Recombinant non-human animals for antibody production

Abstract

The present invention provides genetically modified animals (e.g., mice), humanized heavy chain antibodies, humanized nanobodies, and methods for producing and using them. [Solution] Provided are genetically modified non-human animals (e.g., genetically modified mice) that can be designed to produce heavy chain antibodies that can be used to generate single-domain antibodies or nanobodies. In one embodiment, a genetically modified mouse is provided comprising a germline modification comprising deletion of nucleic acid sequences comprising one or more heavy chain C region genes; the genetically modified mouse expresses an IgG heavy chain antibody and secretes an IgG heavy chain antibody in its serum.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Application No. 63 / 184,384, filed May 5, 2021, and U.S. Provisional Application No. 63 / 184,385, filed May 5, 2021. The disclosures of these prior applications are considered to be a part of the disclosure of this application and are hereby incorporated by reference in their entirety into the disclosure of this application.

[0002] Background 1. Technical Field This document relates to methods and materials involved in the production of antibodies (e.g., single - domain antibodies also known as heavy - chain antibodies and / or nanobodies). For example, genetically - engineered non - human animals (e.g., genetically - engineered mice) having the ability to produce antibodies (e.g., heavy - chain antibodies such as heavy - chain antibodies lacking the CH1 domain and the light chain) are provided. In some examples, single - domain antibodies or nanobodies can be generated using the heavy - chain antibodies obtained as described herein.

Background Art

[0003] 2. Background Information Conventional IgG antibodies consist of four polypeptides: two pairs of identical heavy and light chains. The IgG heavy chain contains one variable domain (VH) and three constant domains (CH1, CH2, and CH3), and the two chains are linked via a disulfide bond at the hinge region (H) between CH1 and CH2. The two light chains contain one variable domain (VL) and one constant domain (CL), and the CL domain is linked to the CH1 domain of the heavy chain via a disulfide bond to form a tetrameric IgG. The two antibody arms (FAb), composed of VH-CH1 and VL-CL, can independently bind to the antigen, and the constant region (Fc) is involved in effector function. Antibody production is initiated by the expression of the B cell receptor (BCR) in pre-B cells by constructing the V (variable region) gene segment, the D (diversity) gene segment, and the J (binding region) gene segment via VDJ recombination of the immunoglobulin heavy chain gene (IgH) to generate diverse VHs. VDJ is spliced ​​into the constant exon of IgM, which then associates with the alternative light chains λ6 and VpreB on the cell surface to form a pre-BCR. This is followed by VJ recombination of immunoglobulin light chain genes (IgK and IgL, both lacking the D gene) and the production of diverse VLs with CL. Pairing of the IgM light and heavy chains results in IgM expression as a complete BCR on immature B cells. V(D)J recombination occurs on both alleles of the heavy and light chain loci, but allele exclusion ensures the expression of only one functional heavy chain and one functional light chain from one of the two alleles in a single B cell. B cells with a successful recombination event subsequently undergo somatic hypermutation, antigen selection, affinity maturation, and class switch recombination to express different isotypes of antibodies (IgG, IgE, and IgA).

[0004] In camels, a subset of IgG has been identified that consists only of two identical heavy chains with variable domains (variable heavy chain homodimers, VHH), but lacks the CH1 domain and associated light chain. These heavy chain-only antibodies (HCAb) arise from splice site mutations in the heavy chain gene, which normally cause the loss of the CH1 exon encoding the CH1 domain in the constant region that binds to the CL of the light chain. Similar light chain HCAb devoid is also found in cartilaginous fish (immunoglobulin neoantigen receptor, IgNAR). The variable domains in camel HCAb-VHH and cartilaginous fish VNAR can function as independent antigen-binding units and may have binding affinities comparable to conventional antibodies. These single-domain antibodies (sdAb) are the smallest antigen-binding antibody fragments and are therefore sometimes called nanobodies (Nb). Many unique properties of sdAbs, such as their small size (11-15 kDa compared to 150 kDa for tetrameric antibodies), strict monomericity, high solubility, efficient folding / refolding, excellent stability, unparalleled target accessibility, effective tissue penetration, rapid blood clearance, superior manufacturability, and low production costs, make them compelling candidates for the development of novel therapeutic and diagnostic agents. One of the most advantageous properties of sdAbs over conventional antibodies is their modularity, which is a crucial characteristic for easier handling of multimeric and multispecific biopharmaceuticals.

[0005] Variable domains (VH and VL) derived from human scaffolds were produced in synthetic sdAb display libraries and tested in vitro against numerous targets (Belanger et al., Protein Eng. Des. Sel., (34):gzab012 (2021)). In contrast to natural sdAbs obtained from immunized animals that have high affinity as a result of somatic hypermutation, sdABs derived from non-immune display libraries are usually low affinity, tend to aggregate, and often require further mutations to improve affinity and function. A solution to both problems is to genetically engineer mice that produce humanized HCAbs, immunize these mice with the target of interest, and isolate highly soluble and high-affinity functional human sdAbs resulting from antigen selection and affinity maturation in vivo. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Belanger et al., Protein Eng. Des. Sel., (34):gzab 012 (2021) [Overview of the project] [Means for solving the problem]

[0007] overview This book relates to recombinant non-human animals (e.g., mice) that produce single-domain antibodies or nanobodies (e.g., mouse single-domain antibodies or mouse nanobodies, or humanized single-domain antibodies or humanized nanobodies), as well as methods for producing such recombinant non-human animals (e.g., mice), and methods for using such recombinant non-human animals (e.g., mice). For example, this book provides genetically modified non-human animals (e.g., genetically modified mice) that can be designed to produce heavy-chain antibodies that can be used to generate single-domain antibodies or nanobodies. In some examples, genetically modified non-human animals (e.g., genetically modified mice) lack the CH1 domain and light chain and can be designed to produce heavy-chain antibodies (e.g., complete mouse heavy-chain IgG antibodies) that can be used to generate single-domain antibodies or nanobodies (e.g., complete mouse single-domain antibodies or complete mouse nanobodies). See, for example, Figure 1. In some cases, genetically modified non-human animals (e.g., genetically modified mice) can be designed to produce chimeric heavy chain antibodies (e.g., human-mouse chimeric heavy chain IgG antibodies) that lack the CH1 domain and light chain and can similarly be chimeric or composed entirely of one species, which can be used to generate single-domain antibodies or nanobodies. For example, a genetically modified mouse can be designed to produce a human-mouse chimeric heavy chain IgG antibody that lacks the CH1 domain and light chain but has a human variable domain and a mouse constant domain. Such human-mouse chimeric heavy chain IgG antibodies obtained from such mice can be used to generate fully human single-domain antibodies or human nanobodies. See, for example, Figure 6. Compositions described herein (e.g., compositions comprising one or more antibodies produced from the recombinant non-human animals (e.g., mice) provided herein) can be used to treat or prevent diseases or disorders, such as inflammatory diseases.

[0008] As described herein, genetically modified non-human animals (e.g., genetically modified mice) can be designed to produce heavy chain antibodies lacking the CH1 domain and light chain (e.g., mouse heavy chain antibodies or chimeric heavy chain antibodies, e.g., human-mouse chimeric heavy chain antibodies, bovine-human-mouse chimeric heavy chain antibodies, alpaca-human-mouse chimeric heavy chain antibodies, or shark-human-mouse chimeric heavy chain antibodies). Such heavy chain antibodies can be used to generate single-domain antibodies or nanobodies (e.g., mouse single-domain antibodies, also referred to herein as mouse nanobodies; non-mouse single-domain antibodies, also referred to herein as non-mouse nanobodies; humanized single-domain antibodies, also referred to herein as humanized nanobodies; human single-domain antibodies, also referred to herein as human nanobodies; bovine-human chimeric single-domain antibodies, also referred to herein as bovine-human chimeric nanobodies; alpaca-human chimeric single-domain antibodies, also referred to herein as alpaca-human chimeric nanobodies; or shark-human chimeric single-domain antibodies, also referred to herein as shark-human chimeric nanobodies).

[0009] Similarly, as described herein, recombinant non-human animals (e.g., mice) provided herein can be used to obtain heavy chain antibodies (e.g., mouse heavy chain antibodies or chimeric heavy chain antibodies) that can be used to produce single-domain antibodies (e.g., mouse single-domain antibodies or non-mouse single-domain antibodies, e.g., humanized single-domain antibodies or human single-domain antibodies).

[0010] In one embodiment, this document provides a genetically modified mouse comprising germline modification including deletion of nucleic acid sequences containing one or more heavy chain C region genes; the genetically modified mouse expresses IgG heavy chain antibodies and secretes IgG heavy chain antibodies in its serum.

[0011] In some embodiments, one or more heavy chain C region genes include an IgM C region gene (Cμ), an IgD C region gene (Cδ), an IgE C region gene (Cε), an IgG3 C region gene (Cγ3), an IgG2b C region gene (Cγ2b), an IgG2c C region gene (Cγ2c), or a combination thereof.

[0012] In some embodiments, the genetically modified mouse further comprises a deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene (Cγ1). In some embodiments, the deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene comprises exon 1.

[0013] In some embodiments, the germline modification further comprises a native nucleic acid sequence encoding a hinge (H) domain, a heavy chain CH2 domain, a heavy chain CH3 domain, or a combination thereof.

[0014] In some embodiments, the germline modification further comprises a native nucleic acid sequence containing an endogenous enhancer. In some embodiments, the enhancer comprises Eμ, 3'RR, 3'γ1E, 5'hsR1, or a combination thereof.

[0015] In some embodiments, the germline modification further comprises a native nucleic acid sequence including a switched tandem repeat element (Sμ) and an Iμ promoter, where Iμ drives the constitutive expression of IgG1 with a cleaved CH1 domain (IgG1ΔCH1).

[0016] In some embodiments, the IgG heavy chain antibody comprises an IgG1 heavy chain antibody. In some embodiments, the IgG1 heavy chain antibody is the IgG1ΔCH1 protein. In some embodiments, the IgG heavy chain antibody lacks a light chain. In some embodiments, the IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

[0017] In some embodiments, the mice do not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, or a combination thereof. In some embodiments, the mice do not express wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or a combination thereof.

[0018] In another embodiment, this document provides a recombinant non-human animal comprising a germline genome containing a recombinant immunoglobulin heavy chain (IgH) allele at an endogenous IgH locus; wherein the recombinant IgH allele lacks an endogenous heavy chain C region gene; and the endogenous heavy chain C region gene comprises Cμ, Cδ, Cε, Cγ3, Cγ2b, Cγ2c, or a combination thereof.

[0019] In some embodiments, the IgH allele contains a deletion in the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene (Cγ1). In some embodiments, the CH1 domain of the IgG1 C region gene contains exon 1.

[0020] In some embodiments, the IgH locus contains a native nucleic acid sequence encoding a hinge (H) domain, a heavy chain CH2 domain, a heavy chain CH3 domain, or a combination thereof.

[0021] In some embodiments, the IgH locus contains a native nucleic acid sequence including an endogenous enhancer. In some embodiments, the enhancer includes Eμ, 3'RR, 3'γ1E, 5'hsR1, or a combination thereof.

[0022] In some embodiments, the IgH locus comprises a native nucleic acid sequence including a switch tandem repeat element (Sμ) and an Iμ promoter, where Iμ drives the constitutive expression of IgG1 with a cleaved CH1 domain (IgG1ΔCH1).

[0023] In some embodiments, non-human animals express IgG heavy chain antibodies. In some embodiments, the IgG heavy chain antibodies include IgG1 heavy chain antibodies.

[0024] In some embodiments, the IgG1 heavy chain antibody is the IgG1ΔCH1 protein.

[0025] In some embodiments, IgG heavy chain antibodies lack a light chain.

[0026] In some embodiments, the IgG heavy chain antibody includes a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

[0027] In some embodiments, non-human animals do not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or any combination thereof.

[0028] In some embodiments, the IgH locus includes an endogenous V, D, or J gene.

[0029] In some embodiments, recombinant non-human animals are homozygous for recombinant IgH alleles.

[0030] In some embodiments, the endogenous IgH gene locus does not contain an exogenous nucleic acid sequence.

[0031] In some embodiments, the endogenous IgH gene locus includes an exogenous nucleic acid sequence. In some embodiments, the exogenous nucleic acid sequence includes a barcode.

[0032] In another embodiment, this book provides recombinant non-human animals, where the non-human animal is a mammal. In some embodiments, the mammal is a mouse.

[0033] In another embodiment, this publication provides a method for producing a genetically modified non-human animal capable of producing heavy chain antibodies, comprising the steps of: (a) deleting an endogenous nucleic acid sequence containing one or more heavy chain C region genes from an endogenous immunoglobulin heavy chain gene locus in stem cells of a non-human animal; (b) transplanting the stem cells into a blastocyst; (c) transplanting the blastocyst into a pseudo-pregnant mouse to obtain a chimeric mouse; (d) mating the chimeric mouse with a wild-type mouse to produce offspring; (e) screening the offspring for heterozygosity; and (f) identifying a founding mouse having the deletion of one or more heavy chain C region genes, wherein the non-human animal is capable of producing heavy chain antibodies.

[0034] In some embodiments, the stem cells are embryonic stem cells.

[0035] In some embodiments, one or more heavy chain C region genes include Cμ, Cδ, Cγ3, Cγ2b, Cγ2c, Cε, or a combination thereof.

[0036] In some embodiments, the method further includes the step of deleting the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene and the CH1 exon of Cγ1. In some embodiments, the deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene includes exon 1.

[0037] In some embodiments, the method further includes the step of preserving a native nucleic acid sequence encoding the hinge (H) domain, heavy chain CH2 domain, and heavy chain CH3 domain of IgG1(Cγ1), or a combination thereof.

[0038] In some embodiments, the method further includes the step of preserving a native nucleic acid sequence containing an endogenous enhancer. In some embodiments, the enhancer includes Eμ, 3'RR, 3'γ1E, 5'hsR1, or a combination thereof.

[0039] In some embodiments, the method further includes the step of preserving a native nucleic acid sequence comprising a switched tandem repeat element (Sμ) and an Iμ promoter, where Iμ drives the constitutive expression of IgG1 with a cleaved CH1 domain (IgG1ΔCH1).

[0040] In some embodiments, the heavy chain antibody is an IgG heavy chain antibody. In some embodiments, the IgG heavy chain antibody includes an IgG1 heavy chain antibody. In some embodiments, the IgG1 heavy chain antibody is an IgG1ΔCH1 protein.

[0041] In some embodiments, the IgG1 heavy-chain antibody lacks a light chain.

[0042] In some embodiments, the IgG1 heavy-chain antibody includes a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

[0043] In some embodiments, non-human animals do not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or any combination thereof.

[0044] In some embodiments, the non-human animal is a mammal. In some embodiments, the mammal is a mouse.

[0045] In some embodiments, the step of deleting an endogenous nucleic acid sequence containing one or more heavy chain C region genes includes CRISPR / Cas9 genome editing.

[0046] In some embodiments, the genetically modified non-human animals are fertile. In some embodiments, the genetically modified non-human animals have normal B cell development and maturation.

[0047] In some embodiments, genetically modified non-human animals do not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or any combination thereof.

[0048] In another embodiment, this document provides a method for producing soluble heavy chain antibodies in recombinant non-human animals, comprising the steps of (a) administering an antigen to a non-human animal; (b) isolating one or more B cells from the non-human animal; (c) isolating mRNA from the one or more B cells; (d) sequencing the mRNA; (e) identifying a clonal type based on the mRNA sequence; and (f) phylogenetic analysis of the clonal type, thereby producing soluble heavy chain antibodies.

[0049] In some embodiments, the non-human animal is a mammal. In some embodiments, the mammal is a mouse.

[0050] In another embodiment, this document provides a method for producing a single-domain antibody (sdAb) identified from a recombinant non-human animal, comprising the steps of expressing a nucleic acid sequence encoding a heavy-chain variable (VH) domain containing V, D, and J in cells, wherein the cells produce the heavy-chain variable domain, and isolating the heavy-chain variable domain from a sample, thereby producing a single-domain antibody. In some embodiments, the single-domain antibody is a mouse single-domain antibody.

[0051] In some embodiments, the single-domain antibody is an IgG1 single-domain antibody derived from an IgG1 heavy-chain antibody. In some embodiments, the IgG1 single-domain antibody is an IgG1ΔCH1 nanobody derived from an IgG1ΔCH1 heavy-chain antibody.

[0052] In another embodiment, this document provides a genetically modified mouse comprising a germline modification including a deletion of a nucleic acid sequence containing one or more heavy chain C region genes; the mouse expresses a humanized IgG heavy chain antibody and secretes a humanized IgG heavy chain antibody in its serum.

[0053] In some embodiments, one or more heavy chain C region genes are IgM C region genes (Cμ), IgD C region genes (Cδ), IgE C region genes (Cε), IgG3 C region genes (Cγ3), IgG2b C region genes (Cγ2b), IgG2c C region genes (Cγ2c), or a combination thereof.

[0054] In some embodiments, the genetically modified mouse further comprises a deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene (Cγ1). In some embodiments, the deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene comprises exon 1.

[0055] In some embodiments, the germline modification further comprises a native nucleic acid sequence encoding the hinge (H) domain, heavy chain CH2 domain, and heavy chain CH3 domain of IgG1(Cγ1), or a combination thereof.

[0056] In some embodiments, the germline modification further comprises a native nucleic acid sequence containing an endogenous enhancer. In some embodiments, the enhancer is Eμ, 3'RR, 3'γ1E, 5'hsR1, or a combination thereof.

[0057] In some embodiments, the germline modification further comprises a native nucleic acid sequence including a switched tandem repeat element (Sμ) and an Iμ promoter, where Iμ drives the constitutive expression of IgG1 with a cleaved CH1 domain (IgG1ΔCH1).

[0058] In some embodiments, the humanized IgG heavy chain antibody includes a humanized IgG1 heavy chain antibody. In some embodiments, the humanized IgG1 heavy chain antibody is the IgG1ΔCH1 protein.

[0059] In some embodiments, humanized IgG heavy chain antibodies lack a light chain.

[0060] In some embodiments, the humanized IgG heavy chain antibody comprises the hinge domain, CH2 domain, and CH3 domain of IgG1(Cγ1), or a combination thereof.

[0061] In some embodiments, the mice do not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, or a combination thereof. In some embodiments, the mice do not express wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or a combination thereof.

[0062] In another embodiment, this document provides a recombinant non-human animal comprising a germline genome containing a recombinant immunoglobulin heavy chain (IgH) allele at an endogenous IgH locus; wherein the recombinant IgH allele lacks an endogenous heavy chain C region gene; and the endogenous heavy chain C region gene comprises Cμ, Cδ, Cε, Cγ3, Cγ2b, Cγ2c, or a combination thereof.

[0063] In some embodiments, the IgH allele contains a deletion in the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene (Cγ1). In some embodiments, the CH1 domain of the IgG1 C region gene contains exon 1.

[0064] In some embodiments, the IgH locus contains a native nucleic acid sequence encoding the hinge (H) domain, heavy chain CH2 domain, and heavy chain CH3 domain of IgG1(Cγ1), or a combination thereof.

[0065] In some embodiments, the IgH locus contains a native nucleic acid sequence that includes an endogenous enhancer. In some embodiments, the enhancer is Eμ, 3'RR, 3'γ1E, 5'hsR1, or a combination thereof.

[0066] In some embodiments, the IgH locus comprises a native nucleic acid sequence including a switch tandem repeat element (Sμ) and an Iμ promoter, where Iμ drives the constitutive expression of IgG1 with a cleaved CH1 domain (IgG1ΔCH1).

[0067] In some embodiments, non-human animals express humanized IgG heavy chain antibodies. In some embodiments, the humanized IgG1 heavy chain antibody comprises humanized IgG1 heavy chain antibodies. In some embodiments, the humanized IgG1 heavy chain antibody is the IgG1ΔCH1 protein.

[0068] In some embodiments, the humanized IgG1 heavy-chain antibody lacks a light chain.

[0069] In some embodiments, the humanized IgG1 heavy-chain antibody includes a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

[0070] In some embodiments, non-human animals do not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, or a combination thereof. In some embodiments, non-human animals do not express wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or a combination thereof.

[0071] In some embodiments, the IgH gene locus includes the human V, D, or J gene.

[0072] In some embodiments, recombinant non-human animals are homozygous for recombinant IgH alleles.

[0073] In some embodiments, the endogenous IgH gene locus includes an exogenous nucleic acid sequence.

[0074] In some embodiments, the exogenous nucleic acid sequence is one or more human V H Gene segment, one or more human D H Gene segment, and one or more J H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is two or more human V H Gene segment, 2 or more human D H Gene segment, and two or more J H Includes gene segments.

[0075] In some embodiments, the exogenous nucleic acid sequence is 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, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 human V HIt contains gene segments. In some embodiments, the exogenous nucleic acid sequence is 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, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 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, 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, or 126 human V H It contains gene segments. In some embodiments, the exogenous nucleic acid sequence is 1 - 5, 6 - 10, 11 - 15, 16 - 20, 21 - 25, 26 - 30, 31 - 35, 36 - 40, 41 - 45, 46 - 50, 51 - 55, 56 - 60, or 60 - 65 human V H It contains gene segments. In some embodiments, the exogenous nucleic acid sequence is 1 - 5, 6 - 10, 11 - 15, 16 - 20, 21 - 25, 26 - 30, 31 - 35, 36 - 40, 41 - 45, 46 - 50, 51 - 55, 56 - 60, 60 - 65, 66 - 70, 71 - 75, 76 - 80, 81 - 85, 86 - 90, 91 - 95, 96 - 100, 101 - 105, 106 - 110, 111 - 115, 116 - 120, or 121 - 126 human V H It contains gene segments. In some embodiments, the exogenous nucleic acid sequence is substantially all human V H It contains gene segments. In some embodiments, the exogenous nucleic acid sequence is about 10, about 20, about 30, about 40, about 50, or about 60 human V HIncludes gene segments. In some embodiments, the exogenous nucleic acid sequence contains approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 human V H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is more than 1, more than 10, more than 20, more than 30, more than 40, more than 50, or more than 60 human V H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is more than 1, more than 10, more than 20, more than 30, more than 40, more than 50, more than 60, more than 70, more than 80, more than 90, more than 100, more than 110, or more than 120 human V H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is 65 human V H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is 126 human V H Includes gene segments.

[0076] In some embodiments, the exogenous nucleic acid sequence is 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 D H It includes a gene segment. In some embodiments, the exogenous nucleic acid sequence is 1-5, 6-10, 11-15, 16-20, 21-25, or 26-27 human D H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is substantially all human D H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is approximately 5, 10, 15, 20, or 25 human D H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is more than 1, more than 5, more than 10, more than 15, more than 20, or more than 25 human D H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is 27 human D H Includes gene segments.

[0077] In some embodiments, the exogenous nucleic acid sequence is 1, 2, 3, 4, 5, or 6 human J H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is 1, 2, 3, 4, 5, 6, 7, 8, or 9 human J H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is 1 to 6 human J H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is 1 to 9 human J H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is substantially all human J H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is approximately 5 human J H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is approximately 9 human J H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is more than 1, more than 2, more than 3, more than 4, or more than 5 human J H Includes gene segments. In some embodiments, the exogenous nucleic acid sequence is more than 1, 2, 3, 4, 5, 6, 7, or 8 human J H Includes a gene segment. In some embodiments, the exogenous nucleic acid sequence is 6 J H Includes gene segments.

[0078] In some embodiments, the exogenous nucleic acid sequence is 65 human V H Gene segment, 27 human D H Gene segment, and 6 J H Includes gene segments.

[0079] In some embodiments, the exogenous nucleic acid sequence is 127 human V H Gene segment, 27 human D H Gene segment, and 9 J H Includes gene segments.

[0080] In some embodiments, the exogenous nucleic acid sequence includes a barcode.

[0081] In some embodiments, the non-human animal is a mammal. In some embodiments, the mammal is a mouse or a rat.

[0082] In another embodiment, this document provides a method for producing a genetically modified non-human animal capable of producing humanized heavy chain antibodies, comprising the steps of: (a) deleting an endogenous nucleic acid sequence containing one or more heavy chain C region genes from an endogenous immunoglobulin heavy chain gene locus in the stem cells of the non-human animal; (b) transplanting the stem cells into a blastocyst; (c) transplanting the blastocyst into a pseudo-pregnant mouse to obtain a chimeric mouse; (d) mating the chimeric mouse with a wild-type mouse to produce offspring; (e) screening the offspring for heterozygosity; and (f) identifying a founding mouse having the deletion of one or more heavy chain C region genes, wherein the non-human animal is capable of producing humanized heavy chain antibodies.

[0083] In some embodiments, the stem cells are embryonic stem cells.

[0084] In some embodiments, one or more heavy chain C region genes include Cμ, Cδ, Cγ3, Cγ2b, Cγ2c, Cε, or a combination thereof.

[0085] In some embodiments, the method further includes the step of deleting the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene. In some embodiments, the deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene includes exon 1.

[0086] In some embodiments, the method further includes the step of preserving a native nucleic acid sequence encoding the hinge (H) domain, heavy chain CH2 domain, heavy chain CH3 domain, or a combination thereof of IgG1(Cγ1).

[0087] In some embodiments, the method further includes the step of preserving a native nucleic acid sequence containing an endogenous enhancer. In some embodiments, the enhancer is Eμ, 3'RR, 3'γ1E, 5'hsR1, or a combination thereof.

[0088] In some embodiments, the method further includes the step of preserving a native nucleic acid sequence comprising a switched tandem repeat element (Sμ) and an Iμ promoter, where Iμ drives the constitutive expression of IgG1 with a cleaved CH1 domain (IgG1ΔCH1).

[0089] In some embodiments, the humanized heavy chain antibody is a humanized IgG1 heavy chain antibody. In some embodiments, the humanized IgG heavy chain antibody includes a humanized IgG1 heavy chain antibody. In some embodiments, the IgG1 heavy chain antibody is the IgG1ΔCH1 protein.

[0090] In some embodiments, the humanized IgG1 heavy-chain antibody lacks a light chain.

[0091] In some embodiments, the humanized IgG1 heavy-chain antibody includes a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

[0092] In some embodiments, non-human animals do not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or any combination thereof.

[0093] In some embodiments, the non-human animal is a mammal. In some embodiments, the mammal is a mouse.

[0094] In some embodiments, the step of deleting an endogenous nucleic acid sequence containing one or more heavy chain C region genes includes CRISPR / Cas9 genome editing.

[0095] In this embodiment, the genetically modified non-human animal is reproductive.

[0096] In some embodiments, genetically modified non-human animals have substantially normal B cell development and maturation.

[0097] In some embodiments, genetically modified non-human animals do not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or any combination thereof.

[0098] In another embodiment, this document provides a method for producing soluble humanized heavy chain antibodies in recombinant non-human animals, comprising the steps of (a) administering an antigen to a non-human animal; (b) isolating one or more B cells from the non-human animal; (c) isolating mRNA from the one or more B cells; (d) sequencing the mRNA; (e) identifying a clone type based on the mRNA sequence; and (f) phylogenetic analysis of the clone type, thereby producing soluble humanized heavy chain antibodies.

[0099] In some embodiments, the non-human animal is a mammal. In some embodiments, the mammal is a mouse or a rat.

[0100] In another embodiment, this document provides a method for producing a humanized single-domain antibody (sdAb) identified from a recombinant non-human animal, comprising the steps of: expressing a nucleic acid sequence encoding a human heavy chain variable (VH) domain comprising V, D, and J in cells, wherein the cells produce the human heavy chain variable domain; and isolating the human heavy chain variable domain from a sample, thereby producing a single-domain antibody.

[0101] In some embodiments, the single-domain antibody is a human single-domain antibody. In some embodiments, the single-domain antibody is an IgG1 single-domain antibody. In some embodiments, the IgG1 single-domain antibody is an IgG1ΔCH1 nanobody.

[0102] In some embodiments, single-domain antibodies lack a light chain.

[0103] In some embodiments, single-domain antibodies lack a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

[0104] In some embodiments, the cells are bacterial cells or human cells.

[0105] In another embodiment, this book features non-human animals, where the genome of the non-human animal includes an immunoglobulin heavy chain (IgH) allele, the IgH allele (or genome) includes an endogenous nucleic acid encoding the CH2 domain or CH3 domain of the IgG subclass, the IgH allele (or genome) lacks a nucleic acid encoding at least a portion of the endogenous CH1 domain of the IgG subclass, and the IgH allele (or genome) lacks an endogenous nucleic acid encoding at least a portion of the IgM constant domain, an endogenous nucleic acid encoding at least a portion of the IgD constant domain, an endogenous nucleic acid encoding at least a portion of the IgE constant domain, or an endogenous nucleic acid encoding at least a portion of the IgA constant domain. The IgH allele (or genome) of a non-human animal may include an endogenous nucleic acid encoding the CH2 and CH3 domains of the IgG subclass. The IgH allele (or genome) of a non-human animal may include an endogenous nucleic acid encoding the hinge domain of the IgG subclass. The IgG subclass may be the IgG2 subclass. An IgG subclass may be an IgG2a, IgG2b, IgG2c, IgG3, or IgG4 subclass. An IgG subclass may be an IgG1 subclass. An IgH allele (or genome) may lack endogenous nucleic acids encoding at least a portion of the IgG2 constant domain, at least a portion of the IgG3 constant domain, or at least a portion of the IgG4 constant domain. An IgH allele (or genome) may lack endogenous nucleic acids encoding at least a portion of the IgG2a constant domain, at least a portion of the IgG2b constant domain, at least a portion of the IgG2c constant domain, at least a portion of the IgG3 constant domain, and at least a portion of the IgG4 constant domain. An IgH allele (or genome) may lack endogenous nucleic acids encoding each of the IgG2 constant domains, each of the IgG3 constant domains, or each of the IgG4 constant domains.An IgH allele (or genome) may lack endogenous nucleic acids encoding each of the IgG2a, IgG2b, IgG2c, IgG3, or IgG4 constant domains. An IgH allele (or genome) may lack endogenous nucleic acids encoding at least a portion of the IgM, IgD, IgE, and IgA constant domains. An IgH allele (or genome) may lack endogenous nucleic acids encoding at least a portion of the IgM, IgD, IgE, or IgA constant domains. An IgH allele (or genome) may lack endogenous nucleic acids encoding each of the IgM constant domains. An IgH allele (or genome) may lack endogenous nucleic acids encoding each of the IgD constant domains. An IgH allele (or genome) may lack endogenous nucleic acids encoding each of the IgE constant domains. An IgH allele (or genome) may lack endogenous nucleic acids encoding the IgA CH1 and CH2 constant domains. An IgH allele (or genome) may lack nucleic acid encoding the endogenous CH1 domain. An IgH allele (or genome) may contain endogenous Eμ. The first nucleic acid sequence encoding the full-length CH2 domain downstream of endogenous Eμ may be the nucleic acid encoding the IgG CH2 domain. The first nucleic acid sequence encoding the full-length CH2 domain downstream of endogenous Eμ may be the nucleic acid encoding the IgG1 CH2 domain. An IgH allele (or genome) may include an endogenous Sμ, an endogenous Iμ promoter, an endogenous Iμ exon, or a combination thereof. The first nucleic acid sequence encoding the full-length CH2 domain downstream of the endogenous Sμ, endogenous Iμ promoter, or endogenous Iμ exon may be the nucleic acid encoding the IgG CH2 domain.The first nucleic acid sequence encoding the endogenous Sμ, the endogenous Iμ promoter, or the full-length CH2 domain downstream of the endogenous Iμ exon may be the nucleic acid encoding the IgG1 CH2 domain. The IgH allele (or genome) may contain endogenous 3'γ1E. The IgH allele (or genome) may lack the endogenous nucleic acid encoding the full-length CH2 domain downstream of endogenous 3'γ1E. The IgH allele (or genome) may contain endogenous 5'hsR1. The first nucleic acid sequence encoding the full-length CH2 domain upstream of endogenous 5'hsR1 may be the nucleic acid encoding the IgG CH2 domain. The first nucleic acid sequence encoding the full-length CH2 domain upstream of endogenous 5'hsR1 may be the nucleic acid encoding the IgG1 CH2 domain. The IgH allele (or genome) may contain endogenous 3'RR. The first nucleic acid sequence encoding the full-length CH2 domain upstream of the endogenous 3'RR may be the nucleic acid encoding the IgG CH2 domain. The first nucleic acid sequence encoding the full-length CH2 domain upstream of the endogenous 3'RR may be the nucleic acid encoding the IgG1 CH2 domain. The IgH allele (or genome) may contain the endogenous 3'CBE. The first nucleic acid sequence encoding the full-length CH2 domain upstream of the endogenous 3'CBE may be the nucleic acid encoding the IgG CH2 domain. The first nucleic acid sequence encoding the full-length CH2 domain upstream of the endogenous 3'CBE may be the nucleic acid encoding the IgG1 CH2 domain. At least one allele of the genome may lack at least a portion of the endogenous Ig heavy chain variable region. At least one allele of the genome may lack all of the exons of the endogenous Ig heavy chain variable region. Both alleles of the genome may lack all of the exons of the endogenous Ig heavy chain variable region. No allele of the genome may contain exogenous exons of the Ig heavy chain variable region. Non-human animals may be non-human animals that do not produce Ig heavy chains. An IgH allele (or genome) may contain one or more exogenous nucleic acids encoding human Ig heavy chain variable region gene segments. An IgH allele (or genome) may contain one or more exogenous human Ig VH gene segments. An IgH allele (or genome) may contain three or more human Ig VH gene segments. An IgH allele (or genome) may contain 26 or more human Ig VH gene segments.An IgH allele (or genome) may contain 65 or more human Ig VH gene segments. An IgH allele (or genome) may contain 126 human Ig VH gene segments. An IgH allele (or genome) may contain 13 or more Ig VD gene segments. An IgH allele (or genome) may contain 27 human Ig VD gene segments. An IgH allele (or genome) may contain 3 or more human Ig VJ gene segments. An IgH allele (or genome) may contain 9 human Ig VJ gene segments. The genome may contain 126 human Ig VH gene segments, 27 or more human Ig VD gene segments, and 9 human Ig VJ gene segments. Non-human animals may produce human-non-human chimeric Ig heavy chain antibodies. The variable region domain of human-non-human chimeric Ig heavy chain antibodies may be fully human. An IgH allele (or genome) may contain one or more exogenous nucleic acids encoding human Ig light chain variable region gene segments. An IgH allele (or genome) may contain one or more exogenous human Igκ variable gene segments. An IgH allele (or genome) may contain 20 or more exogenous human Igκ variable gene segments. An IgH allele (or genome) may contain 40 exogenous human Igκ variable gene segments. An IgH allele (or genome) may contain one or more exogenous human Igλ variable gene segments. An IgH allele (or genome) may contain 10 or more exogenous human Igλ variable gene segments. An IgH allele (or genome) may contain 20 exogenous human Igλ variable gene segments. An IgH allele (or genome) may contain one or more human Igκ VJ gene segments. An IgH allele (or genome) may contain 5 human Igκ VJ gene segments. An IgH allele (or genome) may contain one or more human Igλ VJ gene segments. An IgH allele (or genome) may contain four human Igλ VJ gene segments. An IgH allele (or genome) may contain 40 human Igκ variable gene segments and 5 human Igκ VJ gene segments. An IgH allele (or genome) may contain 20 human Igλ variable gene segments and 4 human Igλ VJ gene segments.Non-human animals can produce human-non-human chimeric Ig heavy chain antibodies. The variable region domain of the human-non-human chimeric Ig heavy chain antibody may be fully human, derived from the light chain. The non-human animal may be a non-human animal of a first non-human species, and the IgH allele (or genome) may contain exogenous nucleic acids encoding one or more Ig heavy chain variable region gene segments of a second non-human species distinct from the first non-human species. The IgH allele (or genome) may contain one or more Ig VH gene segments of the second non-human species. The IgH allele (or genome) may contain ten or more Ig VH gene segments of the second non-human species. The IgH allele (or genome) may contain all of the Ig VH gene segments of the second non-human species. The IgH allele (or genome) may contain three or more Ig VD gene segments of the second non-human species. An IgH allele (or genome) may contain all Ig VD gene segments of a second non-human species. An IgH allele (or genome) may contain three or more Ig VJ gene segments of a second non-human species. An IgH allele (or genome) may contain all Ig VJ gene segments of a second non-human species. An IgH allele (or genome) may contain all Ig VH gene segments, Ig VD gene segments, and Ig VJ gene segments of a second non-human species. A non-human animal can produce a chimeric heavy chain antibody of the first and second species. The variable region domain of the chimeric heavy chain antibody may be entirely the variable region domain of the second species. The first species may be a mouse. The second species may be a cattle, shark, or alpaca. An IgH allele (or genome) may contain at least one exogenous recombinase site-recognizing nucleic acid sequence. At least one exogenous recombinase site-recognizing nucleic acid sequence may be located upstream of an endogenous nucleic acid encoding a CH2 or CH3 domain of an IgG subclass. An IgH allele (or genome) may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different exogenous recombinase site-recognizing nucleic acid sequences. An IgH allele (or genome) may contain at least 3 different exogenous recombinase site-recognizing nucleic acid sequences. An IgH allele (or genome) may contain at least 5 different exogenous recombinase site-recognizing nucleic acid sequences.Each of the different exogenous recombinase site-recognizing nucleic acid sequences is located less than 2.5 Mb upstream of endogenous Eμ. Each of the different exogenous recombinase site-recognizing nucleic acid sequences is located less than 2.0 Mb, less than 1.5 Mb, less than 1.0 Mb, less than 500 kb, or less than 250 kb upstream of endogenous Eμ. Each of the different exogenous recombinase site-recognizing nucleic acid sequences is located less than 200 kb, less than 100 kb, less than 50 kb, less than 25 kb, or less than 10 kb upstream of endogenous Eμ. Each of the different exogenous recombinase site-recognizing nucleic acid sequences may be located less than 500 kb upstream of endogenous Eμ. Each of the different exogenous recombinase site-recognizing nucleic acid sequences may be located less than 250 kb upstream of endogenous Eμ. Each of the different exogenous recombinase site-recognizing nucleic acid sequences may be located less than 200 kb upstream of the endogenous Eμ.

[0106] In another embodiment, this document describes DNA comprising a genetically modified non-human immunoglobulin heavy chain (IgH) allele, wherein the genetically modified non-human IgH allele lacks one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains, including the CH1 constant domain of an IgG subclass, an IgM constant domain, an IgD constant domain, an IgE constant domain, an IgA constant domain, or any combination thereof. The DNA may be germline genomic DNA. The genetically modified non-human IgH allele may lack one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains, including the CH1 constant domain of an IgG subclass. The IgG subclass may include the IgG1, IgG2a, IgG2b, IgG2c, IgG3, or IgG4 subclasses. The IgG subclass may be the IgG1 subclass. The genetically modified non-human IgH allele may include a nucleic acid sequence (Cγ1-ΔCH1) encoding a CH1-cleaved IgG1 constant domain (IgG1ΔCH1). A genetically modified non-human IgH allele may contain nucleic acid sequences encoding the hinge (H) domain, CH2 domain, CH3 domain, or any combination thereof of an IgG subclass. A genetically modified non-human IgH allele may lack one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains, including the IgG2 constant domain, IgG3 constant domain, IgG4 constant domain, or any combination thereof. A genetically modified non-human IgH allele may contain one or more endogenous enhancers, including Eμ, 3'γ1E, 5'hsR1, 3'RR, or any combination thereof. A genetically modified non-human IgH allele may contain the Iμ promoter, Iμ exon, or both. A genetically modified non-human IgH allele may contain a switched tandem repeat element (Sμ). IgG1 expression may be driven by Eμ, the Iμ promoter, Sμ, or any combination thereof. Genetically modified non-human IgH alleles may lack one or more endogenous switch regions, including Sγ3, Sγ1, Sγ2b, Sγ2c, Sε, Sα, or any combination thereof.A genetically modified non-human IgH allele may contain the following components (5' to 3'): Eμ, Iμ promoter, Iμ exon, Sμ, Cγ1-ΔCH1, 3'γ1E, 5'hsR1, and 3'RR. A genetically modified non-human IgH allele may contain a flippase recognition target (frt) site. A genetically modified non-human IgH allele may contain an endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof. A genetically modified non-human IgH allele may lack at least one endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof. A genetically modified non-human IgH allele may contain a docking cassette. The docking cassette may contain left and right homology arms, a frt site, an attB site, a promoter, a loxP site, a nucleic acid sequence encoding a selection marker, or any combination thereof. The docking cassette may contain a nucleic acid sequence encoding a selection marker. The selection marker may include geneticin, hydromycin, puromycin, or any combination thereof. The genetically modified non-human IgH allele may encode an IgG heavy chain antibody. The genetically modified non-human IgH allele may include an exogenous V gene segment, an exogenous D gene segment, an exogenous J gene segment, or any combination thereof. The exogenous gene segment can be selected from the group consisting of human gene segments, mouse gene segments, rat gene segments, bovine gene segments, alpaca gene segments, and shark gene segments. The exogenous gene segment may include a human gene segment. The genetically modified non-human IgH allele may include one or more human VH gene segments, one or more human DH gene segments, and one or more human JH gene segments. The genetically modified non-human IgH allele may include at least 10, 20, 30, 40, 50, 60, 80, 100, 120, or 126 human VH gene segments. A genetically modified non-human IgH allele may contain at least 10, 15, 20, 25, or 27 human DH gene segments. A genetically modified non-human IgH allele may contain at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 human JH gene segments.A genetically modified non-human IgH allele may contain 126 human VH gene segments, 27 human DH gene segments, and 9 human JH gene segments. A genetically modified non-human IgH allele may contain one or more bovine gene segments. One or more bovine gene segments may contain an L1 exon, an L2 exon of IGHV1-7, a coding segment of IGHD8-2, a coding sequence of IGHJ2-4, an IGH2-4 splice donor, or any combination thereof. One or more bovine gene segments may contain IGHD4-1, IGHD5-3, IGHD8-2, IGHD1-3, IGHD7-3, IGHD7-4, IGHD6-3, IGHD3-3, or any combination thereof. One or more bovine gene segments may contain nucleic acid sequences selected from sequence numbers 42-49 and 57. The DNA may contain one or more human VH gene segments. The DNA may contain one or more human JH gene segments. A genetically modified non-human IgH allele may contain one or more alpaca gene segments. These alpaca gene segments may include VHH3-1, VHH3-S1, VHH3-S2, VHH3-S9, VHH3-S10, or any combination thereof. The alpaca gene segments may contain nucleic acid sequences selected from sequence numbers 50-54. The DNA may contain one or more human VH gene segments. The DNA may contain one or more human JH gene segments. A genetically modified non-human IgH allele may contain one or more shark gene segments. These shark gene segments may include VNAR-L38968, VNAR-L38967, or both. The shark gene segments may contain nucleic acid sequences selected from sequence numbers 55-56. The DNA may contain one or more human VH gene segments. The DNA may contain one or more human JH gene segments. A genetically modified non-human IgH allele may encode an IgG heavy chain antibody, which may contain a kappa light chain variable domain, a lambda light chain variable domain, or both. The genetically modified non-human IgH allele may contain one or more exogenous human lambda light chain (LV) gene segments.One or more human LV gene segments may include CH17-262M19, CH17-329P5, CH17-238D3, CH17-261A15, CH17-264L24, CH17-117C7, RP11-1040J16, CH17-320F4, or any combination thereof. Genetically modified non-human IgH alleles may include one or more exogenous human kappa light chain (KV) gene segments. One or more human KV gene segments may include CH17-272M2, CH17-405H5, CH17-140P2, CH17-13E7, CH17-84J8, CH17-53L15, or any combination thereof. DNA may include one or more human VH gene segments. DNA may include one or more human JH gene segments.

[0107] In another embodiment, this document describes genetically modified cells comprising DNA containing a genetically modified non-human immunoglobulin heavy chain (IgH) allele, wherein the genetically modified non-human IgH allele lacks one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains, including the CH1 constant domain of the IgG subclass, the IgM constant domain, the IgD constant domain, the IgE constant domain, the IgA constant domain, or any combination thereof. The DNA may be germline genomic DNA. The genetically modified non-human IgH allele may lack one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains, including the CH1 constant domain of the IgG subclass. The IgG subclass may include the IgG1, IgG2a, IgG2b, IgG2c, IgG3, or IgG4 subclasses. The IgG subclass may be the IgG1 subclass. The genetically modified non-human IgH allele may contain a nucleic acid sequence (Cγ1-ΔCH1) encoding a CH1-cleaved IgG1 constant domain (IgG1ΔCH1). A genetically modified non-human IgH allele may contain nucleic acid sequences encoding the hinge (H) domain, CH2 domain, CH3 domain, or any combination thereof of an IgG subclass. A genetically modified non-human IgH allele may lack one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains, including the IgG2 constant domain, IgG3 constant domain, IgG4 constant domain, or any combination thereof. A genetically modified non-human IgH allele may contain one or more endogenous enhancers, including Eμ, 3'γ1E, 5'hsR1, 3'RR, or any combination thereof. A genetically modified non-human IgH allele may contain the Iμ promoter, Iμ exon, or both. A genetically modified non-human IgH allele may contain a switched tandem repeat element (Sμ). IgG1 expression may be driven by Eμ, the Iμ promoter, Sμ, or any combination thereof. Genetically modified non-human IgH alleles may lack one or more endogenous switch regions, including Sγ3, Sγ1, Sγ2b, Sγ2c, Sε, Sα, or any combination thereof.A genetically modified non-human IgH allele may contain the following components (5' to 3'): Eμ, Iμ promoter, Iμ exon, Sμ, Cγ1-ΔCH1, 3'γ1E, 5'hsR1, and 3'RR. A genetically modified non-human IgH allele may contain a flippase recognition target (frt) site. A genetically modified non-human IgH allele may contain an endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof. A genetically modified non-human IgH allele may lack at least one endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof. A genetically modified non-human IgH allele may contain a docking cassette. The docking cassette may contain left and right homology arms, a frt site, an attB site, a promoter, a loxP site, a nucleic acid sequence encoding a selection marker, or any combination thereof. The docking cassette may contain a nucleic acid sequence encoding a selection marker. The selection marker may include geneticin, hydromycin, puromycin, or any combination thereof. The genetically modified non-human IgH allele may encode an IgG heavy chain antibody. The genetically modified non-human IgH allele may include an exogenous V gene segment, an exogenous D gene segment, an exogenous J gene segment, or any combination thereof. The exogenous gene segment can be selected from the group consisting of human gene segments, mouse gene segments, rat gene segments, bovine gene segments, alpaca gene segments, and shark gene segments. The exogenous gene segment may include a human gene segment. The genetically modified non-human IgH allele may include one or more human VH gene segments, one or more human DH gene segments, and one or more human JH gene segments. The genetically modified non-human IgH allele may include at least 10, 20, 30, 40, 50, 60, 80, 100, 120, or 126 human VH gene segments. A genetically modified non-human IgH allele may contain at least 10, 15, 20, 25, or 27 human DH gene segments. A genetically modified non-human IgH allele may contain at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 human JH gene segments.A genetically modified non-human IgH allele may contain 126 human VH gene segments, 27 human DH gene segments, and 9 human JH gene segments. A genetically modified non-human IgH allele may contain one or more bovine gene segments. One or more bovine gene segments may contain an L1 exon, an L2 exon of IGHV1-7, a coding segment of IGHD8-2, a coding sequence of IGHJ2-4, an IGH2-4 splice donor, or any combination thereof. One or more bovine gene segments may contain IGHD4-1, IGHD5-3, IGHD8-2, IGHD1-3, IGHD7-3, IGHD7-4, IGHD6-3, IGHD3-3, or any combination thereof. One or more bovine gene segments may contain nucleic acid sequences selected from sequence numbers 42-49 and 57. The DNA may contain one or more human VH gene segments. The DNA may contain one or more human JH gene segments. A genetically modified non-human IgH allele may contain one or more alpaca gene segments. These alpaca gene segments may include VHH3-1, VHH3-S1, VHH3-S2, VHH3-S9, VHH3-S10, or any combination thereof. The alpaca gene segments may contain nucleic acid sequences selected from sequence numbers 50-54. The DNA may contain one or more human VH gene segments. The DNA may contain one or more human JH gene segments. A genetically modified non-human IgH allele may contain one or more shark gene segments. These shark gene segments may include VNAR-L38968, VNAR-L38967, or both. The shark gene segments may contain nucleic acid sequences selected from sequence numbers 55-56. The DNA may contain one or more human VH gene segments. The DNA may contain one or more human JH gene segments. A genetically modified non-human IgH allele may encode an IgG heavy chain antibody, which may contain a kappa light chain variable domain, a lambda light chain variable domain, or both. The genetically modified non-human IgH allele may contain one or more exogenous human lambda light chain (LV) gene segments.One or more human LV gene segments may include CH17-262M19, CH17-329P5, CH17-238D3, CH17-261A15, CH17-264L24, CH17-117C7, RP11-1040J16, CH17-320F4, or any combination thereof. Genetically modified non-human IgH alleles may include one or more exogenous human kappa light chain (KV) gene segments. One or more human KV gene segments may include CH17-272M2, CH17-405H5, CH17-140P2, CH17-13E7, CH17-84J8, CH17-53L15, or any combination thereof. DNA may include one or more human VH gene segments. DNA may include one or more human JH gene segments. The cells may be non-human animal cells. The cells may be mammalian cells. Mammalian cells may be from mice, rats, cattle, alpacas, cats, dogs, rabbits, pigs, monkeys, or chimpanzees. The cells may be mouse cells. The cells may be shark cells. The cells may be human cells. The cells may be stem cells. Stem cells may be embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). The cells may be B cells.

[0108] In another aspect, this book features genetically modified non-human animals, where the genetically modified non-human animals include the cells described in the previous paragraph. Non-human animals may be mammals. Mammals may be mice, rats, cattle, alpacas, cats, dogs, rabbits, pigs, monkeys, or chimpanzees. Non-human animals may be mice. Genetically modified non-human animals may include cells expressing IgG heavy chain antibodies. IgG heavy chain antibodies may be secreted into the serum of genetically modified non-human animals. IgG heavy chain antibodies may be CH1-cleaved IgG1 heavy chain antibodies (IgG1ΔCH1). IgG heavy chain antibodies may lack a light chain. IgG heavy chain antibodies may include a hinge domain, a CH2 domain, a CH3 domain, or any combination thereof. Cells expressing IgG heavy chain antibodies may be cells that do not express IgM antibodies, IgD antibodies, IgE antibodies, IgG3 antibodies, IgG2b antibodies, IgG2c antibodies, IgA antibodies, or any combination thereof. IgG heavy chain antibodies may be human IgG heavy chain antibodies. IgG heavy chain antibodies may contain exogenous variable domains selected from the group consisting of human variable domains, mouse variable domains, rat variable domains, bovine variable domains, alpaca variable domains, and shark variable domains. IgG heavy chain antibodies may contain kappa light chain variable domains, lambda light chain variable domains, or both.

[0109] In another aspect, this book features methods for creating genetically modified non-human animals. The above method comprises: (a) deleting one or more nucleic acid sequences from a non-human immunoglobulin heavy chain (IgH) allele, wherein the one or more nucleic acid sequences deleted encode at least a portion of one or more endogenous constant domains, including the CH1 constant domain of the IgG subclass, the IgM constant domain, the IgD constant domain, the IgE constant domain, the IgA constant domain, or any combination thereof, thereby generating a genetically modified non-human IgH allele in germline genomic DNA; (b) transplanting the cells containing the germline genomic DNA into a blastocyst; (c) transplanting the blastocyst into a pseudo-pregnant non-human animal to obtain a chimeric non-human animal; (d) mating the chimeric non-human animal with a wild-type non-human animal to produce offspring; (e) screening the offspring for heterozygosity; and (f) identifying a genetically modified non-human animal having one or more deletions of nucleic acid sequences and capable of producing heavy chain antibodies. A genetically modified non-human animal may be the genetically modified non-human animal described in the preceding paragraph. The step of deleting one or more nucleic acid sequences may include the step of using a CRISPR / Cas genome editing system. The CRISPR / Cas genome editing system may include at least one guide RNA (gRNA) targeting an endogenous heavy chain C region gene and a Cas protein. The Cas protein may include the Cas9 protein. The one or more nucleic acid sequences to be deleted may encode the CH1 constant domain, IgG3 constant domain, IgM constant domain, and IgD constant domain of IgG1. The one or more nucleic acid sequences to be deleted may encode the IgG2 constant domain and IgA constant domain. The step of deleting nucleic acid sequences may include the step of removing a selection marker from a non-human IgH allele using transient expression of Flp recombinase. The one or more nucleic acid sequences to be deleted may encode the CH1 constant domain, IgM constant domain, IgD constant domain, IgE constant domain, and IgA constant domain of an IgG subclass.The above method may include the step of deleting a nucleic acid sequence from a non-human IgH allele, wherein the nucleic acid sequence includes an endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof. The above method may include the step of inserting a docking cassette. The above method may include the step of bringing the docking cassette into contact with a bacterial artificial chromosome (BAC), wherein the BAC is exogenous V. H , D H , and J H The method includes a nucleic acid sequence containing a gene segment. The method may include the step of inserting an exogenous gene segment into a docketing cassette. The exogenous gene segment may be a human gene segment.

[0110] In another embodiment, this book features genetically modified non-human animals, which were created using the method described in the previous paragraph.

[0111] In another embodiment, this document features a method for preparing germline genomic DNA, the method comprising the step of deleting one or more nucleic acid sequences from a non-human immunoglobulin heavy chain (IgH) allele, wherein the one or more nucleic acid sequences to be deleted encode at least a portion of one or more endogenous constant domains, including the CH1 constant domain of an IgG subclass, an IgM constant domain, an IgD constant domain, an IgE constant domain, an IgA constant domain, or any combination thereof, thereby generating a genetically modified non-human IgH allele in germline genomic DNA. The germline genomic DNA may include DNA containing the genetically modified non-human IgH allele. The genetically modified non-human IgH allele may lack one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains, including the CH1 constant domain of an IgG subclass. The IgG subclass may include the IgG1, IgG2a, IgG2b, IgG2c, IgG3, or IgG4 subclasses. The IgG subclass may be the IgG1 subclass. A genetically modified non-human IgH allele may contain a nucleic acid sequence (Cγ1-ΔCH1) encoding a CH1-cleaved IgG1 constant domain (IgG1ΔCH1). A genetically modified non-human IgH allele may contain a nucleic acid sequence encoding a hinge (H) domain, CH2 domain, CH3 domain, or any combination thereof of an IgG subclass. A genetically modified non-human IgH allele may lack one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains, including the IgG2 constant domain, IgG3 constant domain, IgG4 constant domain, or any combination thereof. A genetically modified non-human IgH allele may contain one or more endogenous enhancers, including Eμ, 3'γ1E, 5'hsR1, 3'RR, or any combination thereof. A genetically modified non-human IgH allele may contain an Iμ promoter, an Iμ exon, or both. A genetically modified non-human IgH allele may contain a switched tandem repeat element (Sμ). IgG1 expression can be driven by the Eμ, Iμ promoter, Sμ, or any combination thereof.A genetically modified non-human IgH allele may lack one or more endogenous switch regions, including Sγ3, Sγ1, Sγ2b, Sγ2c, Sε, Sα, or any combination thereof. A genetically modified non-human IgH allele may contain the following components (5' to 3'): Eμ, Iμ promoter, Iμ exon, Sμ, Cγ1-ΔCH1, 3'γ1E, 5'hsR1, and 3'RR. A genetically modified non-human IgH allele may contain a flippase recognition target (frt) site. A genetically modified non-human IgH allele may contain an endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof. A genetically modified non-human IgH allele may lack at least one endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof. A genetically modified non-human IgH allele may contain a docking cassette. The docking cassette may include left and right homology arms, frt site, attB site, promoter, loxP site, nucleic acid sequence encoding a selection marker, or any combination thereof. The docking cassette may include nucleic acid sequence encoding a selection marker. The selection marker may include geneticin, hydromycin, puromycin, or any combination thereof. The genetically modified non-human IgH allele may encode an IgG heavy chain antibody. The genetically modified non-human IgH allele may include an exogenous V gene segment, an exogenous D gene segment, an exogenous J gene segment, or any combination thereof. The exogenous gene segment can be selected from the group consisting of human gene segments, mouse gene segments, rat gene segments, bovine gene segments, alpaca gene segments, and shark gene segments. The exogenous gene segment may include a human gene segment. The genetically modified non-human IgH allele may include one or more human VH gene segments, one or more human DH gene segments, and one or more human JH gene segments. A genetically modified non-human IgH allele may contain at least 10, 20, 30, 40, 50, 60, 80, 100, 120, or 126 human VH gene segments.A genetically modified non-human IgH allele may contain at least 10, 15, 20, 25, or 27 human DH gene segments. A genetically modified non-human IgH allele may contain at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 human JH gene segments. A genetically modified non-human IgH allele may contain 126 human VH gene segments, 27 human DH gene segments, and 9 human JH gene segments. A genetically modified non-human IgH allele may contain one or more bovine gene segments. One or more bovine gene segments may contain an L1 exon, an L2 exon of IGHV1-7, a coding segment of IGH8-2, a coding sequence of IGHJ2-4, an IGH2-4 splice donor, or any combination thereof. One or more bovine gene segments may include IGHD4-1, IGHD5-3, IGHD8-2, IGHD1-3, IGHD7-3, IGHD7-4, IGHD6-3, IGHD3-3, or any combination thereof. One or more bovine gene segments may include nucleic acid sequences selected from SEQ ID NOs. 42-49 and 57. The DNA may include one or more human VH gene segments. The DNA may include one or more human JH gene segments. The genetically modified non-human IgH allele may include one or more alpaca gene segments. One or more alpaca gene segments may include VHH3-1, VHH3-S1, VHH3-S2, VHH3-S9, VHH3-S10, or any combination thereof. The alpaca gene segments may include nucleic acid sequences selected from SEQ ID NOs. 50-54. The DNA may include one or more human VH gene segments. The DNA may contain one or more human JH gene segments. The genetically modified non-human IgH allele may contain one or more shark gene segments. The one or more shark gene segments may contain VNAR-L38968, VNAR-L38967, or both. The shark gene segments may contain nucleic acid sequences selected from SEQ ID NOs. 55-56. The DNA may contain one or more human VH gene segments. The DNA may contain one or more human JH gene segments.A genetically modified non-human IgH allele may encode an IgG heavy chain antibody, which may contain a kappa light chain variable domain, a lambda light chain variable domain, or both. A genetically modified non-human IgH allele may contain one or more exogenous human lambda light chain (LV) gene segments. One or more human LV gene segments may include CH17-262M19, CH17-329P5, CH17-238D3, CH17-261A15, CH17-264L24, CH17-117C7, RP11-1040J16, CH17-320F4, or any combination thereof. A genetically modified non-human IgH allele may contain one or more exogenous human kappa light chain (KV) gene segments. One or more human KV gene segments may include CH17-272M2, CH17-405H5, CH17-140P2, CH17-13E7, CH17-84J8, CH17-53L15, or any combination thereof. The DNA may include one or more human VH gene segments. The DNA may include one or more human JH gene segments. The IgG constant domain may include the constant domain of an IgG subclass. The IgG subclass may include IgG1, IgG2a, IgG2b, IgG2c, IgG3, or IgG4 subclasses.

[0112] In another embodiment, this document features a method for producing IgG heavy chain antibodies in genetically modified non-human animals. The method comprises (a) administering an antigen to any of the genetically modified non-human animals described in the preceding paragraph; (b) isolating one or more B cells from the genetically modified non-human animal; (c) isolating mRNA from the one or more B cells; and (d) producing IgG heavy chain antibodies. The genetically modified non-human animal may include DNA containing a genetically modified non-human immunoglobulin heavy chain (IgH) allele, wherein the genetically modified non-human IgH allele lacks one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains, including the CH1 constant domain of the IgG subclass, the IgM constant domain, the IgD constant domain, the IgE constant domain, the IgA constant domain, or any combination thereof. The DNA may be germline genomic DNA. The genetically modified non-human IgH allele may lack one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains, including the CH1 constant domain of the IgG subclass. The IgG subclass may include the IgG1, IgG2a, IgG2b, IgG2c, IgG3, or IgG4 subclasses. The IgG subclass may be the IgG1 subclass. The genetically modified non-human IgH allele may include a nucleic acid sequence (Cγ1-ΔCH1) encoding the CH1-cleaved IgG1 constant domain (IgG1ΔCH1). The genetically modified non-human IgH allele may include a nucleic acid sequence encoding the hinge (H) domain, CH2 domain, CH3 domain, or any combination thereof of an IgG subclass. The genetically modified non-human IgH allele may lack one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains, including the IgG2 constant domain, IgG3 constant domain, IgG4 constant domain, or any combination thereof. The genetically modified non-human IgH allele may include one or more endogenous enhancers, including Eμ, 3'γ1E, 5'hsR1, 3'RR, or any combination thereof. Genetically modified non-human IgH alleles may contain an Iμ promoter, an Iμ exon, or both. Genetically modified non-human IgH alleles may contain a switched tandem repeat element (Sμ).IgG1 expression may be driven by Eμ, the Iμ promoter, Sμ, or any combination thereof. Modified non-human IgH alleles may lack one or more endogenous switch regions, including Sγ3, Sγ1, Sγ2b, Sγ2c, Sε, Sα, or any combination thereof. Modified non-human IgH alleles may contain the following components (5' to 3'): Eμ, the Iμ promoter, the Iμ exon, Sμ, Cγ1-ΔCH1, 3'γ1E, 5'hsR1, and 3'RR. Modified non-human IgH alleles may contain a flippase recognition target (frt) site. Modified non-human IgH alleles may contain an endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof. Modified non-human IgH alleles may lack at least one endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof. A genetically modified non-human IgH allele may include a docking cassette. The docking cassette may include left and right homology arms, a frt site, an attB site, a promoter, a loxP site, a nucleic acid sequence encoding a selection marker, or any combination thereof. The docking cassette may include a nucleic acid sequence encoding a selection marker. The selection marker may include geneticin, hydromycin, puromycin, or any combination thereof. A genetically modified non-human IgH allele may encode an IgG heavy chain antibody. A genetically modified non-human IgH allele may include an exogenous V gene segment, an exogenous D gene segment, an exogenous J gene segment, or any combination thereof. The exogenous gene segment can be selected from the group consisting of human gene segments, mouse gene segments, rat gene segments, bovine gene segments, alpaca gene segments, and shark gene segments. The exogenous gene segment may include a human gene segment. A genetically modified non-human IgH allele may contain one or more human VH gene segments, one or more human DH gene segments, and one or more human JH gene segments.A genetically modified non-human IgH allele may contain at least 10, 20, 30, 40, 50, 60, 80, 100, 120, or 126 human VH gene segments. A genetically modified non-human IgH allele may contain at least 10, 15, 20, 25, or 27 human DH gene segments. A genetically modified non-human IgH allele may contain at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 human JH gene segments. A genetically modified non-human IgH allele may contain 126 human VH gene segments, 27 human DH gene segments, and 9 human JH gene segments. A genetically modified non-human IgH allele may contain one or more bovine gene segments. One or more bovine gene segments may contain L1 exon, L2 exon of IGHV1-7, coding segment of IGHD8-2, coding sequence of IGHJ2-4, IGH2-4 splice donor, or any combination thereof. One or more bovine gene segments may contain IGHD4-1, IGHD5-3, IGHD8-2, IGHD1-3, IGHD7-3, IGHD7-4, IGHD6-3, IGHD3-3, or any combination thereof. One or more bovine gene segments may contain nucleic acid sequences selected from SEQ ID NOs.42-49 and 57. The DNA may contain one or more human VH gene segments. The DNA may contain one or more human JH gene segments. The genetically modified non-human IgH allele may contain one or more alpaca gene segments. One or more alpaca gene segments may include VHH3-1, VHH3-S1, VHH3-S2, VHH3-S9, VHH3-S10, or any combination thereof. The alpaca gene segments may include nucleic acid sequences selected from SEQ ID NOs. 50-54. The DNA may include one or more human VH gene segments. The DNA may include one or more human JH gene segments. The genetically modified non-human IgH allele may include one or more shark gene segments. One or more shark gene segments may include VNAR-L38968, VNAR-L38967, or both. The shark gene segments may include nucleic acid sequences selected from SEQ ID NOs. 55-56. The DNA may include one or more human VH gene segments.The DNA may contain one or more human JH gene segments. The genetically modified non-human IgH allele may encode an IgG heavy chain antibody, which may contain a kappa light chain variable domain, a lambda light chain variable domain, or both. The genetically modified non-human IgH allele may contain one or more exogenous human lambda light chain (LV) gene segments. One or more human LV gene segments may include CH17-262M19, CH17-329P5, CH17-238D3, CH17-261A15, CH17-264L24, CH17-117C7, RP11-1040J16, CH17-320F4, or any combination thereof. The genetically modified non-human IgH allele may contain one or more exogenous human kappa light chain (KV) gene segments. One or more human KV gene segments may include CH17-272M2, CH17-405H5, CH17-140P2, CH17-13E7, CH17-84J8, CH17-53L15, or any combination thereof. The DNA may include one or more human VH gene segments. The DNA may include one or more human JH gene segments. The above method may include the step of sequencing mRNA isolated from one or more B cells. The above method may include the step of identifying a clonal type based on the above mRNA sequence. The above method may include the step of performing phylogenetic analysis of the above clonal type. The IgG heavy chain antibody may be a humanized IgG heavy chain antibody. The IgG heavy chain antibody may be an IgG heavy chain antibody containing a human variable region and a non-human constant region.

[0113] In another aspect, this book features IgG heavy chain antibodies, which are produced by the method described in the previous paragraph.

[0114] In another embodiment, this work features a recombinant vector system comprising at least one nucleic acid construct encoding a CRISPR / Cas genome editing system comprising a Cas protein and at least one guide RNA (gRNA), wherein the Cas protein and at least one gRNA form a complex that deletes one or more nucleic acid sequences from a non-human immunoglobulin heavy chain (IgH) allele, the one or more nucleic acid sequences to be deleted encoding at least a portion of one or more endogenous constant domains, including the CH1 constant domain of an IgG subclass, an IgM constant domain, an IgD constant domain, an IgE constant domain, an IgA constant domain, or any combination thereof.

[0115] In another embodiment, this document features an antibody comprising a variable region including (a) SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 19, or (b) SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 20. The antibody may bind to the SARS-CoV-2 spike polypeptide. The antibody may be a heavy chain antibody. The antibody may be a single-domain antibody.

[0116] In another embodiment, this document features an antibody containing a variable region including (a) SEQ ID NO: 4, SEQ ID NO: 10, and SEQ ID NO: 19 (SEQ ID NO: 19 is expected to lack the first C residue and the last W residue), or (b) SEQ ID NO: 5, SEQ ID NO: 11, and SEQ ID NO: 20 (SEQ ID NO: 20 is expected to lack the first C residue and the last W residue). The antibody may bind to the SARS-CoV-2 spike polypeptide. The antibody may be a heavy chain antibody. The antibody may be a single-domain antibody.

[0117] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which this disclosure relates. Methods and materials are described herein for use in this disclosure; other preferred methods and materials known in the art may also be used. Materials, methods and examples are illustrative and not intended to limit the scope. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, including definitions, this specification shall prevail.

[0118] Details of one or more embodiments of the present invention are shown in the accompanying drawings and the following specification. Other features, purposes and advantages of the present invention will become apparent from the specification and drawings and from the claims. [Brief explanation of the drawing]

[0119] [Figure 1] Figures 1A-1D show the production of heavy chain-only antibodies from Singularity Musculus mice. (Figure 1A) Genomic structure of the Igh locus in wild-type mice. The mouse VH, DH, and JH, as well as the CH genes, are shown in dark or light colored boxes along with the intronic enhancer Eμ and super-enhancer 3'RR in ovals. (Figure 1B) Recombinant Singularity Musculus (SM) allele with all other CH genes and the CH1 exon deleted. (Figure 1C) Tetrameric mouse IgG1 produced from the WT allele. (Figure 1D) CH1-cleaved heavy chain-only IgG1 is produced from the Singularity Musculus allele, from which nanobodies can be induced. [Figure 2] Figure 2 shows an exemplary genomic locus in mouse Igh. A schematic diagram of the approximately 220kb CH region containing the indicated regulatory element is shown. [Figure 3]Figures 3A-3E show the generation of the Singularity Musculus allele. Figure 3A shows the mouse wild-type IgH locus. Figure 3B shows the constant region of the mouse IgH locus. The region to be deleted in the first round of recombination is indicated by a dotted box. Figure 3C shows the deletion of the Ighm-Ighg1 CH1 exon by CRISPR-mediated NHEJ. The region to be deleted in the second round of recombination is indicated by a dotted box. Figure 3D shows the deletion of Ighg2b-Igha exons 1-3 by CRISPR-mediated HDR. Figure 3E shows the removal of the selective cassette by Flp recombinase expression. [Figure 4] Figure 4 shows exemplary genomic structures of the wild-type mouse IgH allele, the recombinant Singularity Musculus allele, and the recombinant Singularity HyperDock allele. [Figure 5] Figures 5A-5D show the generation of the Singularity HyperDock allele. Figure 5A shows the Singularity Musculus allele. (Figure 5B) The Singularity HyperDock allele is generated by deleting all mouse VH, DH, and JH genes (2.58 Mb) and inserting a docking cassette for sequential RMCE via CRISPR-mediated HDR. Figure 5C shows synteny validation of the Singularity HyperDock allele via Flp recombinase expression. Figure 5D shows removal of the selection marker via ΦC31 recombinase expression. [Figure 6]Figures 6A-6B show the production of human-mouse chimeric heavy chain-only antibodies from exemplary Singularity Sapiens mice. Figure 6A is a schematic diagram of an exemplary human VH-mouse IgG1-ΔCH1 chimeric antibody that can be used to generate human VH nanobodies. Figure 6B shows an exemplary version of a Singularity Sapiens mouse produced by serial introduction of human V, D, and J genes into the Singularity HyperDock allele. Human VH-CH1 cleaved heavy chain-only IgG1 can be produced from the above Singularity Sapiens mice, from which human VH nanobodies can be induced. [Figure 7] Figures 7A-7D show the generation of the Singularity Sapiens allele series (SSV1-3). The Singularity Sapiens allele series is generated by inserting recombinant human IGH BAC1-3 via a sequential RMCE using a list of heterologous lox sites, and then exchanging alternative selection cassettes (neo and hyg) during Cre recombinase expression. [Figure 8] Figures 8A-8D are schematic diagrams of the Singularity Sapiens allele series (SSV4-5), showing the sequential incorporation of human IGH-BAC4 and IGH-BAC5 into clones containing human IGH-BAC1, human IGH-BAC2, and human IGH-BAC3 via RMCE, followed by the removal of the selective marker cassette via the expression of ΦC31 recombinase. [Figure 9-1]Figure 9 shows human IGH BACs based on the GENCODE Genes Track (36th edition, October 2020) human genome GRCh38 / hg38 assembly for the IGH locus, representing human gene segments for variable heavy chain (IGHV), diversity heavy chain (IGHD), binding heavy chain (IGHJ), and constant heavy chains IGHM and IGHD. Five BAC constructs (hIGH BAC1-5, boundaries indicated by dotted boxes) containing human IGHV, IGHD, and IGHJ gene segments were recombined using the corresponding source BACs (solid boxes). The entire human VDJ genomic region was then reconstructed in the Singularity HyperDock allele using the recombinant BACs via RMCE as described herein. The number of V, D, and J gene segments contained in each recombinant BAC construct is shown. [Figure 9-2] This is a continuation of Figure 9-1. [Figure 9-3] This is a continuation of Figure 9-2. [Figure 9-4] This is a continuation of Figure 9-3. [Figure 9-5] This is a continuation of Figure 9-4. [Figure 10] Figure 10 shows an example of BAC recombination. Source BAC is modified by bacterial homologous recombination, and appropriate selectable markers and recombination sites are incorporated at the desired locations. The recombination process of hIgH-BAC1 is shown. [Figure 11] Figure 11 shows VH exon validation, illustrating PCR-based validation of Singularity Sapiens (SSV4) containing 37 functional human VH exons integrated into the IGH locus. PCR results were run on a Qiagen Qiaxel DNA High Resolution cartridge. The upper and lower bands represent the Qiagen QX size marker (100 bp–2.5 kb, catalog No. 929559) and the Qiagen QX alignment marker (15 bp / 3 kb, catalog No. 929522), run in parallel. PCR products were validated by Sanger sequencing to match the corresponding VH genes. [Figure 12] Figure 12 shows an exemplary method for complex BAC recombination. Source BACs are sequentially modified by bacterial homologous recombination, incorporating appropriate selectable markers and recombination sites at desired locations. An example of the recombination process of hIGH-BAC5 from three source BACs is shown. [Figure 13] Figures 13A-13B show recombination in mutant mice lacking the kappa light chain. Figure 13A is a schematic diagram showing deletion of mouse IG kappa and insertion of the docking site by CRISPR-mediated HDR. Figure 13B shows the genotyping PCR results confirming the IGK HyperDock / KO allele in F1 mice. [Figure 14] Figures 14A-14B show recombination in mutant mice lacking the lambda light chain. Figure 14A is a schematic diagram showing the deletion of the entire mouse IG lambda locus by CRISPR-mediated NHEJ. Figure 14B shows the PCR results confirming the generation of the IGL KO allele in ES cells. [Figure 15] Figures 15A-15D show that Singularity Musculus mice produce HcAb with only CH1-cleaved IgG1. Schematic diagrams of the Igh gene locus in WT mice (Figure 15A) and SM mice (Figure 15B). Validation of Singularity Musculus mice is shown by RT-PCR (spleen) (Figure 15C) and Western blot (plasma) (Figure 15D). [Figure 16-1] Figures 16A–16D show that Singularity Sapiens mice produce human-mouse chimeric heavy chain IgG1. Figure 16A shows the Singularity Musculus allele. Figure 16B shows the Singularity Sapiens V1 allele, which contains all human JH genes, all human DH genes, and three human VH genes. (Figure 15C) RT-PCR shows the specific expression of the human-mouse chimeric IgG1ΔCH1 transcript in Singularity Sapiens V1 mice. Figure 16D shows that sequencing confirmed the production of the human-mouse chimeric transcript (SEQ ID NO: 36). [Figure 16-2] This is a continuation of Figure 16-1. [Figure 17] Figure 17A is a schematic diagram of an exemplary human VH-mouse IgG1-ΔCH1 chimeric antibody that can be used to generate human VH nanobodies. Figure 17B shows Western blots of IgM and IgG1 in immunized WT mice and Singularity Sapiens mice (SSV1). [Figure 18] Figures 18A and 18B show the spleen morphology and IgM and IgG expression in B cells of Singularity Musculus mice. Figure 18A shows the spleens of wild-type and Singularity Musculus mice. Figure 18B shows flow cytometry analysis of splenocytes showing the absence of IgM in CD19-positive B cells, but normal IgG expression. [Figure 19] Figures 19A and 19B show B cell markers in Singularity Sapiens mice. Figure 19A shows flow cytometry analysis indicating the presence of IgM+IgD+ B cells in wild-type mice, but their absence in Singularity Sapiens mice (SSV2). Figure 19B shows flow cytometry analysis indicating the differential abundance of IgG1+ B cells in wild-type mice and Singularity Sapiens mice (SSV2). [Figure 20] Figures 20A-20B demonstrate that Singularity Musculus mice initiate a robust humoral immune response upon antigen challenge. Figure 20A shows ELISA results of plasma samples obtained from pre-bleeded wild-type and Singularity Musculus animals. Figure 20B shows ELISA results of plasma samples obtained from terminal bleeds at day 28 of the same animals immunized with SARS-CoV-2 spike protein trimer protein (SAT), compared to a commercially available control antibody (S1 mAb control) against the SARS-CoV-2 spike protein S1 subunit. [Figure 21]Figures 21A and 21B demonstrate that Singularity Musculus mice and Singularity Sapiens mice initiate robust humoral immune responses when subjected to various antigen challenges. Figure 21A shows ELISA results of plasma samples obtained from lethal blood collections at day 51 of wild-type (WT), Singularity Musculus (SM), and Singularity Sapiens (SSV1) animals after antigen challenge against SAT. Figure 21B shows ELISA results of plasma samples obtained from lethal blood collections at day 51 of animals immunized with human PD-L1, compared to a commercially available human PD-L1 antibody. [Figure 22] Figure 22 is a schematic diagram showing the IgG1 transcripts of WT mice and Singularity Musculus mice, and the primer positions for 5'RACE amplification for next-generation sequencing analysis. [Figure 23-1] Figures 23A–23C show that Singularity Musculus mice exhibit antibody diversity comparable to that of wild-type mice. VH diversity (Figure 23A); JH diversity (Figure 23B); and CDR3 length diversity (Figure 23C) for all chronotypes identified from two wild-type mice and two Singularity Musculus mice immunized with SAT are shown. [Figure 23-2] This is a continuation of Figure 23-1. [Figure 23-3] This is a continuation of Figure 23-2. [Figure 24-1] Figures 24A-24C show the chronotype IGHV diversity identified in WT mice and SM mice. Figure 24A shows IGHV utilization in SM mice immunized with the indicated antigen. Figure 24B shows IGHV utilization in SM mice immunized with the indicated antigen. (Figure 24C) SM mice can utilize more IGHV segments than WT mice. [Figure 24-2] This is a continuation of Figure 24-1. [Figure 24-3] This is a continuation of Figure 24-2. [Figure 24-4] This is a continuation of Figure 24-3. [Figure 24-5] This is a continuation of Figure 24-4. [Figure 25] Figures 25A-25B show IGHJ utilization in WT mice and SM mice. Figure 25A shows IGHJ utilization in SM mice immunized with the indicated antigen. (Figure 25B) Different and specific IGHJ segment utilization was observed in SM mice compared to WT mice. [Figure 26-1] Figures 26A and 26B show the distribution of CDR3 length in WT and SM mice. Figure 26A shows the distribution of CDR3 length between chronotypes in SM and WT mice responding to a specified antigen. Figure 26B shows the average CDR3 length observed in SM and WT mice. [Figure 26-2] This is a continuation of Figure 26-1. [Figure 27-1] Figure 27 shows somatic hypermutation in Singularity Musculus mice. The histogram shows the number of amino acid changes at each position in the heavy chain variable region compared to the corresponding germline sequences for the top 100 most abundant nanobody chronotypes identified from one naive Singularity Musculus mouse and three SAT-immunized Singularity Musculus mice. VH residue positions are numbered based on the IMGT scheme. The most significant changes occurred in the CDR region. [Figure 27-2] This is a continuation of Figure 27-1. [Figure 28] Figure 28 shows a flowchart of an exemplary NGS-guided single-cell-independent nanobody discovery process. [Figure 29-1]Figure 29 shows a phylogenetic tree of selected chronotypes identified by next-generation sequencing of the HcAb repertoire in Singularity Musculus mice immunized with SAT. Top-ranked chronotypes (based on abundance) were selected for each immunized animal for high-throughput synthesis, cloning, expression, and ELISA screening for antigen affinity, followed by competitive ELISA for inhibitory (neutralizing) activity against spike-ACE2 receptor binding. Antigen-specific clones are shown in gray shading, and neutralizing clones are shown with asterisks. [Figure 29-2] This is a continuation of Figure 29-1. [Figure 30A] Figures 30A and 30B show the vectors used to express nanobodies. Figure 30A shows the plasmid map of the pFUSE-hIgG1-Fc2 expression vector and the restriction sites (EcoRI and NcoI) for inserting the VH sequence. Figure 30B shows an exemplary Nb-human Fc fusion that can be generated from the pFUSE-hIgG1-Fc2 expression vector. [Figure 30B] Figures 30A and 30B show the vectors used to express nanobodies. Figure 30A shows the plasmid map of the pFUSE-hIgG1-Fc2 expression vector and the restriction sites (EcoRI and NcoI) for inserting the VH sequence. Figure 30B shows an exemplary Nb-human Fc fusion that can be generated from the pFUSE-hIgG1-Fc2 expression vector. [Figure 31] Figure 31 shows an ELISA for screening binders in immunized WT and SM mice. The number of chronotypes screened from WT and SM mice after immunization with the indicated antigen and the number of binders identified from WT and SM mice are provided. The binding results (ELISA result OD450) for each chronotype are provided. [Figure 32]Figure 32 includes pie charts derived from data in Figure 31, showing the percentages of binders with the indicated binding affinity obtained from WT mice and SM mice. Each chart shows the percentage of binders determined by ELISA for nanobodies that bind to the indicated antigen. [Figure 33] Figures 33A-33B show exemplary antibody structures of WT IgG1 and Nb-human Fc fusion under non-reducing and reducing conditions (Figure 33A), and confirmation of size reduction by SDS-PAGE gel for S1 mAb control (WT tetramer IgG1) and purified SAT nanobody-Fc fusion (heavy chain only IgG1) (Figure 33B). The expressed Nb-Fc human fusion is homodimer. [Figure 34] Figures 34A and 34B show the SDS-PAGE gel of purified SAT human nanobody-human Fc fusion (only the heavy chain is IgG1). Figure 34A shows the gellan under non-reducing conditions. Figure 34B shows the gellan under reducing conditions. The expressed human Nb-human Fc fusion is observed as a homodimer. [Figure 35] Figure 35 shows size exclusion chromatography of two human nanobody-human Fc fusion proteins. Purified human Nb-human Fc fusion proteins against the SAT antigen were run through a size exclusion column, and protein aggregation was evaluated. [Figure 36] Figures 36A and 36B show the characterization of the purified SAT Nb-human Fc fusion for antigen binding affinity and the characterization of the SARS-CoV-2 neutralizing titer against the RBD Nb-Fc control (HAb8-S). Figure 36A shows the EC50 value for binding affinity. Figure 36B shows the IC50 value for neutralizing titer. [Figure 37-1] Figures 37A and 37B show the phylogenetic relationships (Figure 37A) and somatic hypermuta analysis (Figure 37B) of closely related VH sequences identified using two SARS-CoV-2 neutralizing nanobodies (indicated by asterisks). Closely related, low-abundance chronotypes were identified for secondary screening of high-affinity and high-titer nanobodies. In Figure 37B, the sequences of the nanobody clones from top to bottom are indicated by sequence numbers 25 to 35, respectively. [Figure 37-2] This is a continuation of Figure 37-1. [Figure 37-3] This is a continuation of Figure 37-2. [Figure 38] Figure 38 is a table showing the binding dynamics of mouse and human SAT nanobody-human Fc fusion molecules. The binding of mouse and human Nb-human Fc fusion proteins to recombinant SAT proteins was assayed using bio-layer interferometry (BLI) with Octet. These results demonstrate that high-affinity mouse nanobodies and high-affinity human nanobodies can be obtained using the recombinant mice provided herein. [Figure 39] Figure 39 shows the binding dynamics of an exemplary human nanobody-human Fc fusion molecule. Sensorogram obtained by BLI of purified human Nb-human Fc fusion protein in the presence of recombinant SAT. [Figure 40] Figure 40 shows the melting peak of the human nanobody-human Fc fusion molecule. Melting curves for the Nb-human Fc fusion protein were generated via a pFUSE-hIgG1-Fc2 expression vector in purified human SAT Expi293F cells. These results suggest that human Nb-human Fc molecules may exhibit similar thermal stability to known natural nanobodies. [Figure 41] Figures 41A and 41B show the results of cell binding assays for mouse nanobody-human Fc fusion molecules and human nanobody-human Fc fusion molecules. Figure 41A is an exemplary result showing positive and negative controls for HEK293-expressing SARS-CoV2 spike protein (upper panel) or HEK293 (lower panel) incubated in the presence of purified mouse or human Nb-human Fc fusion protein. Cell binding was evaluated using a fluorescent secondary antibody against the FC region of the Nb-Fc molecule. Figure 41B shows the aggregated results of cell binding data for mouse and human Nb-human Fc fusion proteins. [Figure 42-1] Figure 42 includes graphs showing cell binding results for all Nb-human Fc fusions in Figures 41A-41B. Top panel: mouse Nb-human Fc; bottom panel: human Nb-human Fc. [Figure 42-2] This is a continuation of Figure 42-1. [Figure 42-3] This is a continuation of Figure 42-2. [Figure 43] Figures 43A-43B show exemplary structures of the Singularity Sapiens-L allele series designed to contain human VL segments. Figure 43A shows RAG1 / RAG2-mediated recombination signal sequences for 12RSS (12nt spacer) and 23RSS (23nt spacer) associated with variable segments of the human loci of IGH, IGK (kappa), and IGL (lambda). (Figure 43B) The Singularity Sapiens DJ dock allele, containing all human DH and JH segments, is used as a platform for incorporating a series of BACs containing human variable light chain segments derived from the human IG-lambda locus on chromosome 22 via sequential RCME. The resulting Singularity Sapiens VL-containing allele can produce antibodies containing human variable light chain segments adjacent to the human DH and JH segments and the subsequent mouse constant region (e.g., mouse IgG1ΔCH1 region). [Figure 44]Figures 44A and 44B show exemplary structures of two different sets of Singularity Sapiens-K allele series expressing human VK segments. Figure 44A is a schematic diagram showing that the Singularity HyperDock allele can be used as a platform for incorporating a series of hIGKVJ-BACs containing human VK and JK segments derived from the human IG kappa locus on chromosome 2 via sequential RCME. The resulting Singularity Sapiens VK-JK-containing allele can produce antibodies containing human variable kappa segments adjacent to the human kappa J segment and the subsequent mouse constant region (e.g., the mouse IgG1ΔCH1 region). Figure 44B is a schematic diagram showing that the Singularity Sapiens allele containing the entire human JH segment can be used as a platform for incorporating a series of recombinant hIGKV-BACs containing human VK segments derived from the human IG kappa locus on chromosome 2 via sequential RCME. The resulting Singularity Sapiens VK-JH-containing allele can produce antibodies containing a human variable kappa segment adjacent to the human JH segment and the subsequent mouse constant region (e.g., the mouse IgG1ΔCH1 region). [Figure 45] Figures 45A–45C show exemplary recombinant designs for Singularity Longhorn. Figure 45A is a schematic diagram of a gene construct (Longhorn VDJ) containing a synthetically constructed bovine DNA sequence (Bos Taurus) that includes a promoter, 5'UTR segment, L1 exon, intron, L2 exons of IGHV1–7, coding segments of IGH8–2, coding sequences of IGHJ2–4, and an IGH2–4 splice donor. Figure 45B is a schematic diagram showing this synthetic construct flanked by different loxP elements and hygromycin selection markers. This construct was incorporated into the IgH locus of the Singularity HyperDock allele by RCME to create the Singularity Longhorn allele. Figure 45C shows PCR confirmation of mice possessing the Singularity Longhorn allele. [Figure 46] Figure 46 shows an exemplary recombination design for Singularity Minotaur. A schematic diagram of a gene construct (Minotaur DH array) containing a DNA sequence synthetically constructed to include a bovine DH segment (e.g., eight longest cow IGVDs, shown inside the box) is shown. To ensure that VDJ recombination occurs, the upstream and downstream sequences of the original human IVD containing the 12RSS signal (labeled under the corresponding bovine IGVD, respectively) are included. This synthetic construct (Minotaur DH array) can be incorporated into the Igh locus of a Singularity Sapiens allele (e.g., SSV5) containing any appropriate number (or all) of human VH, all human DH, and all human JH, for example, by CRISPR / Cas9 targeting, thereby replacing the human IGVD locus with the synthetic Minotaur DH array. [Figure 47] Figures 47A–47B illustrate exemplary recombinant designs for Singularity Sapacos. Figure 47A is a schematic diagram of a gene construct (Sapacos VHH array) containing five VHH elements from alpaca (Vicugna pacos) designed to use human VH elements as a gene scaffold. The individual VHH elements were grafted onto a selective human VH framework containing regulatory elements (e.g., TATA boxes, octamers, and heptamers), leader exon 1, introns, leader exon 2, and an upstream promoter (e.g., a 250 bp upstream promoter) containing a recombinant signal sequence (e.g., 23RSS). Figure 47B is a schematic diagram showing that this synthetic construct (Sapacos VHH array), containing adjacent different lox elements and a selective marker, can be incorporated into the IgH locus of a Singularity Sapiens allele containing all human VD and human VJ elements via RMCE. [Figure 48]Figures 48A–48B show exemplary recombinant designs for Singularity Savnars. Figure 48A is a schematic diagram of a gene construct (Savnars VNAR array) containing two germline VNARs derived from the sablefish shark, designed to use human VH elements as a gene scaffold. Individual VNAR elements were grafted onto a selective human VH framework containing regulatory elements (e.g., TATA boxes, octamers, and heptamers), leader exon 1, introns, leader exon 2, and an upstream promoter (e.g., a 250 bp upstream promoter) containing a recombinant signal sequence (e.g., 23RSS). Figure 48B is a schematic diagram showing that this synthetic construct (Savnars VNAR array), containing adjacent different lox elements and a selective marker, can be incorporated into the IgH locus of the Singularity Sapiens allele, which contains all human VD and human VJ elements, via RMCE. [Modes for carrying out the invention]

[0120] Detailed explanation This book relates to genetically modified or recombinant non-human animals (e.g., genetically modified or recombinant mice) that produce heavy chain antibodies (e.g., mouse heavy chain antibodies, humanized heavy chain antibodies, or chimeric heavy chain antibodies), and methods for producing them. For example, this book provides a genetically modified non-human animal of a specific species (e.g., mouse species) that produces heavy chain antibodies of the same species (e.g., mouse heavy chain antibodies). In another example, this book provides a genetically modified non-human animal (e.g., genetically modified mice) that produces chimeric heavy chain antibodies (e.g., human-mouse chimeric heavy chain antibodies, bovine-human-mouse chimeric heavy chain antibodies, alpaca-human-mouse chimeric heavy chain antibodies, or shark-human-mouse chimeric heavy chain antibodies).

[0121] In some cases, heavy chain antibodies obtained or identified from genetically modified non-human animals (e.g., genetically modified mice) provided herein can be used to produce single-domain antibodies such as mouse single-domain antibodies, non-mouse single-domain antibodies, humanized single-domain antibodies, human single-domain antibodies, or chimeric single-domain antibodies (e.g., bovine-human chimeric single-domain antibodies, alpaca-human chimeric single-domain antibodies, or shark-human chimeric single-domain antibodies).

[0122] This document generally relates to nanobody compositions derived from these genetically modified mice and other sources of nanobody compositions. The compositions described herein can be used to treat or prevent diseases or disorders.

[0123] As described herein, this document provides a method for producing mammalian single-domain antibodies (also known as nanobodies) in vivo. For example, a modified mouse endogenous IgH allele has a constant region C HIt is possible to construct a gene containing only the CH1-cleaved IgG1 gene (IgG1ΔCH1) and removing all other Ig classes or subtypes, resulting in the production of heavy chain-only IgG1 antibodies. This modification rearranges the IgG1-ΔCH1 gene immediately downstream of the Eμ enhancer, Iμ promoter, Iμ exon, and Sμ ​​switch repeat region, while other regulatory elements, including the γ1E, 5'hsR1, 3'RR, and 3'CBE enhancers, are retained intact in the endogenous IgH allele. As a result, it is possible to achieve constitutive high levels of expression of IgG1-based heavy chain antibodies (IgG1 HCAb) instead of inductive expression from native regulatory elements for each Ig subtype, and the entire VH repertoire becomes available to produce IgG1 HCAb regardless of antigen type. The recombinant non-human (e.g., mouse) endogenous IgH alleles described herein can be named Singularity and may be further modified by introducing a docking site that allows for the production of IgG1 HCAb platform-based chimeric antibodies by removing all non-human (e.g., mouse) endogenous variable exons and replacing them with variable exons from human or other mammalian species (or combinations thereof), thereby enabling the derivation of species-specific single-domain antibodies. This specification provides highly efficient methods for introducing sequentially long genomic DNA fragments onto the docking site. As shown herein, these methods can result in the successful generation of Singularity Sapiens alleles containing 91 human VH exons to maximize possible antibody diversity. In some examples, variable exons (VK, VL) from IgK and IgL alleles can be used instead of (or in addition to) VH exons to generate light chain-based single-domain antibodies. Similarly, V from other species can be used. H Segment, Diversity D H Genetic elements corresponding to and / or conjugated JH (or combinations thereof) can be designed and synthesized, and heavy chain antibodies can be produced that can be placed in a singularity allele to create single-domain antibodies with unique properties.

[0124] The recombinant non-human animals (e.g., mice) described herein exhibit normal B cell development and can initiate a robust humoral immune response upon antigen challenge. Using the high-throughput sequence-driven approaches described herein, Ig (e.g., IgG1) HCAbs exhibiting high affinity for immune antigens can be produced. Once antigen immunization is complete, the entire Ig repertoire can be amplified from lymphoid organs (e.g., spleen) and subjected to next-generation sequencing (NGS) to obtain chronotypes for phylogenetic analysis. Candidate chronotypes can be codon-optimized, synthesized, cloned into expression vectors, and expressed as nanobody-Fc fusions and / or nanobodies in 96-well format. The supernatant can be used for ELISA screening to identify antigen-specific heavy chain antibodies and / or nanobodies for large-scale production, purification, and / or characterization. Purified nanobodies, nanobody-Fc fusions, and / or heavy chain antibodies may exhibit high levels of thermal stability, antigen affinity, cell binding, and blockade activity.

[0125] As described herein, non-human animals (e.g., mice) can be designed to produce heavy-chain-only antibodies (HCAbs). In some cases, gene editing (e.g., CRISPR / Cas9) can be used to edit an endogenous IgH allele to generate a Singularity allele (e.g., Singularity Musculus allele) containing only the IgG gene (e.g., Ighg1 gene) in a constant region encoding a CH1-cleaved IgG (e.g., IgG1-ΔCH1). In some cases, all endogenous genes encoding other antibody isotypes (IgM, IgD, IgE, and IgA) and IgG subtypes (IgG2b, IgG2c, and IgG3) can be removed. In some cases, one or more endogenous regulatory elements can be retained to enable efficient and faithful transcription of the mutant IghG1 gene from the endogenous IgH allele. Therefore, in these singularity non-human animals (e.g., mice), class switch recombination can be disabled to avoid any potential mechanisms that may impair IgG1-ΔCH1 expression and to facilitate antibody discovery and purification. The resulting singularity non-human animals (e.g., Singularity Musculus mice) can be viable and fertile without apparent abnormalities, and upon antigen challenge, they can initiate a robust humoral immune response and produce high-affinity IgG1-ΔCH1 heavy chain antibodies. Since knowledge of heavy-light chain pairing is not required, antigen-specific monoclonal heavy chain antibodies can be identified using the much faster and more cost-effective NGS-driven antibody discovery pipeline derived from bulk RNA sequencing (RNA-seq) analysis of splenocytes described herein, where the entire process (e.g., antigen immunization, B cell isolation, bulk sequencing of antibody repertoire, antibody sequence chronotyping, high-throughput cloning, expression, and antigen-binding assay) can be achieved within three months.

[0126] Similarly, as described herein, recombinant non-human animals (e.g., mice) can be designed to produce human and / or chimeric heavy-chain antibodies that can be used to identify therapeutic nanobodies. For example, a Singularity allele (e.g., a Singularity Musculus allele) can be further edited to produce a Singularity HyperDock allele that lacks all mouse VDJ(H) genes and has a docking site for serial introduction of DNA fragments using recombinase-mediated cassette exchange (RMCE). In some examples, clones containing human VDJ(H) fragments (e.g., BAC clones) can be recombined by bacterial homologous recombination such that they incorporate an alternative selection cassette and heterologous lox sites and overlapping genomic fragments are excised. In some examples, recombinant BACs can be used in serial RMCE to stepwise construct the human VDJ gene upstream of a mouse IgH Eμ enhancer. This can lead to the generation of a series of Singularity Sapiens alleles (e.g., SSV1-SSV5) with increased VH diversity until a complete reconstruction of the entire human VDJ genome region is achieved.

[0127] Similarly, a series of Singularity non-human animals (e.g., Singularity mice) can be generated to enable the production of species-specific nanobodies with unique properties for various diagnostic and therapeutic applications.

[0128] definition As used herein, the term “antibody” refers to a molecule that specifically binds to or immunologically reacts with a particular antigen and comprises at least a heavy chain and / or light chain variable domain, and in some examples may comprise at least an immunoglobulin heavy chain variable domain and a light chain variable domain. Antibodies and their antigen-binding fragments, variants, or derivatives include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, primatized antibodies, or chimeric antibodies, heteroconjugate antibodies (e.g., bispecific, trispecific, and tetraspecific antibodies, diabodies, triabodies, and tetrabodies), single-domain antibodies (sdAb), epitope-binding fragments (e.g., Fab, Fab', and F(ab')2, Fd, Fv, single-chain Fv (scFv), recombinant IgG (rlgG), single-chain antibodies (e.g., heavy-chain or light-chain antibodies), disulfide-linked Fv (sdFv), fragments containing either a VL domain or a VH domain, fragments produced by Fab expression libraries, and anti-idiotype (anti-Id) antibodies. The antibody molecules described herein may be antibody molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Furthermore, unless otherwise indicated, the term “monoclonal antibody” (mAb) is intended to encompass both intact molecules capable of specifically binding to a target protein, as well as antibody fragments (e.g., Fab fragments and F(ab')2 fragments). Fab fragments and F(ab')2 fragments lack the Fc fragment of an intact antibody. The term “suppressive antibody” refers to an antibody capable of binding to a target antigen to inhibit or reduce its function and / or attenuating one or more signaling pathways mediated by the antigen. For example, a suppressive antibody may bind to and block the ligand-binding domain of a receptor, or it may bind to the extracellular domain of a transmembrane protein.Suppressive antibody molecules that enter cells can block the function of enzyme antigens or signaling molecule antigens. Suppressive antibodies inhibit or reduce antigen function and / or attenuate one or more antigen-mediated signaling pathways by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more). The term "agonist antibody" refers to an antibody that can bind to a target antigen and increase its activity or function, for example, by increasing or activating one or more antigen-mediated signaling pathways. For example, agonist antibodies can bind to and stimulate the extracellular domain of transmembrane proteins. Agonist antibody molecules that enter cells can enhance the function of enzyme antigens or signaling molecule antigens. Agonist antibodies activate or increase antigen function and / or one or more antigen-mediated signaling pathways by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or more).

[0129] As used herein, the term “antigen” refers to a molecule that, when presented by an MHC molecule, can be bound by an antibody or a T cell receptor (TCR). As used herein, the term “antigen” also includes T cell epitopes. T cell epitopes are recognized by T cell receptors in relation to MHC class I, which is present on all cells of the body except red blood cells, or class II, which is present on immune cells and especially antigen-presenting cells. This recognition event leads to T cell activation and subsequent effector mechanisms, such as T cell proliferation, cytokine secretion, and perforin secretion. Antigens can also be recognized by the immune system and / or induce humoral and / or cellular immune responses that lead to the activation of B lymphocytes and / or T lymphocytes. However, this requires, at least in certain cases, that the antigen contains or is bound to a TH cell epitope and is given in an adjuvant. An antigen may have one or more epitopes (B epitopes and T epitopes). The specific reactions mentioned above mean that an antigen typically reacts very selectively and preferably with its corresponding antibody or TCR, and does not react with a number of other antibodies or TCRs that may be induced by other antigens. Antigens used herein may be mixtures of several individual antigens. Antigens used herein include, but are not limited to, allergens, autoantigens, haptens, cancer antigens (i.e., tumor antigens), and infectious disease antigens, as well as small organic molecules, such as drugs of abuse (e.g., nicotine) and their fragments and derivatives. Furthermore, antigens used for the purposes of this disclosure may include peptides, proteins, domains, sugars, alkaloids, lipids, or small molecules, such as steroid hormones and their fragments and derivatives, autoantibodies and their own cytokines.

[0130] The term "antigen" also refers to a molecule (e.g., a peptide, protein, or non-peptide) containing one or more epitopes (either linear, higher-order structures, or both) that stimulate the host's immune system to produce a humoral and / or cellular antigen-specific response. This term is used interchangeably with the term "immunogen." Typically, B cell epitopes contain at least about 5 amino acids, but can be as small as 3-4 amino acids. T cell epitopes, such as CTL epitopes, contain at least about 7-9 amino acids, and helper T cell epitopes contain at least about 12-20 amino acids. Typically, epitopes contain about 7-15 amino acids, e.g., 9, 10, 12, or 15 amino acids. This term encompasses polypeptides that include modifications (generally naturally conserved modifications) such as deletions, additions, and substitutions compared to the native sequence, as long as the protein maintains its ability to induce an immunological response as defined herein. These modifications may be intentional modifications, such as those caused by site-directed mutagenesis, or accidental modifications, such as those caused by mutations in the antigen-producing host.

[0131] As used herein, the term “antigen-binding fragment” refers to one or more fragments of an immunoglobulin that possess the ability to specifically bind to a target antigen. The antigen-binding function of an immunoglobulin may be exerted by a fragment of a full-length antibody. Antibody fragments may be Fab, F(ab')2, scFv, SMIP, diabody, triabody, aphibody, nanobody, aptamer, or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to, (i) Fab fragments (monovalent fragments consisting of VL, VH, CL, and CH1 domains); (ii) F(ab')2 fragments (bivalent fragments containing two Fab fragments linked by disulfide crosslinking in the hinge region); (iii) Fd fragments consisting of a VH domain and a CH1 domain; (iv) Fv fragments consisting of a VL domain and a VH domain of a single arm of the antibody; (v) dAb containing a VH domain and a VL domain (Ward et al., Nature, 341:544-546 (1989)); (vi) dAb fragments consisting of a VH domain; (vii) dAb consisting of a VH domain or a VL domain; (viii) isolated complementarity-determining regions (CDRs); and (ix) combinations of two or more isolated CDRs that may be optionally linked by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked together using recombination techniques, allowing them to be produced as a single protein chain (known as single-chain Fv (scFv)) where the VL and VH regions form a monovalent molecule. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments can be screened for usefulness in the same way as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in specific cases, by chemical peptide synthesis procedures known in the art.

[0132] As used herein, the terms “antigenic preparation” or “antigenic composition” refer to preparations that, when administered to a subject, for example, a mammal, induce an immune response.

[0133] As used herein, the term “biological sample” refers to a sample isolated from a subject (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., biopsy), pancreatic juice, chorionic villi samples, and cells).

[0134] As used herein, “combination therapy” or “administered in combination” means that two (or more) different drugs or treatments are administered to a subject as part of a prescribed treatment regimen for a particular disease or condition. The treatment regimen specifies the dose and periodicity of administration of each drug so that the effects of the separate drugs on the subject overlap. In some embodiments, the delivery of two or more drugs is simultaneous or parallel, and these drugs may also be co-formulated. In other embodiments, two or more drugs are not co-formulated and are administered sequentially as part of a prescribed regimen. In some embodiments, the administration of a combination of two or more drugs or treatments is such that the reduction in other parameters related to the symptom or disorder exceeds the reduction that would be observed by one drug or treatment delivered alone or in the absence of the other. The effects of the two treatments may be partially additive, fully additive, or more than additive (e.g., synergistic). The sequential or substantially simultaneous administration of each therapeutic agent may be influenced by any suitable route, e.g., but not limited to, oral, intravenous, intramuscular, and direct absorption via mucosal tissue. The therapeutic agents can be administered via the same route or via different routes. For example, the first therapeutic agent in the combination may be administered intravenously, while the second therapeutic agent in the combination may be administered orally.

[0135] As used herein, the terms “effective amount,” “therapeutically effective amount,” and “sufficient amount” of the compositions described herein refer to an amount sufficient to produce a favorable or desired outcome, such as an effect or clinical outcome at the cellular or tissue level, when administered to a subject including mammals (e.g., humans). Therefore, “effective amount” or its synonyms are determined by the context in which they are applied. For example, in the context of cancer treatment, “effective amount” is the amount of composition sufficient to achieve a therapeutic response compared to a response obtained without administration of the composition, antibody, vector construct, viral vector, or cells. The amount of a given composition described herein that corresponds to such an amount will vary depending on various factors, such as a given drug, pharmaceutical formulation, route of administration, type of disease or disorder, subject identity (e.g., age, sex, weight), or host being treated, but can nevertheless be routinely determined by those skilled in the art. Similarly, as used herein, “therapeutically effective amount” of the compositions described herein is the amount that produces a favorable or desired outcome for a subject compared to a control. The therapeutically effective amounts of the compositions described herein, as defined herein, can be readily determined by routine methods known to those skilled in the art. Dosage regimens can be adjusted to provide an optimal therapeutic response.

[0136] The terms “heavy chain antibody,” “heavy-chain antibody,” “heavy-chain only antibody,” and “HCAb” used herein are interchangeable and refer to antibodies that lack a light chain, as is typically found in conventional antibodies. A heavy chain antibody may be any antibody derived from the immunoglobulin heavy chain (IgH) gene locus, for example, an antibody containing one or more heavy chain constant domains. For example, a heavy chain antibody may be an antibody containing one light chain variable domain (VL) and one or more heavy chain constant domains.

[0137] As used herein, the terms “hybrid” or “chimera” refer to a molecule (e.g., a protein or VLP) that contains portions derived from at least two different proteins. For example, a hybrid influenza HA protein refers to a protein that contains at least a portion of an influenza HA protein (e.g., a portion containing one or more antigenic determinants) and a portion of a heterologous protein (e.g., the cytoplasmic and / or transmembrane domains of a different influenza protein or a different viral protein, e.g., an RSV or VSV protein). It will become clear that the hybrid molecules described herein may include full-length proteins fused to further heterologous polypeptides (full length or portion thereof), as well as portions of proteins (full length or portion thereof) fused to further heterologous polypeptides. It will also become clear that a hybrid may contain any one, some, or all of the heterologous domains in wild-type or mutant sequences.

[0138] As used herein, the terms “increase” and “decrease” refer to modulating the function, expression, or activity of a metric compared to a reference, respectively. For example, after administration of an antibody described herein, the amount of a marker of a metric described herein (e.g., cancer cell death or DNA methylation at a target site) may increase or decrease in a subject by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% compared to the amount of the marker before administration. Generally, the metric is measured after administration when the administration has the effect described, for example, at least one week, one month, three months, or six months after the start of the treatment regimen.

[0139] An “immunological response” to an antigen or composition is the occurrence of a humoral and / or cellular immune response to an antigen present in the composition of interest in a subject. For the purposes of this book, “humoral immune response” refers to an immune response mediated by antibody molecules, while “cellular immune response” refers to an immune response mediated by T lymphocytes and / or other leukocytes. One important aspect of cellular immunity involves antigen-specific responses by cytolytic T cells (CTLs). CTLs have specificity for peptide antigens presented with proteins encoded by major histocompatibility complexes (MHC) and expressed on the cell surface. CTLs assist in inducing and promoting the destruction of intracellular microorganisms or assist in the lysis of cells infected with such microorganisms. Another aspect of cellular immunity involves antigen-specific responses by helper T cells. Helper T cells act to stimulate the function of nonspecific effector cells to cells that present peptide antigens with MHC molecules on their surface, and help concentrate their activity. "Cellular immune response" refers to cytokines, chemokines, and other such molecules produced by activated T cells and / or other leukocytes, e.g., CD4 + T cells and CD8 + This also refers to the production of antibodies derived from T cells, etc. Therefore, immunological responses can include one or more of the following effects: antibody production by B cells; and / or activation of suppressor T cells and / or γδ T cells specifically directed to antigens present in the antigen or the composition of interest or vaccine. These responses may play a role in providing protection to the immunized host by neutralizing infectivity and / or mediating antibody complementarity or antibody-dependent cytotoxicity (ADCC). Such responses can be determined using standard immunoassays and neutralization assays well known in the art.

[0140] An "immunogenic composition" is a composition containing an antigenic molecule, wherein administration of the composition to a subject results in the development of a humoral and / or cellular immune response to the antigenic molecule of interest in the subject.

[0141] As used herein, the term "polyvalent" refers to a compound having multiple antigenic proteins against multiple types or strains of infectious agents, such as antigens, antibodies, or virus-like particles (VLPs).

[0142] As used herein, “particle-forming polypeptide” can be derived from specific viral proteins, such as full-length or near-full-length viral proteins, as well as fragments thereof, or viral proteins with internal defects that enable them to form VLPs under conditions favorable to VLP formation. Therefore, polypeptides may include the full-length sequence, fragments, cleaved sequences, and partial sequences of a reference molecule, as well as analog and precursor forms. Thus, the term encompasses deletions, additions, and substitutions to the sequence of the polypeptide, as long as the polypeptide retains its ability to form VLPs. For this reason, the term encompasses native mutations of the identified polypeptide, as mutations often occur in the coat protein between viral isolates. The term also encompasses deletions, additions, and substitutions that do not occur naturally in the reference protein, as long as the protein retains its ability to form VLPs. Preferred substitutions are naturally conserved substitutions, i.e., substitutions occurring within the families of amino acids related to their side chains. Specifically, amino acids are generally divided into four families: (1) acidic - aspartic acid and glutamic acid; (2) basic - lysine, arginine, histidine; (3) nonpolar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) non-charged - glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.

[0143] As used herein, the terms “light chain variable region” and “heavy chain variable region” refer to the variable binding regions derived from the light and heavy chains of the antibody, respectively. The variable binding regions consist of distinct, clearly defined subregions known as “complementarity-determining regions” (CDRs) and “framework regions” (FRs), generally comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in the order from the amino terminus to the carboxyl terminus. In one embodiment, the FRs are humanized. The term “CL” refers to the “immunoglobulin light chain constant region” or “light chain constant region,” i.e., the constant region derived from the antibody light chain. The term “CH” refers to the “immunoglobulin heavy chain constant region” or “heavy chain constant region,” which can be further divided, depending on the antibody isotype, into CH1, hinge, CH2, and CH3 (for IgA, IgD, and IgG), or CH1, CH2, CH3, and CH4 domains (for IgE and IgM).

[0144] As used herein, “pharmaceutical composition” or “pharmaceutical preparation” refers to a composition or preparation that has pharmacological activity or other direct effect in the form of the final dosage or formulation of a disease, and is adapted for human use.

[0145] As used herein, the term “reference” refers to a level, expression level, copy number, sample, or standard used for comparative purposes. For example, a reference sample may be obtained from a healthy individual (e.g., an individual without cancer). A reference level may be the expression level of one or more reference samples. For example, the average expression (e.g., mean expression or median expression) among several individuals (e.g., healthy individuals or individuals without cancer) may be a reference level. In other examples, for example, a reference level may be a predetermined threshold level based on functional expression, which may otherwise be determined by an empirical assay.

[0146] As used herein, the terms “subject” and “patient” refer to animals (e.g., mammals such as humans). Subjects treated according to the methods described herein may be subjects diagnosed with a particular condition or subjects at risk of developing such a condition. Diagnosis can be performed by any method or technique known in the art. Those skilled in the art will understand that subjects treated according to this disclosure may have undergone standard testing, or may have been identified as subjects at risk due to the presence of one or more risk factors associated with the disease or condition, without undergoing testing.

[0147] As used herein, “treatment” and “to treat” refer to the medical management of an object with the intention of improving, relieving, stabilizing (i.e., preventing exacerbation), preventing or curing a disease, condition, or disorder. The term encompasses active treatment (treatment aimed at improving a disease, condition, or disorder), causal treatment (treatment aimed at the cause of the associated disease, condition, or disorder), palliative care (treatment designed to alleviate symptoms), preventive care (treatment aimed at minimizing or partially or completely inhibiting the onset of the associated disease, condition, or disorder), and supportive care (treatment used to complement another treatment). Treatment also encompasses reduction in the severity of the disease or condition, whether detectable or undetectable; prevention of transmission of the disease or condition; delay or slowing the progression of the disease or condition; remission or relief of the disease or condition; and remission (whether partial or complete), whether detectable or undetectable. To “relieve” or “alleviate” a disease or condition means that the degree and / or undesirable clinical symptoms of the disease, disability, or condition are reduced and / or the time course of progression is slowed or prolonged compared to the degree or time course in the absence of treatment. “Treatment” may also mean an extension of survival compared to the survival expected if no treatment is received. Those who require treatment include those who already have a condition or disability, as well as those who are prone to developing a condition or disability, or those who should be prevented from developing a condition or disability.

[0148] It should be understood that all numerical boundaries described for some parameters in this book, such as "approximately," "at least," "less than," and "greater than," necessarily include any range bounded by the stated value. Therefore, for example, the description "at least 1, 2, 3, 4, or 5" also includes the ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, among others.

[0149] As used herein, the terms "a" and "an" should also be understood to refer to "one or more" of the listed components unless otherwise indicated. The use of options (e.g., "or") should be understood to mean one of the options, both, or any combination thereof.

[0150] Singularity non-human animals (e.g., singularity mice) This book provides genetically modified non-human animals (e.g., non-human mammals such as mice) for producing antibodies (e.g., heavy chain antibodies such as mouse heavy chain antibodies or chimeric heavy chain antibodies). For example, a genetically modified non-human animal for producing heavy chain antibodies may be a non-human animal that has a humanized IgG heavy chain (e.g., has been recombinant to have a humanized IgG heavy chain). In some cases, a non-human animal for producing heavy chain antibodies (e.g., heavy chain antibodies such as mouse heavy chain antibodies or chimeric heavy chain antibodies) may have a genome in which the IgG1 C region gene (e.g., C) is located. γ1 A non-human animal may have one or more disruptions in the endogenous nucleic acid sequence encoding the CH1 domain (e.g., it may be genetically modified to have one or more disruptions). In some cases, a non-human animal (e.g., mouse) can be designed to produce heavy chain antibodies (e.g., mouse heavy chain antibodies or chimeric heavy chain antibodies) that lack both the CH1 domain and the light chain. Similarly, methods and materials for constructing and using the non-human animals described herein are provided herein.

[0151] In some cases, one or more germline modifications can be performed to create the non-human animals described herein. Genetic modification can cause non-human animals (e.g., mice) to express IgG heavy chain antibodies and secrete them into their serum. In some cases, IgG heavy chain antibodies can be humanized. For example, the variable region of an IgG heavy chain antibody may be a human variable region, and the constant region of an IgG heavy chain antibody may be a mouse constant region. In some cases, the non-human animals (e.g., mice) described herein can be designed to produce IgG1 heavy chain antibodies, IgG2 heavy chain antibodies, IgG3 heavy chain antibodies, or IgG4 heavy chain antibodies. In some cases, the non-human animals (e.g., mice) described herein can be designed to produce any two or more combinations of (a) IgG1 heavy chain antibodies, (b) IgG2 heavy chain antibodies, (c) IgG3 heavy chain antibodies, and (d) IgG4 heavy chain antibodies.

[0152] In some examples, non-human animals provided herein can be designed to have a deletion in the nucleic acid encoding the CH1 domain of the IgG C region (e.g., the CH1 domain of the IgG1 C region, the CH1 domain of the IgG2a C region, the CH1 domain of the IgG2b C region, and / or the CH1 domain of the IgG3 C region). The CH1 domain may contain multiple exons. In some examples, exon 1 of the CH1 domain of the IgG C region may be deleted so that recombinant non-human animals (e.g., mice) produce IgGΔCH1 heavy chain antibodies.

[0153] When creating one or more gene modifications that delete all or part of the nucleic acids encoding the CH1 domain (e.g., the CH1 domain in the IgG1 C region, the CH1 domain in the IgG2a C region, the CH1 domain in the IgG2b C region, and / or the CH1 domain in the IgG3 C region) in order to enable recombinant non-human animals to produce IgGΔCH1 heavy chain antibodies, the endogenous nucleic acids encoding the hinge domain, heavy chain CH2 domain, and heavy chain CH3 domain may remain intact. For example, in order to create a mouse that produces IgG1ΔCH1 heavy chain antibodies, the genome of that mouse can be modified to delete exon 1 (and / or the added portion) of the CH1 domain of IgG1 while retaining the endogenous mouse nucleic acids required to express the hinge domain, heavy chain CH2 domain, and heavy chain CH3 domain of IgG1, thereby producing a mouse capable of producing IgG1ΔCH1 heavy chain antibodies.

[0154] Further endogenous nucleic acid components that can be deleted from the genome of a non-human animal (e.g., mouse) to produce the non-human animals provided herein include, but are not limited to, introns and / or exons of the IgM constant domain (e.g., the μ constant domain locus), introns and / or exons of the IgD constant domain (e.g., the δ constant domain locus), introns and / or exons of the IgE constant domain (e.g., the ε constant domain locus), and / or introns and / or exons of the IgA constant domain (e.g., the α constant domain locus). For example, the non-human animals provided herein can be designed to lack introns and exons of the IgM constant domain (e.g., the μ constant domain locus), introns and exons of the IgD constant domain (e.g., the δ constant domain locus), introns and exons of the IgE constant domain (e.g., the ε constant domain locus), and introns and exons of the IgA constant domain (e.g., the α constant domain locus).

[0155] In some cases, when designing a non-human animal (e.g., mouse) that produces only IgG1ΔCH1 heavy chain antibodies, the genome of that non-human animal will have (in addition to the lack of endogenous introns and / or exons of the μ constant domain locus, the δ constant domain locus, the ε constant domain locus, and the α constant domain locus) endogenous introns and / or exons of the Igγ3 constant domain (e.g., the γ3 constant domain locus). It is possible to design the genes to lack introns and / or exons (if they are endogenously present), introns and / or exons of the Igγ2a constant domain (e.g., the γ2a constant domain locus), introns and / or exons of the Igγ2b constant domain (e.g., the γ2b constant domain locus), and introns and / or exons of the Igγ2c constant domain (e.g., the γ2c constant domain locus). Examples of recombinant approaches for creating mice that produce only IgG1ΔCH1 heavy chain antibodies are shown in Figures 3A-3E.

[0156] In some cases, when designing a non-human animal (e.g., mouse) to produce only IgG2aΔCH1 heavy chain antibodies, the genome of the non-human animal is (in addition to the lack of endogenous introns and / or exons of the μ constant domain locus, the δ constant domain locus, the ε constant domain locus, and the α constant domain locus) endogenous introns and / or exons of the Igγ3 constant domain (e.g., the γ3 constant domain locus) It is possible to design the gene to lack introns and / or exons (if endogenously present), introns and / or exons (if endogenously present) of the Igγ1 constant domain (e.g., the γ1 constant domain locus), introns and / or exons (if endogenously present) of the Igγ2b constant domain (e.g., the γ2b constant domain locus), and introns and / or exons (if endogenously present) of the Igγ2c constant domain (e.g., the γ2c constant domain locus).

[0157] In some cases, when designing a non-human animal (e.g., mouse) to produce only IgG2bΔCH1 heavy chain antibodies, the genome of the non-human animal is (in addition to the lack of endogenous introns and / or exons of the μ constant domain locus, the δ constant domain locus, the ε constant domain locus, and the α constant domain locus) endogenous introns and / or exons of the Igγ3 constant domain (e.g., the γ3 constant domain locus) It is possible to design the gene to lack introns and / or exons (if endogenously present), introns and / or exons (if endogenously present) of the Igγ2a constant domain (e.g., the γ2a constant domain locus), introns and / or exons (if endogenously present) of the Igγ1 constant domain (e.g., the γ1 constant domain locus), and introns and / or exons (if endogenously present) of the Igγ2c constant domain (e.g., the γ2c constant domain locus).

[0158] In some cases, when designing a non-human animal (e.g., mouse) to produce only IgG2cΔCH1 heavy chain antibodies, the genome of the non-human animal is (in addition to the lack of endogenous introns and / or exons of the μ constant domain locus, the δ constant domain locus, the ε constant domain locus, and the α constant domain locus) endogenous introns and / or exons of the Igγ3 constant domain (e.g., the γ3 constant domain locus) It is possible to design the gene to lack introns and / or exons (if endogenously present), introns and / or exons (if endogenously present) of the Igγ2a constant domain (e.g., the γ2a constant domain locus), introns and / or exons (if endogenously present) of the Igγ2b constant domain (e.g., the γ2b constant domain locus), and introns and / or exons (if endogenously present) of the Igγ1 constant domain (e.g., the γ1 constant domain locus).

[0159] In some cases, when designing a non-human animal (e.g., mouse) to produce only IgG3ΔCH1 heavy chain antibodies, the genome of the non-human animal is (in addition to the lack of endogenous introns and / or exons of the μ constant domain locus, the δ constant domain locus, the ε constant domain locus, and the α constant domain locus) endogenous (in) Igγ1 constant domain (e.g., γ1 constant domain locus) It is possible to design the gene to lack introns and / or exons (if endogenously present), introns and / or exons (if endogenously present) of the Igγ2a constant domain (e.g., the γ2a constant domain locus), introns and / or exons (if endogenously present) of the Igγ2b constant domain (e.g., the γ2b constant domain locus), and introns and / or exons (if endogenously present) of the Igγ2c constant domain (e.g., the γ2c constant domain locus).

[0160] As described herein, by retaining and / or creating novel locations of specific endogenous enhancers or regulatory elements in non-human animals, it is possible to produce non-human animals (e.g., mice) provided herein that efficiently produce a large number and large quantities of diverse heavy chain antibodies (e.g., heavy chain antibodies such as mouse heavy chain antibodies or chimeric heavy chain antibodies). For example, non-human animals (e.g., mice) provided herein can be designed to retain a μ-promoter containing a μ-enhancer (Eμ), a μ-switch region (Sμ), and / or an I-exon (Iμ) endogenously found upstream of the nucleic acid encoding the IgM constant domain. In some examples, non-human animals (e.g., mice) provided herein can be designed to reside in genomic locations such that the retained endogenous Eμ, Sμ, and / or Iμ elements are located downstream of the retained Eμ, Sμ, and / or Iμ elements encoding the full-length endogenous Ig constant domain, and the first nucleic acid sequence downstream of these retained Eμ, Sμ, and / or Iμ elements is a nucleic acid sequence encoding the CH2 domain (e.g., a nucleic acid encoding the full-length IgG1 CH2 domain, a nucleic acid encoding the full-length IgG2a CH2 domain, a nucleic acid encoding the full-length IgG2b CH2 domain, a nucleic acid encoding the full-length IgG2c CH2 domain, or a nucleic acid encoding the full-length IgG3 CH2 domain). Examples of this genomic structure are shown in Figures 1B and 3C, where the nucleic acids of the endogenous mouse Eμ, Sμ, and Iμ elements are rearranged to be upstream of the nucleic acid encoding the endogenous IgG1 CH2 domain.

[0161] In another example, a non-human animal (e.g., mouse) provided herein can be designed to retain a 3'RR and / or 3'CBE element that is endogenously found downstream of a nucleic acid encoding an IgA constant domain. In some examples, a non-human animal (e.g., mouse) provided herein can be designed to be located at a genomic position such that the retained endogenous 3'RR and / or 3'CBE element is located upstream of the retained 3'RR and / or 3'CBE element encoding a full-length endogenous Ig CH2 constant domain, and the first nucleic acid sequence is a nucleic acid sequence encoding an IgG CH2 domain (e.g., a nucleic acid encoding a full-length IgG1 CH2 domain, a nucleic acid encoding a full-length IgG2a CH2 domain, a nucleic acid encoding a full-length IgG2b CH2 domain, a nucleic acid encoding a full-length IgG2c CH2 domain, or a nucleic acid encoding a full-length IgG3 CH2 domain). An example of this genome structure is shown in Figures 2B and 3E, where the nucleic acid of the endogenous mouse 3'RR element is rearranged so that the nucleic acid encoding the endogenous IgG1 CH2 domain is downstream of the nucleic acid encoding the endogenous IgG1 CH2 domain, so that no other nucleic acid encoding the full-length IgG CH2 domain is located between the nucleic acid encoding the endogenous IgG1 CH2 domain and the nucleic acid of the endogenous mouse 3'RR element.

[0162] In some examples, non-human animals (e.g., mice) provided herein can be designed to retain, for example, a 3'γ1E element endogenously located between the IgG1 locus and the IgG2b locus. In some examples, non-human animals (e.g., mice) provided herein can be designed to have a genomic position such that the retained endogenous 3'γ1E element is located between the retained endogenous 3'γ1E element and the retained endogenous 3'RR element and / or the retained endogenous 3'CBE element, where nucleic acids encoding two, one, or zero full-length endogenous Ig CH2 domains are located. An example of this genomic structure is shown in Figure 3E, where the nucleic acid of the endogenous mouse 3'γ1E element is rearranged so that other nucleic acids encoding full-length IgG CH2 domains are located upstream of the retained endogenous 3'RR element, so that they are not located between the endogenous mouse 3'γ1E element and the endogenous 3'RR element.

[0163] In some examples, non-human animals (e.g., mice) provided herein can be designed to retain a 5'hsR1 element endogenously found in the IgA constant domain locus. In some examples, non-human animals (e.g., mice) provided herein can be designed to be located at a genomic position such that the retained endogenous 5'hsR1 element is at a genomic position such that the first nucleic acid sequence upstream of the retained 5'hsR1 element encoding the full-length endogenous Ig CH2 constant domain is a nucleic acid sequence encoding the IgG CH2 domain (e.g., a nucleic acid encoding the full-length IgG1 CH2 domain, a nucleic acid encoding the full-length IgG2a CH2 domain, a nucleic acid encoding the full-length IgG2b CH2 domain, a nucleic acid encoding the full-length IgG2c CH2 domain, or a nucleic acid encoding the full-length IgG3 CH2 domain). An example of this genome structure is shown in Figure 3E, where the nucleic acid of the endogenous mouse 5'hsR1 element is rearranged so that the nucleic acid encoding the endogenous IgG1 CH2 domain is downstream of the nucleic acid encoding the endogenous IgG1 CH2 domain, so that the other nucleic acid encoding the full-length IgG1 CH2 domain is not located between the nucleic acid encoding the endogenous IgG1 CH2 domain and the nucleic acid of the endogenous mouse 5'hsR1 element.

[0164] In some examples, the non-human animals (e.g., mice) provided herein can be designed to have (a) a variable region locus (e.g., mouse variable region locus, non-mouse variable region locus, human variable region locus, or chimeric variable region locus, e.g., bovine-human chimeric variable region locus, alpaca-human chimeric variable region locus, or shark-human chimeric variable region locus), followed by (b) an endogenous Eμ element and / or an endogenous Iμ element and / or an endogenous Sμ element, followed by (c) a nucleic acid encoding an endogenous IgG hinge, CH2 domain and CH3 domain in the absence of the endogenous CH1 domain of its IgG, followed by (e) an endogenous 3'γ1E element, an endogenous 3'RR element and an endogenous 3'CBE element, while lacking an endogenous nucleic acid encoding at least one full-length CH2 domain or CH3 domain of IgM, IgD, IgE, and IgA, respectively. An example of this genome structure is shown in Figure 3E. See also Figures 7, 8, 43B, 44, 45B, 47B, and 48B.

[0165] In some cases, instead of retaining the endogenous enhancers or regulatory elements described herein, one or more exogenous enhancers or regulatory elements can be recombinant in a non-human animal (e.g., a mouse). For example, in some cases, the mouse can be designed as described herein, where the endogenous mouse Eμ element is removed and replaced with a human Eμ element.

[0166] In some cases, recombinant non-human animals provided herein can be designed to have a variable region locus, which is an endogenous variable region locus of a non-human animal. For example, recombinant mice provided herein can be designed to have an endogenous mouse variable region locus. An example of an IgH locus in such a recombinant mouse is shown in Figure 1B.

[0167] In some cases, recombinant non-human animals provided herein can be designed to have a variable region locus that is not endogenous to that non-human animal. For example, recombinant mice provided herein can be designed to have a non-mouse variable region locus (e.g., human variable region locus, alpaca variable region locus, shark variable region locus, bovine variable region locus, goat variable region locus, sheep variable region locus, canine variable region locus, feline variable region locus, rat variable region locus, chicken variable region locus, or rabbit variable region locus). An example of an IgH locus in such a recombinant mouse is shown in Figure 6B.

[0168] In some cases, recombinant non-human animals provided herein can be designed to have a non-endogenous variable region locus in that non-human animal, which contains variable region components from two or more different species, distinct from the variable region components of the aforementioned non-human animal. For example, recombinant mice provided herein can be designed to have a non-mouse variable region locus containing variable region components from humans and alpacas, humans and cattle, humans and sharks, sharks and cattle, alpacas and cattle, humans and goats, humans and sheep, humans and dogs, humans and cats, humans and rats, humans and chickens, or humans and rabbits. Examples of IgH loci in such recombinant mice are shown in Figures 43, 44, 47, and 48.

[0169] In some cases, the recombinant non-human animals provided herein can be designed to have variable region loci that are light chain variable region loci (e.g., kappa light chain locus variable region or lambda light chain locus variable region) as opposed to heavy chain variable region loci. For example, the recombinant mice provided herein can be designed to have light chain variable region loci (e.g., human variable region loci of the kappa or lambda light chain). Examples of IgH loci in such recombinant mice are shown in Figures 43B, 44A, and 44B.

[0170] This publication also provides recombinant non-human animals that can be used to create non-human animals that produce antibodies provided herein (e.g., heavy chain antibodies lacking the CH1 domain). For example, this publication provides recombinant non-human animals that lack the entire set of exons in the endogenous variable region of a heavy chain locus and contain a cloning nucleic acid segment located upstream of the already recombinant constant region described herein. Examples of such recombinant mouse IgH loci are shown in Figures 4, 5D, and 6B, which may be called non-human Singularity HyperDock animals or Singularity HyperDock mice. Non-human Singularity HyperDock animals (e.g., Singularity HyperDock mice) provided herein can be constructed using any suitable cloning nucleic acid segment. For example, non-human Singularity HyperDock animals (e.g., Singularity HyperDock mice) provided herein can be constructed using a cloning nucleic acid segment designed to contain one, two, three, four, or more recombinase site recognition sequences (see, for example, Table 1). In some cases, the non-human Singularity HyperDock animals provided herein (e.g., Singularity HyperDock mice) lack the ability to produce any Ig heavy chain.

[0171] [Table 1]

[0172] Non-human animals provided herein (e.g., non-human animals designed to produce heavy chain antibodies such as the IgG1ΔCH1 heavy chain antibody described herein, and non-human Singularity HyperDock animals, e.g., the Singularity HyperDock mouse described herein) can be produced using any suitable method. For example, non-human animals provided herein can be produced using gene editing techniques (e.g., CRISPR / Cas gene editing, TALEN gene editing, and / or zinc finger-based gene editing), recombination techniques (e.g., serial recombinase-mediated cassette exchange (RMCE)), and combinations thereof. In some examples, non-human animals provided herein can be produced using the recombination techniques described in the examples.

[0173] This publication also provides human nanobodies, humanized nanobodies, heavy chain antibodies lacking the CH1 domain (e.g., complete mouse heavy chain antibodies lacking the CH1 domain), and chimeric heavy chain antibodies (e.g., human-mouse chimeric heavy chain antibodies with or without the CH1 domain). For example, this publication provides complete human nanobodies produced from or derived from non-human animals as described herein. As another example, this publication provides complete mouse heavy chain antibodies lacking the CH1 domain. As yet another example, this publication provides chimeric heavy chain antibodies. Such chimeric heavy chain antibodies may lack the CH1 domain as described herein. In some examples, the chimeric heavy chain antibodies provided herein (e.g., IgGΔCH1 heavy chain antibodies) may comprise one or more variable region components that are human, alpaca, shark, cattle, goat, sheep, dog, cat, rat, chicken, or rabbit, and a constant region component of a different species (e.g., mouse). For example, a chimeric heavy chain antibody (e.g., IgGΔCH1 heavy chain antibody) provided herein may have a human variable region and a mouse constant region. In some examples, a chimeric heavy chain antibody (e.g., IgGΔCH1 heavy chain antibody) provided herein may have an alpaca variable region and a mouse constant region. In some examples, a chimeric heavy chain antibody (e.g., IgGΔCH1 heavy chain antibody) provided herein may have a shark variable region and a mouse constant region. In some examples, a chimeric heavy chain antibody (e.g., IgGΔCH1 heavy chain antibody) provided herein may have a bovine variable region and a mouse constant region. In some examples, a chimeric heavy chain antibody (e.g., IgGΔCH1 heavy chain antibody) provided herein may have at least a portion of the alpaca variable region and at least a portion of the human and mouse constant regions. In some examples, a chimeric heavy chain antibody (e.g., IgGΔCH1 heavy chain antibody) provided herein may have at least a portion of the shark variable region and at least a portion of the human and mouse constant regions. In some cases, the chimeric heavy chain antibodies provided herein (e.g., IgGΔCH1 heavy chain antibodies) may have at least a portion of the variable region of a bovine organism and at least a portion of the constant region of a human organism and a mouse organism.

[0174] The human nanobodies, humanized nanobodies, heavy chain antibodies lacking the CH1 domain (e.g., complete mouse heavy chain antibodies lacking the CH1 domain), and chimeric heavy chain antibodies (e.g., human-mouse chimeric heavy chain antibodies having or lacking the CH1 domain) provided herein can be obtained by any suitable method. For example, the heavy chain antibodies provided herein can be obtained from the plasma of non-human animals provided herein. In some examples, the human nanobodies, humanized nanobodies, heavy chain antibodies lacking the CH1 domain (e.g., complete mouse heavy chain antibodies lacking the CH1 domain), and chimeric heavy chain antibodies (e.g., human-mouse chimeric heavy chain antibodies having or lacking the CH1 domain) provided herein can be obtained using nucleic acid vectors designed to express nanobodies or heavy chain antibodies based on or derived from heavy chain antibodies produced by non-human animals provided herein. For example, human-mouse IgGΔCH1 heavy chain antibodies produced by non-human animals provided herein can be identified and sequenced as having the ability to bind to a target antigen of interest (e.g., SARS-CoV-2 antigen). Using that sequence, nucleic acid vectors can be designed that have the ability to express the same human-mouse IgGΔCH1 heavy chain antibody, or the human variable region of the same human-mouse IgGΔCH1 heavy chain antibody as a human nanobody. In some examples, the sequence can be used to design nucleic acid vectors that can express a fully human full-length heavy chain antibody, which can be used on its own or combined with a fully human light chain to produce a fully tetrameric antibody.

[0175] In some cases, non-human animals provided herein can be immunized with an antigen of interest (e.g., SARS-CoV-2 antigen) so that the non-human animals produce antibodies against the antigen. The nucleic acids encoding the produced heavy chain antibodies (e.g., heavy chain antibodies lacking the CH1 domain) can be isolated. For example, amplification techniques such as PCR or 5'RACE can be used to obtain large amounts of nucleic acids encoding at least a portion of the variable regions (e.g., one or more CDRs, all three CDRs, or the entire variable region) of different heavy chain antibodies produced by the non-human animals. The isolated nucleic acid sequences can be cloned into expression vectors (with or without prior sequencing) to express the obtained nucleic acid sequences in relation to any suitable type of antibody (e.g., nanobody, heavy chain antibody, or complete antibody) that can be evaluated for desired properties (e.g., binding properties, neutralizing properties, and / or solubility properties). Any type of antibody, such as nanobody, can be created using nucleic acid sequences that have the ability to encode antibodies with desired properties.

[0176] Plasma containing nanobodies can be collected from subjects potentially immunized with the antigens described herein, or from non-human animals having a humanized immune system. Nanobodies derived from non-human animals having a humanized immune system can be used in the treatment of human subjects requiring such treatment.

[0177] In some cases, plasma containing chimeric heavy chain antibodies that can be used to generate the nanobodies described herein may be collected from subjects that may have been immunized with the antigens described herein, or from non-human animals provided herein (e.g., non-human animals with a humanized immune system).

[0178] Plasma containing nanobodies can be collected, for example, via plasma exchange. In some examples, plasma containing chimeric heavy chain antibodies described herein can be collected, for example, via plasma exchange. Plasma can be collected multiple times from the same subject, for example, multiple times at intervals after immunization, multiple times after immunization, multiple times during immunization, or any combination thereof.

[0179] Plasma can be collected from non-human animals or human subjects as described herein at any suitable time and amount after immunization, such as the first immunization, the most recent immunization, or an intermediate immunization. Plasma can be collected at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 15 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, or thereafter. In some embodiments, δ is collected for up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 42, 49, or 56 days after immunization. In some embodiments, plasma is collected for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 days or longer after immunization. In some embodiments, the compositions described herein may include plasma collected after administration of the immunogenic composition / antigen described herein.

[0180] Plasma can be frozen (e.g., cryopreserved or transported). In some embodiments, the plasma is kept fresh, or antibodies (e.g., heavy chain antibodies or nanobodies) are purified from fresh plasma.

[0181] Nanobodies are purified from plasma, for example, by affinity purification, using techniques known to those skilled in the art. In some cases, the chimeric heavy chain antibodies described herein can be purified from plasma using any suitable technique, such as affinity purification.

[0182] In some cases, methods for producing the proteins provided herein (e.g., human nanobodies, humanized nanobodies, complete mouse heavy chain antibodies lacking CH1, or chimeric heavy chain antibodies with or without CH1) may involve expression in mammalian cells, but recombinant proteins can also be produced using insect cells, yeast, bacteria, or other cells under the control of an appropriate promoter. In some cases, the antibodies provided herein (e.g., heavy chain antibodies or nanobodies) can be recombinantly produced in prokaryotic hosts, such as Escherichia coli, Bacillus brevis, Bacillus subtilis, Bacillus megaterium, Lactobacillus zeae / casei, or Lactobacillus paracasei.In some cases, the antibodies provided herein (e.g., heavy chain antibodies or nanobodies) are used with eukaryotic hosts such as yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, or Yarrowia lipolytica), Trichoderma (e.g., Trichoderma reesei), and Aspergillus (e.g., Aspergillus niger). It can be recombinantly produced in filamentous fungi such as Aspergillus niger and Aspergillus oryzae, protozoa such as Leishmania tarentolae, insect cells, or mammalian cells (e.g., Chinese hamster ovary (CHO) cells, Per.C6 cells, mouse myeloma NS0 cells, baby hamster kidney (BHK) cells, or mammalian cell lines such as the human embryonic kidney cell line HEK293). For example, see the reference by Frenzel et al. (Front Immunol., 4:217) See (2013). Mammalian expression vectors may include non-transcription elements such as origins of replication, preferred promoters and enhancers, as well as other 5' or 3' adjacent non-transcription sequences and 5' or 3' untranslated sequences, such as essential ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, such as the SV40 origin, early promoter sites, enhancer sites, splice sites, and polyadenylation sites, may be used to provide other gene elements required for the expression of heterologous DNA sequences.Suitable cloning and expression vectors for use with bacteria, fungi, yeasts, and mammalian cell hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Laboratory Press (2012), and can be used to produce antibodies provided herein (e.g., human nanobodies, humanized nanobodies, complete mouse heavy chain antibodies lacking CH1, or chimeric heavy chain antibodies with or without CH1).

[0183] Recombinant proteins or antibodies provided herein (e.g., human nanobodies, humanized nanobodies, complete mouse heavy chain antibodies lacking CH1, or chimeric heavy chain antibodies with or without CH1) can be expressed and manufactured using various mammalian cell culture systems. Examples of mammalian expression systems that can be used include, but are not limited to, CHO cells, COS cells, HeLA, and BHK cell lines. Methods for host cell culture for the manufacture of usable protein therapeutics are described, for example, in Zhou and Kantardjieff (eds.), Mammalian Cell Cultures for Biologics Manufacturing (Advances in Biochemical Engineering / Biotechnology), Springer (2014). Purification of protein therapeutics is described in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010). Formulations of protein-based therapeutic drugs are described in Meyer (ed.), Therapeutic Protein Drug Products: Practical Approaches to Formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012). The compositions described herein may include viral vectors, such as lentiviral vectors, and adenoviruses or adeno-related viruses encoding recombinant proteins. In some embodiments, the vector, such as a viral vector, may include nucleic acids encoding recombinant proteins.In some cases, the methods described herein can be designed to meet standards specified for Good Manufacturing Practices (GMP), including several quality controls and adequate infrastructure, as well as separation of activities to avoid cross-contamination. Finally, the composition can be labeled and distributed worldwide.

[0184] In some embodiments, the therapeutic nanobody preparations described herein may be produced by immunizing a non-human animal having a humanized immune system with an antigen described herein. In some examples, the therapeutic nanobody preparations described herein may be produced by immunizing a recombinant non-human animal described herein with an antigen of interest described herein.

[0185] Non-human animals having a humanized immune system may be ungulates, e.g., donkeys, goats, horses, cows, or pigs; or rodents, e.g., rabbits, rats, or mice. In some embodiments, the non-human animal having a humanized immune system is a cow (bovine). In some embodiments, the non-human animal having a humanized immune system is a chicken. The non-human animal has a humanized immune system, for example, that the immune system includes a humanized immunoglobulin gene locus, or a plurality of humanized immunoglobulin gene loci. In some embodiments, the humanized immunoglobulin gene locus includes germline sequences of human immunoglobulins, enabling the non-human animal to produce humanized antibodies (e.g., fully human antibodies). In some embodiments, the non-human animal having a humanized immune system of this disclosure includes non-human B cells having a humanized immunoglobulin gene locus. The humanized immunoglobulin gene locus undergoes VDJ recombination during B cell development, thereby enabling the generation of B cells with diverse antigen-binding specificities. Immunization with one or more immunogenic compositions described herein results in multiple B cell clones responding to their respective alloantigens, leading to the production of polyclonal antibodies with multiple binding specificities.

[0186] The non-human animals provided herein may be any type of non-human animal. For example, non-human animals designed to express chimeric heavy chain antibodies may be ungulates, e.g., donkeys, goats, horses, cows, or pigs; rodents, e.g., rabbits, rats, or mice. In some embodiments, the non-human animals provided herein (e.g., non-human animals designed to express chimeric heavy chain antibodies) may be cows (bovines). In some embodiments, the non-human animals provided herein (e.g., non-human animals designed to express chimeric heavy chain antibodies) may be chickens.

[0187] In some embodiments, immunization of a non-human animal according to the Disclosure with one or more immunogenic compositions described herein activates at least 1, at least 2, at least 3, at least 4, at least 5, 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 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 non-human B cell clones in the non-human animal. In some embodiments, immunization of a non-human animal of this disclosure with one or more immunogenic compositions described herein results in the production of a polyclonal antiserum containing antibodies (e.g., chimeric heavy chain antibodies) that specifically bind to antigens of at least 1, at least 2, at least 3, at least 4, at least 5, 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 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 immunogenic compositions described herein.

[0188] Various techniques for modifying the genomes of non-human animals (e.g., non-human animals for immunization) can be used to create animals capable of producing antibodies (e.g., humanized antibodies, complete mouse heavy chain antibodies, or chimeric heavy chain antibodies). Non-human animals may be transgenic animals, such as transgenic animals containing all or substantially part of the humanized immunoglobulin gene locus. Non-human animals may be transchromosomal animals, such as non-human animals containing human artificial chromosomes or yeast artificial chromosomes.

[0189] The humanized immunoglobulin gene locus may be present on a vector, such as a human artificial chromosome or yeast artificial chromosome (YAC). A human artificial chromosome (HAC) containing the humanized immunoglobulin gene locus can be introduced into an animal. The vector (e.g., HAC) may contain one or both of the germline repertoire of human antibody heavy chain genes (derived from human chromosome 14) and human antibody light chain genes, such as kappa (derived from human chromosome 2) and lambda (derived from human chromosome 22). The HAC can be transferred into the cells of a non-human animal species to produce transgenic animals by somatic cell nuclear transfer. Transgenic animals can also be bred to produce non-human animals containing the humanized immunoglobulin gene locus.

[0190] In some embodiments, humanized immunoglobulin gene loci are incorporated into the genome of non-human animals. For example, techniques including homologous recombination or homologous recombination repair can be used to modify the animal genome to introduce human nucleotide sequences. Targeted incorporation can be achieved using tools such as CRISPR / Cas, TALEN, and Zn finger nucleases.

[0191] A method for generating non-human animals with a humanized immune system (e.g., non-human animals for immunization with a humanized immune system) is disclosed. For example, a human artificial chromosome can be generated and transferred into cells containing the desired additional genomic modification (e.g., deletion of endogenous non-human immune system genes), and these cells can be used as nuclear donors for generating transgenic non-human animals.

[0192] In some embodiments, the humanized immune system comprises one or more human antibody heavy chains, where each gene encoding an antibody heavy chain is functionally bound to a class switch regulatory element. "Functionally bound" may mean that a first DNA molecule (e.g., a heavy chain gene) is bound to a second DNA molecule (e.g., a class switch regulatory element), where the first and second DNA molecules are arranged such that the first DNA molecule influences the function of the second DNA molecule. These two DNA molecules may or may not be part of a single continuous DNA molecule, and may or may not be adjacent. For example, a promoter is functionally bound to a transcriptionable DNA molecule if the promoter can influence the transcription or translation of the transcriptionable DNA molecule.

[0193] In some embodiments, the humanized immune system comprises one or more human antibody light chains. In some embodiments, the humanized immune system comprises one or more human antibody surrogate light chains.

[0194] In some embodiments, the humanized immune system includes a constant region, such as an amino acid sequence derived from a non-human animal, e.g., a heavy chain constant region or a portion thereof. In some embodiments, the humanized immune system includes a non-human animal-derived IgG (e.g., IgG1) heavy chain constant region (e.g., an ungulate-derived IgG (e.g., IgG1) heavy chain constant region). In some embodiments, at least one class switch regulatory element of a gene encoding one or more human antibody heavy chains is replaced with a non-human (e.g., ungulate-derived) class switch regulatory element to enable antibody class switching, for example, when an antibody is induced against the antigen and / or epitope of the Disclosure in a non-human animal.

[0195] Humanized immunoglobulin gene loci may contain non-human elements incorporated for compatibility with non-human animals. In some embodiments, non-human elements may be present at the humanized immunoglobulin gene loci to reduce recognition by any remaining elements of the immune system of non-human animals. In some embodiments, the immunoglobulin gene may be partially replaced with an amino acid sequence derived from a non-human animal. In some embodiments, non-human regulatory elements may be present at the humanized immunoglobulin gene loci to facilitate expression and regulation of the locus in non-human animals.

[0196] Humanized immunoglobulin gene loci may contain human DNA sequences. Humanized immunoglobulin gene loci can be codon-optimized to facilitate the expression of the included genes (e.g., antibody genes) in non-human animals.

[0197] Non-human animals with a humanized immune system (e.g., non-human animals for immunization with a humanized immune system) may contain or may lack endogenous non-human immune system components. In some embodiments, non-human animals with a humanized immune system may lack non-human antibodies (e.g., lack the ability to produce non-human antibodies). Non-human animals with a humanized immune system may lack, for example, one or more non-human immunoglobulin heavy chain genes, one or more non-human immunoglobulin light chain genes, or a combination thereof.

[0198] Non-human animals with a humanized immune system (e.g., non-human animals for immunization with a humanized immune system) may, for example, harbor non-human immune cells. Non-human animals with a humanized immune system may harbor non-human innate immune system components (e.g., cells, complement, antimicrobial peptides, etc.). In some embodiments, non-human animals with a humanized immune system may harbor non-human T cells. In some embodiments, non-human animals with a humanized immune system may harbor non-human B cells. In some embodiments, non-human animals with a humanized immune system may harbor non-human antigen-presenting cells. In some embodiments, non-human animals with a humanized immune system may harbor non-human antibodies.

[0199] In some embodiments, a non-human animal having a humanized immune system (e.g., a non-human animal for immunization having a humanized immune system) includes any feature or any combination of features or any method of production disclosed in U.S. Patent Application Publication No. 2017 / 0233459 (this document is incorporated herein by reference in its entirety). In some embodiments, non-human animals having a humanized immune system (e.g., non-human animals for immunization having a humanized immune system) are described in Kuroiwa et al., Nat. Biotechnol., 27(2):173-81 (2009); Matsushita et al., PLos ONE, 9(3):e90383 (2014); Hooper et al., Sci. Transl. Med., 6(264):264ra162 (2014); Matsushita et al., PLoS ONE, 10(6):e0130699 (2015); Luke et al., Sci. Transl. Med., 8(326):326ra21 (2016); Dye et al., Sci. Rep., 6:24897 (2016); Gardner et al., J. Virol., 91(14) (2017);Stein et al., Antiviral Res., 146:164-173 (2017);Silver, Clin. Infect. Dis., 66(7):1116-1119 (2018);Beigel et al., Lancet Infect. Dis., 18(4):410-418 (2018);Luke et al., J. Inf. Dis., 218(suppl_5):S636-S648 (2018) (each of which is incorporated herein by reference in its entirety) includes any feature or any combination of features or any method of preparation.

[0200] This publication also provides antibodies (e.g., nanobody or heavy-chain antibodies) containing CDRs described herein (e.g., those described in Table 2, Figure 37, or SEQ ID NOs: 1-24). Such antibodies can be configured to be human antibodies, humanized antibodies, or mouse antibodies. In some examples, the antibodies (e.g., nanobody or heavy-chain antibodies) provided herein may contain CDRs described herein (e.g., those described in Table 2, Figure 37, or SEQ ID NOs: 1-24) and may be monoclonal antibodies (e.g., monoclonal nanobody or monoclonal heavy-chain antibodies).

[0201] In some examples, the antibodies provided herein (e.g., nanobody or heavy chain antibodies) may comprise three CDRs. The first CDR can be selected from the group consisting of SEQ ID NOs. 1-7, or SEQ ID NOs. 1-7 having one, two, or three amino acid modifications (e.g., addition, deletion, or substitution). The second CDR can be selected from the group consisting of SEQ ID NOs. 8-15, or SEQ ID NOs. 8-15 having one, two, or three amino acid modifications (e.g., addition, deletion, or substitution). The third CDR can be selected from the group consisting of SEQ ID NOs. 16-24, or SEQ ID NOs. 16-24 having one, two, or three amino acid modifications (e.g., addition, deletion, or substitution).

[0202] [Table 2]

[0203] In some cases, the antibodies provided herein (e.g., nanobody or heavy chain antibodies) may have or be possessing a heavy chain variable domain having CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 8, and CDR3 having the amino acid sequence of SEQ ID NO: 16. In some cases, the antibodies provided herein (e.g., nanobody or heavy chain antibodies) may have or be possessing a heavy chain variable domain having CDR1 having the amino acid sequence of SEQ ID NO: 2, CDR2 having the amino acid sequence of SEQ ID NO: 8, and CDR3 having the amino acid sequence of SEQ ID NO: 17. In some cases, the antibodies provided herein (e.g., nanobody or heavy chain antibodies) may have or be possessing a heavy chain variable domain having CDR1 having the amino acid sequence of SEQ ID NO: 3, CDR2 having the amino acid sequence of SEQ ID NO: 8, and CDR3 having the amino acid sequence of SEQ ID NO: 18. In some cases, the antibodies provided herein (e.g., nanobody or heavy chain antibodies) may have or be possessing a heavy chain variable domain having CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 9, and CDR3 having the amino acid sequence of SEQ ID NO: 19. In some cases, the antibodies provided herein (e.g., nanobody or heavy chain antibodies) may have or be possessing a heavy chain variable domain having CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 10, and CDR3 having the amino acid sequence of SEQ ID NO: 19. In some cases, the antibodies provided herein (e.g., nanobody or heavy chain antibodies) may have or be possessing a heavy chain variable domain having CDR1 having the amino acid sequence of SEQ ID NO: 5, CDR2 having the amino acid sequence of SEQ ID NO: 11, and CDR3 having the amino acid sequence of SEQ ID NO: 20. In some examples, the antibodies provided herein (e.g., nanobody or heavy chain antibodies) may have or be heavy chain variable domains having CDR1 having the amino acid sequence of SEQ ID NO: 6, CDR2 having the amino acid sequence of SEQ ID NO: 12, and CDR3 having the amino acid sequence of SEQ ID NO: 21.In some cases, the antibodies provided herein (e.g., nanobody or heavy chain antibodies) may have or be possessing a heavy chain variable domain having CDR1 having the amino acid sequence of SEQ ID NO: 5, CDR2 having the amino acid sequence of SEQ ID NO: 13, and CDR3 having the amino acid sequence of SEQ ID NO: 22. In some cases, the antibodies provided herein (e.g., nanobody or heavy chain antibodies) may have or be possessing a heavy chain variable domain having CDR1 having the amino acid sequence of SEQ ID NO: 7, CDR2 having the amino acid sequence of SEQ ID NO: 14, and CDR3 having the amino acid sequence of SEQ ID NO: 23. In some cases, the antibodies provided herein (e.g., nanobody or heavy chain antibodies) may have or be possessing a heavy chain variable domain having CDR1 having the amino acid sequence of SEQ ID NO: 5, CDR2 having the amino acid sequence of SEQ ID NO: 15, and CDR3 having the amino acid sequence of SEQ ID NO: 24.

[0204] In some cases, the CDR3 shown in Table 2 may lack the first C residue and the last W residue.

[0205] As described herein, the amino acid sequences described herein may include amino acid modifications (e.g., amino acid modifications of the number of linked amino acids). Such amino acid modifications include, but are not limited to, amino acid substitutions, amino acid deletions, amino acid additions, and combinations thereof. In some cases, amino acid modifications can be made to improve binding and / or contact with an antigen, and / or to improve the functional activity of antibodies (e.g., nanobody or heavy chain antibodies) provided herein. In some cases, amino acid substitutions in linked sequence identifiers may be conservative amino acid substitutions. For example, a conservative amino acid substitution can be made by substituting one amino acid residue with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0206] In some cases, amino acid substitutions in linked sequence identifiers may be non-conserved amino acid substitutions. Non-conserved amino acid substitutions can be made by substituting one amino acid residue with another amino acid residue having a dissimilar side chain. Examples of non-conserved substitutions include, but are not limited to, (a) substituting a hydrophilic residue (e.g., serine or threonine) with a hydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine, or alanine); (b) substituting cysteine ​​or proline with any other residue; (c) substituting a residue with a basic side chain (e.g., lysine, arginine, or histidine) with a residue with an acidic side chain (e.g., aspartic acid or glutamic acid); and (d) substituting a residue with a bulky side chain (e.g., phenylalanine) with glycine or another residue with a small side chain.

[0207] Methods for generating amino acid sequence variants (e.g., amino acid sequences containing one or more modifications with respect to a linked sequence identifier) ​​include site-directed mutagenesis or random mutagenesis (e.g., by PCR) of nucleic acids encoding an antibody or its fragment. See, for example, Zoller, Curr. Opin. Biotechnol. 3: 348-354 (1992). Both natural and non-natural amino acids (e.g., artificially derivatized amino acids) can be used to generate the amino acid sequence variants provided herein.

[0208] This book also provides pharmaceutical compositions or pharmaceutical formulations which may include any of the antibodies provided herein (e.g., nanobody or heavy chain antibodies). Any of the above pharmaceutical compositions or pharmaceutical formulations may also include further cells or cellular components.

[0209] As described herein, antigens can be administered to non-human animals provided herein to induce the production of antibodies (e.g., heavy chain antibodies such as chimeric heavy chain antibodies). In some embodiments, the antigen is an endogenous antigen or autoantigen for a subject (e.g., mammals, e.g., humans, cows, horses, non-human primates, rabbits, goats, sheep, dogs, pigs, mice, rats).

[0210] In some embodiments, the antigen is a lipid. In some embodiments, the lipid is a membrane lipid and a soluble lipid. Examples of membrane lipids include, but are not limited to, diacylglycerol (DAG), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PtdIns), phosphatidylethanolamine (PE), phosphatidylcholine (PtC), phosphatidylglycerol (PG), sphingomyelin, phosphorylcholine (PC), and cardiolipin. Examples of soluble lipids include, but are not limited to, low-density lipoprotein (LDL), malondialdehyde-LDL (MDA-LDL), oxidized LDL (oxLDL), advanced glycation end product-LDL (AGE-LDL), MDA, and lysophosphatidylcholine (LPC).

[0211] In some embodiments, the antigen is associated with an immune cell (for example, the antigen is a cell surface protein on an immune cell). Examples of immune cells include, but are not limited to, peripheral blood mononuclear cells (PBMCs), macrophages, T cells, dendritic cells, neutrophils, and monocytes.

[0212] In some embodiments, the antigen is a peptide, protein, lipid, molecule, or other biological compound that binds to immune cells.

[0213] In some embodiments, the antigen is associated with damaged cells, dead cells, or dying cells. Cell damage and / or death may be caused by any underlying pathological condition, such as apoptosis, necrosis, or ischemia.

[0214] In some embodiments, the antigen is another immunoglobulin, such as IgG and the like.

[0215] In some embodiments, the antigen can be the antigens listed in Table 3.

[0216]

Table 3

[0217] This document also provides methods for treating or preventing a disease or disorder. In some embodiments, the antibodies provided herein (e.g., nanobodies or heavy chain antibodies) can be used for treating or preventing a disease or disorder. For example, the disease or disorder can be an inflammatory disease, an autoimmune disease, a cardiovascular disease, or a neurodegenerative disease. In some embodiments, the disease or disorder can be diabetes, systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, scleroderma, Crohn's disease, ulcerative colitis, mixed connective tissue disease, Sjogren's syndrome, or polymyositis, dermatomyositis.

[0218] In some examples, a composition comprising the antibodies provided herein (e.g., nanobodies or heavy chain antibodies) can be intended for use in preventing and / or treating a disease or disorder (e.g., an autoimmune disease or an inflammatory disorder). Accordingly, this document further provides a pharmaceutical formulation comprising the antibodies provided herein (e.g., nanobodies or heavy chain antibodies) and a pharmaceutically acceptable carrier therefor. This pharmaceutical formulation can be prepared by conventional techniques as described, for example, in Remington: The Science and Practice of Pharmacy 2005, Lippincott, Williams & Wilkins.

[0219] A pharmaceutically acceptable carrier may be either solid or liquid. Examples of solid preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. The solid carrier may be one or more excipients that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrants, or encapsulating materials.

[0220] Similarly, this also includes solid preparations that are intended to be converted into liquid preparations for oral administration shortly before use. Such liquid preparations include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active ingredient, colorants, flavorings, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers, etc., as described elsewhere, for example (Gervasi et al., Eur. J. Pharmaceutics and Biopharmaceutics, 131:8-24 (2018)).

[0221] Examples of pharmaceutically acceptable carriers that can be used to prepare the pharmaceutical compositions provided herein include, but are not limited to, water, lactic acid, citric acid, sodium chloride, sodium citrate, sodium succinate, sodium phosphate, surfactants (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), dextran 40, or sugars (e.g., sorbitol, mannitol, sucrose, dextrose, or trehalose), and combinations thereof.

[0222] Other components that may be included in the pharmaceutical compositions provided herein include, but are not limited to, amino acids such as glycine or arginine, antioxidants such as ascorbic acid, methionine, or ethylenediaminetetraacetic acid (EDTA), enzalutamide, imatinib, gefitinib, erlotinib, sunitinib, lapatinib, nilotinib, sorafenib, temsirolimus, etc. Examples of anticancer drugs include verolimus, pazopanib, crizotinib, ruxolitinib, axitinib, bosutinib, cabozantinib, ponatinib, regorafenib, ibrutinib, trametinib, perifosin, bortezomib, carfilzomib, batimast, ganetespib, ovatocrax, navitocrax, taxol, paclitaxel, or bevacizumab, as well as combinations thereof.

[0223] In some cases, the antibodies provided herein (e.g., nanobody or heavy-chain antibodies) can be formulated for parenteral administration and may be provided in unit dosage forms with optionally added preservatives in ampoules, pre-filled syringes, small-volume infusions, or multi-dose containers. The compositions may take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, such as solutions in aqueous polyethylene glycol. Examples of oily or non-aqueous carriers, diluents, solvents, or vehicles include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, and may contain agents such as preservatives, wetting agents, emulsifiers or suspenders, stabilizers, and / or dispersants. In some cases, the formulations may contain about 0.5% to 75% by weight of one or more active ingredients together with the remainder consisting of preferred pharmaceutical excipients described herein.

[0224] The compositions provided herein can be administered in an effective amount, whether by oral, rectal, or parenteral (including subcutaneous) routes, in parallel, simultaneously, or together with a pharmaceutically acceptable carrier or diluent, particularly and preferably in the form of the pharmaceutical composition.

[0225] This book also provides pharmaceutical compositions comprising B cells (e.g., B cells isolated from non-human animals provided herein), monoclonal antibodies (e.g., monoclonal heavy chain antibodies or monoclonal nanobodies), and / or polyclonal antibodies (e.g., polyclonal heavy chain antibodies or polyclonal nanobodies). Such pharmaceutical compositions may include adjuvants, buffers, salts, or combinations thereof.

[0226] Adjuvants are pharmacological and / or immunological agents that modify the effects of other drugs. In some embodiments, adjuvants may be added to a composition to modify the immune response by boosting it to provide a larger amount of antibody and / or longer-lasting protection, thereby minimizing the amount of antigenic material injected. Depending on the type of composition, adjuvants may also be used to enhance the potency of the composition by assisting in disrupting the immune response against specific cell types of the immune system (e.g., by activating T cells instead of antibody-secreting B cells). In one embodiment, the composition may contain at least one adjuvant. In another embodiment, the adjuvant may be aluminum-based. Aluminum adjuvants may be aluminum phosphate, aluminum hydroxide, amorphous aluminum hydrogen sulfate phosphate, and / or combinations thereof. Other adjuvants may also be included.

[0227] In another embodiment, the compositions described herein may comprise at least one buffer. In one embodiment, the buffer may be PBS and / or histidine-based. In another embodiment, the buffer may have a pH of 6.0 to 7.5. In one embodiment, the buffer may be isotonic, for example, a 0.6% to 1.8% NaCl buffer.

[0228] Emulsifiers (also known as “emulgents”) are substances that stabilize emulsions by increasing their kinetic stability. One class of emulsifiers is known as “surfactants.” Polysorbates are a class of emulsifiers used in several pharmaceutical and food preparations. Common brand names for polysorbates include Alkest, Canarcel, and Tween. Some examples of polysorbates are polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. In one embodiment, the composition provided herein may contain an emulsifier such as one of the above polysorbates. In one embodiment, the composition may contain 0.001–0.02% polysorbate 80. Other polysorbates or emulsifiers may also be used as described herein.

[0229] In some examples, the pharmaceutical compositions provided herein may comprise an antibody (e.g., a nanobody or heavy chain antibody) provided herein and a pharmaceutically acceptable carrier. In some examples, the pharmaceutical compositions provided herein may comprise an antibody (e.g., a nanobody or heavy chain antibody), a pharmaceutically acceptable carrier, and a buffer. In some examples, the pharmaceutical compositions provided herein may comprise an antibody (e.g., a nanobody or heavy chain antibody), a pharmaceutically acceptable carrier, and an emulsifier. In some examples, the pharmaceutical compositions provided herein may comprise an antibody (e.g., a nanobody or heavy chain antibody), a pharmaceutically acceptable carrier, and an adjuvant. In some examples, the pharmaceutical compositions provided herein may comprise an antibody (e.g., a nanobody or heavy chain antibody), a pharmaceutically acceptable carrier, a buffer, and an adjuvant. In some examples, the pharmaceutical compositions provided herein may comprise an antibody (e.g., a nanobody or heavy chain antibody), a pharmaceutically acceptable carrier, an emulsifier, and an adjuvant. In some examples, the pharmaceutical compositions provided herein may include antibodies (e.g., nanobody or heavy chain antibodies), pharmaceutically acceptable carriers, emulsifiers, buffers, and adjuvants.

[0230] Nucleic acid and polypeptide formulations described herein can be designed and constructed using any suitable method. Recombinant methods are generally applicable. See, for example, Smales & James (eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013). Methods for designing, preparing, evaluating, purifying, and manipulating nucleic acid compositions are described in Green and Sambrook (eds.), Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory Press (2012).

[0231] This book also provides methods for treating diseases or disorders by administering compositions (e.g., pharmaceutical compositions provided herein) containing antibodies (e.g., heavy chain antibodies or nanobodies) provided herein to mammals (e.g., humans). For example, a composition (e.g., pharmaceutical compositions provided herein) containing one or more antibodies provided herein can be administered to a mammal (e.g., human) having an inflammatory disease to treat the mammal. In some examples, administering a composition (e.g., pharmaceutical compositions provided herein) containing one or more antibodies provided herein to a mammal (e.g., human) can reduce the severity of the inflammatory disease in the mammal and / or increase the survival time of the mammal suffering from the inflammatory disease compared to a mammal (e.g., human) that has not been administered the composition.

[0232] The compositions described herein may be administered to a subject by any mode of delivery, for example, parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or intra-articular injection), or by rectal administration, oral administration (e.g., tablets, sprays), vaginal administration, topical administration, transdermal administration (e.g., see International PCT Patent Application Publication WO99 / 27961), or transcutaneous administration (e.g., see International PCT Patent Application Publication WO02 / 074244 and International PCT Patent Application Publication WO02 / 064162), intranasal administration (e.g., see International PCT Patent Application Publication WO03 / 028760), intraocular administration, intraocular administration, pulmonary administration, or other mucosal administration. Multiple doses may be administered via the same or different routes.

[0233] The compositions provided herein (for example, compositions comprising heavy chain antibodies and / or nanobodies produced from non-human animals provided herein) may be administered before, simultaneously with, or after the delivery of other therapeutic agents. Similarly, the administration site may be the same as or different from that of the other therapeutic agents being administered.

[0234] Therapeutic administration using the compositions provided herein may be a single-dose schedule or a multi-dose schedule. A multi-dose schedule is one in which the initial course of vaccine administration may consist of 1 to 10 separate doses, followed by subsequent doses at time intervals selected to maintain and / or enhance the immune response, for example, 1 to 4 months for the second dose, and other doses given several months later as needed. The administration regimen will also be determined, at least in part, by the efficacy of the modality, the delivery method used, the needs of the subject, and by the discretion of the practitioner.

[0235] The above embodiments can be combined to achieve the functional characteristics described above. This is also demonstrated by the following examples illustrating exemplary combinations and the functional characteristics achieved.

Example

[0236] Example 1 - Gene recombination of Singularity mouse Gene recombination procedure Using blastocysts isolated from the mating of 129S6 mice and C57BL / 6N mice, LVGN-YF ES cell lines (40, XY) were established and used for all gene recombinations. This F1 hybrid ES cell line showed robust germline competence after multiple rounds of genetic modification, and the use of the F1 hybrid ES cell line also enabled the use of strain-specific SNPs to identify consecutive genetic modifications occurring on the same chromosome. ES cells were cultured in knockout DMEM supplemented with 20% ES cell-qualified fetal bovine serum (FBS), 0.1 mM MEM non-essential amino acids, 0.1 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 2 mM GlutaMAX-I supplement, 100 units / mL penicillin-streptomycin, 25 nM MEK inhibitor PD98059 (Sigma), 3 nM GSK-3 inhibitor CHIR99021 (Sigma), and 1,000 units / mL mouse leukemia inhibitory factor (LIF, Sigma) on feeder cells. The hygro(®) / neo(®) / puro(®) triple-resistant feeder cell line LVGN-SHNPL was engineered from the SNL76 / 7 feeder cell line and maintained in knockout DMEM supplemented with 10% ES cell-qualified FBS, 0.1 mM MEM non-essential amino acids, 0.1 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 2 mM GlutaMAX-I supplement, and 100 units / mL penicillin-streptomycin. Feeder cells were prepared by treating proliferating cells with 10 mg / mL Mitomycin C (Sigma) for 3 hours.

[0237] All transfections were performed either by lipofection using Lipofectamine LTX (ThermoFisher) or by electroporation using a Bio-Rad Gene Pulser II instrument. For lipofections, the transfection rates were 0.1 to 1 × 10⁻⁶. 6 Dissociated ES cells were mixed with 0.5–2.5 μg of plasmid DNA-lipofectamine complex in Opti-MEM according to the manufacturer's instructions for use, cultured overnight in growth medium, and antibiotic selection was applied after 24 hours as needed. For electroporation, 0.5–1.5 × 10⁻⁶ 7ES cells were mixed with 10–30 μg of plasmid and / or BAC DNA in PBS, electroporated in 4 mm gap cuvettes at 250 V / 500 μF, cultured overnight in growth medium, and antibiotic selection was applied after 24 hours as needed. The antibiotic concentrations used for selection were 250 μg / mL for Geneticin (G418), 200 μg / mL for Hygromycin B, and 5 μg / mL for Puromycin. For CRISPR-mediated gene editing, Cas9 and sgRNA were delivered as separate plasmids, which were co-transfected with the PGK-puro gene or PGK-hygro gene for enrichment of transformants and selected with the corresponding antibiotic for 2 days, or co-transfected with an HDR template and selected with the corresponding antibiotic for 10–14 days to derive stably transfected clones. To remove selection marker cassettes adjacent to recombination sites (lox-lox, frt-frt, or attB-attP), ES cells were first transfected with plasmids expressing the corresponding recombinase or integrase (Cre, Flop, or φC31, respectively), plated at low density, and isolated individual clones. For recombinase-mediated cassette exchange (RMCE), ES cells were co-transfected with an RMCE construct and a Cre-expressing plasmid, and selected with the corresponding antibiotic for 10–14 days. ES cell colonies were picked into 96-well plates for growth, genotyped by PCR, screened for desired mutations, and then Sanger sequenced. Sequence-validated positive clones were grown from 96-well plates to 24-well plates, and then to 6-well plates for cryopreservation.

[0238] Chimeras were produced using positive ES cell lines containing recombinant mutations, following standard procedures. Briefly, 3.5 dpc blastocysts were isolated from superovulated C57BL / 6N females, microinjected with ES cells, and then transferred to the uterus of 2.5 dpc pseudo-pregnant Swiss Webster females for transplantation. High-rate chimeric males were mated with C57BL / 6N females for germline transmission of recombinant mutations. Heterozygous F1 mice were identified by junctional PCR from genomic DNA isolated from biopsies. The F1 mice were then crossed to produce F2 mice homozygous for the same mutation, or crossed with other strains as needed. All recombinant mice were maintained in a mixed 129S6 and C57BL / 6N background.

[0239] Generation of Singularity Musculus Alleles Igh is one of the largest loci in the mouse genome, spanning several megabases (Mb) near the right end of arm 12q. It encodes numerous different elements involved in generating virtually unlimited antibody diversity. The aforementioned locus contains a variable region exceeding 2.5 Mb encoding hundreds of gene segments involved in the majority of antibody diversity, and a much smaller 220 kb constant CH region encoding expression for several antibody classes and subtypes (Figure 1A). The CH region contains eight CH genes encoding different Ig isotypes: Cμ (Ighm), Cδ (Ighd), Cγ3 (Ighg3), Cγ1 (Ighg1), Cγ2b (Ighg2b), Cγ2a / 2c (Ighg2a / 2c), Cε (Ighe), and Cα (Ighha). Regulatory elements adjacent to and further throughout this region are involved in isotype class-switch recombination (CSR) and timely expression (Figure 2).

[0240] In addition to the elements mentioned above, upstream of each Ig isotype (except IgD) are the I promoter / exon and the S switch region. The latter is involved in CSR, while the former is involved in germline transcription of its corresponding Ig isotype. Ig isotype transcription is highly regulated. In dormant B cells, germline transcription (GLT) is limited to Cμ GLT driven by the Eμ enhancer and constitutive Iμ promoter. In activated B cells in response to antigen encounter and cytokines, downstream Ig isotype transcription is activated at its I promoter, which contains each response element. Co-transcription of the activated Ig isotype at the Iμ promoter and downstream I promoter results in AID-mediated CSR.

[0241] A stepwise process was used to generate the Singularity Musculus allele (Figure 1B). Unlike the normal tetrameric antibody produced by wild-type (WT) mice (Figure 1C), homozygous mice for this allele produced only HCAb (Figure 1D), and its genetic makeup and diversity are entirely derived from the mouse's innate immune repertoire.

[0242] The generation of the Singularity Musculus allele was achieved via three rounds of recombination in LVGN-YF ES cells (Figure 3). The Igh locus in these ES cells encoded Cγ2c, which is similar to Cγ2c found in C57BL / 6N mice rather than Cγ2a in the BALB / c lineage (Figure 3A). The first round of modification was performed via CRISPR-mediated non-homologous end joining (NHEJ), which deleted a 92.6Kb genomic DNA fragment across Cμ, Cδ, Cγ3, and the first exon of Cγ1 (encoding the CH1 domain of IgG1) (Figure 3B). The sgRNA was designed to cleave immediately downstream of the Cμ switch region (Sμ) and upstream of the second exon of Cγ1 (encoding the hinge domain of IgG1), thereby placing the cleaved Cγ1 gene under the direct control of the Iμ promoter and Sμ, otherwise constitutively inducing cytokine-induced IgG1 transcription, similar to that of IgM in the WT allele. The second round of modification was carried out via CRISPR-mediated homologous recombination repair (HDR), which removed a 63.2Kb genomic DNA fragment spanning the first three exons of Cγ2b, Cγ2c, Cε, and Cα, while introducing a selective marker cassette (PGK / Em7-neo) adjacent to the frt site (Figure 3C). The CRISPR cleavage sites were selected to avoid removal of the 3'γ1E element downstream of Cγ1 and the 5'hsR1 element in intron 3 of Cα. The third round of recombination utilized transient expression of Flp recombinase, which removed the selection marker cassette, leaving a single frt site for use in synteny validation of subsequent modifications (Figure 3D). Regulatory elements (including Eμ, Iμ, Sμ, 3'γ1E, 5'hsR1, 3'RR, and 3'CBE) were retained intact to enable high levels of constitutive transcription of CH1-cleaved IgG1 (IgG1ΔCH1) from the endogenous Igh allele (Figures 2 and 3E).Therefore, the resulting Singularity Musculus mice produced only HCAb of the IgG1 subtype; all other antibody classes (IgM, IgD, IgE, and IgA), as well as IgG subtypes (IgG2b, IgG2c, and IgG3), were removed to avoid any potential mechanisms that might impair HCAb production and to facilitate nanobody discovery, expression, and purification.

[0243] Singularity HyperDock Allele Generation To extend the versatility of the Singularity platform and generate HCAbs from other species, a Singularity HyperDock allele was generated by deleting a 2.58 Mb genomic DNA fragment (from upstream of Ighv86-1 to downstream of Ighj4) containing all mouse VH, DH, and JH genes, and inserting a docking cassette for sequential RMCE upstream of Eμ via CRISPR-mediated HDR (Figures 4 and 5). The HDR template included a left homology arm, frt site, attB site, PGK promoter, loxP site, Em7-neo cassette, attP site, and lox2272 site followed by a right homology arm. The frt site was incorporated to verify that the introduced RMCE docking cassette was on the same chromosome (C57BL / 6N) as the Singularity Musculus allele during Flp recombinase expression. By selecting a wild-type loxP site instead of other heterologous-specific lox sites and placing it between the PGK promoter and the Em7-neo cassette, we enabled highly efficient RMCE events via selective marker swapping. These modifications resulted in a mouse VDJ null Singularity HyperDock allele containing an RMCE docking site for the serial introduction of synthetic fragments including BACs, cloning constructs, or any combination of V, D, or J gene heavy or light chain alleles from human or other species (Figure 5).

[0244] Generation of the Singularity Sapiens Allele Series Recombinant BACs containing human VH, DH, and JH genes were introduced into the Singularity HyperDock allele to generate the Singularity Sapiens allele series (Figures 6-8). Duplicate IGH BAC clones from the CH17 BAC library and the RPCI-11 library (BACPAC resource) (Figure 9 and Table 4) were modified at both ends by bacterial homologous recombination to incorporate either an Em7-hyg cassette or an Em7-neo cassette to enable selective marker swapping, while simultaneously introducing corresponding heterologous-specific lox sites (Table 1) adjacent to the genomic fragments for sequential RMCE. In short, synthetic gBlocks (IDT or Twist) containing appropriate lox sites and two 75-150 base pair (bp) homology arms adjacent to the antibiotic resistance cassette were electroporated into E. coli strains containing heat-inducible Red recombinase in electroporation cuvettes with a 1 mm gap, using a Bio-Rad GenePulser II instrument at 1.75 kV, 25 μF, and 200 ohms. Next, 1.0 mL of SOC medium was added to each cuvette, then transferred to microcentrifuge tubes, and incubated at 32°C for 1 hour with shaking (200 rpm). The cells were then plated onto LB agar plates containing the corresponding antibiotics. The resulting colonies were screened with junctional primers by PCR and then Sanger sequenced for validation. The recombination process for the first introduced BAC (hIGH-BAC1) is shown in Figure 10, which consists of three human VH genes (two functional genes; IGHV1-2 and IGHV6-1) and 27 human D H Genes, and 9 human J HThe gene was included. The loxP-Em7-hyg-attP-lox5171 cassette was introduced by recombination at the 5' end of the original BAC clone, directly upstream of the human IGHV1-2 gene. Subsequently, the lox2272-aadA cassette was introduced at the 3' end, directly downstream of the human IGHJ6 gene. Then, the recombinant BAC was used for the first round of RMCE (between loxP and lox2272) to introduce three human VH genes, all human DH genes, and all human JH genes directly upstream of the mouse Igh intronic enhancer Eμ, while introducing a different heterologous specific lox site (lox5171) for the next round of RMCE (Figures 7A-7B). Subsequent overlapping BACs were similarly modified using alternative selection markers (Em7-neo and Em7-hyg) and different heterospecific lox sites, and the overlapping fragments were excised to stepwise construct the human VDJ genomic region (Figures 7C-7D and 8). The original BAC clones and heterospecific lox sites used to reconstruct the complete human VDJ region can be found in Tables 4 and 1, respectively. Wild-type loxP sites, which exhibit high recombination efficiency, were used in each round of RMCE and paired with different heterospecific lox sites. All recombinant BACs were confirmed by PacBio SMRT sequencing before transfection into ES cells. No significant mutations were observed, except for a few SNPs and small insertions / deletions (indels) in intergenetic regions. The sequential RMCE process resulted in the generation of a series of humanized singularity alleles (SSV1-5) with enhanced VH diversity. PCR analysis of SSV4 mice using VH-specific primers, followed by Sanger sequencing, confirmed the incorporation of all 37 functional VH elements (Figure 11). hIGH-BAC5 was recombined to include sequences from three source BACs (Figure 12) and introduced into SSV4 ES via RMCE to complete the construction of SSV5 designed to contain the complete human VH repertoire (126 VH genes, 27 DH genes, and 9 JH genes).

[0245] [Table 4]

[0246] Generation of Igk and Igl knockout mice to produce light chain-less singularity mice. To prevent unexplained interference between light chain and HCAb production in singularity mice, the mouse light chain was removed by deleting the V and J gene segments from the IgK and IgL gene loci.

[0247] To remove the kappa light chain, a 3.17 Mb genomic DNA fragment containing the entire mouse VK and JK gene segments was deleted by CRISPR / Cas9-mediated HDR and replaced with a docking cassette (Figure 13A). Similar to the Singularity HyperDock in the Igh allele, the recombinant IgK HyperDock / KO allele contains the attB site, PGK promoter, loxP site, Em7-neo cassette, attP site, and lox2272 site upstream of the 5' enhancer element located at the 5' end of the mouse CK gene, thus enabling sequential RMCE in the Igk allele. Recombinant Igk HyperDock / KO mice were generated and confirmed by PCR and sequencing (Figure 13B).

[0248] To remove the lambda light chain, a 200kb genomic DNA fragment between Olfr164 and Gm10086, containing all lambda loci including the VL1, VL2, VL3 gene segments, as well as the JL and CL gene segments, was deleted by CRISPR / Cas9-mediated NHEJ (Figure 14A). Igl KO ES cells were generated and confirmed by PCR and sequencing (Figure 14B).

[0249] Example 2 - Characterization of Singularity Mouse Singularity mice constitutively express only heavy chain antibodies against CH1-cleaved IgG1. Unlike WT mice (Figure 15A), which can express a complete panel of Ig isotypes, Singularity Musculus mice express only IgG1-ΔCH1 (Figure 15B). To confirm this, transcriptions of different Ig classes and subtypes were analyzed. Total RNA was isolated from the spleens of WT and Singularity Musculus mice using Trizol reagent, and RNA concentration and quality were determined using Bioanalyzer. Reverse transcription was performed using Superscript IV Reverse Transcriptase (ThermoFisher) and oligo(dT)20 primers according to the manufacturer's instructions. Transcriptions of all Ig classes and subtypes were analyzed by RT-PCR with the mouse B cell marker Cd19 as an internal control, following standard procedures. Ighm, Ighd, Ighg3, Ighg1, Ighg2b, Ighg2c, Ighe, and Igha were all detected in WT mice, but Singularity Musculus mice expressed only a reduced-size Ighg1 transcript (Figure 15C) and lacked the CH1 sequence, as verified by sequencing.

[0250] To investigate Ighg1 transcription in Singularity Sapiens mice, RT-PCR was performed on cDNA reverse-transcribed from whole spleen RNA of Singularity Sapiens (SSV1) mice. SSV1 induced from the Singularity Musculus platform (Figure 16A) was compared to human D H and D J Full panel and two functional human V HThe design included (IGHV6-1, IGHV1-2) (Figure 16B). A PCR primer set was designed with forward primers specific to human IGHV6-1, IGHV1-2, and IGHJ3, and a reverse primer specific to mouse Ighg1 CH2. A chimeric transcript of human VDJ-mouse Ighg1-ΔCH1 was detected in Singularity Sapiens (SSV1) mice, but not in Singularity Musculus mice (Figure 16C), and sequencing further verified that the cells were correctly spliced ​​(Figure 16D).

[0251] To investigate the protein expression of different Ig classes and subtypes, immunoglobulins from plasma samples of immunized wild-type and Singularity Musculus mice were purified using protein A / G magnetic beads, separated by reduced SDS-PAGE, and electrophoretically transferred onto Immobilon®-P membranes (Millipore Sigma) according to standard procedures. Immunodetection was performed using HRP-conjugated secondary antibodies and enhanced chemiluminescence, followed by autoradiography using ECL Western blotting reagents. Trepped IgG1 of approximately 40 kDa was detected in Singularity Musculus mice (compared to full-length IgG1 of approximately 50 kDa in wild-type mice), but IgM and IgG2b could not be detected (Figure 15D). Similarly, a truncated IgG1 of approximately 40 kDa, corresponding to human VDJ-mouse IgG1-ΔCH1, was detected in immunized Singularity Sapiens mice (SSV1), but these mice did not express IgM or full-length IgG1, as was observed in wild-type mice (Figure 17).

[0252] Upregulated IgG expression in B cells of Singularity mice The spleen of Singularity Musculus mice was similar in shape and size to that of wild-type mice (Figure 18A). To examine IgM and IgG expression on the B cell membrane, single-cell suspensions were prepared from the spleen, treated with ACK lysis buffer to remove erythrocytes, blocked with an Fc blocker, and stained with rat-anti-mouse IgM (PE-Cy7), rat-anti-mouse IgG (BV421), and rat-anti-mouse CD19 (AF700) in FACS buffer (PBS with 1% FBS). After staining, cells were analyzed by flow cytometry (BD LSR II). IgM expression in Singularity Musculus mice + No B cells were detected, but IgG + B cells are IgG in wild-type mice. + Compared to B cells, IgG1 was detected at a significantly higher rate, consistent with the increased expression level attributable to constitutively high levels of germline transcription of IgG1 (Figure 18B). Similarly, FACS analysis of splenocytes from Singularity Sapiens mice (SSV2) and wild-type mice was performed using the above procedure with rat-anti-mouse IgM (APC), rat-anti-mouse IgG1 (APC), rat-anti-mouse IgD (FITC), and rat-anti-mouse B220 (PerCP-Cy5.5). This analysis showed that in SSV2 mice, IgM + B cells or IgD + Although B cells were not detected, they were found to be abundant in wild-type mice (Figure 19A). IgG1 + The cells were detected at a significantly higher rate in SSV2 mice (Figure 19B).

[0253] Robust humoral immune response in Singularity mice Protein antigens (Table 3) were prepared in phosphate-buffered saline (PBS) and freshly mixed in a 1:1 (v / v) ratio with either complete Freund's adjuvant (Sigma catalog No. 5881, for priming injection) or incomplete Freund's adjuvant (Sigma catalog No. 5506, for boosting injection) by repeatedly passing the mixture through two conjugated syringes until a smooth emulsion was formed. The mixture was injected into male or female mice aged 4-12 weeks using a 1 mL syringe and a 27 gauge needle. Priming and boosting injections were administered subcutaneously (50 μL) and / or intraperitoneally (100 μL) into the left and right inguinal regions at 2-week intervals, using 10-25 μg of antigen protein per mouse. Tail vein blood was collected before each injection. The final boost was administered intraperitoneally at week 4 or 6 with the antigen protein without adjuvant. The animals were slaughtered 3–4 days later for terminal bleeding and tissue extraction. Blood samples were processed into plasma according to standard procedures.

[0254] To assay antibody titers in plasma, ELISA plates were coated overnight at 4°C with 1 μg / mL antigen protein diluted in PBS. After repeated washing with PBST (PBS + 0.05% Tween-20) and blocking with Super Block (Thermo Fisher), plasma samples were serially diluted in dilution buffer and applied to the plates. After removing unbound proteins through multiple washes, bound proteins were detected using the corresponding HRP-conjugated secondary antibody, and the mixture was colored with 3,3',5,5'-tetramethylbenzidine (TMB) substrate (BM Blue, Sigma) and stopped with 50 μL of 1 M H2SO4. Absorbance was read at 450 nm. In Singularity Musculus mice, a robust humoral immune response comparable to that in wild-type mice was observed 4 weeks after SAT immunization (D28) (Figures 20A and 20B), and significantly higher titers were obtained in both Singularity Musculus mice and Singularity Sapiens mice (SSV1) 6 weeks later (D51) (Figure 21A). Similar results were obtained with other immunogens such as PD-L1 (Figure 21B).

[0255] NGS analysis of the VH repertoire in singularity mice Total RNA was isolated from the spleen of wild-type or Singularity Musculus mice immunized with different antigens using Trizol reagent, and RNA quality and concentration were determined using Bioanalyzer. Recombinant variable region (VH) sequences from wild-type or Singularity mice were amplified by 5' rapid amplification (5'RACE) of the cDNA ends for next-generation sequencing (NGS). Briefly, reverse transcription was performed using Superscript IV reverse transcriptionase, oligo(dT)20 primers, and template switch primers containing a 5'RACE adapter and a unique molecular identifier (UMI) sequence (5'-CTACA-CTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNrGrGrGrGrG-3'; SEQ ID NO: 37). Next, the template-switched reverse transcription product was amplified in the first round of PCR using a 5'RACE adapter forward primer (5'-CTACACTC-TTTCCCTACACGACGCTCTTCCGATCT-3'; SEQ ID NO: 38) and a reverse primer specific to the IgG1 CH2 domain (5'-GGTGGTTGTGCAGGCCCTCATG-3'; SEQ ID NO: 39). The purified product was further amplified in the second round of PCR using a 5'RACE adapter forward primer and a reverse primer specific to either the IgG1 CH1 domain (5'-CCATGGAGTTAGTTTGGGCAGCA-3' for wild-type IgG1 transcript; SEQ ID NO: 40) or the IgG1 hinge domain (5'-CAAGGCTTACAACCACAATCCCT-3' for Singularity IgG1 transcript; SEQ ID NO: 41) to ensure that both mouse strains produced amplicons of approximately 600 bp (Figure 22). The resulting nested PCR products were further amplified in the third round of PCR to incorporate Illumina P5 and P7 adapter sequences and barcodes for NGS, enabling sample multiplexing. The final 5' RACE library was purified and sequenced using 2X 300bp pair-end-run on Illumina MiSeq.

[0256] Paired-end sequence reads in fastq format were processed and aligned to VH, DH, and JH reference germline genes based on annotations from the International ImMunoGeneTics Information System (IMGT, imgt.org on the World Wide Web) using KAligner, a special version of the K-mer linkage algorithm (Liao et al., Nucleic Acids Res., 41(10):e108 (2013)), to identify the CDR region. If the CDR3 was identical between sequences and there were two or fewer mismatched nucleotide residues, the full-length in-frame sequence was further constructed into a chronotype. Low-quality sequences were excluded from construction. Chronotypes from each animal were ranked according to their abundance, and chronotypes with fewer than 5 counts were not included in further analysis.

[0257] Eighteen samples from wild-type and Singularity Musculus mice immunized with SARS-CoV-2 spike activity trimer SAT (R&D Systems; Catalog No. 10549-CV), PD-L1 (R&D Systems; Catalog No. 156-B7), rabbit IgG (ThermoFisher; Catalog No. 02-6102), rat IgG (ThermoFisher; Catalog No. 31933), or goat IgG (ThermoFisher; Catalog No. 31245) were processed for NGS to determine their corresponding VH repertoire. Over one million reads were recovered per sample across all samples, and approximately half of these were successfully aligned to the Igh locus (Table 3). Notably, the number of chronotypes for all tested antigens in Singularity Musculus mice was significantly higher than that observed in WT mice, ranging from several to over 20 times. Furthermore, while 79–99 IGHV gene segments were utilized for these antigens in WT mice, a significantly higher number of IGHVs (103–122) were utilized in Singularity Musculus mice, approaching the theoretical limit (125 functional IGHVs based on mouse genome GRCm38 / mm10 annotation) (Figures 23A and 24 and Table 3). The ability of Singularity Musculus mice to utilize a greater number of IGHV segments compared to WT mice was extremely significant across several tested antigens (Figure 24). This ability stems from the high levels of IgG1 GLT in Singularity Musculus mice, as well as the removal of all other Ig classes and subtypes, compared to the inducible cytokine-dependent expression of IgG1 GLT in WT mice, which may have enabled the expression of IgG1 alone, regardless of the immunogen.

[0258] VH sequence analysis showed that Singularity mice exhibited similar diverse use of IGHV gene segments compared to WT mice (Figures 23A and 24A-24B). The IGHV gene segments that resulted in more or fewer chronotypes in wild-type mice were similar in Singularity Musculus mice (Figures 24A-24B). All four IghJ segments were used, but in Singularity Musculus mice, IGHJ3 was distinguished while IGHJ4 was preferred, likely due to structural preference for HCAb formation (Figures 23B and 25). No significant differences were observed in CDR3 size distribution between chronotypes, with an average size of approximately 14 in both wild-type and Singularity Musculus mice (including invariant C and W residues at the CDR3 boundary) (Figures 23C and 26).

[0259] To identify somatic hypermutations, the top 100 chronotype sequences from each naive or SAT-immunized Singularity Musculus mouse were aligned to the corresponding germline IGHV sequence using IgBlast (ncbi.nlm.nih.gov / igblast / on the World Wide Web). Mutation rates at each residue position were calculated and plotted according to the IMGT numbering scheme (Figure 27). Low mutation rates were observed in naive mice, but immunized mice showed significantly higher levels of somatic hypermutation, which were highly enriched in the CDR region (Figure 27). This was further confirmed by subsequent analysis of the complete VH sequence of the validated nanobody binder (Figure 37).

[0260] Example 3 - Discovery of nanobodies using Singularity mice Sequence-driven high-throughput screening for nanobody binders Next-generation sequencing (NGS) and bioinformatics analysis were used to profile and select VH sequences (chronotypes) from immunized Singularity mice, and then nanobody binders were identified by gene synthesis, cloning, expression, and ELISA screening (Figure 28). Alternatively, nanobody binders can be identified by other methods, including, but not limited to, hybridoma, single B cell cloning, single B cell sequencing, and various display approaches, such as bacterial display, yeast display, mammalian cell display, and phage display.

[0261] To select candidate chronotypes for nanobody expression and binder screening, chronotypes from each animal were ranked according to their abundance and somatic hypermutability. Phylogenetic analysis of chronotype sequences was performed using Clustal Omega (ebi.ac.uk / Tools / msa / clustalo on the World Wide Web), and representative sequences were selected from different branches (Figure 29). First, candidate chronotype sequences (VH) from Singularity mice immunized with SAT were human-codon optimized, a cloning adapter was added adjacent to them, and they were synthesized as eBlock gene fragments (IDTs). Next, the synthesized eBlocks were cloned into the pFuse-hIgG1-Fc2 vector (Invivogen) using NEBuilder HiFi DNA assembly to generate in-frame fusions of the IL-2 signal peptide, VH, and the Fc domain (hinge-CH2-CH3) of human IgG1 (Figures 30A-30B). Next, the sequence-validated expression construct was transfected into Expi293F cells (ThermoFisher) in a 96-well format to produce secreted nanobody-Fc fusions according to the manufacturer's instructions. The culture supernatant was collected 6 days after transfection and used for ELISA screening to identify antigen-specific binders.

[0262] Screening of 92 chronotypes selected from Singularity Musculus mice immunized with SAT identified 21 binders (ELISA OD > 0.5) (23%), of which 11 (52%) showed high levels of binding (ELISA OD > 3.0) (Figures 31-32). These VH sequences were then used to query the original chronotype sequence library by phylogenetic analysis to identify homologous VH sequences, which were then used for secondary screening for hit expansion. Of the 30 chronotypes screened, 15 (50%) (most were not included in the primary screening due to low abundance) were binders, of which 11 (73%) showed high affinity (Figures 31-32). In contrast, a control screening using chronotypes derived from wild-type mice selected after the same criteria (high abundance and hypermutability) failed to identify any binders (0 / 29), suggesting that functional nanobodies can only be induced from HCAb produced in Singularity mice, but not from conventional H2L2 antibodies produced in wild-type mice (Figures 31-32). SAT nanobody binder screening using Singularity Sapiens mice (SSV2) identified 14 / 41 (34%) Nb-Fc binders in human VH sequences, suggesting that functional HCAb were produced in humanized mice via the same mechanism (Figures 31-32).

[0263] ELISA screening for nanobodies against PD-L1, goat IgG, rabbit IgG, and rat IgG identified 33%–61% of binders against the corresponding antigens, further demonstrating the highly efficient NGS-driven screening method and its unparalleled suitability for nanobody discovery (Figures 31–32). Due to the single-chain nature of HCAb, each identified chronotype represented a unique antibody, which was identified by bulk RNA-seq without the use of any single-cell approach.

[0264] Biophysical and biochemical properties of purified Nb-Fc A selected set of ELISA-positive SAT Nb-Fc constructs (derived from both mouse and human VH sequences; Table 5) was used to transfect 30 mL Expi293F cell cultures to produce nanobody-Fc fusions, which were then purified using protein A affinity chromatography according to a standard procedure. Briefly, 6 days after transfection, the cell culture supernatant was collected, filtered through a 0.22 μm filter, and loaded onto a 0.5 mL protein A column (MabSelect SuRe, Cytiva) pre-equilibrium with PBS. After washing with 2 mL of PBS, the bound proteins were eluted from the column with 4 mL of citrate buffer (25 mM citrate, 150 mM sodium chloride, pH 3.5) and neutralized with 1 M Tris-HCl (pH 8.8). The final buffer was replaced with PBS using a Vivospin Turbo (30,000 MWCO PES). SDS-PAGE analysis showed that the purified Nb-Fc fusion migrated at approximately 80 kDa under non-reducing conditions and approximately 40 kDa under reducing conditions, in contrast to the conventional antibody (approximately 150 kDa) which has two heavy chains (approximately 50 kDa) and two light chains (approximately 25 kDa). This was consistent with the predicted size of VH-based Nb-Fc as a homodimer (Figures 33-34).

[0265] [Table 5]

[0266] To assess the quality of purified Nb-Fc, size exclusion chromatography (SEC) analysis was performed. In short, 2–10 μL of purified Nb-Fc samples were injected into an ACQUITY UPLC (Waters) Protein BEH SEC 200, 1.7 μm, 4.6 x 150 mm column at a flow rate of 0.3 mL / min for 10 minutes. A mobile phase of 50 mM sodium phosphate, 500 mM NaCl, pH 6.2 was used. The resulting high-concentration Nb-Fc did not show a tendency to aggregate (representative SECs are plotted in Figure 35).

[0267] To evaluate the binding affinity of purified SAT Nb-Fc fusions, ELISA assays were performed using serially diluted protein samples against the SAT antigen. All Nb-Fc tested showed similar binding affinity in the nanomolar and sub-nanomolar ranges to human SAT Nb-Fc control VH-Ab-8 (HAb8-S) (Li et al., Cell, 183:429 (2020)) via ELISA EC assay. 50 This was shown (Figure 36A and Table 5). To access their neutralizing titers, competitive ELISA assays were performed using the COVID-19 spike-ACE2 binding assay kit (Raybiotech) according to the manufacturer's instructions for use. Many Nb-Fc showed strong neutralizing titers for spike-ACE2 binding and IC2. 50 This is the IC of HAb8-S 50 This was equivalent to (Figure 36B; Table 5). Interestingly, many of these were identified through secondary screening using two potent SAT neutralizing nanobodies (LVGN-S3205 and LGVN-S52135) identified from primary screening, further demonstrating the capabilities of the sequence-driven nanobody discovery pipeline (Figures 31 and 37).

[0268] The dynamics of Nb-Fc in relation to SAT binding were analyzed by surface plasmon resonance (SPR) and / or biolayer interferometry (BLI) (Table 5). For BLI, binding experiments were performed on Octet HTX at 25°C. Antibodies were loaded onto anti-human Fc Capture (AHC) sensors and then immersed in serial dilutions of the antigen (starting at 333 nM, 1:3 dilution, 5 points). A reference sample well (buffer) was used for data analysis. Rate constants were calculated using a monovalent (1:1) binding model. Representative dynamics and sensorgrams are shown in Figures 38-39. Most Nb-Fc molecules were one or two orders of magnitude nanomolar (10⁻⁶). -8 -10 -9 M) showed a KD of equivalent to that of HAb8-S (Table 5).

[0269] To evaluate thermal stability, the melting temperature (Tm) of purified Nb-Fc was measured using differential scanning fluorescence (DSF). Briefly, Nb-Fc was mixed with Thermal Shift® Dye (ThermoFisher) for a final concentration of 1 μg / mL, and 10 μL / well of the mixture was transferred to a 384-well plate. This plate was sealed with MicroAmp® Optical Adhesive and loaded onto a Roche LightCycler® 480 instrument. Fluorescence signals were collected as the temperature increased from 20°C to 85°C at a rate of 0.06°C / sec. The most purified Nb-Fc showed high thermal stability, with an average Tm1 = 64.28 ± 0.64°C (PBS, pH 7.4) (Table 5). A representative melting curve is shown in Figure 40.

[0270] FACS-based cell binding assays were performed to investigate the binding properties of Nb-Fc to spike proteins on the cell surface (Figures 41-42). HEK293 parental cells and HEK293-spike cells expressing the SARS-CoV-2 spike (S) protein with an inactivated furin site (293-SARS2-S-dfur, Invivogen No. 293-cov2-sdf) were incubated with 1 μg / mL of individual Nb-Fc at 4°C for 1 hour, washed, and then incubated with goat anti-human IgG-Fc conjugated to DyLight 594 (ThermoFisher) at 4°C for 1 hour. FACS analysis of these samples was performed on BD LSR II, and the geometric mean fluorescence intensity (GMFI) was calculated using FlowJo V10. Of the 18 Nb-Fc molecules that bound to ELISA-formatted SAT, 12 showed cell surface SAT binding above background levels, with GMFI ratios ranging from 4.0 to 42.9 (Figures 41-42).

[0271] Example 4 - Further Singularity-based non-human animals Generation of Singularity Sapiens-L and -K allele series Variable light chain (V LThe gene segment contributes to the immunodiversity of conventional tetrameric antibodies. Human tetrameric antibodies contain either a kappa (κ) light chain or a lambda (λ) light chain. Human immunoglobulin light chains are derived from two distinct loci: IGK and IGL on chromosomes 2 and 22, respectively. Similar to the IGH locus, each locus contains numerous V L It encodes a gene segment. However, unlike the IGH locus, the above light chain locus lacks the diversity D gene segment; recombination at the light chain locus requires the RAG1 / RAG2 protein, but involves direct binding of the VL segment to the JL segment.

[0272] V L To leverage the unique properties of gene segments and expand the immune repertoire of the Singularity Sapiens platform, we used two separate approaches to utilize the diversity provided by light chain variable gene segments. Firstly, the IGLV gene segment is adjacent to a 23RSS signal similar to the 23RSS signal of the IGHV gene segment, so these can precisely pair with the 12RSS signal immediately upstream of the IGHD gene segment, which satisfies the "12 / 23 rule" preferred by RAG1 / RAG2 (Figure 43A). For lambda light chain genes, we first generated the Singularity Sapiens DJ-dock allele by removing the human IGHV segment from the SSV1 allele using CRISPR-Cas9 gene editing, leaving only the human IGHD and IGHJ segments. Next, a panel of human BAC-derived IGLV gene segments (CH17-262M19, CH17-329P5, CH17-238D3, CH17-261A15, CH17-264L24, CH17-117C7, RP11-1040J16, CH17-320F4) is modified (hLGLV-BAC) and sequentially incorporated via RMCE, as previously described (Figure 43B).

[0273] Secondly, because the IGKV gene segment is adjacent to the 12RSS signaling pathway, which does not match the IGHD segment, a different approach is required to introduce the kappa light chain gene into the Singularity allele. First, the Singularity HyperDock allele is used as a platform for incorporating a modified human BAC (hIGKVJ-BAC) containing a series of VK and JK segments (CH17-272M2, CH17-405H5, CH17-140P2, CH17-13E7, CH17-84J8, CH17-53L15) (Figure 44A). Alternatively, the Singularity Sapiens J dock allele was generated by removing the human IGHV and IGHD segments from the SSV1 allele using CRISPR-Cas9 gene editing, leaving only the human IGHJ segment. Next, a panel of IGKV gene segments derived from recombinant human BAC (hIGKV-BAC) derived from CH17-272M2, CH17-405H5, CH17-140P2, CH17-13E7, CH17-84J8, and CH17-53L15 is integrated upstream of the IGHJ gene segment by sequential RMCE, enabling recombination of each individual IGKV with each IGHJ gene segment adjacent to the 23RSS signal upstream (Figure 44B).

[0274] Generation of Singularity Longhorn and Minotaur alleles Bovine antibodies are distinguished by the presence of very long complementarity-determining regions (CDRs) (Berens et al., Int. Immunol., 9(1):189-199 (1997)). These very long CDRs have antibody knob domains that can firmly bind to their antigens as autonomous entities, which have been able to generate ultra-small nanobodies of approximately 3-5 kDa in size (MacPherson et al., PLoS Biol., 18(9):e3000821 (2020)). CDR-H3, which spans 6-20 amino acids in humans and mice, can be as long as 50-70 residues in cows. These very long CDR-H3s are partly due to the extremely long heavy chain diversity encoded in the bovine germline genome (D H ) arises from gene segments (Shojaei et al., Mol. Immunol., 40(1):61-7 (2003); and Ma et al., J. Immunol., 196(10):4358-4366 (2016)). For example, IGHD8-2 has 149 nucleotides and is the longest known D H It is one of these, with at least 50 amino acid residues contributing to bovine CDR-H3, and the combination of IGHV1-7, IGHD8-2, and IGHJ2-4 was mainly observed in isolated extra-long CDR3 bovine antibodies (MacPherson et al., PLoS Biol., 18(9):e3000821 (2020)).

[0275] During construction on the Singularity platform, a synthetic 3252 bp gene fragment was constructed, which contained approximately 2.5 kb of promoter and the upstream 5' UTR region of bovine (Bos Taurus) IGHV1-7, the entire IGHV1-7 leader exon, introns, and coding sequence, followed by IGHD8-2, IGHJ2-4, and a 250 bp sequence immediately downstream of the IGHJ2-4 region containing splice donor sequences (Figure 45A). This construct was incorporated into the Singularity HyperDock allele via RMCE, giving rise to a mouse called Singularity Longhorn (Figure 45B). PCR genotyping and sequencing confirmed the proper incorporation and transduction of the Singularity Longhorn allele in F1 mice (Figure 45C), from which the bovine VDJ-mouse IgG1ΔCH1 transcript was detected.

[0276] TIFF2026062865000007.tif139161TIFF2026062865000008.tif238160TIFF2026062865000009.tif51160

[0277] Singularity Longhorn mouse, the longest known D H Based on successful results demonstrating the expression of bovine-mouse chimeric heavy chain antibodies containing gene segments, we have developed eight longest bovine D2 mice. H A synthetic array consisting of gene segments (IGHD4-1, IGHD5-3, IGHD8-2, IGHD1-3, IGHD7-3, IGHD7-4, IGHD6-3, and IGHD3-3) was constructed and used to replace the human IGHD components in Singularity Sapiens mice. The human framework sequence was based on the genomic region extending from 550 bp upstream of IGHD4-4 to 550 bp downstream of IGHD4-17, and was derived from human D H The intercalating coding sequences of the genes were replaced with their bovine counterparts, while all human gene elements, including the 12RSS signal, were preserved (Figure 46). Synthetic human-bovine D HThe array contained the following sequences, which were used to replace the human IGHD gene fragment in the Singularity Sapiens allele with CRISPR / Cas9-mediated HDR. Mice generated from these genetically modified mice, called Singularity Minotaur for human-cow hybrids, are designed to produce human nanobodies with extra-long CDR-H3 derived from Bos Taurus.

[0278] TIFF2026062865000010.tif72160TIFF2026062865000011.tif213160

[0279] Singularity Sapacos Allele Series Generation The performance of the Singularity platform was extended by constructing a synthetic array (Sapacos VHH) containing five known VHHs from alpaca (Vicugna pacos) (Achour et al., J. Immunol., 181(3):2001-2009 (2008)). Individual VHH elements were grafted onto a framework of selected human VH components, including an upstream human promoter of approximately 250 bp containing regulatory elements involved in VH transcription (e.g., TATA box, octamer, and heptamer), human leader exons 1 and 2, human introns, and human recombinant signaling sequences (RSS) (Figure 47A). Human VHs were selected based on evidence of their high utilization in human and humanized rodent models (e.g., rats and mice). The Sapacos VHH array was designed to include adjacent, different lox elements to facilitate its targeted integration into the Singularity Sapiens IgH locus via RMCE (Figure 47B). The Syn Sapacos array (see sequence below) is inserted into the Singularity Sapiens DJ dock allele via RMCE (Figure 47B). Mice generated from this recombination are called Singularity Sapacos mice and are evaluated for their ability to produce alpaca-human-mouse chimeric heavy chain antibodies (e.g., alpaca-human-mouse chimeric heavy chain IgG1-ΔCH1 antibody).

[0280] Alpaca-human-mouse chimeric heavy chain antibodies produced from Singularity Sapacos mice may possess the naturally optimized nanobody properties of alpacas, allowing for the utilization of further immunodiversity of human D and J elements, enabling rapid humanization for therapeutic use in humans. The Sapacos VHH array offers further VHH from alpacas and other camel species. H It can be easily extended via RMCE-mediated embedding of iterative rounds of arrays including H.

[0281] TIFF2026062865000012.tif67160TIFF2026062865000013.tif232160TIFF2026062865000014.tif137160

[0282] Generation of the Singularity Savnars allele series Cartilaginous fish (e.g., sharks, rays, and rays) produce heavy chain antibodies derived from a special class of immunoglobulins known as variable novel antigen receptors (VNARs) (Greenberg et al., Nature, 374(6518):168-73 (1995)).

[0283] The performance of the Singularity platform was extended by constructing a synthetic shark VNAR array. Individual VNAR elements selected from the germline sequence of the nurse shark (Ginglymostoma cirratum) were grafted onto a framework of selected human VH components, including an approximately 250 bp upstream human promoter containing regulatory elements involved in VH transcription (e.g., TATA boxes, octamers, and heptamers), human leader exons 1 and 2, human introns, and a human recombinant signal sequence (RSS) (Figure 48A). The VNAR array, called Savnars, was synthesized and inserted into the Singularity Sapiens DJ allele via RMCE (Figure 48B). Mice generated from this recombination are called Singularity Savnars mice and are evaluated for their ability to produce shark-human-mouse chimeric heavy chain antibodies (e.g., shark-human-mouse chimeric heavy chain IgG1-ΔCH1 antibody).

[0284] Shark-human-mouse chimeric heavy chain antibodies produced from Singularity Savnars mice may possess the superior biophysical properties of VNARs and allow for the utilization of further immunodiversity of human D and J elements, enabling rapid humanization for therapeutic use in humans. The immunorepertoire of Singularity Savnars mice can be readily expanded through RMCE-mediated incorporation of repeated rounds of arrays containing further VNARs from other shark species.

[0285] TIFF2026062865000015.tif162160

[0286] Example 5 - Exemplary Embodiment Embodiment 1A. A genetically modified mouse comprising germline modification comprising deletion of nucleic acid sequences containing one or more heavy chain C region genes; wherein the mouse expresses IgG heavy chain antibodies and secretes IgG heavy chain antibodies in its serum. Embodiment 2A. A genetically modified mouse of Embodiment 1A, wherein one or more heavy chain C region genes are IgM C region gene (Cμ), IgD C region gene (Cδ), IgE C region gene (Cε), IgG3 C region gene (Cγ3), IgG2b C region gene (Cγ2b), IgG2c C region gene (Cγ2c), or a combination thereof. Embodiment 3A. A genetically modified mouse according to any one of Embodiments 1A to 2A, further comprising a deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene (Cγ1). Embodiment 4A. A genetically modified mouse of Embodiment 3A, wherein the deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene includes exon 1. Embodiment 5A. A genetically modified mouse according to any one of Embodiments 1A to 4A, wherein the germline modification further comprises a native nucleic acid sequence encoding a hinge (H) domain, a heavy chain CH2 domain, a heavy chain CH3 domain, or a combination thereof. Embodiment 6A. A genetically modified mouse according to any one of Embodiments 1A to 5A, wherein the germline modification further comprises a native nucleic acid sequence containing an endogenous enhancer. Embodiment 7A. A genetically modified mouse of Embodiment 6A, wherein the enhancer is Eμ, 3'RR, 3'γ1E, 5'hsR1, or a combination thereof. Embodiment 8A. A genetically modified mouse of any one of Embodiments 1A to 7A, wherein the germline modification further comprises a native nucleic acid sequence containing a switched tandem repeat element (Sμ), where Sμ drives IgG1 expression. Embodiment 9A. A genetically modified mouse according to any one of Embodiments 1A to 8A, wherein the IgG heavy chain antibody contains an IgG1 heavy chain antibody. Embodiment 10A. A genetically modified mouse of Embodiment 9A, wherein the IgG1 heavy chain antibody is the IgG1ΔCH1 protein. Embodiment 11A. A genetically modified mouse according to any one of Embodiments 1A to 10A, wherein the IgG heavy chain antibody lacks the light chain. Embodiment 12A. A genetically modified mouse according to any one of Embodiments 1A to 11A, wherein the IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof. Embodiment 13A. A genetically modified mouse according to any one of Embodiments 1A to 12A, wherein the mouse does not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, or any combination thereof. Embodiment 14A. A genetically modified mouse according to any one of Embodiments 1A to 14A, wherein the mouse does not express wild-type Ig A protein, wild-type IgG2b protein, wild-type IgG2c protein, or a combination thereof. Embodiment 15A. A recombinant non-human animal comprising a germline genome containing a recombinant immunoglobulin heavy chain (IgH) allele at an endogenous IgH locus; wherein the recombinant IgH allele lacks an endogenous heavy chain C region gene; and the endogenous heavy chain C region gene contains Cμ, Cδ, Cε, Cγ3, Cγ2b, Cγ2c, or a combination thereof. Embodiment 16A. A recombinant non-human animal of Embodiment 15A, wherein the IgH allele contains a deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene (Cγ1). Embodiment 17A. A recombinant non-human animal of Embodiment 16A, wherein the CH1 domain of the IgG1 C region gene contains exon 1. Embodiment 18A. A recombinant non-human animal of any one of Embodiments 15A to 17A, wherein the IgH gene locus contains a native nucleic acid sequence encoding a hinge (H) domain, a heavy chain CH2 domain, a heavy chain CH3 domain, or a combination thereof. Embodiment 19A. A recombinant non-human animal of any one of Embodiments 15A to 18A, wherein the IgH gene locus contains a native nucleic acid sequence including an endogenous enhancer. Embodiment 20A. A recombinant non-human animal of Embodiment 19A, wherein the enhancer is Eμ, 3'RR, 3'γ1E, 5'hsRI, or a combination thereof. Embodiment 21A. A recombinant non-human animal of any one of Embodiments 15A to 20A, wherein the IgH gene locus comprises a native nucleic acid sequence containing a switch tandem repeat element (Sμ), where Sμ drives IgG1 expression. Embodiment 22A. A recombinant non-human animal according to any one of Embodiments 15A to 21A, which expresses an IgG heavy chain antibody. Embodiment 23A. A recombinant non-human animal of Embodiment 22A, wherein the IgG heavy chain antibody comprises an IgG1 heavy chain antibody. Embodiment 24A. A recombinant non-human animal according to any one of Embodiments 22A to 23A, wherein the IgG1 heavy chain antibody is the IgG1ΔCH1 protein. Embodiment 25A. A recombinant non-human animal from any one of Embodiments 22A to 24A, wherein the IgG heavy chain antibody lacks the light chain. Embodiment 26A. A recombinant non-human animal from any one of Embodiments 22A to 25A, wherein the IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof. Embodiment 27A. A recombinant non-human animal according to any one of Embodiments 15A to 26A, wherein the non-human animal does not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, or a combination thereof. Embodiment 28A. A recombinant non-human animal according to any one of Embodiments 15A to 27A, wherein the non-human animal does not express wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or a combination thereof. Embodiment 29A. A recombinant non-human animal according to any one of Embodiments 15A to 28A, wherein the IgH gene locus contains an endogenous V, D, or J gene. Embodiment 30A. A recombinant non-human animal from any one of Embodiments 15A to 29A, wherein the recombinant non-human animal is homozygous for the recombinant IgH allele. Embodiment 31A. A recombinant non-human animal according to any one of Embodiments 15A to 30A, wherein the endogenous IgH gene locus does not contain an exogenous nucleic acid sequence. Embodiment 32A. A recombinant non-human animal according to any one of Embodiments 15A to 30A, wherein the endogenous IgH gene locus contains an exogenous nucleic acid sequence. Embodiment 33A. A recombinant non-human animal of Embodiment 32A, wherein the exogenous nucleic acid sequence includes a barcode. Embodiment 34A. A recombinant non-human animal from any one of Embodiments 15A to 33A, wherein the non-human animal is a mammal. Embodiment 35A. A recombinant non-human animal of Embodiment 34A, wherein the mammal is a mouse or a rat. Embodiment 36A. A method for producing a genetically modified non-human animal capable of producing heavy chain antibodies, comprising: (a) deleting an endogenous nucleic acid sequence containing one or more heavy chain C region genes from an endogenous immunoglobulin heavy chain gene locus in stem cells of a non-human animal; (b) transplanting the stem cells into a blastocyst; (c) transplanting the blastocyst into a pseudo-pregnant mouse to obtain a chimeric mouse; (d) mating the chimeric mouse with a wild-type mouse to produce offspring; (e) screening the offspring for heterozygosity; and (f) identifying a founding mouse having a deletion of one or more heavy chain C region genes, wherein the non-human animal is capable of producing heavy chain antibodies. Embodiment 37A. The method according to Embodiment 36A, wherein the stem cells are embryonic stem cells. Embodiment 38A. One of the methods of Embodiments 36A to 37A, wherein one or more heavy chain C region genes include Cμ, Cδ, Cγ3, Cγ2b, Cγ2c, Cε, or a combination thereof. Embodiment 39A. Any one of Embodiments 36A to 38A, further comprising the step of deleting the nucleic acid sequences encoding the CH1 domain of the IgG1 C region gene and the CH1 exon of Cγ1. Embodiment 40A. The method of Embodiment 39A, wherein the deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene includes exon 1. Embodiment 41A. Any one of Embodiments 36A to 40A, further comprising the step of preserving a native nucleic acid sequence encoding a hinge (H) domain, a heavy chain CH2 domain, a heavy chain CH3 domain, or a combination thereof. Embodiment 42A. Any one of Embodiments 36A to 41A, further comprising the step of preserving a natural nucleic acid sequence containing an endogenous enhancer. Embodiment 43A. The method of Embodiment 42A, wherein the enhancer is Eμ, 3'RR, 3'γ1E, 5'hsRI, or a combination thereof. Embodiment 44A. Any one of Embodiments 36A to 43A, further comprising the step of preserving a native nucleic acid sequence containing a switched tandem repeat element (Sμ), wherein Sμ drives IgG1 expression. Embodiment 45A. Any one of Embodiments 36A to 44A, wherein the heavy chain antibody is an IgG heavy chain antibody. Embodiment 46A. The method of Embodiment 45A, wherein the IgG heavy chain antibody comprises an IgG1 heavy chain antibody. Embodiment 47A. The method of Embodiment 46A, wherein the IgG1 heavy chain antibody is the IgG1ΔCH1 protein. Embodiment 48A. Any one of Embodiments 45A to 47A, wherein the IgG heavy chain antibody lacks a light chain. Embodiment 49A. One of the methods of Embodiments 45A to 48A, wherein the IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof. Embodiment 50A. One of the methods of Embodiments 36A to 49A, wherein a non-human animal does not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, or a combination thereof. Embodiment 51A. One of the methods of Embodiments 36A to 50A, wherein a non-human animal does not express wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or a combination thereof. Embodiment 52A. Any one of Embodiments 36A to 51A, wherein the non-human animal is a mammal. Embodiment 53A. The method of Embodiment 52A, wherein the mammal is a mouse or a rat. Embodiment 54A. Any one of Embodiments 36A to 53A, wherein the step of deleting an endogenous nucleic acid sequence containing one or more heavy chain C region genes includes CRISPR / Cas9 genome editing. Embodiment 55A. One of the methods of Embodiments 36A to 54A, wherein the genetically modified non-human animal is reproductive. Embodiment 56A. Any one of Embodiments 36A to 55A, wherein a genetically modified non-human animal has substantially normal B cell development and maturation. Embodiment 57A. One of the methods of Embodiments 36A to 56A, wherein a genetically modified non-human animal does not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or a combination thereof. Embodiment 58A. A method for producing a soluble heavy chain antibody in any one recombinant non-human animal of Embodiments 36A to 57A, comprising the steps of (a) administering an antigen to a non-human animal; (b) isolating one or more B cells from the non-human animal; (c) isolating mRNA from the one or more B cells; (d) sequencing the mRNA; (e) identifying a clonal type based on the mRNA sequence; and (f) performing phylogenetic analysis of the clonal type, thereby producing a soluble heavy chain antibody. Embodiment 59A. The method of Embodiment 58A, wherein the non-human animal is a mammal. Embodiment 60A. The method of Embodiment 59A, wherein the mammal is a mouse or a rat. Embodiment 61A. A method for producing a single-domain antibody (sdAb) identified from any one recombinant non-human animal of Embodiments 36A to 57A, wherein (a) a heavy chain variable (V) containing V, D, and J is produced in cells. H (b) a method comprising the steps of expressing a nucleic acid sequence encoding a domain, wherein the cells produce a heavy chain variable domain; and (b) isolating the heavy chain variable domain from a sample, thereby producing a single-domain antibody. Embodiment 62A. The method of Embodiment 61A, wherein the single-domain antibody is a single-domain mouse antibody. Embodiment 63A. The method of Embodiment 62A, wherein the single-domain antibody is an IgG1 single-domain antibody. Embodiment 64A. The method of Embodiment 63A, wherein the IgG1 single-domain antibody is an IgG1ΔCH1 nanobody. Embodiment 65A. Any one of Embodiments 61A to 64A, wherein the single-domain antibody lacks a light chain. Embodiment 66A. One of the methods of Embodiments 61A to 65A, wherein the single-domain antibody lacks a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

[0287] Embodiment 1B. A genetically modified mouse comprising germline modification comprising deletion of nucleic acid sequences containing one or more heavy chain C region genes; wherein the mouse expresses humanized IgG heavy chain antibodies and secretes humanized IgG heavy chain antibodies in its serum. Embodiment 2B. A genetically modified mouse of Embodiment 1B, wherein one or more heavy chain C region genes are IgM C region gene (Cμ), IgD C region gene (Cδ), IgE C region gene (Cε), IgG3 C region gene (Cγ3), IgG2b C region gene (Cγ2b), IgG2c C region gene (Cγ2c), or a combination thereof. Embodiment 3B. A genetically modified mouse according to any one of Embodiments 1B to 2B, further comprising a deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene (Cγ1). Embodiment 4B. A genetically modified mouse of Embodiment 3B, wherein the deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene includes exon 1. Embodiment 5B. A genetically modified mouse according to any one of Embodiments 1B to 4B, wherein the germline modification further comprises a native nucleic acid sequence encoding a hinge (H) domain, a heavy chain CH2 domain, a heavy chain CH3 domain, or a combination thereof. Embodiment 6B. A genetically modified mouse according to any one of Embodiments 1B to 5B, wherein the germline modification further comprises a native nucleic acid sequence containing an endogenous enhancer. Embodiment 7B. A genetically modified mouse of Embodiment 6B, wherein the enhancer is Eμ, 3'RR, 3'γ1E, 5'hsRI, or a combination thereof. Embodiment 8B. A genetically modified mouse of any one of Embodiments 1B to 7B, wherein the germline modification further comprises a native nucleic acid sequence containing a switched tandem repeat element (Sμ), where Sμ drives IgG1 expression. Embodiment 9B. A genetically modified mouse according to any one of Embodiments 1B to 8B, wherein the humanized IgG heavy chain antibody comprises a humanized IgG1 heavy chain antibody. Embodiment 10B. A genetically modified mouse of Embodiment 9B, wherein the humanized IgG1 heavy chain antibody is the IgG1ΔCH1 protein. Embodiment 11B. A genetically modified mouse according to any one of Embodiments 1B to 10B, wherein the humanized IgG heavy chain antibody lacks a light chain. Embodiment 12B. A genetically modified mouse according to any one of Embodiments 1B to 11B, wherein the humanized IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof. Embodiment 13B. A genetically modified mouse according to any one of Embodiments 1B to 12B, wherein the mouse does not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, or any combination thereof. Embodiment 14B. A genetically modified mouse according to any one of Embodiments 1B to 14B, wherein the mouse does not express wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or a combination thereof. Embodiment 15B. A recombinant non-human animal comprising a germline genome containing a recombinant immunoglobulin heavy chain (IgH) allele at an endogenous IgH locus; wherein the recombinant IgH allele lacks an endogenous heavy chain C region gene; and the endogenous heavy chain C region gene contains Cμ, Cδ, Cε, Cγ3, Cγ2b, Cγ2c, or a combination thereof. Embodiment 16B. A recombinant non-human animal of Embodiment 15B, wherein the IgH allele contains a deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene (Cγ1). Embodiment 17B. A recombinant non-human animal of Embodiment 16B, wherein the CH1 domain of the IgG1 C region gene contains exon 1. Embodiment 18B. A recombinant non-human animal of any one of Embodiments 15B to 17B, wherein the IgH locus contains a native nucleic acid sequence encoding a hinge (H) domain, a heavy chain CH2 domain, a heavy chain CH3 domain, or a combination thereof. Embodiment 19B. A recombinant non-human animal of any one of Embodiments 15B to 18B, wherein the IgH gene locus contains a native nucleic acid sequence including an endogenous enhancer. Embodiment 20B. A recombinant non-human animal of Embodiment 19B, wherein the enhancer is Eμ, 3'RR, 3'γ1E, 5'hsRI, or a combination thereof. Embodiment 21B. A recombinant non-human animal of any one of Embodiments 15B to 20B, wherein the IgH gene locus comprises a native nucleic acid sequence containing a switched tandem repeat element (Sμ), where Sμ drives IgG1 expression. Embodiment 22B. A recombinant non-human animal according to any one of Embodiments 15B to 21B, wherein the non-human animal expresses a humanized IgG heavy chain antibody. Embodiment 23B. A recombinant non-human animal of Embodiment 22B, wherein the humanized IgG heavy chain antibody comprises a humanized IgG1 heavy chain antibody. Embodiment 24B. A recombinant non-human animal according to any one of Embodiments 22B to 23B, wherein the humanized IgG1 heavy chain antibody is the IgG1ΔCH1 protein. Embodiment 25B. A recombinant non-human animal from any one of Embodiments 22B to 24B, wherein the humanized IgG heavy chain antibody lacks a light chain. Embodiment 26B. A recombinant non-human animal of any one of Embodiments 22B to 25B, wherein the humanized IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof. Embodiment 27B. A recombinant non-human animal according to any one of Embodiments 15B to 26B, wherein the non-human animal does not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, or a combination thereof. Embodiment 28B. A recombinant non-human animal according to any one of Embodiments 15B to 27B, wherein the non-human animal does not express wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or a combination thereof. Embodiment 29B. A recombinant non-human animal of any one of Embodiments 15B to 28B, wherein the IgH gene locus contains the human V, D, or J gene. Embodiment 30B. A recombinant non-human animal from any one of Embodiments 15B to 29B, wherein the recombinant non-human animal is homozygous for the recombinant IgH allele. Embodiment 31B. A recombinant non-human animal according to any one of Embodiments 15B to 30B, wherein the endogenous IgH gene locus contains an exogenous nucleic acid sequence. Embodiment 32B. Human V, which contains one or more exogenous nucleic acid sequences. H Gene segment, one or more human D H Gene segment, and one or more J H A recombinant non-human animal according to any one of embodiments 15B to 31B, comprising a gene segment. Embodiment 33B. The exogenous nucleic acid sequence comprises 65 human V H A recombinant non-human animal according to any one of embodiments 15B to 32B, comprising a gene segment. Embodiment 34B. The exogenous nucleic acid sequence is 27 human D H A recombinant non-human animal according to any one of embodiments 15B to 32B, comprising a gene segment. Embodiment 35B. The exogenous nucleic acid sequence has 6 J H A recombinant non-human animal according to any one of embodiments 15B to 32B, comprising a gene segment. Embodiment 36B. The exogenous nucleic acid sequence comprises 65 human V H Gene segment, 27 human D H Gene segment, and 6 J H A recombinant non-human animal according to any one of embodiments 15B to 35B, comprising a gene segment. Embodiment 37B. A recombinant non-human animal of Embodiment 31B, wherein the exogenous nucleic acid sequence includes a barcode. Embodiment 38B. A recombinant non-human animal from any one of Embodiments 15B to 37B, wherein the non-human animal is a mammal. Embodiment 39B. A recombinant non-human animal of Embodiment 38B, wherein the mammal is a mouse or a rat. Embodiment 40B. A method for producing a genetically modified non-human animal capable of producing humanized heavy chain antibodies, comprising: (a) deleting an endogenous nucleic acid sequence containing one or more heavy chain C region genes from an endogenous immunoglobulin heavy chain gene locus in stem cells of a non-human animal; (b) transplanting the stem cells into a blastocyst; (c) transplanting the blastocyst into a pseudo-pregnant mouse to obtain a chimeric mouse; (d) mating the chimeric mouse with a wild-type mouse to produce offspring; (e) screening the offspring for heterozygosity; and (f) identifying a founding mouse having a deletion of one or more heavy chain C region genes, wherein the non-human animal is capable of producing humanized heavy chain antibodies. Embodiment 41B. The method of Embodiment 40B, wherein the stem cells are embryonic stem cells. Embodiment 42B. Any one of Embodiments 40B to 41B, wherein one or more heavy chain C region genes include Cμ, Cδ, Cγ3, Cγ2b, Cγ2c, Cε, or a combination thereof. Embodiment 43B. Any one of Embodiments 40B to 42B, further comprising the step of deleting the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene (Cγ1). Embodiment 44B. The method of Embodiment 43B, wherein the deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene includes exon 1. Embodiment 45B. Any one of Embodiments 40B to 44B, further comprising the step of preserving a natural nucleic acid sequence encoding a hinge (H) domain, a heavy chain CH2 domain, a heavy chain CH3 domain, or a combination thereof. Embodiment 46B. Any one of Embodiments 40B to 45B, further comprising the step of preserving a natural nucleic acid sequence containing an endogenous enhancer. Embodiment 47B. The method of Embodiment 46B, wherein the enhancer is Eμ, 3'RR, 3'γ1E, 5'hsRI, or a combination thereof. Embodiment 48B. Any one of Embodiments 40B to 47B, further comprising the step of preserving a native nucleic acid sequence containing a switched tandem repeat element (Sμ), wherein Sμ drives IgG1 expression. Embodiment 49B. Any one of Embodiments 40B to 48B, wherein the humanized heavy chain antibody is a humanized IgG heavy chain antibody. Embodiment 50B. The method of Embodiment 49B, wherein the humanized IgG heavy chain antibody comprises a humanized IgG1 heavy chain antibody. Embodiment 51B. The method of Embodiment 50B, wherein the IgG1 heavy chain antibody is the IgG1ΔCH1 protein. Embodiment 52B. Any one of Embodiments 50B to 51B, wherein the humanized IgG heavy chain antibody lacks a light chain. Embodiment 53B. Any one of Embodiments 49B to 52B, wherein the humanized IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof. Embodiment 54B. Any one of Embodiments 40B to 53B, wherein the non-human animal does not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, or a combination thereof. Embodiment 55B. Any one of Embodiments 40B to 54B, wherein the non-human animal does not express wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or a combination thereof. Embodiment 56B. Any one of Embodiments 40B to 55B, wherein the non-human animal is a mammal. Embodiment 57B. The method of Embodiment 56B, wherein the mammal is a mouse or a rat. Embodiment 58B. Any one of Embodiments 40B to 57B, wherein the step of deleting an endogenous nucleic acid sequence containing one or more heavy chain C region genes includes CRISPR / Cas9 genome editing. Embodiment 59B. Any one of Embodiments 40B to 58B, wherein the genetically modified non-human animal is reproductive. Embodiment 60B. Any one of Embodiments 40B to 59B, wherein a genetically modified non-human animal has substantially normal B cell development and maturation. Embodiment 61B. Any one of Embodiments 40B to 60B, wherein the genetically modified non-human animal does not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or any combination thereof. Embodiment 62B. A method for producing a soluble humanized heavy chain antibody in any one recombinant non-human animal of Embodiments 40B to 61B, comprising the steps of (a) administering an antigen to a non-human animal; (b) isolating one or more B cells from the non-human animal; (c) isolating mRNA from the one or more B cells; (d) sequencing the mRNA; (e) identifying a clonal type based on the mRNA sequence; and (f) performing phylogenetic analysis of the clonal type, thereby producing a soluble humanized heavy chain antibody. Embodiment 63B. The method of Embodiment 62B, wherein the non-human animal is a mammal. Embodiment 64B. The method of Embodiment 63B, wherein the mammal is a mouse or a rat. Embodiment 65B. A method for producing a humanized single-domain antibody (sdAb) identified from any one recombinant non-human animal of Embodiments 40 to 61, comprising the steps of (a) expressing a nucleic acid sequence encoding a human heavy chain variable (VH) domain comprising V, D, and J in cells, wherein the cells produce the human heavy chain variable domain; and (b) isolating the human heavy chain variable domain from a sample, thereby producing a single-domain antibody. Embodiment 66B. The method of Embodiment 65B, wherein the single-domain antibody is a human single-domain antibody. Embodiment 67B. The method of Embodiment 66B, wherein the single-domain antibody is an IgG1 single-domain antibody. Embodiment 68B. The method of Embodiment 67B, wherein the IgG1 single-domain antibody is an IgG1ΔCH1 nanobody. Embodiment 69B. Any one of Embodiments 65B to 68B, wherein the single-domain antibody lacks a light chain. Embodiment 70B. Any one of Embodiments 65B to 69B, wherein the single-domain antibody lacks a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof. Embodiment 71B. Any one of Embodiments 65B to 70B, wherein the cells are bacterial cells or human cells.

[0288] Embodiment 1C. DNA comprising a genetically modified non-human immunoglobulin heavy chain (IGH) allele, wherein the genetically modified non-human IgH allele lacks one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains, including the CH1 constant domain of the IgG subclass, the IgM constant domain, the IgD constant domain, the IgE constant domain, the IgA constant domain, or any combination thereof. Embodiment 2C. DNA of Embodiment 2C, wherein the DNA is germline genomic DNA. Embodiment 3C. Any one of Embodiments 1C to 2C, wherein the genetically modified non-human IgH allele lacks one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains, including the CH1 constant domain of the IgG subclass. Embodiment 4C. DNA of Embodiment 3C, wherein the IgG subclass includes the IgG1, IgG2a, IgG2b, IgG2c, IgG3, or IgG4 subclass. Embodiment 5C. DNA of Embodiment 3C, wherein the IgG subclass is the IgG1 subclass. Embodiment 6C. Any one of Embodiments 1C to 5C, wherein the genetically modified non-human IgH allele contains a nucleic acid sequence (Cγ1-ΔCH1) encoding a CH1-cleaved IgG1 constant domain (IgG1ΔCH1). Embodiment 7C. Any one of Embodiments 1C to 6C, wherein the genetically modified non-human IgH allele contains a nucleic acid sequence encoding a hinge (H) domain, CH2 domain, CH3 domain, or any combination thereof of an IgG subclass. Embodiment 8C. Any one of Embodiments 1C to 7C, wherein the genetically modified non-human IgH allele lacks one or more nucleic acid sequences encoding at least a portion of one or more endogenous constant domains, including the IgG2 constant domain, the IgG3 constant domain, the IgG4 constant domain, or any combination thereof. Embodiment 9C. Any one of Embodiments 1C to 8C, wherein the genetically modified non-human IgH allele comprises one or more endogenous enhancers including Eμ, 3'γ1E, 5'hsR1, 3'RR, or any combination thereof. Embodiment 10C. A DNA from any one of Embodiments 1C to 9C, wherein the genetically modified non-human IgH allele includes an Iμ promoter, an Iμ exon, or both. Embodiment 11C. A DNA from any one of Embodiments 1C to 10C, wherein the genetically modified non-human IgH allele contains a switch tandem repeat element (Sμ). Embodiment 12C. One DNA from any of Embodiments 1C to 11C, in which IgG1 expression is driven by Eμ, Iμ promoter, Sμ, or any combination thereof. Embodiment 13C. Any one of the DNAs from Embodiments 1C to 12C, wherein the genetically modified non-human IgH allele lacks one or more endogenous switch regions, including Sγ3, Sγ1, Sγ2b, Sγ2c, Sε, Sα, or any combination thereof. Embodiment 14C. Any one of Embodiments 1C to 13C, wherein the genetically modified non-human IgH allele comprises the following components (5' to 3'): Eμ, Iμ promoter, Iμ exon, Sμ, Cγ1-ΔCH1, 3'γ1E, 5'hsR1, and 3'RR. Embodiment 15C. One DNA from any of Embodiments 1C to 14C, wherein the genetically modified non-human IgH allele contains a flippase recognition target (frt) site. Embodiment 16C. One DNA from any of Embodiments 1C to 15C, wherein the genetically modified non-human IgH allele includes an endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof. Embodiment 17C. A DNA from any one of Embodiments 1C to 16C, wherein the genetically modified non-human IgH allele lacks at least one endogenous V gene segment, D gene segment, J gene segment, or any combination thereof. Embodiment 18C. Any one of the DNAs from Embodiments 1C to 17C, wherein a genetically modified non-human IgH allele is included in a docking cassette. Embodiment 19C. DNA of Embodiment 18C, wherein the docking cassette comprises left and right homology arms, a frt site, an attB site, a promoter, a loxP site, a nucleic acid sequence encoding a selection marker, or any combination thereof. Embodiment 20C. DNA of Embodiment 18C, wherein the docking cassette contains a nucleic acid sequence encoding a selection marker. Embodiment 21C. DNA of Embodiment 20C, wherein the selection marker comprises geneticin, hydromycin, puromycin, or any combination thereof. Embodiment 22C. One of the DNAs from Embodiments 1C to 21C, wherein a genetically modified non-human IgH allele encodes an IgG heavy chain antibody. Embodiment 23C. Any one of the DNAs from Embodiments 1C to 22C, wherein the genetically modified non-human IgH allele includes an exogenous V gene segment, an exogenous D gene segment, an exogenous J gene segment, or any combination thereof. Embodiment 24C. The DNA of Embodiment 23C, wherein the exogenous gene segment is selected from the group consisting of human gene segments, mouse gene segments, rat gene segments, bovine gene segments, alpaca gene segments, and shark gene segments. Embodiment 25C. DNA of Embodiment 23C, wherein the exogenous gene segment includes a human gene segment. Embodiment 26C. A DNA from any one of Embodiments 1C to 25C, wherein the genetically modified non-human IgH allele includes one or more human VH gene segments, one or more human DH gene segments, and one or more human JH gene segments. Embodiment 27C. Any one of Embodiments 1C to 26C, wherein the genetically modified non-human IgH allele contains at least 10, 20, 30, 40, 50, 60, 80, 100, 120, or 126 human VH gene segments. Embodiment 28C. A DNA from any one of Embodiments 1C to 27C, wherein the genetically modified non-human IgH allele contains at least 10, 15, 20, 25, or 27 human DH gene segments. Embodiment 29C. Any one of Embodiments 1C to 28C, wherein the genetically modified non-human IgH allele contains at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 human JH gene segments. Embodiment 30C. A DNA from any one of Embodiments 1C to 29C, wherein the genetically modified non-human IgH allele comprises 126 human VH gene segments, 27 human DH gene segments, and 9 human JH gene segments. Embodiment 31C. A DNA from any one of Embodiments 1C to 30C, wherein the genetically modified non-human IgH allele contains one or more bovine gene segments. Embodiment 32C. DNA of Embodiment 31C, wherein one or more bovine gene segments include an L1 exon, an L2 exon of IGHV1-7, a coding segment of IGH8-2, a coding sequence of IGHJ2-4, an IGH2-4 splice donor, or any combination thereof. Embodiment 33C. One DNA from any of Embodiments 31C-32C, wherein one or more bovine gene segments include IGHD4-1, IGHD5-3, IGHD8-2, IGHD1-3, IGHD7-3, IGHD7-4, IGHD6-3, IGHD3-3, or any combination thereof. Embodiment 34C. One DNA from any of Embodiments 31C to 33C, wherein one or more bovine gene segments contain nucleic acid sequences selected from SEQ ID NOs. 42 to 49 and 57. Embodiment 35C. One DNA from any of Embodiments 32C to 34C, wherein the DNA contains one or more human VH gene segments. Embodiment 36C. One DNA from any of Embodiments 32C to 35C, wherein the DNA contains one or more human JH gene segments. Embodiment 37C. Any one of Embodiments 1C to 36C, wherein the genetically modified non-human IgH allele contains one or more alpaca gene segments. Embodiment 38C. DNA of Embodiment 37C, wherein one or more alpaca gene segments include VHH3-1, VHH3-S1, VHH3-S2, VHH3-S9, VHH3-S10, or any combination thereof. Embodiment 39C. One of the DNAs from Embodiments 37C to 38C, wherein the alpaca gene segment contains a nucleic acid sequence selected from SEQ ID NOs. 50 to 54. Embodiment 40C. One DNA from any of Embodiments 37C to 39C, wherein the DNA contains one or more human VH gene segments. Embodiment 41C. One DNA from any of Embodiments 37C to 40C, wherein the DNA contains one or more human JH gene segments. Embodiment 42C. A DNA from any one of Embodiments 1C to 41C, wherein the genetically modified non-human IgH allele contains one or more shark gene segments. Embodiment 43C. DNA of Embodiment 42C, wherein one or more shark gene segments contain VNAR-L38968, VNAR-L38967, or both. Embodiment 44C. A DNA from any one of Embodiments 42C to 43C, wherein the shark gene segment contains a nucleic acid sequence selected from SEQ ID NOs. 55 to 56. Embodiment 45C. One DNA from any of Embodiments 42C to 44C, wherein the DNA contains one or more human VH gene segments. Embodiment 46C. Any one of the DNAs from Embodiments 42C to 45C, wherein the DNA contains one or more human JH gene segments. Embodiment 47C. A DNA from any one of Embodiments 1C to 46C, wherein a genetically modified non-human IgH allele encodes an IgG heavy chain antibody, and the IgG heavy chain antibody includes a kappa light chain variable domain, a lambda light chain variable domain, or both. Embodiment 48C. DNA of Embodiment 47C, wherein the genetically modified non-human IgH allele contains one or more exogenous human lambda light chain (LV) gene segments. Embodiment 49C. DNA of Embodiment 48C, wherein one or more human LV gene segments include CH17-262M19, CH17-329P5, CH17-238D3, CH17-261A15, CH17-264L24, CH17-117C7, RP11-1040J16, CH17-320F4, or any combination thereof. Embodiment 50C. One DNA from any of Embodiments 47C to 49C, wherein the genetically modified non-human IgH allele contains one or more exogenous human kappa light chain (KV) gene segments. Embodiment 51C. DNA of Embodiment 50C, wherein one or more human KV gene segments include CH17-272M2, CH17-405H5, CH17-140P2, CH17-13E7, CH17-84J8, CH17-53L15, or any combination thereof. Embodiment 52C. One DNA from any of Embodiments 47C to 51C, wherein the DNA contains one or more human VH gene segments. Embodiment 53C. Any one of the DNAs from Embodiments 47C to 52C, wherein the DNA contains one or more human JH gene segments. Embodiment 54C. A genetically modified cell containing one DNA from any one of Embodiments 1C to 53C. Embodiment 55C. The cells of Embodiment 54C, wherein the cells are non-human animal cells. Embodiment 56C. The cells of Embodiment 54C, wherein the cells are mammalian cells. Embodiment 57C. The cells of Embodiment 56C, wherein the mammalian cells are cells from a mouse, rat, cow, alpaca, cat, dog, rabbit, pig, monkey, or chimpanzee. Embodiment 58C. The cells of Embodiment 54C, wherein the cells are mouse cells. Embodiment 59C. The cells of Embodiment 54C, wherein the cells are shark cells. Embodiment 60C. The cells of Embodiment 54C, wherein the cells are human cells. Embodiment 61C. One of the cells from Embodiments 54C to 60C, wherein the cell is a stem cell. Embodiment 62C. The cells of Embodiment 61C, wherein the stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Embodiment 63C. One of the cells from Embodiments 54C to 60C, wherein the cell is a B cell. Embodiment 64C. A genetically modified non-human animal comprising a cell from any one of Embodiments 54C to 63C. Embodiment 65C. A genetically modified non-human animal according to Embodiment 64C, wherein the non-human animal is a mammal. Embodiment 66C. A genetically modified non-human animal according to Embodiment 65C, wherein the mammal is a mouse, rat, cow, alpaca, cat, dog, rabbit, pig, monkey, or chimpanzee. Embodiment 67C. A genetically modified non-human animal of Embodiment 64C, wherein the non-human animal is a mouse. Embodiment 68C. A genetically modified non-human animal according to any one of Embodiments 64C to 67C, wherein the genetically modified non-human animal includes cells that express IgG heavy chain antibodies. Embodiment 69C. A genetically modified non-human animal according to Embodiment 68C, in which IgG heavy chain antibodies are secreted into the serum of the genetically modified non-human animal. Embodiment 70C. A genetically modified non-human animal according to any one of Embodiments 68C to 69C, wherein the IgG heavy chain antibody is a CH1-cleaved IgG1 heavy chain antibody (IgG1ΔCH1). Embodiment 71C. A genetically modified non-human animal according to any one of Embodiments 68C to 70C, wherein the IgG heavy chain antibody lacks a light chain. Embodiment 72C. A genetically modified non-human animal of any one of Embodiments 68C to 71C, wherein the IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or any combination thereof. Embodiment 73C. A genetically modified non-human animal according to any one of Embodiments 68C to 72C, wherein cells expressing IgG heavy chain antibodies do not express IgM antibodies, IgD antibodies, IgE antibodies, IgG3 antibodies, IgG2b antibodies, IgG2c antibodies, IgA antibodies, or any combination thereof. Embodiment 74C. A genetically modified non-human animal according to any one of Embodiments 68C to 73C, wherein the IgG heavy chain antibody is a human IgG heavy chain antibody. Embodiment 75C. A genetically modified non-human animal according to any one of Embodiments 68C to 74C, wherein the IgG heavy chain antibody includes an exogenous variable domain selected from the group consisting of a human variable domain, a mouse variable domain, a rat variable domain, a bovine variable domain, an alpaca variable domain, and a shark variable domain. Embodiment 76C. A genetically modified non-human animal according to any one of Embodiments 68C to 75C, wherein the IgG heavy chain antibody comprises a kappa light chain variable domain, a lambda light chain variable domain, or both. Embodiment 77C. A method for preparing germline genomic DNA, comprising the step of deleting one or more nucleic acid sequences from a non-human immunoglobulin heavy chain (IgH) allele, wherein the one or more nucleic acid sequences deleted encode at least a portion of one or more endogenous constant domains, including the CH1 constant domain of an IgG subclass, the IgM constant domain, the IgD constant domain, the IgE constant domain, the IgA constant domain, or any combination thereof, thereby generating a genetically modified non-human IgH allele in germline genomic DNA. Embodiment 78C. The method of Embodiment 77C, wherein the germline genomic DNA comprises one of the DNAs from Embodiments 1C to 53C. Embodiment 79C. Any one of Embodiments 77C to 78C, wherein the IgG constant domain includes a constant domain of an IgG subclass. Embodiment 80C. The method of Embodiment 79C, wherein the IgG subclass includes the IgG1, IgG2a, IgG2b, IgG2c, IgG3, or IgG4 subclass. Embodiment 81C. A method for producing a genetically modified non-human animal, comprising the following steps: (a) The step of deleting one or more nucleic acid sequences from a non-human immunoglobulin heavy chain (IGH) allele, wherein the one or more nucleic acid sequences to be deleted encode at least a portion of one or more endogenous constant domains, including the CH1 constant domain of an IgG subclass, the IgM constant domain, the IgD constant domain, the IgE constant domain, the IgA constant domain, or any combination thereof, thereby generating a genetically modified non-human IgH allele in germline genomic DNA; (b) The step of transplanting cells containing germline genomic DNA into a blastocyst; (c) The step of obtaining a chimeric non-human animal by transplanting the above blastocyst into a pseudo-pregnant non-human animal; (d) The step of mating the above-mentioned chimeric non-human animal with a wild-type non-human animal to produce offspring; (e) A step of screening the above offspring for heterozygosity; and (f) A step of identifying a genetically modified non-human animal that has one or more nucleic acid sequence deletions and is capable of producing heavy chain antibodies. The above method, including. Embodiment 82C. The method of Embodiment 81C, wherein the genetically modified non-human animal is one of the genetically modified non-human animals of Embodiments 64C to 76C. Embodiment 83C. A method according to any one of Embodiments 81C to 82C, wherein the step of deleting one or more nucleic acid sequences includes a step of using a CRISPR / Cas genome editing system. Embodiment 84C. The method of Embodiment 83C, wherein the CRISPR / Cas genome editing system comprises at least one guide RNA (gRNA) targeting an endogenous heavy chain C region gene and a Cas protein. Embodiment 85C. Any one of Embodiments 83C to 84C, wherein the Cas protein contains the Cas9 protein. Embodiment 86C. One of the methods of Embodiments 81C to 85C, wherein one or more nucleic acid sequences to be deleted encode the CH1 constant domain, IgG3 constant domain, IgM constant domain, and IgD constant domain of IgG1. Embodiment 87C. Any one of Embodiments 81C to 86C, wherein one or more nucleic acid sequences to be deleted encode the IgG2 constant domain and the IgA constant domain. Embodiment 88C. Any one of Embodiments 81C to 87C, wherein the step of deleting a nucleic acid sequence includes a step of removing a select marker from a non-human IgH allele using transient expression of Flp recombinase. Embodiment 89C. One of the methods of Embodiments 81C to 88C, wherein one or more nucleic acid sequences to be deleted encode the CH1 constant domain, IgM constant domain, IgD constant domain, IgE constant domain, and IgA constant domain of the IgG subclass. Embodiment 90C. A method according to any one of Embodiments 81C to 89C, comprising the step of deleting a nucleic acid sequence from a non-human IgH allele, wherein the nucleic acid sequence includes an endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof. Embodiment 91C. Any one of Embodiments 81C to 90C, comprising the step of inserting a docking cassette. Embodiment 92C. Includes the step of bringing a docking cassette into contact with a bacterial artificial chromosome (BAC), wherein the BAC is exogenous V H , D H , and J H The method of Embodiment 91C, comprising a nucleic acid sequence containing a gene segment. Embodiment 93C. The method of Embodiment 92C, comprising the step of inserting an exogenous gene segment into a docketing cassette. Embodiment 94C. Any one of Embodiments 92C to 93C, wherein the exogenous gene segment is a human gene segment. Embodiment 95C. A genetically modified non-human animal prepared using any one of the methods of Embodiments 81C to 94C. Embodiment 96C. A method for producing IgG heavy chain antibodies in a genetically modified non-human animal, comprising the following steps: (a) The step of administering the antigen to a genetically modified non-human animal according to any one of embodiments 64C to 76C; (b) The step of isolating one or more B cells from a genetically modified non-human animal; (c) the step of isolating mRNA from one or more B cells; and (d) Step of producing IgG heavy chain antibodies The above method, including. Embodiment 97C. The method according to Embodiment 96C, wherein the genetically modified non-human animal contains one DNA from any one of Embodiments 1C to 53C. Embodiment 98C. Any one of Embodiments 96C to 97C, comprising the step of sequencing mRNA isolated from one or more B cells. Embodiment 99C. Any one of Embodiments 96C to 98C, comprising the step of identifying a clone type based on an mRNA sequence. Embodiment 100C. The method of Embodiment 99C, comprising the step of performing a clonal phylogenetic analysis. Embodiment 101C. Any one of Embodiments 96C to 100C, wherein the IgG heavy chain antibody is a humanized IgG heavy chain antibody. Embodiment 102C. Any one of Embodiments 96C to 100C, wherein the IgG heavy chain antibody is an IgG heavy chain antibody comprising a human variable region and a non-human constant region. Embodiment 103C. An IgG heavy chain antibody produced by any one of the methods of Embodiments 96C to 102C. Embodiment 104C. A recombinant vector system comprising at least one nucleic acid construct encoding a CRISPR / Cas genome editing system comprising a Cas protein and at least one guide RNA (gRNA), wherein the Cas protein and at least one gRNA form a complex that deletes one or more nucleic acid sequences from a non-human immunoglobulin heavy chain (IgH) allele, and the one or more nucleic acid sequences deleted thereby encode at least a portion of one or more endogenous constant domains, including the CH1 constant domain of an IgG subclass, an IgM constant domain, an IgD constant domain, an IgE constant domain, an IgA constant domain, or any combination thereof.

[0289] Other Embodiments While the present invention is described in connection with its detailed description, the foregoing description is intended to illustrate, not to limit, the scope of the invention as defined by the appended claims, and it is understood that other aspects, advantages, and modifications are within the scope of the following claims.

[0290] All references, publications, patents, and patent applications referenced in this specification are incorporated herein by reference to the same extent as each individual publication, patent, and patent application is specifically and individually indicated and incorporated herein by reference.

[0291] While this disclosure is specifically shown and described in relation to embodiments of its examples, those skilled in the art will understand that various modifications of form and detail can be made in its embodiments without departing from the scope of the invention as encompassed in the appended claims.

[0292] [Sequence List] SEQUENCE LISTING <110> Leveragen, Inc. <120> ENGINEERED NON-HUMAN ANIMALS FOR PRODUCING ANTIBODIES <130> PA25-583 <150> US 63 / 184,385 <151> 2021-05-05 <150> US 63 / 184,384 <151> 2021-05-05 <160> 57 <170> PatentIn version 3.5 <210> 1 <211> 12 <212> PRT <213> Artificial <220> <223> CDR of a heavy chain antibody having the ability to bind to a SARS-CoV2 Spike antigen <400> 1 Tyr Thr Phe Thr Asp Tyr Tyr Met Lys Trp Leu Lys 1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial <220> <223> CDR of a heavy chain antibody having the ability to bind to a SARS-CoV2 Spike antigen <400> 2 Tyr Thr Phe Thr Asp Tyr Tyr Val Lys Trp Glu Lys 1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial <220> <223> CDR of a heavy chain antibody having the ability to bind to a SARS-CoV2 Spike antigen <400> 3 Tyr Thr Phe Thr Asp Tyr Tyr Val Lys Trp Lys Lys 1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial <220> <223> CDR of a heavy chain antibody having the ability to bind to a SARS-CoV2 Spike antigen <400> 4 Tyr Thr Phe Thr Asp Tyr Tyr Ile Lys Trp Glu Lys 1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial <220> <223> CDR of a heavy chain antibody having the ability to bind to a SARS-CoV2 Spike antigen <400> 5 Tyr Thr Phe Thr Asp Tyr Tyr Met Lys Trp Val Lys 1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial <220> <223> CDR of a heavy chain antibody having the ability to bind to a SARS-CoV2 Spike antigen <400> 6 Tyr Thr Phe Thr Asp Tyr Tyr Met Lys Trp Ala Lys 1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial <220> <223> CDR of a heavy chain antibody having the ability to bind to a SARS-CoV2 Spike antigen <400> 7 Tyr Thr Phe Thr Asp Tyr Tyr Met Lys Trp Glu Lys 1 5 10 <210> 8 <211> 10 <212> PRT <213> Artificial <220> <223> CDR of a heavy chain antibody having the ability to bind to a SARS-CoV2 Spike antigen <400> 8 Gly Asp Thr Phe Tyr Asn Gln Lys Phe Lys 1 5 10 <210> 9 <211> 10 <212> PRT <213> Artificial <220> <223> CDR of a heavy chain antibody having the ability to bind to a SARS-CoV2 Spike antigen <400> 9 Gly Asp Ile Phe Tyr Asn Pro Gln Phe Lys 1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial <220> <223> CDR of a heavy chain antibody having the ability to bind to a SARS-CoV2 Spike antigen <400> 10 Gly Glu Thr Phe Tyr Asn Gln Gln Phe Lys 1 5 10 <210> 11 <211> 10 <212> PRT <213> Artificial <220> <223> CDR of a heavy chain antibody having the ability to bind to a SARS-CoV2 Spike antigen <400> 11 Gly Asp Thr Asn Tyr Ser Gln Asn Phe Lys 1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial <220> <223> CDR of a heavy chain antibody having the ability to bind to a SARS-CoV2 Spike antigen <400> 12 Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys 1 5 10 <210> 13 <211> 10 <212> PRT <213> Artificial <220> <223> CDR of a heavy chain antibody having the ability to bind to a SARS-CoV2 Spike antigen <400> 13 Gly Gly Ala Arg Tyr Asn Gln Lys Phe Lys 1 5 10 <210> 14 <211> 10 <212> PRT <213> Artificial <220> <223> CDR of a heavy chain antibody having the ability to bind to a SARS-CoV2 Spike antigen <400> 14 Gly Gly Thr Arg Tyr Asn Gln Lys Phe Arg 1 5 10 <210> 15 <211> 10 <212> PRT <213> Artificial <220> <223> CDR of a heavy ch...

Claims

1. A genetically modified mouse comprising germline alteration including deletion of nucleic acid sequences containing one or more heavy chain C region genes; The mouse expresses IgG heavy chain antibodies and secretes IgG heavy chain antibodies into its serum. The aforementioned genetically modified mouse.

2. The genetically modified mouse according to claim 1, wherein one or more heavy chain C region genes are IgM C region genes (Cμ), IgD C region genes (Cδ), IgE C region genes (Cε), IgG3 C region genes (Cγ3), IgG2b C region genes (Cγ2b), IgG2c C region genes (Cγ2c), or a combination thereof.

3. A genetically modified mouse according to any one of claims 1 to 2, further comprising a deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene (Cγ1).

4. A genetically modified mouse according to claim 3, wherein the deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene includes exon 1.

5. The genetically modified mouse according to any one of claims 1 to 4, wherein the germline modification further comprises a native nucleic acid sequence encoding a hinge (H) domain, a heavy chain CH2 domain, a heavy chain CH3 domain, or a combination thereof.

6. The genetically modified mouse according to any one of claims 1 to 5, wherein the germline modification further comprises a natural nucleic acid sequence containing an endogenous enhancer.

7. The genetically modified mouse according to claim 6, wherein the enhancer is Eμ, 3'RR, 3'γ1E, 5'hsR1, or a combination thereof.

8. The genetically modified mouse according to any one of claims 1 to 7, wherein the germline modification further comprises a native nucleic acid sequence containing a switched tandem repeat element (Sμ), where Sμ drives IgG1 expression.

9. A genetically modified mouse according to any one of claims 1 to 8, wherein the IgG heavy chain antibody comprises an IgG1 heavy chain antibody.

10. The genetically modified mouse according to claim 9, wherein the IgG1 heavy chain antibody is the IgG1ΔCH1 protein.

11. A genetically modified mouse according to any one of claims 1 to 10, wherein the IgG heavy chain antibody lacks a light chain.

12. A genetically modified mouse according to any one of claims 1 to 11, wherein the IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

13. A genetically modified mouse according to any one of claims 1 to 12, wherein the mouse does not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, or a combination thereof.

14. A genetically modified mouse according to any one of claims 1 to 14, wherein the mouse does not express wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or a combination thereof.

15. A genetically modified mouse comprising germline modification including deletion of nucleic acid sequences containing one or more heavy chain C region genes; the genetically modified mouse expressing a humanized IgG heavy chain antibody and secreting the humanized IgG heavy chain antibody into its serum.

16. The genetically modified mouse according to claim 15, wherein one or more heavy chain C region genes are IgM C region genes (Cμ), IgD C region genes (Cδ), IgE C region genes (Cε), IgG3 C region genes (Cγ3), IgG2b C region genes (Cγ2b), IgG2c C region genes (Cγ2c), or a combination thereof.

17. A genetically modified mouse according to any one of claims 15 to 16, further comprising a deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene (Cγ1).

18. The genetically modified mouse according to claim 17, wherein the deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene includes exon 1.

19. The genetically modified mouse according to any one of claims 15 to 18, wherein the germline modification further comprises a native nucleic acid sequence encoding a hinge (H) domain, a heavy chain CH2 domain, a heavy chain CH3 domain, or a combination thereof.

20. The genetically modified mouse according to any one of claims 15 to 19, wherein the germline modification further comprises a natural nucleic acid sequence containing an endogenous enhancer.

21. The genetically modified mouse according to claim 20, wherein the enhancer is Eμ, 3'RR, 3'γ1E, 5'hsR1, or a combination thereof.

22. The genetically modified mouse according to any one of claims 15 to 21, wherein the germline modification further comprises a native nucleic acid sequence containing a switched tandem repeat element (Sμ), where Sμ drives IgG1 expression.

23. A genetically modified mouse according to any one of claims 15 to 22, wherein the humanized IgG heavy chain antibody comprises a humanized IgG1 heavy chain antibody.

24. The genetically modified mouse according to claim 23, wherein the humanized IgG1 heavy chain antibody is the IgG1ΔCH1 protein.

25. A genetically modified mouse according to any one of claims 15 to 24, wherein the humanized IgG heavy chain antibody lacks a light chain.

26. A genetically modified mouse according to any one of claims 15 to 25, wherein the humanized IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

27. A genetically modified mouse according to any one of claims 15 to 26, wherein the mouse does not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, or a combination thereof.

28. A genetically modified mouse according to any one of claims 15 to 17, wherein the mouse does not express wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or a combination thereof.

29. A recombinant non-human animal comprising a germline genome containing a recombinant immunoglobulin heavy chain (IgH) allele at an endogenous IgH locus; wherein the recombinant IgH allele lacks an endogenous heavy chain C region gene; and the endogenous heavy chain C region gene comprises Cμ, Cδ, Cε, Cγ3, Cγ2b, Cγ2c, or a combination thereof.

30. The recombinant non-human animal according to claim 29, wherein the IgH allele comprises a deletion of the nucleic acid sequence encoding the CH1 domain of the IgG1 C region gene (Cγ1).

31. The recombinant non-human animal according to claim 30, wherein the CH1 domain of the IgG1 C region gene contains exon 1.

32. A recombinant non-human animal according to any one of claims 29 to 31, wherein the IgH locus comprises a native nucleic acid sequence encoding a hinge (H) domain, a heavy chain CH2 domain, a heavy chain CH3 domain, or a combination thereof.

33. A recombinant non-human animal according to any one of claims 29 to 32, wherein the IgH gene locus comprises a natural nucleic acid sequence containing an endogenous enhancer.

34. The recombinant non-human animal according to claim 33, wherein the enhancer is Eμ, 3'RR, 3'γ1E, 5'hsR1, or a combination thereof.

35. The recombinant non-human animal according to any one of claims 29 to 34, wherein the IgH gene locus comprises a native nucleic acid sequence containing a switch tandem repeat element (Sμ), where Sμ drives IgG1 expression.

36. A recombinant non-human animal according to any one of claims 29 to 35, wherein the IgH gene locus comprises an endogenous V, D, or J gene.

37. A recombinant non-human animal according to any one of claims 29 to 35, wherein the non-human animal expresses a humanized IgG heavy chain antibody.

38. The recombinant non-human animal according to claim 37, wherein the humanized IgG heavy chain antibody comprises a humanized IgG1 heavy chain antibody.

39. A recombinant non-human animal according to any one of claims 37 to 38, wherein the humanized IgG1 heavy chain antibody is the IgG1ΔCH1 protein.

40. A recombinant non-human animal according to any one of claims 37 to 39, wherein the humanized IgG heavy chain antibody lacks a light chain.

41. A recombinant non-human animal according to any one of claims 37 to 40, wherein the humanized IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

42. A recombinant non-human animal according to any one of claims 37 to 41, wherein the IgH gene locus comprises the human V, D, or J gene.

43. A recombinant non-human animal according to any one of claims 37 to 42, wherein the endogenous IgH gene locus comprises an exogenous nucleic acid sequence.

44. Human V with one or more exogenous nucleic acid sequences H Gene segment, one or more human D H Gene segment, and one or more J H A recombinant non-human animal according to claim 43, comprising a gene segment.

45. The exogenous nucleic acid sequence contains 65 human V H A recombinant non-human animal according to any one of claims 43 to 44, comprising a gene segment.

46. The exogenous nucleic acid sequence contains 27 human D H A recombinant non-human animal according to any one of claims 43 to 45, comprising a gene segment.

47. The exogenous nucleic acid sequence has 6 J H A recombinant non-human animal according to any one of claims 43 to 46, comprising a gene segment.

48. The exogenous nucleic acid sequence comprises 65 human V H gene segments, 27 human D H gene segments, and 6 J H gene segments, and the recombinant non-human animal according to any one of claims 43 to 47.

49. A recombinant non-human animal according to any one of claims 43 to 48, wherein the exogenous nucleic acid sequence includes a barcode.

50. A recombinant non-human animal according to any one of claims 29 to 49, wherein the non-human animal does not express wild-type IgM protein, wild-type IgD protein, wild-type IgE protein, wild-type IgG3 protein, or a combination thereof.

51. A recombinant non-human animal according to any one of claims 29 to 50, wherein the non-human animal does not express wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or a combination thereof.

52. The recombinant non-human animal according to any one of claims 29 to 51, wherein the recombinant non-human animal is homozygous for the recombinant IgH allele.

53. A recombinant non-human animal according to any one of claims 29 to 52, wherein the non-human animal is a mammal.

54. The recombinant non-human animal according to claim 53, wherein the mammal is a mouse or a rat.

55. A method for producing a genetically modified non-human animal capable of producing heavy chain antibodies, comprising: (a) deleting an endogenous nucleic acid sequence containing one or more heavy chain C region genes from an endogenous immunoglobulin heavy chain gene locus in a stem cell of a non-human animal; (b) transplanting the stem cell into a blastocyst; (c) transplanting the blastocyst into a pseudo-pregnant mouse to obtain a chimeric mouse; (d) mating the chimeric mouse with a wild-type mouse to produce offspring; (e) screening the offspring for heterozygosity; and (f) identifying a founding mouse having a deletion of one or more heavy chain C region genes, wherein the non-human animal is capable of producing heavy chain antibodies.

56. A method for producing a genetically modified non-human animal capable of producing humanized heavy chain antibodies, comprising: (a) deleting an endogenous nucleic acid sequence containing one or more heavy chain C region genes from an endogenous immunoglobulin heavy chain gene locus in the stem cells of the non-human animal; (b) transplanting the stem cells into a blastocyst; (c) transplanting the blastocyst into a pseudo-pregnant mouse to obtain a chimeric mouse; (d) mating the chimeric mouse with a wild-type mouse to produce offspring; (e) screening the offspring for heterozygosity; and (f) identifying a founding mouse having a deletion of one or more heavy chain C region genes, wherein the non-human animal is capable of producing humanized heavy chain antibodies.

57. The method according to any one of claims 55 to 56, wherein the stem cells are embryonic stem cells.

58. A method for producing a soluble heavy chain antibody in a recombinant non-human animal according to claim 55, comprising the steps of (a) administering an antigen to a non-human animal; (b) isolating one or more B cells from the non-human animal; (c) isolating mRNA from the one or more B cells; (d) sequencing the mRNA; (e) identifying a clone type based on the mRNA sequence; and (f) performing phylogenetic analysis of the clone type, thereby producing a soluble heavy chain antibody.

59. A method for producing a single-domain antibody (sdAb) identified from a recombinant non-human animal according to claim 55, comprising: (a) a heavy chain variable (V) containing V, D, and J in cells H (b) a method comprising the steps of expressing a nucleic acid sequence encoding a domain, wherein the cell produces a heavy chain variable domain; and (b) isolating the heavy chain variable domain from a sample, thereby producing a single-domain antibody.

60. The method according to claim 59, wherein the single-domain antibody is a single-domain antibody of mouse.

61. A method for producing a soluble humanized heavy chain antibody in a recombinant non-human animal according to any one of claims 56 to 57, comprising: (a) administering an antigen to a non-human animal; (b) isolating one or more B cells from the non-human animal; (c) isolating mRNA from the one or more B cells; (d) sequencing the mRNA; (e) identifying a clonal type based on the mRNA sequence; and (f) performing phylogenetic analysis of the clonal type, thereby producing a soluble humanized heavy chain antibody.

62. A method for producing a humanized single-domain antibody (sdAb) identified from a recombinant non-human animal according to any one of claims 56 to 57, comprising: (a) a human heavy chain variable (V) containing V, D and J in cells H (b) a method comprising the steps of expressing a nucleic acid sequence encoding a domain, wherein the cell produces a human heavy chain variable domain; and (b) isolating a human heavy chain variable domain from a sample, thereby producing a single-domain antibody.

63. The method according to claim 62, wherein the single-domain antibody is a human single-domain antibody.

64. The method according to claim 62, wherein the cells are bacterial cells or human cells.

65. A non-human animal wherein the genome of the non-human animal comprises an immunoglobulin heavy chain (IgH) allele, wherein the IgH allele comprises an endogenous nucleic acid encoding a CH2 domain or a CH3 domain of an IgG subclass, the IgH allele lacks a nucleic acid encoding at least a portion of the endogenous CH1 domain of the IgG subclass, and the IgH allele also lacks an endogenous nucleic acid encoding at least a portion of the IgM constant domain, an endogenous nucleic acid encoding at least a portion of the IgD constant domain, an endogenous nucleic acid encoding at least a portion of the IgE constant domain, or an endogenous nucleic acid encoding at least a portion of the IgA constant domain.

66. The non-human animal according to claim 65, wherein the IgH allele of the non-human animal comprises an endogenous nucleic acid encoding the CH2 domain and the CH3 domain of the IgG subclass.

67. The non-human animal according to any one of claims 65 to 66, wherein the IgH allele of the non-human animal comprises an endogenous nucleic acid encoding the hinge domain of the IgG subclass.

68. The non-human animal according to any one of claims 65 to 67, wherein the IgG subclass is the IgG2 subclass.

69. The non-human animal according to any one of claims 65 to 67, wherein the IgG subclass is IgG2a, IgG2b, IgG2c, IgG3, or IgG4 subclass.

70. The non-human animal according to any one of claims 65 to 67, wherein the IgG subclass is the IgG1 subclass.

71. The non-human animal according to claim 70, wherein the IgH allele lacks an endogenous nucleic acid encoding at least a portion of the IgG2 constant domain, an endogenous nucleic acid encoding at least a portion of the IgG3 constant domain, or an endogenous nucleic acid encoding at least a portion of the IgG4 constant domain.

72. The non-human animal according to claim 70, wherein the IgH allele lacks an endogenous nucleic acid encoding at least a portion of the IgG2a constant domain, an endogenous nucleic acid encoding at least a portion of the IgG2b constant domain, an endogenous nucleic acid encoding at least a portion of the IgG2c constant domain, an endogenous nucleic acid encoding at least a portion of the IgG3 constant domain, and an endogenous nucleic acid encoding at least a portion of the IgG4 constant domain.

73. The non-human animal according to claim 70, wherein the IgH allele lacks endogenous nucleic acids encoding each of the IgG2 constant domains, endogenous nucleic acids encoding each of the IgG3 constant domains, or endogenous nucleic acids encoding each of the IgG4 constant domains.

74. The non-human animal according to claim 70, wherein the IgH allele lacks endogenous nucleic acids encoding each of the IgG2a constant domain, each of the IgG2b constant domain, each of the IgG2c constant domain, each of the IgG3 constant domain, or each of the IgG4 constant domain.

75. The non-human animal according to any one of claims 65 to 74, wherein the IgH allele lacks an endogenous nucleic acid encoding at least a portion of the IgM constant domain, an endogenous nucleic acid encoding at least a portion of the IgD constant domain, an endogenous nucleic acid encoding at least a portion of the IgE constant domain, and an endogenous nucleic acid encoding at least a portion of the IgA constant domain.

76. The non-human animal according to any one of claims 65 to 74, wherein the IgH allele lacks endogenous nucleic acids encoding each of the IgM constant domains, endogenous nucleic acids encoding each of the IgD constant domains, endogenous nucleic acids encoding each of the IgE constant domains, or endogenous nucleic acids encoding each of the IgA constant domains.

77. The non-human animal according to any one of claims 65 to 74, wherein the IgH allele lacks endogenous nucleic acids encoding each of the IgM constant domains.

78. The non-human animal according to any one of claims 65 to 77, wherein the IgH allele lacks endogenous nucleic acids encoding each of the IgD constant domains.

79. The non-human animal according to any one of claims 65 to 78, wherein the IgH allele lacks endogenous nucleic acids encoding each of the IgE constant domains.

80. The non-human animal according to any one of claims 65 to 79, wherein the IgH allele lacks endogenous nucleic acids encoding the IgA CH1 constant domain and the CH2 constant domain.

81. The non-human animal according to any one of claims 65 to 80, wherein the IgH allele lacks the nucleic acid encoding the endogenous CH1 domain.

82. The non-human animal according to any one of claims 65 to 81, wherein the IgH allele includes endogenous Eμ.

83. The non-human animal according to any one of claims 65 to 82, wherein the first nucleic acid sequence encoding the full-length CH2 domain downstream of the endogenous Eμ is a nucleic acid encoding the IgG CH2 domain.

84. The non-human animal according to any one of claims 65 to 83, wherein the first nucleic acid sequence encoding the full-length CH2 domain downstream of the endogenous Eμ is a nucleic acid encoding the IgG1 CH2 domain.

85. The non-human animal according to any one of claims 65 to 84, wherein the IgH allele comprises an endogenous Sμ, an endogenous Iμ promoter, an endogenous Iμ exon, or a combination thereof.

86. The non-human animal according to any one of claims 65 to 85, wherein the first nucleic acid sequence encoding the endogenous Sμ, the endogenous Iμ promoter, or the full-length CH2 domain downstream of the endogenous Iμ exon is a nucleic acid encoding an IgG CH2 domain.

87. The non-human animal according to any one of claims 65 to 86, wherein the first nucleic acid sequence encoding the endogenous Sμ, the endogenous Iμ promoter, or the full-length CH2 domain downstream of the endogenous Iμ exon is a nucleic acid encoding the IgG1 CH2 domain.

88. The non-human animal according to any one of claims 65 to 87, wherein the IgH allele contains endogenous 3'γ1E.

89. The non-human animal according to any one of claims 65 to 88, wherein the IgH allele lacks an endogenous nucleic acid encoding the full-length CH2 domain downstream of the endogenous 3'γ1E.

90. The non-human animal according to any one of claims 65 to 89, wherein the IgH allele comprises endogenous 5'hsR1.

91. The non-human animal according to any one of claims 65 to 90, wherein the first nucleic acid sequence encoding the full-length CH2 domain upstream of the endogenous 5'hsR1 is a nucleic acid encoding the IgG CH2 domain.

92. The non-human animal according to any one of claims 65 to 91, wherein the first nucleic acid sequence encoding the full-length CH2 domain upstream of the endogenous 5'hsR1 is a nucleic acid encoding the IgG1 CH2 domain.

93. The non-human animal according to any one of claims 65 to 92, wherein the IgH allele contains endogenous 3'RR.

94. The non-human animal according to any one of claims 65 to 93, wherein the first nucleic acid sequence encoding the full-length CH2 domain upstream of the endogenous 3'RR is a nucleic acid encoding the IgG CH2 domain.

95. The non-human animal according to any one of claims 65 to 94, wherein the first nucleic acid sequence encoding the full-length CH2 domain upstream of the endogenous 3'RR is a nucleic acid encoding the IgG1 CH2 domain.

96. The non-human animal according to any one of claims 65 to 95, wherein the IgH allele contains endogenous 3'CBE.

97. The non-human animal according to any one of claims 65 to 96, wherein the first nucleic acid sequence encoding the full-length CH2 domain upstream of the endogenous 3'CBE is a nucleic acid encoding the IgG CH2 domain.

98. The non-human animal according to any one of claims 65 to 97, wherein the first nucleic acid sequence encoding the full-length CH2 domain upstream of the endogenous 3'CBE is a nucleic acid encoding the IgG1 CH2 domain.

99. A non-human animal according to any one of claims 65 to 98, wherein at least one allele of the genome lacks at least a portion of the endogenous Ig heavy chain variable region.

100. A non-human animal according to any one of claims 65 to 99, wherein at least one allele of the genome lacks all exons of the endogenous Ig heavy chain variable region.

101. The non-human animal according to any one of claims 65 to 100, wherein both alleles of the genome lack all exons of the endogenous Ig heavy chain variable region.

102. A non-human animal according to any one of claims 65 to 100, wherein none of the alleles of the genome contain exogenous exons of the Ig heavy chain variable region.

103. The non-human animal according to claim 102, wherein the non-human animal does not produce an Ig heavy chain.

104. The non-human animal according to any one of claims 65 to 101, wherein the IgH allele comprises an exogenous nucleic acid encoding one or more human Ig heavy chain variable region gene segments.

105. The non-human animal according to claim 104, wherein the IgH allele comprises one or more exogenous human IgVH gene segments.

106. The non-human animal according to claim 104, wherein the IgH allele comprises three or more human IgVH gene segments.

107. The non-human animal according to claim 104, wherein the IgH allele comprises 26 or more human IgVH gene segments.

108. The non-human animal according to claim 104, wherein the IgH allele comprises 65 or more human IgVH gene segments.

109. The non-human animal according to claim 104, wherein the IgH allele comprises 126 human IgVH gene segments.

110. The non-human animal according to any one of claims 104 to 109, wherein the IgH allele comprises 13 or more IgVD gene segments.

111. The non-human animal according to any one of claims 104 to 109, wherein the IgH allele comprises 27 human IgVD gene segments.

112. The non-human animal according to any one of claims 104 to 111, wherein the IgH allele comprises three or more human Ig VJ gene segments.

113. The non-human animal according to any one of claims 104 to 112, wherein the IgH allele comprises nine human Ig VJ gene segments.

114. The non-human animal according to claim 104, wherein the genome comprises 126 human Ig VH gene segments, 27 or more human Ig VD gene segments, and 9 human Ig VJ gene segments.

115. The non-human animal according to any one of claims 104 to 114, wherein the non-human animal produces a human-non-human chimeric Ig heavy chain antibody.

116. The non-human animal according to claim 115, wherein the variable region domain of the human-non-human chimeric Ig heavy chain antibody is fully human.

117. The non-human animal according to any one of claims 65 to 101 and 104 to 116, wherein the IgH allele comprises an exogenous nucleic acid encoding one or more human Ig light chain variable region gene segments.

118. The non-human animal according to claim 117, wherein the IgH allele comprises one or more exogenous human Igκ variable gene segments.

119. The non-human animal according to any one of claims 117 to 118, wherein the IgH allele comprises 20 or more exogenous human Igκ variable gene segments.

120. The non-human animal according to any one of claims 117 to 119, wherein the IgH allele comprises 40 exogenous human Igκ variable gene segments.

121. The non-human animal according to any one of claims 117 to 120, wherein the IgH allele comprises one or more exogenous human Igλ variable gene segments.

122. The non-human animal according to any one of claims 117 to 121, wherein the IgH allele comprises 10 or more exogenous human Igλ variable gene segments.

123. The non-human animal according to any one of claims 117 to 122, wherein the IgH allele comprises 20 exogenous human Igλ variable gene segments.

124. The non-human animal according to any one of claims 117 to 123, wherein the IgH allele comprises one or more human Igκ VJ gene segments.

125. The non-human animal according to any one of claims 117 to 124, wherein the IgH allele comprises five human Igκ VJ gene segments.

126. The non-human animal according to any one of claims 117 to 125, wherein the IgH allele comprises one or more human Igλ VJ gene segments.

127. The non-human animal according to any one of claims 117 to 126, wherein the IgH allele comprises four human Igλ VJ gene segments.

128. The non-human animal according to any one of claims 117 to 127, wherein the IgH allele comprises 40 human Igκ variable gene segments and 5 human Igκ VJ gene segments.

129. The non-human animal according to any one of claims 117 to 128, wherein the IgH allele comprises 20 human Igλ variable gene segments and 4 human Igλ VJ gene segments.

130. The non-human animal according to any one of claims 117 to 129, wherein the non-human animal produces a human-non-human chimeric Ig heavy chain antibody.

131. The non-human animal according to claim 130, wherein the variable region domain of the human-non-human chimeric Ig heavy chain antibody is entirely human, derived from the light chain.

132. The non-human animal according to any one of claims 65 to 101, 104 to 115, and 117 to 130, wherein the non-human animal is a non-human animal of a first non-human species, and the IgH allele comprises an exogenous nucleic acid encoding one or more Ig heavy chain variable region gene segments of a second non-human species different from the first non-human species.

133. The non-human animal according to claim 132, wherein the IgH allele comprises one or more IgVH gene segments of the second non-human species.

134. The non-human animal according to claim 132, wherein the IgH allele comprises 10 or more IgVH gene segments of the second non-human species.

135. The non-human animal according to claim 132, wherein the IgH allele comprises all IgVH gene segments of the second non-human species.

136. The non-human animal according to any one of claims 132 to 135, wherein the IgH allele comprises three or more IgVD gene segments of the second non-human species.

137. The non-human animal according to any one of claims 132 to 136, wherein the IgH allele comprises all Ig VD gene segments of the second non-human species.

138. The non-human animal according to any one of claims 132 to 137, wherein the IgH allele comprises three or more Ig VJ gene segments of the second non-human species.

139. The non-human animal according to any one of claims 132 to 138, wherein the IgH allele comprises all Ig VJ gene segments of the second non-human species.

140. The non-human animal according to claim 132, wherein the IgH allele comprises all of the IgVH gene segments, IgVD gene segments, and IgVJ gene segments of the second non-human species.

141. The non-human animal according to any one of claims 132 to 140, wherein the non-human animal produces chimeric heavy chain antibodies of the first and second species.

142. The non-human animal according to claim 141, wherein the variable region domain of the chimeric heavy chain antibody is entirely the variable region domain of the second species.

143. The non-human animal according to any one of claims 132 to 142, wherein the first species is a mouse species.

144. The non-human animal according to any one of claims 132 to 143, wherein the second species is a cattle species, a shark species, or an alpaca species.

145. The non-human animal according to any one of claims 65 to 144, wherein the IgH allele comprises at least one exogenous recombinase site-recognizing nucleic acid sequence.

146. The non-human animal according to claim 145, wherein the at least one exogenous recombinase site-recognizing nucleic acid sequence is located upstream of the endogenous nucleic acid encoding the CH2 domain or CH3 domain of the IgG subclass.

147. The non-human animal according to any one of claims 145 to 146, wherein the IgH allele comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different exogenous recombinase site-recognizing nucleic acid sequences.

148. The non-human animal according to any one of claims 145 to 146, wherein the IgH allele comprises at least three different exogenous recombinase site-recognizing nucleic acid sequences.

149. The non-human animal according to any one of claims 145 to 146, wherein the IgH allele comprises at least five different exogenous recombinase site-recognizing nucleic acid sequences.

150. The non-human animal according to any one of claims 147 to 149, wherein each of the different exogenous recombinase site-recognizing nucleic acid sequences is located less than 2.5 Mb upstream of endogenous Eμ.

151. A non-human animal according to any one of claims 147 to 149, wherein each of the different exogenous recombinase site-recognizing nucleic acid sequences is located less than 2.0 Mb, less than 1.5 Mb, less than 1.0 Mb, less than 500 kb, or less than 250 kb upstream of endogenous Eμ.

152. A non-human animal according to any one of claims 147 to 149, wherein each of the different exogenous recombinase site-recognizing nucleic acid sequences is located less than 200 kb, less than 100 kb, less than 50 kb, less than 25 kb, or less than 10 kb upstream of endogenous Eμ.

153. below: (a) Sequence ID 4, Sequence ID 10, and Sequence ID 19, or (b) Sequence ID 5, Sequence ID 11, and Sequence ID 20 An antibody containing a variable region.

154. The antibody according to claim 153, wherein the antibody binds to the SARS-CoV-2 spike polypeptide.

155. The antibody according to any one of claims 153 to 154, wherein the antibody is a heavy chain antibody.

156. The antibody according to any one of claims 153 to 154, wherein the antibody is a single-domain antibody.

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