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

Abstract

The present invention 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, where the modified immunoglobulin heavy chain locus comprises an IgG constant region gene, where the IgG constant region gene encodes an IgG heavy chain constant region lacking a CH1 domain, where 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

[0001] Priority declaration

[0002] This application claims priority to PCT / CN2022 / 119188 filed on September 16, 2022 and PCT / CN2022 / 136246 filed on December 2, 2022. All of the above are incorporated herein by reference. Technical Field

[0003] The present invention relates to genetically modified animals and methods for producing heavy chain antibodies. The present invention also relates to anti-TFR1 antibodies, antigen-binding fragments and uses thereof. Background of the Invention

[0005] Therapeutic antibodies are the fastest growing class of therapeutic compounds, outpacing small molecule drugs. For example, monoclonal antibodies have revolutionized cancer therapy. However, the large size of conventional antibodies hampers delivery to tumor cells in vivo. The minimal target recognition module of conventional antibodies consists of two non-covalently bound variable domains (VH and VL). The inherent hydrophobic interactions of the VH and VL domains limit the stability and solubility of engineered antibodies, often causing aggregation and / or mispairing of the V-domains.

[0006] The discovery of heavy-chain antibodies has brought unprecedented opportunities to impact cancer treatment. These unique forms of camelid-derived antibodies lack complete light chains and CH1 domains and consist only of a single variable domain called VHH. Recombinant VHH are small (15-20 kDa) and strictly monomeric; they bind to their targets with nano-affinity and are stable over a wide range of pH and temperature. Molecular manipulation is also easier with VHH; this facilitates the generation of monoclonal antibodies in multivalent form compared to conventional recombinant antibodies and their fragments, which are problematic due to aggregation and reduced affinity. In addition, VHHs typically bind to epitopes that are less immunogenic than conventional antibodies.

[0007] Typically, therapeutic antibodies are human or humanized antibodies. Human or humanized antibodies can be produced by humanization of rodent antibodies (such as mouse antibodies) or by using phage libraries. However, these animals or phage libraries are usually unable to produce heavy chain antibodies. Instead, heavy chain antibodies are usually obtained from heavy chain antibodies of camelids. These heavy chain antibodies of camelids need humanization. The humanization process may have an adverse effect on binding affinity and introduce immunogenic epitopes into antibodies. Repeated and time-consuming experiments are usually required to improve the properties of these antibodies. In some cases, these antibodies may also be immunogenic in patients, causing their efficacy to weaken over time. Therefore, an efficient and reliable platform is needed to produce human or humanized heavy chain antibodies and nanobodies. Summary of the invention

[0008] The present invention relates to genetically modified animals and cells, which have humanized immunoglobulin heavy chain variable region loci and truncated immunoglobulin heavy chain constant region loci. For example, the CH1 coding region in the IGHG1 gene can be knocked out so that the expressed IgG does not include the CH1 domain. In some embodiments, immunoglobulin light chain (e.g., κ chain and λ chain) loci are also knocked out. After immunization, these animals can produce heavy chain antibodies with high affinity / diversity. In some embodiments, the heavy chain antibody can be further processed to generate nano antibodies.

[0009] In one aspect, the disclosure relates to a genetically modified non-human animal comprising a modified immunoglobulin heavy chain locus, in some embodiments, the modified immunoglobulin heavy chain locus comprises an IgG constant region gene, in some embodiments, the IgG constant region gene encodes an IgG heavy chain constant region lacking a CH1 domain, in some embodiments, the genetically modified non-human animal expresses a heavy chain antibody. In some embodiments, the animal comprises only 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 a CH2 domain and a CH3 domain and an optional hinge region, or consists of a CH2 domain and a CH3 domain and an optional hinge region.

[0010] 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 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 also includes a deletion of an endogenous IGHE gene at the endogenous immunoglobulin heavy chain locus. In some embodiments, the genetic modification further includes a deletion of the endogenous Sγ2b, Sγ2c, and Sε conversion regions of the endogenous immunoglobulin heavy chain locus. In some embodiments, the modified immunoglobulin heavy chain locus comprises a modified IGHG1 gene that is deleted of a sequence encoding a CH1 domain, and in some embodiments, the modified IGHG1 gene comprises a sequence having at least 80%, 90%, 95%, or 99% identity to SEQ ID NO: 1. In some embodiments, the genetic modification further includes a deletion of the endogenous Sγ3 conversion region of the endogenous immunoglobulin heavy chain locus. In some embodiments, the genome of the animal comprises an endogenous Sμ, Sγ1, Sα switch region, a modified IGHG1 gene lacking a sequence encoding a CH1 domain, and endogenous IGHM, IGHδ, IGHA genes. In some embodiments, the genetic modification further comprises a deletion of endogenous IGHM and IGHδ genes at the endogenous immunoglobulin heavy chain locus. In some embodiments, the genome of the animal comprises an endogenous Sμ, Sγ1, Sα switch region, a modified IGHG1 gene lacking a sequence encoding a CH1 domain, and endogenous

[0011] IGHA gene. In some embodiments, the Sμ and Sγ1 switch regions are linked to a sequence having at least 80%, 90%, 95% or 99% identity to SEQ ID NO: 8. In some embodiments, the genetic modification further includes the deletion of the CH1 coding 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 that deletes the sequence encoding the CH1 domain, a modified IGHG1 gene that deletes the sequence 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 having at least 80%, 90%, 95% or 99% identity to SEQ ID NO: 10. In some embodiments, the genetic modification also includes the deletion of the CH1 exon of the IGHM gene at the endogenous immunoglobulin heavy chain locus and the deletion of the IGHδ gene. In some embodiments, the genome of the animal comprises an endogenous Sμ, Sγ1, Sα conversion region, a modified IGHM gene that deletes a sequence encoding a CH1 domain, a modified IGHG1 gene that deletes a sequence encoding a CH1 domain, and an endogenous IGHA gene. In some embodiments, the genetic modification also includes deletion of the CH1 exon of the IGHM gene at the endogenous immunoglobulin heavy chain locus and deletion of the CH1 coding sequence of the IGHδ gene. In some embodiments, the genome of the animal comprises an endogenous Sμ, Sγ1, Sα conversion region, a modified IGHM gene that deletes a sequence encoding a CH1 domain, a modified IGHδ gene that deletes a sequence encoding a CH1 domain, a modified IGHG1 gene that deletes a sequence encoding a CH1 domain, and an endogenous IGHA gene. In some embodiments, the modified IGHM gene is linked to a sequence having at least 80%, 90%, 95% or 99% identity to SEQ ID NO: 10, and the modified IGHδ gene comprises a sequence having at least 80%, 90%, 95% or 99% identity to SEQ ID NO: 41. In some embodiments, the modified IGHM gene comprises a sequence having at least 80%, 90%, 95% or 99% identity to SEQ ID NO: 13. In some embodiments, the genetic modification further comprises a deletion of the endogenous Sγ1 switch region of the endogenous immunoglobulin heavy chain locus. In some embodiments, the genome of the animal comprises an endogenous Sμ, Sγ3, Sα switch region, a modified IGHG1 gene lacking a sequence encoding a CH1 domain, and endogenous IGHM, IGHδ, IGHA genes. In some embodiments, the genetic modification further comprises a deletion of the endogenous Sγ3 switch region of the endogenous immunoglobulin heavy chain locus.In some embodiments, the genetic modification further includes the deletion of endogenous IGHM and IGHδ genes at the endogenous immunoglobulin heavy chain locus. In some embodiments, the genome of the animal comprises an endogenous Sμ, Sα switch region, a modified IGHG1 gene lacking a sequence encoding a CH1 domain, and an endogenous IGHA gene. In some embodiments, the Sμ switch region and the modified IGHG1 gene are connected to a sequence having at least 80%, 90%, 95% or 99% identity to SEQ ID NO: 9. In some embodiments, the modified genome comprises a functional IGHM gene.

[0012] In some embodiments, an animal may still have an endogenous sequence when a sequence in the genome is replaced with the same sequence or a sequence from the same animal.

[0013] In one aspect, the present disclosure relates to a genetically modified non-human animal whose genome comprises the following elements in 5' to 3' order at the endogenous immunoglobulin heavy chain locus: Sμ switch region, IGHM gene, IGHδ gene, Sγ1 switch region, IGHG1 gene lacking a sequence encoding a CH1 domain, Sα switch region, and 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: Sμ switch region, IGHM gene, IGHδ gene, Sγ1 switch region, IGHG1 gene lacking a sequence encoding a CH1 domain, Sα switch region, and IGHA gene.

[0014] In one aspect, the present disclosure relates to a genetically modified non-human animal whose genome comprises the following elements in 5' to 3' order at the endogenous immunoglobulin heavy chain locus: Sμ switch region, IGHM gene, IGHδ gene, Sγ3 switch region, IGHG1 gene lacking a sequence encoding a CH1 domain, Sα switch region, and 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: Sμ switch region, IGHM gene, IGHδ gene, Sγ3 switch region, IGHG1 gene lacking a sequence encoding a CH1 domain, Sα switch region, and IgHA gene.

[0015] In one aspect, the present disclosure relates to a genetically modified non-human animal whose genome comprises the following elements in 5' to 3' order on 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. 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 a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene.

[0016] In one aspect, the present disclosure relates to a genetically modified non-human animal whose genome comprises the following elements in 5' to 3' order on 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 a switch region: an Sμ switch region, an IGHG1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene.

[0017] In one aspect, the present disclosure relates to a genetically modified non-human animal whose genome comprises the following elements in 5' to 3' order at the endogenous immunoglobulin heavy chain locus: Sμ switch region, IGHM gene lacking a sequence encoding a CH1 domain, IGHδ gene, Sγ1 switch region, IGHG1 gene lacking a sequence encoding a CH1 domain, Sα switch region, and 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: Sμ switch region, IGHM gene lacking a sequence encoding a CH1 domain, IGHδ gene, Sγ1 switch region, IGHG1 gene lacking a sequence encoding a CH1 domain, Sα switch region, and IGHA gene.

[0018] In one aspect, the present disclosure relates to a genetically modified non-human animal whose genome comprises the following elements in 5' to 3' order at the endogenous immunoglobulin heavy chain locus: Sμ switch region, IGHM gene lacking a sequence encoding a CH1 domain, Sγ1 switch region, IGHG1 gene lacking a sequence encoding a CH1 domain, Sα switch region, and 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: Sμ switch region, IGHM gene lacking a sequence encoding a CH1 domain, Sγ1 switch region, IGHG1 gene lacking a sequence encoding a CH1 domain, Sα switch region, and IGHA gene.

[0019] In one aspect, the present disclosure relates to a genetically modified non-human animal whose genome comprises the following elements in 5' to 3' order at the endogenous immunoglobulin heavy chain locus: Sμ switch region, IGHM gene lacking a sequence encoding a CH1 domain, IGHδ gene lacking a sequence encoding a CH1 domain, Sγ1 switch region, IGHG1 gene lacking a sequence encoding a CH1 domain, Sα switch region, and 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: Sμ switch region, IGHM gene lacking a sequence encoding a CH1 domain, IGHδ gene lacking a sequence encoding a CH1 domain, Sγ1 switch region, IGHG1 gene lacking a sequence encoding a CH1 domain, Sα switch region, and IGHA gene.

[0020] In some embodiments, the animal expresses a heavy chain antibody comprising an IgG heavy chain constant region lacking a CH1 domain. In some embodiments, the heavy chain antibody is expressed at less than 10 -7 M, less than 10 -8 M or less than 10 -9 In some embodiments, the heavy chain antibody comprises a variable region, a CH2 domain and a CH3 domain 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).

[0021] 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 the endogenous immunoglobulin heavy chain locus, and in some embodiments, the human IGHV genes, human IGHD genes, and human IGHJ genes are operably linked and can undergo 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 the endogenous immunoglobulin heavy chain locus of human chromosome 14 of a human subject. In some embodiments, the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at the endogenous immunoglobulin heavy chain locus of human chromosome 14 in a human cell. In some embodiments, the animal is a mouse, and the genetic modification in the endogenous immunoglobulin heavy chain locus of the animal includes the 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 in the endogenous heavy chain immunoglobulin locus of the animal includes the deletion of a continuous sequence starting from the mouse IGHV1-85 gene to the mouse IGHJ4 gene. In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin locus, and in some embodiments, the unmodified human sequence is at least 800 kb. In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin locus starting from human IGHV (III) -82 to human IGHV1-2. In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin locus starting from human IGHV (III) -82 to human IGHV6-1. In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin locus starting from human IGHD1-1 to human IGHJ6. In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin locus starting from human IGHV(III)-82 to human IGHJ6.

[0022] In some embodiments, the genome of the genetically modified non-human animal described herein comprises at the endogenous immunoglobulin heavy chain locus: replacing one or more endogenous IGHV, endogenous IGHD and endogenous IGHJ genes with one or more human IGHV, human IGHD and human IGHJ genes, in some embodiments, the human IGHV, human IGHD and human IGHJ genes are operably linked to one or more of the endogenous IGHM, IGHδ, IGHG1 and IGHA genes lacking the sequence encoding the CH1 domain. In some embodiments, one or more endogenous IGHV, endogenous IGHD and endogenous IGHJ genes are replaced by at least 150 human IGHV genes in Table 1, at least 20 human IGHD genes in Table 2, and at least 5 human IGHJ genes in Table 3. In some embodiments, the animal is a mouse, and at least 180 mouse IGHV genes in Table 4, all mouse IGHD genes in Table 5, and all mouse IGHJ genes in 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 the endogenous immunoglobulin light chain locus. In some embodiments, the animal lacks an endogenous immunoglobulin heavy chain variable region locus that can rearrange and form a nucleic acid sequence encoding an endogenous heavy chain variable domain. In some embodiments, the animal can produce 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.

[0023] In one aspect, the disclosure relates to cells obtained from a genetically modified non-human animal as described herein. In some embodiments, the cell is a B cell expressing a chimeric immunoglobulin heavy chain comprising a rearranged immunoglobulin heavy chain variable domain derived from one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes, 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.

[0024] In one aspect, the present disclosure relates to a method for preparing an antibody that specifically binds to an antigen, the method comprising a) exposing the genetically modified non-human animal to the antigen; b) generating a hybridoma from cells collected from the animal; and c) collecting the heavy chain antibodies produced by the hybridoma. In some embodiments, the method further comprises sequencing the genome of the hybridoma.

[0025] In one aspect, the present disclosure relates to a method for preparing an antibody that specifically binds to an antigen, the method comprising a) exposing the genetically modified non-human animal to the antigen; b) sequencing a nucleic acid encoding a human immunoglobulin heavy chain variable region in a cell expressing a heavy chain antibody that specifically binds to the antigen; and c) operably linking a nucleic acid encoding a human immunoglobulin heavy chain variable region to a nucleic acid encoding a human immunoglobulin heavy chain constant region in the cell.

[0026] In one aspect, the present disclosure relates to a method for preparing 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 an antigen, in some embodiments, the cell is obtained by exposing a genetically modified non-human animal described herein to an antigen; b) operably linking a nucleic acid encoding a 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 a cell, thereby obtaining the antibody.

[0027] In one aspect, the present disclosure relates to a method for obtaining a nucleic acid encoding an antibody binding domain that specifically binds to an antigen, the method comprising a) exposing a genetically modified non-human animal as described herein to the antigen; and b) sequencing a nucleic acid encoding a human immunoglobulin heavy chain variable region in a cell expressing a heavy chain antibody that specifically binds to the antigen.

[0028] In one aspect, the present disclosure relates to a method for preparing an antibody that specifically binds to an antigen, the method comprising a) exposing a genetically modified non-human animal as described herein to the antigen; b) constructing a phage plasmid library using RNA prepared from an immune cell (e.g., a spleen cell) of the animal; c) screening the phage plasmid library and d) sequencing nucleic acids encoding human immunoglobulin heavy chain variable regions from phage plasmids, the phage plasmid encoding a heavy chain antibody that specifically binds to the antigen. In some embodiments, the screening comprises separating phages expressing immunoglobulin heavy chain variable regions based on binding affinity to the antigen.

[0029] In one aspect, the present disclosure relates to a method for obtaining a sample, the method comprising a) exposing a genetically modified non-human animal as described herein to an antigen; and b) collecting a sample from the animal. In some embodiments, the sample is an immune cell, lymphoid tissue, spleen tissue, spleen cell or B cell.

[0030] In one aspect, the invention 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, 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%, 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%, 98%, 99%, or 100% identical to a selected VHH CDR3 amino acid sequence; in some embodiments, the selected VHH CDRs The amino acid sequences of 1, 2 and 3 are one of the following:

[0031] (1) The amino acid sequences of the selected VHH CDRs 1, 2, and 3 are shown in SEQ ID NOs: 42, 43, and 44, respectively;

[0032] (2) The amino acid sequences of the selected VHH CDRs 1, 2, and 3 are shown in SEQ ID NOs: 45, 46, and 47, respectively;

[0033] (3) The amino acid sequences of the selected VHH CDRs 1, 2, and 3 are shown in SEQ ID NOs: 48, 49, and 50, respectively;

[0034] (4) The amino acid sequences of the selected VHH CDRs 1, 2, and 3 are shown in SEQ ID NOs: 51, 52, and 53, respectively;

[0035] (5) The amino acid sequences of the selected VHH CDRs 1, 2, and 3 are shown in SEQ ID NOs: 54, 55, and 56, respectively;

[0036] (6) The amino acid sequences of the selected VHH CDRs 1, 2, and 3 are shown in SEQ ID NOs: 57, 58, and 59, respectively;

[0037] (7) the amino acid sequences of the selected VHH CDRs 1, 2, and 3 are shown in SEQ ID NOs: 60, 61, and 62, respectively; and

[0038] (8) The amino acid sequences of the selected VHH CDRs 1, 2, and 3 are shown in SEQ ID NOs: 63, 64, and 65, respectively.

[0039] In some embodiments, the VHH comprises CDRs 1, 2, 3 of the amino acid sequences shown in SEQ ID NOs: 42, 43, and 44, respectively. In some embodiments, the VHH comprises CDRs 1, 2, 3 of the amino acid sequences shown in SEQ ID NOs: 45, 46, and 47, respectively. In some embodiments, the VHH comprises CDRs 1, 2, 3 of the amino acid sequences shown in SEQ ID NOs: 48, 49, and 50, respectively. In some embodiments, the VHH comprises CDRs 1, 2, 3 of the amino acid sequences shown in SEQ ID NOs: 51, 52, and 53, respectively.

[0040] In one aspect, the present invention relates to an antibody or antigen-binding fragment thereof that binds to TFR1, comprising a heavy chain single variable region (VHH), the VHH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with a selected VHH sequence, in some embodiments, the selected VHH sequence is selected from SEQ ID NO: 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 (eg, a bispecific antibody).

[0041] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof comprising the VHH CDRs 1, 2, 3 of the antibody or antigen-binding fragment thereof as described herein.

[0042] In some embodiments, the antibody or antigen-binding fragment comprises a human IgG Fc (e.g., a human IgG1 Fc). In some embodiments, according to EU numbering, the human IgG Fc comprises a non-asparagine residue (e.g., alanine) at position 297. In some embodiments, the antibody or antigen-binding fragment comprises two or more heavy chain single variable domains.

[0043] In one aspect, the disclosure relates to a nucleic acid comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof as described herein. In some embodiments, the nucleic acid is a cDNA.

[0044] In one aspect, the invention relates to a vector comprising one or more nucleic acids described herein.

[0045] In one aspect, the disclosure relates to a cell comprising a vector as described herein. In some embodiments, the cell is a CHO cell. In one aspect, the disclosure relates to a cell comprising one or more nucleic acids described herein.

[0046] In one aspect, the present disclosure relates to a method of producing an antibody or an antigen-binding fragment thereof, comprising (a) culturing a cell as described herein under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment thereof; and (b) collecting the antibody or the antigen-binding fragment thereof produced by the cell.

[0047] In one aspect, the disclosure relates to an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof as described herein covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0048] In one aspect, the present disclosure relates to a method for treating a subject with 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 as described herein, or an antibody-drug conjugate. In some embodiments, the antibody or antigen-binding fragment thereof, or the antibody-drug conjugate can cross the blood-brain barrier (BBB) ​​of the subject.

[0049] On the one hand, the present disclosure relates to a method for treating a subject suffering from cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an antibody or its antigen-binding fragment as described herein, or an antibody-drug conjugate. In some embodiments, the cancer is brain cancer, lung cancer, gastric cancer, colorectal cancer, liver cancer, ovarian cancer, prostate cancer, leukemia or breast cancer. On the one hand, the present disclosure relates to a method for identifying a subject suffering from a brain disease (e.g., brain cancer), the method comprising detecting a sample collected from a subject suffering from a brain disease by an antibody or its antigen-binding fragment as described herein, thereby identifying a subject suffering from a brain disease. In some embodiments, the sample is a brain parenchyma sample from a subject. In some embodiments, the subject described herein is a human subject.

[0050] In one aspect, the present disclosure relates to a method for delivering an agent to cross 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 an antibody drug conjugate. In some embodiments, the agent is an anti-amyloid antibody.

[0051] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described herein and a pharmaceutically acceptable carrier. In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody drug conjugate as described herein and a pharmaceutically acceptable carrier.

[0052] In one aspect, the invention relates to antibodies or antigen-binding fragments thereof that cross-compete with the antibodies or antigen-binding fragments thereof described herein.

[0053] In one aspect, the present disclosure provides a method for preparing an antibody that specifically binds to an antigen. The method includes exposing an animal as described herein to an antigen; obtaining a nucleic acid sequence encoding a human heavy chain immunoglobulin variable region in a cell expressing a chimeric heavy chain antibody that specifically binds to an antigen (e.g., by sequencing); and operably linking a nucleic acid encoding a human heavy chain immunoglobulin variable region to a nucleic acid encoding a human heavy chain immunoglobulin constant region in a cell.

[0054] The present invention also relates to offspring of non-human mammals. In some embodiments, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

[0055] The disclosure also provides cells comprising targeting vectors as described herein. The disclosure also relates to cells (e.g., stem cells, embryonic stem cells, immune cells, B cells, T cells or hybridomas) or cell lines or primary cell cultures thereof from non-human mammals or their progeny. The invention also relates to tissues, organs or cultures thereof from non-human mammals or their progeny.

[0056] The present invention also relates to the use of non-human mammals or their offspring, animal models produced by the methods described herein, in developing products related to immune processes, producing human antibodies, or in model systems for pharmacology, immunology, microbiology and medical research.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not restrictive. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

[0058] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 It is a schematic diagram of a heavy chain antibody.

[0060] Figure 2 Schematic representation of the structures of the IgM, IgD, IgG, IgE, and IgA immunoglobulin isotypes is shown. Mouse and human immunoglobulin heavy chain loci genes are aligned and labeled with corresponding names.

[0061] Figure 3 Schematic diagram showing the constant region genes of the mouse immunoglobulin heavy chain locus. The Cγ1 gene structure including exons CH1, H, CH2, CH3, M1 and M2 is shown.

[0062] Figure 4A-4B Mutant alleles following genetic modification at the mouse immunoglobulin heavy chain constant region locus are shown.

[0063] Figure 5 The gene structure of Cγ1ΔCH1 is shown. The CH1 coding region is deleted (left) or replaced by the NEO cassette (right).

[0064] Figure 6 The procedure for genetic modification using targeting vector V1 is shown (Example 3). The 100883 bp sequence from Sγ3 to Cε at the mouse immunoglobulin heavy chain constant region locus was replaced in one step with a 16076 bp sequence including mouse Sγ1 and Cγ1ΔCH1 knock-in sequences to generate mutant allele 1.

[0065] Figure 7 A scheme for genetic modification using targeting vector V2 is shown (Example 3). A 92859 bp sequence from Cγ3 to Cε at the mouse immunoglobulin heavy chain constant region locus was replaced in a single step with a sequence including the Cγ1ΔCH1 knock-in sequence to generate mutant allele 1′.

[0066] Figure 8 The scheme for genetic modification using targeting vector V3 is shown (Example 3). 16434 bp of Cμ and Cδ sequences were knocked out from mutant allele 1 to generate mutant allele 2.

[0067] Fig. 9 The scheme for genetic modification using the targeting vector V4 is shown (Example 3). The sequences including Cμ, Cδ and Sγ1 are knocked out from mutant allele 1, so that Sμ and Cγ1ΔCH1' are directly linked, generating mutant allele 2'.

[0068] Fig.10 A scheme for genetic modification using targeting vector V5 is shown (Example 3). The CH1 coding sequence in Cμ was knocked out from mutant allele 1 to generate mutant allele 3.

[0069] Fig.11 The scheme for genetic modification using the targeting vector V6 is shown (Example 3). The CH1 coding sequence and the entire Cδ in Cμ were knocked out from mutant allele 1, generating mutant allele 4.

[0070] Fig.12 The scheme for genetic modification using the targeting vector V7 is shown (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.

[0071] Figures 13A-13B The results of PCR detection using primer pairs L-GT-F1 / L-GT-R1 and R-GT-F2 / R-GT-R2 are shown to verify the genotype of mutant allele 1. WT is the wild-type control. 2 O is a blank control. M is a marker.

[0072] Figure 14A shows the Southern blot results of mutant allele 1 positive clones digested with BclI and hybridized with LR probe. M is a marker. WT is wild type.

[0073] Figure 14B shows the 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.

[0074] Figure 14C shows the Southern blot results of mutant allele 1 positive clones digested with XmnI and hybridized with probe A. M is marker. WT is wild type.

[0075] Figure 14D shows the Southern blot results of mutant allele 1 positive clones digested with BglII and hybridized with the 5' probe. M is marker. WT is wild type.

[0076] Fig.15 The results of PCR detection using primers DE-F1 and DE-R1 to verify the sequence from Cμ to Cδ in the knockout mutant allele 2 are shown.

[0077] Fig.16 Shown are the results of PCR detection using primers GT-Mut-F, GT-Mut-R, and GT-WT-R to verify the sequence from Cμ to Sγ1 in the 2′ of the knockout mutant allele.

[0078] Fig.17 The results of PCR detection of the CH1 coding sequence of Cμ in knockout mutant allele 3 using primers GT-3F and GT-3R are shown.

[0079] Fig.18AThe results of PCR detection using primers Mut-F and Mut-R to verify the CμΔCH1 sequence in mutant allele 4 are shown. WT is the wild-type control. 2 O is the blank control.

[0080] Fig.18B The results of PCR testing using primers F4 and R4 to verify the absence of Cδ in mutant allele 4 are shown.

[0081] Figures 19A-19B The results of PCR detection using primer pairs Mut-F / Mut-R and F3 / R3 to verify the CμΔCH1 and CδΔCH1 sequences in mutant allele 5 are shown. WT is the wild-type control. 2 O is the blank control.

[0082] Fig. 20 is an exemplary flow chart of a method for introducing human immunoglobulin genes into the mouse genome.

[0083] Fig.21 is an overview of replacing the mouse immunoglobulin heavy chain variable region locus sequence with the human immunoglobulin heavy chain variable region locus sequence.

[0084] Fig. 22 The length distribution of CDR3 in the heavy chain variable region of antibodies produced by immunizing heterozygous Mut3 mice with Antigen A is shown.

[0085] Fig.23 Germline gene usage of variable region genes for the heavy chain mutant allele 3 genotype in heterozygous mice is shown.

[0086] Fig.24 The distribution of KD values ​​for antibodies raised against Antigen A in heterozygous Mut3 mice (H / -) is shown.

[0087] Fig.25 The distribution of KD values ​​for antibodies raised against Antigen A in homozygous Mut2 mice is shown.

[0088] Fig.26 Germline gene usage of variable region genes in homozygous Mut2 mice is shown.

[0089] Fig. 27 is a schematic diagram showing the human immunoglobulin heavy chain (IGH) locus on chromosome 14 (14q32.33).

[0090] Fig.28 is a schematic diagram showing the IGH locus on chromosome 12 (12F2) of mouse (Mus musculus) (strain C57BL / 6).

[0091] Fig.29The IMGT repertoire of the human heavy chain immunoglobulin locus (IGH) is listed.

[0092] Fig.30 The IMGT lineages of mouse IGH are listed.

[0093] Fig.31 Sequences discussed in this disclosure are listed.

[0094] Fig.32 Western blot results of serum IgG levels of mice with mutant allele 2', mutant allele 3, and mutant allele 4 genotypes are shown. Biot.Ladder is a protein marker. WT represents wild-type mice.

[0095] Fig.33 Shown is the length distribution of CDR3 in the heavy chain variable region of antibodies generated by immunizing heterozygous Mut3 mice with human 4-1BB.

[0096] Fig.34 Germline gene usage of variable region genes in mice homozygous for the heavy chain mutant allele 3 genotype is shown.

[0097] Fig.35 Shown is the length distribution of CDR3 in the heavy chain variable region of antibodies generated by immunizing hybrid Mut3 mice with human CD3ED and cynomolgus CD3ED.

[0098] Fig.36 Germline gene usage of variable region genes in mice homozygous for the heavy chain mutant allele 3 genotype is shown.

[0099] Fig.37 The CDR sequences of the heavy chain variable regions of the anti-TFR1 antibodies are listed according to Kabat numbering.

[0100] Fig.38 The CDR sequences of the heavy chain variable regions of the anti-TFR1 antibodies are listed according to IMGT numbering.

[0101] Fig.39 The amino acid sequences of the heavy chain variable regions of anti-TFR1 antibodies are listed.

[0102] Fig.40A Shown are antibody concentrations in total brain protein of hTFR1 mice within 72 hours following intravenous (iv) administration of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6).

[0103] Fig.40BShown are the ratios of antibody concentrations in total brain protein to serum antibody concentrations of hTFR1 mice within 72 hours of intravenous (iv) administration of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6).

[0104] Fig.40C Shown are antibody concentrations in the brain parenchyma of hTFR1 mice within 72 hours following intravenous (iv) administration of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6).

[0105] Fig.40D Shown are the ratios of antibody concentrations in the brain parenchyma to serum antibody concentrations of hTFR1 mice within 72 hours of intravenous (iv) administration of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5), or 24C9-N (G6).

[0106] Fig.41A Shown are the results of antibody concentration detection in brain parenchyma 24 hours after intravenous (iv) administration of hIgG1 (G1), JR141-N (G2), 23B8-N (G3), 24A1-N (G4), or 24G5-N (G5).

[0107] Fig.41B The results of antibody concentration detection of 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).

[0108] Fig.42 The results show the antibody concentrations 6 or 24 hours after intravenous (iv) administration of JR141-N (G2-G4) or 24G5-N (G5-G7). hIgG1 was used as a negative control;

[0109] Fig.43 Germline gene usage of variable region genes in mice homozygous for the heavy chain mutant allele 3 genotype is shown;

[0110] Fig.44 represents germline gene usage of variable region genes in heterozygous Mut2′ mice (H / -);

[0111] Fig.45 represents the distribution of KD values ​​of anti-human serum albumin antibodies produced in heterozygous Mut2' mice (H / -);

[0112] Fig.46 Germline gene usage of variable region genes in mice homozygous for the heavy chain mutant allele 4 genotype is shown;

[0113] Fig.47 Germline gene usage of variable region genes in mice homozygous for the heavy chain mutant allele 5 genotype is shown.

[0114] Detailed description

[0115] The present invention relates to genetically modified animals and methods for producing heavy chain antibodies.

[0116] Heavy chain antibodies (or heavy chain-only antibodies) are antibodies that have only heavy chains (usually two heavy chains) and lack the two light chains usually found in antibodies. Naturally occurring heavy chain antibodies are found in cartilaginous fish (such as sharks) and camelids (such as llamas). For example, in cartilaginous fish, the immunoglobulin new antigen receptor (IgNAR) is a heavy chain antibody. IgNAR displays significant structural differences from other antibodies. It has five constant regions (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 site of binding to the light chain in other antibodies. These differences, combined with the phylogenetic age of cartilaginous fish, led to the hypothesis that IgNAR may be closer to primitive antigen binding proteins than mammalian immunoglobulins.

[0117] The only mammals with heavy chain (IgG-like) antibodies are camelids, such as dromedaries, camels, llamas and alpacas. Like all mammals, camelids (e.g., llamas) can produce conventional antibodies (e.g., IgG1) composed of two heavy chains and two light chains bound together by Y-shaped disulfide bonds. However, they also produce two unique IgG subclasses: IgG2 and IgG3, also known as heavy chain IgG. These antibodies consist of only two heavy chains, which lack the CH1 region but still have an antigen binding domain (e.g., VHH) at their N-termini. Conventional Ig requires the combination of variable regions from heavy and light chains to allow for a high diversity of antigen-antibody interactions. Although isolated heavy and light chains still exhibit this ability, they exhibit very low affinity compared to paired heavy and light chains. The unique feature of heavy chain IgG is the ability of its monomeric antigen binding region to bind antigen, with specificity, affinity and, in particular, diversity comparable to conventional antibodies, without the need to pair with another region. This feature is mainly due to a pair of major variations in the amino acid sequence of the variable regions of the two heavy chains, which induce profound conformational changes when compared to conventional Ig. The major substitutions in the variable regions prevent the light chain from binding to the heavy chain, but also prevent the unbound heavy chain from being recycled by the immunoglobulin binding protein.

[0118] The single variable domain of these heavy chain antibodies (named VHH, sdAb or nanobody) is the minimum antigen binding domain produced by the adaptive immune system. It is usually found that the complementary determining region 3 (CDR3) of the variable region of these antibodies is twice as long as that of conventional antibodies. This leads to an increase in the interaction surface with the antigen and an increase in the diversity of antigen-antibody interactions, which compensates for the lack of light chain. With a long complementary determining region 3 (CDR3), VHH can extend into gaps on proteins that conventional antibodies cannot approach, including sites of interest in function, such as the active site of an enzyme or receptor binding valleys on the surface of a virus. In addition, additional cysteine ​​residues make the structure more stable, thereby increasing the strength of the interaction.

[0119] Compared with conventional antibodies carrying the variable domains (VH and VL) of conventional antibodies, VHH provides many other advantages, including higher stability, solubility, expression yield and refolding ability, and better in vivo tissue penetration. In addition, in contrast to the VH domains of conventional antibodies, VHH does not show an intrinsic tendency to bind to light chains. This helps to induce heavy chain antibodies in the presence of functional light chain loci. In addition, since VHH does not bind to the VL domain, it is easier to reorganize VHH into a bispecific antibody construct than a construct containing a conventional VH-VL pair or a single domain based on the VH domain.

[0120] The significant difference between camel VHH and human VH domains is the length and direction of the CDR3 loop. CDR3 corresponds to a unique region of the antibody molecule, which is encoded by a newly generated DNA element during the development of B cells. Genetic recombination results in the fusion of D-elements with flanking V- and J-elements. In the recombination process, further genetic diversity is produced by adding and / or deleting nucleotides at the junction. Therefore, the CDR3 loop provides a major contribution to antibody diversity and specificity. In some early transgenic heavy chain antibody animals, a limited number of variable region genes (IGHV, IGHD and IGHJ) cause some antigens to be unable to be recognized by these animals, although wild-type animals can produce effective antigenic responses (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 complete human heavy chain antibody library. Therefore, the variable domains produced by these animals can have the greatest possible diversity of human heavy chain variable domains, thereby maximizing the chance of obtaining fully humanized heavy chain antibodies.

[0121] In addition, because the entire sequence of the human immunoglobulin locus is introduced into the animal genome (without modification or with only limited modification), these genes can undergo V (D) J rearrangement in a very similar manner to that occurring in humans, reducing the risk of producing new immunogenic epitopes that can be recognized in the human immune system, thereby reducing immunogenicity. Immunogenicity can lead to the production of anti-drug antibodies and can include efficacy. Here, endogenous IGHV, IGHD and IGHJ genes have been effectively deleted. The antibodies produced by the antibody library are less likely to be immunogenic in humans. In addition, due to effective V (D) J recombination, antibody production can be very efficient and have a production rate similar to the normal production rate.

[0122] Therefore, the antibodies are more suitable as therapeutic agents for humans.Therefore, genetically modified animals provide a favorable platform for producing humanized heavy chain antibodies.

[0123] In addition, IgG1 is the most abundant antibody subtype in serum, with long serum half-life, strong FcγR affinity, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) activity, etc. IgG1 has unique advantages in the field of antibody drug development. Therefore, in one aspect, the present invention particularly relates to the preparation of humanized mice that can produce IgG1 subtype heavy chain antibodies. At the same time, 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.

[0124] As used herein, the term "antibody" refers to any antigen binding molecule containing at least one (e.g., one, two, three, four, five or six) complementary determining regions (CDRs) (e.g., any one of the three CDRs from an immunoglobulin light chain or any one of the three CDRs from an immunoglobulin heavy chain) and 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, the antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives, such as bispecific antibodies, single-chain antibodies, double antibodies, linear antibodies, and multispecific antibodies formed by antibody fragments.

[0125] As used herein, the term "antigen binding fragment" refers to a portion of a full-length antibody, wherein the 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 variable domain of a heavy chain or a variable domain of a light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0126] As used herein, the term "human antibody" refers to an antibody encoded by a nucleic acid present in a human (e.g., a rearranged human immunoglobulin heavy chain or light chain locus). In some embodiments, human antibodies are collected from humans or produced in human cell cultures (e.g., human hybridoma cells). In some embodiments, human antibodies are produced in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are produced in bacteria or yeast cells. In some embodiments, human antibodies are produced in transgenic non-human animals (e.g., mice) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy chain or light chain human immunoglobulin loci).

[0127] As used herein, the term "chimeric antibody" refers to an antibody containing sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, such as human and mouse antibodies). A non-limiting example of a chimeric antibody is an antibody containing a variable domain sequence of a human antibody (e.g., all or part of a light chain and / or heavy chain variable domain sequence) and a constant domain of a non-human antibody. Other examples of chimeric antibodies are described herein and are known in the art.

[0128] As used herein, the term "humanized antibody" refers to a non-human antibody that contains sequences derived from non-human (eg, mouse) immunoglobulin and sequences derived from human immunoglobulin.

[0129] As used herein, the term "single-chain antibody" refers to a single polypeptide containing at least two immunoglobulin variable domains (eg, variable domains of a mammalian immunoglobulin heavy or light chain) capable of specific binding to an antigen.

[0130] As used herein, the term "heavy chain antibody" refers to an antibody molecule consisting only of heavy chains (usually two) and without any light chains.

[0131] As used herein, the term "VHH" refers to a variable domain derived from a heavy chain antibody. VHH can specifically recognize an antigen without the need to pair with VL. In some embodiments, VHH described herein (also referred to as sdAb or nano antibodies) are derived from any humanized heavy chain antibodies described herein. In some embodiments, VHH, sdAb or nano antibodies described herein are derived from heavy chain antibodies produced by any genetically modified non-human animals described herein.

[0132] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and describe animals, humans or non-humans. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adults or adolescents (e.g., people under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Including, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domestic, farm, and zoo animals.

[0133] As used herein, the phrases "specific binding" and "specific binding" when referring to antibodies refer to the interaction of an antibody with its target molecule as opposed to other molecules because the interaction is dependent on the presence of a specific structure (i.e., an antigenic determinant or epitope) on the target molecule; in other words, the agent recognizes and binds to molecules that include a specific structure, rather than all molecules in general. An antibody that specifically binds to a target molecule may be referred to as a target-specific antibody.

[0134] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to amino acid polymers of at least two amino acids of any length.

[0135] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein to refer to a polymer of nucleotides of any length of at least two nucleotides, and include but are not limited to DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0136] As used herein, the term "unmodified human sequence" refers to a sequence derived from a human subject, a human cell, a cultured human cell, or a human cell line, wherein the sequence is identical to the genetic sequence of the human subject, the human cell, the cultured human cell, or the human cell line.

[0137] Immunoglobulin heavy chain constant region locus

[0138] The heavy chain immunoglobulin locus, also called IGH or immunoglobulin heavy locus, is a region on a chromosome (e.g., mouse chromosome 12) that contains genes for antibody (or immunoglobulin) heavy chains. It includes sequences encoding heavy chain variable regions and heavy chain constant regions.

[0139] like Figure 2As shown, the mouse immunoglobulin heavy chain constant region gene includes (as shown in the following order): immunoglobulin heavy chain constant region μ (IGHM, or Cμ), immunoglobulin heavy chain constant region δ (IGHδ, or Cδ), immunoglobulin heavy chain constant region γ3 (IGHG3, or Cγ3), immunoglobulin heavy chain constant region γ1 (IGHG1, or Cγ1), immunoglobulin heavy chain constant region γ2b (IGHG2b, or Cγ2b), immunoglobulin heavy chain constant region γ2c (IGHG2c, or Cγ2c), immunoglobulin heavy chain constant region ε (IGHE, or Cε) and immunoglobulin heavy chain constant region α (IGHA, or Cα) genes. In some embodiments, the immunoglobulin heavy chain constant region γ2a (IGHG2a) is located at the position of IGHG2c. In contrast, human immunoglobulin constant region genes include (as shown in the following order): immunoglobulin heavy chain constant region μ (IGHM, or Cμ), immunoglobulin heavy chain constant region δ (IGHδ, Cδ), immunoglobulin heavy chain constant region γ3 (IGHG3, or Cγ3), immunoglobulin heavy chain constant region γ1 (IGHG1, or Cγ1), immunoglobulin heavy chain constant region εP1 (pseudogene) (IGHEP1, or ψCε), immunoglobulin heavy chain constant region α1 (IGHA1, or Cα1), immunoglobulin heavy chain constant region γP (non-functional) (IGHGP, or CγP; not shown), immunoglobulin heavy chain constant region γ2 (IGHG2 or Cγ2), immunoglobulin heavy chain constant region γ4

[0140] (IGHG4, Cγ4), immunoglobulin heavy chain constant region epsilon (IGHE or Cε) and immunoglobulin heavy chain constant region alpha 2 (IGHA2 or Cα2) genes.

[0141] Immunoglobulin isotype switching (or isotype switching, or isotype switching, or class switch recombination (CSR)) is a biological mechanism that changes antibodies produced by B cells from one type to another; for example, 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 unchanged. Since the variable region does not change, class switching does not affect antigen specificity. Instead, the antibody retains affinity for the same antigen, but can interact with different effector molecules. 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) binding. Class switch recombination is a biological mechanism that allows the class of antibodies produced by activated B cells to change in a process called isotype or class switching. During CSR, parts of the antibody-heavy chain locus are removed from the chromosome, and the gene segments surrounding the missing parts are reconnected to retain functional antibody genes that produce antibodies of different isotypes. Double-strand breaks are generated at conserved nucleotide motifs in the DNA, called switch (S) regions, located upstream of the gene segments encoding the antibody heavy chain constant regions; these occur near all heavy chain constant region genes except Cδ. The DNA is nicked and broken at two selected switch regions by the activity of a series of enzymes, including activation-induced (cytidine) deaminase (AID), uracil DNA glycosylase, and apyrimidinic / apurinic (AP)-endonucleases. The intervening DNA between the switch regions is subsequently deleted from the chromosome, for example, removing the unwanted Cμ or Cδ heavy chain constant region sequences and allowing replacement of the 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 connect the variable domain exons to the desired downstream constant domain exons 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 that favors microhomologous joining. With the exception of the Cμ and Cδ genes, B cells express only one class of antibodies at any point in time. Figure 3 The position of each switch region (e.g., Sμ, Sγ3, Sγ1, Sγ2b, Sγ2c, Sε, and Sα) in the mouse immunoglobulin heavy chain constant region locus is shown. Specifically, the Cγ1 gene contains exons encoding the CH1 domain, hinge region, CH2 domain, CH3 domain, and two transmembrane domains. Exons are labeled CH1, H, CH2, CH3, M1, and M2, respectively.

[0142] The five major classes of immunoglobulins are IgM, IgD, IgG, IgE, and IgA, each of which can occur as either a transmembrane antigen receptor or a secreted antibody. In humans, IgG is found in four subclasses (IgG1, IgG2, IgG3, and IgG4), named in descending order of their abundance in serum, and IgA antibodies are found in two subclasses (IgA1 and IgA2). The different heavy chains that define these classes are called isotypes, and are denoted by the lowercase Greek letters μ (IgM), δ (IgD), γ (IgG), ε (IgE), and α (IgA), respectively.

[0143] Although the position and number of disulfide bonds between IgG1-IgG4 are different, the structures of the four IgG subtypes are very similar. In fact, as the most abundant IgG subtype in plasma, IgG1 is widely used to prepare recombinant therapeutic antibodies. In contrast, IgG3 is rarely used in antibody drug development because it has a weaker binding affinity to FcRn and a shorter half-life (about 9 days). Therefore, antibody drugs based on IgG3 must be administered more frequently due to pharmacokinetic reasons. In addition, the antibody levels of different subtypes can change during physical development, which is described in, for example, 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), which is incorporated herein by reference in its entirety. IgG1 is the most abundant antibody subtype in serum, with a long serum half-life, strong FcγR affinity, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) activity, etc. IgG1 has unique advantages in the field of antibody drug development. Therefore, in one aspect, the present disclosure relates to the preparation of humanized mice that can produce heavy chain antibodies of the IgG1 subtype. In one aspect, the present invention particularly relates to the preparation of humanized mice that can produce heavy chain antibodies of the IgG1 subtype. In some embodiments, the heavy chain antibodies can be further processed to produce nanobodies.

[0144] In one aspect, the present disclosure relates to a genetically modified non-human animal comprising a modified immunoglobulin heavy chain locus, wherein the modified immunoglobulin heavy chain locus comprises an IgG constant region gene, wherein the IgG constant region gene encodes an IgG heavy chain constant region lacking a CH1 domain, wherein the genetically modified non-human animal expresses heavy chain antibodies.

[0145] In some embodiments, the IgG constant region gene is one of the following: 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 encodes 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 IGHG3, IGHG2a, IGHG2b and / or IGHG2c genes.

[0146] 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 in IGHG is deleted. In some embodiments, the IgHG gene is operably linked to Sγ3, Sγ1, Sγ2a, Sγ2b or Sγ2c (e.g., Sγ3 or Sγ1).

[0147] In some embodiments, IGHM is a complete 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 a complete 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.

[0148] In some embodiments, the IGHE is a complete functional endogenous IGHE gene. In some embodiments, the sequence encoding the CH1 domain in IGHE is deleted. In some embodiments, the IGHE is deleted.

[0149] In some embodiments, IGHA is a complete functional endogenous IGHA gene. In some embodiments, the sequence encoding the CH1 domain in IGHA is deleted. In some embodiments, IGHA is deleted.

[0150] In various embodiments, the humanized heavy chain antibody includes a unique immunoglobulin constant region (Fc), which lacks at least a CH1 domain. In some embodiments, it also lacks the hinge region of a human Fc. In some embodiments, the heavy chain antibody includes the CH2 and CH3 regions of an immunoglobulin G (IgG) heavy chain constant region. In some embodiments, the constant region of the heavy chain antibody includes the hinge, CH2, and CH3 regions of an IgG heavy chain Fc.

[0151] In some embodiments, a suitable number of rearranged heavy chain variable regions are required, and when present during B cell development, the rearranged heavy chain variable regions can effectively survive selection. On the one hand, the disclosure provides a transgenic animal comprising a germline genetic modification, wherein the germline genetic modification comprises the deletion of a nucleotide sequence encoding the CH1 domain of IgG, wherein the animal expresses a functional IgM and the animal expresses an IgG heavy chain antibody (e.g., having IgG1 heavy chain CH2 and CH3 domains) in its serum. In some embodiments, IgM comprises two heavy chains, and they are combined with two λ or κ light chains to recognize antigens. In some embodiments, functional IgM includes the CH1 domain. In some embodiments, functional IgM does not include the CH1 domain. Without being bound by any theory, it is believed that deleting the sequence encoding the CH1 domain from the endogenous IGHM gene does not substantially change the function of IgM.

[0152] 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 the function of IgD.

[0153] Immunoglobulin heavy chain variable region locus

[0154] In order to produce humanized heavy chain antibodies (HCAb), the heavy chain immunoglobulin variable region locus in animals 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 (connection) and C (constant) regions. The genes in the V region form the V gene cluster (also referred to as the IGHV gene cluster). The genes in the D region form the D gene cluster (also referred to as the IGHD gene cluster). The genes in the J region form the J gene cluster (also referred to as the IGHJ gene cluster).

[0155] During B cell development, recombination events at the DNA level join a single D segment (also called the IGHD gene) to a J segment (also called the IGHJ gene); the fused DJ exons of this partially rearranged DJ region are then 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; 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 μ or δ heavy chains. Mature B cells in the lymph nodes undergo switch recombination, which brings the fused VDJ gene segment into proximity with one of the IGHG, IGHA, or IGHE gene segments, and each cell expresses a γ, α, or ε heavy chain. The potential recombination of many different IGHV genes with several IGHJ genes provides a wide range of antigen recognition. Additional diversity can be obtained through the junctional diversity generated by the random addition of nucleotides by terminal deoxynucleotidyl transferase, and 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 encode proteins and are considered to be pseudogene segments (often simply referred to as pseudogenes).

[0156] The human heavy chain immunoglobulin locus is located on human chromosome 14 ( Fig. 27 and Fig.29 ). Table 1 lists the IGHV genes and their relative order in this locus.

[0157] Table 1 List of IGHV genes on human chromosome 14

[0158]

[0159]

[0160] RPS8P1, ADAM6 and KIAA0125 are also located in 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 the IGHD genes on human chromosome 14 and their relative order. Table 3 lists all the IGHJ genes on human chromosome 14 and their relative order. The genes of the immunoglobulin constant region are located after the IGHV, IGHD and IGHJ genes. These genes include (as shown in the following order): immunoglobulin heavy chain constant region μ (IGHM), immunoglobulin heavy chain constant region δ (IGHδ), immunoglobulin heavy chain constant region γ3 (IGHG3), immunoglobulin heavy chain constant region γ1

[0161] (IGHG1), immunoglobulin heavy chain constant region epsilon p1 (pseudogene) (IGHEP1), immunoglobulin heavy chain constant region alpha 1 (IGHA1), immunoglobulin heavy chain constant region gamma P (non-functional) (IGHGP), immunoglobulin heavy chain constant region gamma 2 (IGHG2), immunoglobulin heavy chain region gamma 4 (IGHG4), immunoglobulin heavy chain constant region epsilon (IGHE), and immunoglobulin heavy chain constant region alpha 2 (IGHA2). These genes and the sequences of these genes are also shown in Fig. 27 and Fig.29 middle.

[0162] Table 2 List of IGHD genes on human chromosome 14

[0163] Gene name order Gene name order Gene name order Gene name order IGHD1-1 165 IGHD2-8 172 IGHD2-15 179 IGHD3-22 186 IGHD2-2 166 IGHD3-9 173 IGHD3-16 180 IGHD4-23 187 IGHD3-3 167 IGHD3-10 174 IGHD4-17 181 IGHD5-24 188 IGHD4-4 168 IGHD4-11 175 IGHD5-18 182 IGHD6-25 189 IGHD5-5 169 IGHD5-12 176 IGHD6-19 183 IGHD1-26 190 IGHD6-6 170 IGHD6-13 177 IGHD1-20 184 * IGHD1-7 171 IGHD1-14 178 IGHD2-21 185 IGHD7-27 192

[0164] Table 3 List of IGHJ genes on human chromosome 14

[0165]

[0166]

[0167] The mouse heavy chain immunoglobulin locus is located on mouse chromosome 12 ( Fig.28 and Fig.30 ). Table 4 lists the IGHV genes and their relative order in this locus.

[0168] Table 4 List of IGHV genes on mouse chromosome 12

[0169]

[0170]

[0171] 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. The genes of the immunoglobulin constant region are located after the IGHV, IGHD and IGHJ genes. These genes include (as shown in the following order): immunoglobulin heavy chain constant region μ (IGHM), immunoglobulin heavy chain constant region δ (IGHδ), immunoglobulin heavy chain constant region γ3 (IGHG3), immunoglobulin heavy chain constant region γ1 (IGHG1), immunoglobulin heavy chain constant region γ2b (IGHG2b), immunoglobulin heavy chain constant region γ2c (IGHG2c), immunoglobulin heavy chain constant region ε (IGHE) and immunoglobulin heavy chain constant region α (IGHA) genes. In some embodiments, the immunoglobulin heavy chain constant region γ2a (IGHG2a) is located at the position of IGHG2c. These genes and the order of these genes are also shown in Fig.28 and Fig.30 .

[0172] Table 5 List of IGHD genes on mouse chromosome 12

[0173] Gene name order Gene name order Gene name order IGHD5-1 183 IGHD2-5 191 IGHD5-5 198 IGHD3-1 184 IGHD5-3 192 IGHD2-8 199 IGHD1-1 185 IGHD5-7 193 IGHD5-6 200 IGHD6-1 186 IGHD2-6 194 IGHD3-2 201 IGHD2-3 187 IGHD5-4 195 IGHD4-1 202 IGHD6-2 188 IGHD5-8 196 IGHD2-4 189 IGHD2-7 197

[0174] Table 6 List of IGHJ genes on mouse chromosome 12

[0175] Gene name order Gene name order IGHJ1 203 IGHJ3 205 IGHJ2 204 IGHJ4 206

[0176] The present disclosure provides a genetically modified non-human animal 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 together and can undergo VDJ rearrangement. In some embodiments, the human IGHV genes, human IGHD genes and human IGHJ genes are located at an endogenous heavy chain immunoglobulin locus.

[0177] 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., as shown in Table 1).

[0178] 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 human IGHV genes in Table 1 except IGHV2-10, IGHV3-9, and IGHV1-8, all human IGHD genes in Table 2, and all human IGHJ genes in Table 3. In some embodiments, the animal comprises all human IGHV genes in Table 1 except IGHV5-10-1 and IGHV3-64D, all human IGHD genes in Table 2, and all human IGHJ genes in Table 3. In some embodiments, the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at the endogenous heavy chain immunoglobulin locus of human chromosome 14 of a human subject. In some embodiments, the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at the endogenous heavy chain immunoglobulin locus of human chromosome 14 of a human cell (e.g., a somatic cell, a cultured cell, a non-immune cell, a cell without any V(D)J rearrangement).

[0179] 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.

[0180] 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.

[0181] In some embodiments, the animal comprises an unmodified human sequence comprising a gene starting 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 at 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 starting from human IGHV(III)-82 to human IGHV1-2. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus starting 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 starting from human IGHV (III) -82 to human IGHV-6-1.

[0182] 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., as 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.

[0183] 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., genes as 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.

[0184] In some embodiments, the animal comprises an unmodified human sequence comprising a gene starting with 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 starting from human IGHD1-1 to human IGHJ6.

[0185] In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus starting from human IGHD1-1 to human IGHD7-27.

[0186] In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus starting from human IGHJ1P to human IGHJ6. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus starting from human IGHJ1 to human IGHJ6.

[0187] In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus starting from human IGHV(III)-82 to human IGHJ6.

[0188] In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus starting from human IGHV1-2 to human IGHJ6. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus starting from human IGHV (II) -1-1 to human IGHJ6. In some embodiments, the unmodified human sequence is derived from the human heavy chain immunoglobulin locus starting from human IGHV6-1 to human IGHJ6.

[0189] In some embodiments, the animal may have one, two, three, four, five, six, seven, eight, nine, or ten unmodified human sequences. In some embodiments, the unmodified human sequences have about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,

[0190] 900 or 1000kb in length.

[0191] In some aspects, the present disclosure relates to a genetically modified animal comprising a first sequence at an endogenous heavy chain immunoglobulin locus, the 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, the second sequence, and the third sequence are operably linked.

[0192] 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.

[0193] In some embodiments, the first sequence is an unmodified sequence derived from a human heavy chain immunoglobulin locus. In 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.

[0194] 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.

[0195] 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 human IGHD genes in Table 2, and all human IGHJ genes in Table 3.

[0196] In some embodiments, the animal comprises one or more endogenous genes selected from the group consisting of immunoglobulin heavy chain constant region μ (IGHM), immunoglobulin heavy chain constant region δ (IGHδ), immunoglobulin heavy chain constant region γ3 (IGHG3), immunoglobulin heavy chain constant region γ1 (IGHG1), immunoglobulin heavy chain constant region γ2b (IGHG2b), immunoglobulin heavy chain constant region γ2c (IGHG2c), immunoglobulin heavy chain constant region ε (IGHE), and immunoglobulin heavy chain constant region α (IGHA) genes. In some embodiments, immunoglobulin heavy chain constant region γ2a (IGHG2a) is located at the position of IGHG2c. In some embodiments, these endogenous genes are operably linked together. In some embodiments, these endogenous genes have the same order as wild-type animals. In some embodiments, isotype switching (immunoglobulin class switching) can occur in animals.

[0197] In some embodiments, IGHV gene, IGHD gene and / or IGHJ gene are operably linked together. VDJ recombination can occur between these genes and produce functional antibodies. In some embodiments, these genes are arranged in an order similar to the order in the human heavy chain immunoglobulin locus. This arrangement provides various advantages, for example, the arrangement of these genes allows the production of heavy chain variable domains with diversity, which is very similar to the diversity of human heavy chain variable domains. Since some random sequences can be inserted into the sequence during VDJ recombination, in some embodiments, the complete human antibody library without modification or minimal modification can reduce the possibility of inserting non-human sequences during VDJ recombination.

[0198] In some embodiments, the IGHV gene, IGHD gene and / or IGHJ gene are operably linked to one or more genes (e.g., all genes) selected from the group consisting of IGHM, IGHδ, IGHG3, IGHG1, IGHG2a, IGHG2b, IGHG2c, IGHE and IGHA genes.

[0199] In some embodiments, the animal comprises a disruption in the animal's endogenous heavy chain immunoglobulin locus. In some embodiments, the disruption of 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.

[0200] In some embodiments, the animal is a mouse. Disruption of the animal's endogenous heavy chain immunoglobulin loci comprises 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,

[0201] 179, 180, 181, or 182 mouse IGHV genes (e.g., as shown in Table 4). In some embodiments, the disruption comprises deleting 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 includes 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).

[0202] 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.

[0203] In some embodiments, the disruption of 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., as 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 includes 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.

[0204] In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, or 4 mouse IGHJ genes selected from IGHJ1, IGHJ2, IGHJ3, and IGHJ4. In some embodiments, the mouse still comprises about or at least 1, 2, 3, or 4 mouse IGHJ genes selected from IGHJ1, IGHJ2, IGHJ3, and IGHJ4.

[0205] In some embodiments, the disruption of 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 the endogenous sequence.

[0206] In some embodiments, the deletion sequence is from IGHV1-86 to IGHJ4, from IGHV1-85 to IGHJ4, from IGHV1-84 to IGHJ4, from IGHV1-83 to IGHJ4, or from IGHV1-82 to IGHJ4 (e.g., from IGHV1-85 to IGHJ4).

[0207] 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 sequence has a length of 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 some embodiments, the sequence starts from human IGHV(III)-82 to IGHV1-2. In some embodiments, the sequence starts from human IGHV7-81 to IGHV1-2. In some embodiments, the sequence starts from human IGHV(II)-1-1 to IGHJ6. In some embodiments, the sequence starts from human IGHV6-1 to IGHJ6.

[0208] Human IGHV gene, human IGHD gene and human IGHJ gene are operably connected together and can undergo VDJ rearrangement. In some embodiments, the modified mouse has a complete human IGHV, IGHD and IGHJ gene library (for example, including all non-pseudo-human IGHV, IGHD and IGHJ genes). Therefore, the modified mouse can produce a complete human antibody library. In some embodiments, after VDJ recombination, an IGHV gene (for example, IGHV3-21 or IGHV3-74) constitutes a sequence encoding an antibody heavy chain variable region. An IGHD gene constitutes a sequence encoding an antibody heavy chain variable region. And an IGHJ gene constitutes a sequence encoding an antibody heavy chain variable region. In some embodiments, the IGHV gene is IGHV3-21 or IGHV3-74.

[0209] In some embodiments, one IGHV gene (e.g., IGHV3-30, IGHV3-33, IGHV4-39 or IGHV4-34) constitutes a sequence encoding an antibody heavy chain variable region. One IGHD gene (e.g., IGHD6-19) constitutes a sequence encoding an antibody heavy chain variable region. One IGHJ gene (e.g., IGHJ4 or IGHJ6) constitutes a sequence encoding an antibody heavy chain variable region.

[0210] Furthermore, in some cases, the entire mouse IGHV gene, IGHD gene, and IGHJ gene (e.g., including all non-pseudogenes) are knocked out, and the heavy chain variable region will not have any sequence encoded by a sequence from the mouse, thereby minimizing immunogenicity in humans.

[0211] 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 camelids). 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 camelids). 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 camelids). These non-human exogenous genes help to increase the diversity of VHH domains.

[0212] For example, various modifications at the heavy chain immunoglobulin locus are described in WO2020169022A1, which is incorporated herein by reference in its entirety.

[0213] Immunoglobulin light chain locus

[0214] In some embodiments, the animal has an intact kappa and / or lambda chain immunoglobulin locus. In some embodiments, the animal has a disrupted kappa and / or lambda chain immunoglobulin locus.

[0215] The kappa chain immunoglobulin locus (also referred to as IGK or immunoglobulin kappa locus) is a region of a chromosome (e.g., chromosome 6) that contains human antibody (or immunoglobulin) light chain genes. Similarly, immunoglobulin light chain genes can also undergo a series of rearrangements, resulting in the production of mature immunoglobulin light chain nucleic acids (e.g., kappa chains).

[0216] The connection of the V segment (also called IGKV gene) and the J segment (also called IGKJ gene) produces a continuous exon encoding the entire light chain variable domain. In unrearranged DNA, the V gene segment (or IGKV gene cluster) is located relatively far away from the C region. The J gene segment (or IGKJ gene cluster) is located near the C region. The connection of the V segment to the J gene segment also brings the V gene close to the C region sequence. The J gene segment of the rearranged V region is separated from the C region sequence only by introns. In order to prepare a complete immunoglobulin light chain messenger RNA, the V region exons are connected to the C region sequence by RNA splicing after transcription.

[0217] In some embodiments, the animal comprises a disruption in an endogenous light chain immunoglobulin locus of the animal. In some embodiments, the disruption of 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.

[0218] In some embodiments, the animal is a mouse. The disruption of the animal's endogenous kappa 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 includes 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.

[0219] 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.

[0220] In some embodiments, the disruption comprises a deletion of about or at least 1, 2, 3, 4, or 5 mouse IGKJ genes selected from IGKJ1, 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 IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5 (e.g., IGKJ5).

[0221] In some embodiments, the disruption of 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 the endogenous sequence.

[0222] In some embodiments, the deleted sequence starts from IGKV2-137 to IGKJ4, starts from IGKV1-136 to IGKJ4, starts from IGKV1-135 to IGKJ4, starts from IGKV2-137 to IGKJ5, starts from IGKV1-136 to IGKJ5, or starts from IGKV1-135 to IGKJ5 (e.g., starts from IGKV2-137 to IGKJ5).

[0223] In some embodiments, the animal comprises a disruption in an endogenous lambda light chain immunoglobulin locus of the animal. In some embodiments, the disruption of the endogenous light chain immunoglobulin locus of the animal comprises a deletion of one or more endogenous IGLV genes, one or more endogenous IGLJ genes, and / or one or more immunoglobulin lambda constant (IGLC) genes (e.g., IGLC1, IGLC2, IGLC3, and IGLC4).

[0224] The destruction of the animal's endogenous lambda light chain immunoglobulin locus includes 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 includes about or at least 1, 2, 3 or 4 mouse IGLC genes selected from IGLC1, IGLC2, IGLC3 and IGLC4. In some embodiments, the destruction includes about or at least 1, 2 or 3 mouse IGLV genes selected from IGLV1, IGLV2 and IGLV3. In some embodiments, the destruction includes a deletion of about or at least 1, 2, 3, 4 or 5 mouse IGLJ genes selected from IGLJ1, IGLJ2, IGLJ3, IGLJ3P and IGLJ4.

[0225] In some embodiments, the disruption of the animal's endogenous lambda light chain immunoglobulin locus comprises 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 lambda light chain immunoglobulin gene.

[0226] In some embodiments, the deleted sequence starts from IGLV2 to IGLC1, starts from IGLV3 to IGLC1, or starts from IGLJ2 ​​to IGLC1.

[0227] For example, various modifications on the light chain immunoglobulin locus are described in WO2020169022A1, the entire contents of which are incorporated herein by reference.

[0228] Genetically modified animals

[0229] The application provides a genetically modified non-human animal that produces an antibody having only a heavy chain, i.e., an antibody lacking a light chain. In some embodiments, the genetically modified non-human animal does not produce an IgG (e.g., IgG1) molecule comprising a light chain. In some embodiments, the genetically modified non-human animal does not produce any conventional IgG molecules, i.e., an IgG antibody having two heavy chains and two light chains.

[0230] In some embodiments, the genetically modified non-human animal has a fully functional endogenous light chain locus. In some embodiments, the immunoglobulin heavy chain locus of the animal comprises fully functional IGHM, IGHδ and / or IGHA genes. In some embodiments, the genome of the animal does not comprise exogenous sequences (e.g., human immunoglobulin heavy chain constant region genes) in the endogenous immunoglobulin heavy chain constant region locus.

[0231] In some embodiments, the modified immunoglobulin heavy chain locus lacks the CH1 exon of an endogenous IGHG gene (e.g., an IGHG1 gene). In some embodiments, the immunoglobulin heavy chain locus of an animal comprises fully functional H, CH2, CH3, M1 and / or M2 exons of an IGHG1 gene.

[0232] In some embodiments, the immunoglobulin heavy chain locus of the animal has only a modification of the CH1 exon of the endogenous IGHG1 gene. In some embodiments, the modification does not include a mutation, such as a deletion or loss-of-function mutation, of a 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 of the CH2 or CH3 exons of the IGHG1 gene. In some embodiments, the immunoglobulin heavy chain locus does not have a mutation or modification of the CH1 exon of the IGHM gene. In some embodiments, the genetically modified non-human animal has a functional gene fragment encoding the CH1 domain of IgM at the endogenous IGHM locus.

[0233] In some embodiments, the genetically modified non-human animal has fully functional genes encoding other heavy chain constant isotypes (e.g., 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 include any mutations to endogenous IGHM, IGHδ, and / or IGHA genes. In some embodiments, the endogenous immunoglobulin heavy chain locus has complete endogenous IGHM, IGHδ, or IGHA genes.

[0234] In some embodiments, the genetically modified non-human animal expresses wild-type IgM, IgD, and / or IgA proteins.

[0235] In some embodiments, the immunoglobulin heavy chain locus includes a mutation (e.g., deletion) or modification of 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 an IgM with a missing CH1 domain and / or an IgD with a missing CH1 domain.

[0236] In some embodiments, the introduction of modifications to the endogenous immunoglobulin heavy chain loci in a genetically modified non-human animal can maintain the health of the animal, including substantially normal B cell development and maturation. In some embodiments, the introduction of modifications to the endogenous immunoglobulin heavy chain loci in a genetically modified non-human animal reduces or avoids the immunogenicity of the exogenous sequence. In some embodiments, the introduction of minimal changes to the endogenous immunoglobulin heavy chain loci in a genetically modified non-human animal retains the normal function of the endogenous immunoglobulin heavy chain loci, including VDJ rearrangement, classical switch recombination, and somatic hypermutation.

[0237] In some embodiments, the genetically modified non-human animal has one or more fully functional light chain loci, such as a lambda light chain locus and / or a kappa light chain locus. In some embodiments, the genetically modified non-human animal has an unchanged endogenous light chain locus. In some embodiments, no mutation is introduced into the endogenous light chain locus of the genetically modified non-human animal. In some embodiments, the lambda and / or kappa light chain variable region loci of the genetically modified non-human animal are functional, rather than silent. In some embodiments, the genetically modified non-human animal expresses wild-type lambda light chains and / or wild-type kappa light chains. In some embodiments, the genetically modified non-human animal expresses functional IgM molecules comprising light chains. In some embodiments, the genetically modified non-human animal expresses functional IgA, IgD and / or IgM molecules comprising 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 lambda and / or kappa light chain variable region loci of the genetically modified non-human animal can be knocked out.

[0238] In some embodiments, the modified immunoglobulin heavy chain locus does not comprise a rearranged gene (e.g., a rearranged IGHV, IGHD and / or IGHJ gene). In some embodiments, the modified immunoglobulin heavy chain locus comprises all unrearranged human IGHV, IGHD and IGHJ genes.

[0239] In some embodiments, the modified immunoglobulin heavy chain locus comprises a functional splice site immediately following the CH1 exon of the endogenous IGHG1 gene. In some embodiments, the modified immunoglobulin heavy chain locus comprises a wild-type splice site immediately following the CH1 exon of the endogenous IGHG1 gene.

[0240] In some embodiments, the animals described herein express a membrane-bound IgG1 lacking the CH1 domain. In some embodiments, the animals described herein express a soluble IgG1 lacking the CH1 domain.

[0241] 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 an endogenous immunoglobulin locus.

[0242] 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 an endogenous κ or λ light chain immunoglobulin locus of the animal. In some embodiments, the animal does not have a disruption in an endogenous κ or λ light chain immunoglobulin locus of the animal.

[0243] The genetically modified non-human animal can be various animals, such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, rhesus monkeys). For non-human animals that are not easy to obtain suitable genetically modified embryonic stem (ES) cells, other methods are used to prepare non-human animals comprising genetic modifications. Such methods include, for example, modifying non-ES cell genomes (e.g., fibroblasts or induced pluripotent cells) and using nuclear transplantation to transfer the modified genome to suitable cells, such as oocytes, and incubating the modified cells (e.g., modified oocytes) in non-human animals under suitable conditions to form embryos. These methods are known in the art and are described in, for example, A. Nagy et al. "Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition)", Cold Spring Harbor Laboratory Press, 2003, the entire contents of which are incorporated herein by reference. Therefore, in various embodiments, human V, D and / or J segments can be operably linked to non-human animal (e.g., rodent, mouse, rat, hamster) constant region gene sequences. During B cell development, these rearranged human V, D and / or J segments are linked to non-human animal immunoglobulin constant regions.

[0244] In one aspect, the animal is a mammal, for example, a mammal of the superfamily Diplopodae or Muroidea. In some embodiments, the transgenic animal is a rodent. Rodents can be selected from mice, rats and hamsters. In some embodiments, the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, white-tailed rats, Madagascar rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., moles, bamboo rats and zokors). In some embodiments, the genetically modified rodent is selected from a mouse or rat (Muridae), a gerbil, a spiny mouse, and a whiskered rat. In some embodiments, the non-human animal is a mouse.

[0245] 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 strains of 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 Mouse, 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 entirety. In some embodiments, the genetically modified mouse is a hybrid strain of 129 strain and C57BL / 6 strain. In some embodiments, the genetically modified mouse is a hybrid strain of 129 strain or a hybrid strain of BL / 6 strain. In some embodiments, the mouse is a BALB strain, such as a BALB / c strain. In some embodiments, the mouse is a hybrid strain 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 having 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.

[0246] Different animals have different germline organizations and genes at their endogenous immunoglobulin heavy chain (IgH) loci. The IgH loci of many species have been sequenced. The gene location and exon / intron organization of the IgH loci in mice, rats and rabbits can be found in, for example, the IMGT library and the NCBI database, the entire contents of which are incorporated herein by reference.

[0247] 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 hybrid strain of two or more strains selected from Wistar, LEA, Sprague Dawley, Fischer, F344, F6 and Dark Agouti.

[0248] The animal may have one or more additional genetic modifications and / or other modifications as appropriate for the particular purpose of making the humanized animal.

[0249] A genetically modified non-human animal comprising a modification of an endogenous non-human immunoglobulin locus. In some embodiments, the modification may comprise a human nucleic acid sequence encoding at least a portion of a human protein (e.g., having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to a human heavy chain variable domain or light chain variable domain sequence). Although genetically modified cells (e.g., ES cells, somatic cells) that may comprise modifications described herein are also provided, in many embodiments, the genetically modified non-human animal comprises a modification of an endogenous locus in the animal germline.

[0250] Genetically modified animals can express humanized antibodies and / or chimeric antibodies from endogenous mouse loci, wherein one or more endogenous mouse immunoglobulin genes have been replaced by human immunoglobulin genes and / or nucleotide sequences, the nucleotide sequences having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to human immunoglobulin gene sequences (e.g., IGHV, IGHD, IGHJ, IGKV and / or IGKJ genes). In various embodiments, the endogenous non-human immunoglobulin loci are modified in whole or in part to contain human nucleic acid sequences.

[0251] The above-mentioned non-human mammals can be subjected to genetic, molecular and behavioral analysis. The present disclosure also relates to offspring produced by mating the non-human mammals provided by the present disclosure with the same genotype 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.

[0252] The present disclosure also provides cell lines or primary cell cultures derived from non-human mammals or their progeny. Cell culture-based models can be prepared, for example, by the following methods. Cell cultures can be obtained by separation from non-human mammals, or cells can be obtained from cell cultures established using the same constructs and standard cell transfection techniques. The integration of genetic constructs comprising DNA sequences encoding human or humanized immunoglobulins can be detected by a variety of methods.

[0253] There are many analytical methods that can be used to detect modifications on exogenous DNA or genomic DNA, including methods at the nucleic acid level (including mRNA quantification methods using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blots, and in situ hybridization) and protein level methods (including histochemistry, immunoblotting analysis, and in vitro binding studies). In addition, the expression level of the target gene can be quantified by ELISA technology well known to those skilled in the art. Many standard analytical methods can be used to complete quantitative measurements. For example, RT-PCR and hybridization methods (including RNA enzyme protection, Southern blot analysis, RNA dot analysis (RNAdot) analysis) can be used to measure transcription levels. Immunohistochemical staining, flow cytometry, and Western blot analysis can also be used to assess the presence of people or humanized proteins.

[0254] Antibodies and antigen-binding fragments

[0255] The present invention provides antibodies and antigen-binding fragments thereof (eg, heavy chain antibodies, humanized heavy chain antibodies, or multispecific antibodies) produced by the methods described herein.

[0256] Typically, conventional antibodies are composed of two types of polypeptide chains, namely light chains and heavy chains. The non-limiting antibodies of the present invention can be complete four-chain immunoglobulin antibodies comprising 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 subclasses, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. The antibody may contain two identical copies of the light chain and two identical copies of the heavy chain. Each heavy chain containing a variable region (or variable region, VH) and multiple constant regions (or constant regions) is bound to each other by disulfide bonds in its constant region to form the "stem" of the antibody. Each light chain contains a variable domain (or variable region, VL) and a constant domain (or constant region), and each light chain is bound to a heavy chain by disulfide bonding. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound. The variable regions of both the light and heavy chains are comprised of three hypervariable regions sandwiched between more conserved framework regions (FR).

[0257] These hypervariable regions, called complementarity determining regions (CDRs), form loops that include the major antigen binding surface of the antibody. The four framework regions primarily adopt a β-sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, together with the CDRs from the other chain, contribute to the formation of the antigen binding region.

[0258] Methods for identifying the CDR regions of antibodies by analyzing the amino acid sequence of the antibody are well known, and a number of definitions of CDRs are commonly used: the Kabat definition is based on sequence variability, while the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described, for example, in Martin et al. "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): 3832-3839; Wu, TT and 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.

[0259] CDR is important for recognizing the epitope of an antigen. As used herein, an "epitope" is 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 about three, four, five, six or seven amino acids, but these amino acids do not need to be in a continuous linear sequence of the primary structure of the antigen, because the epitope may depend on the three-dimensional configuration of the antigen based on the secondary and tertiary structures of the antigen.

[0260] In some embodiments, the antibody is a complete immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differing in their constant regions, particularly their hinge and upper CH2 domains. The sequences and differences of IgG subclasses are known in the art and are described in, for example, 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 et al. 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 in the heavy chain antibody can be from any immunoglobulin molecule described herein (e.g., IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgM, IgD, IgE, IgA).

[0261] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, rats, camels). Antibodies disclosed herein also include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, and chimeric antibodies comprising immunoglobulin binding domains fused to another polypeptide. The term "antigen binding domain" or "antigen binding fragment" is a part of an antibody that retains the specific binding activity of a complete antibody, that is, any part of an antibody that can specifically bind to an epitope on a target molecule of a complete antibody. It includes, for example, Fab, Fab', F(ab')2 and variants of these fragments. Therefore, in some embodiments, an antibody or its antigen binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, double antibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed by antibody fragments, and any polypeptide including an antibody binding domain or a binding domain homologous to an antibody binding domain. Non-limiting examples of antigen binding domains include, e.g., heavy and / or light chain CDRs of an intact antibody, heavy and / or light chain variable regions of an intact antibody, the full length heavy or light chain of an intact antibody, or a single CDR from a heavy or light chain of an intact antibody.

[0262] In some embodiments, the antigen binding fragment may form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a VHH fusion as described herein, fused to a CD3-ζ transmembrane domain and an endodomain.

[0263] Antibodies and antigen-binding fragments thereof (e.g., humanized antibodies or chimeric antibodies) produced by the methods described herein have various advantages. In some embodiments, no further optimization is required to obtain desired properties (e.g., binding affinity, thermal stability and / or limited aggregation).

[0264] In some embodiments, the antibody (or antigen-binding fragment thereof) is detected in less than 0.1 s -1 , less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 or less than 0.00001s -1 The dissociation rate (koff) of the target is specific. In some embodiments, the dissociation rate (koff) is greater than 0.01s -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 or greater than 0.000001s -1 .

[0265] In some embodiments, the kinetic association rate (kon) is greater than 1 x 10 2 / Ms, greater than 1x 10 3 / Ms, greater than 1x10 4 / Ms, greater than 1x 10 5 / Ms or greater than 1x 10 6 / Ms. In some embodiments, the kinetic association rate (kon) is less than 1 x 10 5 / Ms, less than 1x 10 6 / Ms or less than 1x 10 7 / Ms.

[0266] Affinity can be derived from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, KD is less than 1 x 10 -6 M, less than 1x 10 -7 M, less than 1x 10 -8 M, less than 1x 10 -9 M or less than 1x 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 greater than 1 x 10 -7 M, greater than 1x 10 -8 M, greater than 1x 10 -9 M, greater than 1x 10 -10 M, greater than 1x 10 -11 M or greater than 1x 10 -12 M. In some embodiments, the antibody binds to the target with a KD of less than or equal to 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.

[0267] In some embodiments, thermal stability is determined. The Tm of the antibodies or antigen-binding fragments described herein may be higher than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C or 95°C.

[0268] In various embodiments, the parental heavy chain antibody sequence is replaced to prepare a variant heavy chain antibody. Typically, the heavy chain antibody variant of the parental heavy chain antibody has 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 10000%) parental heavy chain antibody binding affinity to a specific antigen. In some embodiments, compared with the parental heavy chain antibody, the variant heavy chain antibody will comprise a single replacement. However, in other embodiments, compared with the parental heavy chain antibody sequence derived from other human heavy chain sequences sharing identity at a given position, several amino acids (e.g., up to about 5 or 10 or more amino acids) are replaced. In various embodiments, the resulting variant heavy chain antibody is tested to confirm that the required binding affinity and / or specificity are not significantly reduced by the replaced residue. In some embodiments, improved variant heavy chain antibodies are produced by replacing amino acids from different human heavy chain sequences. In various embodiments, the VHH has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the parent VHH.

[0269] The VHH described herein can be used to prepare multispecific (e.g., bispecific antibodies). In one aspect, the present invention provides a multispecific antibody 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 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.

[0270] 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 and a light chain, the heavy chain comprising VH, and the light chain comprising 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 moiety is a Fab-like domain comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising a first VHH fused to a CH1 domain, and the second polypeptide chain comprising a second VHH fused to a CL domain.

[0271] 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 VHH binds to the same epitope. In some embodiments, the VHH binds to different epitopes.

[0272] In some embodiments, the antibody or antigen-binding fragment thereof has four or more VHHs. In some embodiments, in order to increase developability, at least four VHHs are combined without adding an IgG Fc domain to construct a tetraspecific VHH. Compared to bispecific and trispecific VHH-Fc, these molecules have the additional advantage of increased affinity and avidity for antigens despite the lack of Fc effector functions.

[0273] In some embodiments, the antibodies or antigen-binding fragments thereof (e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) have a functional Fc.

[0274] In some embodiments, the heavy chain antibodies produced by the genetically modified non-human animals described herein have a VHH domain comprising CDR1, CDR2 and CDR3. In some embodiments, the CDR3 length is between 6-23, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23. In some embodiments, the CDR3 length 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.

[0275] Method for preparing transgenic animals

[0276] Genetically modified animals can be prepared by modifying the immunoglobulin loci. Figure 6-12 The schemes of genetic modification using targeting vectors V1-V7 are shown respectively. Fig. 20 A method for preparing a humanized animal is shown. In some embodiments, the method first involves modifying the human immunoglobulin locus on a human chromosome. The modified human chromosome is then introduced into a mouse recipient cell. The human immunoglobulin variable region is then introduced into the corresponding region 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 prepare chimeric mice. Subsequent breeding can be performed to obtain mice containing complete humanized immunoglobulin loci.

[0277] Several other techniques can be used to produce genetically modified animals, including, for example, non-homologous end joining (NHEJ), homologous recombination (HR), zinc finger nucleases (ZFNs), nucleases (TALENs) based on transcription activator-like effectors, and regular clustered short palindromic repeats (CRISPR)-Cas systems. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to produce genetically modified animals. Many gene editing techniques in these genome editing techniques are known in the art, and are described in, for example, Yin et al., "Delivery technologies for genome editing," Nature Reviews Drug Discovery 16.6 (2017): 387-399, which is incorporated herein by reference in its entirety. Many other methods are also provided, which can be used for genome editing, for example, by microinjecting a genetically modified nucleus into an enucleated oocyte, and fusing the enucleated oocyte with another genetically modified cell.

[0278] The genetic modification process may include replacing endogenous sequences with human sequences by homologous recombination. In some embodiments, cutting upstream and downstream of the target site (e.g., by zinc finger nucleases, TALENs or CRISPR) may result in double-stranded DNA breaks, and homologous recombination is used to replace endogenous sequences with human sequences.

[0279] In some embodiments, the method of deleting the CH1 sequence in the IGHG gene includes one or a combination of the following methods. These modifications can be performed in various cells. In some embodiments, the cell is a stem cell, an embryonic stem cell, or a fertilized egg cell.

[0280] In some embodiments, the sequence from the Sγ3 switch region to Cε is deleted, and then a sequence including the Sγ1 switch region and the Cγ1 sequence without CH1 (abbreviated as Cγ1ΔCH1) is inserted. As a result, Cγ1ΔCH1 is operably linked to Sγ1.

[0281] In some embodiments, a sequence comprising the Sγ1 switch region and the Cγ1 sequence without CH1 (abbreviated as Cγ1ΔCH1) is used to directly replace all sequences starting from the Sγ3 switch region to Cε ( Figure 6 ). As a result, Cγ1ΔCH1 and Sγ1 are operably linked.

[0282] In some embodiments, the Sγ3 switch region and the Cγ3 sequence are first knocked out, and then the Cγ1 sequence without CH1 (abbreviated as Cγ1ΔCH1) is used to replace all sequences from Cγ1 to Cε. As a result, Cγ1ΔCH1 is operably linked to Sγ1.

[0283] In some embodiments, the Cγ1 sequence without CH1 (abbreviated as Cγ1ΔCH1) is used to directly replace all sequences starting from Cγ3 to Cε ( Figure 7 ). As a result, Cγ1ΔCH1 and Sγ3 are operably linked.

[0284] In some embodiments, based on Figure 6 The allele shown here contains sequences including Cμ and Cδ ( Figure 8 ). As a result, both IGHM and IGHδ genes were knocked out, and Cγ1ΔCH1 and Sγ1 were operably linked.

[0285] In some embodiments, based on Figure 6 The allele shown here knocks out the sequences including Cμ, Cδ and Sγ1 ( Fig. 9 ). As a result, both IGHM and IGHδ genes were knocked out, and Cγ1ΔCH1′ was operably linked to Sμ.

[0286] In some embodiments, based on Figure 6In the indicated alleles, the CH1 coding region of Cμ was knocked out ( Fig.10 ). As a result, the modified locus includes sequences encoding IgM, IgD and IgG1 lacking the CH1 domain. In addition, Cγ1ΔCH1 is operably linked to Sγ1.

[0287] In some embodiments, based on Figure 6 The allele shown, the Cμ sequence without the CH1 coding region (abbreviated as CμΔCH1) was used to direct the replacement of the sequence containing Cμ and Cδ ( Fig.11 ). As a result, the modified locus includes sequences encoding IgM lacking the CH1 domain and IgG1 lacking the CH1 domain. In addition, Cγ1ΔCH1 is operably linked to Sγ1.

[0288] In some embodiments, based on Figure 6 The allele shown was directed to replace the sequence including Cμ and Cδ with a sequence including CμΔCH1 and Cδ without the CH1 coding region (abbreviated as CδΔCH1) Fig.12 ). As a result, the modified locus includes sequences encoding IgM lacking a CH1 domain, IgD lacking a CH1 domain, and IgG1 lacking a CH1 domain. In addition, Cγ1ΔCH1 is operably linked to Sγ1.

[0289] In some embodiments, provided herein are genetically modified non-human animals comprising a modified immunoglobulin heavy chain constant region locus. In some embodiments, the modified immunoglobulin heavy chain constant region locus comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of Cγ1ΔCH1, Cγ1ΔCH1', CμΔCH1 or CδΔCH1. In some embodiments, the sequence of Cγ1ΔCH1' comprises at least 1, at least 2, at least 3 or at least 4 nucleotides deleted at the 5' end of the Cγ1ΔCH1 (SEQ ID NO: 1) sequence.

[0290] The present invention also relates to a genetically modified non-human animal comprising a nucleic acid sequence, wherein the nucleic acid sequence may be selected from:

[0291] a) a nucleic acid sequence shown in SEQ ID NO: 1, 8, 9, 10, 13 or 41;

[0292] b) a nucleic acid sequence that can hybridize to the nucleotide sequence shown in SEQ ID NO: 1, 8, 9, 10, 13 or 41 under low stringency conditions or high stringency conditions;

[0293] c) a nucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, the nucleotide sequence shown in SEQ ID NO: 1, 8, 9, 10, 13 or 41; and

[0294] d) a nucleic acid sequence encoding an amino acid sequence, wherein the amino acid sequence has at least 90% homology or at least 90% identity with the amino acid sequence of endogenous IgG, IgM, IgD or IgA.

[0295] The method also includes transplanting the genetically modified cells into the fallopian tube or uterus of a recipient female non-human mammal, allowing the cells to develop in 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.

[0296] In some embodiments, the method for preparing a genetically modified humanized animal may also include a step of replacing a nucleic acid (e.g., V, D, J region or V, J region) with a corresponding region of a human sequence at an endogenous locus (or site). The sequence may include a region (e.g., a partial 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.

[0297] 5' homology arm and / or 3' homology arm can have desired length to promote homologous recombination.In some embodiments, homology arm is about or at least 1,2,3,4,5,6,7,8,9,10,20,30,40 or 50kb (for example, about 3kb).In some embodiments, homology arm is less than 1,2,3,4,5,6,7,8,9,10,20,30,40 or 50kb.

[0298] 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.).

[0299] 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.

[0300] The present invention also provides a method for establishing a humanized animal model, which comprises the following steps:

[0301] (a) providing a cell (e.g., a fertilized egg cell) according to the method described herein;

[0302] (b) culturing the cells in a liquid culture medium;

[0303] (c) transplanting the cultured cells into the fallopian tube or uterus of a recipient female non-human mammal, allowing the cells to develop in the uterus of the female non-human mammal;

[0304] (d) identifying germline transmission of the genetically modified humanized non-human mammal in offspring of the pregnant female of step (c).

[0305] In some embodiments, the non-human mammal in the foregoing methods is a mouse (eg, a C57 mouse, a BALB / c mouse, or a C57BL / 6 mouse).

[0306] In some embodiments, the non-human mammal in step (c) is a pseudopregnant (or pseudo-pregnant) female animal.

[0307] In some embodiments, the fertilized eggs used in the above methods are C57BL / 6 fertilized eggs. Other fertilized eggs that can also 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.

[0308] The fertilized egg can be from any non-human animal, such as any non-human animal 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 DNA microinjection. For example, by culturing the fertilized egg after microinjection, the cultured fertilized egg can be transferred to a pseudopregnant non-human animal, and then the non-human animal produces a non-human mammal, thereby producing the non-human mammal mentioned in the above method.

[0309] Also provided are cells, tissues, and animals (eg, mice) comprising the nucleotide sequences described herein, as well as cells, tissues, and animals (eg, mice) expressing humanized or chimeric antibodies from endogenous non-human loci.

[0310] The present disclosure also provides various targeting vectors (e.g., vectors that can be used to prepare genetically modified animals). In some embodiments, the vector may include: a) a DNA fragment homologous to the 5' end of the region to be changed (5' homology arm); b) a sequence comprising a desired genetic element (e.g., loxP recognition site, drug resistance gene and / or reporter gene, etc.); and c) a second DNA fragment homologous to the 3' end of the region to be changed (3' homology arm). The present disclosure also relates to cells comprising a targeting vector as described herein.

[0311] In some embodiments, the gene in the cell is heterozygous. In some embodiments, the gene in the cell is homozygous.

[0312] In some embodiments, the non-human mammal cell is a mouse cell.In some embodiments, the cell is a fertilized egg cell.

[0313] The present invention also provides a nucleic acid sequence having 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% identity to any of the nucleotide sequences described herein, and an amino acid sequence having 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% identity to any of the amino acid sequences described herein.

[0314] 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,

[0315] 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

[0316] In some embodiments, the disclosure relates to a nucleotide sequence encoding any peptide described herein, or any amino acid sequence encoded by any nucleotide sequence 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.

[0317] 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.

[0318] 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.

[0319] In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (e.g., for optimal alignment, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences may be ignored for the purpose of comparison). The length of the reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, in some embodiments at least 90%, 95%, or 100%. 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 at that position are identical (as used herein, "identity" of amino acids or nucleic acids is equivalent to "homology" of amino acids or nucleic acids). Taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences. For purposes of the present invention, comparison of sequences and determination of percent identity between two sequences can be accomplished using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0320] The percentage (homology percentage) of conservative residues (e.g., leucine and isoleucine) with similar physicochemical properties 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), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In many cases, the homology percentage is higher than the identity percentage. Thus, the disclosure also provides amino acid sequences having 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% homology to any of the amino acid sequences described herein, or nucleic acids encoding these amino acid sequences.

[0321] Methods of using genetically modified animals

[0322] Genetically modified animals can be used to produce heavy chain antibodies that can specifically bind to a target. In some embodiments, the target (e.g., a protein or protein fragment) is used as an immunogen to produce antibodies in these animals using standard techniques for polyclonal and monoclonal antibody preparation. In some embodiments, the genetically modified animal is exposed to a selected antigen for a period of time under conditions that allow the animal to produce antigen-specific antibodies.

[0323] Polyclonal antibodies can be produced in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of antigenic peptides or proteins. In some embodiments, the antigenic peptides or proteins are injected together with at least one adjuvant. In some embodiments, the antigenic peptides or proteins can be conjugated with an agent that is immunogenic in the species to be immunized. Animals can be injected with antigenic peptides or proteins more than once (e.g., twice, three times, or four times).

[0324] The full-length polypeptide or protein can be used, or, alternatively, an antigenic peptide fragment thereof can be used as an immunogen. The antigenic peptide of the protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence and contains an epitope of the protein, such that antibodies generated against the peptide form specific immune complexes with the protein.

[0325] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a genetically modified animal as described herein). Suitable immunogenic preparations may include, for example, recombinantly expressed or chemically synthesized polypeptides (e.g., fragments of proteins). The preparations may also include an adjuvant, such as complete or incomplete Freund's adjuvant, or a similar immunostimulant.

[0326] Antibody titers in immunized subjects can be monitored over time by standard techniques, such as enzyme-linked immunosorbent assay (ELISA) using fixed polypeptides or peptides. If necessary, antibody molecules can be separated from mammals (e.g., from blood), and further purified by known techniques (such as protein A of protein G chromatography) to obtain IgG components. At the appropriate time after immunization, for example, when the specific antibody titer is the highest, cells producing antibodies can be obtained from subjects, and by standard techniques such as hybridoma techniques described initially by Kohler et al. (Nature 256: 495-497, 1975), human B cell hybridoma techniques (Kozbor et al., Immunol. Today 4: 72, 1983), EBV-hybridoma techniques (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 77-96 pages, 1985) or tri-source hybridoma techniques are used to prepare monoclonal antibodies. Techniques for producing hybridomas are well known (see generally, Current Protocols in Immunology, 1994, Coligan et al. (eds.), John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing monoclonal antibodies are detected by, for example, screening hybridoma culture supernatants for antibodies that bind to the polypeptide or epitope of interest using a standard ELISA assay.

[0327] On the one hand, the disclosure provides a mouse comprising a modification of an endogenous immunoglobulin heavy chain locus, wherein the mouse produces a B cell comprising a rearranged immunoglobulin sequence operably connected to a heavy chain constant region gene sequence. In some embodiments, the rearranged immunoglobulin sequence operably connected 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 CH1, hinge, CH2, CH3 and a combination thereof.

[0328] On the one hand, the present invention relates to a method for preparing a somatically mutated heavy chain antibody in an animal. The method includes immunizing an animal with an antigen, maintaining the animal under conditions sufficient to initiate an immune response to the antigen; and, isolating a somatically mutated heavy chain antibody from the animal, comprising a variable region gene fragment derived from the human or endogenous heavy chain immunoglobulin variable region gene fragment, and wherein the somatically mutated heavy chain antibody specifically binds to the antigen. In some embodiments, the animal comprises an unrearranged human or endogenous heavy chain immunoglobulin variable region gene fragment, and wherein the animal lacks a nucleotide sequence encoding at least one allele of a functional IgG CH1 domain, and wherein the animal expresses an IgM comprising a CH1 domain.

[0329] The mouse B cell or splenocyte may comprise, for example, a rearranged non-mouse immunoglobulin variable gene sequence operably linked to a mouse immunoglobulin constant region gene. Determine the sequence encoding the human heavy chain variable region and the human light chain variable region. The sequence may be determined by, for example, sequencing the target hybridoma or B cell. In some embodiments, a single B cell is used for screening. It can screen natural antibody pedigrees without hybridoma fusion and combination display. For example, B cells can be mixed with a small group of DNA bar-coded antigens so that one or more antigen barcodes and B cell receptor (BCR) sequences of a single B cell can be recovered by a single cell sequencing scheme.

[0330] The antibody can be further modified to obtain a humanized or human antibody by, for example, operably linking a sequence encoding a human heavy chain variable region to a sequence encoding a human heavy chain constant region.

[0331] In some embodiments, if the mouse expresses a protein that is very similar to the target antigen, it may be difficult to induce an immune response in the mouse. This is because during the development of immune cells, B cells and T cells that recognize MHC molecules bound to self-derived peptides are deleted from the lineage of immune cells. In these cases, humanized mice can be further modified. The corresponding gene in the mouse can be knocked out, and then the mouse is exposed to the target antigen. Because the mouse does not perform negative selection on the gene product, the mouse can produce antibodies that are easy to specifically bind to the target.

[0332] The present disclosure also provides methods for preparing antibodies, nucleic acids, cells, tissues (e.g., spleen tissue). In some embodiments, the method includes exposing an animal as described herein to an antigen. Antibodies (e.g., chimeric antibodies), nucleic acids encoding antibodies, cells and / or tissues (e.g., spleen tissue) can be obtained from animals. In some embodiments, nucleic acids encoding human heavy chain and light chain immunoglobulin variable regions can be determined, for example, by sequencing. In some embodiments, nucleic acids encoding human heavy chain immunoglobulin variable regions can be operably connected to nucleic acids encoding human heavy chain immunoglobulin constant regions. In some embodiments, nucleic acids encoding human light chain immunoglobulin variable regions can be operably connected to nucleic acids encoding human light chain immunoglobulin constant regions. In some embodiments, cells containing nucleic acids as described herein are cultivated and antibodies are collected.

[0333] In some embodiments, no mouse immunoglobulin V, D, J genes (e.g., no mouse IGHV, IGHD, IGHJ, IGKV or IGKJ genes) contribute to the heavy chain variable region sequence. In some embodiments, the heavy chain variable region sequence produced by the animal is completely human and is completely provided by human immunoglobulin V, D, J genes (e.g., human IGHV, IGHD, IGHJ, IGKV and IGKJ genes). In some embodiments, the rearranged VDJ sequence can further undergo somatic hypermutation.

[0334] Variants of antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into DNA encoding human, humanized or chimeric antibodies or antigen-binding fragments thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence constituting the antigen-binding site or antigen-binding domain of the antibody. In such a population of variants, some antibodies or antigen-binding fragments have increased affinity for the target protein. Any combination of deletions, insertions and / or combinations can be performed to obtain antibodies or antigen-binding fragments thereof with increased binding affinity for the target. Amino acid changes introduced into antibodies or antigen-binding fragments can also change or introduce new post-translational modifications into antibodies or antigen-binding fragments, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation sites (e.g., so that different sugars are connected to glycosylation sites under the action of intracellular enzymes), or introducing new glycosylation sites.

[0335] The antibodies found herein can be from any animal species, including mammals. Non-limiting examples of natural antibodies include antibodies from humans, primates (e.g., monkeys and monkeys), cattle, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits) (including transgenic rodents that are genetically engineered to produce human antibodies).

[0336] Human antibodies and humanized antibodies include antibodies having variable and constant regions derived from human germline immunoglobulin sequences or having the same amino acid sequence as the variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may, for example, include amino acid residues not encoded by human germline immunoglobulin sequences in CDR (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutations in vivo).

[0337] The antibody or antigen binding fragment may be further modified. For example, one or more cysteine ​​residues may be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The homodimeric antibodies thus produced may have any extended half-life in vitro and / or in vivo. Homodimeric antibodies with extended half-life in vitro and / or in vivo may also be prepared using heterobifunctional cross-linking agents, and the method for preparing antibodies with heterobifunctional cross-linking agents is described in, for example, Wolff et al. (Cancer Res. 53: 2560-2565, 1993). Alternatively, the antibody may be engineered to have dual Fc regions (see, for example, Stevenson et al., Anti-Cancer Drug Design 3: 219-230, 1989).

[0338] In some embodiments, the antibody or its antigen-binding fragment can be covalently modified. These covalent modifications can be produced 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 the target amino acid residues of the antibody or antibody fragment with an organic derivatizing agent that is capable of reacting with selected side chains or N or C terminal residues.

[0339] Transferrin receptor 1 (TFR1)

[0340] Transferrin receptor 1 (TFR1), also known as cluster of differentiation 71 (CD71), is widely expressed and binds to transferrin (Tf) with high affinity. Human TFR1 is a 90 kDa type II transmembrane protein composed of 760 amino acids that exists as a disulfide-linked dimer (180 kDa) on the cell surface. The TFR1 monomer consists of a large extracellular C-terminal domain of 671 amino acids containing the Tf binding site, a transmembrane region (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, which are required for the receptor to fully function.

[0341] Transferrin (Tf) is an 80 kDa glycoprotein composed of two 40 kDa subunits, called the N- and C-domains, separated by a short linker sequence. Each subunit is able to bind to one free ferric iron (Fe 3+ ) and thus, Tf can bind up to two iron atoms. Tf in its iron-free form, apo-Tf, binds Fe efficiently in the blood. 3+ As a membrane protein that regulates iron import, TFR1 is a member of the TFR family that interacts with Fe 3+The bound transferrin (Tf) exhibits nanomolar affinity. The Tf-TFR1 complex is internalized via clathrin-mediated endocytosis, and when the pH drops to 5.5, Fe 3+ Dissociate from Tf. At this pH, Apo-Tf and TFR1 remain bound and are recycled to the cell surface with physiological pH, so the former is released.

[0342] Transferrin receptor uptake of iron is an important pathway for tumor cells to take up iron. There is increasing evidence that TFR1 is involved in the occurrence and development of tumors, and its expression is significantly dysregulated in many tumors. The relationship between TFR1 and cancer has been revealed, making TFR1 a valuable drug target for cancer intervention.

[0343] TFR1, expressed on endothelial cells of the blood-brain barrier, is also used in preclinical studies to allow delivery of macromolecules, including antibodies, into the brain. TFR1-targeting antibodies can cross the blood-brain barrier without interfering with iron uptake.

[0344] A detailed description of TFR1, Tf and their functions can be found in, for example, 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 newsight for cancer therapy." American Journal of Cancer Research 8.6 (2018): 916; each of these patents is incorporated by reference in its entirety.

[0345] Heavy chain single variable domain (VHH) antibodies

[0346] Monoclonal antibodies and recombinant antibodies are important tools in medicine and biotechnology. Like all mammals, camelids (e.g., llamas) can produce Y-shaped conventional antibodies (e.g., IgG1) formed by two heavy chains and two light chains bound together with disulfide bonds. However, they also produce two unique IgG subclasses: IgG2 and IgG3, also referred to as heavy chain IgG. These antibodies consist of only two heavy chains that lack the CH1 region, which are called VHH (or nanobodies), and their N-termini still have antigen-binding domains. Conventional Ig requires the combination of the variable regions from heavy and light chains to allow for the high diversity of antigen-antibody interactions. Although the separated heavy and light chains still show this ability, they show very low affinity compared to paired heavy and light chains. The unique feature of heavy chain IgG is the ability of its monomeric antigen-binding region to bind antigens, and its specificity, affinity and especially diversity are comparable to conventional antibodies, without the need to pair with another region. This feature is mainly due to a set of major changes in the amino acid sequence of the variable regions of the two heavy chains, which induce profound conformational changes when compared to conventional Ig. The major substitutions in the variable regions prevent the light chain from binding to the heavy chain, but also prevent the unbound heavy chain from being recycled by the immunoglobulin binding protein.

[0347] The single variable domain of these antibodies (named VHH, sdAb or nanobody) is the minimum antigen binding domain produced by the adaptive immune system. It has been found that the third complementary determining region (CDR3) of the variable region of these antibodies is twice as long as that of conventional antibodies. This leads to an increase in the interaction surface with the antigen and an increase in the diversity of antigen-antibody interactions, which compensates for the absence of light chains. With a long complementary determining region 3 (CDR3), VHH can extend into gaps on proteins that conventional antibodies cannot access, including sites of functional interest, such as the active site of an enzyme or a receptor binding canyon on the surface of a virus.

[0348] Compared to conventional antibodies carrying the variable domains (VH and VL) of conventional antibodies, VHHs offer many other advantages, including higher stability, solubility, expression yield and refolding ability, as well as better in vivo tissue penetration and internalization. In addition, in contrast to the VH domains of conventional antibodies, VHHs do not show an intrinsic tendency to bind to light chains. Since VHHs do not bind to VL domains, it is much easier to reformat VHHs into multispecific (e.g., bispecific antibody) constructs than constructs containing conventional VH-VL pairs or single domains based on VH domains.

[0349] The present invention provides, for example, anti-TFR1 antibodies, modified antibodies thereof, chimeric antibodies thereof, and humanized antibodies thereof.

[0350] The CDR sequences of 23B8 and 23B8-derived antibodies (e.g., humanized antibodies) include CDRs of VHH domains as defined by Kabat numbering, as shown in SEQ ID NOs: 42, 43, and 44, respectively. CDRs can also be defined by the IMGT system. Under IMGT numbering, the CDRs of VHH domains are shown in SEQ ID NOs: 54, 55, and 56, respectively.

[0351] The CDR sequences of 24A1 and 24A1-derived antibodies (e.g., humanized antibodies) include CDRs of VHH domains as defined by Kabat numbering, as shown in SEQ ID NOs: 45, 46, and 47, respectively. CDRs can also be defined by the IMGT system. Under IMGT numbering, the CDRs of VHH domains are shown in SEQ ID NOs: 57, 58, and 59, respectively.

[0352] The CDR sequences of 24C9 and 24C9-derived antibodies (e.g., humanized antibodies) include CDRs of VHH domains defined by Kabat numbering, as shown in SEQ ID NOs: 48, 49, and 50, respectively. CDRs can also be defined by the IMGT system. Under IMGT numbering, the CDRs of VHH domains are shown in SEQ ID NOs: 60, 61, and 62, respectively.

[0353] The CDR sequences of 24G5 and 24G5-derived antibodies (e.g., humanized antibodies) include CDRs of VHH domains as defined by Kabat numbering, as shown in SEQ ID NOs: 51, 52, and 53, respectively. CDRs can also be defined by the IMGT system. Under IMGT numbering, the CDRs of VHH domains are shown in SEQ ID NOs: 63, 64, and 65, respectively.

[0354] 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.

[0355] Various modified or humanized VHH amino acid sequences are also provided. Due to the presence of different ways to modify or humanize heavy chain antibodies (e.g., different amino acids can be used to replace the modified sequence), the VHH domain of the heavy chain antibody can have more than one form of humanized sequence. In some embodiments, the humanized VHH domain has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any sequence of SEQ ID NO: 66-69.

[0356] In addition, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may also contain one, two or three VHH domain CDRs selected from SEQ ID NOs: 42-44, SEQ ID NOs: 45-47, SEQ ID NOs: 48-50, SEQ ID NOs: 51-53, SEQ ID NOs: 54-56, SEQ ID NOs: 57-59, SEQ ID NOs: 60-62, and SEQ ID NOs: 63-65.

[0357] In some embodiments, the antibody may have a heavy chain single variable domain (VHH) comprising complementarity determining regions (CDR) 1, 2, 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. The selected VHH CDR 1, 2, 3 amino acid sequences are as follows. Fig.37 and Fig.38 shown.

[0358] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing 1, 2 or 3 VHH CDR1s with 0, 1 or 2 amino acid insertions, deletions or substitutions; VHH CDR2s with 0, 1 or 2 amino acid insertions, deletions or substitutions; and VHH CDR3s with 0, 1 or 2 amino acid insertions, deletions or substitutions, wherein VHH CDR1, VHH CDR2 and VHH CDR3 are selected from Fig.39 .

[0359] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing 1, 2 or 3 CDRs: 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; SEQ ID NO:44 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0360] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing 1, 2 or 3 CDRs: SEQ ID NO:45 having 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO:46 having 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO:47 having 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0361] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing 1, 2 or 3 CDRs: 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; SEQ ID NO:50 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0362] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing 1, 2 or 3 CDRs: 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; SEQ ID NO:53 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0363] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing 1, 2 or 3 CDRs: SEQ ID NO:54 having 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO:55 having 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO:56 having 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0364] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing 1, 2 or 3 CDRs: 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; SEQ ID NO:59 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0365] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing 1, 2 or 3 CDRs: 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; SEQ ID NO:62 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0366] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain single variable domain (VHH) containing 1, 2 or 3 CDRs: SEQ ID NO:63 having 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO:64 having 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO:65 having 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0367] Insertions, deletions and substitutions can be within a CDR sequence, or at one or both ends of a CDR sequence. In some embodiments, CDRs are determined based on the Kabat numbering scheme. In some embodiments, CDRs are determined based on the Chothia numbering scheme. In some embodiments, CDRs are determined based on a combined numbering scheme. In some embodiments, CDRs are determined based on the IMGT numbering scheme.

[0368] The present invention also provides an antibody or antigen-binding fragment thereof that binds to TFR1 (human TFR1). The antibody or antigen-binding fragment thereof contains a heavy chain single variable region (VHH), and the VHH comprises or consists 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.

[0369] In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, these sequences are compared for the purpose of optimal comparison (e.g., a gap can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal comparison, and non-homologous sequences can be ignored for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the 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 (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). Taking into account the number of gaps that need to be introduced to achieve the best comparison of the two sequences and the length of each gap, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences. For example, the comparison of sequences and the determination of the percent identity between the two sequences can be completed using the Blossum 62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0370] The present disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides comprising immunoglobulin heavy chain single variable domains (VHH). VHHs include, for example, Fig.37 and Fig.38 The CDRs shown, or having Fig.39 sequence shown.

[0371] Antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multispecific (e.g., bispecific) antibodies or antibody fragments. Other antibodies provided herein are polyclonal antibodies, monoclonal antibodies, multispecific (multimer, e.g., bispecific) antibodies, human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, antibodies prepared in cells (i.e., intrabodies) and antigen-binding fragments thereof.

[0372] In some embodiments, the antibody or its antigen-binding fragment comprises an Fc domain, which can be derived from various types (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), categories (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclasses. In some embodiments, the Fc domain is derived from an IgG antibody or its antigen-binding fragment. In some embodiments, the Fc domain comprises one, two, three, four, or more heavy chain constant regions.

[0373] The present invention also provides antibodies or their antigen binding fragments that cross-compete with any antibody or antigen binding fragment described herein. Cross-competition analysis is known in the art and is described in, for example, Moore et al., "Antibody Cross-Competition Analysis of the Human Immunodeficiency Virus type 1gp120exterior envelope glycoprotein." Journal of Virology 70.3 (1996): 1863-1872, which is incorporated herein by reference in its entirety. On the one hand, the present invention also provides antibodies or their antigen binding fragments that bind to the same epitope or region as any antibody or antigen binding fragment described herein. Epitope combination analysis is known in the art and is described in, for example, Estep et al., "High throughput solution-based measurement ofantibody-antigen affinity and epitope binning". MAbs. Vol. 5. No. 2. Taylor & Francis, 2013, which is incorporated herein by reference in its entirety.

[0374] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR1 selected from SEQ ID NO: 42, 45, 48, 51, 54, 57, 60 and 63.

[0375] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR2 selected from SEQ ID NO: 43, 46, 49, 52, 55, 58, 61 and 64.

[0376] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain single variable domain (VHH) CDR3 selected from SEQ ID NO: 44, 47, 50, 53, 56, 59, 62, and 65.

[0377] Antibody characteristics

[0378] TFR1 plays a key role in cellular iron uptake through its interaction with iron-binding TFs. Iron is required for a variety of cellular processes and is essential for DNA synthesis and cell proliferation. Due to its central role in cancer cell pathology, malignant cells often overexpress TFR1, and this increased expression may be associated with poor prognosis in different types of cancer. The increased expression levels of TFR1 on malignant cells, combined with its extracellular accessibility, internalization capacity, and central role in cancer cell pathology, make this receptor an attractive target for antibody-mediated therapy.

[0379] In some embodiments, the antibodies or antigen-binding fragments thereof described herein cannot block the binding between TFR1 and TF. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can block the binding between TFR1 and TF. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can be conjugated to anticancer agents that are internalized by receptor-mediated endocytosis. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can disrupt the function of the receptor. In some embodiments, the antibodies or antigen-binding fragments thereof described herein cannot induce Fc effector functions, thereby preventing or ameliorating their negative effects on normal cells.

[0380] The present disclosure provides antibodies or antigen-binding fragments thereof comprising a human Fc domain, which can induce an enhancement of Fc-dependent effector function by 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 the antibody or antigen-binding fragment thereof described in the present invention.

[0381] The present disclosure provides antibodies or antigen-binding fragments thereof comprising a human Fc domain, which induce an enhanced host immune response by 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 the antibody or antigen-binding fragment thereof described in the present invention.

[0382] The present disclosure provides antibodies or antigen-binding fragments thereof that can be internalized into human brain cells (e.g., cortical microvascular endothelial cells) with an internalization 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 internalization 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 compared to an isotype control antibody.

[0383] In some embodiments, antibodies or antigen-binding fragments thereof comprising a single heavy chain are provided herein. In some embodiments, antibodies or antigen-binding fragments thereof comprising a pair of heavy chains are provided herein. In some embodiments, the heavy chain pairs are connected by disulfide bonds. In some embodiments, the heavy chain pairs include knob-and-hole modifications. In some embodiments, the heavy chain comprises the Fc domain of human IgG. 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 to the same epitope. In some embodiments, the VHH domains in each heavy chain specifically bind to different epitopes. In some embodiments, the VHH domains in each heavy chain bind to at least 1, 2, 3, 4 or 5 different epitopes.

[0384] 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 4, 5 or 6 antigens.

[0385] In some embodiments, the antibody (or antigen-binding fragment thereof) is induced at a rate of less than 0.1 s -1 Less than 0.01s -1 , less than 0.001s -1 , less than 0.0001s -1 or less than 0.00001s -1 In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001s -1 , greater than 0.0001s -1 , greater than 0.00001s -1 or greater than 0.000001s -1 .

[0386] In some embodiments, the kinetic binding rate (kon) is greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic binding rate (kon) is less than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0387] Affinity can be derived from the quotient of the kinetic rate constants (KD = koff / kon). In some embodiments, KD is less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M or less than 1×10 -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 greater than 1×10 -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M, greater than 1×10 -10 M, greater than 1×10 -11 M or greater than 1×10 -12 M.

[0388] Techniques for measuring the affinity of an antibody for 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.

[0389] In some embodiments, the antibody does not bind human TFR1, monkey TFR1, mouse TFR1, or chimeric TFR1.

[0390] In some embodiments, thermal stability is determined. The antibodies or antigen-binding fragments described herein can have a thermal stability 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 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135 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.

[0391] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.

[0392] 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.

[0393] In some embodiments, the antibody or antigen-binding fragment does not have an Fc region. For example, the antibody (or its antigen-binding fragment) is a polypeptide comprising one or more VHH domains interconnected by a connecting peptide. 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.

[0394] In some embodiments, the antibody or its antigen-binding fragment 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, according to EU numbering, the Fc region has a mutation (e.g., N297A) at position 297. In some embodiments, the mutated human IgG1 Fc region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 76.

[0395] In some embodiments, the concentration of an antibody or antigen-binding fragment thereof described herein in the brain (e.g., whole brain or brain parenchyma) can be greater than 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of its concentration immediately after administration (e.g., 0.5 hour) 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours after administration of the drug to a subject. In some embodiments, the concentration of the antibody or antigen-binding fragment thereof described herein in the brain (e.g., whole brain or brain 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 higher than the concentration of a control antibody (e.g., hIgG1 or JR141-N) or the concentration in the serum of the subject 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours after administration of the drug to the subject.

[0396] Method for preparing anti-TFR1 antibodies

[0397] Variants of antibodies or antigen-binding fragments described herein can be prepared by introducing suitable nucleotide variants into DNA encoding human, humanized or chimeric antibodies or their antigen-binding fragments, or by peptide synthesis. Such variants include, for example, deletions, insertions or replacements of residues in the amino acid sequence constituting the antigen-binding site or antigen-binding domain of the antibody. In the population of these variants, some antibodies or antigen-binding fragments enhance affinity for target proteins (e.g., TFR1). Any combination of deletions, insertions and / or combinations can be performed to obtain antibodies or their antigen-binding fragments with higher binding affinity to the target. Amino acid changes introduced into antibodies or antigen-binding fragments can also change or introduce new post-translational modifications into antibodies or antigen-binding fragments, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation sites (e.g., changing the amino acid sequence so that different sugars are connected by enzymes present in the cell), or introducing new glycosylation sites. In some embodiments, the heavy chain antibodies or their antigen-binding fragments described herein are obtained by immunizing any genetically modified animals described herein (e.g., heavy chain mutant allele 3 genotype homozygous mice).

[0398] Humanized antibodies include antibodies with variable and constant regions of human germline immunoglobulin sequences derived from human immunoglobulin scaffold sequences (or with amino acid sequences identical to sequences derived therefrom). 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 mutations in vivo). Therefore, "humanized" antibodies are chimeric antibodies in which sequences from non-human species are replaced by corresponding human sequences.

[0399] Typically, the amino acid sequence variants of a human, humanized or chimeric anti-TFRl antibody will contain an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence present in the VHH domain of the original antibody.

[0400] Identity or homology to a native sequence is typically the percentage of amino acid residues present in a candidate sequence that are identical to the sequence 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, and not considering any conservative substitutions as part of the sequence identity.

[0401] The antibody or antigen binding fragment may be further modified. For example, one or more cysteine ​​residues may be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The homodimeric antibodies thus produced may have any increased in vitro and / or in vivo half-life. Homodimeric antibodies with increased in vitro and / or in vivo half-life may also be prepared using heterobifunctional cross-linkers as described, for example, by Wolff et al. (Cancer Res. 53: 2560-2565, 1993). Alternatively, an antibody having two Fc regions may be engineered.

[0402] In some embodiments, anti-TFR1 antibodies or antigen-binding fragments thereof may be covalently modified. These covalent modifications may be performed by chemical or enzymatic synthesis or by enzymatic cleavage or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting the targeted amino acid residues of the antibody or fragment with an organic derivatizing agent that is capable of reacting with selected side chains or N-terminal or C-terminal residues.

[0403] In some embodiments, antibody variants are provided, which have a carbohydrate structure lacking fucose (directly or indirectly) connected to the Fc region. For example, the amount of fucose in such antibodies 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 sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) connected to Asn297, as measured by MALDI-TOF mass spectrometry (e.g., as described in WO 2008 / 077546). Asn297 refers to an asparagine residue located at approximately position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering); however, due to minor sequence variations in antibodies, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function.In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody may be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0404] The present disclosure also provides a recombinant vector (e.g., an expression vector) comprising an isolated polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein), a host cell into which the recombinant vector has been introduced (i.e., such that the host cell contains the polynucleotide and / or a vector comprising the polynucleotide), and production of a recombinant antibody polypeptide or fragment thereof by recombinant technology.

[0405] As used herein, "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. "Expression vector" can deliver one or more polynucleotides of interest and express them as encoded polypeptides in a host cell that has been introduced into the expression vector. Therefore, in the expression vector, by being operably connected with a regulatory element (such as a promoter, enhancer and / or poly-A tail) at or near or on both sides of the integration site of the polynucleotide of interest in the vector or in the genome of the host cell, the polynucleotide of interest is positioned in the vector for expression so that the polynucleotide of interest will be translated in the host cell introduced into the expression vector.

[0406] The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection and infection and / or transduction (e.g., with a recombinant virus). Therefore, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0407] 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., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of non-pathogenic (defective), replication-competent viruses, or replication-defective viruses may be used. In the latter case, viral propagation typically occurs only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. NY 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., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisle 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 of ordinary skill in the art. The 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 increased by coating the DNA on biodegradable beads that are efficiently transported into cells.

[0408] For expression, the DNA insert comprising the polynucleotide encoding the antibody or polypeptide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the promoter of bacteriophage λPL, E. coli lac, trp and tac, SV40 early and late promoters, and the promoter of retroviral LTR, etc. Other suitable promoters are known to the skilled person. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. The expression construct may further contain a transcription initiation and termination site, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct may include a translation starting at the start and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0409] As noted, the expression vector may include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culture in Escherichia coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells, such as Escherichia coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells, such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Suitable culture media and conditions for host cells described herein are known in the art.

[0410] 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, 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 apparent to those skilled in the art.

[0411] Suitable non-limiting bacterial promoters include E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, lambda PR and PL promoters and trp promoter. Suitable eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, promoters of retroviral LTRs, such as the promoter of Rous sarcoma virus (RSV), and metallothionein promoters, such as mouse metallothionein-I promoter.

[0412] In Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters are available, such as those for α-factor, alcohol oxidase, and PGH.

[0413] The construct can be introduced into the host cell by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. These methods are 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.

[0414] By inserting an enhancer sequence into the vector, the transcription of the DNA encoding the antibodies of the present disclosure by higher eukaryotes can be increased. An enhancer is a cis-acting element of DNA, usually about 10 to 300 bp, that acts to increase the 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, base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer located on the late side of the replication origin, and adenovirus enhancers.

[0415] In order to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, an appropriate secretion signal can be incorporated into the expressed polypeptide. The signal can be an endogenous signal of the polypeptide or a heterologous signal.

[0416] Polypeptides (e.g., antibodies) can be expressed in a modified form, such as a fusion protein (e.g., GST fusion) or with a histidine tag, and can include not only a secretion signal, but also additional heterologous functional regions. For example, during purification or during subsequent processing and storage, additional amino acids, particularly regions of charged amino acids, can be added to the N-terminus of the polypeptide to improve stability and durability in the host cell. In addition, peptide moieties can be added to the polypeptide to promote purification. Such regions can be removed before the final preparation of the polypeptide. Adding peptide moieties to the polypeptide to produce secretion or excretion, improve stability, and promote purification, etc., is a conventional technique familiar to the art.

[0417] The present invention also provides a nucleic acid sequence, which has 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% similarity to any nucleotide sequence described herein. The invention relates to any nucleotide sequence encoding any peptide described herein, and the amino acid sequence has 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% identity with any amino acid sequence described herein. In some embodiments, the present disclosure relates to a nucleotide sequence encoding any peptide described herein, or any amino acid sequence encoded by any nucleotide sequence 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.

[0418] 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.

[0419] 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.

[0420] In some embodiments, the antibody or antigen-binding fragment thereof is expressed in yeast, insect cells, or mammalian cells (eg, CHO cells).

[0421] Treatment and diagnostic methods

[0422] The anti-TFR1 antibodies or antigen-binding fragments thereof of the present invention can be used for various therapeutic purposes. On the one hand, the present disclosure provides a method for treating a brain disease (e.g., brain cancer, dementia, or Alzheimer's disease) in a subject, a method for identifying a subject suffering from a brain disease (e.g., brain cancer, dementia, or Alzheimer's disease), a method for reducing the risk of brain disease development, or a method for reducing the risk of developing other symptoms in a subject. In some embodiments, treatment can stop, slow, delay, or inhibit the progression of a brain disease (e.g., brain cancer, dementia, or Alzheimer's disease). In some embodiments, treatment can result in a reduction in 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.

[0423] In one aspect, the disclosure features methods including administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (eg, a subject having, identified or diagnosed as having, a brain disease).

[0424] In one aspect, the disclosure features methods of carrying 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.

[0425] In some embodiments, the compositions and methods disclosed herein can be used to treat patients with risk of brain diseases (e.g., brain cancer, dementia, or Alzheimer's disease). Patients suffering from brain diseases (e.g., brain cancer, dementia, or Alzheimer's disease) can be identified using various methods known in the art.

[0426] In some embodiments, the brain disease is brain cancer.

[0427] In one aspect, the present disclosure relates to a method of reducing tumor growth rate, comprising contacting a tumor cell with an effective amount of a composition comprising an antibody or antigen binding fragment thereof, or an antibody-drug conjugate as described herein. In one aspect, the present disclosure relates to a method of killing a tumor cell, comprising contacting a tumor cell with an effective amount of a composition comprising an antibody or antigen binding fragment thereof, or an antibody-drug conjugate as described herein.

[0428] As used herein, "effective amount" refers to an amount or dosage sufficient to achieve beneficial or desired results, including terminating, slowing, delaying 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, antibody drug conjugate, antibody encoding polynucleotide, vector comprising the polynucleotide and / or a composition thereof is to be administered, the severity of the symptoms, and the route of administration, and therefore can be determined on an individual basis.

[0429] The effective amount can be applied in one or more administrations. For example, the effective amount of an antibody, an Fab or an antibody drug conjugate is enough to improve, terminate, stabilize, reverse, inhibit, slow down and / or delay the amount of the progress of an autoimmune disease or cancer in a patient, or is enough to improve, terminate, stabilize, reverse, slow down and / or delay the amount of a cell (e.g., a biopsy cell, any one of the cancer cells described herein or a cell line (e.g., a cancer cell line)) in vitro propagation. As understood in the art, the effective amount of an antibody, an Fab or an antibody drug conjugate can be based on, especially the patient's medical history and other factors (such as the type (and / or dosage) of the antibody used) change.

[0430] The effective amount and dosing regimen of the antibodies, polynucleotides encoding the antibodies, antibody drug conjugates and / or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of the art. It will be appreciated by those skilled in the art that the dosage that must be administered will vary depending on, for example, the mammal to which the antibodies, polynucleotides encoding the antibodies, antibody drug conjugates and / or compositions disclosed herein will be administered, the route of administration, the specific type of antibodies, polynucleotides encoding the antibodies, antigen-binding fragments, antibody drug conjugates and / or compositions disclosed herein used, and other drugs administered to the mammal. Guidance for selecting appropriate doses of antibodies or antigen-binding fragments can be found in the literature on the therapeutic use of antibodies and antigen-binding fragments, such as 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.

[0431] The typical daily dosage of an effective amount of an antibody is 0.01 mg / kg to 100 mg / kg. In some embodiments, the dosage may 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 dosage may 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 dosage 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.

[0432] In any of the methods described herein, at least one antibody, antigen binding fragment thereof, or pharmaceutical composition (e.g., any antibody, antigen binding fragment, or pharmaceutical composition described herein) and optionally at least one additional therapeutic agent can be administered 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 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 in the form of a sustained-release oral formulation.

[0433] In some embodiments, one or more additional therapeutic agents may be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, antibody drug conjugate, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, antibody drug conjugate, 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 biological activity period of the one or more additional therapeutic agents and at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.

[0434] In some embodiments, 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) may be administered to a subject over an extended period of time (e.g., over 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, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or tracking the effectiveness of treatment (e.g., observing at least one symptom of the disease). As described herein, a skilled medical professional may also change the type and amount (e.g., increase or decrease) of the antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject, and may also adjust the dose or frequency of administration of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject based on an assessment of the effectiveness of the treatment (e.g., increase or decrease).

[0435] In some embodiments, one or more additional therapeutic agents may be administered to a subject. Additional therapeutic agents may include one or more selected from B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K / mTOR dual inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and inhibitors of isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors.

[0436] In some embodiments, the additional therapeutic agent may include one or more inhibitors selected from the group consisting of: a HER3 inhibitor, a LSD1 inhibitor, a MDM2 inhibitor, a BCL2 inhibitor, a CHK1 inhibitor, an inhibitor of activated hedgehog signaling pathway, and an agent that selectively degrades estrogen receptors.

[0437] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, zykadia, sutent, temsirolimus, axitinib ), sorafenib, Votrient, IMA-901, AGS-003, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

[0438] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the following: adjuvants, TLR agonists, tumor necrosis factor (TNF) α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, CX3CL1-targeted therapies, CXCL9-targeted therapies, CXCL10-targeted therapies, CCL5-targeted therapies, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0439] In some embodiments, the subject is administered carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0440] 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.

[0441] Pharmaceutical compositions and routes of administration

[0442] Also provided herein are pharmaceutical compositions containing at least one (e.g., one, two, three, or four) antibody or antigen-binding fragment described herein. Two or more (e.g., two, three, or four) of any antibody or antigen-binding fragment described herein may be present in the pharmaceutical composition in any combination. The pharmaceutical composition may be formulated in any manner known in the art.

[0443] Pharmaceutical compositions are formulated to be compatible with their intended routes of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous or peritoneal). Compositions may include sterile diluents (e.g., sterile water or saline), fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, antibacterial or antifungal agents (such as benzyl alcohol or methyl paraben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (such as ascorbic acid or sodium bisulfite), chelating agents (such as ethylenediaminetetraacetic acid), buffers (such as acetate, citrate or phosphate) and isotonic agents (such as sugars (e.g., glucose), polyols (e.g., mannitol or sorbitol) or salts (e.g., sodium chloride)), or any combination thereof. Liposomal suspensions may also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). The preparation of the composition may be formulated and encapsulated in ampoules, disposable syringes or multi-dose vials. When necessary (e.g., in injectable formulations), appropriate fluidity can be maintained, for example, by using a coating (such as lecithin) or a surfactant. Absorption of the antibody or its antigen-binding fragment can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled 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 and Nova Pharmaceuticals).

[0444] 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 dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound to facilitate administration and uniformity of dosage).

[0445] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical methods in cell culture or experimental animals (e.g., monkeys). For example, the LD50 (the dose that causes 50% of the population to be lethal) and the ED50 (the dose that is therapeutically effective in 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. When an agent exhibits adverse side effects, care should be taken to minimize potential damage (i.e., reduce adverse side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0446] The data obtained from cell culture assays and animal studies can be used to formulate the appropriate dose of any given medicament for a subject (e.g., a person). The therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or their antigen-binding fragments (e.g., any antibody or antibody fragment described herein) will be the treatment of the disease in the subject, reducing the severity, frequency, and / or duration of one or more symptoms of the disease in the subject (e.g., a person) of the subject or identified as being at risk of developing the disease. The effectiveness and dosage of any antibody or antigen-binding fragment described herein can be determined by a health care professional or veterinary professional using methods known in the art and by observing one or more disease symptoms of a subject (e.g., a person). Certain factors may affect the dosage and time required for the effective treatment of a subject (e.g., the severity of the disease or illness, previous treatment, the general health status and / or age of the subject, and the presence of other diseases).

[0447] Exemplary dosages include milligram or microgram amounts of any of the antibodies or antigen-binding fragments described 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). Although these dosages cover a wide range, those of ordinary skill in the art will appreciate that the efficacy of therapeutic agents, including antibodies and antigen-binding fragments thereof, varies, and an effective amount can be determined by methods known in the art. Typically, a relatively low dose is first administered, and then the attending health care professional or veterinary professional (in the case of therapeutic applications) or researcher (when still working in the development phase) can then gradually increase the dose until an appropriate response is obtained. Furthermore, it should be understood that the specific dosage level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the age, weight, general health, sex and diet of the subject, the time of administration, the route of administration, the rate of excretion and the in vivo half-life of the antibody or antibody fragment.

[0448] The pharmaceutical composition may be contained in a container, pack or dispenser together with instructions for administration. The present invention also provides methods for preparing antibodies or antigen-binding fragments thereof for the various uses described herein.

[0449] Additional embodiments

[0450] It should be understood that although the invention has been described in conjunction with the detailed description of the invention, this description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the claims. Other aspects, advantages and modifications are within the scope of the claims. Additional embodiments are also provided.

[0451] Embodiment 1 is a genetically modified rodent comprising a modified immunoglobulin heavy chain locus, wherein the modified immunoglobulin heavy chain locus comprises an IgG constant region gene, wherein the IgG constant region gene encodes an IgG heavy chain constant region lacking a CH1 domain, wherein the genetically modified rodent expresses heavy chain antibodies.

[0452] Embodiment 2 is the genetically modified rodent of embodiment 1, wherein said rodent comprises exactly one IgG constant region gene.

[0453] Embodiment 3 is the genetically modified rodent described in embodiment 1 or 2, wherein the IgG heavy chain constant region gene is IGHG1.

[0454] Embodiment 4 is the genetically modified rodent of any one of embodiments 1-3, wherein the IgG heavy chain constant region comprises or consists of a CH2 domain and a CH3 domain and optionally a hinge region.

[0455] 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 at the rodent immunoglobulin heavy chain locus.

[0456] Embodiment 6 is the rodent of embodiment 5, wherein the germline genetic modification further comprises a deletion of the IGHE gene at the rodent immunoglobulin heavy chain locus.

[0457] Embodiment 7 is the rodent of embodiment 5 or 6, wherein the genetic modification further comprises a deletion of Sγ2b, Sγ2c and Sε switch region at the rodent immunoglobulin heavy chain locus.

[0458] Embodiment 8 is the rodent of any one of embodiments 5-7, wherein the modified immunoglobulin heavy chain locus comprises a modified IGHG1 gene lacking a sequence encoding a CH1 domain, wherein the modified IGHG1 gene comprises a sequence that is at least 80%, 90%, 95% or 99% identical to SEQ ID NO:1.

[0459] Embodiment 9 is the rodent of any one of embodiments 5-8, wherein the genetic modification further comprises a deletion of the rodent Sγ3 switch region at the rodent immunoglobulin heavy chain locus.

[0460] Embodiment 10 is the rodent of any one of embodiments 5-9, wherein the genome of the rodent comprises a rodent Sμ, Sγ1, Sα switch region, a modified rodent IGHG1 gene lacking a sequence encoding a CH1 domain, and rodent IGHM, IGHδ, IGHA genes.

[0461] Embodiment 11 is the rodent of any one of embodiments 5-9, wherein the genetic modification further comprises a deletion of the rodent IGHM and IGHδ genes at the rodent immunoglobulin heavy chain locus.

[0462] Embodiment 12 is the rodent of any one of embodiments 5-9 and 11, wherein the rodent genome comprises a rodent Sμ, Sγ1, Sα switch region, a modified IGHG1 gene lacking a sequence encoding a CH1 domain, and a rodent IGHA gene.

[0463] 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.

[0464] Embodiment 14 is the rodent of any one of embodiments 5-9, wherein the genetic modification further comprises a deletion of the CH1 coding sequence of the IGHM gene at the rodent immunoglobulin heavy chain locus.

[0465] Embodiment 15 is the rodent of any one of embodiments 5-9 and 14, wherein the genome of the rodent comprises rodent Sμ, Sγ1, Sα switching regions, a modified rodent IGHM gene lacking a sequence encoding a CH1 domain, a modified IGHG1 gene lacking a sequence encoding a CH1 domain, and rodent IGHδ, IGHA genes.

[0466] Embodiment 16 is the rodent of embodiment 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.

[0467] Embodiment 17 is the rodent of any one of embodiments 5-9, 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 rodent immunoglobulin heavy chain locus.

[0468] Embodiment 18 is the rodent of any one of embodiments 5-9 and 17, wherein the genome of the rodent comprises a rodent Sμ, Sγ1, Sα switch region, a modified IGHM gene lacking a sequence encoding a CH1 domain, a modified IGHG1 gene lacking a sequence encoding a CH1 domain, and a rodent IgHA gene.

[0469] Embodiment 19 is the rodent of any one of embodiments 5-9, 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 rodent immunoglobulin heavy chain locus.

[0470] Embodiment 20 is a rodent of any one of embodiments 5-9 and 19, wherein the genome of the rodent comprises a rodent Sμ, Sγ1, Sα switch region, 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.

[0471] 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.

[0472] Embodiment 22 is the rodent of any one of embodiments 14-21, wherein the modified IGHM gene comprises a sequence that is at least 80%, 90%, 95% or 99% identical to SEQ ID NO:13.

[0473] Embodiment 23 is the rodent of any one of embodiments 5-8, wherein the genetic modification further comprises a deletion of the rodent Sγ1 switch region at the rodent immunoglobulin heavy chain locus.

[0474] Embodiment 24 is the rodent of any one of embodiments 5-8 and 23, wherein the genome of the rodent comprises a rodent Sμ, Sγ3, Sα switch region, a modified IGHG1 gene lacking a sequence encoding a CH1 domain, and rodent IGHM, IGHδ, IGHA genes.

[0475] Embodiment 25 is the rodent of any one of embodiments 5-8 and 23, wherein the genetic modification further comprises a deletion of the rodent Sγ3 switch region at the rodent immunoglobulin heavy chain locus.

[0476] Embodiment 26 is the rodent of any one of embodiments 5-8, 23 and 25, wherein the genetic modification further comprises a deletion of the rodent IGHM and IGHδ genes at the rodent immunoglobulin heavy chain locus.

[0477] Embodiment 27 is the rodent of any one of embodiments 5-8, 23, 25 or 26, wherein the genome of the rodent comprises a rodent Sμ, Sα switch region, a modified IGHG1 gene lacking a sequence encoding a CH1 domain, and a rodent IGHA gene.

[0478] Embodiment 28 is the rodent of embodiment 27, wherein the Sμ switch region and the modified IGHG1 gene are linked by a sequence that is at least 80%, 90%, 95% or 99% identical to SEQ ID NO:9.

[0479] Embodiment 29 is the rodent of any one of embodiments 5-10, 23 and 24, wherein the modified genome comprises a functional IGHM gene. Example

[0480] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0481] Example 1: Overview

[0482] The immunoglobulin heavy chain locus of non-human animals is modified by gene editing. For example, in order to obtain mice that can express heavy chain antibodies, the immunoglobulin heavy chain constant region locus in mouse chromosome 12 is modified. The genetically modified mice can express Figure 1 In addition, all endogenous VDJ sequences in the heavy chain variable region locus on chromosome 12 are replaced by human VDJ sequences, so that the variable region in the heavy chain antibody expressed by the mouse has a completely humanized sequence. In some embodiments, mice with human VDJ sequences are genetically modified in the immunoglobulin heavy chain constant region locus.

[0483] Example 2: Modification of the Mouse Immunoglobulin Heavy Chain Constant Region Locus

[0484] As Figure 2 shown, the mouse (C57BL / 6) immunoglobulin constant region genes include (in the following order): immunoglobulin heavy chain constant region μ (IGHM, or Cμ), immunoglobulin heavy chain constant region δ (IGHδ, or Cδ), immunoglobulin heavy chain constant region γ3 (IGHG3, or Cγ3), immunoglobulin heavy chain constant region γ1 (IGHG1, or Cγ1), immunoglobulin heavy chain constant region γ2b (IGHG2b, or Cγ2b), immunoglobulin heavy chain constant region γ2c (IGHG2c, or Cγ2c), immunoglobulin heavy chain constant region ε (IGHE, or Cε), and immunoglobulin heavy chain constant α (IGHA, or Cα) genes. As Figure 3 shown, the switch regions (such as 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 includes sequences encoding the CH1, H (hinge), CH2, CH3, M1, and M2 regions of IgG1 from the N-terminus to the C-terminus. The mouse immunoglobulin heavy chain constant region locus was modified by various methods discussed below. The experiments were performed in mice with fully humanized VDJ sequences. Details of the VDJ region humanized mice can be found, for example, in WO2020169022A1 and US20200390073A1; the entire contents of each patent are incorporated herein by reference.

[0485] To obtain mice that express only heavy chain antibodies, the Cγ3, Cγ1, Cγ2b, Cγ2c, and Cε gene loci were modified to obtain a truncated Cγ1 sequence (Cγ1ΔCH1) that retains only the region without the CH1 coding region, to obtain Figure 4A the mutant alleles shown as mutant allele 1 or mutant allele 1' in. Based on mutant allele 1, different modifications were made to the switch regions (such as 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' (further lacking Sγ1). Alternatively, the regions to be knocked out were selected from: the CH1 coding region of Cμ (the resulting allele is shown as mutant allele 3); the CH1 coding region of Cμ and the entire Cδ (the resulting allele is shown as mutant allele 4); and the CH1 coding region of Cμ and the CH1 coding region of Cδ (the resulting allele is shown as mutant allele 5).

[0486] Example 3: Construction of Targeting Vectors

[0487] Targeting vectors such as V1, V2, V3, V4, V5, V6, and V7 were used in the modification.

[0488] Vector V1

[0489] like Figure 5 As shown, vector V1 includes a Cγ1ΔCH1 knock-in sequence (SEQ ID NO: 1). The Cγ1ΔCH1 sequence does not have a CH1 coding sequence. The CH1 coding region is deleted or replaced by a Neo cassette. The Neo cassette includes a Neo gene sequence flanked by two Frt (or LoxP) sequences. Flp transgenic mice are mated with mice carrying the Neo cassette to remove the cassette.

[0490] The targeting vector V1 includes the following characteristic sequences in 5' to 3' order: upstream homology arm (5' homology arm), mouse Sγ1 promoter sequence, mouse Sγ1, Cγ1ΔCH1 sequence and downstream homology arm (3' homology arm). The targeting vector may further include an antibiotic resistance gene (e.g., neomycin phosphotransferase gene, or Neo) for positive clone screening, and two Frt recombination sites flanking the antibiotic resistance gene. In addition, a coding gene with a negative selection marker (a gene encoding diphtheria toxin A subunit (DTA)) may also be inserted into the targeting vector.

[0491] A 16076 bp sequence containing mouse Sγ1 promoter, mouse Sγ1 and Cγ1ΔCH1 knock-in sequence (SEQ ID NO: 1) was cloned from mouse bacterial artificial chromosome (RP23-38K22 or RP23-265p18). The following two sets of primer pairs were used to obtain sequences containing mouse Sγ1 promoter, mouse Sγ1 and Cγ1ΔCH1 knock-in sequences for construction of V1 vector. The sequences amplified from V1-F1 and V1-R1 and the sequences amplified from V1-F2 and V1-R2 were then connected.

[0492] V1-F1(SEQ ID NO:2):5'-GTGGTTCTGGCTACAAGATAGAGTCCTGTCAATGATGTTTGCAGAGACTACA-3'

[0493] V1-R1(SEQ ID NO:3):5'-CTCCCTATACGTCCTCTCACCTACAAGAAAAAGTATATGTGATTACACTGTCAGACAG-3'

[0494] V1-F2(SEQ ID NO:4):5'-GTGTAATCACATATACTTTTTCTTGTAGGTGAGAGGACGTATAGGGAGGAGGGGTTC-3'

[0495] V1-R2(SEQ ID NO:5):5'-CGTCTAGTCCTTGCCCACGTGTCGACCCCATAGGGAGGACAGACTGAGG-3'

[0496] like Figure 6 As shown, the V1 vector was used to replace a 100883 bp sequence (nucleic acids 113232142 to 113333024 of NCBI reference sequence NC_0000787) of the mouse heavy chain constant region locus (from Sγ3 to Cε) in a single step.

[0497] Vector V2

[0498] like Figure 7 As shown, the V2 vector was used to replace a 92859 bp sequence (nucleic acid 113232142 to 113325000 of NCBI reference sequence NC_0000787) of the mouse heavy chain constant region (spanning Cγ3 to Cε) in a single step.

[0499] The method also includes using a pair of primers to clone from a mouse bacterial artificial chromosome (BAC) to obtain a

[0500] The knock-in sequence of Cγ1ΔCH1 gene (SEQ ID NO: 1) was used. The following primers were used:

[0501] V2-F1 (SEQ ID NO:6):

[0502] 5'-TCTGAACTACTTCGTCGACGTGAGAGGACGTATAGGGAGGAGGG-3'

[0503] V2-R1 (SEQ ID NO:7):

[0504] 5'-CACGTGGATCCGCGGCCGCCCATAGGGAGGACAGACTGAGGAC-3'

[0505] Vector V3

[0506] like Figure 8 As shown, 16434 bp of nucleotides including Cμ and Cδ were knocked out from the chromosome using the V3 vector. The junction sequence between Sμ and Sγ1 in the recombinant mutant allele 2 is shown in SEQ ID NO:8.

[0507] Vector V4

[0508] like Fig. 9As shown, the targeting vector V4 includes the following characteristic sequences in the order of 5' to 3': an upstream homology arm (5' homology arm), a portion of mouse Sμ, and a downstream homology arm (3' homology arm). The targeting vector also includes an antibiotic resistance gene (neomycin phosphotransferase gene, or Neo) for positive clone screening, and two Frt recombination sites flanking the antibiotic resistance gene. In addition, a coding gene (a gene encoding diphtheria toxin A subunit (DTA)) with a negative selection marker is inserted into the targeting vector. Specifically, in the recombinant mutant allele 2', Sμ is directly connected to Cγ1ΔCH1' (i.e., there is no other conversion region or immunoglobulin gene sequence). The connection sequence is shown in SEQ ID NO:9.

[0509] Carrier V5

[0510] like Fig.10 As shown in FIG. 1 , the CH1 coding sequence in Cμ is knocked out using the V5 vector. After the CH1 coding sequence in Cμ is knocked out, the connection sequence is shown in SEQ ID NO: 10.

[0511] Carrier V6

[0512] like Fig.11 As shown, the targeting vector V6 includes the following characteristic sequences in the order of 5' to 3': upstream homology arm (5' homology arm), part of the mouse Sμ region, mouse CμΔCH1 sequence and downstream homology arm (3' homology arm). The targeting vector also includes an antibiotic resistance gene (neomycin phosphotransferase gene, or Neo) for positive clone screening, and two Frt recombination sites flanking the antibiotic resistance gene. In addition, a coding gene (a gene encoding diphtheria toxin A subunit (DTA)) with a negative selection marker is inserted into the targeting vector.

[0513] The sequence including the mouse CμΔCH1 sequence was cloned from mouse somatic cells. The following primer pair was used to obtain the sequence including the mouse CμΔCH1 (or CμΔCH1 knock-in sequence; SEQ ID NO: 13) for the construction of V6 vector.

[0514] V6-F1 (SEQ ID NO: 11):

[0515] 5'-ATCCCTCTCTGGTCCTAACCAAACCCTCCCAGCAGGGGTG-3'

[0516] V6-R1 (SEQ ID NO: 12):

[0517] 5'-TTGACCCATCTCAGTTTACATGGTGAATGACTACAATATATCTGGAATTTGG-3'

[0518] Vector V7

[0519] like Fig.12 As shown, the targeting vector V7 includes the following characteristic sequences from 5' to 3': upstream homology arm (5' homology arm), CμΔCH1 sequence, CδΔCH1 sequence (or CδΔCH1 knock-in sequence; SEQ ID NO: 41) and downstream homology arm (3' homology arm). The targeting vector also includes an antibiotic resistance gene (neomycin phosphotransferase gene, or Neo) for positive clone screening, and two Frt recombination sites flanking the antibiotic resistance gene. In addition, a coding gene with a negative selection marker (a gene encoding diphtheria toxin A subunit (DTA)) is inserted into the targeting vector.

[0520] The mouse Sμ switch region sequence, CμΔCH1 sequence, and CδΔCH1 sequence were cloned from the mouse genome. The following primer pairs were used to obtain knock-in sequences including mouse CμΔCH1 and CδΔCH1 upstream nucleotides for the construction of V7 vectors.

[0521] V7-F1 (SEQ ID NO: 14):

[0522] 5'-ATCCCTCTCTGGTCCTAACCAAACCCTCCCAGCAGGGGTG-3'

[0523] V7-R1 (SEQ ID NO: 15):

[0524] 5'-TTCTGCATGGTCCAGGGATTGATCAGACAGATAGTGAAGTTCTGAGGACA-3'

[0525] Example 4: Verification of genetic modification

[0526] Mutant allele 1

[0527] PCR and Southern Blot were used to detect the genotype of mutant allele 1. First, two sets of primers, L-GT-F1 / L-GT-R1 and R-GT-F2 / R-GT-R2, were used to perform PCR identification on positive clones. Figures 13A-13B The sequences of the primers are shown in the table below.

[0528] Table 7

[0529]

[0530] Next, the positive clones were verified by Southern Blot (digested with BclI, ScaI, XmnI and BglII, respectively, and then hybridized with 4 corresponding probes) to screen out 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 following table.

[0531] Table 8

[0532] Restriction enzymes Probe WT size Target size BclI LR Probe 6.1kb 18.4kb ScaI 3'Probe 9.7kb 12.8kb Xgf A Probe 9.7kb 16.6kb BglII 5'Probe 7.2kb 4.3kb

[0533] Table 9

[0534]

[0535] Exemplary test results are as follows Figures 14A-14D As shown. 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.

[0536] Mutant allele 2

[0537] Primers DE-F1 and DE-R1 were used to confirm the Cμ to Cδ sequence in the knockout mutant allele 2. The test results are shown in Fig.15 As shown. 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 sequences of the primers are shown in the table below.

[0538] Table 10

[0539]

[0540] Mutant allele 2'

[0541] Primers GT-Mut-F, GT-Mut-R, and GT-WT-R were used to confirm the sequence from Cμ to Sγ1 in the 2' of the knockout mutant allele. Fig.16 As shown. Mice numbered F1-2 and F1-4 were identified as positive heterozygous mice. The sequences of the primers are shown in the table below.

[0542] Table 11

[0543]

[0544] Mutant allele 3

[0545] Primers GT-3F and GT-3R were used to confirm the CH1 coding sequence of Cμ in the knockout mutant allele 3. The test results are shown in Fig.17 As shown. Mice numbered F1-2, F1-3 and F1-6 were identified as positive heterozygous mice. The sequences of the primers are shown in the table below.

[0546] Table 12

[0547]

[0548] Mutant allele 4

[0549] Primers Mut-F and Mut-R were used to confirm the sequence of CμΔCH1 in mutant allele 4, and primers F4 and R4 were used to confirm the deletion of Cδ in mutant allele 4. The test results were as follows: Figures 18A-18B As shown. 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 sequences of the primers are shown in the table below.

[0550] Table 13

[0551]

[0552] Mutant allele 5

[0553] Primers Mut-F and Mut-R were used to determine the sequence of CμΔCH1 in mutant allele 5, and primers F3 and R3 were used to determine the sequence of CδΔCH1 in mutant allele 5. The test results were as follows: Figures 19A-19B As shown. 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 sequences of the primers are shown in the table below.

[0554] Table 14

[0555]

[0556] Example 5: Modification of mouse immunoglobulin heavy chain variable region loci

[0557] Experiments were conducted to introduce human immunoglobulin genes into the mouse genome to produce mice expressing humanized antibodies. Fig. 20 A method for preparing humanized mice is shown. The method first involves modifying the human immunoglobulin region on a human chromosome. The modified human chromosome is then introduced into a mouse recipient cell.

[0558] By directly replacing (for example, homologous recombination or Cre-mediated recombination) the mouse immunoglobulin variable region is replaced with the human immunoglobulin variable region. In some cases, the human immunoglobulin variable region can be introduced into the mouse genome by a stepwise method. Then, the correctly replaced recipient cells are screened. The cells are then injected into the blastocyst to prepare chimeric mice. Breeding is subsequently performed to obtain mice containing people or humanized immunoglobulin variable regions.

[0559] The immunoglobulin heavy chain locus is located on mouse chromosome 12, and two recombination sites are introduced on both sides of the immunoglobulin heavy chain variable region locus.

[0560] Experiments were also conducted to generate modified human chromosomes. Two recombination sites were introduced on either side of the variable region of the heavy chain immunoglobulin locus. The modified human chromosome was then introduced into mouse cells, and the cells were screened to select only those containing only one human chromosome. The 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 ( Fig.21 ).

[0561] Positive cloned cells are microinjected into blastocysts of BALB / c mice. Embryo microinjection is performed according to the method described in A.Nagy et al., "Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition)," ColdSpring Harbor Laboratory Press, 2003. The injected fertilized eggs are then transferred to a culture medium for short-term culture and then transplanted into the oviducts of recipient mice to produce genetically modified humanized mice (F0 generation). The mice are then bred with mice with a C57BL / 6 background. PCR analysis is performed on the DNA obtained from the tail of the mice. The mice are further bred several times (e.g., at least 5 times) with mice with a BALB / c background to obtain humanized heterozygous mice with heavy chain immunoglobulin loci on a BALB / c background.

[0562] The heterozygous mice are then mated with each other to obtain homozygous mice. A detailed description of how to prepare mice homozygous for humanized heavy chain immunoglobulins is provided in WO2020169022A1 and US20200390073A1; all contents of each patent are incorporated herein by reference.

[0563] Example 6: Modification of the mouse immunoglobulin light chain locus

[0564] Kappa (κ) light chain knockout mice

[0565] 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 in, for example, WO2020169022A1 and US20200390073A1; Zou, X. et al. "Subtledifferences inantibody responses and hypermutation ofλlight chains in mice with a disruptedχconstant region." European Journal of Immunology 25.8(1995):2154-2162. Zou, YR et al. "Gene targeting in 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 chain intron enhancer abolishes kappa chain gene rearrangement in cis but not lambda chain gene rearrangement in trans." The EMBO Journal 12.6 (1993): 2329-2336; These patents are all incorporated herein by reference.

[0566] Lambda (λ) light chain knockout mice

[0567] Immunoglobulin lambda light chain (λ) is located on mouse chromosome 16. Mouse chromosome 16 is modified by knocking out the entire sequence of the immunoglobulin lambda light chain variable region locus. Detailed knockout methods can be found in, for example, 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, which is incorporated herein by reference in its entirety.

[0568] The mice described herein can be interbred to obtain mice having human immunoglobulin heavy chain VDJ regions, modified mouse immunoglobulin heavy chain constant region loci (lacking the CH1 coding region of Cγ1), and lacking all or part of the mouse immunoglobulin light chain loci.

[0569] Example 7: Heavy chain antibodies from mice carrying a modified IgG1 gene

[0570] The mice lacking the CH1 coding region of Cγ1 identified above (mice having mutant allele 2', mutant allele 3, and mutant allele 4 genotypes, respectively) and wild-type (WT) mice were bled to obtain serum samples. Serum samples were prepared for Western blot analysis to identify any expressed IgG in the serum using anti-mIgG1 antibody (Cat. No.: ab190481, Abcam). Fig.32 As shown, the results showed mixed bands: a band of about 75 kD (the expected size of dimeric IgG1 lacking the CH1 domain), and a band of about 150 kD (the expected size of wild-type IgG). The results show that mice generated using the methods described herein can express IgG1 lacking the CH1 domain in peripheral blood.

[0571] Mice can be immunized by injection of the immunogen, followed by screening by various methods (e.g., hybridoma, phage display screening, 10x Genomics single cell technology or The present invention screens antibodies with specific binding using an optofluidic system. Preliminary results show that the mice prepared 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). Mice with mutant allele 3 (Mut3)

[0572] Humanized mice (heterozygous heavy chain mutant allele 3 genotype, κ light chain locus knockout, λ light chain locus non-knockout) were immunized with antigen A (5 mice). After 3 immunizations, serum titers increased by 10% as detected by FACS. 4 times. Then use Plasma cells that produce antigen-specific monoclonal antibodies were isolated by an optofluidic system. Expression vectors for each antibody were constructed and transferred into host cells. The number of positive cells confirmed by FACS was 63. Further analysis of the antibody sequences revealed that the CDR3 length of the heavy chain variable region (defined by IMGT) ranged from 6 to 23 ( Fig. 22 In addition, 94% of clones (59 / 63) had CDR3 length greater than or equal to 12, with a total of 35 unique CDR3 sequences. Fig.23 The germline gene usage of the variable region genes is shown. In addition, the affinity of some antibodies was tested. Fig.24 As shown, the KD of these antibodies against antigen A reached 10 -9 M, indicating good binding affinity.

[0573] In another experiment, humanized mice (4 mice were homozygous heavy chain mutant allele 3 genotype, homozygous κ light chain locus deletion and homozygous λ light chain locus deletion; 3 mice were homozygous heavy chain mutant allele 3 genotype, homozygous κ light chain locus deletion and homozygous λ light chain locus deletion) were immunized with human 4-1BB (Cat. No. 41B-H5258, ACROBiosystems). After 4 immunizations, the serum titer detected by FACS increased by 10 4 The number of antigen-specific clones confirmed by FACS was 67. Further analysis of the antibody sequences revealed that the length of the CDR3 of the heavy chain variable region (defined by IMGT) ranged from 7 to 19 ( Fig.33 ). Fig.34 Germline gene usage of the variable region genes is shown. In addition, the affinity of some antibodies was tested.

[0574] In another experiment, humanized mice (19 mice were homozygous for heavy chain mutant allele 3 genotype, homozygous kappa light chain locus deletion, and homozygous lambda light chain locus deletion) were immunized with antigens human CD3ED and cynomolgus macaque CD3ED. After 4 immunizations, the serum titer detected by FACS increased by 10 5 The number of antigen-specific clones confirmed by FACS was 273. Further analysis of the antibody sequences revealed that the length of the CDR3 of the heavy chain variable region (defined by IMGT) ranged from 6 to 25 ( Fig.35 ). Fig.36 Germline gene usage of the variable region genes is shown. In addition, the affinity of some antibodies was tested.

[0575] Humanized mice (homozygous heavy chain mutant allele 3 genotype, κ light chain locus and λ light chain locus knockout) were immunized with the antigen human serum albumin (10 mice). Plasma cells that produce antigen-specific monoclonal antibodies were isolated by an optofluidic system. The number of positive cells confirmed by FACS was 84. Further analysis of the antibody sequences revealed that the CDR3 length of the heavy chain variable region (defined by IMGT) was between 12 and 17. Fig.43 Germline gene usage of variable region genes is shown.

[0576] Mice with mutant allele 2 (Mut2)

[0577] Humanized mice (homozygous heavy chain mutant allele 2 genotype, κ light chain locus knockout, λ light chain locus non-knockout) were immunized with antigen A (10 mice). The optofluidic system isolated plasma cells that produced antigen-specific monoclonal antibodies. The number of positive cells confirmed by FACS was 40. Further analysis of the antibody sequences showed a total of 14 unique CDR3 sequences. In addition, the affinity of some antibodies was tested. Fig.25 As shown, the KD of these antibodies against antigen A reached 10 -8 M, indicating good binding affinity.

[0578] In addition, spleen tissue from immunized Mut2 mice was collected to extract total RNA from spleen cells. The immunoglobulin variable region loci can be cloned by PCR and then inserted into a phage plasmid to construct a phage recombinant plasmid library. In one experiment, the constructed library was subjected to 2 rounds of panning and screening, and a total of 202 ELISA-positive clones were obtained. After removing redundant sequences, the number of positive cells confirmed by FACS was 94. Further analysis of the sequences of these antibodies showed that the CDR3 length of their heavy chain variable regions was between 8 and 18 (defined by IMGT). Fig.26 Germline gene usage of variable region genes is shown.

[0579] Mice with mutant allele 2' (Mut2')

[0580] Humanized mice (heterozygous heavy chain mutant allele 2' genotype, knockout of kappa light chain and lambda light chain loci) were immunized with the antigen human serum albumin (12 mice). The optofluidic system isolated plasma cells that produced antigen-specific monoclonal antibodies. The number of positive cells confirmed by FACS was 96. Further analysis of the antibody sequences revealed that the CDR3 length of the heavy chain variable region (defined by IMGT) ranged from 5 to 22. In addition, 78.1% of the clones (75 / 96) had a CDR3 length greater than or equal to 12. Fig.44The germline gene usage of the variable region genes is shown. In addition, the affinity of some antibodies was tested. Fig.45 As shown, the KD of some antibodies reached 10 -9 M, showing good affinity. Mice with mutant allele 4 (Mut4)

[0581] Humanized mice (homozygous heavy chain mutant allele 4 genotype, κ light chain and λ light chain loci knockout) were immunized with the antigen human serum albumin (10 mice). Plasma cells that produce antigen-specific monoclonal antibodies were isolated by an optofluidic system. The number of positive cells confirmed by FACS was 41. Further analysis of the antibody sequences showed that the CDR3 length of the heavy chain variable region (defined by IMGT) ranged from 8 to 23, with 65.9% of the clones (27 / 41) having a CDR3 length greater than or equal to 12. Fig.46 Germline gene usage of variable region genes is shown.

[0582] Mice with mutant allele 5 (Mut5)

[0583] Humanized mice (homozygous heavy chain mutant allele 5 genotype, κ light chain and λ light chain loci knockout) were immunized with antigen A. Plasma cells that produce antigen-specific monoclonal antibodies were isolated by optofluidics. The number of positive cells confirmed by FACS was 20. Further sequence analysis showed that the length of CDR3 of the heavy chain variable region (defined by IMGT) was between 10-18. Fig.47 The germline gene usage of the variable region genes is shown. In addition, the affinity of some antibodies was tested. These antibodies have a KD of 10 for antigen A. -9 M, indicating good binding affinity.

[0584] B cell development

[0585] Experiments were conducted to compare the immune systems of modified IgG1 mice and wild-type mice. RenMab mice (humanized heavy chain immunoglobulin loci) and modified IgG1 mice aged 6-8 weeks were selected. Among them, the modified IgG1 mice had similar weight, appearance and vitality compared with RenMab mice. Peripheral blood, spleen, lymph nodes and bone marrow tissues were taken from mice, and no obvious anatomical changes were found.

[0586] Example 8 Production of human anti-TFR1 antibodies

[0587] Mice (5 mice with homozygous heavy chain mutant allele 3 genotype, homozygous kappa light chain locus deletion, and homozygous lambda light chain locus deletion) were immunized with His-tagged human TFR1 (transferrin receptor 1) protein (hTFR1-His, ACROBiosystems, catalog number: CD1-H5243) to obtain anti-TFR1 antibodies. Before immunization, retro-orbital blood was 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 were performed (once every two weeks). One week after the third immunization, orbital blood was collected and serum antibody titers were detected by flow cytometry.

[0588] A booster immunization was also performed at least 14 days after the previous immunization, with TFR1 protein injected intraperitoneally and CHO-S cells expressing human TFR1 antigen injected via the tail vein.

[0589] Isolate antigen-specific immune cells from immunized mice to further obtain anti-TFR1 antibodies or obtain the heavy chain variable region sequence of anti-TFR1 antibodies. For example, using single cell technology (e.g., using Optofluidics system, Digital Cell Biology, Inc.) to screen and discover plasma cells that secrete antigen-specific monoclonal antibodies. The antibody variable region sequence was obtained by reverse transcription and PCR sequencing. The obtained variable region sequence was used for antibody expression to verify the binding affinity with TFR1 using FACS. Because of the lack of the CH1 domain, the heavy chain variable region (VH) of the obtained antibody is also called the heavy chain single variable domain (VHH).

[0590] Specifically, the obtained VHH sequences are linked to human IgG1 constant regions (e.g., hinge region, CH2 domain, and CH3 domain), respectively. Exemplary antibodies obtained by this method include: 23B8, 24A1, 24C9, and 24G5. The heavy chain CDR1-3 sequences are as follows: Fig.37 and Fig.38 The VHH region sequences of 23B8, 24A1, 24C9 and 24G5 are shown in Fig.39 shown.

[0591] The constant region of the antibody can be further engineered using alanine to replace asparagine at position 297 (N297A). For example, when the N297A mutation was introduced into the constant region of 24G5, the resulting antibody was designated 24G5-N.

[0592] Example 9 Cross-species binding of anti-TFR1 antibodies

[0593] CHO-S-hTFR1 cells or CHO-S-fasTFR1 cells were respectively 5The cells were transferred to a 96-well plate at a density of 10 cells / well. Serially diluted samples of anti-TFR1 antibody were added to the 96-well plate and incubated at 4°C for 30 minutes. PBS was used as a negative control (NC). Then, the cells were incubated with the secondary antibody anti-hIgG-Fc-Alex Flour TM 647 (Jackson ImmunoResearch Laboratories, Cat. No. 109-606-170) was incubated at 4°C in the dark for 15 min before flow cytometric analysis.

[0594] 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 cynomolgus monkey (Macaca fascicularis) TFR1 amino acid sequence (fasTFR1, SEQ ID NO: 71), respectively. The test results are shown in the following table.

[0595] JR141 is a humanized IgG1 antibody targeting human TFR1 conjugated to human iduronate-2-sulfatase, which was first approved in Japan in March 2021 for intravenous treatment of mucopolysaccharidosis type II. The VH and VL sequences of JR141 are shown in SEQ ID NO:72 and SEQ ID NO:73, respectively. For the positive control (JR141-N), the VH and VL of JR141 were linked to the human IgG1 constant region with the N297A mutation.

[0596] Table 15

[0597]

[0598] Example 10. Binding affinity of anti-TFR1 antibodies

[0599] In a Biacore equipped with a pre-immobilized protein A sensor chip TM Surface plasmon resonance (SPR) was used to measure the binding affinity of anti-TFR1 antibodies to His-tagged TFR1 proteins of human (hTFR1-His, ACROBiosystems, Catalog No.: CD1-H5243) or monkey (fasTFR1-His, ACROBiosystems, Catalog No.: TFR-C524a) on a Biacore 8K biosensor (Biacore, Piscataway, NJ).

[0600] Purified anti-TFR1 antibody was captured on a Protein A chip (Series S Sensor Chip Protein A) for detection. 1 μg / mL purified anti-TFR1 antibody was loaded at a rate of 10 μL / min to bind hTFR1-His and fasTFR1-His (200 nM). The flow rate was 30 μL / min. The association and dissociation times were set to 180 seconds and 600 seconds, respectively. After the last injection of each round of titration, the chip was regenerated with glycine solution (pH 2.0) at a rate of 30 μL / min for 30 seconds.

[0601] Data analysis in Biacore TM The 8K evaluation software 3.0 was used to fit the 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994, Methods Enzymology 6, 99-110), and the kinetic association rate (kon) and dissociation rate (koff) were obtained simultaneously. The affinity value was derived from the quotient of the kinetic rate constant (KD = koff / kon).

[0602] As will be appreciated by one of ordinary skill in the art, the same method was used for each anti-TFR1 antibody tested, with appropriate adjustments to parameters (e.g., antibody concentration). The results of the tested 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.

[0603] Table 16

[0604]

[0605] Example 11 Epitope Analysis of Anti-TFR1 Antibodies

[0606] The relative position of the target protein epitope between a pair of purified anti-TFR1 antibodies was analyzed by biolayer interferometry (BLI) at 30°C using the ForteBio Octet system. 1×HBS-EP+ buffer (10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150mM NaCl, 3mM ethylenediaminetetraacetic acid (EDTA), and 0.05% P20, pH 7.4) diluted from HBS-EP+ buffer (10×) was used as the running buffer for the entire experiment. Approximately 10μg / mL of hTFR1-His protein was captured by HIS1K (Anti-Penta-HIS) for 200 seconds, and 200nM of antibody (analyte 1) was injected at a flow rate of 30μL / min to bind to the ligand. Another antibody (analyte 2) was injected under the same conditions to determine whether the binding of different antibodies interfered with each other. The binding time for each antibody was 300 seconds.

[0607] The binding values ​​for each antibody were obtained using Data Analysis HT 12.0. In order to quantify the interference of one antibody with the binding of another antibody, the binding ratio was calculated to compare each pair of antibodies. The binding rate is defined as the binding value of the second antibody (analyte 2) divided by the binding rate of the first antibody (analyte 1). The binding rates of each antibody pair are summarized in the table below. Specifically, if analyte 1 exhibits a blocking effect on analyte 2, the binding ratio is between 0.0 and 0.5; if analyte 1 does not exhibit a blocking effect on analyte 2, the binding ratio is between 0.5-1.1. Typically, antibody pairs that interfere with each other have identical or overlapping epitopes.

[0608] Epitope binding experiments showed that 24A1 and 24G5 recognized the same epitope, while 23B8, 24C9 and JR141-N recognized different epitopes.

[0609] Table 17

[0610]

[0611] Example 12 Internalization of anti-TFR1 antibodies

[0612] Anti-TFR1 antibody was added to human cortical microvascular endothelial cells (hCMEC / D cells) together with pHAb-goat anti-human IgG secondary antibody 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 antibody was calculated. Human IgG1 protein (CrownBio, Cat. No.: C0001) was used as an isotype control (ISO). The results are shown in the table below, indicating that all four antibodies showed good endocytosis activity in human cortical microvascular endothelial cells.

[0613] Table 18

[0614] Antibody MFI Positive group ISO 4288 0.9％ 23B8 11392 67.0％ 24A1 26972 96.6％ 24C9 37086 96.0％ 24G5 26406 96.0％

[0615] Example 13. Stability analysis of anti-TFR1 antibodies

[0616] The stability of anti-TFR1 antibodies 23B8, 24A1, 24C9 and 24G5 was evaluated. Specifically, the following tests were performed: (1) observing the appearance of the solution and the presence of visible insoluble matter; (2) detecting the purity change of the antibody by size exclusion ultra-high performance liquid chromatography (SEC-UPLC) (expressed as the percentage of the main peak area to the sum of all peak areas (purity, %)); (3) using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) to detect the change in the apparent hydrophobicity of the antibody (expressed as the retention time of the main peak (HIC, min)); (4) detecting the charge variable in the antibody by capillary isoelectric focusing (cIEF) (the percentage of the epitope as the main component, the acidic component and the basic component); and (5) detecting the thermal stability of the antibody by the UNcle system (expressed as the melting temperature (Tm) and the aggregation temperature (Tagg)).

[0617] In the SEC-UPLC experiment, an Agilent 1290 chromatography system (with XBridge TM Protein BEH SEC column ( Waters Corporation). 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 plate temperature: 6°C; run time: 7 minutes.

[0618] In the HIC-HPLC experiments, an Agilent 1260 chromatography system (with ProPac TM HIC-10 column (4.6×100 mm, Thermo Scientific) was connected), and the sample was diluted to 0.5 mg / mL using 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 plate temperature: about 6°C; run time: 45 min.

[0619] In the cIEF experiment, the Maurice cIEF method development kit (Protein Simple, Cat. No.: 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 Maurice cIEF pI marker-4.05, 1 μL Maurice cIEF pI marker-9.99, 35 μL 1% methylcellulose solution, 2 μL Maurice cIEF 500mM arginine, 4 μL ampholytes (Pharmalyte pH range 3-10) and water (added to make the final volume 100 μL). On the Maurice analyzer (Protein Simple, Santa Clara, CA), the imaging capillary isoelectric focusing spectra were generated using the Maurice cIEF box (PS-MC02-C). The samples were focused for a total of 10 minutes. The analysis software installed on the instrument was used to analyze the absorbance of the focused protein at 280nm.

[0620] In the thermal stability experiment, a 60 mg / mL antibody solution was heated from 25°C to 95°C in 1°C increments with a 1 minute equilibration time before each measurement.

[0621] The detailed results are shown in the table below. The results show that all four antibodies have good stability and physicochemical properties.

[0622] Table 19

[0623]

[0624] Example 14. Pharmacokinetic (PK) Analysis

[0625] The humanized TFR1 mouse model (hTFR1 mouse) was modified to express a chimeric TFR1 protein (SEQ ID NO: 74), wherein the mouse TFR1 protein extracellular region was replaced by the corresponding human TFR1 extracellular region. A detailed description of the humanized TFR1 mouse model can be found in PCT application PCT / CN2022 / 105924, which is incorporated herein by reference in its entirety.

[0626] The concentration of anti-TFR1 antibodies was determined in hTFR1 mice. Specifically, mice were divided into different groups (8 mice per group) and given approximately equimolar doses of JR141-N (G2), 23B8-N (G3), 24A1-N (G4), 24G5-N (G5) or 24C9-N (G6) by intravenous (iv) injection. The control group (G1) mice were given human IgG1 (hIgG1). The detailed dosing regimen is shown in the table below.

[0627] Table 20

[0628]

[0629] Blood samples and brain samples were collected at 0.5, 6, 24 and 72 hours after administration. Two mice were sampled at each time point, and the mice were anesthetized after retroorbital blood sampling. In order to avoid interference from residual blood in the brain, mice were perfused with saline for 10 minutes at room temperature. Specifically, saline was perfused through the systemic circulation from the left ventricle to the right ventricle. Brain samples were removed and divided into two hemibrains through the sagittal plane. The left hemibrain was quantified for injected antibodies, while the right hemibrain was fixed with formalin and embedded in paraffin for serial sectioning. Brain samples were cut into small pieces and homogenized with DPBS (Dulbecco's phosphate buffered saline) containing 1× mixed protease inhibitors. The brain homogenate was divided equally to extract protein, and then antibody quantification was performed by electrochemiluminescence. For the remaining homogenate, the capillary was removed by gradient density centrifugation at 5400g for 15 minutes using 15% dextran. After centrifugation, the part at the top of the centrifuge tube was preserved as parenchyma, and protein extraction and antibody quantification were performed. Figures 40A-40D The antibody concentration in total brain protein is shown ( Fig.40A ), the ratio of antibody concentration in total brain protein to that in serum ( Fig.40B ), antibody concentration in brain parenchyma ( Fig.40C ), and the ratio of the antibody concentration in the brain parenchyma to the antibody concentration in the serum at each time point ( Fig.40D ). These results indicate that 24G5-N (G5 group) is the most abundant in both the brain parenchyma and the whole brain.

[0630] In a similar experiment, hTFR1 mice were divided into 5 groups (3 mice per group) and given 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) by intravenous injection (1 dose in total). The control group (G1) mice were given hIgG1 (G1). 24 hours after administration, brain samples were collected to determine the concentration of anti-TFR1 antibodies. Figures 41A-41B The results of antibody concentration tests in brain parenchyma and total brain protein are shown separately. The concentrations of all tested antibodies in the brain were higher than hIgG1(G1), indicating that 23B8-N(G3) and 24G5-N(G5) can better cross the blood-brain barrier and enter the brain parenchyma compared with the positive control JR141-N(G2).

[0631] In another similar experiment, hTFR1 mice were divided into 7 groups (6 mice per group) and given JR141-N (G2-G4) or 24G5-N (G5-G7) by intravenous (iv) injection. The control group (G1) mice were given hIgG1. The detailed dosing schedule is shown in the table below.

[0632] Table 21

[0633] Group Number of mice Antibody Dosage (mg / kg) Dosage frequency G1 6 hIgG1 5.52mg / kg iv Single dose G2 6 JR141-N 1.84mg / kg iv Single dose G3 6 JR141-N 5.52mg / kg iv Single dose G4 6 JR141-N 18.4mg / kg iv Single dose G5 6 24G5-N 1mg / kg iv Single dose G6 6 24G5-N 3mg / kg iv Single dose G7 6 24G5-N 10mg / kg iv Single dose

[0634] At 6 and 24 hours after administration, blood and brain samples were collected using the above method. Three mice were sampled at each time point. Tissue processing and antibody quantification were also performed as described above. The results of the determination of humanized anti-TFR1 antibody concentration in the brain parenchyma are shown in Fig.42 As shown. The results showed that under each dose condition, the antibody concentration accumulated by 24G5-N in the brain parenchyma was significantly higher than that of hIgG1. In addition, the concentrations of JR141-N and 24G5-N in the brain parenchyma showed a dose-dependent trend.

[0635] In order to detect the distribution of humanized anti-TFR1 antibody 24G5-N in the mouse brain, immunofluorescence assay was performed by staining hIgG, hTFR1 and mCD31 on the right hemisphere sections of the mice used in the above experiment. The results showed that mCD31 was well labeled in microvessels. hTFR1 was also detected on microvessels, where it colocalized with mCD31. In addition, the expression of hTFR1 was also detected on some neurons in the brain parenchyma. In particular, the anti-TFR1 antibody 24G5-N was detected by colocalization with 488-conjugated secondary anti-IgG antibody staining. Similar to hTFR1, 24G5-N was detected in microvessels and parenchyma, and its signal overlapped with the hTFR1 signal. Therefore, for the quantification of 24G5-N in whole brain or brain parenchyma, or the visual evidence of 24G5-N immunofluorescence in brain parenchyma, the results indicate that the humanized anti-TFR1 antibody 24G5-N can effectively cross the blood-brain barrier (BBB).

[0636] Example 15 Blocking Test

[0637] Biolayer interferometry (BLI) using ForteBio System, the blocking effect of anti-TFR1 antibodies 23B8, 24A1, 24C9 and 24G5 on the binding of TFR1 to TF (transferrin) was detected at 30°C. Specifically, 1×HBS-EP+ buffer (10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150mMNaCl, 3mM EDTA and 0.05% surfactant P20, pH 7.4) diluted from HBS-EP+ buffer (10×) was used as the running buffer for the entire experiment. About 10μg / mL of antibody was captured by AHC (anti-human IgG FC capture) for 200 seconds, and 800nM hTFR1-His (ACROBiosystems, Catalog No.: CD1-H5243) and HTF-His (human transferrin, Kactus Biosystems, Catalog No.: TFN-HM101) were injected to bind to the ligand. The binding time of each antibody was 300 seconds. The binding value of each antibody was obtained using Data Analysis HT 12.0. The results showed that none of the 4 antibodies could block the binding of TFR1 to TF. Therefore, this non-blocking antibody is unlikely to interfere with the TFR1-TF interaction in normal cells.

Claims

1. A genetically modified non-human animal comprising a modified immunoglobulin heavy chain locus, wherein the modified immunoglobulin heavy chain locus comprises an IgG constant region gene, wherein the IgG constant region gene encodes an IgG heavy chain constant region lacking a CH1 domain, wherein the genetically modified non-human animal expresses heavy chain antibodies.

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

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

4. The genetically modified non-human animal of 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 at the endogenous immunoglobulin heavy chain locus.

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. The animal of claim 5 or 6, wherein the genetic modification further comprises deletion of endogenous Sγ2b, Sγ2C and Sε switch regions at the endogenous immunoglobulin heavy chain locus.

8. The animal of any one of claims 5-7, wherein the genetically modified immunoglobulin heavy chain locus comprises a modified IGHG1 gene lacking a sequence encoding a CH1 domain, 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 5-8, wherein the genetic modification further comprises a deletion of an endogenous Sγ3 switch region at the endogenous immunoglobulin heavy chain locus.

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

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

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

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

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

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 5-9, 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. The animal of any one of claims 5-9 and 17, 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 IGHG1 gene lacking a sequence encoding a CH1 domain, and an endogenous IGHA gene.

19. The animal of any one of claims 5-9, 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. The animal of any one of claims 5-9 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, a modified IGHG1 gene lacking a sequence encoding a CH1 domain, and an endogenous IGHA gene.

21. The animal of claim 19 or 20, wherein 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.

22. The animal of any one of claims 14-21, 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 5-8, wherein the genetic modification further comprises a deletion of an endogenous Sγ1 switch region at the endogenous immunoglobulin heavy chain locus.

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

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

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

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

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

30. A genetically modified non-human animal whose genome comprises the following elements in 5' to 3' order at the endogenous immunoglobulin heavy chain locus: 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 the elements are operably linked.

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

32. A genetically modified non-human animal whose genome comprises the following elements in 5' to 3' order at the endogenous immunoglobulin heavy chain locus: 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 the elements are operably linked.

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

34. A genetically modified non-human animal whose genome comprises the following elements in 5' to 3' order at the 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; wherein the elements are operably linked.

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

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

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

38. A genetically modified non-human animal whose genome comprises the following elements in 5' to 3' order at the 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 IGHG1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene; wherein the elements are operably linked.

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

40. A genetically modified non-human animal whose genome comprises the following elements in 5' to 3' order at the endogenous immunoglobulin heavy chain locus: 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; wherein the elements are operably linked.

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

42. A genetically modified non-human animal whose genome comprises the following elements in 5' to 3' order at the 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 IGHG1 gene lacking a sequence encoding a CH1 domain, an Sα switch region, and an IGHA gene; wherein the elements are operably linked.

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

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

45. The animal of claim 44, wherein the heavy chain antibody is expressed in an amount less than 10 -7 M, less than 10 -8 M or less than 10 -9 The KD of M binds to its target antigen.

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. The animal of any one of claims 1-47, wherein the genetically modified non-human animal does not express light chain-containing IgG antibodies.

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

50. The genetically modified non-human animal of any one of claims 1-49, 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, human IGHD genes, and human IGHJ genes are operably linked and can undergo VDJ rearrangement.

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. 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 of human chromosome 14 of a human subject.

53. 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 of human chromosome 14 in a human cell.

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

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 a contiguous sequence starting from the mouse IGHV1-85 gene to the mouse IGHJ4 gene.

56. The genetically modified non-human animal of claim 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. The genetically modified non-human animal of claim 50, wherein the animal comprises unmodified human sequences derived from the human heavy chain immunoglobulin locus starting from human IGHV(III)-82 to human IGHV1-2.

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

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

60. The genetically modified non-human animal of claim 50, wherein the animal comprises a polypeptide derived from human IGHV(III)-82 Unmodified human sequence of the human heavy chain immunoglobulin locus starting at human IGHJ6.

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

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

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 in Table 4, all mouse IGHD genes in Table 5, and all mouse IGHJ genes in Table 6 are replaced.

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

65. The genetically modified non-human animal of any one of claims 1-63, wherein the animal is heterozygous for an immunoglobulin heavy chain gene.

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

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

68. A genetically modified non-human animal according to any one of claims 1-67, wherein the animal lacks an endogenous immunoglobulin heavy chain variable region locus that is capable of rearranging and forming a nucleic acid sequence encoding an endogenous heavy chain variable domain.

69. The genetically modified non-human animal of any one of claims 1-68, wherein the animal can produce humanized antibodies.

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

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

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

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

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

75. The cell of claim 74, wherein the cell is a B cell that expresses a chimeric immunoglobulin heavy chain comprising a rearranged immunoglobulin heavy chain variable domain derived from 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. The cell of claim 74 or 75, wherein the cell is an embryonic stem (ES) cell.

77. A method for preparing an antibody that specifically binds to an antigen, the method comprising: include: a) exposing the genetically modified non-human animal of any one of claims 1 to 73 to an antigen; b) producing hybridomas from cells collected from the animal; and C) collecting the heavy chain antibodies produced by the hybridoma.

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

79. A method for preparing an antibody that specifically binds to an antigen, the method include: a) exposing the genetically modified non-human animal of any one of claims 1 to 73 to an antigen; b) sequencing of nucleic acids encoding human immunoglobulin heavy chain variable regions in cells expressing heavy chain antibodies that specifically bind to an antigen; and c) In the cell, a nucleic acid encoding a human immunoglobulin heavy chain variable region is operably linked to a nucleic acid encoding a human immunoglobulin heavy chain constant region.

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

81. A method for obtaining a nucleic acid encoding an antibody binding domain that specifically binds to an antigen, the method comprising: include: a) exposing the genetically modified non-human animal of any one of claims 1 to 73 to an antigen; and 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.

82. A method for preparing an antibody that specifically binds to an antigen, the method comprising: include: a) exposing the genetically modified non-human animal of any one of claims 1 to 73 to an antigen; b) constructing a phage plasmid library using RNA prepared from immune cells (e.g., spleen cells) of an animal; c) screening of phage plasmid libraries, and d) sequencing a 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.

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

84. A method for obtaining a sample, the method include: a) exposing the genetically modified non-human animal of any one of claims 1 to 73 to an antigen; and b) Collect samples from animals.

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

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