ) to link the variable domain exon to the desired downstream constant domain exon of the antibody heavy chain. In the absence of nonhomologous end joining, the free ends of the DNA can be rejoined by an alternative pathway biased toward microhomology joining.With the exception of the Cμ and Cδ genes, only one antibody class is expressed by a B cell at any given time. Figure 3 shows the location of each switch region (e.g., Sμ, Sγ3, Sγ1, Sγ2b, Sγ2c, Sε, and Sα) in the mouse immunoglobulin heavy chain constant region locus. Specifically, the Cγ1 gene contains exons encoding the CH1 domain, hinge region, CH2 domain, CH3 domain, and two transmembrane domains. The exons are labeled CH1, H, CH2, CH3, M1, and M2, respectively.

[0073] The five major classes of immunoglobulins are IgM, IgD, IgG, IgE, and IgA, each of which exists as a transmembrane antigen receptor or a secreted antibody. In humans, IgG is divided into four subclasses (IgG1, IgG2, IgG3, and IgG4) named in descending order of their abundance in serum, and IgA antibodies are divided into two subclasses (IgA1 and IgA2). The different heavy chains that define these classes are called isotypes and are designated by the lowercase Greek letters μ (IgM), δ (IgD), γ (IgG), ε (IgE), and α (IgA), respectively.

[0074] Although the location and number of disulfide bonds differ among IgG1–IgG4 subtypes, the structures of the four IgG subtypes are very similar. Indeed, as the most abundant IgG subtype in plasma, IgG1 is widely used to generate recombinant therapeutic antibodies. In contrast, IgG3 is rarely used in antibody drug development due to its weak binding affinity to FcRn and short half-life (approximately 9 days). Therefore, IgG3-based antibody drugs require more frequent administration for pharmacokinetic reasons. Furthermore, antibody levels of different subtypes may change during physical development, as described, for example, in Elena Blanco et al., “Age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood,” Journal of Allergy and Clinical Immunology, Volume 141, Issue 6 (2018), incorporated herein by reference in its entirety. IgG1 is the most abundant antibody subtype in serum, has a long serum half-life, strong FcγR affinity, and possesses antibody-dependent cellular cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and other activities. IgG1 has unique advantages in the field of antibody drug development. Thus, in one aspect, the present disclosure relates to the preparation of humanized mice capable of producing heavy chain antibodies of the IgG1 subtype. In one aspect, the present disclosure particularly relates to the preparation of humanized mice capable of producing heavy chain antibodies of the IgG1 subtype. In some embodiments, heavy chain antibodies can be further processed to generate nanobodies.

[0075] In one aspect, the present disclosure relates to a genetically modified non-human animal comprising an engineered immunoglobulin heavy chain locus, wherein said engineered immunoglobulin heavy chain locus comprises an IgG constant region gene, wherein said IgG constant region gene encodes an IgG heavy chain constant region lacking a CH1 domain, and wherein said genetically modified non-human animal expresses a heavy chain antibody.

[0076] In some embodiments, the IgG constant region gene is one of IGHG3, IGHG1, IGHG2a, IGHG2b, and IGHG2c. In some embodiments, the modified immunoglobulin heavy chain locus has one, two, three, four, or five IgG constant region genes, none of which encode a CH1 domain. In some embodiments, only one, two, or three IGHG genes do not encode a CH1 domain, and at least one, two, or three remaining IGHG genes can encode a CH1 domain. In some embodiments, the modified immunoglobulin heavy chain locus has only one (e.g., exactly one) IgG constant region gene, e.g., IGHG1. In some embodiments, the modified immunoglobulin heavy chain locus does not include an IGHG3, IGHG2a, IGHG2b, and / or IGHG2c gene.

[0077] In some embodiments, the modified immunoglobulin heavy chain locus has only one IGHG gene (e.g., IGHG3, IGHG1, IGHG2a, IGHG2b, or IGHG2c) and the sequence encoding the CH1 domain within IGHG is deleted. In some embodiments, the IGHG gene is operably linked to Sγ3, Sγ1, Sγ2a, Sγ2b, or Sγ2c (e.g., Sγ3 or Sγ1).

[0078] In some embodiments, IGHM is an intact, functional endogenous IGHM gene. In some embodiments, the sequence encoding the CH1 domain in IGHM is deleted. In some embodiments, IGHM is deleted. In some embodiments, IGHδ is an intact, functional endogenous IGHδ gene. In some embodiments, the sequence encoding the CH1 domain in IGHδ is deleted. In some embodiments, IGHδ is deleted. In some embodiments, both IGHM and IGHδ are deleted.

[0079] In some embodiments, the IGHE is an intact, functional endogenous IGHE gene. In some embodiments, the sequence encoding the CH1 domain within IGHE is deleted. In some embodiments, the IGHE is deleted.

[0080] In some embodiments, IGHA is an intact, functional endogenous IGHA gene. In some embodiments, the sequence encoding the CH1 domain within IGHA is deleted. In some embodiments, IGHA is deleted.

[0081] In various embodiments, the humanized heavy chain antibody comprises a unique immunoglobulin constant region (Fc) lacking at least the CH1 domain. In some embodiments, the hinge region of the human Fc is also lacking. In some embodiments, the heavy chain antibody comprises the CH2 and CH3 regions of the immunoglobulin G (IgG) heavy chain constant region. In some embodiments, the constant region of the heavy chain antibody comprises the hinge, CH2, and CH3 regions of the IgG heavy chain Fc.

[0082] In some embodiments, an appropriate number of rearranged heavy chain variable regions is required to effectively survive selection when presented during B cell development. In one aspect, the present disclosure provides transgenic animals comprising a germline genetic modification comprising a deletion of a nucleotide sequence encoding the CH1 domain of IgG, wherein the animal expresses functional IgM, and the animal expresses IgG heavy chain antibodies (e.g., comprising IgG1 heavy chain CH2 and CH3 domains) in serum. In some embodiments, the IgM comprises two heavy chains, which recognize antigens in association with two lambda or kappa light chains. In some embodiments, the functional IgM comprises a CH1 domain. In some embodiments, the functional IgM does not comprise a CH1 domain. Without wishing to be bound by any theory, it is believed that deleting the sequence encoding the CH1 domain from the endogenous IGHM gene does not substantially alter IgM function.

[0083] In some embodiments, the modified immunoglobulin heavy chain constant region locus comprises a modified IGHδ gene that lacks a sequence encoding a CH1 domain. In some embodiments, the modified IGHδ expresses functional IgD. Without being bound by any theory, it is believed that deleting the sequence encoding the CH1 domain from the endogenous IGHδ gene does not substantially alter IgD function.

[0084] Immunoglobulin heavy chain variable region locus To produce humanized heavy chain antibodies (HCAbs), an animal's heavy chain immunoglobulin variable region locus can be humanized. The heavy chain immunoglobulin variable region represents the germline organization of the heavy chain locus. The locus includes V (variable), D (diversity), J (joining), and C (constant) segments. Genes in the V region form the V gene cluster (also called the IGHV gene cluster). Genes in the D region form the D gene cluster (also called the IGHD gene cluster). Genes in the J region form the J gene cluster (also called the IGHJ gene cluster).

[0085] During B cell development, a recombination event at the DNA level joins a single D segment (also called the IGHD gene) to a J segment (also called the IGHJ gene), and then the fused DJ exons of this partially rearranged DJ region are joined to a V segment (also called the IGHV gene). The rearranged VDJ region containing the fused VDJ exons is then transcribed and fused to the IGHM constant region at the RNA level, and this transcript encodes the μ heavy chain. Later in development, B cells produce VDJ-Cμ-Cδ pre-messenger RNA, which is alternatively spliced ​​to encode either the μ or δ heavy chain. Mature B cells in lymph nodes undergo switch recombination, whereby the fused VDJ gene segment is adjacent to one of the IGHG, IGHA, or IGHE gene segments, allowing each cell to express either the gamma, alpha, or epsilon heavy chain. Potential recombination between many different IGHV genes and several IGHJ genes provides a broad range of antigen recognition. Further diversity is achieved by junctional diversity resulting from random addition of nucleotides by terminal deoxynucleotidyl transferase and by somatic hypermutation that occurs during B-cell maturation in the spleen and lymph nodes. Some V, D, J, and C segments are known to be unable to code for proteins and are considered pseudogene segments (often simply called pseudogenes).

[0086] The human heavy chain immunoglobulin locus is located on human chromosome 14 (Figures 27 and 29). Table 1 lists the IGHV genes and their relative order at this locus.

[0087] [Table 1] TIFF2025532609000003.tif238161

[0088] RPS8P1, ADAM6, and KIAA0125 are also located at this locus. The relative order of RPS8P1 is 160, the relative order of ADAM6 is 161, and the relative order of KIAA0125 is 164. Table 2 lists all IGHD genes on human chromosome 14 and their relative order. Table 3 lists all IGHJ genes on human chromosome 14 and their relative order. Genes for immunoglobulin constant domains are located after the IGHV, IGHD, and IGHJ genes. These genes include (in the following order): immunoglobulin heavy constant μ (IGHM), immunoglobulin heavy constant delta (IGHδ), immunoglobulin heavy constant gamma 3 (IGHG3), immunoglobulin heavy constant gamma 1 (IGHG1), immunoglobulin heavy constant epsilon P1 (pseudogene) (IGHEP1), immunoglobulin heavy constant alpha 1 (IGHA1), immunoglobulin heavy constant gamma P (nonfunctional) (IGHGP), immunoglobulin heavy constant gamma 2 (IGHG2), immunoglobulin heavy constant gamma 4 (IGHG4), immunoglobulin heavy constant epsilon (IGHE), and immunoglobulin heavy constant alpha 2 (IGHA2). These genes and the order of these genes are also shown in Figures 27 and 29.

[0089] [Table 2]

[0090] [Table 3]

[0091] The mouse heavy chain immunoglobulin locus is located on mouse chromosome 12 (Figures 28 and 30). Table 4 lists the IGHV genes and their relative order at this locus.

[0092] [Table 4] TIFF2025532609000007.tif96161

[0093] Table 5 lists all IGHD genes on mouse chromosome 12 and their relative order. Table 6 lists all IGHJ genes on mouse chromosome 12 and their relative order. Genes for the immunoglobulin constant domain are located after the IGHV, IGHD, and IGHJ genes. These genes include (in the following order): immunoglobulin heavy constant μ (IGHM), immunoglobulin heavy constant delta (IGHδ), immunoglobulin heavy constant gamma 3 (IGHG3), immunoglobulin heavy constant gamma 1 (IGHG1), immunoglobulin heavy constant gamma 2b (IGHG2b), immunoglobulin heavy constant gamma 2c (IGHG2c), immunoglobulin heavy constant epsilon (IGHE), and immunoglobulin heavy constant alpha (IGHA) genes. In some embodiments, immunoglobulin heavy constant gamma 2a (IGHG2a) is located at the IGHG2c position. These genes and their order are also shown in Figures 28 and 30.

[0094] [Table 5]

[0095] [Table 6]

[0096] The present disclosure provides genetically modified non-human animals comprising one or more human IGHV genes, one or more human IGHD genes, and / or one or more human IGHJ genes. In some embodiments, the human IGHV genes, human IGHD genes, and human IGHJ genes are operably linked and capable of undergoing VDJ rearrangement. In some embodiments, the human IGHV genes, human IGHD genes, and human IGHJ genes are located at endogenous heavy chain immunoglobulin loci.

[0097] In some embodiments, the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, or 161 human IGHV genes (e.g., genes shown in Table 1).

[0098] In some embodiments, the animal comprises about or at least 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, or 161 human IGHV genes selected from Table 1, about or at least 20, 21, 22, 23, 24, 25, 26, or 27 human IGHD genes selected from Table 2, and about or at least 5, 6, 7, 8, or 9 human IGHJ genes selected from Table 3. In some embodiments, the animal comprises all of the human IGHV genes in Table 1 except IGHV2-10, IGHV3-9, and IGHV1-8, all of the human IGHD genes in Table 2, and all of the human IGHJ genes in Table 3. In some embodiments, the animal comprises all of the human IGHV genes in Table 1 except for IGHV5-10-1 and IGHV3-64D, all of the human IGHD genes in Table 2, and all of the human IGHJ genes in Table 3. In some embodiments, the animal comprises all of the human IGHV genes, all of the human IGHD genes, and all of the human IGHJ genes at an endogenous heavy chain immunoglobulin locus on human chromosome 14 in a human subject. In some embodiments, the animal comprises all of the human IGHV genes, all of the human IGHD genes, and all of the human IGHJ genes at an endogenous heavy chain immunoglobulin locus on human chromosome 14 in a human cell (e.g., a somatic cell, a cultured cell, a non-immune cell, a cell without any V(D)J rearrangement).

[0099] In some embodiments, the animal comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes selected from IGHV(III)-82, IGHV7-81, IGHV4-80, IGHV3-79, IGHV(II)-78-1, IGHV5-78, IGHV7-77, IGHV(III)-76-1, IGHV3-76, and IGHV3-75.

[0100] In some embodiments, the animal comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes selected from IGHV(III)-5-2, IGHV(III)-5-1, IGHV2-5, IGHV7-4-1, IGHV4-4, IGHV1-3, IGHV(III)-2-1, IGHV1-2, IGHV(II)-1-1, and IGHV6-1.

[0101] In some embodiments, the animal comprises an unmodified human sequence that includes a sequence beginning with a gene selected from IGHV(III)-82, IGHV7-81, IGHV4-80, IGHV3-79, IGHV(II)-78-1, IGHV5-78, IGHV7-77, IGHV(III)-76-1, IGHV3-76, and IGHV3-75, and ending with a gene selected from IGHV(III)-5-2, IGHV(III)-5-1, IGHV2-5, IGHV7-4-1, IGHV4-4, IGHV1-3, IGHV(III)-2-1, IGHV1-2, IGHV(II)-1-1, and IGHV6-1. In some embodiments, the unmodified human sequence is derived from a human heavy chain immunoglobulin locus from human IGHV(III)-82 through human IGHV1-2. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGHV(III)-82 to human IGHV(II)-1-1. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGHV(III)-82 to human IGHV-6-1.

[0102] In some embodiments, the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 human IGHD genes (e.g., the genes shown in Table 2). In some embodiments, the animal comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes selected from IGHD1-1, IGHD2-2, IGHD3-3, IGHD4-4, IGHD5-5, IGHD4-23, IGHD5-24, IGHD6-25, IGHD1-26, and IGHD7-27.

[0103] In some embodiments, the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 human IGHJ genes (e.g., the genes shown in Table 3). In some embodiments, the animal comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 human IGHJ genes selected from IGHJ1P, IGHJ1, IGHJ2, IGHJ2P, IGHJ3, IGHJ4, IGHJ5, IGHJ3P, and IGHJ6.

[0104] In some embodiments, the animal comprises an unmodified human sequence that includes a sequence beginning with a gene selected from IGHD1-1, IGHD2-2, IGHD3-3, IGHD4-4, IGHD5-5, IGHD4-23, IGHD5-24, IGHD6-25, IGHD1-26, and IGHD7-27 and ending with a gene selected from IGHJ1P, IGHJ1, IGHJ2, IGHJ2P, IGHJ3, IGHJ4, IGHJ5, IGHJ3P, and IGHJ6. In some embodiments, the unmodified human sequence is derived from a human heavy chain immunoglobulin locus from human IGHD1-1 through human IGHJ6.

[0105] In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGHD1-1 through human IGHD7-27.

[0106] In some embodiments, the unmodified human sequence is derived from a human heavy chain immunoglobulin locus from human IGHJ1P to human IGHJ6. In some embodiments, the unmodified human sequence is derived from a human heavy chain immunoglobulin locus from human IGHJ1 to human IGHJ6.

[0107] In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus from human IGHV(III)-82 to human IGHJ6.

[0108] In some embodiments, the unmodified human sequence is derived from a human heavy chain immunoglobulin locus from human IGHV1-2 to human IGHJ6. In some embodiments, the unmodified human sequence is derived from a human heavy chain immunoglobulin locus from human IGHV(II)-1-1 to human IGHJ6. In some embodiments, the unmodified human sequence is derived from a human heavy chain immunoglobulin locus from human IGHV6-1 to human IGHJ6.

[0109] In some embodiments, the animal may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified human sequences. In some embodiments, the length of the unmodified human sequences is about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 kb.

[0110] In some aspects, the present disclosure relates to a genetically modified animal that comprises, at an endogenous heavy chain immunoglobulin locus, a first sequence comprising one or more human IGHV genes, a second sequence comprising an endogenous sequence, and a third sequence comprising one or more human IGHD genes and one or more human IGHJ genes, wherein the first sequence, second sequence, and third sequence are operably linked.

[0111] In some embodiments, the first sequence comprises about or at least 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, or 161 human IGHV genes selected from Table 1. In some embodiments, the first sequence comprises about or at least 20, 21, 22, 23, 24, 25, 26, or 27 human IGHD genes selected from Table 2.

[0112] In some embodiments, the first sequence is an unmodified sequence derived from a human heavy chain immunoglobulin locus, hi some embodiments, the first sequence is about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 kb.

[0113] In some embodiments, the second sequence comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 kb of endogenous sequence.

[0114] In some embodiments, the third sequence comprises about or at least 20, 21, 22, 23, 24, 25, 26, or 27 human IGHD genes selected from Table 2. In some embodiments, the third sequence comprises about or at least 5, 6, 7, 8, or 9 human IGHJ genes selected from Table 3. In some embodiments, the third sequence comprises all of the human IGHD genes in Table 2 and all of the human IGHJ genes in Table 3.

[0115] In some embodiments, the animal comprises one or more endogenous genes selected from the group consisting of immunoglobulin heavy constant μ (IGHM), immunoglobulin heavy constant δ (IGHδ), immunoglobulin heavy constant γ3 (IGHG3), immunoglobulin heavy constant γ1 (IGHG1), immunoglobulin heavy constant γ2b (IGHG2b), immunoglobulin heavy constant γ2c (IGHG2c), immunoglobulin heavy constant ε (IGHE), and immunoglobulin heavy constant α (IGHA) genes. In some embodiments, immunoglobulin heavy constant γ2a (IGHG2a) is located at the position of IGHG2c. In some embodiments, these endogenous genes are operably linked. In some embodiments, these endogenous genes have the same order as in a wild-type animal. In some embodiments, isotype switching (immunoglobulin class switching) can occur in the animal.

[0116] In some embodiments, the IGHV, IGHD, and / or IGHJ genes are operably linked. VDJ recombination can occur between these genes to produce functional antibodies. In some embodiments, these genes are arranged in an order similar to that of the human heavy chain immunoglobulin locus. This arrangement has various advantages, for example, it allows for the production of heavy chain variable domains with a diversity that closely resembles that of human heavy chain variable domains. Because some random sequences may be inserted into the sequence during VDJ recombination, in some embodiments, a fully human antibody repertoire with no or minimal modifications can reduce the possibility of non-human sequences being inserted during VDJ recombination.

[0117] In some embodiments, the IGHV gene, IGHD gene, and / or IGHJ gene is operably linked to one or more genes (e.g., all of the genes) selected from the IGHM, IGHδ, IGHG3, IGHG1, IGHG2a, IGHG2b, IGHG2c, IGHE, and IGHA genes.

[0118] In some embodiments, the animal comprises a disruption in the animal's endogenous heavy chain immunoglobulin locus, hi some embodiments, the disruption in the animal's endogenous heavy chain immunoglobulin locus comprises a deletion of one or more endogenous IGHV genes, one or more endogenous IGHD genes, and one or more endogenous IGHJ genes.

[0119] In some embodiments, the animal is a mouse. The disruption in the animal's endogenous heavy chain immunoglobulin locus comprises a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, or 182 mouse IGHV genes (e.g., the genes shown in Table 4). In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHV genes selected from IGHV1-86, IGHV1-85, IGHV1-84, IGHV1-83, IGHV1-82, IGHV1-81, IGHV1-80, IGHV1-79, IGHV1-78, and IGHV1-77. In some embodiments, the mouse still comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHV genes selected from IGHV1-86, IGHV1-85, IGHV1-84, IGHV1-83, IGHV1-82, IGHV1-81, IGHV1-80, IGHV1-79, IGHV1-78, and IGHV1-77 (e.g., IGHV1-86).

[0120] In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHV genes selected from IGHV5-6, IGHV5-5, IGHV2-3, IGHV6-1, IGHV5-4, IGHV5-3, IGHV2-2, IGHV5-2, IGHV2-1, and IGHV5-1. In some embodiments, the mouse still comprises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHV genes selected from IGHV5-6, IGHV5-5, IGHV2-3, IGHV6-1, IGHV5-4, IGHV5-3, IGHV2-2, IGHV5-2, IGHV2-1, and IGHV5-1.

[0121] In some embodiments, the disruption in the animal's endogenous heavy chain immunoglobulin locus comprises a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mouse IGHD genes (e.g., the genes shown in Table 5). In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHD genes selected from IGHD5-1, IGHD3-1, IGHD1-1, IGHD6-1, IGHD2-3, IGHD2-7, IGHD2-8, IGHD5-6, IGHD3-2, and IGHD4-1. In some embodiments, the mouse still comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHD genes selected from IGHD5-1, IGHD3-1, IGHD1-1, IGHD6-1, IGHD2-3, IGHD2-7, IGHD2-8, IGHD5-6, IGHD3-2, and IGHD4-1.

[0122] In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, or 4 mouse IGHJ genes selected from IGHJl, IGHJ2, IGHJ3, and IGHJ4, hi some embodiments, the mouse still comprises about or at least 1, 2, 3, or 4 mouse IGHJ genes selected from IGHJl, IGHJ2, IGHJ3, and IGHJ4.

[0123] In some embodiments, the disruption in the animal's endogenous heavy chain immunoglobulin locus comprises a deletion of about or at least 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, 1500 kb, 2000 kb, 2500 kb, or 3000 kb of endogenous sequence.

[0124] In some embodiments, the deleted sequence is IGHV1-86 to IGHJ4, IGHV1-85 to IGHJ4, IGHV1-84 to IGHJ4, IGHV1-83 to IGHJ4, or IGHV1-82 to IGHJ4 (e.g., IGHV1-85 to IGHJ4).

[0125] In some embodiments, the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence in a human heavy chain immunoglobulin locus. In some embodiments, the sequences are about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, or 3500 kb in length. In some embodiments, the sequence is human IGHV(III)-82 through IGHV1-2. In some embodiments, the sequence is human IGHV7-81 through IGHV1-2. In some embodiments, the sequence is human IGHV(II)-1-1 through IGHJ6. In some embodiments, the sequence is human IGHV6-1 through IGHJ6.

[0126] The human IGHV gene, human IGHD gene, and human IGHJ gene are operably linked and can undergo VDJ rearrangement. In some embodiments, the modified mouse has a complete human IGHV, IGHD, and IGHJ gene repertoire (e.g., including all non-pseudohuman IGHV, IGHD, and IGHJ genes). Thus, the modified mouse can produce a complete human antibody repertoire. In some embodiments, after VDJ recombination, one IGHV gene (e.g., IGHV3-21 or IGHV3-74) contributes a sequence encoding an antibody heavy chain variable region. One IGHD gene contributes a sequence encoding an antibody heavy chain variable region. And one IGHJ gene contributes a sequence encoding an antibody heavy chain variable region. In some embodiments, the IGHV gene is IGHV3-21 or IGHV3-74.

[0127] In some embodiments, one IGHV gene (e.g., IGHV3-30, IGHV3-33, IGHV4-39, or IGHV4-34) contributes sequences encoding an antibody heavy chain variable region, one IGHD gene (e.g., IGHD6-19) contributes sequences encoding an antibody heavy chain variable region, and one IGHJ gene (e.g., IGHJ4 or IGHJ6) contributes sequences encoding an antibody heavy chain variable region.

[0128] Additionally, in some cases, the entire mouse IGHV, IGHD, and IGHJ genes (e.g., including all non-pseudogenes) are knocked out, such that the heavy chain variable region does not contain sequences encoded by mouse-derived sequences, thereby minimizing immunogenicity in humans.

[0129] In some embodiments, the locus may have about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, or 40 non-human exogenous IGHV genes (e.g., from a camelid). In some embodiments, the locus may have about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, or 40 non-human exogenous IGHD genes (e.g., from a camelid). In some embodiments, the locus may have about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, or 40 non-human exogenous IGHJ genes (e.g., from a camelid). These non-human exogenous genes may be useful for improving the diversity of VHH domains.

[0130] Various modifications at the heavy chain immunoglobulin locus are described, for example, in WO2020169022A1, which is incorporated herein by reference in its entirety.

[0131] immunoglobulin light chain locus In some embodiments, the animal has intact kappa and / or lambda chain immunoglobulin loci, hi some embodiments, the animal has disrupted kappa and / or lambda chain immunoglobulin loci.

[0132] The kappa chain immunoglobulin locus (also called the IGK or immunoglobulin kappa locus) is a region on a chromosome (e.g., chromosome 6) that contains the genes for the light chain of human antibodies (or immunoglobulins). Similarly, immunoglobulin light chain genes can also undergo a series of rearrangements that result in the production of mature immunoglobulin light chain nucleic acids (e.g., kappa chains).

[0133] The joining of the V segment (also called the IGKV gene) and the J segment (also called the IGKJ gene) creates a contiguous exon sequence that encodes the entire light chain variable domain. In unrearranged DNA, the V gene segment (or IGKV gene cluster) is located relatively far from the C region. The J gene segment (or IGKJ gene cluster) is located close to the C region. The joining of the V segment to the J gene segment also brings the V gene closer to the C region sequence. The J gene segment of the rearranged V region is separated from the C region sequence only by introns. To create the complete immunoglobulin light chain messenger RNA, the V region exons are joined to the C region sequence by RNA splicing after transcription.

[0134] In some embodiments, the animal comprises a disruption in the animal's endogenous light chain immunoglobulin locus, hi some embodiments, the disruption in the animal's endogenous light chain immunoglobulin locus comprises a deletion of one or more endogenous IGKV genes and one or more endogenous IGKJ genes.

[0135] In some embodiments, the animal is a mouse. The disruption in the animal's endogenous κ chain immunoglobulin locus comprises a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, or 163 mouse IGKV genes. In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGKV genes selected from IGKV2-137, IGKV1-136, IGKV1-135, IGKV14-134-1, IGKV17-134, IGKV1-133, IGKV1-132, IGKV1-131, IGKV14-130, and IGKV9-129. In some embodiments, the mouse still comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGKV genes selected from IGKV2-137, IGKV1-136, IGKV1-135, IGKV14-134-1, IGKV17-134, IGKV1-133, IGKV1-132, IGKV1-131, IGKV14-130, and IGKV9-129.

[0136] In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGKV genes selected from IGKV3-10, IGKV3-9, IGKV3-8, IGKV3-7, IGKV3-6, IGKV3-5, IGKV3-4, IGKV3-3, IGKV3-2, and IGKV3-1. In some embodiments, the mouse still comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGKV genes selected from IGKV3-10, IGKV3-9, IGKV3-8, IGKV3-7, IGKV3-6, IGKV3-5, IGKV3-4, IGKV3-3, IGKV3-2, and IGKV3-1.

[0137] In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, or 5 mouse IGKJ genes selected from IGKJl, IGKJ2, IGKJ3, IGKJ4, and IGKJ5. In some embodiments, the mouse still comprises about or at least 1, 2, 3, 4, or 5 mouse IGKJ genes selected from IGKJl, IGKJ2, IGKJ3, IGKJ4, and IGKJ5 (e.g., IGKJ5).

[0138] In some embodiments, the disruption in the animal's endogenous κ light chain immunoglobulin locus comprises a deletion of about or at least 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, 1500 kb, 2000 kb, 2500 kb, 3000 kb, or 3500 kb of endogenous sequence.

[0139] In some embodiments, the deleted sequence is IGKV2-137 to IGKJ4, IGKV1-136 to IGKJ4, IGKV1-135 to IGKJ4, IGKV2-137 to IGKJ5, IGKV1-136 to IGKJ5, or IGKV1-135 to IGKJ5 (e.g., IGKV2-137 to IGKJ5).

[0140] In some embodiments, the animal comprises a disruption in the animal's endogenous λ light chain immunoglobulin locus, hi some embodiments, the disruption in the animal's endogenous light chain immunoglobulin locus comprises a deletion of one or more endogenous IGLV genes, one or more endogenous IGLJ genes, and / or one or more immunoglobulin λ constant (IGLC) genes (e.g., IGLC1, IGLC2, IGLC3, and IGLC4).

[0141] The disruption in the animal's endogenous λ light chain immunoglobulin locus comprises a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mouse IGLV, IGLJ, and IGLC genes. In some embodiments, the deletion comprises about or at least 1, 2, 3, or 4 mouse IGLC genes selected from IGLC1, IGLC2, IGLC3, and IGLC4. In some embodiments, the disruption comprises a deletion of about or at least 1, 2, or 3 mouse IGLV genes selected from IGLV1, IGLV2, and IGLV3. In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, or 5 mouse IGLJ genes selected from IGLJ1, IGLJ2, IGLJ3, IGLJ3P, and IGLJ4.

[0142] In some embodiments, the disruption in the animal's endogenous λ light chain immunoglobulin locus comprises a deletion of about or at least 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 110 kb, 120 kb, 130 kb, 140 kb, 150 kb, 160 kb, 170 kb, 180 kb, 190 kb, 200 kb, 210 kb, 220 kb, 230 kb, 240 kb, 250 kb, 260 kb, 270 kb, 280 kb, 290 kb, 300 kb, 350 kb, 400 kb, 450 kb, 500 kb, or 1000 kb of nucleotides. In some embodiments, there is no disruption in the animal's endogenous λ light chain immunoglobulin gene.

[0143] In some embodiments, the deleted sequence is from IGLV2 to IGLC1, from IGLV3 to IGLC1, or from IGLJ2 ​​to IGLC1.

[0144] Various modifications of the light chain immunoglobulin locus are described, for example, in WO2020169022A1, which is incorporated herein by reference in its entirety.

[0145] Genetically modified animals The present application provides genetically modified non-human animals that produce heavy chain-only antibodies, i.e., antibodies lacking light chains. In some embodiments, the genetically modified non-human animals do not produce IgG molecules that contain light chains (e.g., IgG1). In some embodiments, the genetically modified non-human animals do not produce any conventional IgG molecules, i.e., IgG antibodies that have two heavy chains and two light chains.

[0146] In some embodiments, the genetically modified non-human animal has a fully functional endogenous light chain locus. In some embodiments, the animal's immunoglobulin heavy chain locus comprises a fully functional IGHM, IGHδ, and / or IGHA gene. In some embodiments, the animal's genome does not comprise an exogenous sequence (e.g., a human immunoglobulin heavy chain constant region gene) within the endogenous immunoglobulin heavy chain constant region locus.

[0147] In some embodiments, the modified immunoglobulin heavy chain locus lacks the CH1 exon of the endogenous IGHG gene (e.g., the IGHG1 gene). In some embodiments, the immunoglobulin heavy chain locus of the animal comprises fully functional H, CH2, CH3, M1, and / or M2 exons of the IGHG1 gene.

[0148] In some embodiments, the immunoglobulin heavy chain locus of the animal has a modification only in the CH1 exon of the endogenous IGH1 gene. In some embodiments, the modification does not include a mutation, such as a deletion or loss-of-function mutation, in the gene encoding the hinge region of IgG1. In some embodiments, the modification does not include a mutation, such as a deletion or loss-of-function mutation, in the CH2 or CH3 exons of the IGH1 gene. In some embodiments, the immunoglobulin heavy chain locus does not have a mutation or modification in the CH1 exon of the IGHM gene. In some embodiments, the genetically modified non-human animal has a functional gene segment encoding the CH1 domain of IgM at the endogenous IGHM locus.

[0149] In some embodiments, the genetically modified non-human animal has fully functional genes encoding other heavy chain constant isotypes, such as IgM, IgD, and / or IgA. In some embodiments, the genetically modified non-human animal has fully functional IGHM, IGHδ, and / or IGHA genes. In some embodiments, the modified immunoglobulin heavy chain locus has wild-type IGHM, IGHδ, and / or IGHA genes. In some embodiments, the modified immunoglobulin heavy chain locus does not contain any mutations to the endogenous IGHM, IGHδ, and / or IGHA genes. In some embodiments, the endogenous immunoglobulin heavy chain locus has an intact endogenous IGHM, IGHδ, or IGHA gene. In some embodiments, the genetically modified non-human animal expresses wild-type IgM, IgD, and / or IgA proteins.

[0150] In some embodiments, the immunoglobulin heavy chain locus includes a mutation (e.g., a deletion) or recombination to the CH1 exon of the IGHM and / or IGHδ gene. In some embodiments, the endogenous immunoglobulin heavy chain locus has a modified IGHM gene and / or a modified IGHδ gene. In some embodiments, the genetically modified non-human animal does not express wild-type IgM and / or IgD. In some embodiments, the genetically modified non-human animal expresses IgM lacking a CH1 domain and / or IgD lacking a CH1 domain.

[0151] In some embodiments, the introduction of modifications into an endogenous immunoglobulin heavy chain locus in a genetically modified non-human animal maintains the health of the animal, including substantially normal B-cell development and maturation. In some embodiments, the introduction of modifications into an endogenous immunoglobulin heavy chain locus in a genetically modified non-human animal reduces or avoids the immunogenicity of the exogenous sequence. In some embodiments, the introduction of minimal changes into an endogenous immunoglobulin heavy chain locus in a genetically modified non-human animal maintains the normal function of the endogenous immunoglobulin heavy chain locus, including VDJ recombination, class switch recombination, and somatic hypermutation.

[0152] In some embodiments, the genetically modified non-human animal has one or more fully functional light chain loci, e.g., a λ light chain locus and / or a κ light chain locus. In some embodiments, the genetically modified non-human animal has an unchanged endogenous light chain locus. In some embodiments, no mutations are introduced into the endogenous light chain loci of the genetically modified non-human animal. In some embodiments, the λ and / or κ light chain variable region loci of the genetically modified non-human animal are functional and not silenced. In some embodiments, the genetically modified non-human animal expresses wild-type λ light chains and / or wild-type κ light chains. In some embodiments, the genetically modified non-human animal expresses functional IgM molecules comprising the light chains. In some embodiments, the genetically modified non-human animal expresses functional IgA, IgD, and / or IgM molecules comprising the light chains. In some embodiments, the genetically modified non-human animal does not have an exogenous light chain gene or gene cluster. For example, the λ and / or κ light chain variable region loci of the genetically modified non-human animal can be knocked out.

[0153] In some embodiments, the modified immunoglobulin heavy chain locus does not comprise rearranged genes (e.g., rearranged IGHV, IGHD, and / or IGHJ genes). In some embodiments, the modified immunoglobulin heavy chain locus comprises a complete set of unrearranged human IGHV, IGHD, and IGHJ genes.

[0154] In some embodiments, the modified immunoglobulin heavy chain locus comprises a functional splice site immediately after the CH1 exon of the endogenous IGHG1 gene. In some embodiments, the modified immunoglobulin heavy chain locus comprises a wild-type splice site immediately after the CH1 exon of the endogenous IGHG1 gene.

[0155] In some embodiments, the animals described herein express membrane-bound IgG1 that lacks the CH1 domain. In some embodiments, the animals described herein express soluble IgG1 that lacks the CH1 domain.

[0156] In one aspect, the present disclosure provides a genetically modified non-human animal comprising a humanized heavy chain immunoglobulin locus. In some embodiments, the animal comprises one or more human IGHV genes, one or more human IGHD genes, and / or one or more human IGHJ genes. In some embodiments, these genes are located at the endogenous immunoglobulin locus.

[0157] In some embodiments, the animal comprises an endogenous κ or λ chain immunoglobulin locus. In some embodiments, the animal does not comprise an endogenous κ or λ chain immunoglobulin locus. In some embodiments, the animal comprises a disruption in the animal's endogenous κ or λ light chain immunoglobulin locus. In some embodiments, the animal does not have a disruption in the animal's endogenous κ or λ light chain immunoglobulin locus.

[0158] Genetically modified non-human animals can be a variety of animals, such as mice, rats, rabbits, pigs, bovines (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, and primates (e.g., marmosets and rhesus monkeys). For non-human animals for which suitable, genetically modifiable embryonic stem (ES) cells are not readily available, other methods are used to generate non-human animals containing genetic modifications. Such methods include, for example, modifying the genome of a non-ES cell (e.g., a fibroblast or induced pluripotent cell) and using nuclear transfer to transfer the modified genome into a suitable cell, such as an oocyte, and gestation of the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo. These methods are well known in the art and are described, for example, in A. Nagy, et al., "Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition)," Cold Spring Harbor Laboratory Press, 2003, which is incorporated herein by reference in its entirety. Thus, in various embodiments, human V, D, and / or J segments can be operably linked to constant region gene sequences of a non-human animal (e.g., a rodent, mouse, rat, hamster). During B cell development, these rearranged human V, D, and / or J segments are linked to the immunoglobulin constant region of the non-human animal.

[0159] In one aspect, the animal is a mammal, e.g., a superfamily of Jerboidea or Muridea. In some embodiments, the genetically modified animal is a rodent. The rodent can be selected from a mouse, a rat, and a hamster. In some embodiments, the genetically modified animal is from a family selected from Macropus punctatus (e.g., Macropus punctatus), Cricetidae (e.g., hamsters, New World rats and mice, field mice), Muridae (True mice and rats, gerbils, spiny mice, crested rats), Tetranychus punctatus (climbing mice, rock mice, white-tailed mice, Malagasy rats and mice), Dormice punctatus (e.g., spiny dormice), and Mole rats (e.g., mole rats, bamboo rats, and zokor). In some embodiments, the genetically modified rodent is selected from True mice or rats (family Muridae), gerbils, spiny mice, and crested rats. In some embodiments, the non-human animal is a mouse.

[0160] In some embodiments, the animal is a mouse of a C57BL strain selected from C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. In some embodiments, the mouse is a 129 strain selected from the group consisting of strains that are 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / SvIm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1, 129T2. These mice are described, for example, in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10:836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129 / SvEv- and C57BL / 6-Derived Mouse Embryonic Stem Cell Lines (2000), both of which are incorporated herein by reference in their entireties. In some embodiments, the genetically modified mice are a mix of 129 and C57BL / 6 strains. In some embodiments, the mice are a mix of 129 strains or a mix of BL / 6 strains. In some embodiments, the mice are a BALB strain, e.g., a BALB / c strain. In some embodiments, the mice are a mix of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid strain (e.g., 50% BALB / c-50% 12954 / Sv; or 50% C57BL / 6-50% 129). In some embodiments, the non-human animal is a rodent.In some embodiments, the non-human animal is a mouse with a BALB / c, A, A / He, A / J, A / WySN, AKR, AKR / A, AKR / J, AKR / N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA / 2, KM, NIH, ICR, CFW, FACA, C57BL / A, C57BL / An, C57BL / GrFa, C57BL / KaLwN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL (C57BL / 10Cr, and C57BL / Ola), C58, CBA / Br, CBA / Ca, CBA / J, CBA / st, or CBA / H background.

[0161] Different animals have different germline tissues and genes at their endogenous immunoglobulin heavy chain (IgH) loci. The IgH loci of many species have been sequenced. The gene locations and exon / intron organization of the IgH loci of mouse, rat, and rabbit can be found, for example, in the IMGT repertoire and NCBI databases, which are incorporated herein by reference in their entireties.

[0162] In some embodiments, the animal is a rat. The rat can be selected from Wistar rats, LEA strains, Sprague Dawley strains, Fischer strains, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

[0163] The animal may have one or more other genetic modifications and / or other modifications suitable for the particular purpose for which the humanized animal is being generated.

[0164] Genetically modified non-human animals comprising modifications of endogenous non-human immunoglobulin loci. In some embodiments, the modification comprises a human nucleic acid sequence encoding at least a portion of a human protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a human heavy chain variable domain or light chain variable domain sequence). Genetically modified cells (e.g., ES cells, somatic cells) that can comprise the modifications described herein are also provided, although in many embodiments, the genetically modified non-human animals comprise modifications of endogenous loci in the germline of the animal.

[0165] Genetically modified animals can express humanized and / or chimeric antibodies from endogenous mouse loci, in which one or more endogenous mouse immunoglobulin genes have been replaced with human immunoglobulin genes and / or nucleotide sequences that are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to human immunoglobulin gene sequences (e.g., IGHV, IGHD, IGHJ, IGKV, and / or IGKJ genes). In various embodiments, endogenous non-human immunoglobulin loci are modified in whole or in part to comprise human nucleic acid sequences.

[0166] Genetic, molecular, and behavioral analyses of the non-human mammals described above can be performed. The present disclosure also relates to offspring produced by the non-human mammals provided herein when bred with the same or other genotypes. The non-human mammal can be any non-human animal known in the art and can be used in the methods described herein. Preferred non-human mammals are mammals (e.g., rodents). In some embodiments, the non-human mammal is a mouse.

[0167] The present disclosure also provides cell lines or primary cell cultures derived from non-human mammals or their descendants. Cell culture-based models can be prepared, for example, by the following methods. Cell cultures can be obtained by isolation from non-human mammals, or cells can be obtained from established cell cultures using the same construct and standard cell transfection techniques. Integration of gene constructs containing DNA sequences encoding human or humanized immunoglobulins can be detected in a variety of ways.

[0168] Many analytical methods are available for detecting alterations in exogenous or genomic DNA, including methods at the nucleic acid level (including mRNA quantification approaches using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, and in situ hybridization) and methods at the protein level (including histochemistry, immunoblot analysis, and in vitro binding studies). Furthermore, the expression level of a gene of interest can be quantified using ELISA techniques well known to those skilled in the art. Quantitative measurements can be completed using many standard analytical methods. For example, transcript levels can be measured using RT-PCR and hybridization methods, including RNase protection, Southern blot analysis, and RNA dot analysis. Immunohistochemical staining, flow cytometry, and Western blot analysis can also be used to assess the presence of human or humanized proteins.

[0169] Antibodies and antigen-binding fragments The present disclosure provides antibodies and antigen-binding fragments thereof (e.g., heavy chain antibodies, humanized heavy chain antibodies, or multispecific antibodies) produced by the methods described herein.

[0170] Generally, traditional antibodies are composed of two types of polypeptide chains, light chains and heavy chains. Non-limiting antibodies of the present disclosure can be intact four immunoglobulin chain antibodies, including two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype, including IgM, IgG, IgE, IgA, or IgD, or a subclass, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. An antibody can include two identical copies of the light chain and two identical copies of the heavy chain. The heavy chains, each containing one variable domain (or variable region, VH) and multiple constant domains (or constant regions), are bound to each other via disulfide bonds within their constant domains to form the antibody "stem." Each light chain, which contains one variable domain (or variable region, VL) and one constant domain (or constant region), is bound to one heavy chain via disulfide bonds. The variable region of each light chain aligns with the variable region of the heavy chain to which it is bound. Both the light and heavy chain variable regions contain three hypervariable regions sandwiched between more conserved framework regions (FR).

[0171] The hypervariable regions, known as complementarity-determining regions (CDRs), form the loops that comprise the principal antigen-binding surface of an antibody. The four framework regions largely conform to a β-sheet structure, and the CDRs form connecting loops that, in some cases, form part of the β-sheet structure. The CDRs of each chain are held in close proximity by the framework regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding region.

[0172] Methods for identifying CDR regions of antibodies by analyzing their amino acid sequences are well known, and several definitions of CDRs are commonly used: the Kabat definition is based on sequence variability, and the Chothia definition is based on the location of structural loop regions. These methods and definitions are described, for example, by Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody engineering, Springer Berlin Heidelberg, 2001.422-439; Abhinandan, et al. Kabat,EA(1970)J.Exp.Med.132:211-250;Martin et al.,Methods Enzymol.203:121-53(1991);Morea et al.,Biophys Chem.68(1-3):9-16(Oct.1997);Morea et al.,J Mol Biol.275(2):269-94(Jan.1998);Chothia et al., Nature 342(6252):877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007), each of which is incorporated herein by reference in its entirety.

[0173] CDRs are important for recognizing the epitope of an antigen. As used herein, "epitope" refers to the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be approximately 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be in a contiguous linear sequence in the primary structure of the antigen, as the epitope may depend on the three-dimensional structure of the antigen based on the secondary and tertiary structure of the antigen.

[0174] In some embodiments, antibodies are intact immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved and differ in their constant regions, particularly the hinge and upper CH2 domains. The sequences and differences between IgG subclasses are well known in the art and are described, for example, in Vidarsson, et al., "IgG subclasses and allotypes: from structure to effector functions," Frontiers in Immunology 5 (2014); Irani, et al., "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases," Molecular Immunology 67.2 (2015):171-182; Shakib, Farouk, ed., The human IgG subclasses: molecular analysis of structure, function, and regulation. Elsevier, 2016, each of which is incorporated herein by reference in its entirety. The heavy chain constant region of the heavy chain antibody can be derived from any of the immunoglobulin molecules described herein (e.g., IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgM, IgD, IgE, IgA).

[0175] An antibody can also be an immunoglobulin molecule from any species (e.g., human, rodent, mouse, rat, camelid). Antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" refers to any portion of an antibody that retains the specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specifically binding to an epitope on the intact antibody's target molecule. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or antigen-binding fragment thereof can be, for example, an scFv, Fv, Fd, dAb, diabody, bispecific scFv, diabody, linear antibody, single-chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide comprising a binding domain that is, or is homologous to, an antibody-binding domain. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0176] In some embodiments, the antigen-binding fragment can form part of a chimeric antigen receptor (CAR), which in some embodiments is a fusion of a VHH described herein fused to a CD3-zeta transmembrane domain and endodomain.

[0177] The antibodies and antigen-binding fragments thereof (e.g., humanized or chimeric antibodies) produced by the methods described herein have various advantages. In some embodiments, no further optimization is required to obtain the desired properties (e.g., binding affinity, thermal stability, and / or limited aggregation).

[0178] In some embodiments, the antibody (or antigen-binding fragment thereof) is -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Less than or 0.00001s -1 Specifically binds to a target with a k of less than 0.01 s. In some embodiments, the k is less than 0.01 s. -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.00001s -1 Over or 0.000001s -1 It's super.

[0179] In some embodiments, the kinetic association rate (k) is 1×10 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or more than 1 × 10 6 In some embodiments, the kinetic association rate (k) is greater than 1×10 5 / Ms less than 1 × 10 6 / Ms or less than 1 x 10 7 / Ms is less than.

[0180] Affinity can be estimated from the quotient of the kinetic rate constants (K = k / k). In some embodiments, K is greater than or equal to 1 x 10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M or 1 x 10 -10 In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is less than 1 x 10 -7 Super M, 1×10 -8 Super M, 1×10 -9 Super M, 1×10 -10 Super M, 1×10-11 More than M or 1 x 10 -12 In some embodiments, the antibody binds to the target with a KD of about 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM or less.

[0181] In some embodiments, thermal stability is measured. The antibodies or antigen-binding fragments described herein may have a Tm of greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C.

[0182] In various embodiments, substitutions are made to the parent heavy chain antibody sequence to generate a mutant heavy chain antibody. Generally, the heavy chain antibody variant of the parent heavy chain antibody has an antigen-binding affinity of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% (e.g., at least 150%, at least 200%, at least 500%, at least 1000%, or up to at least 10,000%) of the parent heavy chain antibody's binding affinity for a particular antigen. In some embodiments, the mutant heavy chain antibody contains a single substitution compared to the parent heavy chain antibody. However, in other embodiments, several amino acids, e.g., up to about 5 or 10 or more, are substituted compared to the parent heavy chain antibody sequence derived from another human heavy chain sequence that shares identity at a particular position. In various embodiments, the resulting mutant heavy chain antibody is tested to ensure that the substituted residues do not significantly reduce the desired binding affinity and / or specificity. In some embodiments, improved mutant heavy chain antibodies are produced by substitution of amino acids from different human heavy chain sequences. In various embodiments, the VHH is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the parent VHH.

[0183] The VHHs described herein can be used to generate multispecific antibodies (e.g., bispecific antibodies). In one aspect, the present disclosure provides multispecific antibodies comprising a first antigen-binding portion and a second antigen-binding portion. In some embodiments, the first antigen-binding portion comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and VL together form an antigen-binding site that specifically binds to a first epitope. In some embodiments, the first antigen-binding portion comprises a VHH that specifically binds to a first epitope. In some embodiments, the second antigen-binding portion comprises a VHH that specifically binds to a second epitope. In some embodiments, the first epitope and the second epitope are from the same antigen. In some embodiments, the first epitope and the second epitope are from different antigens.

[0184] In some embodiments, the first antigen-binding moiety is a full-length antibody consisting of two heavy chains and two light chains. In some embodiments, the first antigen-binding moiety is an antibody fragment comprising a heavy chain comprising a VH and a light chain comprising a VL. In some embodiments, the second antigen-binding moiety comprises a single polypeptide chain. In some embodiments, the C-terminus of the second antigen-binding moiety is fused to the N-terminus of at least one heavy chain of the first antigen-binding moiety. In some embodiments, the C-terminus of the second antigen-binding moiety is fused to the N-terminus of at least one light chain of the first antigen-binding moiety. In some embodiments, the N-terminus of the second antigen-binding moiety is fused to the C-terminus of at least one heavy chain of the first antigen-binding moiety. In some embodiments, the N-terminus of the second antigen-binding moiety is fused to the C-terminus of at least one light chain of the first antigen-binding moiety. In some embodiments, the second antigen-binding portion is a Fab-like domain comprising a first polypeptide chain comprising a first VHH fused to a CH1 domain and a second polypeptide chain comprising a second VHH fused to a CL domain.

[0185] In some embodiments, the antibody or antigen-binding fragment thereof is a trispecific antibody. In some embodiments, the trispecific antibody is a trispecific VHH-Fc. In some embodiments, the trispecific antibody comprises the same VHH. In some embodiments, the trispecific antibody comprises different VHH. In some embodiments, the VHHs bind to the same epitope. In some embodiments, the VHHs bind to different epitopes.

[0186] In some embodiments, an antibody or antigen-binding fragment thereof comprises four or more VHHs. In some embodiments, to enhance developability, at least four VHHs are combined to construct a tetraspecific VHH without the addition of an IgG Fc domain. These molecules lack Fc effector functions but have the added advantage of improved affinity and avidity for antigen compared to bispecific and trispecific VHH-Fc.

[0187] In some embodiments, these antibodies or antigen-binding fragments thereof (e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) have a functional Fc.

[0188] In some embodiments, a heavy chain antibody produced by a genetically modified non-human animal described herein has a VHH domain comprising CDR1, CDR2, and CDR3. In some embodiments, the length of CDR3 is 6 to 23, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the length of CDR3 is at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23.

[0189] Methods for producing genetically modified animals Genetically modified animals can be produced by modifying immunoglobulin loci. Figures 6-12 show the workflow for genetic modification using targeting vectors V1-V7, respectively. Figure 20 shows a method for producing humanized animals. In some embodiments, the method first involves modifying human immunoglobulin loci on human chromosomes. The modified human chromosomes are then introduced into mouse recipient cells. Human immunoglobulin variable regions are then introduced into the corresponding regions of the mouse genome by direct replacement. The recipient cells are then screened. In some embodiments, the cells do not contain human chromosomes. The cells are then injected into blastocysts to produce chimeric mice. Subsequent breeding can be performed to obtain mice containing intact humanized immunoglobulin loci.

[0190] Several other techniques can also be used to generate genetically modified animals, including non-homologous end joining (NHEJ), homologous recombination (HR), zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to generate genetically modified animals. Many of these genome editing techniques are well known in the art and are described, for example, in Yin et al., "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6(2017):387-399, incorporated by reference in its entirety. Many other methods are also provided and can be used for genome editing, such as microinjecting a genetically modified nucleus into an enucleated oocyte and fusing the enucleated oocyte with another genetically modified cell.

[0191] The genetic modification process may involve replacing endogenous sequences with human sequences by homologous recombination. In some embodiments, cleavage can be performed upstream and downstream of the target site (e.g., by zinc finger nucleases, TALENs, or CRISPRs), resulting in a DNA double-strand break, and homologous recombination is used to replace the endogenous sequence with the human sequence.

[0192] In some embodiments, methods for deleting the CH1 sequence in the IGHG gene include one or a combination of the following methods. These modifications can be performed in a variety of cells. In some embodiments, the cell is a stem cell, an embryonic stem cell, or a fertilized egg cell.

[0193] In some embodiments, the sequence from the Sγ3 switch region to Cε is knocked out, and then a sequence comprising the Sγ1 switch region and a Cγ1 sequence without CH1 (abbreviated as Cγ1ΔCH1) is inserted, such that Cγ1ΔCH1 is operably linked to Sγ1.

[0194] In some embodiments, a sequence comprising the Sγ1 switch region and a Cγ1 sequence lacking CH1 (abbreviated as Cγ1ΔCH1) is used to directly replace all of the sequence from the Sγ3 switch region through Cε ( FIG. 6 ), such that Cγ1ΔCH1 is operably linked to Sγ1.

[0195] In some embodiments, the Sγ3 switch region and Cγ3 sequence are first knocked out, and then a Cγ1 sequence without CH1 (abbreviated as Cγ1ΔCH1) is used to replace all sequences from Cγ1 to Cε, such that Cγ1ΔCH1 is operably linked to Sγ1.

[0196] In some embodiments, the CH1-less Cγ1 sequence (abbreviated as Cγ1ΔCH1) is used to directly replace all sequences from Cγ3 through Cε ( FIG. 7 ), such that Cγ1ΔCH1 is operably linked to Sγ3.

[0197] In some embodiments, sequences including Cμ and Cδ are knocked out (FIG. 8), based on the alleles shown in FIG. 6. As a result, both the IGHM and IGHδ genes are knocked out, and Cγ1ΔCH1 is operably linked to Sγ1.

[0198] In some embodiments, sequences including Cμ, Cδ, and Sγ1 are knocked out (FIG. 9), based on the alleles shown in FIG. 6. As a result, both the IGHM and IGHδ genes are knocked out, and Cγ1ΔCH1′ is operably linked to Sμ.

[0199] In some embodiments, the CH1 coding region of Cμ is knocked out (FIG. 10), based on the allele shown in FIG. 6. The resulting modified locus includes sequences encoding IgM lacking the CH1 domain, IgD, and IgG1 lacking the CH1 domain. Additionally, Cγ1ΔCH1 is operably linked to Sγ1.

[0200] In some embodiments, based on the allele shown in Figure 6, a Cμ sequence lacking the CH1 coding region (abbreviated as CμΔCH1) is used to directly replace the sequence containing Cμ and Cδ (Figure 11). The resulting modified locus contains sequences encoding an IgM lacking the CH1 domain and an IgG1 lacking the CH1 domain. Additionally, Cγ1ΔCH1 is operably linked to Sγ1.

[0201] In some embodiments, based on the allele shown in Figure 6, a sequence comprising CμΔCH1 and Cδ lacking the CH1 coding region (abbreviated as CδΔCH1) is used to directly replace a sequence comprising Cμ and Cδ (Figure 12). The resulting modified locus contains sequences encoding IgM lacking the CH1 domain, IgD lacking the CH1 domain, and IgG1 lacking the CH1 domain. Additionally, Cγ1ΔCH1 is operably linked to Sγ1.

[0202] In some embodiments, provided herein are genetically modified non-human animals comprising an engineered immunoglobulin heavy chain constant region locus. In some embodiments, the engineered immunoglobulin heavy chain constant region locus comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of Cγ1ΔCH1, Cγ1ΔCH1', CμΔCH1, or CδΔCH1. In some embodiments, the sequence of Cγ1ΔCH1' comprises a deletion of at least 1, at least 2, at least 3, or at least 4 nucleotides at the 5' end of the sequence of Cγ1ΔCH1 (SEQ ID NO: 1).

[0203] The present disclosure also relates to a genetically modified non-human animal comprising a nucleic acid sequence, wherein the nucleic acid sequence can be selected from the group consisting of: a) a nucleic acid sequence set forth in SEQ ID NO: 1, 8, 9, 10, 13, or 41; b) a nucleic acid sequence capable of hybridizing under low stringency or strict stringency conditions to the nucleotide sequence set forth in SEQ ID NO: 1, 8, 9, 10, 13, or 41; c) a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO: 1, 8, 9, 10, 13, or 41; and d) A nucleic acid sequence encoding an amino acid sequence, wherein said amino acid sequence has at least 90% homology or is at least 90% identical to the amino acid sequence of endogenous IgG, IgM, IgD, or IgA.

[0204] The method further comprises implanting the genetically modified cells into the oviduct or uterus of a recipient female non-human mammal and allowing the cells to grow within the uterus of the female non-human mammal. In some embodiments, experiments are performed to identify germline transmission of the genetically modified gene in offspring.

[0205] In some embodiments, methods for producing genetically modified humanized animals can also include replacing a nucleic acid (e.g., a V, D, J region, or a V, J region) at an endogenous locus (or site) with a corresponding region of a human sequence. The sequence can include a region (e.g., a portion or entire region) of an IGHV, IGHD, IGHJ, IGKV, and / or IGKJ gene. In some embodiments, the replacement is mediated by homologous recombination. In some embodiments, the replacement is mediated by Cre recombinase.

[0206] The 5'-end homology arm and / or the 3'-end homology arm can have a desired length to facilitate homologous recombination. In some embodiments, the homology arm is about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 kb (e.g., about 3 kb). In some embodiments, the homology arm is less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 kb.

[0207] In some embodiments, the vector may also optionally include a reporter protein, such as luciferase (eg, Gluc) or a fluorescent protein (eg, EGFP, BFP, etc.).

[0208] These modifications can be performed in a variety of cells. In some embodiments, the cells are stem cells, embryonic stem cells, or fertilized egg cells.

[0209] The present disclosure provides: (a) providing a cell (e.g., a fertilized egg cell) according to the methods described herein; (b) culturing the cells in a liquid medium; (c) transplanting the cultured cells into the oviduct or uterus of a recipient female non-human mammal and allowing the cells to grow within the uterus of the female non-human mammal; (d) identifying germline transmission in the offspring genetically modified humanized non-human mammals of the pregnant females of step (c); Further provided is a method for establishing a humanized animal model, comprising:

[0210] In some embodiments, the non-human mammal in the aforementioned methods is a mouse (eg, a C57 mouse, a BALB / c mouse, or a C57BL / 6 mouse).

[0211] In some embodiments, the non-human mammal in step (c) is a pseudopregnant (or phantom pregnant) female.

[0212] In some embodiments, the fertilized eggs for the above-described methods are C57BL / 6 fertilized eggs. Other fertilized eggs that can be used in the methods described herein include, but are not limited to, FVB / N fertilized eggs, BALB / c fertilized eggs, DBA / 1 fertilized eggs, and DBA / 2 fertilized eggs.

[0213] The fertilized egg can be derived from any non-human animal, for example, any of the non-human animals described herein. In some embodiments, the fertilized egg cell is derived from a rodent. The gene construct can be introduced into the fertilized egg by microinjection of DNA. For example, the fertilized egg can be cultured after microinjection, and the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, which then produces a non-human mammal, thereby generating the non-human mammal referred to in the above-mentioned method.

[0214] Cells, tissues, and animals (e.g., mice) comprising the nucleotide sequences described herein, as well as cells, tissues, and animals (e.g., mice) that express humanized or chimeric antibodies derived from endogenous non-human loci, are also provided.

[0215] The present disclosure also provides various targeting vectors (e.g., vectors useful for generating genetically modified animals). In some embodiments, the vectors can include: a) a DNA fragment homologous to the 5' end of the region to be modified (5' homology arm); b) a sequence containing a desired genetic element (e.g., a LoxP recognition site, a drug resistance gene, and / or a reporter gene); and c) a second DNA fragment homologous to the 3' end of the region to be modified (3' homology arm). The present disclosure also relates to cells containing the targeting vectors described herein.

[0216] In some embodiments, the gene in the cell is heterozygous. In some embodiments, the gene in the cell is homozygous.

[0217] In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell.

[0218] The present disclosure also provides nucleic acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the nucleotide sequences described herein, and Also provided are amino acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the amino acid sequences described herein.

[0219] In some embodiments, the disclosure relates to a nucleotide sequence encoding any of the peptides described herein or any amino acid sequence encoded by any of the nucleotide sequences described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

[0220] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences described herein.

[0221] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences described herein.

[0222] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are considered identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences. For illustrative purposes, sequence comparison and percent identity determination between two sequences can be performed using the Blossum62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5, as appropriate.

[0223] The percentage of residues that conserve similar physicochemical properties (percent homology), such as leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues with similar physicochemical properties have been defined in the art. These families include, for example, amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the percentage homology is higher than the percentage identity. Accordingly, the present disclosure also provides amino acid sequences having a percentage homology of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any of the amino acid sequences described herein, or nucleic acids encoding these amino acid sequences.

[0224] Uses of genetically modified animals Genetically modified animals can be used to generate heavy chain antibodies capable of specifically binding to a target. In some embodiments, the target (e.g., a protein or a fragment of a protein) can be used as an immunogen to generate antibodies in these animals using standard techniques for polyclonal and monoclonal antibody preparation. In some embodiments, the genetically modified animals are exposed to a selected antigen for a time and under conditions that allow the animal to produce antibodies specific for the antigen.

[0225] Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal) of the antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species being immunized. Animals can be injected with the antigenic peptide or protein more than once (e.g., two, three, or four times).

[0226] The full-length polypeptide or protein can be used, or alternatively, an antigenic peptide fragment thereof can be used as an immunogen. An antigenic peptide of a protein contains at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of an amino acid sequence that encompasses an epitope of the protein such that antibodies raised against the peptide form specific immune complexes with the protein.

[0227] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a genetically modified animal described herein). An appropriate immunogenic preparation can contain, for example, a recombinantly expressed or chemically synthesized polypeptide (e.g., a fragment of a protein). The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0228] Antibody titers in immunized subjects can be monitored over time by standard techniques, such as enzyme-linked immunosorbent assay (ELISA) using immobilized polypeptide or peptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein G or protein A chromatography, to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titer is at its highest, antibody-producing cells are obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or the trioma technique. Hybridoma production techniques are well known (see generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY)). For example, hybridoma cells producing monoclonal antibodies are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest using a standard ELISA assay.

[0229] In one aspect, the disclosure provides a mouse comprising a modification of an endogenous immunoglobulin heavy chain locus, wherein the mouse produces B cells comprising a rearranged immunoglobulin sequence operably linked to a heavy chain constant region gene sequence. In some embodiments, the rearranged immunoglobulin sequence operably linked to the heavy chain constant region gene sequence comprises a human heavy chain V, D, and / or J sequence. In some embodiments, the heavy chain constant region gene sequence comprises a human or mouse heavy chain sequence selected from the group consisting of a CH1, hinge, CH2, CH3, and a combination thereof.

[0230] In one aspect, the disclosure relates to a method for producing a somatically mutated heavy chain antibody in an animal, the method comprising: immunizing the animal with an antigen and maintaining the animal under conditions sufficient to mount an immune response to the antigen; and isolating from the animal a somatically mutated heavy chain antibody comprising a variable domain derived from a human or endogenous heavy chain immunoglobulin variable region gene segment, wherein the somatically mutated heavy chain antibody specifically binds to the antigen. In some embodiments, the animal comprises unrearranged human or endogenous heavy chain immunoglobulin variable region gene segments, wherein the animal lacks a nucleotide sequence of at least one allele encoding a functional IgG CH1 domain, and the animal expresses an IgM comprising the CH1 domain.

[0231] The B cells or spleen cells may contain, for example, rearranged non-mouse immunoglobulin variable gene sequences operably linked to mouse immunoglobulin constant region genes. The sequence encoding the human heavy chain variable region is determined. The sequence can be determined, for example, by sequencing the hybridoma or B cells of interest. In some embodiments, single B cell screening is used, which can screen the natural antibody repertoire without the need for hybridoma fusion or combinatorial display. For example, by mixing B cells with a panel of DNA-barcoded antigens, both the antigen barcode and B cell receptor (BCR) sequence of each B cell can be recovered by single-cell sequencing protocols.

[0232] Antibodies can be further modified to obtain humanized or human antibodies, for example, by operably linking a sequence encoding a human heavy chain variable region to a sequence encoding a human heavy chain constant region.

[0233] In some embodiments, if a mouse expresses a protein highly similar to an antigen of interest, it may be difficult to induce an immune response in the mouse. This is because during immune cell development, B cells and T cells that recognize MHC molecules bound to self-derived peptides are deleted from the immune cell repertoire. In such cases, the humanized mouse can be further modified. The corresponding gene can be knocked out in the mouse, and the mouse is then exposed to the antigen of interest. Because the mouse is not subject to negative selection against the gene product, it is easy to generate antibodies that can specifically bind to the target.

[0234] The present disclosure also provides methods of producing antibodies, nucleic acids, cells, and tissues (e.g., spleen tissue). In some embodiments, the methods include exposing an animal described herein to an antigen. Antibodies (e.g., hybrid antibodies), nucleic acids encoding the antibodies, cells, and / or tissues (e.g., spleen tissue) can be obtained from the animal. In some embodiments, nucleic acids encoding variable regions are determined, for example, by sequencing. In some embodiments, nucleic acids encoding human heavy chain immunoglobulin variable regions can be operably linked to nucleic acids encoding human heavy chain immunoglobulin constant regions. In some embodiments, cells containing the nucleic acids described herein are cultured and the antibodies are collected.

[0235] In some embodiments, no mouse immunoglobulin V, D, or J genes (e.g., mouse IGHV, IGHD, IGHJ, IGKV, or IGKJ genes) contribute to the heavy chain variable region sequence. In some embodiments, the heavy chain variable region sequences produced by the animal are fully human, contributed entirely by human immunoglobulin V, D, and J genes (e.g., human IGHV, IGHD, IGHJ, IGKV, and IGKJ genes). In some embodiments, the rearranged VDJ sequences may further undergo somatic hypermutation.

[0236] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into DNA encoding a human, humanized, or chimeric antibody, or an antibody or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletion, insertion, or substitution of residues within the amino acid sequence that makes up the antigen-binding site or domain of the antibody. Within such a population of variants, some antibodies or antigen-binding fragments may have increased affinity for the target protein. Any combination of deletion, insertion, and / or a combination can be achieved in antibodies or antigen-binding fragments thereof that have increased binding affinity for the target. Amino acid changes introduced into the antibody or antigen-binding fragment, such as changing the number (e.g., increasing or decreasing) of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that different sugars are attached by enzymes present in cells), or introducing new glycosylation sites, can alter the antibody or antigen-binding fragment or introduce new post-translational modifications into the antibody or antigen-binding fragment.

[0237] The antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates, such as monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents that have been genetically engineered to produce human antibodies.

[0238] Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequences derived from) human germline immunoglobulin sequences. Human antibodies can include, for example, amino acid residues within the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0239] Additional modifications can be made to the antibody or antigen-binding fragment. For example, cysteine ​​residues can be introduced in the Fc region to allow interchain disulfide bond formation in this region. The homodimeric antibody thus generated may optionally have increased in vitro and / or in vivo half-lives. Homodimeric antibodies with increased in vitro and / or in vivo half-lives can also be prepared using, for example, heterobifunctional cross-linkers as described by Wolff et al. (Cancer Res. 53:2560-2565, 1993). Alternatively, antibodies can be engineered with dual Fc regions (see, e.g., Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0240] In some embodiments, antibodies or antigen-binding fragments thereof can be covalently modified. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with organic derivatizing agents capable of reacting with selected side chains or with the N- or C-terminal residues.

[0241] Transferrin receptor 1 (TFR1) TFR1, also known as cluster of differentiation 71 (CD71), is ubiquitously expressed and can bind transferrin (Tf) with high affinity. Human TFR1 is a 90-kDa type II transmembrane glycoprotein consisting of 760 amino acids found as a dimer (180 kDa) held together by disulfide bonds on the cell surface. The TFR1 monomer consists of a large extracellular C-terminal domain of 671 amino acids, which contains the Tf-binding site, a transmembrane domain (28 amino acids), and an intracellular N-terminal domain (61 amino acids). The C-terminal extracellular domain contains three N-linked glycosylation sites at asparagine residues 251, 317, and 727, and one O-linked glycosylation site at threonine 104, all of which are required for full receptor function.

[0242] Transferrin (Tf) is an 80 kDa glycoprotein composed of two 40 kDa subunits, known as the N- and C-lobes, separated by a short linker sequence. Each subunit can bind one free ferric ion (Fe3+), so Tf can have up to two bound iron atoms. Tf in its iron-free form, apoTf, is highly efficient at transporting Fe3+ in the blood. 3+ bound to Fe 3+Upon interaction with TFR1, TFR1 transports the iron to the cell surface for internalization. As a membrane protein regulating iron import, TFR1 is a member of the TFR family that exhibits nanomolar affinity for Fe3+-bound transferrin (Tf). The Tf-TFR1 complex is internalized by clathrin-mediated endocytosis and transports Fe3+ to the cell surface when the pH is reduced to 5.5. 3+ At this pH, apoTf and TFR1 still associate and are recycled to the cell surface at physiological pH, releasing apoTf.

[0243] Because iron uptake via the transferrin receptor is an important method for cancer cells to absorb iron, accumulating evidence demonstrates that TFR1 is involved in tumor initiation and progression, and its expression is significantly dysregulated in many cancers. The relationship between TFR1 and cancer has become clear, making TFR1 a valuable pharmaceutical target for cancer intervention.

[0244] TFR1, which is expressed on the endothelial cells of the blood-brain barrier, can also be used in preclinical studies to deliver macromolecules, including antibodies, to the brain. Antibodies targeting TFR1 can cross the blood-brain barrier without interfering with iron uptake.

[0245] A detailed description of TFR1, Tf, and their functions can be found, for example, in Candelaria, PV, et al. "Antibodies targeting the transferrin receptor 1 (TfR1) as direct anti-cancer agents." Frontiers in Immunology 12(2021):607692; and Shen, Y., et al. "Transferrin receptor 1 in cancer: a new sight for cancer therapy." American Journal of Cancer Research 8.6(2018):916, each of which is incorporated by reference in its entirety.

[0246] Heavy chain single variable domain (VHH) antibodies Monoclonal and recombinant antibodies are important tools in medicine and biotechnology. Like all mammals, camelids (e.g., llamas) can produce conventional antibodies (e.g., IgG1) consisting of two heavy chains and two light chains linked in a Y-shape by disulfide bonds. However, they also produce two unique subclasses of IgG (also called heavy-chain IgGs): IgG2 and IgG3. These antibodies lack the CH1 region but still consist of only two heavy chains with antigen-binding domains at their N-termini, called VHHs (or nanobodies). Conventional IgGs require the combination of both heavy and light chain variable regions to enable high diversity in antigen-antibody interactions. While single heavy and light chains still exhibit this ability, they exhibit significantly lower affinity compared to paired heavy and light chains. A unique feature of heavy-chain IgGs is the ability of their monomeric antigen-binding regions to bind antigens with specificity, affinity, and especially diversity comparable to conventional antibodies, without the need for pairing. This feature is mainly due to several key mutations in the amino acid sequences of the variable regions of the two heavy chains, which cause major structural changes compared to conventional Igs. The key substitutions in the variable regions prevent the light chains from binding to the heavy chains, but also prevent the unbound heavy chains from being recycled by immunoglobulin-binding proteins.

[0247] The single variable domains of these antibodies (designated VHHs, sdAbs, or nanobodies) are the smallest antigen-binding domains generated by the adaptive immune system. It is well known that the third complementarity-determining region (CDR3) of the variable regions of these antibodies is twice as long as that of conventional antibodies. This results in an expanded interaction surface with the antigen, increasing the diversity of antigen-antibody interactions and compensating for the lack of a light chain. The long complementarity-determining region 3 (CDR3) allows VHHs to reach niches on proteins inaccessible to conventional antibodies, including functionally interesting sites such as the active site of an enzyme or the receptor-binding canyon on the surface of a virus.

[0248] Compared to conventional antibodies with conventional antibody variable domains (VH and VL), VHHs offer numerous other advantages, including higher stability, solubility, expression yield, and refolding ability, as well as better in vivo tissue penetration and internalization. Furthermore, in contrast to the VH domain of conventional antibodies, VHHs do not exhibit an intrinsic tendency to bind to light chains. Because VHHs do not bind to VL domains, it is much easier to reformat VHHs into multispecific (e.g., bispecific) constructs than constructs containing conventional VH-VL pairs or single domains based on VH domains.

[0249] The present disclosure provides, for example, anti-TFR1 antibodies, modified antibodies thereof, chimeric antibodies thereof, and humanized antibodies thereof.

[0250] The CDR sequences of 23B8 and 23B8-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as defined by Kabat numbering and set forth in SEQ ID NOs: 42, 43, and 44, respectively. CDRs can also be defined by the IMGT system, in which the CDRs of the VHH domain are set forth in SEQ ID NOs: 54, 55, and 56, respectively.

[0251] The CDR sequences of 24A1 and 24A1-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domains set forth in SEQ ID NOs: 45, 46, and 47, respectively, as defined by Kabat numbering. CDRs can also be defined by the IMGT system, in which the CDRs of the VHH domains are set forth in SEQ ID NOs: 57, 58, and 59, respectively.

[0252] The CDR sequences of 24C9 and 24C9-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as defined by Kabat numbering and set forth in SEQ ID NOs: 48, 49, and 50, respectively. CDRs can also be defined by the IMGT system, in which the CDRs of the VHH domain are set forth in SEQ ID NOs: 60, 61, and 62, respectively.

[0253] The CDR sequences of 24G5 and 24G5-derived antibodies (e.g., humanized antibodies) include the CDRs of the VHH domain as defined by Kabat numbering and set forth in SEQ ID NOs: 51, 52, and 53, respectively. CDRs can also be defined by the IMGT system, in which the CDRs of the VHH domain are set forth in SEQ ID NOs: 63, 64, and 65, respectively.

[0254] The amino acid sequence of the VHH domain of the 23B8 antibody is shown in SEQ ID NO: 66. The amino acid sequence of the VHH domain of the 24A1 antibody is shown in SEQ ID NO: 67. The amino acid sequence of the VHH domain of the 24C9 antibody is shown in SEQ ID NO: 68. The amino acid sequence of the VHH domain of the 24G5 antibody is shown in SEQ ID NO: 69.

[0255] The amino acid sequences of various modified or humanized VHHs are also provided. Because there are different methods for modifying or humanizing heavy chain antibodies (e.g., sequences can be modified with different amino acid substitutions), multiple versions of humanized sequences may exist for the VHH domain of a heavy chain antibody. In some embodiments, the humanized VHH domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 66-69.

[0256] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may also contain one, two, or three VHH domain CDRs selected from the group consisting of SEQ ID NOs: 42-44, 45-47, 48-50, 51-53, 54-56, 57-59, 60-62, and 63-65.

[0257] In some embodiments, the antibody may have a heavy chain single variable domain (VHH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VHH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VHH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VHH CDR3 amino acid sequence. Selected VHH CDR1, 2, and 3 amino acid sequences are shown in Figures 37 and 38.

[0258] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy chain single variable domain (VHH) comprising one, two, or three of: a VHH CDR1 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; a VHH CDR2 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; and a VHH CDR3 with 0, 1, or 2 amino acid insertions, deletions, or substitutions, wherein VHH CDR1, VHH CDR2, and VHH CDR3 are selected from the CDRs in Figure 39.

[0259] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of SEQ ID NO: 42 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 43 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 44 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0260] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of SEQ ID NO: 45 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 46 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 47 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0261] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of SEQ ID NO: 48 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 49 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 50 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0262] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of SEQ ID NO: 51 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 52 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 53 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0263] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of SEQ ID NO: 54 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 55 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 56 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0264] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of SEQ ID NO: 57 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 58 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 59 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0265] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of SEQ ID NO: 60 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 61 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 62 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0266] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing one, two, or three of the CDRs of SEQ ID NO: 63 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 64 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 65 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0267] Insertions, deletions, and substitutions can occur within the CDR sequences or at either or both ends of the CDR sequences. In some embodiments, the CDRs are determined according to the Kabat numbering scheme. In some embodiments, the CDRs are determined according to the Chothia numbering scheme. In some embodiments, the CDRs are determined according to a combined numbering scheme. In some embodiments, the CDRs are determined according to the IMGT numbering scheme.

[0268] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to TFR1 (human TFR1). The antibody or antigen-binding fragment thereof includes a heavy chain single variable region (VHH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VHH sequence. In some embodiments, the selected VHH sequence is SEQ ID NO: 66. In some embodiments, the selected VHH sequence is SEQ ID NO: 67. In some embodiments, the selected VHH sequence is SEQ ID NO: 68. In some embodiments, the selected VHH sequence is SEQ ID NO: 69.

[0269] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences. By way of example, sequence comparison and percent identity determination between two sequences can be performed using, for example, the Blossum62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0270] The present disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides comprising immunoglobulin heavy chain single variable domains (VHHs), wherein the VHHs comprise the CDRs as shown in Figures 37 and 38, or have a sequence as shown in Figure 39.

[0271] The antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multispecific (multimeric, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and antigen-binding fragments thereof.

[0272] In some embodiments, the antibody or antigen-binding fragment thereof comprises an Fc domain, which can be derived from various types (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), classes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclasses. In some embodiments, the Fc domain is derived from an IgG antibody or antigen-binding fragment thereof. In some embodiments, the Fc domain comprises one, two, three, four, or more heavy chain constant regions.

[0273] The present disclosure also provides antibodies or antigen-binding fragments thereof that cross-compete with any of the antibodies or antigen-binding fragments described herein. Cross-competition assays are well known in the art and are described, for example, in Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein," Journal of Virology 70.3 (1996):1863-1872, the entire contents of which are incorporated herein by reference. In one aspect, the present disclosure also provides antibodies or antigen-binding fragments thereof that bind to the same epitope or region as any of the antibodies or antigen-binding fragments described herein. Epitope binding assays are well known in the art and are described, for example, in Estep et al., "High throughput solution-based measurement of antibody-antigen affinity and epitope binning," MAbs. Vol. 5. No. 2. Taylor & Francis, 2013, the entire contents of which are incorporated herein by reference.

[0274] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR1 selected from SEQ ID NOs: 42, 45, 48, 51, 54, 57, 60, and 63.

[0275] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR2 selected from SEQ ID NOs: 43, 46, 49, 52, 55, 58, 61, and 64.

[0276] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR3 selected from SEQ ID NOs: 44, 47, 50, 53, 56, 59, 62, and 65.

[0277] Antibody characteristics TFR1 plays a key role in cellular iron uptake through its interaction with iron-binding TF. Iron is required for multiple cellular processes and is essential for DNA synthesis and, therefore, cell proliferation. Due to its central role in cancer cytopathology, malignant cells often overexpress TFR1, and this increased expression may be associated with poor prognosis in different types of cancer. The elevated expression levels of TfR1 in malignant cells, its extracellular accessibility, internalization ability, and its central role in cancer cytopathology make this receptor an attractive target for antibody-mediated therapy.

[0278] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are unable to block the binding between TFR1 and TF. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are capable of blocking the binding between TFR1 and TF. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be conjugated to anti-cancer drugs that are internalized by receptor-mediated endocytosis. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are capable of disrupting receptor function. In some embodiments, the antibodies or antigen-binding fragments thereof described herein are unable to induce Fc effector function, thus preventing or ameliorating their adverse effects on normal cells.

[0279] The present disclosure provides antibodies or antigen-binding fragments thereof comprising a human Fc domain that induce Fc-dependent effector function by at least or about at least or about 1-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, or at least or about 100-fold compared to the absence of an antibody or antigen-binding fragment thereof described herein.

[0280] The present disclosure provides antibodies or antigen-binding fragments thereof comprising a human Fc domain that induce a host immune response at least or about at least or about 1-fold, at least or about 2-fold, at least or about 3-fold, at least or about 4-fold, at least or about 5-fold, at least or about 6-fold, at least or about 7-fold, at least or about 8-fold, at least or about 9-fold, at least or about 10-fold, at least or about 20-fold, at least or about 30-fold, at least or about 40-fold, at least or about 50-fold, or at least or about 100-fold compared to the absence of an antibody or antigen-binding fragment thereof described herein.

[0281] The present disclosure provides antibodies or antigen-binding fragments thereof capable of internalizing into human brain cells (e.g., cortical microvascular endothelial cells) with an endocytosis rate of at least 50%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, the endocytosis rate of the antibodies or antigen-binding fragments thereof described herein is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold higher than that of an isotype control antibody.

[0282] In some embodiments, provided herein are antibodies or antigen-binding fragments thereof comprising a single heavy chain. In some embodiments, provided herein are antibodies or antigen-binding fragments thereof comprising a pair of heavy chains. In some embodiments, the heavy chain pair is linked by a disulfide bond. In some embodiments, the heavy chain pair comprises a knobs-in-holes modification. In some embodiments, the heavy chain comprises a human IgG Fc domain. In some embodiments, the antibody or antigen-binding fragment thereof comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 VHH domains in each heavy chain. In some embodiments, the VHH domains in each heavy chain specifically bind the same epitope. In some embodiments, the VHH domains in each heavy chain specifically bind different epitopes. In some embodiments, the VHH domains in each heavy chain bind at least 1, 2, 3, 4, or 5 different epitopes.

[0283] In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody or a trispecific antibody. In some embodiments, the antibody or antigen-binding fragment thereof can specifically bind to at least four, five, or six antigens.

[0284] In some embodiments, the antibody (or antigen-binding fragment thereof) is -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 In some embodiments, the koff is less than 0.01 s or less than 0.00001 s. -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.00001s -1 Over or 0.000001s -1 It's super.

[0285] In some embodiments, the kinetic association rate (k) is 1×10 2 / Ms super, 1×103 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or more than 1 × 10 6 In some embodiments, the kinetic association rate (k) is greater than 1×10 5 / Ms less than 1 × 10 6 / Ms or less than 1 x 10 7 / Ms is less than.

[0286] Affinity can be estimated from the quotient of the kinetic rate constants (K = k / k). In some embodiments, K is greater than or equal to 1 x 10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M or 1 x 10 -10 In some embodiments, the KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is less than 1 x 10 -7 Super M, 1×10 -8 Super M, 1×10 -9 Super M, 1×10 -10 Super M, 1×10 -11 Over M or 1 x 10 -12 It's over M.

[0287] Common techniques for measuring the affinity of an antibody to an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibody binds to human TFR1, monkey TFR1, mouse TFR1, or chimeric TFR1. In some embodiments, the antibody does not bind to human TFR1, monkey TFR1, mouse TFR1, or chimeric TFR1.

[0288] In some embodiments, thermal stability is measured. The antibodies or antigen-binding fragments described herein may have a Tm (melting temperature) of greater than 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C. In some embodiments, the Tm is less than 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C. The antibodies or antigen-binding fragments described herein can have a Tag (aggregation temperature, e.g., Tag at 266 nm (Tagg266) or Tag at 473 nm (Tagg473)) greater than 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C. In some embodiments, Tagg is less than 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C.

[0289] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.

[0290] In some embodiments, the antibody or antigen-binding fragment thereof has a functional Fc region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a human IgG1 Fc region. In some embodiments, the human IgG1 Fc region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 75.

[0291] In some embodiments, the antibody or antigen-binding fragment does not have an Fc region. For example, an antibody (or antigen-binding fragment thereof) is a polypeptide comprising one or more VHH domains interconnected by linker peptides. In some embodiments, the antibody comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 VHH domains. In some embodiments, the VHH domains specifically bind to the same epitope. In some embodiments, the VHH domains bind to different epitopes. In some embodiments, the VHH domains bind to at least 1, 2, 3, 4, or 5 different epitopes.

[0292] In some embodiments, the antibody or antigen-binding fragment thereof does not have a functional Fc region. In some embodiments, the Fc region has a LALA mutation (L234A and L235A mutations in EU numbering) or a LALA-PG mutation (L234A, L235A, P329G mutations in EU numbering). In some embodiments, the Fc region has a mutation at position 297 according to EU numbering (e.g., N297A). In some embodiments, the mutated human IgG1 Fc region comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 76.

[0293] In some embodiments, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours after administration to a subject, the concentration of an antibody or antigen-binding fragment thereof described herein in the brain (e.g., whole brain or parenchyma) may be greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of its concentration immediately after administration (e.g., 0.5 hours). In some embodiments, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours after administration to a subject, the concentration of an antibody or antigen-binding fragment thereof described herein in the brain (e.g., whole brain or parenchyma) can be at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold, or 10000-fold the concentration of a control antibody (e.g., hIgG1 or JR141-N) or the concentration in the serum of the subject.

[0294] Method for producing anti-TFR1 antibodies Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into DNA encoding a human, humanized, or chimeric antibody, or an antibody or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletion, insertion, or substitution of residues within the amino acid sequence that makes up the antigen-binding site or domain of the antibody. In a population of such variants, some antibodies or antigen-binding fragments have increased affinity for a target protein, e.g., TFR1. Any combination of deletion, insertion, and / or a combination can be achieved in an antibody or antigen-binding fragment thereof with increased binding affinity for the target. Antibodies or antigen-binding fragments can be altered or new post-translational modifications can be introduced into the antibody or antigen-binding fragment by introducing amino acid changes into the antibody or antigen-binding fragment, such as changing the number (e.g., increasing or decreasing) of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that different sugars are attached by enzymes present in cells), or introducing new glycosylation sites. In some embodiments, the heavy chain antibodies or antigen-binding fragments thereof described herein are obtained by immunizing any of the genetically modified animals described herein (e.g., mice homozygous for the heavy chain mutant allele 3 genotype).

[0295] Humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequences as) human germline immunoglobulin sequences of a human immunoglobulin scaffold sequence. Humanized antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). Thus, "humanized" antibodies are chimeric antibodies in which sequences from a non-human species are replaced by corresponding human sequences.

[0296] Typically, an amino acid sequence variant of a human, humanized, or chimeric anti-TFR1 antibody contains an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% percent identity with the sequence present in the VHH domain of the original antibody.

[0297] Identity or homology to the original sequence is typically the percentage of amino acid residues present in the candidate sequence that are identical to sequences present in a human, humanized, or chimeric anti-TFR1 antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity.

[0298] Further modifications can be made to the anti-TFR1 antibody or antigen-binding fragment. For example, cysteine ​​residues can be introduced into the Fc region to allow interchain disulfide bond formation in this region. The homodimeric antibody thus generated may optionally have increased in vitro and / or in vivo half-lives. Homodimeric antibodies with increased in vitro and / or in vivo half-lives can also be prepared using, for example, heterobifunctional cross-linkers as described by Wolff et al. ("Monoclonal antibody homodimers: enhanced antitumor activity in nude mice," Cancer research 53.11 (1993):2560-2565). Alternatively, antibodies having dual Fc regions can be engineered.

[0299] In some embodiments, covalent modifications can be made to anti-TFR1 antibodies or antigen-binding fragments thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with organic derivatizing agents capable of reacting with selected side chains or the N- or C-terminal residues.

[0300] In some embodiments, antibody variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibody compositions can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycans (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 in the Fc region (position 314 in the EU numbering or Kabat numbering of Fc region residues). However, Asn297 may also be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variation in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, the Fc region of the antibody can be further modified to replace the asparagine at position 297 with alanine (N297A) to reduce glycan heterogeneity.

[0301] The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such that the host cell contains the polynucleotides and / or vectors comprising the polynucleotides), and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

[0302] As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector is introduced. Thus, in an expression vector, a polynucleotide of interest is positioned for expression in the vector by being operably linked to control elements, such as a promoter, enhancer, and / or polyA tail, at, near, or adjacent to the integration site of the polynucleotide of interest, either within the vector or in the genome of the host cell, such that the polynucleotide of interest is translated in a host cell into which the expression vector is introduced.

[0303] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0304] In some embodiments, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., variola or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication-competent virus, or may employ a replication-defective virus, in which case viral propagation generally occurs only in complementary viral packaging cells. Suitable systems are described, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. N.Y. Acad. Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al. al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. DNA can also be "naked," as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. Uptake of naked DNA can be enhanced by coating the DNA onto biodegradable beads that are efficiently transported into cells.

[0305] For expression, a DNA insert containing an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter). Some examples include the phage lambda PL promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and promoters of retroviral long terminal repeats. Other suitable promoters are known to those of skill in the art. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. The expression construct can further contain sites for transcription initiation and termination, and, within the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct can include a translation initiation codon at the beginning and a termination codon (UAA, UGA, or UAG) positioned approximately at the end of the polypeptide to be translated.

[0306] As indicated, the expression vector can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance genes for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila melanogaster S2 and Spodoptera litura Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells, and plant cells. Appropriate culture media and conditions for the host cells described herein are well known in the art.

[0307] Non-limiting vectors for use in bacteria include pQE70, pQE60, and pQE-9 available from Qiagen, pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene, and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those of skill in the art.

[0308] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, promoters of retroviral LTRs such as those of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0309] In the yeast Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, can be used.

[0310] Introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0311] Transcription of DNA encoding the antibodies of the present disclosure in more eukaryotic organisms can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that serve to increase transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0312] For secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide. The signals can be endogenous to the polypeptide or they can be heterologous signals.

[0313] Polypeptides (e.g., antibodies) can be expressed in modified forms, such as fusion proteins (e.g., GST fusions) or with histidine tags, and can contain not only secretion signals but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of a polypeptide to improve stability and durability in host cells during purification or during subsequent handling and storage. Peptide moieties can also be added to polypeptides to facilitate purification. Such regions can be removed before final preparation of the polypeptide. The addition of peptide moieties to polypeptides to, inter alia, cause secretion or excretion, improve stability, and facilitate purification is well known and routine in the art.

[0314] The present disclosure also provides nucleic acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of the nucleotide sequences described herein, and Also provided are amino acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence described herein. In some embodiments, the present disclosure relates to a nucleotide sequence encoding any of the peptides described herein or any amino acid sequence encoded by any of the nucleotide sequences described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.

[0315] In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences described herein.

[0316] In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences described herein.

[0317] In some embodiments, the antibody or antigen-binding fragment thereof is expressed in yeast, insect cells, or mammalian cells (eg, CHO cells).

[0318] Treatment and diagnostic methods The anti-TFR1 antibodies of the present disclosure, or antibodies or antigen-binding fragments thereof, can be used for various therapeutic purposes. In one aspect, the present disclosure provides methods for treating a brain disease (e.g., brain cancer, dementia, or Alzheimer's disease) in a subject, methods for identifying a subject with a brain disease (e.g., brain cancer, dementia, or Alzheimer's disease), methods for reducing the risk of developing a brain disease, or methods for reducing the risk of developing additional symptoms in a subject. In some embodiments, treatment can halt, slow, delay, or inhibit the progression of the brain disease (e.g., brain cancer, dementia, or Alzheimer's disease). In some embodiments, treatment can reduce the number, severity, and / or duration of one or more symptoms of a brain disease (e.g., brain cancer, dementia, or Alzheimer's disease) in a subject.

[0319] In one aspect, the disclosure features a method that includes administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a brain disorder).

[0320] In one aspect, the disclosure features a method of delivering a therapeutic agent across the blood-brain barrier. In some embodiments, an antibody or antigen-binding fragment thereof described herein is linked to a therapeutic agent. In some embodiments, the therapeutic agent is an antibody, an antigen-binding fragment thereof, a small molecule, or an antibody-drug conjugate.

[0321] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for brain disorders (e.g., brain cancer, dementia, or Alzheimer's disease). Patients with brain disorders (e.g., brain cancer, dementia, or Alzheimer's disease) can be identified by a variety of methods known in the art.

[0322] In some embodiments, the brain disease is brain cancer.

[0323] In one aspect, the present disclosure relates to a method of reducing tumor growth rate, comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof, or an antibody drug conjugate described herein. In one aspect, the present disclosure relates to a method of killing tumor cells, comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof, or an antibody drug conjugate described herein.

[0324] As used herein, "effective amount" means an amount or dosage sufficient to bring about a beneficial or desired result, including halting, slowing, retarding, or inhibiting the progression of a disease, e.g., cancer. The effective amount will vary depending on, for example, the age and weight of the subject to whom the antibody, antigen-binding fragment, polynucleotide encoding the antibody, vector comprising the polynucleotide, and / or composition is administered, the severity of the symptoms, and the route of administration, and thus, dosing can be determined on an individual basis.

[0325] An effective amount can be administered in one or more administrations. By way of example, an effective amount of an antibody or antigen-binding fragment thereof is an amount sufficient to ameliorate, halt, stabilize, reverse, inhibit, slow, and / or delay the progression of a patient's disease. As understood in the art, an effective amount of an antibody or antigen-binding fragment may vary depending on other factors, such as, inter alia, the patient's medical history, as well as the type (and / or dosage) of antibody used.

[0326] Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of one in the art. One of skill in the art will understand that the dosage required to be administered will vary depending, for example, on the mammal receiving the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein, the route of administration, the particular type of antibody, antibody-encoding polynucleotide, antigen-binding fragment, and / or composition disclosed herein used, and other agents administered to the mammal. Guidance for selecting appropriate doses for antibodies or antigen-binding fragments can be found in literature on the therapeutic use of antibodies and antigen-binding fragments, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.

[0327] A typical daily dose of an effective amount of antibody is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose can be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dose can be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dose is about 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0328] In any of the methods described herein, at least one antibody, antigen-binding fragment thereof, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein), and optionally at least one additional therapeutic agent, can be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered as a sustained release oral formulation.

[0329] In some embodiments, one or more additional therapeutic agents can be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, the one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to a subject such that there is overlap in the period of biological activity of the one or more additional therapeutic agents with the period of biological activity of the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.

[0330] In some embodiments, a subject can be administered at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) for an extended period of time (e.g., for a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional can determine the length of treatment period using any of the methods described herein to diagnose or track the effectiveness of the treatment (e.g., by observing at least one symptom of the disease). As described herein, a skilled medical professional can also vary (e.g., increase or decrease) the identity and number of antibodies or antigen-binding antibody fragments (and / or one or more additional therapeutic agents) administered to a subject, and can adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) to a subject based on an evaluation of the effectiveness of the treatment (e.g., using any of the methods described herein and well known in the art).

[0331] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agents can include one or more inhibitors selected from the group consisting of inhibitors of B-Raf, EGFR inhibitors, inhibitors of MEK, inhibitors of ERK, inhibitors of K-Ras, inhibitors of c-Met, inhibitors of anaplastic lymphoma kinase (ALK), inhibitors of phosphatidylinositol 3-kinase (PI3K), inhibitors of Akt, inhibitors of mTOR, dual PI3K / mTOR inhibitors, inhibitors of Bruton's tyrosine kinase (BTK), and inhibitors of isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2).

[0332] In some embodiments, the additional therapeutic agent can comprise one or more inhibitors selected from the group consisting of inhibitors of HER3, inhibitors of LSD1, inhibitors of MDM2, inhibitors of BCL2, inhibitors of CHK1, inhibitors of the activated hedgehog signaling pathway, and agents that selectively degrade the estrogen receptor.

[0333] In some embodiments, the additional therapeutic agent is trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, leolysin, Alimta, Dicaida, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-00 The present invention may include one or more therapeutic agents selected from the group consisting of 3, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0334] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a CX3CL1 targeted therapy, a CXCL9 targeted therapy, a CXCL10 targeted therapy, a CCL5 targeted therapy, an LFA-1 agonist, an ICAM1 agonist, and a selectin agonist.

[0335] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0336] In some embodiments, the additional therapeutic agent is an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody.

[0337] Pharmaceutical Compositions and Routes of Administration Also provided herein are pharmaceutical compositions containing at least one (e.g., 1, 2, 3, or 4) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., 2, 3, or 4) of any of the antibodies or antigen-binding fragments described herein can be present in the pharmaceutical composition, in any combination. Pharmaceutical compositions can be formulated in any manner known in the art.

[0338] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). Compositions may contain a sterile diluent (e.g., sterile water or saline), fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol, methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetate, citrate, or phosphate), and isotonic agents (e.g., sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Pat. No. 4,522,811). The composition preparation can be formulated and enclosed in ampoules, disposable syringes, or multiple-dose vials. Where necessary (e.g., in injectable formulations), proper fluidity can be maintained, for example, by the use of a coating such as lecithin or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, sustained release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0339] Compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in unit dosage form (i.e., physically discrete units containing a predetermined amount of active compound(s) for ease of administration and uniformity of dosage).

[0340] The toxicity and therapeutic efficacy of a composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). For example, the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Drugs that exhibit a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care should be taken to minimize the potential for harm (i.e., reduce the undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0341] Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dose of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) is an amount that treats a disease in a subject, a subject, or a subject identified as being at risk for developing the disease, and reduces the severity, frequency, and / or duration of one or more symptoms of the disease in a subject (e.g., a human). The efficacy and administration of any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods well known in the art, as well as by observing one or more symptoms of the disease in a subject (e.g., a human). Certain factors can affect the dose and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the subject's overall health and / or age, and the presence of other diseases).

[0342] Exemplary doses include amounts (milligrams or micrograms) of any of the antibodies or antigen-binding fragments described herein per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg, about 100 μg / kg to about 500 mg / kg, about 100 μg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, about 10 μg / kg to about 0.5 mg / kg, or about 1 μg / kg to about 50 μg / kg). While these doses cover a wide range, those of skill in the art will appreciate that the efficacy and effective amounts of therapeutic agents comprising antibodies and antigen-binding fragments thereof can be determined by methods well known in the art. Typically, a relatively low dose is administered initially, and the dose can be subsequently and gradually increased by the attending health care professional or veterinary professional (for therapeutic uses) or by a researcher (if still working in the development phase) until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the subject's age, weight, general health, sex, and diet, time of administration, route of administration, rate of excretion, and half-life of the antibody or antibody fragment in vivo.

[0343] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.The present disclosure also provides methods for producing antibodies or antigen-binding fragments thereof for the various uses described herein.

[0344] Additional Embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the claims. Additional embodiments are also provided.

[0345] Embodiment 1 is a genetically modified rodent comprising an engineered immunoglobulin heavy chain locus, wherein said engineered immunoglobulin heavy chain locus comprises an IgG constant region gene, said IgG constant region gene encoding an IgG heavy chain constant region lacking a CH1 domain, and wherein said genetically modified rodent expresses a heavy chain antibody.

[0346] Embodiment 2 is the genetically modified rodent of embodiment 1, wherein said rodent contains exactly one IgG constant region gene.

[0347] Embodiment 3 is the genetically modified rodent of embodiment 1 or 2, wherein the IgG heavy chain constant region gene is IGHG1.

[0348] Embodiment 4 is the genetically modified rodent of any one of Embodiments 1 to 3, wherein the IgG heavy chain constant region comprises or consists of a CH2 domain and a CH3 domain, and optionally a hinge region.

[0349] Embodiment 5 is a genetically modified rodent whose genome comprises a germline genetic modification comprising a deletion of the IGHG3, IGHG2b, and IGHG2c genes and a deletion of the CH1 exon of the IGHG1 gene in the rodent immunoglobulin heavy chain locus.

[0350] Embodiment 6 is the rodent of embodiment 5, wherein the germline genetic modification further comprises a deletion of the rodent IGHE gene at the rodent immunoglobulin heavy chain locus.

[0351] Embodiment 7 is the rodent of embodiment 5 or 6, wherein the genetic modification further comprises a deletion of rodent Sγ2b, Sγ2c, and Sε switch regions in the rodent immunoglobulin heavy chain locus.

[0352] Embodiment 8 is a rodent according to any one of embodiments 5 to 7, wherein the modified immunoglobulin heavy chain locus comprises a modified IGHG1 gene lacking a sequence encoding a CH1 domain, and the modified IGHG1 gene comprises a sequence at least 80%, 90%, 95% or 99% identical to SEQ ID NO:1.

[0353] Embodiment 9 is the rodent of any one of Embodiments 5 to 8, wherein the genetic modification further comprises a deletion of a rodent Sγ3 switch region in the rodent immunoglobulin heavy chain locus.

[0354] Embodiment 10 is a rodent according to any one of embodiments 5 to 9, wherein the genome of the rodent comprises a modified rodent IGH1 gene lacking sequences encoding rodent Sμ, Sγ1, and Sα switch regions, a CH1 domain, and rodent IGHM, IGHδ, and IGHA genes.

[0355] Embodiment 11 is the rodent of any one of Embodiments 5 to 9, wherein the genetic modification further comprises a deletion of the rodent IGHM gene and the IGHδ gene in the rodent immunoglobulin heavy chain locus.

[0356] Embodiment 12 is a rodent according to any one of embodiments 5 to 9 and 11, wherein the rodent genome comprises rodent Sμ, Sγ1, Sα switch regions, a modified IGHG1 gene lacking sequences encoding the CH1 domain, and a rodent IGHA gene.

[0357] Embodiment 13 is the rodent of embodiment 12, wherein the Sμ and Sγ1 switch regions are linked to a sequence that is at least 80%, 90%, 95%, or 99% identical to SEQ ID NO:8.

[0358] Embodiment 14 is the rodent of any one of Embodiments 5 to 9, wherein the genetic modification further comprises a deletion of the CH1 coding sequence of the IGHM gene in the rodent immunoglobulin heavy chain locus.

[0359] Embodiment 15 is a rodent according to any one of embodiments 5 to 9 and 14, wherein the genome of the rodent comprises rodent Sμ, Sγ1, and Sα switch regions, a modified rodent IGHM gene lacking a sequence encoding the CH1 domain, a modified IGHG1 gene lacking a sequence encoding the CH1 domain, and rodent IGHδ and IGHA genes.

[0360] Embodiment 16 is the rodent of embodiment 15, wherein the Sμ switch region and the modified IGHM gene are linked to a sequence at least 80%, 90%, 95%, or 99% identical to SEQ ID NO:10.

[0361] Embodiment 17 is the rodent of any one of embodiments 5 to 9, wherein the genetic modification further comprises a deletion of the CH1 exon of the IGHM gene and a deletion of the IGHδ gene in the rodent immunoglobulin heavy chain locus.

[0362] Embodiment 18 is a rodent according to any one of embodiments 5 to 9 and 17, wherein the genome of the rodent comprises rodent Sμ, Sγ1, Sα switch regions, a modified IGHM gene lacking sequences encoding a CH1 domain, a modified IGHG1 gene lacking sequences encoding a CH1 domain, and a rodent IGHA gene.

[0363] Embodiment 19 is the rodent of any one of embodiments 5 to 9, wherein the genetic modification further comprises a deletion of the CH1 exon of the IGHM gene and a deletion of the CH1 coding sequence of the IGHδ gene in the rodent immunoglobulin heavy chain locus.

[0364] Embodiment 20 is a rodent according to any one of embodiments 5 to 9 and 19, wherein the genome of the rodent comprises rodent Sμ, Sγ1, Sα switch regions, a modified IGHM gene lacking a sequence encoding a CH1 domain, a modified IGHδ gene lacking a sequence encoding a CH1 domain, a modified IGHG1 gene lacking a sequence encoding a CH1 domain, and a rodent IGHA gene.

[0365] Embodiment 21 is the rodent of embodiment 19 or 20, wherein the modified IGHM gene is linked to a sequence that is at least 80%, 90%, 95% or 99% identical to SEQ ID NO: 10, and the modified IGHδ gene comprises a sequence that is at least 80%, 90%, 95% or 99% identical to SEQ ID NO: 41.

[0366] Embodiment 22 is the rodent of any one of embodiments 14 to 21, wherein the modified IGHM gene comprises a sequence at least 80%, 90%, 95%, or 99% identical to SEQ ID NO:13.

[0367] Embodiment 23 is the rodent of any one of embodiments 5 to 8, wherein the genetic modification further comprises a deletion of a rodent Sγ1 switch region in the rodent immunoglobulin heavy chain locus.

[0368] Embodiment 24 is a rodent according to any one of embodiments 5 to 8 and 23, wherein the genome of the rodent comprises a modified IGH1 gene lacking sequences encoding rodent Sμ, Sγ3, and Sα switch regions, a CH1 domain, and rodent IGHM, IGHδ, and IGHA genes.

[0369] Embodiment 25 is the rodent of any one of embodiments 5 to 8 and 23, wherein the genetic modification further comprises a deletion of a rodent Sγ3 switch region in the rodent immunoglobulin heavy chain locus.

[0370] Embodiment 26 is the rodent of any one of embodiments 5 to 8, 23, and 25, wherein the genetic modification further comprises a deletion of rodent IGHM and IGHδ genes in the rodent immunoglobulin heavy chain locus.

[0371] Embodiment 27 is a rodent according to any one of embodiments 5 to 8, 23, 25 or 26, wherein the genome of the rodent comprises a rodent Sμ, an Sα switch region, a modified IGHG1 gene lacking sequences encoding the CH1 domain, and a rodent IGHA gene.

[0372] Embodiment 28 is the rodent of embodiment 27, wherein the Sμ switch region and the modified IGHG1 gene are linked to a sequence at least 80%, 90%, 95%, or 99% identical to SEQ ID NO:9.

[0373] Embodiment 29 is the rodent of any one of embodiments 5 to 10, 23 and 24, wherein the modified genome comprises a functional IGHM gene. [Example]

[0374] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0375] Example 1: Overview The immunoglobulin heavy chain locus of a non-human animal was modified by gene editing. For example, to obtain a mouse capable of expressing a heavy chain antibody, the immunoglobulin heavy chain constant region locus in mouse chromosome 12 was modified. The genetically modified mouse was capable of expressing a heavy chain antibody, as shown in Figure 1. Furthermore, all endogenous VDJ sequences in the heavy chain variable region locus on chromosome 12 were replaced with human VDJ sequences, thereby resulting in a fully humanized variable region of the heavy chain antibody expressed by the mouse. In some experiments, genetic modification of the immunoglobulin heavy chain constant region locus was performed in mice with human VDJ sequences.

[0376] Example 2: Modification of the mouse immunoglobulin heavy chain constant region locus As shown in Figure 2, mouse (C57BL / 6) immunoglobulin constant region genes include (in the following order): immunoglobulin heavy constant μ (IGHM or Cμ), immunoglobulin heavy constant δ (IGHδ or Cδ), immunoglobulin heavy constant γ3 (IGHG3 or Cγ3), immunoglobulin heavy constant γ1 (IGHG1 or Cγ1), immunoglobulin heavy constant γ2b (IGHG2b or Cγ2b), immunoglobulin heavy constant γ2c (IGHG2c or Cγ2c), immunoglobulin heavy constant ε (IGHE or Cε), and immunoglobulin heavy constant α (IGHA or Cα) genes. As shown in Figure 3, switch regions (e.g., Sμ, Sγ3, Sγ1, Sγ2b, Sγ2c, Sε, and Sα) and their respective promoters are located upstream of the corresponding constant region genes. In particular, the Cγ1 gene contains, from the N-terminus to the C-terminus, sequences encoding the CH1, H (hinge), CH2, CH3, M1, and M2 regions of IgG1.

[0377] Mouse immunoglobulin heavy chain constant region loci were modified by various methods described below. Experiments were performed in mice with fully humanized VDJ sequences. Details of VDJ region-humanized mice can be found, for example, in WO2020169022A1 and US20200390073A1, each of which is incorporated herein by reference in its entirety.

[0378] To obtain mice expressing only heavy chain antibodies, the Cγ3, Cγ1, Cγ2b, Cγ2c, and Cε loci were modified to retain only the truncated Cγ1 sequence without the CH1 coding region (Cγ1ΔCH1), resulting in mutant alleles shown as mutant allele 1 or mutant allele 1' in Figure 4A. Based on mutant allele 1, different modifications were made to the switch regions (e.g., Sμ and Sγ1), Cμ, and / or Cδ sequences. For example, the entire Cμ and Cδ sequences were knocked out, and the resulting alleles are shown as mutant allele 2 and mutant allele 2' (additionally lacking Sγ1). Alternatively, the regions to be knocked out were selected from the CH1 coding region of Cμ (with the resulting allele shown as mutant allele 3), the CH1 coding region of Cμ and the entire Cδ (with the resulting allele shown as mutant allele 4), and both the CH1 coding region of Cμ and the CH1 coding region of Cδ (with the resulting allele shown as mutant allele 5).

[0379] Example 3: Construction of targeting vector Targeting vectors such as V1, V2, V3, V4, V5, V6, and V7 were used for the modifications.

[0380] Vector V1 Vector V1 contained the Cγ1ΔCH1 knock-in sequence (SEQ ID NO: 1) as shown in Figure 5. The Cγ1ΔCH1 sequence does not contain the coding sequence for CH1. The CH1 coding region was either deleted or replaced with a Neo cassette. The Neo cassette contains the Neo gene sequence flanked by two Frt (or LoxP) sequences. Flp transgenic mice were bred with mice carrying the Neo cassette to remove the cassette.

[0381] The targeting vector V1 contained the following characteristic sequences, in 5' to 3' order: an upstream homologous arm (5' homologous arm), a mouse Sγ1 promoter sequence, a mouse Sγ1, Cγ1ΔCH1 sequence, and a downstream homologous arm (3' homologous arm). The targeting vector may also contain an antibiotic resistance gene (e.g., neomycin phosphotransferase gene, or Neo) for screening positive clones and two Frt recombination sites flanking the antibiotic resistance gene. Additionally, a coding gene with a negative selection marker (e.g., a gene encoding diphtheria toxin A subunit (DTA)) can also be inserted into the targeting vector.

[0382] A 16,076-bp sequence containing the mouse Sγ1 promoter, mouse Sγ1, and Cγ1ΔCH1 knock-in sequence (SEQ ID NO: 1) was cloned from a mouse bacterial artificial chromosome (RP23-38K22 or RP23-265P18). The following two sets of primer pairs were used to obtain sequences containing the mouse Sγ1 promoter, mouse Sγ1, and Cγ1ΔCH1 knock-in sequence for construction of the V1 vector. Next, the sequences amplified from V1-F1 and V1-R1 and the sequences amplified from V1-F2 and V1-R2 were ligated. V1-F1 (SEQ ID NO: 2): 5'-GTGGTTCTGGCTACAAGATAGAGCTCTGTCAATGATGTTTGCAGAGACTACA-3' V1-R1 (SEQ ID NO: 3): 5'-CTCCCTATACGTCCTTCTCACCTACAAGAAAAAAGTATATGTGATTACACTGTCAGACAG-3' V1-F2 (SEQ ID NO: 4): 5'-GTGTAATCACATATACTTTTTCTTGTAGGTGAGAGGACGTATAGGGAGGAGGGGTTC-3' V1-R2 (SEQ ID NO: 5): 5'-CGTCTAGTCCTTGCCCACGTGTCGACCCCATAGGGAGGACAGACTGAGG-3'

[0383] As shown in Figure 6, the V1 vector was used to replace a 100883 bp sequence (nucleic acids 113232142 to 113333024 of the NCBI reference sequence NC_000078.7) of the mouse heavy chain constant region locus (from Sγ3 to Cε) in one step.

[0384] Vector V2 As shown in Figure 7, the V2 vector was used to replace the 92859 bp sequence of the mouse heavy chain constant region (spanning Cγ3 to Cε) (nucleic acids 113232142 to 113325000 of the NCBI reference sequence NC_000078.7) in one step.

[0385] The method involved cloning from a mouse bacterial artificial chromosome (BAC) using a pair of primers to obtain a knock-in sequence containing the Cγ1ΔCH1 knock-in sequence (SEQ ID NO: 1). The following primers were used: V2-F1 (SEQ ID NO: 6): 5'-TCTGAACTACTTCGTCGACGTGAGAGGACGTATAGGGAGGAGGG-3' V2-R1 (SEQ ID NO: 7): 5'-CACGTGGATCCGCGGCGCCCATAGGGAGGACAGACTGAGGAC-3'

[0386] Vector V3 A total of 16,434 bp of nucleotides were knocked out from the chromosome, including Cμ and Cδ, using the V3 vector, as shown in Figure 8. The junction sequence between Sμ and Sγ1 in the recombinant mutant allele 2 is shown in SEQ ID NO:8.

[0387] Vector V4 As shown in Figure 9, targeting vector V4 contained the following characteristic sequences, in 5' to 3' order: an upstream homologous arm (5' homologous arm), a portion of mouse Sμ, and a downstream homologous arm (3' homologous arm). The targeting vector also contained an antibiotic resistance gene (neomycin phosphotransferase gene, or Neo) for screening positive clones and two Frt recombination sites flanking the antibiotic resistance gene. Additionally, a coding gene carrying a negative selection marker (the gene encoding diphtheria toxin A subunit (DTA)) was also inserted into the targeting vector. Specifically, Sμ was directly linked to Cγ1ΔCH1' of the recombinant mutant allele 2' (i.e., there were no other switch regions or immunoglobulin gene sequences in between). The linked sequence is shown in SEQ ID NO:9.

[0388] Vector V5 The V5 vector was used to knock out the CH1 coding sequence in Cμ, as shown in Figure 10. The CH1 coding sequence of Cμ was deleted, and the ligated sequence is shown in SEQ ID NO:10.

[0389] Vector V6 As shown in Figure 11, targeting vector V6 contained the following characteristic sequences, in 5' to 3' order: an upstream homologous arm (5' homologous arm), a portion of the mouse Sμ region, a mouse CμΔCH1 sequence, and a downstream homologous arm (3' homologous arm). The targeting vector also contained an antibiotic resistance gene (neomycin phosphotransferase gene, or Neo) for screening positive clones and two Frt recombination sites flanking the antibiotic resistance gene. Additionally, a coding gene with a negative selection marker (the gene encoding diphtheria toxin A subunit (DTA)) was also inserted into the targeting vector.

[0390] A sequence containing the mouse CμΔCH1 sequence was cloned from mouse somatic cells. The following primer pair was used to obtain a sequence containing mouse CμΔCH1 (or CμΔCH1 knock-in sequence, SEQ ID NO: 13) for construction of the V6 vector: V6-F1 (SEQ ID NO: 11): 5'-ATCCCTCTCTGGTCTAACCAAACCCTCCAGCAGGGGTG-3' V6-R1 (SEQ ID NO: 12): 5'-TTGACCCATCTCAGTTTACATGGTGAATGACTACAATATATCTGGAATTTGG-3'

[0391] Vector V7 As shown in Figure 12, targeting vector V7 contained the following characteristic sequences, in 5' to 3' order: an upstream homologous arm (5' homologous arm), a CμΔCH1 sequence, a CδΔCH1 sequence (or a CδΔCH1 knock-in sequence, SEQ ID NO: 41), and a downstream homologous arm (3' homologous arm). The targeting vector also contained an antibiotic resistance gene (neomycin phosphotransferase gene, or Neo) for screening positive clones and two Frt recombination sites flanking the antibiotic resistance gene. In addition, a coding gene with a negative selection marker (the gene encoding diphtheria toxin A subunit (DTA)) was also inserted into the targeting vector.

[0392] A sequence containing the mouse Sμ switch region, CμΔCH1 sequence, and CδΔCH1 sequence was cloned from the mouse genome. The following primer pair was used to obtain a knock-in sequence containing nucleotides upstream of mouse CμΔCH1 and CδΔCH1 for construction of the V7 vector: V7-F1 (SEQ ID NO: 14): 5'-ATCCCTCTCTGGTCTAACCAAACCCTCCAGCAGGGGTG-3' V7-R1 (SEQ ID NO: 15): 5'-TTCTGCATGGTCCAGGGATTGATCAGACAGATAGTGAAGTTCTGAGGACA-3'

[0393] Example 4: Verification of genetic modification Mutant allele 1 The genotype of mutant allele 1 was detected using PCR and Southern blot. First, positive clone cells were identified by PCR using two sets of primer pairs: L-GT-F1 / L-GT-R1 and R-GT-F2 / R-GT-R2. Exemplary results are shown in Figures 13A-13B, respectively. The primer sequences are listed in the table below.

[0394] [Table 7]

[0395] Next, positive clones were verified by Southern blot (digestion with BclI, ScaI, XmnI, and BglII, respectively, followed by hybridization with the four corresponding probes) to select the correct positive clone cells. The Southern blot detection strategy (including restriction enzymes, probes, and target fragment sizes) and probe primers are shown in the table below.

[0396] [Table 8]

[0397] [Table 9]

[0398] Exemplary detection results are shown in Figures 14A-14D. Based on the PCR and Southern blot results, mice numbered F1-012, F1-017, F1-018, and F1-019 were identified as heterozygous mice positive for mutant allele 1. No random insertions were detected in mutant allele 1.

[0399] Mutant allele 2 Primers DE-F1 and DE-R1 were used to confirm knockout of the Cμ to Cδ sequence of mutant allele 2. The detection results are shown in Figure 15. Mice numbered F1-1, F1-2, F1-3, F1-4, F1-5, F1-6, F1-7, and F1-8 were identified as positive heterozygous mice. The primer sequences are shown in the table below.

[0400] [Table 10]

[0401] Mutant allele 2' Primers GT-Mut-F, GT-Mut-R, and GT-WT-R were used to confirm knockout of the sequence from Cμ to Sγ1 of the mutant allele 2'. The detection results are shown in Figure 16. Mice numbered F1-2 and F1-4 were identified as positive heterozygous mice. The primer sequences are shown in the table below.

[0402] [Table 11]

[0403] Mutant allele 3 Primers GT-3F and GT-3R were used to confirm knockout of the CH1 coding sequence of Cμ in mutant allele 3. The detection results are shown in Figure 17. Mice numbered F1-2, F1-3, and F1-6 were identified as positive heterozygous mice. The primer sequences are shown in the table below.

[0404] [Table 12]

[0405] Mutant allele 4 Primers Mut-F and Mut-R were used to confirm the sequence of CμΔCH1 in mutant allele 4. Primers F4 and R4 were used to confirm the absence of Cδ in mutant allele 4. The detection results are shown in Figures 18A-18B, respectively. Mice numbered F1-1, F1-2, F1-3, F1-4, F1-5, F1-6, F1-7, F1-8, F1-9, and F1-10 were identified as positive heterozygous mice. The primer sequences are shown in the table below.

[0406] [Table 13]

[0407] Mutant allele 5 For mutant allele 5, primers Mut-F and Mut-R were used to confirm the sequence of CμΔCH1, and primers F3 and R3 were used to confirm the sequence of CδΔCH1. The detection results are shown in Figures 19A-19B, respectively. Mice numbered F1-1, F1-2, F1-3, F1-4, F1-5, F1-6, F1-7, and F1-8 were identified as positive heterozygous mice. The primer sequences are shown in the table below.

[0408] [Table 14]

[0409] Example 5: Modification of mouse immunoglobulin heavy chain variable region loci Experiments were conducted to introduce human immunoglobulin genes into the mouse genome and generate mice that express humanized antibodies. Figure 20 illustrates the method for generating humanized mice. This method involves first modifying the human immunoglobulin region on a human chromosome. The modified human chromosome was then introduced into a mouse recipient cell.

[0410] Mouse immunoglobulin variable region locus sequences were replaced with human immunoglobulin variable region locus sequences by direct replacement (e.g., homologous recombination or Cre-mediated recombination). In some cases, human immunoglobulin variable region genes can be introduced into the mouse genome by a stepwise approach. Recipient cells were then screened for the correct replacement. The cells were then injected into blastocysts to generate chimeric mice. Subsequent breeding was performed to obtain mice containing human or humanized immunoglobulin variable region locus sequences.

[0411] The immunoglobulin heavy chain locus is located on mouse chromosome 12. Two recombination sites were introduced on either side of the immunoglobulin heavy chain variable region locus.

[0412] Experiments were also performed to generate modified human chromosomes. Two recombination sites were introduced on either side of the variable region of the immunoglobulin heavy chain locus. The modified human chromosomes were then introduced into mouse cells. The cells were then screened to select for cells containing only one human chromosome. Cre recombinase then mediated the replacement of the V, D, and J regions on the mouse chromosome with the V, D, and J regions on the human chromosome (Figure 21).

[0413] Positive cloned cells were injected into BALB / c mouse blastocysts by microinjection. Embryo microinjection was performed according to the method described, for example, in A. Nagy, et al., "Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition)," Cold Spring Harbor Laboratory Press, 2003. The injected fertilized eggs were transferred to culture medium, cultured for a short period, and then implanted into the oviducts of recipient mice to generate genetically modified humanized mice (F0 generation). The mice were then mated with mice on a C57BL / 6 background. PCR analysis was performed on DNA obtained from the mouse tails. The mice were further mated (e.g., at least five times) with mice on a BALB / c background to obtain heavy chain immunoglobulin locus humanized heterozygous mice on a BALB / c background.

[0414] The heterozygous mice were then bred with each other to obtain homozygous mice. A detailed description of how to generate immunoglobulin heavy chain locus humanized homozygous mice is provided in WO2020169022A1 and US20200390073A1, each of which is incorporated by reference in its entirety.

[0415] Example 6: Modification of the mouse immunoglobulin light chain locus Kappa (κ) light chain locus knockout mice The immunoglobulin kappa light chain (κ) locus is located on mouse chromosome 6. Mouse chromosome 6 was modified by knocking out the entire sequence of the immunoglobulin kappa light chain variable region locus. Detailed knockout methods can be found, for example, in WO2020169022A1 and US20200390073A1; Zou, X., et al. the Ig kappa locus:efficient generation of lambda chain-expressing B cells,independent of gene rearrangements in Ig kappa.” The EMBO Journal 12.3(1993):811-820;Takeda,S.,et al. “Deletion of the immunoglobulin kappa chain intron enhancer abolishes kappa chain gene rearrangement in cis but not lambda chain gene rearrangement in trans.” Journal 12.6(1993):2329-2336, each of which is incorporated herein by reference in its entirety.

[0416] Lambda (λ) light chain locus knockout mice The immunoglobulin λ light chain (λ) is located on mouse chromosome 16. Mouse chromosome 16 was modified by knocking out the entire sequence of the immunoglobulin λ light chain variable region locus. Detailed knockout methods are described, for example, in Zou, X., et al. "Block in development at the pre-B-II to immature B cell stage in mice without Igκ and Igλ light chain," The Journal of Immunology 170.3(2003):1354-1361, the entire contents of which are incorporated herein by reference.

[0417] The mice described herein can be bred with each other to obtain mice having a human immunoglobulin heavy chain VDJ region, a modified mouse immunoglobulin heavy chain constant region locus (lacking the CH1 coding region of Cγ1), and lacking all or part of the mouse immunoglobulin light chain locus.

[0418] Example 7: Mouse heavy chain antibodies with modified IgG1 genes Serum samples were obtained by blood collection from mice lacking the CH1-coding region of Cγ1 identified above (mice with the genotypes of mutant allele 2', mutant allele 3, and mutant allele 4, respectively) and wild-type (WT) mice. The serum samples were prepared for Western blotting analysis to identify any expressed IgG in the serum using an anti-mIgG1 antibody (catalog number: ab190481, Abcam). As shown in Figure 32, the results revealed a mixture of bands: one band at approximately 75 kD (the expected size of dimeric IgG1 lacking the CH1 domain) and one band at approximately 150 kD (the expected size of wild-type IgG). The results demonstrate that mice generated using the methods described herein can express IgG1 lacking the CH1 domain in their peripheral blood.

[0419] Mice can be immunized by injecting an immunogen and then screened for specific-binding antibodies by various methods (e.g., hybridoma, phage screening, single-cell technology by 10x Genomics, or the Beacon® Optofluidic System). Preliminary results indicate that mice generated by the methods described herein can be used to obtain antibodies with high affinity, high diversity, good functionality (e.g., high endocytosis activity), and good developability (e.g., high hydrophilicity and good thermal stability).

[0420] Mice carrying mutant allele 3 (Mut3) Humanized mice (heterozygous for the heavy chain mutant allele 3 genotype, with a kappa light chain locus knockout and no lambda light chain locus knockout) were immunized with antigen A (5 mice). After three immunizations, serum titers were measured at 10, as detected by FACS. 4 The number of antibodies produced increased 2-fold. Next, we used the Beacon® Optofluidic System to isolate plasma cells capable of producing antigen-specific monoclonal antibodies. Expression vectors for each antibody were constructed and introduced into host cells. Sixty-three positive cells were identified by FACS. Further analysis of the antibody sequences showed that the CDR3 length (as defined by IMGT) of the heavy chain variable region ranged from 6 to 23 (Figure 22). Furthermore, 94% (59 / 63) of the clones had a CDR3 length of 12 or more, with a total of 35 unique CDR3 sequences. Figure 23 shows the germline gene usage of the variable region genes. Furthermore, the affinity of some antibodies was also tested. As shown in Figure 24, the KD of these antibodies against antigen A was 10 -9 M, indicating good binding affinity.

[0421] In another experiment, humanized mice (four mice homozygous for the heavy chain mutant allele 3 genotype, homozygous for a deletion of the κ light chain locus, and heterozygous for a deletion of the λ light chain locus; three mice homozygous for the heavy chain mutant allele 3 genotype, homozygous for a deletion of the κ light chain locus, and homozygous for a deletion of the λ light chain locus) were immunized with human 4-1BB (catalog number: 41B-H5258, ACROBiosystems). After four immunizations, serum titers were measured at 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 320, 240, 280, 360, 380, 400, 420, 440, 460, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 95 4 The number of antigen-specific clones confirmed by FACS was 67. Further analysis of the antibody sequences showed that the CDR3 length (as defined by IMGT) of the heavy chain variable region was 7 to 19 (Figure 33). Figure 34 shows the germline gene usage of the variable region genes. Furthermore, the affinity of some antibodies was also tested.

[0422] In another experiment, humanized mice (19 mice homozygous for the heavy chain mutant allele 3 genotype, homozygous for a deletion of the kappa light chain locus, and heterozygous for a deletion of the lambda light chain locus) were immunized with the antigens human CD3ED and cynomolgus CD3ED. After four immunizations, serum titers were measured at 10, as detected by FACS. 5 The number of antigen-specific clones confirmed by FACS was 273. Further analysis of the antibody sequences showed that the CDR3 length (as defined by IMGT) of the heavy chain variable region was 6 to 25 (Figure 35). Figure 36 shows the germline gene usage of the variable region genes. Furthermore, the affinity of some antibodies was also tested.

[0423] Humanized mice (homozygous for the heavy chain mutant allele 3 genotype, with kappa and lambda light chain loci knockout) were immunized with the antigen human serum albumin (10 mice). Plasma cells capable of producing antigen-specific monoclonal antibodies were then isolated using the Beacon® Optofluidic System. The number of positive cells confirmed by FACS was 84. Further analysis of the antibody sequence indicated that the CDR3 length of the heavy chain variable region (as defined by IMGT) was 12 to 17. Figure 43 shows the germline gene usage of the variable region genes.

[0424] Mice carrying mutant allele 2 (Mut2) Humanized mice (homozygous for heavy chain mutant allele 2 genotype, κ light chain locus knockout, no λ light chain locus knockout) were immunized with antigen A (10 mice). Next, using the Beacon® Optofluidic System, plasma cells capable of producing antigen-specific monoclonal antibodies were isolated. The number of positive cells confirmed by FACS was 40. Further analysis of the antibody sequences revealed a total of 14 unique CDR3 sequences. In addition, the affinity of some antibodies was also tested. As shown in Figure 25, the KD of these antibodies against antigen A was 10 -8 M, indicating good binding affinity.

[0425] Furthermore, spleen tissue from immunized Mut2 mice was collected, and total RNA was extracted from the spleen cells. Immunoglobulin variable region loci can be cloned by PCR and then inserted into phage plasmids to construct a phage recombinant plasmid library. In one experiment, a total of 202 ELISA-positive clones were obtained after two rounds of panning and screening of the constructed library. After removing redundant sequences, 94 positive cells were identified by FACS. Further analysis of the sequences of these antibodies indicated that the CDR3 length of the heavy chain variable region was 8–18 (as defined by IMGT). Figure 26 shows the germline gene usage of the variable region genes.

[0426] Mice carrying the mutant allele 2' (Mut2') Humanized mice (heterozygous for the heavy chain mutant allele 2' genotype, with kappa and lambda light chain loci knockout) were immunized with the antigen human serum albumin (12 mice). Plasma cells capable of producing antigen-specific monoclonal antibodies were then isolated using the Beacon® Optofluidic System. The number of positive cells confirmed by FACS was 96. Further analysis of the antibody sequences showed that the CDR3 length (as defined by IMGT) of the heavy chain variable region was 5 to 22. Furthermore, 78.1% (75 / 96) of the clones had a CDR3 length of 12 or more. Figure 44 shows the germline gene usage of the variable region genes. Furthermore, the affinity of several antibodies was also tested. As shown in Figure 45, the KD of several antibodies against human serum albumin was 10. -9 M, indicating good binding affinity.

[0427] Mice carrying mutant allele 4 (Mut4) Humanized mice (homozygous for the heavy chain variant allele 4 genotype, with kappa and lambda light chain loci knockout) were immunized with the antigen human serum albumin (10 mice). Plasma cells capable of producing antigen-specific monoclonal antibodies were then isolated using the Beacon® Optofluidic System. The number of positive cells confirmed by FACS was 41. Further analysis of the antibody sequences showed that the CDR3 length (as defined by IMGT) of the heavy chain variable region ranged from 8 to 23, with 65.9% (27 / 41) of the clones having a CDR3 length of 12 or more. Figure 46 shows the germline gene usage of the variable region genes.

[0428] Mice carrying mutant allele 5 (Mut5) Humanized mice (homozygous for the heavy chain mutant allele 5 genotype, kappa and lambda light chain loci knockout) were immunized with antigen A. Next, plasma cells capable of producing antigen-specific monoclonal antibodies were isolated using the Beacon® Optofluidic System. The number of positive cells confirmed by FACS was 20. Further analysis of the sequence showed that the length of CDR3 (as defined by IMGT) of the heavy chain variable region was 10 to 18. Figure 47 shows the germline gene usage of the variable region genes. In addition, the affinity of several antibodies was also tested. The KD of these antibodies against antigen A was 10 -9 M, indicating good binding affinity.

[0429] B cell development We conducted an experiment to compare the immune systems of modified IgG1 mice and wild-type mice. Six- to eight-week-old RenMab mice (humanized heavy chain immunoglobulin locus) and modified IgG1 mice were selected. The modified IgG1 mice were similar in weight, appearance, and vitality to the RenMab mice. Peripheral blood, spleen, lymph node, and bone marrow tissues were collected from these mice, and no obvious anatomical changes were found.

[0430] Example 8. Generation of human anti-TFR1 antibodies Mice (five mice were homozygous for the heavy chain mutant allele 3 genotype, homozygous for a deletion of the κ light chain locus, and heterozygous for a deletion of the λ light chain locus) were immunized with His-tagged human TFR1 (transferrin receptor 1) protein (hTFR1-His, ACROBiosystems, catalog number: CD1-H5243) to obtain anti-TFR1 antibodies. Prior to immunization, retro-orbital blood samples were collected as a negative control. Complete Freund's adjuvant (CFA) was used for the first immunization, and incomplete Freund's adjuvant (IFA) was used for the second and third immunizations. A total of three immunizations (every two weeks) were performed. One week after the third immunization, retro-orbital blood samples were collected, and serum antibody titers were detected by FACS.

[0431] A booster immunization procedure was also performed at least 14 days after the previous immunization: TFR1 protein was injected intraperitoneally, and CHO-S cells expressing human TFR1 antigen were injected via the tail vein.

[0432] Antigen-specific immune cells were isolated from immunized mice, and anti-TFR1 antibodies were obtained, or heavy chain variable region sequences of anti-TFR1 antibodies were obtained. For example, single-cell technology (e.g., using the Beacon® Optofluidic System, Berkeley Lights Inc.) was used to screen for plasma cells secreting antigen-specific monoclonal antibodies. Antibody variable region sequences were obtained using reverse transcription and PCR sequencing. The obtained variable region sequences were used for antibody expression, and binding affinity to TFR1 was verified using FACS. Because the CH1 domain is missing, the heavy chain variable region (VH) of the obtained antibody is also called a heavy chain single variable domain (VHH).

[0433] Specifically, the resulting VHH sequences were each ligated to a human IgG1 constant region (e.g., hinge region, CH2 domain, and CH3 domain). Exemplary antibodies obtained by this method included 23B8, 24A1, 24C9, and 24G5. The heavy chain CDR1-3 sequences are shown in Figures 37 and 38. The VHH region sequences of 23B8, 24A1, 24C9, and 24G5 are shown in Figure 39.

[0434] The constant region of the antibody can be further modified to replace the asparagine at position 297 with alanine (N297A). For example, if the N297A mutation is introduced into the constant region of 24G5, the resulting antibody is designated 24G5-N.

[0435] Example 9. Cross-species binding of anti-TFR1 antibodies CHO-S-hTFR1 cells or CHO-S-fasTFR1 cells were cultured at 10 5The cells were transferred to a 96-well plate at a density of 1000 cells / well. Serially diluted sample anti-TFR1 antibodies were added to the 96-well plate and incubated at 4°C for 30 minutes. PBS was used as a negative control (NC). The cells were then incubated with the secondary antibody anti-hIgG-Fc-Alex Flour™ 647 (Jackson ImmunoResearch Laboratories, Cat. No. 109-606-170) in the dark for 15 minutes at 4°C before flow cytometry analysis.

[0436] CHO-S-hTFR1 cells or CHO-S-fasTFR1 cells were obtained by transfecting CHO-S cells with vectors expressing human TFR1 (hTFR1, SEQ ID NO: 70) or Macaca fascicularis (cynomolgus monkey) TFR1 amino acid sequence (fasTFR1, SEQ ID NO: 71), respectively. The test results are shown in the table below.

[0437] JR141, a humanized IgG1 antibody targeting human TFR1 conjugated to human iduronate-2-sulfatase, was the first approved for the intravenous treatment of mucopolysaccharidosis type II in Japan in March 2021. The VH and VL sequences of JR141 are shown in SEQ ID NO: 72 and SEQ ID NO: 73, respectively. In the positive control (JR141-N), the VH and VL of JR141 were ligated to a human IgG1 constant region with the N297A mutation.

[0438] [Table 15]

[0439] Example 10. Binding affinity of anti-TFR1 antibodies The binding affinity of anti-TFR1 antibodies to human (hTFR1-His, ACROBiosystems, catalog number: CD1-H5243) or monkey (fasTFR1-His, ACROBiosystems, catalog number: TFR-C524a) His-tagged TFR1 proteins was verified using surface plasmon resonance (SPR) on a Biacore™ (Biacore, Inc., Piscataway, NJ) 8K biosensor equipped with a pre-immobilized Protein A sensor chip.

[0440] Purified anti-TFR1 antibodies were captured on a Protein A chip (Series S SensorChip Protein A) for detection. Purified anti-TFR1 antibodies (1 μg / mL) were loaded at 10 μL / min to bind to hTFR1-His and fasTFR1-His (200 nM). The flow rate was 30 μL / min. The association and dissociation times were set to 180 and 600 seconds, respectively. After the last injection of each titration solution, the chip was regenerated with glycine solution (pH 2.0) at 30 μL / min for 30 seconds.

[0441] The kinetic association rate (k) and dissociation rate (k) were obtained simultaneously by fitting the entire data set to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using Biacore™ 8K Evaluation software 3.0. The affinity was estimated from the quotient of the kinetic rate constants (K = k / k).

[0442] As will be understood by those skilled in the art, the same method was performed for each test antibody, adjusting parameters (e.g., antibody concentration) appropriately. The results for the test antibodies are summarized in the table below. The results show that all four anti-TFR1 antibodies can bind to human and monkey TFR1 with high affinity.

[0443] [Table 16]

[0444] Example 11. Epitope analysis of anti-TFR1 antibodies The relative location of target protein epitopes between pairs of purified anti-TFR1 antibodies was analyzed by biolayer interferometry (BLI) using a ForteBio Octet system at 30°C. 1x HBS-EP+ buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% P20, pH 7.4), diluted from HBS-EP+ buffer (10x), was used as the running buffer throughout the experiment. Approximately 10 μg / mL hTFR1-His protein was captured with HIS1K (anti-PentaHIS) for 200 s, and 200 nM antibody (analyte 1) was injected at a flow rate of 30 μL / min to bind to the ligand. To determine whether the binding of different antibodies interfered with each other, another antibody (analyte 2) was injected under the same conditions. The binding time was 300 seconds for each antibody.

[0445] Binding values ​​for each antibody were obtained using Data Analysis HT 12.0. To quantify the interference of one antibody with another, binding ratios were calculated and compared for each pair of antibodies. The binding ratio was defined as the binding value of the second antibody (sample 2) divided by the binding value of the first antibody (sample 1). The binding ratios for each antibody pair are summarized in the matrix table below. Specifically, if sample 1 had an inhibitory effect on sample 2, the binding ratio was 0.0 to 0.5; if sample 1 had no blocking effect on sample 2, the binding ratio was 0.5 to 1.1. Generally, antibody pairs that interfere with each other have identical or overlapping epitopes.

[0446] The results of the epitope binding assay indicate that 24A1 and 24G5 can recognize the same epitope, while 23B8, 24C9 and JR141-N can recognize different epitopes.

[0447] [Table 17]

[0448] Example 12. Internalization of anti-TFR1 antibodies Anti-TFR1 antibody and pHAb-goat anti-human IgG secondary antibody were added to human cortical microvascular endothelial cells (hCMEC / D cells) and incubated for 3 hours. After incubation, the cells were centrifuged and washed with FACS buffer. The mean fluorescence intensity (MFI) was measured using a flow cytometer. The endocytosis rate of the antibodies was calculated. Human IgG1 protein (CrownBio, catalog number: C0001) was used as an isotype control (ISO). The results are shown in the table below, which indicates that all four antibodies exhibited good endocytosis activity in human cortical microvascular endothelial cells.

[0449] [Table 18]

[0450] Example 13. Stability analysis of anti-TFR1 antibodies The stability of anti-TFR1 antibodies 23B8, 24A1, 24C9, and 24G5 was evaluated by the following tests: (1) observing the appearance of the solution and the presence of visible insoluble material; (2) detecting changes in antibody purity by size-exclusion ultra-performance liquid chromatography (SEC-UPLC) (expressed as the percentage of the main peak area relative to the sum of all peak areas (purity, %)); (3) detecting changes in apparent hydrophobicity by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) (expressed as the retention time of the main peak (HIC, minutes)); (4) detecting charge variants of the antibody by capillary isoelectric focusing (cIEF) (expressed as the percentage of the main, acidic, and alkaline components); and (5) detecting thermal stability of the antibody by the UNcle system (expressed as the melting temperature (Tm) and aggregation temperature (Tag)).

[0451] For SEC-UPLC experiments, an Agilent 1290 chromatographic system coupled with an XBridge™ Protein BEH SEC column (200 Å, Waters Corporation) was used. Antibody samples were diluted to 1 mg / mL with purified water. The following parameters were used: mobile phase: 25 mM phosphate buffer (PB) (pH 6.8) + 0.3 M NaCl; flow rate: 1.8 mL / min; column temperature: 25°C; detection wavelength: 280 nm; injection volume: 10 μL; sample tray temperature: 6°C; and run time: 7 min.

[0452] For HIC-HPLC experiments, an Agilent 1260 chromatographic system coupled with a ProPac™ HIC-10 column (4.6 × 100 mm, Thermo Scientific) was used, and the sample was diluted to 0.5 mg / mL with mobile phase A. The following parameters were used: mobile phase A: 0.9 M ammonium sulfate, 0.1 M PB, 10% acetonitrile (pH 6.5); mobile phase B: 0.1 M PB, 10% acetonitrile (pH 6.5); flow rate: 0.8 mL / min; gradient: 0 min 100% A, 2 min 100% A, 32 min 100% B, 34 min 100% B, 35 min 100% A, and 45 min 100% A; column temperature: 30°C; detection wavelength: 280 nm; injection volume: 10 μg; sample tray temperature: approximately 6°C; and run time: 45 min.

[0453] For cIEF experiments, a Maurice cIEF Method Development Kit (Protein Simple, catalog number: PS-MDK01-C) was used for sample preparation. Specifically, 40 μg of protein sample was mixed with the following reagents in the kit: 1 μL of Maurice cIEF pI Marker-4.05, 1 μL of Maurice cIEF pI Marker-9.99, 35 μL of 1% methylcellulose solution, 2 μL of Maurice cIEF 500 mM arginine, 4 μL of ampholytes (Pharmalyte pH range 3–10), and water (to a final volume of 100 μL). Imaging capillary isoelectric focusing spectra were generated using a Maurice cIEF cartridge (PS-MC02-C) on a Maurice analyzer (Protein Simple, Santa Clara, CA). The sample was focused for a total of 10 min. The absorbance of the proteins, focused at 280 nm, was analyzed using the analytical software installed on the instrument.

[0454] For thermal stability experiments, 60 mg / mL antibody solutions were heated from 25°C to 95°C in 1°C increments, with a 1 minute equilibration period before each measurement.

[0455] Detailed results are shown in the table below. The results show that all four antibodies have good stability and physical and chemical properties.

[0456] [Table 19]

[0457] Example 14. Pharmacokinetic (PK) Analysis A humanized TFR1 mouse model (hTFR1 mouse) was engineered to express a chimeric TFR1 protein (SEQ ID NO: 74) in which the extracellular domain of the mouse TFR1 protein was replaced with the corresponding human TFR1 extracellular domain. A detailed description of the humanized TFR1 mouse model can be found in PCT Application No. PCT / CN2022 / 105924, which is incorporated herein by reference in its entirety.

[0458] The concentration of anti-TFR1 antibodies was determined in hTFR1 mice. Specifically, mice were placed in different groups (8 mice per group) and administered approximately equimolar amounts of JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6) intravenously (iv). Control group (G1) mice were administered human IgG1 (hIgG1). The details of the administration scheme are shown in the table below.

[0459] [Table 20]

[0460] Blood and brain samples were collected 0.5, 6, 24, and 72 hours after administration. Two mice were sampled at each time point. After retroorbital blood collection, the mice were anesthetized. To avoid interference from residual blood in the brain, the mice were perfused with saline for 10 minutes at room temperature. Specifically, saline was perfused from the left ventricle to the right ventricle via the systemic circulation. Brain samples were excised and divided into two hemibrains along the sagittal plane. The left hemibrain was subjected to quantification of the injected antibodies, while the right hemibrain was fixed in formalin and embedded in paraffin for serial sectioning. Brain samples were minced and homogenized in Dulbecco's phosphate-buffered saline (DPBS) containing 1x mixed protease inhibitors. Aliquots of the brain homogenate were used for protein extraction, followed by antibody quantification using electrochemiluminescence. The remaining homogenate was subjected to 15-minute gradient density centrifugation at 5400 g using 15% dextran to remove capillaries. After centrifugation, the upper fraction of the centrifuge tube was saved as the parenchyma and was also used for protein extraction and antibody quantification. Figures 40A-40D show the antibody concentration in total brain protein (Figure 40A), the ratio of antibody concentration in total brain protein to serum antibody concentration (Figure 40B), the antibody concentration in the brain parenchyma (Figure 40C), and the ratio of antibody concentration in the brain parenchyma to serum antibody concentration at each time point (Figure 40D). These results demonstrated that 24G5-N (group G5) was most abundant in either the parenchyma or the whole brain.

[0461] In a similar experiment, hTFR1 mice were divided into five groups (three mice per group) and administered 18.4 mg / kg JR141-N (G2), 10 mg / kg 23B8-N (G3), 10 mg / kg 24A1-N (G4), or 10 mg / kg 24G5-N (G5) intravenously (one total dose). Mice in the control group (G1) were administered hIgG1 (G1). Brain samples were collected 24 hours after administration to determine the concentration of anti-TFR1 antibodies. Figures 41A-41B show the results of antibody concentration tests in the brain parenchyma and total brain protein, respectively. All tested antibodies showed higher concentrations in the brain than hIgG1 (G1), and compared to the positive control JR141-N (G2), 23B8-N (G3) and 24G5-N (G5) showed better ability to cross the blood-brain barrier and enter the brain parenchyma.

[0462] In another similar experiment, hTFR1 mice were divided into seven groups (six mice per group) and administered JR141-N (G2-G4) or 24G5-N (G5-G7) intravenously (iv). Control group (G1) mice were administered hIgG1. The details of the administration scheme are shown in the table below.

[0463] [Table 21]

[0464] Six and 24 hours after administration, blood and brain samples were collected using the methods described above. Three mice were sampled at each time point. Tissue processing and antibody quantification were also performed as described above. Measurement results of the humanized anti-TFR1 antibody concentration in the brain parenchyma are shown in Figure 42. The results show that under each administration condition, the antibody concentration accumulated in the brain parenchyma of 24G5-N was significantly higher than that of hIgG1. Furthermore, the concentrations of both JR141-N and 24G5-N showed a dose-dependent trend in the brain parenchyma.

[0465] To detect the distribution of the humanized anti-TFR1 antibody 24G5-N in the mouse brain, we performed immunofluorescence assays by staining for hIgG, hTFR1, and mCD31, respectively, in right hemibrain sections from the mice used in the above experiments. The results showed that mCD31 was well labeled in microvessels. hTFR1 was also detected in microvessels and colocalized with mCD31. Furthermore, hTFR1 expression was also detected in some neurons within the parenchyma. In particular, the anti-TFR1 antibody 24G5-N was stained with a secondary anti-IgG antibody conjugated with DyLight® 488. Similar to hTFR1, 24G5-N was detected in microvessels and the parenchyma, and its signal overlapped with the hTFR1 signal. Thus, whether by quantification of 24G5-N in the whole brain or parenchyma, or by visual evidence of immunofluorescence of 24G5-N in the parenchyma, the results demonstrate that the humanized anti-TFR1 antibody 24G5-N can efficiently cross the blood-brain barrier (BBB).

[0466] Example 15. Blocking assay Blocking of TFR1 binding to TF (transferrin) by anti-TFR1 antibodies 23B8, 24A1, 24C9, and 24G5 was tested by biolayer interferometry (BLI) using a ForteBio Octet® system at 30° C. Specifically, 1× HBS-EP+ buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20, pH 7.4) diluted from HBS-EP+ buffer (10×) was used as the running buffer throughout the experiment. Approximately 10 μg / mL of antibody was captured with AHC (anti-human IgG Fc capture) for 200 seconds, and 800 nM of hTFR1-His (ACROBiosystems, catalog number: CD1-H5243) and hTF-His (human transferrin protein, Kactus Biosystems, catalog number: TFN-HM101) were injected to allow binding to the ligands. The binding time was 300 seconds for each antibody. Binding values ​​for each antibody were obtained using Data Analysis HT 12.0. The results showed that these four antibodies did not block the binding of TFR1 to TF. Therefore, such non-blocking antibodies are unlikely to interfere with TFR1-TF interaction in normal cells.

Claims

1. 1. A genetically modified non-human animal comprising an engineered immunoglobulin heavy chain locus, wherein said engineered immunoglobulin heavy chain locus comprises an IgG constant region gene, said IgG constant region gene encoding an IgG heavy chain constant region lacking a CH1 domain, and wherein said genetically modified non-human animal expresses a heavy chain antibody.

2. 2. The genetically modified non-human animal of claim 1, wherein the animal comprises exactly one IgG constant region gene.

3. The genetically modified non-human animal according to claim 1 or 2, wherein the IgG heavy chain constant region gene is IGHG1.

4. 4. The genetically modified non-human animal according to any one of claims 1 to 3, wherein the IgG heavy chain constant region comprises or consists of a CH2 domain and a CH3 domain, and optionally a hinge region.

5. A genetically modified non-human animal, whose genome comprises a germline genetic modification comprising a deletion of the IGHG3, IGHG2b, and IGHG2c genes and a deletion of the CH1 exon of the IGHG1 gene in the endogenous immunoglobulin heavy chain locus.

6. 6. The animal of claim 5, wherein the germline genetic modification further comprises a deletion of an endogenous IGHE gene at the endogenous immunoglobulin heavy chain locus.

7. 7. The animal of claim 5 or 6, wherein the genetic modification further comprises a deletion of endogenous Sγ2b, Sγ2c, and Sε switch regions in the endogenous immunoglobulin heavy chain locus.

8. 8. The animal of any one of claims 5 to 7, wherein the modified immunoglobulin heavy chain locus comprises a modified IGHG1 gene that lacks a sequence encoding a CH1 domain, and wherein the modified IGHG1 gene comprises a sequence that is at least 80%, 90%, 95% or 99% identical to SEQ ID NO:

1.

9. The animal of any one of claims 5 to 8, wherein the genetic modification further comprises a deletion of an endogenous Sγ3 switch region in the endogenous immunoglobulin heavy chain locus.

10. 10. The animal of any one of claims 5 to 9, wherein the genome of the animal comprises a modified rodent IGHG1 gene lacking sequences encoding endogenous Sμ, Sγ1, Sα switch regions, and CH1 domain, and endogenous IGHM, IGHδ, and IGHA genes.

11. The animal of any one of claims 5 to 9, wherein the genetic modification further comprises a deletion of an endogenous IGHM gene and an IGHδ gene at the endogenous immunoglobulin heavy chain locus.

12. 12. The animal of any one of claims 5 to 9 and 11, wherein the animal genome comprises the modified IGHG1 gene lacking sequences encoding endogenous Sμ, Sγ1, Sα switch regions, CH1 domain, and an endogenous IGHA gene.

13. 13. The animal of claim 12, wherein the Sμ and Sγ1 switch regions are linked to a sequence that is at least 80%, 90%, 95%, or 99% identical to SEQ ID NO:

8.

14. 10. The animal of any one of claims 5 to 9, wherein the genetic modification further comprises a deletion of the CH1 coding sequence of the IGHM gene in the endogenous immunoglobulin heavy chain locus.

15. 15. The animal of any one of claims 5 to 9 and 14, wherein the genome of the animal comprises endogenous Sμ, Sγ1, Sα switch regions, a modified endogenous IGHM gene lacking sequences encoding a CH1 domain, the modified IGHG1 gene lacking sequences encoding a CH1 domain, and endogenous IGHδ and IGHA genes.

16. 16. The animal of claim 15, wherein the Sμ switch region and the modified IGHM gene are linked to a sequence that is at least 80%, 90%, 95%, or 99% identical to SEQ ID NO:

10.

17. The animal of any one of claims 5 to 9, wherein the genetic modification further comprises a deletion of the CH1 exon of the IGHM gene and a deletion of the IGHδ gene at the endogenous immunoglobulin heavy chain locus.

18. 18. The animal of any one of claims 5 to 9 and 17, wherein the genome of the animal comprises endogenous Sμ, Sγ1, Sα switch regions, a modified IGHM gene lacking sequences encoding a CH1 domain, the modified IGHG1 gene lacking sequences encoding a CH1 domain, and an endogenous IGHA gene.

19. 10. The animal of any one of claims 5 to 9, wherein the genetic modification further comprises a deletion of the CH1 exon of the IGHM gene and a deletion of the CH1 coding sequence of the IGHδ gene at the endogenous immunoglobulin heavy chain locus.

20. 20. The animal of any one of claims 5 to 9 and 19, wherein the genome of the animal comprises endogenous Sμ, Sγ1, Sα switch regions, a modified IGHM gene lacking a sequence encoding a CH1 domain, a modified IGHδ gene lacking a sequence encoding a CH1 domain, the modified IGHG1 gene lacking a sequence encoding a CH1 domain, and an endogenous IGHA gene.

21. 21. The animal of claim 19 or 20, wherein the modified IGHM gene is linked to a sequence that is at least 80%, 90%, 95% or 99% identical to SEQ ID NO: 10, and the modified IGHδ gene comprises a sequence that is at least 80%, 90%, 95% or 99% identical to SEQ ID NO:

41.

22. 22. The animal of any one of claims 14 to 21, wherein the modified IGHM gene comprises a sequence that is at least 80%, 90%, 95% or 99% identical to SEQ ID NO:

13.

23. The animal of any one of claims 5 to 8, wherein the genetic modification further comprises a deletion of an endogenous Sγ1 switch region in the endogenous immunoglobulin heavy chain locus.

24. 24. The animal of any one of claims 5 to 8 and 23, wherein the genome of the animal comprises the modified IGHG1 gene lacking sequences encoding endogenous Sμ, Sγ3, Sα switch regions, CH1 domain, and endogenous IGHM, IGHδ, and IGHA genes.

25. 24. The animal of any one of claims 5 to 8 and 23, wherein the genetic modification further comprises a deletion of an endogenous Sγ3 switch region in the endogenous immunoglobulin heavy chain locus.

26. 26. The animal of any one of claims 5 to 8, 23 and 25, wherein the genetic modification further comprises a deletion of endogenous IGHM and IGHδ genes at the endogenous immunoglobulin heavy chain locus.

27. 27. The animal of any one of claims 5 to 8, 23, 25 or 26, wherein the genome of the animal comprises the modified IGHG1 gene lacking sequences encoding endogenous Sμ, Sα switch regions, CH1 domain, and an endogenous IGHA gene.

28. 28. The animal of claim 27, wherein the Sμ switch region and the modified IGHG1 gene are linked to a sequence that is at least 80%, 90%, 95%, or 99% identical to SEQ ID NO:

9.

29. 25. The animal of any one of claims 5 to 10, 23 and 24, wherein the modified genome comprises a functional IGHM gene.

30. 1. A genetically modified non-human animal, whose genome comprises, in 5' to 3' order at an endogenous immunoglobulin heavy chain locus, the following elements: an Sμ switch region, an IGHM gene, an IGHδ gene, an Sγ1 switch region, an IGHG1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene, wherein said elements are operably linked.

31. 31. The animal of claim 30, wherein the endogenous immunoglobulin heavy chain constant region locus consists of the following functional genes and switch regions: an Sμ switch region, an IGHM gene, an IGHδ gene, an Sγ1 switch region, an IGHG1 gene lacking sequences encoding a CH1 domain, an Sα switch region, and an IGHA gene.

32. 1. A genetically modified non-human animal, whose genome comprises, in 5' to 3' order at an endogenous immunoglobulin heavy chain locus, the following elements: an Sμ switch region, an IGHM gene, an IGHδ gene, an Sγ3 switch region, an IGHG1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene, wherein said elements are operably linked.

33. 33. The animal of claim 32, wherein the endogenous immunoglobulin heavy chain constant region locus consists of the following functional genes and switch regions: an Sμ switch region, an IGHM gene, an IGHδ gene, an Sγ3 switch region, an IGHG1 gene lacking sequences encoding a CH1 domain, an Sα switch region, and an IGHA gene.

34. 1. A genetically modified non-human animal, whose genome comprises, in 5' to 3' order, the following elements at an endogenous immunoglobulin heavy chain locus: an Sμ switch region, an Sγ1 switch region, an IGHG1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene, said elements being operably linked.

35. 35. The animal of claim 34, wherein the endogenous immunoglobulin heavy chain constant region locus consists of the following functional genes and switch regions: an Sμ switch region, an Sγ1 switch region, an IGHG1 gene lacking sequences encoding a CH1 domain, an Sα switch region, and an IGHA gene.

36. 1. A genetically modified non-human animal, whose genome comprises, in 5' to 3' order at an endogenous immunoglobulin heavy chain locus, the following elements: an Sμ switch region, an IGHG1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene, said elements being operably linked.

37. 37. The animal of claim 36, wherein the endogenous immunoglobulin heavy chain constant region locus consists of the following functional genes and switch regions: an Sμ switch region, an IGHG1 gene lacking sequences encoding a CH1 domain, an Sα switch region, and an IGHA gene.

38. 1. A genetically modified non-human animal, whose genome comprises, in 5' to 3' order at an endogenous immunoglobulin heavy chain locus, the following elements: an Sμ switch region, an IGHM gene lacking a sequence encoding a CH1 domain, an IGHδ gene, an Sγ1 switch region, an IGHG1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene, wherein said elements are operably linked.

39. 39. The animal of claim 38, wherein the endogenous immunoglobulin heavy chain constant region locus consists of the following functional genes and switch regions: an Sμ switch region, an IGHM gene lacking sequences encoding a CH1 domain, an IGHδ gene, an Sγ1 switch region, an IGHG1 gene lacking sequences encoding a CH1 domain, an Sα switch region, and an IGHA gene.

40. 1. A genetically modified non-human animal, whose genome comprises, in 5' to 3' order at an endogenous immunoglobulin heavy chain locus, the following elements: an Sμ switch region, an IGHM gene lacking a sequence encoding a CH1 domain, an Sγ1 switch region, an IGHG1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene, said elements being operably linked.

41. 41. The animal of claim 40, wherein the endogenous immunoglobulin heavy chain constant region locus consists of the following functional genes and switch regions: an Sμ switch region, an IGHM gene lacking a sequence encoding a CH1 domain, an Sγ1 switch region, an IGHG1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene.

42. 1. A genetically modified non-human animal, whose genome comprises, in 5' to 3' order at an endogenous immunoglobulin heavy chain locus, the following elements: an Sμ switch region, an IGHM gene lacking a sequence encoding a CH1 domain, an IGHδ gene lacking a sequence encoding a CH1 domain, an Sγ1 switch region, an IGHG1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene, wherein said elements are operably linked.

43. 43. The animal of claim 42, wherein the endogenous immunoglobulin heavy chain constant region locus consists of the following functional genes and switch regions: an Sμ switch region, an IGHM gene lacking sequences encoding a CH1 domain, an IGHδ gene lacking sequences encoding a CH1 domain, an Sγ1 switch region, an IGHG1 gene lacking sequences encoding a CH1 domain, an Sα switch region, and an IGHA gene.

44. 44. The animal of any one of claims 1 to 43, wherein the animal expresses a heavy chain antibody comprising an IgG heavy chain constant region lacking a CH1 domain.

45. The heavy chain antibody －７ Less than M, 10 －８ Less than M or 10 －９ 45. The animal of claim 44, which binds to a target antigen with a KD of less than M.

46. 46. ​​The animal of claim 44 or 45, wherein the heavy chain antibody comprises or consists of a variable region, a CH2 domain and a CH3 domain.

47. The animal of any one of claims 44 to 46, wherein the heavy chain antibody further comprises a transmembrane domain and / or a cytoplasmic domain.

48. 48. The animal of any one of claims 1 to 47, wherein the genetically modified non-human animal does not express an IgG antibody comprising a light chain.

49. 49. The animal of any one of claims 1 to 48, wherein the animal expresses IgM, IgD, and / or IgA (e.g., functional IgM, IgD, and / or IgA).

50. 50. The genetically modified non-human animal of any one of claims 1 to 49, wherein the animal comprises one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes at an endogenous immunoglobulin heavy chain locus, wherein the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operably linked and capable of undergoing VDJ rearrangement.

51. 51. The genetically modified non-human animal of claim 50, wherein the animal comprises at least 150 human IGHV genes selected from Table 1, at least 20 human IGHD genes selected from Table 2, and at least 5 human IGHJ genes selected from Table 3.

52. 51. The genetically modified non-human animal of claim 50, wherein the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at the endogenous immunoglobulin heavy chain locus on human chromosome 14 of the human subject.

53. 51. The genetically modified non-human animal of claim 50, wherein the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at an endogenous immunoglobulin heavy chain locus on human chromosome 14 in a human cell.

54. 51. The genetically modified non-human animal of claim 50, wherein the animal is a mouse, and wherein the genetic modification at an endogenous immunoglobulin heavy chain locus of the animal comprises a deletion of one or more mouse IGHV genes of Table 4, one or more mouse IGHD genes of Table 5, and / or one or more mouse IGHJ genes of Table 6.

55. 55. The genetically modified non-human animal of claim 54, wherein the animal is a mouse and the genetic modification in the animal's endogenous heavy chain immunoglobulin locus comprises a deletion of the contiguous sequence from the mouse IGHV1-85 gene to the mouse IGHJ4 gene.

56. 51. The genetically modified non-human animal of any one of claims 50, wherein the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin locus, wherein the unmodified human sequence is at least 800 kb.

57. 51. The genetically modified non-human animal of claim 50, wherein said animal comprises an unmodified human sequence derived from the human heavy chain immunoglobulin locus from human IGHV(III)-82 to human IGHV1-2.

58. 51. The genetically modified non-human animal of claim 50, wherein said animal comprises an unmodified human sequence derived from the human heavy chain immunoglobulin locus from human IGHV(III)-82 to human IGHV6-1.

59. 51. The genetically modified non-human animal of claim 50, wherein the animal comprises unmodified human sequences derived from the human heavy chain immunoglobulin loci from human IGHD1-1 to human IGHJ6.

60. 51. The genetically modified non-human animal of claim 50, wherein the animal comprises an unmodified human sequence derived from the human heavy chain immunoglobulin locus from human IGHV(III)-82 to human IGHJ6.

61. 50. The genetically modified non-human animal of any one of claims 1 to 49, wherein the genome comprises a replacement of one or more endogenous IGHV, IGHD, and IGHJ genes with one or more human IGHV, IGHD, and IGHJ genes at the endogenous immunoglobulin heavy chain locus, wherein the human IGHV, IGHD, and IGHJ genes are operably linked to one or more of endogenous IGHM, IGHδ, IGHG1 lacking a sequence encoding a CH1 domain, and IGHA genes.

62. 62. The genetically modified non-human animal of claim 61, wherein one or more endogenous IGHV genes, endogenous IGHD genes, and endogenous IGHJ genes have been replaced with at least 150 human IGHV genes of Table 1, at least 20 human IGHD genes of Table 2, and at least 5 human IGHJ genes of Table 3.

63. 63. The genetically modified non-human animal of claim 61 or 62, wherein the animal is a mouse and at least 180 mouse IGHV genes of Table 4, all mouse IGHD genes of Table 5, and all mouse IGHJ genes of Table 6 have been replaced.

64. 64. The genetically modified non-human animal of any one of claims 1 to 63, wherein the animal is homozygous for the immunoglobulin heavy chain locus.

65. 64. The genetically modified non-human animal of any one of claims 1 to 63, wherein said animal is heterozygous for said immunoglobulin heavy chain locus.

66. 66. The genetically modified non-human animal of any one of claims 1 to 65, wherein the animal comprises an endogenous light chain immunoglobulin locus.

67. 66. The genetically modified non-human animal of any one of claims 1 to 65, wherein said animal comprises a disruption in said endogenous immunoglobulin light chain locus.

68. 68. The genetically modified non-human animal of any one of claims 1 to 67, wherein the animal lacks an endogenous immunoglobulin heavy chain variable region locus capable of rearranging and forming nucleic acid sequences encoding endogenous heavy chain variable domains.

69. 69. The genetically modified non-human animal of any one of claims 1 to 68, wherein said animal is capable of producing humanized antibodies.

70. 70. The genetically modified non-human animal of any one of claims 1 to 53, 56 to 62, and 64 to 69, wherein the animal is a mammal.

71. 71. The genetically modified non-human animal of claims 11 to 53, 56 to 62, and 64 to 70, wherein the animal is a rodent.

72. The genetically modified non-human animal of claims 1 to 53, 56 to 62, and 64 to 71, wherein the animal is a mouse.

73. 73. The genetically modified non-human animal of any one of claims 1 to 72, wherein said animal has substantially normal B-cell development and maturation.

74. A cell obtained from a genetically modified non-human animal according to any one of claims 1 to 73.

75. 75. The cell of claim 74, wherein the cell is a B cell that expresses a chimeric immunoglobulin heavy chain comprising an immunoglobulin heavy chain variable domain derived from a rearrangement of one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, wherein the immunoglobulin heavy chain variable domain is operably linked to a non-human heavy chain constant region.

76. 76. The cell of claim 74 or 75, wherein the cell is an embryonic stem (ES) cell.

77. 1. A method for producing an antibody that specifically binds to an antigen, comprising: a) exposing a genetically modified non-human animal according to any one of claims 1 to 73 to said antigen; b) producing hybridomas from cells taken from said animal; c) harvesting the heavy chain antibodies produced by the hybridoma; A method comprising:

78. 78. The method of claim 77, wherein the method further comprises sequencing the genome of the hybridoma.

79. 1. A method for producing an antibody that specifically binds to an antigen, comprising: a) exposing a genetically modified non-human animal according to any one of claims 1 to 73 to said antigen; b) sequencing nucleic acid encoding a human immunoglobulin heavy chain variable region in a cell that expresses a heavy chain antibody that specifically binds to the antigen; c) operably linking said nucleic acid encoding said human immunoglobulin heavy chain variable region to a nucleic acid encoding a human immunoglobulin heavy chain constant region in a cell; A method comprising:

80. 1. A method for producing an antibody that specifically binds to an antigen, comprising: a) obtaining a nucleic acid sequence encoding a human immunoglobulin heavy chain variable region in a cell that expresses a heavy chain antibody that specifically binds to the antigen, the cell being obtained by exposing the genetically modified non-human animal of any one of claims 1 to 73 to the antigen; b) operably linking said nucleic acid encoding said human immunoglobulin heavy chain variable region to a nucleic acid encoding a human immunoglobulin heavy chain constant region; c) expressing the nucleic acid in the cell, thereby obtaining said antibody; A method comprising:

81. 1. A method for obtaining nucleic acid encoding an antibody binding domain that specifically binds to an antigen, comprising: a) exposing a genetically modified non-human animal according to any one of claims 1 to 73 to said antigen; b) sequencing nucleic acid encoding a human immunoglobulin heavy chain variable region in a cell that expresses a heavy chain antibody that specifically binds to the antigen; A method comprising:

82. 1. A method for producing an antibody that specifically binds to an antigen, comprising: a) exposing a genetically modified non-human animal according to any one of claims 1 to 73 to said antigen; b) constructing a phage plasmid library using RNA prepared from immune cells (e.g., spleen cells) of said animal; c) screening the phage plasmid library; and d) sequencing nucleic acid encoding a human immunoglobulin heavy chain variable region from a phage plasmid encoding a heavy chain antibody that specifically binds to the antigen; A method comprising:

83. 83. The method of claim 82, wherein said screening comprises isolating phage expressing an immunoglobulin heavy chain variable region based on binding affinity to said antigen.

84. 1. A method for obtaining a sample, comprising: a) exposing a genetically modified non-human animal according to any one of claims 1 to 73 to said antigen; b) taking a sample from said animal; A method comprising:

85. 85. The method of claim 84, wherein the sample is an immune cell, lymphoid tissue, spleen tissue, spleen cells, or B cells.

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