CN117651486A

CN117651486A - Engineered non-human animals for antibody production

Info

Publication number: CN117651486A
Application number: CN202280047822.8A
Authority: CN
Inventors: W·V·陈
Original assignee: Liwei Ruijian Co ltd
Current assignee: Liwei Ruijian Co ltd
Priority date: 2021-05-05
Filing date: 2022-05-05
Publication date: 2024-03-05

Abstract

This document provides genetically modified animals (e.g., mice), humanized heavy chain antibodies, humanized nanobodies, and their preparation and use. For example, genetically engineered non-human animals (e.g., genetically engineered mice) are provided that can be designed to produce heavy chain antibodies that can be used to produce single domain antibodies or nanobodies.

Description

Engineered non-human animals for antibody production

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional application Ser. No. 63/184,384, filed 5/2021, and U.S. provisional application Ser. No. 63/184,385, filed 5/2021. The disclosure of the prior application is considered to be part of the disclosure of the present application, the entire contents of which are incorporated herein by reference.

Background

1. Technical field

Related methods and materials for producing antibodies, e.g., heavy chain antibodies and/or single domain antibodies (also referred to as nanobodies), are described herein. For example, genetically engineered non-human animals (e.g., genetically engineered mice) are provided that have the ability to produce antibodies (e.g., heavy chain antibodies, such as heavy chain antibodies that lack CHl domains and light chains). In some cases, the heavy chain antibodies obtained as described herein can be used to generate single domain antibodies or nanobodies.

2. Background art

Conventional IgG antibodies consist of four polypeptides: two pairs of identical heavy and light chains. The heavy chain of IgG contains one variable domain (VH) and three constant domains (CH 1, CH2 and CH 3), the two chains being linked by disulfide bonds at the hinge region (H) between CH1 and CH 2. The two light chains contain one variable domain (VL) and one constant domain (CL), where the CL domain is linked to the CH1 domain of the heavy chain by disulfide bonds to form a tetrameric IgG. Two antibody arms (Fab) consisting of VH-CH1 and VL-CL can bind antigen independently, the constant region (Fc) being responsible for effector function. Antibody production begins with the expression of B Cell Receptor (BCR) in pre-B cells, producing different VH by VDJ recombination assembly of V (variable), D (diversity) and J (junction) gene segments of immunoglobulin heavy chain genes (IgH). VDJ is spliced into the constant exons of IgM and then combined with the cell surface alternate light chain λ6 and VbreB to form pre-BCR. Followed by VJ recombination of immunoglobulin light chain genes (IgK and IgL, both lacking the D gene) and producing a different VL with CL. Pairing the light and heavy chains of IgM causes IgM to be expressed as a complete BCR on immature B cells. Although V (D) J recombination occurs at both alleles of the heavy and light chain loci, allelic exclusion ensures that only one functional heavy chain and one functional light chain are expressed by one of the two alleles in a single B cell. B cells carrying successful recombination events will then undergo somatic hypermutation, antigen selection, affinity maturation and type switching recombination to express the different isotypes of antibodies (IgG, igE and IgA).

In camelids, it has been confirmed that only two identical heavy chains with variable domains (variable heavy homodimers, VHHs) are comprised, but a subset of IgG lacking the CH1 domain and the associated light chain. These heavy chain-only antibodies (hcabs) result from splice site mutations in the heavy chain gene resulting in the elimination of the CH1 exon, which encodes the CH1 domain of the constant region, which is normally associated with CL of the light chain. Similar hcabs (immunoglobulin neoantigen receptor, igNAR) lacking light chains are also found in cartilaginous fish. The variable domains in hcabs (VHH in camelids and VNAR in cartilaginous fish) can function as independent antigen binding units and have binding affinities comparable to conventional antibodies. These single domain antibodies (sdabs) are the smallest antigen-binding antibody fragments and are therefore sometimes referred to as nanobodies (Nb). sdabs have many unique properties, including small size (11-15 KDa while tetrameric antibodies are 150 KDa), stringent uniqueness, high solubility, efficient folding/refolding, excellent stability, unparalleled target accessibility, efficient tissue penetration, rapid blood clearance, excellent manufacturability, and low cost make them powerful candidates for developing new therapeutic and diagnostic agents. One of the most advantageous properties of sdabs compared to conventional antibodies is their modularity, a key property for easier engineering of multimers and multispecific biologies.

Variable domains derived from the human scaffold (VH and VL) have been generated in synthetic sdAb display libraries and tested in vitro against a number of targets (belanger et al, protein eng. Des. Sel., (34): gzab012 (2021)). Unlike naturally occurring sdabs obtained from immunized animals that have high affinity due to somatic hypermutation, sdabs derived from non-immune display libraries are generally lower in affinity and prone to aggregation, and often require further mutations to improve affinity and function. One approach to solve both of these problems is to genetically engineer mice that can be immunized with a target of interest to isolate functional human sdabs with high solubility and affinity due to in vivo antigen selection and affinity maturation to produce humanized hcabs.

Disclosure of Invention

This document relates to engineered 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 of making such engineered non-human animals (e.g., mice) and methods of using such engineered non-human animals (e.g., mice). For example, provided herein are genetically engineered non-human animals (e.g., genetically engineered mice) that can be designed to produce heavy chain antibodies that can be used to produce single domain antibodies or nanobodies. In some cases, genetically engineered non-human animals (e.g., genetically engineered mice) can be designed to produce heavy chain antibodies (e.g., fully mouse heavy chain IgG antibodies) that lack CH1 domains and light chains, and can be used to produce single domain antibodies or nanobodies (e.g., fully mouse single domain antibodies or fully mouse nanobodies). See, for example, fig. 1. In some cases, genetically engineered non-human animals (e.g., genetically engineered mice) can be designed to produce chimeric heavy chain antibodies (e.g., fully mouse heavy chain IgG antibodies) lacking the CH1 domain and light chain, which can be used to produce single domain antibodies or nanobodies, which can also be chimeric or can be fully single species. For example, genetically engineered mice can be designed to produce human-mouse chimeric heavy chain IgG antibodies that lack the CH1 domain and light chain, but have 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, fig. 6. The compositions described herein (e.g., compositions containing one or more antibodies produced by an engineered non-human animal (e.g., a mouse) provided herein) can be used to treat or prevent a disease or disorder (e.g., an inflammatory disease).

As described herein, genetically engineered non-human animals (e.g., genetically engineered mice) can be designed to produce heavy chain antibodies (e.g., mouse heavy chain antibodies or chimeric heavy chain antibodies such as 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) that lack CH1 domains and light chains. 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)).

As also described herein, the engineered 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 generate single domain antibodies (e.g., mouse single domain antibodies or non-mouse single domain antibodies (such as humanized single domain antibodies or human single domain antibodies)

In one embodiment, provided herein is a genetically engineered mouse comprising a germline modification comprising a deletion of a nucleic acid sequence comprising one or more heavy chain C-region genes; wherein the mouse expresses and secretes IgG heavy chain antibodies into its serum.

In some embodiments, one or more heavy chain C region genes comprise an IgM C region gene (cμ), an IgD C region gene (cδ), an IgE C region gene (cε), an IgG 3C region gene (cγ3), an IgG2b C region gene (cγ2b), an IgG2C C region gene (cγ2c), or a combination thereof.

In some embodiments, the genetically engineered mouse further comprises a deletion of a nucleic acid sequence encoding a CH1 domain of an IgG 1C region gene (cγ1). In some embodiments, the deletion of the nucleic acid sequence encoding the CHl domain of the IgGl C region gene comprises exon 1.

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.

In some embodiments, the germline modification further comprises a native nucleic acid sequence comprising an endogenous enhancer. In some embodiments, the enhancer comprises E μ, 3' rr, 3' γ1e, 5' hsr1, or a combination thereof.

In some embodiments, the germline modification further comprises a native nucleic acid sequence comprising a switch tandem repeat element (sμ) and an iμ promoter, wherein iμ drives constitutive expression of IgG1 (igg1Δch1) truncated to the CH1 domain.

In some embodiments, the IgG heavy chain antibody comprises an IgG1 heavy chain antibody. In some embodiments, the IgG1 heavy chain antibody is an igg1Δch1 protein. In some embodiments, igG heavy chain antibodies lack light chains. In some embodiments, the IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

In some embodiments, 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. In some embodiments, the mouse does not express wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or a combination thereof.

In another embodiment, provided herein is an engineered non-human animal comprising a germline genome comprising an engineered immunoglobulin heavy chain (IgH) allele at an endogenous IgH locus; wherein the engineered IgH allele lacks an endogenous heavy chain C region gene; and wherein the endogenous heavy chain C region gene comprises cμ, cδ, cε, cγ3, cγ2b, cγ2c, or a combination thereof.

In some embodiments, the IgH allele comprises a deletion of a nucleic acid sequence encoding a CH1 domain of an IgG 1C region gene (cγ1). In some embodiments, the CHl domain of the IgGl C region gene comprises exon 1.

In some embodiments, 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.

In some embodiments, the IgH locus comprises a native nucleic acid sequence comprising an endogenous enhancer. In some embodiments, the enhancer comprises E μ, 3' rr, 3' γ1e, 5' hsr1, or a combination thereof.

In some embodiments, the IgH locus comprises a native nucleic acid sequence comprising a switch tandem repeat element (sμ) and an iμ promoter, wherein iμ drives constitutive expression of IgG1 (igg1Δch1) truncated to the CH1 domain.

In some embodiments, the non-human animal expresses an IgG heavy chain antibody. In some embodiments, the IgG heavy chain antibody comprises an IgG1 heavy chain antibody.

In some embodiments, the IgG1 heavy chain antibody is an igg1Δch1 protein.

In some embodiments, igG heavy chain antibodies lack light chains.

In some embodiments, the IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

In some embodiments, the 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.

In some embodiments, the IgH locus comprises an endogenous V, D or J gene.

In some embodiments, the engineered non-human animal is homozygous for the engineered IgH allele.

In some embodiments, the endogenous IgH locus does not comprise an exogenous nucleic acid sequence.

In some embodiments, the endogenous IgH locus comprises an exogenous nucleic acid sequence. In some embodiments, the exogenous nucleic acid sequence comprises a barcode.

In another embodiment, provided herein is an engineered non-human animal, wherein the non-human animal is a mammal. In some embodiments, the mammal is a mouse.

In another embodiment, provided herein is a method of making a genetically modified non-human animal capable of producing heavy chain antibodies, comprising (a) deleting an endogenous nucleic acid sequence comprising one or more heavy chain C region genes from an endogenous immunoglobulin heavy chain locus in a stem cell of the non-human animal; (b) implanting stem cells into the blastula; (c) Implanting blastula into pseudopregnant mice to obtain chimeric mice; (d) Crossing the chimeric mice with wild-type mice to produce offspring; (e) screening the offspring for heterozygosity; and (f) identifying a founder mouse (founder mouse) carrying one or more heavy chain C region gene deletions; and wherein the non-human animal is capable of producing heavy chain antibodies.

In some embodiments, the stem cell is an embryonic stem cell.

In some embodiments, the one or more heavy chain C region genes comprise cμ, cδ, cγ3, cγ2b, cγ2c, cε, or a combination thereof.

In some embodiments, the method further comprises deleting a nucleic acid sequence encoding the CH1 domain of the IgG 1C region gene and the CH1 exon of cγ1. In some embodiments, the deletion of the nucleic acid sequence encoding the CHl domain of the IgGl C region gene comprises exon 1.

In some embodiments, the method further comprises retaining a native nucleic acid sequence encoding a hinge (H) domain, a heavy chain CH2 domain, a heavy chain CH3 domain, or a combination thereof of IgG1 (cγ1).

In some embodiments, the method further comprises retaining the native nucleic acid sequence comprising the endogenous enhancer. In some embodiments, the enhancer comprises E μ, 3' rr, 3' γ1e, 5' hsr1, or a combination thereof.

In some embodiments, the method further comprises retaining a native nucleic acid sequence comprising a switch tandem repeat element (sμ) and an iμ promoter, wherein iμ drives constitutive expression of IgG1 (igg1Δch1) truncated to the CH1 domain.

In some embodiments, the heavy chain antibody is an IgG heavy chain antibody. In some embodiments, the IgG heavy chain antibody comprises an IgG1 heavy chain antibody. In some embodiments, the IgG1 heavy chain antibody is an igg1Δch1 protein.

In some embodiments, the IgG1 heavy chain antibody lacks a light chain.

In some embodiments, the IgG1 heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

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

In some embodiments, deleting an endogenous nucleic acid sequence comprising one or more heavy chain C region genes comprises CRISPR/Cas9 genome editing.

In some embodiments, the genetically modified non-human animal is fertility. In some embodiments, the genetically modified non-human animal has normal B cell development and maturation.

In some embodiments, 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 a combination thereof.

In another embodiment, provided herein is a method of making a soluble heavy chain antibody in an engineered non-human animal comprising (a) administering an antigen to the 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) confirming clonotype based on mRNA sequence, and (f) phylogenetic analysis of the clonotype; thereby producing soluble heavy chain antibodies.

In another embodiment, provided herein is a method of making a single domain antibody (sdAb) identified from an engineered non-human animal comprising expressing in a cell a nucleic acid encoding a heavy chain variable (V) comprising V, D and J _H ) Domain nucleic acid sequences, wherein the cell produces a heavy chain variable domain, and isolating the heavy chain variable domain from the sample, thereby producing a single domain antibody. In some embodiments, the single domain antibody is a murine single domain antibody.

In some embodiments, the single domain antibody is an IgGl single domain antibody derived from an IgGl heavy chain antibody. In some embodiments, the IgGl single domain antibody is an igg1Δch1 nanobody derived from an igg1Δch1 heavy chain antibody.

In another embodiment, provided herein is a genetically engineered mouse comprising a germline modification comprising a deletion of a nucleic acid sequence comprising one or more heavy chain C-region genes; wherein the mouse expresses the humanized IgG heavy chain antibody and secretes the humanized IgG heavy chain antibody into its serum.

In some embodiments, the one or more heavy chain C region genes are IgM C region genes (cμ), igD C region genes (cδ), igE C region genes (cε), igG 3C region genes (cγ3), igG2b C region genes (cγ2b), igG2C C region genes (cγ2c), or a combination thereof.

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 of IgG1 (cγ1).

In some embodiments, the germline modification further comprises a native nucleic acid sequence comprising an endogenous enhancer. In some embodiments, the enhancer is E μ, 3' rr, 3' γ1e, 5' hsr1, or a combination thereof.

In some embodiments, the humanized IgG heavy chain antibody comprises a humanized IgG1 heavy chain antibody. In some embodiments, the humanized IgG1 heavy chain antibody is an igg1Δch1 protein.

In some embodiments, the humanized IgG heavy chain antibody lacks a light chain.

In some embodiments, the humanized IgG heavy chain antibody comprises a hinge domain, a CH2 domain, and a CH3 domain of IgG1 (cγ1), or a combination thereof.

In some embodiments, the IgH locus comprises a native nucleic acid sequence encoding a hinge (H) domain of IgG1 (cγ1), a heavy chain CH2 domain, a heavy chain CH3 domain, or a combination thereof.

In some embodiments, the IgH locus comprises a native nucleic acid sequence comprising an endogenous enhancer. In some embodiments, the enhancer is E μ, 3' rr, 3' γ1e, 5' hsr1, or a combination thereof.

In some embodiments, the non-human animal expresses a humanized IgG heavy chain antibody. In some embodiments, the humanized IgG1 heavy chain antibody comprises a humanized IgG1 heavy chain antibody. In some embodiments, the humanized IgG1 heavy chain antibody is an igg1Δch1 protein.

In some embodiments, the humanized IgG1 heavy chain antibody lacks a light chain.

In some embodiments, the humanized IgG1 heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

In some embodiments, 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. In some embodiments, the non-human animal does not express wild-type IgA protein, wild-type IgG2b protein, wild-type IgG2c protein, or a combination thereof.

In some embodiments, the IgH locus comprises a human V, D or J gene.

In some embodiments, the endogenous IgH locus comprises an exogenous nucleic acid sequence.

In some embodiments, the exogenous nucleic acid sequence comprises one or more human V _H Gene segment, one or more humans D _H Gene segments and one or more J _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises two or more individuals V _H Gene segment, two or more individuals D _H Gene segments and two or more J _H A gene segment.

In some embodiments, the exogenous nucleic acid sequence comprises 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, 3 9. 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 person V _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises 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, 102, 103, 105, 106, 107, 108, 109, 111, 113, 115, 116, 112, 122, 112, 118, 122, or 126 _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises 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 A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises 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 person V _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises substantially all of human V _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises about 10, about 20, about 30, about 40, about 50, or about 60 human V _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, or about 120V _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises 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 A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises 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 person V _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises 65 human V _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises 126 human V _H A gene segment.

In some embodiments, the exogenous nucleic acid sequence comprises 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 A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises 1-5, 6-10, 11-15, 16-20, 21-25, or 26-27 human D _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises substantially all of human D _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises about 5, about 10, about 15, about 20, or about 25 person D _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises more than 1, more than 5, more than 10, more than 15, more than 20, or more than 25 human D _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises 27 human D _H A gene segment.

In some embodiments, the exogenous nucleic acid sequence comprises 1, 2, 3, 4, 5, or 6 person J _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 person J _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises 1-6 individuals J _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises 1-9 individuals J _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises substantially all human J _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises about 5 human J _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises about 9 human J _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises more than 1, more than 2, more than 3, more than 4, or more than 5 human J _H A gene segment. In some embodimentsIn which the exogenous nucleic acid sequence comprises more than 1, more than 2, more than 3, more than 4, more than 5, more than 6, more than 7 or more than 8 human J _H A gene segment. In some embodiments, the exogenous nucleic acid sequence comprises 6 individuals J _H A gene segment.

In some embodiments, the exogenous nucleic acid sequence comprises 65 human V _H Gene segment, 27 person D _H Gene segment and 6J _H A gene segment.

In some embodiments, the exogenous nucleic acid sequence comprises 127 person V _H Gene segment, 27 person D _H Gene segment and 9J _H A gene segment.

In some embodiments, the exogenous nucleic acid sequence comprises a barcode.

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

In another embodiment, provided herein is a method of making a genetically modified non-human animal capable of producing a humanized heavy chain antibody, comprising (a) deleting an endogenous nucleic acid sequence comprising one or more heavy chain C region genes from an endogenous immunoglobulin heavy chain locus in a non-human animal stem cell; (b) implanting stem cells into the blastula; (c) Implanting blastula into pseudopregnant mice to obtain chimeric mice; (d) Crossing the chimeric mice with wild-type mice to produce offspring; (e) screening the offspring for heterozygosity; and (f) identifying a first-established mouse carrying one or more heavy chain C region gene deletions; and wherein the non-human animal is capable of producing humanized heavy chain antibodies.

In some embodiments, the stem cell is an embryonic stem cell.

In some embodiments, the method further comprises deleting a nucleic acid sequence encoding a CH1 domain of an IgG 1C region gene. In some embodiments, the deletion of the nucleic acid sequence encoding the CHl domain of the IgGl C region gene comprises exon 1.

In some embodiments, the method further comprises retaining the native nucleic acid sequence comprising the endogenous enhancer. In some embodiments, the enhancer is E μ, 3' rr, 3' γ1e, 5' hsr1, or a combination thereof.

In some embodiments, the method further comprises retaining a native nucleic acid sequence comprising a switch tandem repeat element (sμ) and an iμ promoter, wherein iμ drives constitutive expression of the CH1 domain truncated IgG1 (igg1Δch1).

In some embodiments, the humanized heavy chain antibody is a humanized IgG1 heavy chain antibody. In some embodiments, the humanized IgG heavy chain antibody comprises a humanized IgG1 heavy chain antibody. In some embodiments, the IgG1 heavy chain antibody is an igg1Δch1 protein.

In embodiments, the genetically modified non-human animal is fertility.

In some embodiments, the genetically modified non-human animal has substantially normal B cell development and maturation.

In another embodiment, provided herein is a method of making a soluble humanized heavy chain antibody in an engineered non-human animal comprising (a) administering an antigen to the 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) confirming clonotype based on the mRNA sequence, and (f) phylogenetic analysis of the clonotype; thereby producing soluble humanized heavy chain antibodies.

In another embodiment, provided herein is a method of making a humanized single domain antibody (sdAb) identified from an engineered non-human animal comprising expressing in a cell a nucleic acid encoding a human heavy chain variable (V) comprising V, D and J _H ) Domain nucleic acid sequences, wherein the cell produces a human heavy chain variable domain, and isolating the human heavy chain variable domain from the sample, thereby producing a single domain antibody.

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.

In some embodiments, the single domain antibody lacks a light chain.

In some embodiments, the single domain antibody lacks a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

In some embodiments, the cell is a bacterial cell or a human cell.

In another aspect, described herein is a non-human animal, wherein the genome of the non-human animal comprises an immunoglobulin heavy chain (IgH) allele, wherein the IgH allele (or genome) comprises an endogenous nucleic acid encoding a CH2 or CH3 domain of an IgG subclass, wherein the IgH allele (or genome) lacks a nucleic acid encoding at least a portion of an endogenous CH1 domain of the IgG subclass, and wherein the IgH allele (or genome) lacks an endogenous nucleic acid encoding at least a portion of an IgM constant domain, an endogenous nucleic acid encoding at least a portion of an IgD constant domain, an endogenous nucleic acid encoding at least a portion of an IgE constant domain, or an endogenous nucleic acid encoding at least a portion of an IgA constant domain. The IgH allele (or genome) of the non-human animal may comprise endogenous nucleic acids encoding the CH2 domain and the CH3 domain of the IgG subclass. The IgH allele (or genome) of the non-human animal may comprise an endogenous nucleic acid encoding a hinge domain of an IgG subclass. The IgG subclass may be an IgG2 subclass. The IgG subclass may be an IgG2a, igG2b, igG2c, igG3 or IgG4 subclass. The IgG subclass may be an IgG1 subclass. The IgH allele (or genome) may lack an endogenous nucleic acid encoding at least part of an IgG2 constant domain, an endogenous nucleic acid encoding at least part of an IgG3 constant domain, or an endogenous nucleic acid encoding at least part of an IgG4 constant domain. The IgH allele (or genome) may lack endogenous nucleic acid encoding at least part of an IgG2a constant domain, endogenous nucleic acid encoding at least part of an IgG2b constant domain, endogenous nucleic acid encoding at least part of an IgG2c constant domain, endogenous nucleic acid encoding at least part of an IgG3 constant domain, and endogenous nucleic acid encoding at least part of an IgG4 constant domain. The IgH allele (or genome) lacks the endogenous nucleic acid encoding each IgG2 constant domain, the endogenous nucleic acid encoding each IgG3 constant domain, or the endogenous nucleic acid encoding each IgG4 constant domain. The IgH allele (or genome) lacks the endogenous nucleic acid encoding each IgG2a constant domain, the endogenous nucleic acid encoding each IgG2b constant domain, the endogenous nucleic acid encoding each IgG2c constant domain, the endogenous nucleic acid encoding each IgG3 constant domain, or the endogenous nucleic acid encoding each IgG4 constant domain. The IgH allele (or genome) lacks an endogenous nucleic acid encoding at least a portion of an IgM constant domain, an endogenous nucleic acid encoding at least a portion of an IgD constant domain, an endogenous nucleic acid encoding at least a portion of an IgE constant domain, and an endogenous nucleic acid encoding at least a portion of an IgA constant domain. The IgH allele (or genome) lacks the endogenous nucleic acid encoding each IgM constant domain, the endogenous nucleic acid encoding each IgD constant domain, the endogenous nucleic acid encoding each IgE constant domain, or the endogenous nucleic acid encoding each IgA constant domain. The IgH allele (or genome) may lack the endogenous nucleic acid IgH allele (or genome) encoding each IgM constant domain, and may lack the endogenous nucleic acid encoding each IgD constant domain. The IgH allele (or genome) may lack the endogenous nucleic acid encoding each IgE constant domain. The IgH allele (or genome) may lack endogenous nucleic acids encoding IgA CH1 and CH2 constant domains. The IgH allele (or genome) may lack nucleic acid encoding an endogenous CH1 domain. The IgH allele (or genome) may comprise endogenous E μ. The first nucleic acid sequence encoding an endogenous E mu downstream full length CH2 domain may be a nucleic acid encoding an IgG CH2 domain. The first nucleic acid sequence encoding the endogenous E mu downstream full length CH2 domain may be a nucleic acid encoding an IgG1CH2 domain. The 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 endogenous sμ, endogenous iμ promoter, or the downstream full length CH2 domain of the endogenous iμ exon may be a nucleic acid encoding an IgG CH2 domain. The first nucleic acid sequence encoding the endogenous sμ, endogenous iμ promoter, or the downstream full length CH2 domain of the endogenous iμ exon may be a nucleic acid encoding an IgG CH2 domain. The IgH allele (or genome) may comprise endogenous 3' γ1e. The IgH allele (or genome) may lack endogenous nucleic acid encoding the full length CH2 domain downstream of the endogenous 3' γ1e. The IgH allele (or genome) may comprise endogenous 5' γ1e. The first nucleic acid sequence encoding the full length CH2 domain upstream of the endogenous 5' hsR1 may be a nucleic acid encoding an IgG CH2 domain. The first nucleic acid sequence encoding the endogenous 5' hsr1 upstream full length CH2 domain may be a nucleic acid encoding an IgG1CH2 domain. The IgH allele (or genome) may comprise an endogenous 3' rr. The first nucleic acid sequence encoding the full length CH2 domain upstream of the endogenous 3' rr may be a nucleic acid encoding an IgG CH2 domain. The first nucleic acid sequence encoding the full length CH2 domain upstream of the endogenous 3' rr may be a nucleic acid encoding an IgG1CH2 domain. The IgH allele (or genome) may comprise an endogenous 3' cbe. The first nucleic acid sequence encoding the full length CH2 domain upstream of the endogenous 3' cbe may be a nucleic acid encoding an IgG CH2 domain. The first nucleic acid sequence encoding the full length CH2 domain upstream of the endogenous 3' cbe may be a nucleic acid encoding an IgG1CH2 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 exons of the endogenous Ig heavy chain variable region. Both alleles of the genome may lack all exons of the endogenous Ig heavy chain variable region. Neither allele of the genome can contain exogenous exons of the Ig heavy chain variable region. The non-human animal may be a non-human animal that does not produce Ig heavy chains. The IgH allele (or genome) may comprise exogenous nucleic acid encoding one or more human Ig heavy chain variable region gene segments. The IgH allele (or genome) may comprise one or more exogenous human Ig VH gene segments. The IgH allele (or genome) may comprise three or more human Ig VH gene segments. The IgH allele (or genome) may comprise 26 or more human Ig VH gene segments. The IgH allele (or genome) may comprise 65 or more human Ig VH gene segments. The IgH allele (or genome) may comprise 126 human Ig VH gene segments. The IgH allele (or genome) can comprise 13 or more human Ig VD gene segments. The IgH allele (or genome) can comprise 27 human Ig VD gene segments. The IgH allele (or genome) may comprise three or more human Ig VJ gene segments. The IgH allele (or genome) may comprise 9 Ig VJ gene segments. The genome may comprise 126 Ig VH gene segments, 27 or more Ig VD gene segments, and 9 Ig VJ gene segments. The non-human animal may produce human-non-human chimeric Ig heavy chain antibodies. The variable region domain of a human-non-human chimeric Ig heavy chain antibody can be entirely human. The IgH allele (or genome) may comprise exogenous nucleic acid encoding one or more human Ig light chain variable region gene segments. The IgH allele (or genome) may comprise one or more exogenous human igκ variable gene segments. The IgH allele (or genome) may comprise 20 or more exogenous human igκ variable gene segments. The IgH allele (or genome) may comprise 40 exogenous human igκ variable gene segments. The IgH allele (or genome) may comprise one or more exogenous human iglambda variable gene segments. The IgH allele (or genome) may comprise 10 or more exogenous human iglambda variable gene segments. The IgH allele (or genome) may comprise 20 exogenous human igλ variable gene segments. The IgH allele (or genome) may comprise one or more human igκvj gene segments. The IgH allele (or genome) may comprise five human igκvj gene segments. The IgH allele (or genome) may comprise one or more human igλvj gene segments. The IgH allele (or genome) may comprise four human igλvj gene segments. The IgH allele (or genome) may comprise 40 human igκ variable gene segments and 5 human igκvj gene segments. The IgH allele (or genome) may comprise 20 human iglambda variable gene segments and four human iglambda VJ gene segments. The non-human animal may produce human-non-human chimeric Ig heavy chain antibodies. The variable region domains of human-non-human chimeric Ig heavy chain antibodies can be entirely of human light chain origin. The non-human animal may be a first non-human species and the IgH allele (or genome) may comprise 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. The IgH allele (or genome) may comprise one or more Ig VH gene segments of the second non-human species. The IgH allele (or genome) may comprise 10 or more Ig VH gene segments of the second non-human species. The IgH allele (or genome) may comprise all Ig VH gene segments of the second non-human species. The IgH allele (or genome) can comprise three or more Ig VD gene segments of the second non-human species. The IgH allele (or genome) can comprise all Ig VD gene segments of the second non-human species. The IgH allele (or genome) may comprise three or more Ig VJ gene segments of the second non-human species. The IgH allele (or genome) may comprise all Ig VJ gene segments of the second non-human species. The IgH allele (or genome) may comprise all of the Ig VH gene segments, ig VD gene segments, and Ig VJ gene segments of the second non-human species. The non-human animal may produce chimeric heavy chain antibodies 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 species. The second species may be a bovine species, a shark species or an alpaca species. The IgH allele (or genome) may comprise at least one exogenous recombinase site recognition nucleic acid sequence. The at least one exogenous recombinase site recognition nucleic acid sequence may be located upstream of an endogenous nucleic acid encoding a CH2 or CH3 domain of the IgG subclass. The IgH allele (or genome) may comprise one, two, three, four, five, six, seven, eight, nine or ten different exogenous recombinase site recognition nucleic acid sequences. The IgH allele (or genome) may comprise at least three different exogenous recombinase site recognition nucleic acid sequences. The IgH allele (or genome) may comprise at least five different exogenous recombinase site recognition nucleic acid sequences. The different exogenous recombinase site recognition nucleic acid sequences are each located less than 2.5Mb upstream of the endogenous E. The different exogenous recombinase site recognition nucleic acid sequences are each located less than 2.0Mb, less than 1.5Mb, less than 1.0Mb, less than 500kb, or less than 250kb upstream of the endogenous E.mu. The different exogenous recombinase site recognition nucleic acid sequences are each located less than 200kb, less than 100kb, less than 50kb, less than 25kb, or less than 10kb upstream of the endogenous eμ. The different exogenous recombinase site recognition nucleic acid sequences may each be located less than 500kb upstream of the endogenous E. Different exogenous recombinase site recognition nucleic acid sequences may each be located less than 250kb upstream of the endogenous E. Different exogenous recombinase site recognition nucleic acid sequences may each be located less than 200kb upstream of the endogenous E.

In another aspect, described herein is a 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 comprising a CH1 constant domain, an IgM constant domain, an IgD constant domain, an IgE constant domain, an IgA constant domain, or any combination thereof of an IgG subclass. 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 part of one or more endogenous constant domains comprising a CH1 constant domain of the IgG subclass. The IgG subclass may comprise an IgG1, igG2a, igG2b, igG2c, igG3, or IgG4 subclass. The IgG subclass may be an IgG1 subclass. The genetically modified non-human IgH allele may comprise a nucleic acid sequence (Cγ1-. DELTA.CH1) encoding a CH1 truncated IgG1 constant domain (IgG 1-. DELTA.CH1). The genetically modified non-human IgH allele may comprise a nucleic acid sequence encoding a hinge (H) domain, a CH2 domain, a CH3 domain of an IgG subclass, or any combination thereof. 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 comprising an IgG2 constant domain, an IgG3 constant domain, an IgG4 constant domain, or any combination thereof. The genetically modified non-human IgH allele may comprise one or more endogenous enhancers comprising E μ, 3' γ1e, 5' hsr1, 3' rr, or any combination thereof. The genetically modified non-human IgH allele may comprise an I μ promoter, an I μ exon, or both. The genetically modified non-human IgH allele may comprise a switch tandem repeat element (sμ). IgG1 expression may be driven by E.mu.I.mu.promoter, S.mu.or any combination thereof. The genetically modified non-human IgH allele may lack one or more endogenous switching regions comprising sγ3, sγ1, sγ2b, sγ2c, sε, sα, or any combination thereof. The genetically modified non-human IgH allele may comprise the following composition (from 5 'to 3'): eμ, Iμ promoter, Iμ exon, Sμ, Cγ1-. DELTA.CH1, 3' γ1E, 5' hsR1 and 3' RR. The genetically modified non-human IgH allele may comprise a invertase recognition target (frt) site. The genetically modified non-human IgH allele may comprise an endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof. The genetically modified non-human IgH allele may lack at least one endogenous V gene segment, D gene segment, J gene segment, or any combination thereof. The genetically modified non-human IgH allele may comprise a docking cassette (docking cassette). The docking cassette may comprise left and right homology arms, a frt site, an attB site, a promoter, a loxP site, a nucleic acid sequence encoding a selectable marker, or any combination thereof. The docking cassette may comprise a nucleic acid sequence encoding a selectable marker. The selectable marker may comprise 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 can comprise an exogenous V gene segment, an exogenous D gene segment, an exogenous J gene segment, or any combination thereof. The exogenous gene segment may be selected from the group consisting of: human, mouse, rat, bovine, alpaca, and shark gene segments. The exogenous gene segment may comprise a human gene segment. The genetically modified non-human IgH allele may comprise 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 comprise at least 10, 20, 30, 40, 50, 60, 80, 100, 120 or 126 human VH gene segments. The genetically modified non-human IgH allele may comprise at least 10, 15, 20, 25, or 27 human DH gene segments. The genetically modified non-human IgH allele can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 human JH gene segments. The genetically modified non-human IgH allele may comprise 126 human VH gene segments, 27 human DH gene segments, and 9 human JH gene segments. The genetically modified non-human IgH allele may comprise one or more bovine gene segments. The one or more bovine gene segments may comprise the L1 exon, the L2 exon, the coding segment for IGHD8-2, the coding sequence for IGHJ2-4, the IGH2-4 splice donor, or any combination thereof, of IGHV 1-7. One or more bovine gene segments may comprise IGHD4-1, IGHD5-3, IGHD8-2, IGHD1-3, IGHD7-4, IGHD6-3, IGHD3-3, or any combination thereof. One or more of the bovine gene segments may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOS 42-49 and 57. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments. The genetically modified non-human IgH allele may comprise one or more alpaca gene segments. The one or more alpaca gene segments may comprise VHH3-1, VHH3-S2, VHH3-S9, VHH3-S10 or any combination thereof. The alpaca gene segment may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs 50-54. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments. The genetically modified non-human IgH allele may comprise one or more shark gene segments. One or more of the shark gene segments may comprise VNAR-L38968, VNAR-L38967 or both of the shark gene segments may comprise a nucleic acid sequence selected from SEQ ID NOs 55-56. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments. The genetically modified non-human IgH allele can encode an IgG heavy chain antibody, and the IgG heavy chain antibody can comprise a kappa light chain variable domain, a lambda light chain variable domain, or both. The genetically modified non-human IgH allele may comprise one or more exogenous human lambda light chain (LV) gene segments. The one or more human LV gene segments may comprise 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 comprise one or more exogenous human kappa light chain (LV) gene segments. One or more human KV gene segments may comprise CH17-272M2, CH17-405H5, CH17-140P2, CH17-13E7, CH17-84J8, CH17-53L15, or any combination thereof. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments.

In another aspect, described herein is a genetically modified cell comprising 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 part of one or more endogenous constant domains comprising a CH1 constant domain, igM constant domain, igD constant domain, igE constant domain, igA constant domain, or any combination thereof of an IgG subclass. 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 part of one or more endogenous constant domains comprising a CH1 constant domain of the IgG subclass. The IgG subclass may comprise an IgG1, igG2a, igG2b, igG2c, igG3, or IgG4 subclass. The IgG subclass may be an IgG1 subclass. The genetically modified non-human IgH allele may comprise a nucleic acid sequence (Cγ1-. DELTA.CH1) encoding a CH1 truncated IgG1 constant domain (IgG 1-. DELTA.CH1). The genetically modified non-human IgH allele may comprise a nucleic acid sequence encoding a hinge (H) domain, a CH2 domain, a CH3 domain of an IgG subclass, or any combination thereof. 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 comprising an IgG2 constant domain, an IgG3 constant domain, an IgG4 constant domain, or any combination thereof. The genetically modified non-human IgH allele may comprise one or more endogenous enhancers comprising E μ, 3' γ1e, 5' hsr1, 3' rr, or any combination thereof. The genetically modified non-human IgH allele may comprise an I μ promoter, an I μ exon, or both. The genetically modified non-human IgH allele may comprise a switch tandem repeat element (sμ). IgG1 expression may be driven by E.mu.I.mu.promoter, S.mu.or any combination thereof. The genetically modified non-human IgH allele may lack one or more endogenous switching regions comprising sγ3, sγ1, sγ2b, sγ2c, sε, sα, or any combination thereof. The genetically modified non-human IgH allele may comprise the following composition (from 5 'to 3'): eμ, Iμ promoter, Iμ exon, Sμ, Cγ1-. DELTA.CH1, 3' γ1E, 5' hsR1 and 3' RR. The genetically modified non-human IgH allele may comprise a invertase recognition target (frt) site. The genetically modified non-human IgH allele may comprise an endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof. The genetically modified non-human IgH allele may lack at least one endogenous V gene segment, D gene segment, J gene segment, or any combination thereof. The genetically modified non-human IgH allele may comprise a docking box. The docking cassette may comprise left and right homology arms, a frt site, an attB site, a promoter, a loxP site, a nucleic acid sequence encoding a selectable marker, or any combination thereof. The docking cassette may comprise a nucleic acid sequence encoding a selectable marker. The selectable marker may comprise 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 can comprise an exogenous V gene segment, an exogenous D gene segment, an exogenous J gene segment, or any combination thereof. The exogenous gene segment may be selected from the group consisting of: human, mouse, rat, bovine, alpaca, and shark gene segments. The exogenous gene segment may comprise a human gene segment. The genetically modified non-human IgH allele may comprise 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 comprise at least 10, 20, 30, 40, 50, 60, 80, 100, 120 or 126 human VH gene segments. The genetically modified non-human IgH allele may comprise at least 10, 15, 20, 25, or 27 human DH gene segments. The genetically modified non-human IgH allele can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 human JH gene segments. The genetically modified non-human IgH allele may comprise 126 human VH gene segments, 27 human DH gene segments, and 9 human JH gene segments. The genetically modified non-human IgH allele may comprise one or more bovine gene segments. The one or more bovine gene segments may comprise the L1 exon, the L2 exon, the coding segment for IGHD8-2, the coding sequence for IGHJ2-4, the IGH2-4 splice donor, or any combination thereof, of IGHV 1-7. One or more bovine gene segments may comprise IGHD4-1, IGHD5-3, IGHD8-2, IGHD1-3, IGHD7-4, IGHD6-3, IGHD3-3, or any combination thereof. One or more of the bovine gene segments may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOS 42-49 and 57. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments. The genetically modified non-human IgH allele may comprise one or more alpaca gene segments. The one or more alpaca gene segments may comprise VHH3-1, VHH3-S2, VHH3-S9, VHH3-S10 or any combination thereof. The alpaca gene segment may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs 50-54. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments. The genetically modified non-human IgH allele may comprise one or more shark gene segments. One or more of the shark gene segments may comprise VNAR-L38968, VNAR-L38967 or both of the shark gene segments may comprise a nucleic acid sequence selected from SEQ ID NOs 55-56. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments. The genetically modified non-human IgH allele can encode an IgG heavy chain antibody, and the IgG heavy chain antibody can comprise a kappa light chain variable domain, a lambda light chain variable domain, or both. The genetically modified non-human IgH allele may comprise one or more exogenous human lambda light chain (LV) gene segments. The one or more human LV gene segments may comprise 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 comprise one or more exogenous human kappa light chain (LV) gene segments. One or more human KV gene segments may comprise CH17-272M2, CH17-405H5, CH17-140P2, CH17-13E7, CH17-84J8, CH17-53L15, or any combination thereof. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments. The cells may be non-human animal cells. The cell may be a mammalian cell. The mammalian cell may be a mouse, rat, bovine, alpaca, cat, dog, rabbit, pig, monkey or chimpanzee cell. The cells may be mouse cells. The cells may be shark cells. The cells may be human cells. The cells may be stem cells. The stem cells may be Embryonic Stem Cells (ESCs) or Induced Pluripotent Stem Cells (iPSCs). The cells may be B cells.

In another aspect, described herein is a genetically modified non-human animal, wherein the genetically modified non-human animal comprises the cells described in the preceding paragraph. The non-human animal may be a mammal. The mammal may be a mouse, rat, cow, alpaca, cat, dog, rabbit, pig, monkey or chimpanzee. The non-human animal may be a mouse. The genetically modified non-human animal may comprise cells expressing IgG heavy chain antibodies. IgG heavy chain antibodies can be secreted into the serum of genetically modified non-human animals. The IgG heavy chain antibody may be a CH1 truncated IgG1 heavy chain antibody (igg1Δch1). IgG heavy chain antibodies may lack light chains. The IgG heavy chain antibody may comprise a hinge domain, a CH2 domain, a CH3 domain, or any combination thereof. The cells expressing the IgG heavy chain antibodies may be cells not expressing IgM antibodies, igD antibodies, igE antibodies, igG3 antibodies, igG2b antibodies, igG2c antibodies, igA antibodies, or any combination thereof. The IgG heavy chain antibody may be a human IgG heavy chain antibody. IgG heavy chain antibodies may comprise exogenous variable domains selected from the group consisting of: human, mouse, rat, bovine, alpaca, and shark variable domains. IgG heavy chain antibodies may comprise a kappa light chain variable domain, a lambda light chain variable domain, or both.

In another aspect, described herein is a method of making a genetically modified non-human animal. The method comprises (a) deleting one or more nucleic acid sequences from a non-human immunoglobulin heavy chain (IgH) allele, wherein the deleted one or more nucleic acid sequences encode at least a portion of one or more endogenous constant domains comprising a CH1 constant domain, igM constant domain, igD constant domain, igE constant domain, igA constant domain, or any combination thereof of an IgG subclass, thereby generating a genetically modified non-human IgH allele in germline genomic DNA; (b) Implanting cells comprising germline genomic DNA into a blastocyst; (c) Implanting the blastocyst into a pseudo-pregnant non-human animal to obtain a chimeric non-human animal; (d) Crossing the chimeric non-human animal with a wild-type non-human animal to produce offspring; (e) screening the offspring for heterozygosity; (f) A genetically modified non-human animal carrying a deletion of one or more nucleic acid sequences and capable of producing heavy chain antibodies is identified. The genetically modified non-human animal may be a genetically modified non-human animal as described in the preceding paragraph. Deleting one or more sequences may include using a CRISPR/Cas genome editing system. The CRISPR/Cas genome editing system can comprise at least one guide RNA (gRNA) targeting an endogenous heavy chain C region gene and a Cas protein. The Cas protein may comprise a Cas9 protein. The deleted one or more nucleic acid sequences may encode a CH1 constant domain, an IgG3 constant domain, an IgM constant domain, and an IgD constant domain of IgG 1. The deleted one or more nucleic acid sequences may encode an IgG2 constant domain and an IgA constant domain. Deletion of the nucleic acid sequence may include removal of the selectable marker from the non-human IgH allele using transient expression of the Flp recombinase. The deleted one or more nucleic acid sequences may encode a CH1 constant domain, an IgM constant domain, an IgD constant domain, an IgE constant domain, and an IgA constant domain of the IgG subclass. The method may comprise deleting a nucleic acid sequence from the non-human IgH allele, wherein the nucleic acid sequence comprises Containing endogenous V gene segments, D gene segments, J gene segments, or any combination thereof. The method may include inserting a docking box. The method may comprise contacting the docking cassette with a Bacterial Artificial Chromosome (BAC), wherein the BAC comprises a polypeptide comprising an exogenous V _H 、D _H And J _H Nucleic acid sequence of the gene segment. The method may comprise inserting the exogenous gene segment into a docking box. The exogenous gene segment may comprise a human gene segment.

In another aspect, described herein is a genetically modified non-human animal, wherein the genetically modified non-human animal is prepared using the method described in the preceding paragraph.

In another aspect, described herein is a method for preparing germline genomic DNA, wherein the method comprises deleting one or more nucleic acid sequences from a non-human immunoglobulin heavy chain (IgH) allele, wherein the deleted one or more nucleic acid sequences encode at least a portion of one or more endogenous constant domains (comprising a CH1 constant domain, igM constant domain, igD constant domain, igE constant domain, igA constant domain, or any combination thereof of an IgG subclass) to thereby produce a hereditary constant domain. The germline genomic DNA may comprise a DNA genetically modified non-human IgH allele comprising a genetically modified non-human IgH allele may lack one or more nucleic acid sequences encoding at least part of one or more endogenous constant domains comprising a CH1 constant domain of an IgG subclass. The IgG subclass may comprise an IgG1, igG2a, igG2b, igG2c, igG3, or IgG4 subclass. The IgG subclass may be an IgG1 subclass. The genetically modified non-human IgH allele may comprise a nucleic acid sequence (Cγ1-. DELTA.CH1) encoding a CH1 truncated IgG1 constant domain (IgG 1-. DELTA.CH1). The genetically modified non-human IgH allele may comprise a nucleic acid sequence encoding a hinge (H) domain, a CH2 domain, a CH3 domain of an IgG subclass, or any combination thereof. 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 comprising an IgG2 constant domain, an IgG3 constant domain, an IgG4 constant domain, or any combination thereof. The genetically modified non-human IgH allele may comprise one or more endogenous enhancers comprising E μ, 3' γ1e, 5' hsr1, 3' rr, or any combination thereof. The genetically modified non-human IgH allele may comprise an I μ promoter, an I μ exon, or both. The genetically modified non-human IgH allele may comprise a switch tandem repeat element (sμ). IgG1 expression may be driven by E.mu.I.mu.promoter, S.mu.or any combination thereof. The genetically modified non-human IgH allele may lack one or more endogenous switching regions comprising sγ3, sγ1, sγ2b, sγ2c, sε, sα, or any combination thereof. The genetically modified non-human IgH allele may comprise the following composition (from 5 'to 3'): eμ, Iμ promoter, Iμ exon, Sμ, Cγ1-. DELTA.CH1, 3' γ1E, 5' hsR1 and 3' RR. The genetically modified non-human IgH allele may comprise a invertase recognition target (frt) site. The genetically modified non-human IgH allele may comprise an endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof. The genetically modified non-human IgH allele may lack at least one endogenous V gene segment, D gene segment, J gene segment, or any combination thereof. The genetically modified non-human IgH allele may comprise a docking box. The docking cassette may comprise left and right homology arms, a frt site, an attB site, a promoter, a loxP site, a nucleic acid sequence encoding a selectable marker, or any combination thereof. The docking cassette may comprise a nucleic acid sequence encoding a selectable marker. The selectable marker may comprise 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 can comprise an exogenous V gene segment, an exogenous D gene segment, an exogenous J gene segment, or any combination thereof. The exogenous gene segment may be selected from the group consisting of: human, mouse, rat, bovine, alpaca, and shark gene segments. The exogenous gene segment may comprise a human gene segment. The genetically modified non-human IgH allele may comprise 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 comprise at least 10, 20, 30, 40, 50, 60, 80, 100, 120 or 126 human VH gene segments. The genetically modified non-human IgH allele may comprise at least 10, 15, 20, 25, or 27 human DH gene segments. The genetically modified non-human IgH allele can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 human JH gene segments. The genetically modified non-human IgH allele may comprise 126 VH gene segments, 27 DH gene segments, and 9 JH gene segments. The genetically modified non-human IgH allele may comprise one or more bovine gene segments. The one or more bovine gene segments may comprise the L1 exon, the L2 exon, the coding segment for IGHD8-2, the coding sequence for IGHJ2-4, the IGH2-4 splice donor, or any combination thereof, of IGHV 1-7. One or more bovine gene segments may comprise IGHD4-1, IGHD5-3, IGHD8-2, IGHD1-3, IGHD7-4, IGHD6-3, IGHD3-3, or any combination thereof. One or more of the bovine gene segments may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOS 42-49 and 57. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments. The genetically modified non-human IgH allele may comprise one or more alpaca gene segments. The one or more alpaca gene segments may comprise VHH3-1, VHH3-S2, VHH3-S9, VHH3-S10 or any combination thereof. The alpaca gene segment may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs 50-54. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments. The genetically modified non-human IgH allele may comprise one or more shark gene segments. One or more of the shark gene segments may comprise VNAR-L38968, VNAR-L38967 or both of the shark gene segments may comprise a nucleic acid sequence selected from SEQ ID NOs 55-56. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments. The genetically modified non-human IgH allele can encode an IgG heavy chain antibody, and the IgG heavy chain antibody can comprise a kappa light chain variable domain, a lambda light chain variable domain, or both. The genetically modified non-human IgH allele may comprise one or more exogenous human lambda light chain (LV) gene segments. The one or more human LV gene segments may comprise 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 comprise one or more exogenous human kappa light chain (LV) gene segments. One or more human KV gene segments may comprise CH17-272M2, CH17-405H5, CH17-140P2, CH17-13E7, CH17-84J8, CH17-53L15, or any combination thereof. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments. The IgG constant domain may comprise a constant domain of the IgG subclass. The IgG subclass may comprise an IgG1, igG2a, igG2b, igG2c, igG3, or IgG4 subclass.

In another aspect, described herein is a method of producing IgG heavy chain antibodies in a genetically modified non-human animal. The method comprises (a) administering an antigen to the genetically modified non-human animal of any one of the preceding paragraphs; (b) Isolating one or more B cells from the genetically modified non-human animal; (c) isolating mRNA from one or more B cells; (d) producing IgG heavy chain antibodies. The genetically modified non-human animal may comprise 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 comprising a CH1 constant domain, an IgM constant domain, an IgD constant domain, an IgE constant domain, an IgA constant domain, or any combination thereof of an IgG subclass. 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 part of one or more endogenous constant domains comprising a CH1 constant domain of the IgG subclass. The IgG subclass may comprise an IgG1, igG2a, igG2b, igG2c, igG3, or IgG4 subclass. The IgG subclass may be an IgG1 subclass. The genetically modified non-human IgH allele may comprise a nucleic acid sequence (Cγ1-. DELTA.CH1) encoding a CH1 truncated IgG1 constant domain (IgG 1-. DELTA.CH1). The genetically modified non-human IgH allele may comprise a nucleic acid sequence encoding a hinge (H) domain, a CH2 domain, a CH3 domain of an IgG subclass, or any combination thereof. 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 comprising an IgG2 constant domain, an IgG3 constant domain, an IgG4 constant domain, or any combination thereof. The genetically modified non-human IgH allele may comprise one or more endogenous enhancers comprising E μ, 3' γ1e, 5' hsr1, 3' rr, or any combination thereof. The genetically modified non-human IgH allele may comprise an I μ promoter, an I μ exon, or both. The genetically modified non-human IgH allele may comprise a switch tandem repeat element (sμ). IgG1 expression may be driven by E.mu.I.mu.promoter, S.mu.or any combination thereof. The genetically modified non-human IgH allele may lack one or more endogenous switching regions comprising sγ3, sγ1, sγ2b, sγ2c, sε, sα, or any combination thereof. The genetically modified non-human IgH allele may comprise the following composition (from 5 'to 3'): eμ, Iμ promoter, Iμ exon, Sμ, Cγ1-. DELTA.CH1, 3' γ1E, 5' hsR1 and 3' RR. The genetically modified non-human IgH allele may comprise a invertase recognition target (frt) site. The genetically modified non-human IgH allele may comprise an endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof. The genetically modified non-human IgH allele may lack at least one endogenous V gene segment, D gene segment, J gene segment, or any combination thereof. The genetically modified non-human IgH allele may comprise a docking box. The docking cassette may comprise left and right homology arms, a frt site, an attB site, a promoter, a loxP site, a nucleic acid sequence encoding a selectable marker, or any combination thereof. The docking cassette may comprise a nucleic acid sequence encoding a selectable marker. The selectable marker may comprise 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 can comprise an exogenous V gene segment, an exogenous D gene segment, an exogenous J gene segment, or any combination thereof. The exogenous gene segment may be selected from the group consisting of: human, mouse, rat, bovine, alpaca, and shark gene segments. The exogenous gene segment may comprise a human gene segment. The genetically modified non-human IgH allele may comprise 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 comprise at least 10, 20, 30, 40, 50, 60, 80, 100, 120 or 126 human VH gene segments. The genetically modified non-human IgH allele may comprise at least 10, 15, 20, 25, or 27 human DH gene segments. The genetically modified non-human IgH allele can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 human JH gene segments. The genetically modified non-human IgH allele may comprise 126 VH gene segments, 27 DH gene segments, and 9 JH gene segments. The genetically modified non-human IgH allele may comprise one or more bovine gene segments. The one or more bovine gene segments may comprise the L1 exon, the L2 exon, the coding segment for IGHD8-2, the coding sequence for IGHJ2-4, the IGH2-4 splice donor, or any combination thereof, of IGHV 1-7. One or more bovine gene segments may comprise IGHD4-1, IGHD5-3, IGHD8-2, IGHD1-3, IGHD7-4, IGHD6-3, IGHD3-3, or any combination thereof. One or more of the bovine gene segments may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOS 42-49 and 57. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments. The genetically modified non-human IgH allele may comprise one or more alpaca gene segments. The one or more alpaca gene segments may comprise VHH3-1, VHH3-S2, VHH3-S9, VHH3-S10 or any combination thereof. The alpaca gene segment may comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs 50-54. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments. The genetically modified non-human IgH allele may comprise one or more shark gene segments. One or more of the shark gene segments may comprise VNAR-L38968, VNAR-L38967 or both of the shark gene segments may comprise a nucleic acid sequence selected from SEQ ID NOs 55-56. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments. The genetically modified non-human IgH allele can encode an IgG heavy chain antibody, and the IgG heavy chain antibody can comprise a kappa light chain variable domain, a lambda light chain variable domain, or both. The genetically modified non-human IgH allele may comprise one or more exogenous human lambda light chain (LV) gene segments. The one or more human LV gene segments may comprise 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 comprise one or more exogenous human kappa light chain (LV) gene segments. One or more human KV gene segments may comprise CH17-272M2, CH17-405H5, CH17-140P2, CH17-13E7, CH17-84J8, CH17-53L15, or any combination thereof. The DNA may comprise one or more human VH gene segments. The DNA may comprise one or more human JH gene segments. The method may comprise sequencing mRNA isolated from one or more B cells. The method may include confirming clonotypes based on mRNA sequences. The method may comprise performing phylogenetic analysis of the clonotypes. 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 comprising a human variable region and a non-human constant region.

In another aspect, described herein are IgG heavy chain antibodies, wherein the IgG heavy chain antibodies are produced by the methods described in the preceding paragraphs.

In another aspect, described herein is 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 capable of deleting one or more nucleic acid sequences from a non-human immunoglobulin heavy chain (IgH) allele, wherein the deleted one or more nucleic acid sequences encode at least a portion of one or more endogenous constant domains comprising CHl constant domains, igM constant domains, igD constant domains, igE constant domains, igA constant domains, or any combination thereof of an IgG subclass.

In another aspect, described herein are antibodies comprising a variable region comprising (a) SEQ ID NO. 4, SEQ ID NO. 10, and SEQ ID NO. 19, or (b) SEQ ID NO. 5, SEQ ID NO. 19. 11, and SEQ ID NO. 20. The antibody can bind to SARS-CoV2 spike polypeptide. The antibody may be a heavy chain antibody. The antibody may be a single domain antibody.

In another aspect, described herein are antibodies comprising a variable region comprising (a) SEQ ID NO. 4, SEQ ID NO. 10, and SEQ ID NO. 19, but SEQ ID NO. 19 lacks the first C residue and the last W residue, or (b) SEQ ID NO. 5, SEQ ID NO. 11, and SEQ ID NO. 20, but SEQ ID NO. 20 lacks the first C residue and the last W residue. The antibody can bind to SARS-CoV2 spike polypeptide. The antibody may be a heavy chain antibody. The antibody may be a single domain antibody.

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

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Drawings

FIGS. 1A-1D show the production of heavy chain-only antibodies from an odd murine line (Singularity Musculus) mouse. (FIG. 1A) genomic structure of Igh locus of wild-type mice. Mouse V _H 、D _H 、J _H And C _H Genes are represented by dark or light boxes, with the intronic enhancers Eμ and super enhancer 3' RR in ovals. (FIG. 1B) an engineered odd murine (SM) allele in which all other CH genes are deleted, as well as the CH1 exon of IgG1. (FIG. 1C) tetrameric mouse IgG1 produced by WT allele. (FIG. 1D) IgG1, which has only CH1 truncated heavy chains, was generated from the odd murine allele from which nanobodies can be derived.

FIG. 2 illustrates an exemplary genomic locus for mouse Igh. The expression cassette shows 220kb C containing the regulatory elements shown _H Schematic representation of the region.

FIGS. 3A-3E illustrate the generation of an odd murine allele. FIG. 3A shows the wild-type Igh locus of the mouse. FIG. 3B shows the constant region of the mouse Igh locus. The area to be removed in the first round of engineering is shown as a dashed box. FIG. 3C shows deletion of Ighm-Ighg1 CH1 exons by CRISPR mediated NHEJ. The area to be removed in the second round of engineering is shown as a dashed box. FIG. 3D shows removal of Ighg2b-Igha exons 1-3 by CRISPR mediated HDR. FIG. 3E shows the removal of the selection cassette by expression of Flp recombinase.

Fig. 4 shows an exemplary genomic structure of a wild-type mouse Igh allele and an engineered odd murine line and an odd hyper-berth allele.

FIGS. 5A-5D illustrate the generation of an odd murine allele. Figure 5A shows the odd murine allele. (FIG. 5B) by removing all mice V _H 、D _H 、J _H Gene (2.58 Mb) and inserts a poise cassette for sequential RMCE via CRISPR mediated HDR to generate an odd hyper-poise allele. FIG. 5C shows the identity (Synteny) verification of the odd-superposition allele by expression of Flp recombinase. FIG. 5D shows the removal of the selectable marker by expression of the ΦC31 recombinase.

Fig. 6A-6B illustrate the generation of human-mouse only chimeric heavy chain antibodies from exemplary human (Singularity Sapiens) mice. FIG. 6A is a schematic representation of an exemplary human VH-mouse IgG 1-. DELTA.CH1 chimeric antibody that can be used to generate human VH nanobodies. Fig. 6B shows an exemplary version of an odd human mouse generated by sequential introduction of the human V, D, J gene into an odd hyper-berth (Singularity HyperDock) allele. Human VH nanobodies can be derived from odd human mice by generating only human VH-CH1 truncated heavy chain IgG 1.

FIGS. 7A-7D show the generation of an odd human allele series (SSVs 1-3). The odd human allele series was generated by using a series of bispecific lox sites, inserting engineered human IGH BAC1-3 via sequential RMCE, and exchanging the alternative selection cassettes (neo and hyg) when expressing Cre recombinase.

FIGS. 8A-8D are schematic representations of the odd human allele series (SSV 4-5) showing the sequential integration of human IGH-BAC4 and IGH-BAC5 by RMCE into clones containing human IGH-BAC1, human IGH-BAC2 and human IGH-BAC3, followed by removal of the selectable marker cassette by expression of the ΦC31 recombinase.

FIG. 9 shows human IGH BAC based on human genome GRCh38/hg38 assembly GENCODE gene locus (GENCODE Genes Track) (36 th edition, 10 month 2020), showing variable heavy chain (IGHV), diverse heavy chain (IGHD), human gene fragment linked to heavy chain (IGHJ), and constant weight IGHM and IGHD. Five BAC constructs (hIGH BAC1-5, border marked with dotted boxes) carrying human IGHV, IGHD and IGHJ gene fragments were engineered by recombinant engineering (recombination) with the corresponding source BAC (solid boxes). The entire human V-D-J genomic region was then reconstructed by RMCE into an odd superposition allele using engineered BACs as described herein. The number of V, D and J gene fragments contained in each engineered BAC construct is indicated.

FIG. 10 shows an example of BAC recombination engineering. The source BAC is modified by bacterial homologous recombination (recombinant engineering) to incorporate appropriate selectable markers and recombination sites at the desired locations. Shown is the engineering process of hIgH-BAC 1.

FIG. 11 shows VH exon verification, which shows PCR-based verification of an odd human line (SSV 4) containing 37 functional human VH exons integrated at the IGH locus. The PCR results were run on a Qiagen Qiaxel DNA high resolution kit. The top and bottom bands represent the Qiagen QX alignment marker 15bp/3kb (catalog number 929522), which runs with the Qiagen QX size marker 100bp-2.5kb (catalog number 929559). PCR products were verified by sanger sequencing to match the corresponding VH genes.

FIG. 12 illustrates an exemplary method of composite BAC recombination engineering. The source BACs are sequentially modified by bacterial homologous recombination (recombinant engineering) to incorporate appropriate selectable markers and recombination sites at the desired locations. Examples of engineering of hIGH-BAC5 from three sources of BAC are shown.

Figures 13A-13B show engineering of mutant mice lacking kappa light chains. Fig. 13A is a schematic showing HDR insertion docking sites mediated by CRISPR and deletion of mouse IG kappa. FIG. 13B is a genotyping PCR result confirming the IGK hyper-berth/KO allele in F1 mice.

Fig. 14A-14B show engineering of mutant mice lacking lambda light chains. Fig. 14A is a schematic showing deletion of the entire mouse IG lambda locus by CRISPR-mediated NHEJ. FIG. 14B shows the result of PCR for confirming the production of IGL KO alleles in ES cells.

Figures 15A-15D show that the odd murine mice produced hcabs of only CH1 truncated IgG1. Schematic representation of Igh loci in WT (fig. 15A) and SM (fig. 15B) mice. Verification of the odd murine mice was shown by RT-PCR (spleen) (fig. 15C) and Western blot (plasma) (fig. 15D).

FIGS. 16A-16D show that the odd human mice produce human-mouse chimeric heavy chain IgG1. Figure 16A shows the odd murine allele. FIG. 16B shows an odd human V1 allele, which contains all of human J _H Owner D _H And 3 people V _H And (3) a gene. (FIG. 16C) RT-PCR shows the specific expression of human-mouse chimeric IgG1. DELTA.CH1 transcripts in the odd human V1 mice. FIG. 16D shows that sequencing verifies the production of human-mouse chimeric transcripts (SEQ ID NO: 36).

Fig. 17A-17B. FIG. 17A is a schematic diagram of an exemplary human VH-mouse IgG 1-. DELTA.CH1 chimeric antibody that can be used to generate human VH nanobodies. FIG. 17B shows Western blots of IgM and IgG1 in immunized WT and odd human mice (SSV 1).

FIGS. 18A-18B show spleen morphology and IgM and IgG expression in B cells of an odd murine mouse. Fig. 18A shows spleens of wild-type and odd murine mice. Fig. 18B shows flow cytometry analysis of spleen cells, which indicated that IgM was absent but normal IgG expression was present in CD19 positive B cells.

Figures 19A-19B show B cell markers in an odd human mouse. FIG. 19A shows a flow cytometry analysis, demonstrating IgM ⁺ IgD ⁺ B cells are present in wild-type mice but not in the odd human line mice (SSV 2). FIG. 19B shows a flow cytometry analysis demonstrating IgG1 in wild-type mice and in odd-type human mice (SSV 2) ⁺ B cell abundance differences.

Figures 20A-20B show that the odd murine mice developed a strong humoral immune response following antigen challenge. Fig. 20A shows ELISA results from plasma samples of wild-type and odd murine animals prior to blood collection. FIG. 20B shows ELISA results for plasma samples collected at the end of day 28 of the same animals immunized with SARS-CoV2 spike activity trimeric protein (SAT) as compared to a commercially available control antibody against the S1 subunit of SARS-CoV2 spike protein (S1 mAb control).

Figures 21A-21B show that both the odd murine and the odd human mice develop a strong humoral immune response following challenge with multiple antigens. FIG. 21A shows ELISA results of wild-type (WT), odd murine (SM) and odd human (SSV 1) animals on end-of-day 51-day blood plasma samples after challenge with SAT antigen. FIG. 21B shows ELISA results from end-of-day blood plasma samples from animals immunized with human PD-L1 compared to commercially available human PD-L1 antibodies.

FIG. 22 shows a schematic representation of the primer positions for 5' RACE amplification of WT and odd murine transcripts.

Figures 23A-23C show that the odd murine mice exhibited antibody diversity comparable to wild type mice. Showing V _H Diversity (fig. 23A); j (J) _H Diversity (fig. 23B); and CDR3 length diversity of all clonotypes confirmed from two wild-type and two odd murine mice immunized with SAT (fig. 23C).

FIGS. 24A-24C show IGHV diversity for clonotypes confirmed in WT and SM mice. FIGS. 24A and B show IGHV use in SM mice immunized with the indicated antigens. (FIG. 24C) SM mice had more accessible IGHV segments than WT mice.

FIGS. 25A-25B show IGHJ utilization in WT and SM mice. FIG. 25A shows IGHJ use in SM mice immunized with the indicated antigens. (FIG. 25B) a different use of certain IGHJ segments in SM mice compared to WT mice was observed.

FIGS. 26A-26B show CDR3 length distributions in WT and SM mice. FIG. 26A shows the distribution of CDR3 lengths in clonotypes of SM mice and WT mice in response to the antigen. Fig. 26B shows the average CDR3 lengths observed in SM and WT mice.

Figure 27 shows somatic hypermutations in the odd murine mice. The histogram shows the top 100 most abundant confirmed from one naive mouse and three curious murine mice immunized with SAT compared to the corresponding germline sequences The number of amino acid changes at each position of the heavy chain variable region of the nanobody clonotype. V (V) _H Residue position numbering is based on IMGT scheme. The most significant changes occur in the CDR regions.

Figure 28 shows a flow chart of an exemplary NGS-directed, single-cell independent nanobody discovery process.

Figure 29 shows a phylogenetic tree of selected clonotypes confirmed by next generation sequencing of HcAb libraries of odd murine mice immunized with SAT. The top (abundance-based) clonotypes are selected for each immunized animal, high-throughput synthesis, cloning, expression and ELISA screening for antigen affinity is performed, and then competitive ELISA is performed to detect inhibitor (neutralization) activity of spike ACE2 receptor binding. Antigen-specific clones are shaded in grey and neutralizing clones are indicated with asterisks.

FIGS. 30A-30B illustrate vectors for expression of nanobodies. FIG. 30A shows a plasmid map of the pFUSE-hIgG1-Fc2 expression vector and restriction sites (EcoRI and NcoI) for insertion into the VH sequence. FIG. 30B shows an exemplary Nb-human Fc fusion that can be generated from a pFUSE-hIgG1-Fc2 expression vector.

Figure 31 illustrates ELISA screening for binders in immunized WT and SM mice. The clonotypes screened and the number of confirmed binders from WT and SM mice after immunization with the indicated antigens are provided. Provides binding results for each clonotype (ELISA results OD ₄₅₀ )。

Figure 32 contains a pie chart from the data in figure 31 showing the proportion of binders with indicated binding affinities obtained from WT and SM mice. Each graph shows the proportion of binders bound by nanobody to the indicated antigen as determined by ELISA.

FIGS. 33A-33B show exemplary antibody structures of WT IgGl and Nb-human Fc fusions under unreduced and reduced conditions (FIG. 33A), and size reduction was confirmed with S1 mAb control (WT tetramer IgGl) and purified SAT nanobody-Fc fusions (heavy chain IgG1 only) (FIG. 33B). The expressed Nb-Fc human fusion is homodimer.

FIGS. 34A-34B show SDS-PAGE gels of purified SAT human nanobody-human Fc fusions (heavy chain IgG1 only). FIG. 34A shows gel run under non-reducing conditions. Fig. 34B shows the gel run under reducing conditions. Expressed human Nb-human Fc fusion was observed to be homodimeric.

Figure 35 shows size exclusion chromatography of two human nanobody-human Fc fusion proteins. Purified human Nb-human Fc fusion proteins against SAT antigens were passed through size exclusion columns to assess protein aggregation.

FIGS. 36A-36B show characterization of purified SAT Nb-human Fc fusion for antigen binding affinity and SARS-CoV2 neutralization potency against RBD Nb-Fc control (HAb 8-S). FIG. 36A shows binding affinity EC ₅₀ Values. FIG. 36B shows the neutralizing effect of IC ₅₀ Values.

FIGS. 37A-37B show the phylogenetic relationship (FIG. 37A) and somatic hypermutation analysis (FIG. 37B) of closely related VH sequences confirmed using two SARS-CoV2 neutralizing nanobodies (indicated by asterisks). Closely related low abundance clonotypes were identified for secondary screening of high affinity and high potency nanobodies. The sequences of the nanobody clones of FIG. 37B from top to bottom are shown in SEQ ID NOS 25-35, respectively.

FIG. 38 is a table presenting the binding kinetics of mouse and human SAT nanobody-human Fc fusion molecules. Binding of the mouse and human Nb-human Fc fusion proteins to the recombinant SAT proteins was determined by Biological Layer Interferometry (BLI) using Octet. These results demonstrate that the engineered mice provided herein can be used to obtain high affinity mouse nanobodies and high affinity human nanobodies.

Fig. 39 shows the binding kinetics of an exemplary human nanobody-human Fc fusion molecule. A sensorgram of purified human Nb-human Fc fusion protein was obtained by BLI in the presence of recombinant SAT.

FIG. 40 illustrates melting peaks of human nanobody-human Fc fusion molecules. Melting curves of purified human SAT Nb-human Fc fusion proteins were generated in an Expi293F cell by pFUSE-hIgG1-Fc2 expression vector. These results indicate that the human Nb-human Fc molecules can exhibit similar thermostability as known natural nanobodies.

FIGS. 41A-41B show cell binding assays for mouse nanobody-human Fc fusion molecules and human nanobody-human Fc fusion molecules. FIG. 41A is a graph showing exemplary results of HEK293 (upper panel) or positive and negative controls of HEK293 expressing SARS-Cov2 spike protein incubated in the presence of purified mouse or human Nb-human Fc fusion protein. Cell binding was assessed using a fluorescent secondary antibody directed against the Fc region of the Nb-Fc molecule. FIG. 41B shows the summary of cell binding data for mouse and human Nb-human Fc fusion proteins.

FIG. 42 contains graphs showing the cell binding results of all Nb-human Fc fusions of FIGS. 41A-41B. Upper panel, mouse Nb-human Fc; the lower panel, human Nb-human Fc.

43A-43B show exemplary constructs of an odd human-L allele series designed to include a human VL segment. FIG. 43A shows RAG1/RAG2 mediated recombination signal sequences of 12RSS (12 nt spacer) and 23RSS (23 nt spacer) associated with variable segments at human loci of IGH, IGK (kappa) and IGL (lambda). (FIG. 43B) will contain the owner D _H And J _H The segment's idiosyncratic human DJ docking allele serves as a platform to integrate a series of BACs comprising human variable light chain segments from the human IG lambda locus of chromosome 22 by sequential RCME. The resulting allele containing the odd-numbered human VL can be produced to contain the same amino acid sequence as human D _H Segment and person J _H Human variable light chain segments adjacent to the segment are followed by antibodies to a mouse constant region (e.g., a mouse igg1Δch1 region).

FIGS. 44A-44B show exemplary constructions of two different sets of the odd human-K allele series expressing human VK segments. FIG. 44A is a schematic diagram showing that an odd superposition allele can be used as a platform to integrate a series of hIGKVJ BACs by sequential RCME, comprising human VK and JK fragments from the human IGK locus of chromosome 2. The resulting allele containing the odd human VK-JK can produce antibodies containing a human variable kappa segment adjacent to a human kappa J segment, followed by a mouse constant region (e.g., a mouse igg1Δch1 region). FIG. 44B is a schematic diagram showing that an odd human allele containing all human JH segments can be used as a platform to integrate a series of engineered hIGKV-BACs containing human VK segments from the human IGκ locus of chromosome 2 by sequential RCME. The obtained product contains the odd human series VK-JHAlleles can produce antibodies containing antibodies to human J _H Human variable kappa segments adjacent to the segment are followed by a mouse constant region (e.g., a mouse igg1Δch1 region).

45A-45C illustrate an exemplary engineered design of an odd long horn bovine (Longhorn). FIG. 45A is a schematic representation of a genetic construct (bovine longhorn VDJ) comprising a synthetically assembled bovine DNA sequence (Bos Taurus) comprising a promoter, a 5' UTR segment, an L1 exon, an intron, an L2 exon of IGHV1-7, a coding segment of IGHD8-2, a coding sequence of IGHJ2-4, and an IGH2-4 splice donor. FIG. 45B is a schematic showing the synthetic construct flanked by different loxP elements and hygromycin selection markers. This construct was integrated into the Igh locus of the odd superpositioned allele by RCME to create the odd long horn bovine allele. FIG. 45C shows PCR confirmation of mice bearing the odd-horned calf allele.

Fig. 46 shows an exemplary engineering design of the curiosity Mi Nuotao (Minotaur). A schematic representation of a genetic construct (Mi Nuotao DH array) is shown comprising a synthetically assembled DNA sequence comprising bovine DH segments (e.g., the 8 longest cow IGVD shown in boxes). To ensure that VDJ recombination occurs, upstream and downstream sequences of the original human IGVD containing the 12RSS signal (labeled below the corresponding bovine IGVD, respectively) are also included. The synthetic construct (Mi Nuotao DH array) can be targeted for integration into the Igh locus comprising any suitable number (or all) of the human VH, all human DH, and the odd human allele of all human JH (e.g., SSV 5) by, for example, CRISPR/Cas9, thereby replacing the human IGVD locus with a synthetic Mi Nuotao DH array.

Fig. 47A-47B illustrate exemplary engineering designs for the sapaco (Sapacos). FIG. 47A is a graph containing 5V of alpaca (Vicugna pacos) _H Schematic representation of genetic constructs of H (saparaceae VHH array) designed to use human VH elements as genetic scaffold. Subjects V _H The H element is grafted onto a framework of a selective human VH comprising an upstream promoter (e.g.250 bp upstream promoter), a leader exon 1, an intron, a leader exon 2, comprising regulatory elements (e.g.TATA box, octamer and heptamer) And recombinant signal sequences (e.g., 23 RSS) figure 47B shows that synthetic constructs (saparate VHH array) containing flanking different lox elements and selectable markers can be integrated by RMCE into the Igh locus containing the odd human alleles of all human VD and VJ elements.

Fig. 48A-48B illustrate exemplary engineering designs of the odd savland. Fig. 48A is a schematic of a genetic construct (savland VNAR array) comprising two germline VNARs from nurse sharks, designed to use human VH elements as genetic scaffolds. Individual VNAR elements are transplanted onto a framework of a selective human VH, which framework includes an upstream promoter (e.g., 250bp upstream promoter) containing regulatory elements (e.g., TATA boxes, octamers, and heptamers), a leader exon 1, an intron, a leader exon 2, and a recombination signal sequence (e.g., 23 RSS). FIG. 48B is a schematic showing that synthetic constructs (Savalan VNAR arrays) containing flanking different lox elements and selectable markers can be integrated by RMCE into Igh loci containing the idiopathic human alleles of all human VD and VJ elements.

Detailed Description

The present disclosure relates to genetically modified or engineered non-human animals (e.g., genetically modified or engineered mice) that produce heavy chain antibodies (e.g., mouse heavy chain antibodies, humanized heavy chain antibodies, or chimeric heavy chain antibodies) and methods of making the same. For example, provided herein are genetically engineered non-human animals of a particular species (e.g., a mouse species) that produce heavy chain antibodies (e.g., mouse heavy chain antibodies) of the same species. In another example, provided herein are genetically engineered non-human animals (e.g., genetically engineered mice) that produce chimeric heavy chain antibodies (e.g., human-mouse chimeric heavy chain antibodies, bovine-human-mouse chimeric heavy chain antibodies, alpaca-human chimeric heavy chain antibodies, or shark-human-mouse chimeric heavy chain antibodies).

In some cases, heavy chain antibodies obtained or validated from genetically engineered non-human animals (e.g., genetically engineered mice) provided herein can be used to generate 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).

The present disclosure also relates generally to nanobody compositions and other sources of nanobody compositions from these genetically modified mice. The compositions described herein are useful for treating or preventing a disease or disorder.

As described herein, this document provides methods for producing mammalian single domain antibodies (also referred to as nanobodies) in vivo. For example, a modified mouse endogenous IgH allele may be constructed such that the constant region C _H Only the CH1 truncated IgG1 gene (igg1Δch1) was contained and all other Ig classes or subtypes were removed, resulting in heavy chain-only IgG1 antibodies. By this modification, the IgG1- Δch1 gene is repositioned downstream of the E μ enhancer, I μ promoter, I μ exon, and S μ switch repeat, and other regulatory elements including γ1e, 5' hsr1, 3' rr, and 3' cbe enhancers remain unchanged in the endogenous IgH allele. Thus, high level expression of IgG 1-based heavy chain antibodies (IgG 1 hcabs) can be achieved constitutively, rather than inducing expression from the natural regulatory elements of each Ig subtype, and the entire VH pool becomes prone to IgG1 hcabs regardless of antigen type. The engineered non-human (e.g., mouse) endogenous IgH alleles described herein can be referred to as idiosyncratic (Singularity) and can be further modified by removing all non-human (e.g., mouse) endogenous variable exons and introducing docking sites to allow replacement of variable exons from human or other mammalian species (or combinations thereof) to produce chimeric antibodies based on the IgG1 HCAb platform, which can be used to derive species-specific single domain antibodies. Provided herein is an efficient method for sequentially introducing long genomic DNA fragments into docking sites. As demonstrated herein, these methods can successfully generate an odd human (Singularity Sapiens) allele containing 91 human VH exons, thereby maximizing possible antibody diversity. In some cases, variable exons of IgK and IgL (VK, VL) alleles may be used instead of (or in addition to) VH exons to generate light chain-based single domain antibodies. In a similar manner, V from other species can be designed and synthesized _H Segment, diversity D _H And/or connect J _H (or a combination thereof) and placing it into an odd allele to produce a heavy chain antibody, which can be used to make a single domain antibody with unique properties.

The engineered non-human animals (e.g., mice) described herein can exhibit normal B cell development and develop a strong humoral immune response upon antigen challenge. The high throughput sequence driven methods described herein can be used to generate Ig (e.g., igG 1) hcabs that exhibit high affinity for immune antigens. After antigen immunization is complete, the entire Ig pool can be amplified from lymphoid organs (e.g., spleen) and Next Generation Sequencing (NGS) performed to obtain clonotypes for phylogenetic analysis. Candidate clonotypes may be codon optimized, synthesized, cloned into an expression vector and expressed as nanobody-Fc fusions and/or nanobodies in 96-well format. The supernatant can be used in ELISA screens to confirm antigen-specific heavy chain antibodies and/or nanobodies for mass production, purification and/or characterization. Purified nanobodies, nanobody-Fc fusions, and/or heavy chain antibodies can exhibit high levels of thermal stability, antigen affinity, cell binding, and blocking activity.

As described herein, a non-human animal (e.g., a mouse) can be designed to produce heavy chain only antibodies (hcabs). In some cases, gene editing (e.g., CRISPR/Cas 9) can be used to edit endogenous IgH alleles to generate an odd allele (e.g., an odd murine allele) that contains only IgG genes (e.g., ighg1 genes) in the constant region encoding CH1 truncated IgG (e.g., igG1- Δch1), in some cases all endogenous genes encoding other antibody isotypes (IgM, igD, igE and IgA) and IgG subtypes (IgG 2b, igG2c, and IgG 3) can be excluded. In some cases, one or more endogenous regulatory elements may be maintained to allow efficient and reliable transcription of the mutant IgH g1 gene from the endogenous IgH allele. Class switching recombination can therefore be eliminated in these odd non-human animals (e.g., mice) to avoid any potential mechanisms that might impair IgG1- Δch1 expression and facilitate antibody discovery and purification. The resultant odd-numbered non-human animals (e.g., odd-numbered mice) can survive and have fertility without significant abnormalities, and they can produce a robust humoral immune response upon antigen challenge and produce IgG1- Δch1 heavy chain antibodies with high affinity. Because knowledge of heavy chain-light chain pairing is not required, the faster and more cost-effective NGS-driven antibody discovery procedure in spleen cell batch RNA sequencing (RNA-seq) analysis described herein can be used to identify antigen-specific monoclonal antibody heavy chain antibodies, and the entire process (e.g., antigen immunization, B cell isolation, batch sequencing of antibody libraries, antibody sequence cloning typing, high throughput cloning, expression, and antigen binding assays) can be completed within 3 months.

As also described herein, an engineered non-human animal (e.g., a mouse) can be designed to produce human and/or chimeric heavy chain antibodies that can be used to confirm therapeutic nanobodies. For example, an odd allele (e.g., an odd murine allele) can be further edited to generate an odd hyper-berth allele that lacks all of the mouse VDJ (H) genes and carries a docking site for sequential introduction of DNA fragments using recombinase-mediated cassette exchange (RMCE). In some cases, clones containing human VDJ (H) fragments (e.g., BAC clones) can be engineered by bacterial homologous recombination (recombinant engineering) to incorporate alternating selection cassettes and a heterologous Lox site, with overlapping genomic fragments trimmed away. In some cases, the engineered BACs can be used for sequential RMCE to assemble the human VDJ gene upstream of the mouse IgH E μ enhancer in a stepwise fashion. This can lead to the generation of a series of odd human alleles (e.g., SSV1-SSV 5) with increased VH diversity until a complete reconstruction of the entire human VDJ genomic region is obtained.

In a similar manner, a series of odd non-human animals (e.g., odd mice) are generated to produce species-specific nanobodies with unique properties for a variety of diagnostic and therapeutic applications.

Definition of the definition

As used herein, the term "antibody" refers to a molecule that specifically binds to or immunoreacts with a particular antigen, and includes at least the variable domains of the heavy and/or light chains, and in some cases mayAt least the variable domains comprising immunoglobulin heavy and light chains. Antibodies and antigen binding fragments, variants or derivatives thereof include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, heteroconjugate antibodies (e.g., bispecific and tetraspecific antibodies, diabodies, triabodies and tetrabodies), single domain antibodies (sdabs), epitope-binding fragments (e.g., fab ', and F (ab') ₂ Fd, fv, single chain Fv (scFv), recombinant IgG (rlgG), single chain antibodies (e.g., heavy or light chain antibodies), disulfide-linked Fv (sdFv), fragments comprising a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotype (anti-Id) antibodies. The antibody molecules described herein can be of any type (e.g., igG, igE, igM, igD, igA and IgY), class (e.g., igG) of immunoglobulin molecules ₁ 、IgG ₂ 、IgG ₃ 、IgG ₄ 、IgA ₁ And IgA ₂ ) Or subclasses. Furthermore, unless otherwise indicated, the term "monoclonal antibody" (mAb) is intended to include intact molecules as well as antibody fragments (such as, for example, fab and F (ab') 2 fragments) capable of specifically binding to a target protein. Fab and F (ab') 2 fragments lack the Fc fragment of the intact antibody. The term "inhibitory antibody" refers to an antibody that is capable of binding to a target antigen and inhibiting or reducing its function and/or attenuating one or more signal transduction pathways mediated by that antigen. For example, an inhibitory antibody may bind to and block the ligand binding domain of a receptor, or the extracellular region of a transmembrane protein. Inhibitory antibody molecules that enter cells may block the function of enzyme antigens or signal molecule antigens. Inhibitory antibodies inhibit or reduce antigen function and/or attenuate one or more antigen-mediated signal transduction 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 is capable of binding to a target antigen and enhancing its activity or function, e.g., an antibody that enhances or activates one or more signal transduction pathways mediated by the antigen. For example, agonist antibodies may bind to and activate extracellular regions of transmembrane proteins. Agonist antibody molecules entering the cell may enhance the function of the enzyme antigen or signal molecule antigen. Excitation device The agonist antibody activates or enhances antigen function and/or one or more antigen-mediated signal transduction pathways by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or more).

As used herein, the term "antigen" refers to a molecule that, if presented by an MHC molecule, is capable of being bound by an antibody or a T Cell Receptor (TCR). As used herein, the term "antigen" also encompasses T cell epitopes. T cell epitopes are recognized by T cell receptors in the context of MHC class I, which is present on all cells of the body except erythrocytes, while class II epitopes are present on immune cells, particularly antigen presenting cells. This recognition event results in activation of T cells and subsequent effector mechanisms such as T cell proliferation, cytokine secretion, perforin secretion, etc. Antigens can also be recognized by the immune system and/or can induce humoral and/or cellular immune responses, resulting in activation of B-and/or T-lymphocytes. However, in at least some cases, this may require that the antigen contain or be linked to a TH cell epitope and be administered in an adjuvant. The antigen may have one or more epitopes (B-and T-epitopes). The specific reactions mentioned above are intended to indicate that an antigen preferably reacts with its corresponding antibody or TCR in a highly selective manner in general, rather than with a large number of other antibodies or TCRs that may be induced by other antigens. An antigen as used herein may also be a mixture of several individual antigens. Antigens as used herein include, but are not limited to, allergens, autoantigens, haptens, cancer antigens (i.e., tumor antigens) and infectious disease antigens, and small organic molecules such as drugs of abuse (e.g., nicotine) and fragments and derivatives thereof. Furthermore, antigens for use in the present disclosure may be peptides, proteins, domains, carbohydrates, alkaloids, lipids or small molecules such as, for example, steroid hormones and fragments and derivatives thereof, autoantibodies and cytokines themselves.

An "antigen" also refers to a molecule (e.g., peptide, protein, or non-peptide) that contains one or more epitopes (linear, conformational, or both) that will stimulate the immune system of the host to produce humoral and/or cellular antigen-specific responses. The term is used interchangeably with the term "immunogen". Typically, a B cell epitope will comprise at least about 5 amino acids, but may be as small as 3-4 amino acids. T cell epitopes (such as CTL epitopes) will comprise at least about 7-9 amino acids and helper T cell epitopes will comprise at least about 12-20 amino acids. Typically, an epitope will comprise about 7 to 15 amino acids (such as 9, 10, 12 or 15 amino acids). As defined herein, the term includes polypeptides that comprise modifications, such as deletions, additions and substitutions (typically conservative in nature), as compared to the native sequence, provided that the protein retains the ability to elicit an immune response. These modifications may be deliberate (e.g., by site-directed mutagenesis) or may be accidental (such as by mutation of the host producing the antigen).

As used herein, the term "antigen binding fragment" refers to one or more fragments of an immunoglobulin that retain the ability to specifically bind a target antigen. The antigen binding function of an immunoglobulin may be performed by a fragment of a full-length antibody. Antibody fragments may be Fab, F (ab') ₂ scFv, SMIP, diabody, triabody, affibody, 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) A Fab fragment, a monovalent fragment consisting of VL, VH, CL and CHl domains; (ii) F (ab') ₂ Fragments, which are bivalent fragments, comprising two Fab fragments linked at the hinge region by a disulfide bridge; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of an antibody single arm, (v) dAbs comprising the VH and VL domains (Ward et al Nature,341:544-546 (1989)); (vi) a dAb fragment consisting of a VH domain; (vii) a dAb consisting of a VH or VL domain; (viii) an isolated Complementarity Determining Region (CDR); and (ix) a combination of two or more isolated CDRs, which may optionally be 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 joined, using recombinant methods, as a single protein chain by a linker, in which the VL and VH regions pair to form monovalent molecules, known as single chain Fv (scFv). These antibody fragments can be obtained using conventional techniques known to those of ordinary skill in the art and screened for their presence with the intact antibody Fragments of the same application. Antigen binding fragments may be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some cases, by chemical peptide synthesis methods known in the art.

As used herein, the term "antigen formulation" or "antigen composition" refers to an agent that induces an immune response when administered to a subject (e.g., a mammal).

As used herein, the term "biological sample" refers to a sample (e.g., blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., biopsy sample), pancreatic fluid, chorionic villus sample, and cells isolated from a subject).

As used herein, "combination therapy" or "combination administration" refers to the administration of two (or more) different agents or treatments to a subject as part of a prescribed treatment regimen for a particular disease or condition. The treatment regimen defines the dose and period of administration of each agent such that the effects of the different agents on the subject overlap. In some embodiments, delivery of two or more agents is simultaneous or concurrent and the agents may be co-formulated. In other embodiments, two or more agents are not co-formulated and administered in a sequential manner as part of a prescribed regimen. In some embodiments, the combined administration of two or more agents or treatments reduces/lowers other parameters associated with a symptom or condition to a greater extent than would be observed if one agent or treatment was delivered alone and not the other. The effect of the two treatments may be partially additive, fully additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of the individual therapeutic agents may be by any suitable route including, but not limited to, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue. The therapeutic agents may be administered by the same route or by different routes. For example, a first therapeutic agent of a combination may be administered by intravenous injection, while a second therapeutic agent of the combination may be administered orally.

As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of a composition as described herein refer to an amount sufficient to produce a beneficial or desired result (including cellular level, tissue level, or clinical result) when administered to a subject (including a mammal (e.g., a human)), and thus "effective amount" or synonyms thereof depend on the context of the application for which it is intended. For example, in the case of treating cancer, it is an amount of the composition sufficient to produce a treatment response compared to the case where the composition, antibody, vector construct, viral vector, or cell is not administered. The corresponding amounts of a given composition described herein will vary depending upon various factors such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, identity of the host or subject to be treated (e.g., age, sex, weight), etc., but can be routinely determined by one of skill in the art. Furthermore, as used herein, a "therapeutically effective amount" of a composition described herein is an amount that produces a beneficial or desired result in a subject as compared to a control. As defined herein, a therapeutically effective amount of a composition described herein can be readily determined by one of ordinary skill by conventional methods known in the art. The dosage regimen may be adjusted to provide optimal therapeutic relief.

As used herein, the terms "heavy chain antibody (heavy chain antibody)", "heavy chain antibody" (heavy-chain antibody), heavy chain-only antibody "and" HCAb "are used interchangeably and refer to antibodies that lack the light chain common to conventional antibodies. The heavy chain antibody may be any antibody derived from an immunoglobulin heavy chain (IgH) locus, such as an antibody comprising one or more heavy chain constant domains. For example, a heavy chain antibody can be an antibody comprising one light chain variable domain VL and one or more heavy chain constant domains.

As used herein, the term "hybrid" or "chimera" refers to a molecule (e.g., a protein or VLP) that contains portions thereof from at least two different proteins. For example, a hybrid influenza HA protein refers to cytoplasmic and/or transmembrane domains comprising 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., a different influenza protein or a different viral protein (e.g., RSV or VSV protein). It will be apparent that the hybrid molecules described herein may include full length proteins fused to additional heterologous polypeptides (full length or portions thereof) as well as portions of proteins fused to additional heterologous polypeptides (full length or portions thereof). It is also apparent that a hybrid may include wild-type sequences or mutant sequences in any, some or all of the heterologous domains.

As used herein, the terms "increase" and "decrease" refer to a greater or lesser modulation of the amount of function, expression, or activity, respectively, that results in a metric relative to a reference. For example, subsequent administration of an antibody described herein can increase or decrease the amount of a marker of a metric described herein (e.g., cancer cell death or DNA methylation of a target site) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more relative to the amount of the marker administered. In general, the metric is measured after administration, i.e., when the effect has been produced (e.g., at least one week, one month, 3 months, or 6 months after the initiation of the treatment regimen).

An "immunological response" to an antigen or composition is a humoral and/or cellular immune response in a subject to an antigen present in a target composition. For purposes herein, "humoral immune response" refers to an immune response mediated by antibody molecules, while "cellular immune response" is an immune response mediated by T lymphocytes and/or other leukocytes. An important aspect of cellular immunity involves antigen-specific responses of cytolytic T Cells (CTLs). CTLs are specific for peptide antigens that are presented in association with proteins encoded by the Major Histocompatibility Complex (MHC) and expressed on the cell surface. CTLs help induce and promote the destruction of intracellular microorganisms, or lysis of cells infected with such microorganisms. Another important aspect of cellular immunity relates to antigen-specific responses of helper T cells. Helper T cells function to help stimulate the function of non-specific effector cells and concentrate their activity against cells displaying peptide antigens associated with MHC molecules on the surface. "cellular immune response" also refers to the activation of T cells and/or other white blood cells Cells (including derived from CD 4) ⁺ And CD8 ⁺ Those of T cells), and the production of cytokines, chemokines, and other such molecules. Thus, the immune response may include one or more of the following effects: b cells produce antibodies; and/or activating suppressor T cells and/or γδ T cells that specifically target one or more antigens present in the composition or vaccine of interest. These reactions can be used to neutralize infectivity, and/or mediate antibody complement or Antibody Dependent Cellular Cytotoxicity (ADCC), thereby providing protection for the immunized host. Such a reaction can be determined using standard immunoassays and neutralization assays well known in the art.

An "immunogenic composition" is a composition comprising an antigenic molecule, wherein administration of the composition to a subject results in a humoral and/or cellular immune response in the subject against the antigenic molecule of interest.

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

As used herein, a "particle-forming polypeptide" may be derived from a particular viral protein, including full-length or near-full-length viral proteins and fragments thereof, or have an internal deletion of viral proteins that has the ability to form VLPs under conditions conducive to VLP formation. Thus, polypeptides may comprise full-length sequences, fragments, truncated and partial sequences, as well as analogs and precursor forms of the reference molecule. Thus, the term includes deletions, additions and substitutions of sequences, provided that the polypeptide retains the ability to form VLPs. Thus, the term includes natural variations of the particular polypeptide, as variations in capsid proteins are often present between viral isolates. The term also includes non-naturally occurring deletions, additions and substitutions in the reference protein, so long as the protein retains the ability to form VLPs. Preferred substitutions are those which are conservative in nature, i.e., those which occur within a family of side chain related amino acids. Specifically, amino acids are generally divided into 4 families: (1) acidity-aspartic acid and glutamic acid; (2) alkaline-lysine, arginine, histidine; (3) Nonpolar-alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; (4) Uncharged polarity-glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.

As used herein, the terms "light chain variable region" and "heavy chain variable region" refer to variable binding regions from an antibody light chain and heavy chain, respectively. The variable binding region consists of discrete, well-defined subregions, termed "complementarity determining regions" (CDRs) and "framework regions" (FRs), which are typically sequenced from amino-terminus to carboxy-terminus as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In one embodiment, the FR is humanized. The term "CL" refers to an "immunoglobulin light chain constant region" or "light chain constant region," i.e., a constant region from an antibody light chain. The term "CH" refers to an "immunoglobulin heavy chain constant region" or "heavy chain constant region" which can be further divided into CH1, hinge, CH2 and CH3 (for IgA, igD and IgG) or CH1, CH2, CH3 and CH4 domains (for IgE and IgM) depending on the antibody isotype.

As used herein, a "pharmaceutical composition" or "pharmaceutical preparation" is a composition or preparation that has pharmacological activity or other direct effect in alleviating, treating or preventing a disease, and/or a finished dosage form or formulation thereof, and which is intended for human use.

As used herein, the term "reference" refers to a level, expression level, copy number, sample or standard for comparison purposes. For example, a reference sample may be obtained from a healthy individual (e.g., an individual not suffering from cancer). The reference level may be the expression level of one or more reference samples. For example, the average expression (e.g., average expression or median expression) between a plurality of individuals (e.g., healthy individuals or individuals not suffering from cancer) may be a reference level. In other cases, the reference level may be a predetermined threshold level, e.g., based on the functional expression as otherwise determined (e.g., through empirical testing).

As used herein, the terms "subject" and "patient" refer to an animal (e.g., a mammal, such as a human). According to the methods described herein, the subject to be treated may be a subject that has been diagnosed with a particular condition, or is at risk of developing such a condition. Diagnosis may be made by any method or technique known in the art. Those of skill in the art will appreciate that a subject to be treated according to the present disclosure may have received standard testing or may be determined to be at risk without examination due to the presence of one or more risk factors associated with a disease or condition.

As used herein, "treatment" and "treatment" refer to medical management of a subject with the aim of improving, alleviating, stabilizing (i.e., not worsening), preventing or curing a disease, pathological condition, or disorder. The term includes active treatment (treatment for ameliorating a disease, pathological condition or disorder), causal treatment (treatment for the cause of the associated disease, pathological condition or disorder), palliative treatment (treatment intended to alleviate symptoms), prophylactic treatment (treatment intended to minimize or partially or completely inhibit the development of the associated disease, pathological condition or disorder), and supportive treatment (treatment for supplementing another therapy). Treatment also includes reducing the extent of a disease or condition; preventing the spread of a disease or condition; delay or slow the progression of the disease or condition; improvement or alleviation of a disease or condition; remission (whether partial or total) is detectable or undetectable. By "ameliorating" or "alleviating" a disease or condition is meant a slowing or extension of the extent of a disease, disorder or condition and/or the time course of an adverse clinical manifestation to decrease and/or progress as compared to the extent or time course in the absence of treatment. "treatment" may also refer to an extended survival period compared to the expected survival without treatment. Thus, those (subjects) in need of treatment include those (subjects) already with a condition or disorder; those prone to have a condition or disorder (subject); and those (subjects) in need of prevention of a condition or disorder.

It should be understood that for all numerical limits of some of the parameters described herein, such as "about," "at least," "less than/less than," and "greater than/more than," the description also necessarily encompasses any range defined by the recited values. Thus, for example, a description of "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, 4-5, and the like.

It is to be understood that the terms "a" and "an" as used herein refer to "one or more" of the recited components unless otherwise specified. The use of table alternatives (e.g., "or") is understood to mean one, both, or any combination thereof.

Odd non-human animals (e.g. odd mice)

Provided herein are genetically engineered 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, the genetically engineered non-human animal used to produce heavy chain antibodies can be a non-human animal having (e.g., engineered to have) humanized IgG heavy chains. In some cases, a non-human animal used to produce a heavy chain antibody (e.g., a heavy chain antibody such as a mouse heavy chain antibody or a chimeric heavy chain antibody) may be one whose genome encodes an IgGl C region gene (e.g., C) _γ1 ) Has (e.g., is genetically engineered to have) one or more disrupted non-human animals in the endogenous nucleic acid sequence of the CHl domain. In some cases, a non-human animal (e.g., a mouse) may be designed to produce a heavy chain antibody (e.g., a heavy chain antibody (such as a mouse heavy chain antibody or chimeric heavy chain antibody)) that lacks the CHl domain and that lacks the light chain. Also provided herein are methods and materials for making and using the non-human animals described herein.

In some cases, one or more genetic modifications may be made to create the non-human animals described herein. The genetic modification may result in the non-human animal (e.g., mouse) expressing and secreting IgG heavy chain antibodies into its serum. In some cases, igG heavy chain antibodies may be humanized. For example, the variable region of an IgG heavy chain antibody can be a human variable region, and the constant region of an IgG heavy chain antibody can be a mouse constant region. In some cases, a non-human animal (e.g., a mouse) described herein can be designed to produce an IgGl heavy chain antibody, an IgG2 heavy chain antibody, an IgG3 heavy chain antibody, or an IgG4 heavy chain antibody. In some cases, a non-human animal (e.g., a mouse) described herein can be designed to produce a combination of two or more of the following: (a) IgGl heavy chain antibody, (b) IgG2 heavy chain antibody, (c) IgG3 heavy chain antibody and (d) IgG4 heavy chain antibody.

In some cases, the non-human animals provided herein can be designed with deletions of nucleic acids encoding the CHl domain of the IgG C region (e.g., the CHl domain of the IgGl C region, the CHl domain of IgG2a, the CH1 domain of the IgG2b C region, and/or the CH1 domain of the IgG 3C region). The CH1 domain may contain multiple exons. In some cases, exon 1 of the CH1 domain of the IgG C region can be removed, such that the engineered non-human animal (e.g., mouse) produces an IgG Δch1 heavy chain antibody.

When one or more genetic modifications are made to remove all or part of the nucleic acid encoding a CH1 domain (e.g., the CH1 domain of the IgG 1C region, the CH1 domain of the IgG2a C region, the CH1 domain of the IgG2b C region, and/or the CH1 domain of the IgG 3C region) such that the engineered non-human animal produces an IgG Δch1 heavy chain antibody, the endogenous nucleic acid domains encoding the hinge domain, heavy chain CH2 domain, and heavy chain CH3 domain may remain intact. For example, to make an IgG1 Δch1 heavy chain antibody-producing mouse, the genome of the mouse may lack exon 1 (and/or other portions) of the IgG1 CH1 domain, while retaining endogenous mouse nucleic acids required to express the hinge domain, heavy chain CH2 domain, and heavy chain CH3 domain of IgG1, thereby resulting in a mouse capable of producing an IgG1 Δch1 heavy chain antibody.

Additional endogenous nucleic acid components may be removed from the genome of a non-human animal (e.g., a mouse) to create a non-human animal provided herein, including, but not limited to, introns and/or exons of IgM constant domain (μ constant domain locus), introns and/or exons of IgD constant domain (e.g., δ constant domain locus), introns and/or exons of IgE constant domain (e.g., ε constant domain locus), and/or introns and/or exons of IgA constant domain (e.g., α constant domain locus). For example, the non-human animals provided herein can be designed to lack introns and exons of IgM constant domains (e.g., μ constant domain loci), introns and exons of IgD constant domains (e.g., δ constant domain loci), introns and exons of IgE constant domains (e.g., epsilon constant domain loci), and introns and exons of IgA constant domains (e.g., α constant domain loci).

In some cases, when a non-human animal (e.g., a mouse) is designed to produce an IgG1 Δch1 heavy chain antibody alone, the genome of the non-human animal can be designed to lack (in addition to the endogenous introns and/or exons of the μ constant domain locus, the endogenous introns and/or exons of the δ constant domain locus, the endogenous introns and/or exons of the ε constant domain locus, and the endogenous introns and/or exons of the α constant domain locus) igγ3 constant domain (e.g., γ3 constant domain locus), if endogenous, the endogenous (if endogenous) introns and/or exons of the igγ2a constant domain (e.g., γ2a constant domain locus), the endogenous (if endogenous) introns and/or exons of the γ2b constant domain (e.g., γ2b constant domain locus), and the endogenous (e.g., γ2a constant domain locus), if endogenous (e.g., γ2a constant domain locus). Figures 3A-3E show examples of genetic engineering methods for creating mice producing IgG1 Δch1 heavy chain antibodies only.

In some cases, when a non-human animal (e.g., a mouse) is designed to produce an IgG2a Δch1 heavy chain antibody alone, the genome of the non-human animal can be designed to lack (in addition to lack of endogenous introns and/or exons of the mu constant domain locus, endogenous introns and/or exons of the delta constant domain locus, endogenous introns and/or exons of the epsilon constant domain locus, and endogenous introns and/or exons of the alpha constant domain locus, igy 3 constant domain (e.g., gamma 3 constant domain locus), endogenous (if endogenous) and/or exons, igy 1 constant domain (e.g., gamma 1 constant domain locus), endogenous (if endogenous) introns and/or exons, igy 2b constant domain (e.g., gamma 2b constant domain locus), and igy 2c constant domain (e.g., gamma 2b constant domain locus), endogenous (endogenous) and/or igy 2c constant domain (endogenous) if endogenous).

In some cases, when a non-human animal (e.g., a mouse) is designed to produce IgG2b Δch1 heavy chain antibody alone, the genome of the non-human animal can be designed to lack (in addition to lack of endogenous introns and/or exons of the mu constant domain locus, endogenous introns and/or exons of the delta constant domain locus, endogenous introns and/or exons of the epsilon constant domain locus, and endogenous introns and/or exons of the alpha constant domain locus) igγ3 constant domain (e.g., γ3 constant domain locus), endogenous (if endogenous) introns and/or exons of the igγ2a constant domain (e.g., γ2a constant domain locus), endogenous (if endogenous) introns and/or exons of the gamma 1 constant domain locus, and Ig 2c constant domain (e.g., γ2a constant domain locus), endogenous (if endogenous) and/or endogenous (e.g., γ2c constant domain locus).

In some cases, when a non-human animal (e.g., a mouse) is designed to produce an IgG2c Δch1 heavy chain antibody alone, the genome of the non-human animal can be designed to lack (in addition to lack of endogenous introns and/or exons of the mu constant domain locus, endogenous introns and/or exons of the delta constant domain locus, endogenous introns and/or exons of the epsilon constant domain locus, and endogenous introns and/or exons of the alpha constant domain locus) igy 3 constant domain (e.g., gamma 3 constant domain locus), endogenous (if endogenous) introns and/or exons of the igy 2a constant domain (e.g., gamma 2a constant domain locus), endogenous (if endogenous) introns and/or exons of the gamma 2b constant domain (e.g., gamma 2b constant domain locus), and endogenous (e.g., gamma 1) constant domain (endogenous) introns and/or endogenous (gamma 1) constant domain locus.

In some cases, when a non-human animal (e.g., a mouse) is designed to produce an IgG3 Δch1 heavy chain antibody alone, the genome of the non-human animal can be designed to lack (in addition to lack of endogenous introns and/or exons of the μ constant domain locus, endogenous introns and/or exons of the δ constant domain locus, endogenous introns and/or exons of the ε constant domain locus, and endogenous introns and/or exons of the αconstant domain locus) igγ1 constant domain (e.g., γ1 constant domain locus), endogenous (if present) introns and/or exons of the igγ2a constant domain (e.g., γ2a constant domain locus), endogenous (if present) introns and/or exons of the igγ2b constant domain (e.g., γ2b constant domain locus), and endogenous (e.g., γ2c) constant domain locus, endogenous (if present) and/or endogenous (if present) constant domain locus.

As described herein, retaining and/or establishing a new location for certain endogenous enhancers or regulatory elements of a non-human animal can result in the non-human animal (e.g., a mouse) provided herein effectively producing a large number of different heavy chain antibodies (e.g., heavy chain antibodies such as mouse heavy chain antibodies or chimeric heavy chain antibodies) for example, the non-human animal (e.g., a mouse) provided herein can be designed to retain a mu enhancer (e.g., E mu), a mu switching region (S mu) and/or a mu promoter containing an I-exon (I mu) present endogenously upstream of a nucleic acid encoding an IgM constant domain, in some cases, the non-human animal (e.g., a mouse) provided herein can be designed such that the retained endogenous E mu, S mu and/or I mu elements are in genomic positions such that the retained e.g., E mu, S mu and/or I mu elements encoding full length endogenous constant domains are located downstream of the nucleic acid sequence encoding a CH2 domain (e.g., E mu, S mu and/or I mu elements encoding a full length IgG2 CH2 nucleic acid, C2 nucleic acid encoding an IgG2 domain, e.g., a full length IgG2 CH2 nucleic acid, C2 nucleic acid set up to a full length C2 CH2 nucleic acid encoding an IgG2 domain.

In another example, a non-human animal (e.g., a mouse) provided herein can be designed to remain in the 3'rr and/or 3' cbe elements that are endogenously present downstream of the nucleic acid encoding the IgA constant domain. In some cases, the non-human animals (e.g., mice) provided herein can be designed such that the retained endogenous 3'rr and/or 3' cbe elements are in genomic positions such that the first nucleic acid sequence upstream of the retained 3'rr and/or 3' cbe elements encoding the full length endogenous Ig CH2 constant domain is a sequence encoding the Ig CH2 domain (e.g., a nucleic acid encoding the full length IgGl 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 such genomic arrangements are shown in fig. 2B and 3E, in which the nucleic acid of the endogenous mouse 3'rr element is relocated downstream of the nucleic acid encoding the endogenous IgGl CH2 domain such that no other nucleic acid encoding the full length IgG CH2 domain is located between the nucleic acid encoding the endogenous IgGl CH2 domain and the nucleic acid of the endogenous mouse 3' rr element.

In some cases, a non-human animal (e.g., a mouse) provided herein can be designed to retain the 3' γ1e element endogenously present, for example, between IgGl and IgG2b loci. In some cases, the non-human animals (e.g., mice) provided herein can be designed such that the retained endogenous 3'γ1e elements are in genomic positions such that nucleic acids encoding two, one, or a non-full length endogenous Ig CH2 domains are located between the retained endogenous 3' γ1e elements and the retained endogenous 3'rr elements and/or the retained endogenous 3' cbe elements. An example of such a genomic arrangement is shown in fig. 3E, where the nucleic acid of the endogenous mouse 3'γ1e element is relocated upstream of the retained endogenous 3' rr element such that there is no other nucleic acid encoding the full length IgG CH2 domain between the endogenous mouse 3'γ1e element and the endogenous 3' rr element.

In some cases, a non-human animal (e.g., a mouse) provided herein can be designed to retain the 5' hsr1 element endogenously present within the IgG constant domain locus. In some cases, a non-human animal (e.g., a mouse) provided herein can be designed such that the retained endogenous 5'hsr1 element is in 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 sequence encoding the Ig CH2 domain (e.g., a nucleic acid encoding the full length IgGl 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 such a genomic arrangement is shown in fig. 3E, in which the nucleic acid of the endogenous mouse 5'hsr1 element is relocated downstream of the nucleic acid encoding the endogenous IgGl CH2 domain such that no other nucleic acid encoding the full length IgG CH2 domain is located between the nucleic acid encoding the endogenous IgGl CH2 domain and the nucleic acid of the endogenous mouse 5' hsr1 element.

In some cases, a non-human animal (e.g., a mouse) provided herein can be designed with (a) a variable region locus (e.g., a mouse variable region locus, a non-mouse variable region locus, a human variable region locus, or a chimeric variable region locus (such as a bovine-human chimeric variable region locus, a alpaca-human chimeric variable region locus, or a shark-human chimeric variable region locus)) followed by (c) a nucleic acid encoding an endogenous IgG hinge, CH2, and CH3 domain in the absence of the endogenous CH1 domain of the IgG, followed by (E) an endogenous 3' γ1e element, an endogenous 3' rr element, and an endogenous 3' cbe element, while lacking endogenous nucleic acids encoding at least one full-length CH2 or CH3 domain of each IgM, igD, igE and IgA. An example of such a genomic arrangement is shown in FIG. 3E. See also fig. 7, 8, 43B, 44, 45B, 47B, and 48B.

In some cases, one or more exogenous enhancers or regulatory elements may be engineered into a non-human animal (e.g., a mouse) rather than retaining endogenous enhancers or regulatory elements as described herein. For example, in some cases, mice can be designed as described herein, with endogenous mouse eμ elements removed and replaced with human eμ elements.

In some cases, the engineered non-human animals provided herein can be designed to have variable region loci that are endogenous variable region loci of the non-human animal. For example, the engineered mice provided herein can be designed to have endogenous mouse variable region loci. An example of such an engineered mouse IgH locus is shown in figure 1B.

In some cases, the engineered non-human animals provided herein can be designed to have variable region loci that are non-endogenous to the non-human animal. For example, the engineered mice provided herein can be designed with a non-mouse variable region locus (e.g., a human variable region locus, an alpaca variable region locus, a shark variable region locus, a bovine variable region locus, a goat variable region locus, a sheep variable region locus, a dog variable region locus, a cat variable region locus, a rat variable region locus, a chicken variable region locus, or a rabbit variable region locus). An example of such an engineered mouse IgH locus is shown in figure 6B.

In some cases, the engineered non-human animals provided herein can be designed to have variable region loci that are non-endogenous to the non-human animal such that they include variable region components from two or more different species that are different from the non-human animal. For example, the engineered mice provided herein can be designed to have non-mouse variable region loci, including human and alpaca, human and bovine, human and shark, shark and bovine, alpaca and bovine, human and goat, human and sheep, human and dog, human and cat, human and mouse, human and chicken, or human and rabbit variable region loci. Examples of IgH loci for such engineered mice are shown in fig. 43, 44, 47 and 48.

In some cases, the engineered non-human animals provided herein can be designed to have a variable region locus that is a variable region locus of a light chain (e.g., a kappa light chain locus variable region or a lambda light chain locus variable region) rather than a heavy chain variable region locus. For example, the engineered mice provided herein can be designed as variable region loci having a light chain locus (e.g., a human variable region locus of a kappa or lambda light chain). Examples of IgH loci for such engineered mice are shown in fig. 43B, 44A and 44B.

This document also provides engineered non-human animals useful for creating antibodies provided herein (e.g., heavy chain antibodies lacking CHl domains). For example, the engineered non-human animals provided herein lack the entire set of exons of the endogenous variable region of the heavy chain locus and contain cloned nucleic acid segments at positions upstream of the engineered constant regions described herein. Examples of such engineered mice IgH loci are shown in FIGS. 4, 5D and 6B, which may be referred to as non-human odd-super-berth animals or odd-super-berth mice. Any suitable cloned nucleic acid segment can be used to prepare a non-human odd superpositioned animal (e.g., an odd superpositioned mouse) provided herein. For example, cloned nucleic acid segments designed to contain one, two, three, four or more recombinase site recognition sequences (see, e.g., table 1) can be used to prepare the non-human odd-super-berth animals provided herein (e.g., odd-super-berth mice). In some cases, the non-human odd-superpositioned animals provided herein (e.g., odd-superpositioned mice) lack the ability to produce any Ig heavy chain.

Table 1. Exemplary recombination site recognition sequences.

The non-human animals provided herein (e.g., non-human animals designed to produce heavy chain antibodies, such as IgGl Δch1 heavy chain antibodies described herein, and non-human odd-superphagous animals, such as odd-superphagous mice described herein) may be prepared using any suitable method, e.g., gene editing techniques (e.g., CRISPR/Cas gene editing, TALEN gene editing, and/or zinc finger-based gene editing), recombination techniques (e.g., sequential recombinase-mediated cassette exchange (RMCE)), and combinations thereof, may be used to prepare the non-human animals provided herein.

This document also provides human nanobodies, humanized nanobodies, heavy chain antibodies lacking a CH1 domain (e.g., a fully mouse heavy chain antibody lacking a CH1 domain), and chimeric heavy chain antibodies (e.g., a human-mouse chimeric heavy chain antibody with or without a CH1 domain). For example, provided herein are fully human nanobodies produced or derived from non-human animals described herein. As another example, provided herein are full mouse heavy chain antibodies lacking a CH1 domain. As another example, provided herein are chimeric heavy chain antibodies. Such chimeric heavy chain antibodies may lack the CH1 domains described herein. In some cases, a chimeric heavy chain antibody provided herein (e.g., an IgG Δch1 heavy chain antibody) can contain one or more variable region components that are constant region components of humans, alpacas, sharks, cattle, goats, sheep, dogs, cats, rats, chickens or rabbits, as well as different species (e.g., mice). For example, a chimeric heavy chain antibody provided herein (e.g., an IgG Δch1 heavy chain antibody) can have a human variable region and a mouse constant region. In some cases, a chimeric heavy chain antibody provided herein (e.g., an IgG Δch1 heavy chain antibody) can have an alpaca variable region and a mouse constant region. In some cases, a chimeric heavy chain antibody provided herein (e.g., an IgG Δch1 heavy chain antibody) can have a shark variable region and a mouse constant region. In some cases, a chimeric heavy chain antibody provided herein (e.g., an IgG Δchl heavy chain antibody) can have a bovine variable region and a mouse constant region. In some cases, chimeric heavy chain antibodies provided herein (e.g., igG Δchl heavy chain antibodies) can have at least a portion of alpaca variable regions and at least a portion of human and mouse constant regions. In some cases, a chimeric heavy chain antibody provided herein (e.g., an IgG Δchl heavy chain antibody) can have at least a portion of a shark variable region and at least a portion of human and mouse constant regions. In some cases, a chimeric heavy chain antibody provided herein (e.g., an IgG Δchl heavy chain antibody) can have at least a portion of a bovine variable region and at least a portion of a human and mouse constant region.

The human nanobodies, humanized nanobodies, heavy chain antibodies lacking a CH1 domain (e.g., a fully mouse heavy chain antibody lacking a CH1 domain), and chimeric heavy chain antibodies (e.g., a human-mouse chimeric heavy chain antibody with or without a CH1 domain) provided herein can be obtained using any suitable method. For example, the heavy chain antibodies provided herein can be obtained from the plasma of a non-human animal provided herein. In some cases, the human nanobodies, humanized nanobodies, heavy chain antibodies lacking a CH1 domain (e.g., a fully mouse heavy chain antibody lacking a CH1 domain), and chimeric heavy chain antibodies (e.g., a human-mouse chimeric heavy chain antibody with or without a CH1 domain) provided herein can be obtained using nucleic acid vectors designed for expression of the nanobodies or heavy chain antibodies that are based on or derived from the heavy chain antibodies provided herein that are produced by non-human animals. For example, a human-mouse IgG Δch1 heavy chain antibody produced by a non-human animal provided herein can be identified as having the ability to bind to a target antigen of interest (e.g., SARS-CoV-2 antigen) and can be sequenced. The sequences can be used to design nucleic acid vectors that have the ability to express the human variable region of the same human-mouse IgG Δch1 heavy chain antibody or the same human-mouse IgG Δch1 heavy chain antibody as the human nanobody. In some cases, this sequence can be used to design nucleic acid vectors that can express fully human full length heavy chain antibodies, which can be used alone or in combination with fully human light chains to create fully tetrameric antibodies.

In some cases, the non-human animals provided herein can be immunized with an antigen of interest (e.g., SARS-CoV-2 antigen) such that the non-human animal produces antibodies to the antigen. Nucleic acids encoding the produced heavy chain antibodies (e.g., heavy chain antibodies lacking a CH1 domain) can be isolated. For example, amplification techniques such as PCR or 5' race can be used to obtain a large collection of nucleic acids encoding at least a portion (e.g., one or more CDRs, all three CDRs, or the entire variable region) of different variable regions of a heavy chain antibody produced by a non-human animal. The isolated nucleic acid sequences may be cloned (with or without pre-sequencing) into an expression vector to express the resulting nucleic acid sequences in the context of any suitable type of antibody (e.g., nanobody, heavy chain antibody, or whole antibody) that can be evaluated for desired properties (e.g., binding properties, neutralizing properties, and/or lytic properties). Those nucleic acid sequences capable of encoding antibodies having the desired properties can be used to create any type of antibody (such as nanobodies).

The plasma comprising nanobodies may be collected from a subject or from a non-human animal having a humanized immune system that may have been immunized with an antigen as described herein. Nanobodies from non-human animals having a humanized immune system are useful for treating human subjects in need thereof.

In some cases, plasma comprising chimeric heavy chain antibodies useful for generating nanobodies described herein may be collected from a subject or a non-human animal provided herein (e.g., a non-human animal having a humanized immune system) that may have been immunized with an antigen described herein.

The plasma containing nanobodies may be collected by, for example, plasmapheresis. In some cases, plasma comprising the chimeric heavy chain antibodies described herein can be collected by, for example, plasmapheresis. Plasma may be collected multiple times from the same subject, e.g., multiple times during each given period of time after immunization, multiple times between immunization, or any combination thereof.

Plasma may be collected from the non-human animal or human subject described herein at any suitable amount of time after immunization (e.g., first immunization, last immunization, or intermediate immunization). The plasma may 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, or at least 30 days or more after immunization. In some embodiments, plasma may be collected at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 15, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 35, at most 42, at most 49, or at most 56 days after immunization. In some embodiments, plasma may be collected about 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 after immunization. In some embodiments, the compositions described herein may include plasma collected after administration of the immunogenic compositions/antigens described herein.

The plasma may be frozen (e.g., for frozen storage or transport). In some embodiments, the plasma is kept fresh, or antibodies (e.g., heavy chain antibodies or nanobodies) are purified from fresh plasma.

Nanobodies are purified from plasma using techniques known to those skilled in the art (e.g., by affinity purification). In some cases, the chimeric heavy chain antibodies described herein can be purified from plasma using any suitable technique (such as by affinity purification).

In some cases, the methods for producing a protein provided herein (e.g., a human nanobody, a humanized nanobody, a fully mouse heavy chain antibody lacking CH1, or a chimeric heavy chain antibody with or without CH 1) may involve expression in mammalian cells, although insect cells, yeast, bacteria, or other cells under the control of an appropriate promoter may also be used to produce the recombinant protein. In some cases, the antibodies (e.g., heavy chain antibodies or nanobodies) provided herein can be recombinantly produced in a prokaryotic host, such as e.coli, bacillus brevis (Bacillus brevis), bacillus subtilis (Bacillus subtilis), bacillus megaterium (Bacillus megaterium), lactobacillus zeae/lactobacillus casei (Lactobacillus zeae/casei), or lactobacillus paracasei (Lactobacillus paracasei). In some cases, the antibodies (e.g., heavy chain antibodies or nanobodies) provided herein can be produced in a eukaryotic host such as a filamentous fungus of the genus Trichoderma (e.g., trichoderma reesei) and Aspergillus (e.g., aspergillus niger (a. Nigers) and Aspergillus oryzae (a. Oryzae)), a protozoa (e.g., talent leishmania), an insect cell or a mammalian cell (e.g., mammalian cell line such as Chinese Hamster Ovary (CHO) cells, per. C6 cells, mouse myeloma NS0 cells, baby Hamster Kidney (BHK) cells or human embryonic kidney cell line HEK 293), a yeast (e.g., pichia pastoris), saccharomyces cerevisiae (Saccharomyces cerevisiae), hansenula polymorpha (Hansenula polymorpha), pompe schizosaccharomyces (Schizosaccharomyces pombe), schwannoma (Schwanniomyces occidentalis), kluyveromyces lactis (Kluyveromyces lactis) or yarrowia lipolytica (Yarrowia lipolytica). See, for example, frenzel et al (Front immunol.,4:217 (2013)). Mammalian expression vectors may contain non-transcriptional elements such as origins of replication, suitable promoters and enhancers, and other 5 'or 3' flanking non-transcribed sequences, and 5 'or 3' non-translated sequences such as the necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor (receptor) sites, and termination sequences. DNA sequences derived from the SV40 viral genome (e.g., SV40 origin, early promoter, enhancer, splice and polyadenylation sites) may be used to provide other genetic elements necessary for expression of heterologous DNA sequences. Suitable cloning and expression vectors for bacterial, fungal, yeast and mammalian cell hosts are described in Green and Sambrook, molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual) (fourth edition), cold spring harbor laboratory press (2012), and can be used to produce antibodies provided herein (e.g., human nanobodies, humanized nanobodies, fully mouse heavy chain antibodies lacking CH1, or chimeric heavy chain antibodies with or without CH 1).

Various mammalian cell culture systems can be employed to express and make recombinant proteins or antibodies provided herein (e.g., human nanobodies, humanized nanobodies, fully mouse heavy chain antibodies lacking CH1, or chimeric heavy chain antibodies with or without CH 1). Examples of mammalian expression systems that may be used include, but are not limited to, CHO cells, COS cells, heLA and BHK cell lines. Host cell culture procedures for the production of useful protein therapeutics are described, for example, in Zhou and kantadjiiff (editors), "mammalian cell culture for bioproduct manufacture (progress of biochemical engineering/Biotechnology), (Mammalian Cell Cultures for Biologics Manufacturing (Advances in Biochemical Engineering/Biotechnology)), schpringer publishing (Springer) (2014). Franks, protein biotechnology: isolation, characterization and stabilization (Protein Biotechnology: isolation, characterization, and Stabilization), humana Press (2013); and Cutler, protein purification protocol (methods of molecular biology) (Protein Purification Protocols (Methods in Molecular Biology)), purification of protein therapeutics is described in the sumac press (2010). Meyer (editorial) in therapeutic protein pharmaceutical products: practical formulation methods in laboratory, manufacturing and clinical (Therapeutic Protein Drug Products: practical Approaches to formulation in the Laboratory, manufacturing, and the Clinic), wu Dehai De Kogyo Serial (2012). The compositions described herein may include a vector, such as a viral vector (e.g., a lentiviral vector encoding a recombinant protein, an adenovirus, or an adeno-associated virus). In some embodiments, a vector (e.g., a viral vector) may comprise a nucleic acid encoding a recombinant protein. In some cases, the processes described herein may be designed to meet Good Manufacturing Practice (GMP) defined standards, which would involve multiple quality controls and appropriate infrastructure and activity separation to avoid cross-contamination. Finally, the composition may be labeled and distributed worldwide.

In some embodiments, the therapeutic nanobody formulations described herein can be produced by immunizing a non-human animal having a humanized immune system with an antigen described herein. In some cases, the therapeutic nanobody formulations described herein can be produced by immunizing an engineered non-human animal described herein with an antigen of interest described herein.

The non-human animal having a humanized immune system may be an ungulate, such as a donkey, goat, horse, cow or pig; rodents, such as rabbits, rats or mice. In some embodiments, the non-human animal having a humanized immune system is a cow (cow). In some embodiments, the non-human animal having a humanized immune system is a chicken. The non-human animal has a humanized immune system, e.g., its immune system comprises a humanized immunoglobulin locus, or multiple humanized immunoglobulin loci. In some embodiments, the humanized immunoglobulin loci comprise human immunoglobulin germline sequences that allow a non-human animal to produce humanized antibodies (e.g., fully human antibodies). In some embodiments, the non-human animal having the humanized immune system of the present disclosure comprises non-human B cells having a humanized immunoglobulin locus. The humanized immunoglobulin loci undergo VDJ recombination during B cell development, producing B cells with multiple antigen binding specificities. Upon immunization with one or more of the immunogenic compositions described herein, a plurality of B cell clones react to their respective cognate antigens, resulting in the production of polyclonal antibodies with a variety of binding specificities.

The non-human animal provided herein may be any type of non-human animal. For example, the non-human animal designed to express the chimeric heavy chain antibody may be an ungulate, such as donkey, goat, horse, cow or pig; rodents, such as rabbits, rats or mice. In some embodiments, a non-human animal provided herein (e.g., a non-human animal designed to express a chimeric heavy chain antibody) can be a cow (bovine). In some embodiments, a non-human animal provided herein (e.g., a non-human animal designed to express a chimeric heavy chain antibody) can be a chicken.

In some embodiments, immunization of a non-human animal of the present 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 the disclosure with one or more immunogenic compositions described herein results in the production of polyclonal antisera comprising 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 antigens that specifically bind to an immunogenic composition described herein.

Various techniques for modifying the genome of a non-human animal (e.g., a non-human animal for immunization) can be employed to develop an animal capable of producing an antibody (e.g., a humanized antibody, a full mouse heavy chain antibody, or a chimeric heavy chain antibody). The non-human animal may be a transgenic animal, for example a transgenic animal comprising all or a substantial portion of one or more humanized immunoglobulin loci. The non-human animal may be a transchromosomal animal, for example a non-human animal comprising a human artificial chromosome or a yeast artificial chromosome.

The humanized immunoglobulin loci may be present on a vector, such as a human artificial chromosome or a Yeast Artificial Chromosome (YAC). Human Artificial Chromosomes (HACs) comprising humanized immunoglobulin loci can be introduced into animals. The vector (e.g., HAC) may contain a germline pool of human antibody heavy chain genes (from human chromosome 14) and human antibody light chain genes (e.g., one or both of kappa (from human chromosome 2) and lambda (from human chromosome 22)). HACs can be transferred into cells of non-human animal species and transgenic animals can be produced by somatic cell nuclear transfer. Transgenic animals can also be bred to produce non-human animals comprising humanized immunoglobulin loci.

In some embodiments, the humanized immunoglobulin locus is integrated into the genome of a non-human animal. For example, techniques involving homologous recombination or homology directed repair can be employed to modify the genome of an animal to introduce a human nucleotide sequence. Tools such as CRISPR/Cas, TALENs and zinc finger nucleases can be used for targeted integration.

Methods of generating non-human animals having a humanized immune system (e.g., non-human animals for immunization having a humanized immune system) have been disclosed. For example, a human artificial chromosome can be generated and transferred into a cell that contains other genomic modifications of interest (e.g., a deletion of endogenous non-human immune system genes), and the cell can be used as a nuclear donor to generate a transgenic chromosomal non-human animal.

In some embodiments, the humanized immune system comprises one or more human antibody heavy chains, wherein each gene encoding an antibody heavy chain is operably linked to a type-switching regulatory element. Operatively linked may refer to a first DNA molecule (e.g., a heavy chain gene) being linked to a second DNA molecule (e.g., a type-switching regulatory element), wherein the first and second DNA molecules are arranged such that the first DNA molecule can affect the function of the second DNA molecule. The two DNA molecules may or may not be part of a single continuous DNA molecule and may or may not be contiguous. For example, a promoter is operably linked to a transcribable DNA molecule if it is capable of affecting the transcription or translation of the transcribable DNA molecule.

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 antibodies in place of the light chain.

In some embodiments, the humanized immune system comprises an amino acid sequence derived from a non-human animal, for example a constant region (such as a heavy chain constant region or portion thereof). In some embodiments, the humanized immune system comprises an IgG (e.g., igGl) heavy chain constant region from a non-human animal (e.g., an ungulate-derived IgG (e.g., igGl) heavy chain constant region). In some embodiments, at least one type of switching regulatory element of a gene encoding one or more human antibody heavy chains is replaced with, for example, a non-human (e.g., ungulate-derived) type switching regulatory element to allow for antibody class switching when antibodies to the antigens and/or epitopes of the disclosure are produced in a non-human animal.

The humanized immunoglobulin loci may comprise non-human elements that are incorporated to be compatible with non-human animals. In some embodiments, non-human elements may be present in the humanized immunoglobulin loci to reduce recognition of any remaining elements in the non-human animal immune system. In some embodiments, immunoglobulin genes may be partially replaced by amino acid sequences from non-human animals. In some embodiments, a non-human regulatory element may be present in a humanized immunoglobulin locus to facilitate expression and regulation of the locus in a non-human animal.

The humanized immunoglobulin locus may comprise a human DNA sequence. The humanized immunoglobulin loci may be codon optimized to facilitate expression of the encompassed genes (e.g., antibody genes) in non-human animals.

A non-human animal having a humanized immune system (e.g., a non-human animal for immunization having a humanized immune system) may comprise or lack endogenous non-human immune system components. In some embodiments, a non-human animal having a humanized immune system may lack non-human antibodies (e.g., lack the ability to produce non-human antibodies). The non-human animal having 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.

A non-human animal with a humanized immune system (e.g., a non-human animal for immunization with a humanized immune system) may retain, for example, non-human immune cells. A non-human animal with a humanized immune system may retain non-human innate immune system components (e.g., cells, complement, antimicrobial peptides, etc.). In some embodiments, a non-human animal having a humanized immune system may retain non-human T cells. In some embodiments, a non-human animal with a humanized immune system may retain non-human B cells. In some embodiments, a non-human animal having a humanized immune system may retain non-human antigen presenting cells. In some embodiments, a non-human animal having a humanized immune system may retain non-human antibodies.

In some embodiments, a non-human animal having a humanized immune system (e.g., a non-human animal having a humanized immune system for immunization) comprises any feature or any combination of features or any method of preparation as disclosed in U.S. patent application publication No. 2017/023459, the entire contents of which are incorporated herein by reference. In some embodiments, a non-human animal having a humanized immune system (e.g., a non-human animal having a humanized immune system for immunization) comprises any feature or any combination of features or any method of preparation as disclosed below: 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); matsushit et al, PLoS ONE,10 (6): e 013699 (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, anti-viral Res.,146:164-173 (2017); silver, clin. Effect. Dis.,66 (7): 1116-1119 (2018); beigel et al, lancet effect. Dis.,18 (4): 410-418 (2018); luke et al, j.inf.dis.,218 (journal_5): S636-S648 (2018), each of which is incorporated herein by reference in its entirety.

Also provided herein are antibodies (e.g., nanobodies or heavy chain antibodies) comprising CDRs described herein (e.g., as set forth in table 2, fig. 37, or SEQ ID NOs: 1-24). Such antibodies may be provided as human, humanized or mouse antibodies. In some cases, an antibody provided herein (e.g., a nanobody or a heavy chain antibody) can include CDRs as described herein (e.g., as set forth in table 2, fig. 37, or SEQ ID NOs: 1-24) and can be a monoclonal antibody (e.g., a monoclonal nanobody or a monoclonal heavy chain antibody).

In some cases, an antibody provided herein (e.g., a nanobody or a heavy chain antibody) can include three CDRs. The first CDR may 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., additions, deletions or substitutions). The second CDR may be selected from the group consisting of: SEQ ID NOS 8-15 or SEQ ID NOS 8-15 with one, two or three amino acid modifications (e.g., additions, deletions or substitutions). The third CDR may be selected from the group consisting of: SEQ ID NOS 16-24 or SEQ ID NOS 16-24 with one, two or three amino acid modifications (e.g., additions, deletions or substitutions).

Table 2. Exemplary CDRs of heavy chain antibodies capable of binding SARS-CoV2 spike antigen.

In some cases, an antibody provided herein (e.g., a nanobody or a heavy chain antibody) can be or can have a heavy chain variable domain having CDR1 with the amino acid sequence of SEQ ID No. 1, CDR2 with the amino acid sequence of SEQ ID No. 8, and CDR3 with the amino acid sequence of SEQ ID No. 16. In some cases, an antibody provided herein (e.g., a nanobody or a heavy chain antibody) can be or can have a heavy chain variable domain having CDR1 with the amino acid sequence of SEQ ID No. 2, CDR2 with the amino acid sequence of SEQ ID No. 8, and CDR3 with the amino acid sequence of SEQ ID No. 17. In some cases, an antibody provided herein (e.g., a nanobody or a heavy chain antibody) can be or can have a heavy chain variable domain having CDR1 with the amino acid sequence of SEQ ID No. 3, CDR2 with the amino acid sequence of SEQ ID No. 8, and CDR3 with the amino acid sequence of SEQ ID No. 18. In some cases, an antibody provided herein (e.g., a nanobody or a heavy chain antibody) can be or can have a heavy chain variable domain having CDR1 with the amino acid sequence of SEQ ID No. 4, CDR2 with the amino acid sequence of SEQ ID No. 9, and CDR3 with the amino acid sequence of SEQ ID No. 19. In some cases, an antibody provided herein (e.g., a nanobody or a heavy chain antibody) can be or can have a heavy chain variable domain having CDR1 with the amino acid sequence of SEQ ID No. 4, CDR2 with the amino acid sequence of SEQ ID No. 10, and CDR3 with the amino acid sequence of SEQ ID No. 19. In some cases, an antibody provided herein (e.g., a nanobody or a heavy chain antibody) can be or can have a heavy chain variable domain having CDR1 with the amino acid sequence of SEQ ID No. 5, CDR2 with the amino acid sequence of SEQ ID No. 11, and CDR3 with the amino acid sequence of SEQ ID No. 20. In some cases, an antibody provided herein (e.g., a nanobody or a heavy chain antibody) can be or can have a heavy chain variable domain having CDR1 with the amino acid sequence of SEQ ID No. 6, CDR2 with the amino acid sequence of SEQ ID No. 12, and CDR3 with the amino acid sequence of SEQ ID No. 21. In some cases, an antibody provided herein (e.g., a nanobody or a heavy chain antibody) can be or can have a heavy chain variable domain having CDR1 with the amino acid sequence of SEQ ID No. 5, CDR2 with the amino acid sequence of SEQ ID No. 13, and CDR3 with the amino acid sequence of SEQ ID No. 22. In some cases, an antibody provided herein (e.g., a nanobody or a heavy chain antibody) can be or can have a heavy chain variable domain having CDR1 with the amino acid sequence of SEQ ID No. 7, CDR2 with the amino acid sequence of SEQ ID No. 14, and CDR3 with the amino acid sequence of SEQ ID No. 23. In some cases, an antibody provided herein (e.g., a nanobody or a heavy chain antibody) can be or can have a heavy chain variable domain having CDR1 with the amino acid sequence of SEQ ID No. 5, CDR2 with the amino acid sequence of SEQ ID No. 15, and CDR3 with the amino acid sequence of SEQ ID No. 24.

In some cases, CDR3 shown in table 2 may lack the first C residue and may lack the last W residue.

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

In some cases, the amino acid substitutions within the linker sequence identifier may be non-conservative amino acid substitutions. Non-conservative amino acid substitutions may be made by substituting one amino acid residue for another amino acid residue having a different side chain. Examples of non-conservative substitutions include, but are not limited to: (a) Substitution of a hydrophobic residue (e.g., leucine, isoleucine, phenylalanine, valine, or alanine) with a hydrophilic residue (e.g., serine or threonine); (b) Substitution of cysteine or proline for any other residue; (c) Substitution of a residue with an acidic side chain (e.g., amino acid or glutamic acid) with a residue with a basic side chain (e.g., lysine, arginine, or histidine) and (d) substitution of glycine or other residue with a small side chain with a residue with a large side chain (e.g., phenylalanine).

Methods for producing amino acid sequence variants (e.g., comprising one or more modified amino acid sequences relative to a linker sequence identifier) may include site-specific mutagenesis or random mutagenesis (e.g., by PCR) of a nucleic acid encoding the antibody or fragment thereof. See, e.g., zoller, curr. Opin. Biotechnol.3:348-354 (1992). Both naturally occurring and non-naturally occurring amino acids (e.g., amino acids of artificial origin) can be used to generate the amino acid sequence variants provided herein

Also provided herein are pharmaceutical compositions or pharmaceutical formulations, which can include any of the antibodies provided herein (e.g., nanobodies or heavy chain antibodies). Any pharmaceutical composition or pharmaceutical formulation may also include other cells or cell components.

As described herein, an antigen can be administered to a non-human animal provided herein to produce an antibody (e.g., a heavy chain antibody (such as a chimeric heavy chain antibody)). In some embodiments, the antigen is an endogenous antigen or autoantigen of a subject (e.g., a mammal, such as a human, cow, horse, non-human primate, rabbit, goat, sheep, dog, pig, mouse, rat).

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 products-LDL (AGE-LDL), MDA and Lysophosphatidylcholine (LPC).

In some embodiments, the antigen is associated with an immune cell (e.g., 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.

In some embodiments, the antigen is a peptide, protein, lipid, molecule, or other biological compound that binds to an immune cell.

In some embodiments, the antigen is associated with a damaged cell, a dead cell, or a dying cell. Cell damage and/or death may be caused by any potential pathology (such as apoptosis, necrosis, ischemia, etc.).

In some embodiments, the antigen is another immunoglobulin, such as IgG.

In some embodiments, the antigen may be an antigen listed in table 3.

Table 3. Examples of antigens useful for immunizing non-human animals provided herein.

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Also provided herein are methods of treating or preventing a disease or disorder. In some embodiments, the antibodies provided herein (e.g., nanobodies or heavy chain antibodies) can be used to treat or prevent a disease or disorder. For example, the disease or disorder may be an inflammatory disease, an autoimmune disease, a cardiovascular disease, or a neurodegenerative disease. In some embodiments, the disease or condition may be diabetes, systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, scleroderma, crohn's disease, ulcerative colitis, mixed connective tissue disease, sjogren's syndrome syndrome) or polymyositis, dermatomyositis.

In some cases, compositions comprising an antibody provided herein (e.g., a nanobody or heavy chain antibody) are intended for use in the prevention and/or treatment of a disease or disorder (e.g., an autoimmune disease or inflammatory disorder). Accordingly, further provided herein is a pharmaceutical formulation comprising an antibody (e.g., nanobody or heavy chain antibody) provided herein and a pharmaceutically acceptable carrier thereof. Pharmaceutical formulations may be prepared by conventional techniques, e.g., as in "leimington: described in pharmaceutical science and practice (Remington: the Science and Practice of Pharmacy) 2005, LWW Press (Lippincot, williams & Wilkins).

The pharmaceutically acceptable carrier may be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. The solid carrier may be one or more excipients which may also function as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents, or an encapsulating material.

Also included are solid form preparations for conversion to oral liquid form preparations immediately prior to use. Such liquid forms include solutions, suspensions and emulsions. These formulations may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like, as described elsewhere (Gervasi et al, eur. J. Pharmaceuticals and Biopharmaceutics,131:8-24 (2018)).

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.

Other ingredients 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 ethylenediamine tetraacetic acid (EDTA), anticancer agents such as enzalutamide (enzalutamide), imatinib (imatinib), gefitinib (gefitinib), erlotinib (erlotini), sunitinib (supininib), lapatinib (lapatinib), nilotinib (nilotinib), sorafenib (sorafenib), temsirolimus (temsirolimus), everolimus (everolimus), pazopanib (pazopanib), crizotinib (ruxolitinib), acetinib (axitinib), bosutinib (botinib), cabatinib (cabatinib), bostinib (lapatinib), oxatinib (opium), or combinations thereof, and combinations thereof.

In some cases, the antibodies provided herein (e.g., nanobodies or heavy chain antibodies) can be formulated for parenteral administration and can be presented in unit dosage form in ampoules, prefilled syringes, small volume infusions, or multi-dose containers, optionally with the addition of a preservative. The composition may take the form of a suspension, solution or emulsion, such as in an oily or aqueous vehicle, for example a solution in aqueous polyethylene glycol. Examples of oily or nonaqueous vehicles, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters, and may contain agents such as preservatives, wetting agents, emulsifying or suspending agents, stabilizing and/or dispersing agents. In some cases, the formulation may contain about 0.5% to 75% by weight of the active ingredient, with the remainder being composed of suitable pharmaceutical excipients as described herein.

The compositions provided herein may be administered simultaneously (concurrently), simultaneously (simultaneously) or together with a pharmaceutically acceptable carrier or diluent, whether by oral, rectal or parenteral (including subcutaneous) route, particularly and preferably in the form of a pharmaceutical composition thereof, in an effective amount.

Also provided herein are compositions comprising B cells (e.g., B cells isolated from a non-human animal 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 comprise adjuvants, buffers, salts, or combinations thereof.

Adjuvants are pharmacological and/or immunological agents that modify the effects of other agents. In some embodiments, adjuvants may be added to the composition to alter the immune response by boosting it to provide more antibodies and/or longer lasting protection, thereby minimizing the amount of antigenic material injected. Adjuvants may also be used to enhance the efficacy of the composition by helping to disrupt an immune response against a particular cell type of the immune system, e.g., activating T cells instead of B cells secreting antibodies depending on the type of composition. In one embodiment, the composition may comprise at least one adjuvant. In another embodiment, the adjuvant may be aluminum-based. The aluminum adjuvant may be aluminum phosphate, aluminum hydroxide, amorphous aluminum hydroxy phosphate sulfate, and/or combinations thereof. Other adjuvants may also be included.

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 embodiments, the buffer may be isotonic (such as 0.6% -1.8% NaCl buffer).

An emulsifier (emulgent) is a substance that stabilizes an emulsion by improving its kinetic stability. One type of emulsifier is known as "surfactants" (surface active agents) or surfactants). Polysorbates are a class of emulsifiers used in some pharmaceutical and food formulations. Common brand names for polysorbates include Alkest, canarcel and Tween. Some examples of polysorbates are polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80. In one embodiment, the compositions provided herein may comprise an emulsifier (such as one of the polysorbates described above). In one embodiment, the composition may comprise 0.001-0.02% polysorbate 80. Other polysorbates or emulsifiers may also be used as described herein.

In some cases, a pharmaceutical composition provided herein can comprise an antibody (e.g., a nanobody or a heavy chain antibody) provided herein and a pharmaceutically acceptable carrier. In some cases, a pharmaceutical composition provided herein can comprise an antibody (e.g., a nanobody or a heavy chain antibody) provided herein, a pharmaceutically acceptable carrier, and a buffer. In some cases, a pharmaceutical composition provided herein can comprise an antibody (e.g., a nanobody or a heavy chain antibody) provided herein, a pharmaceutically acceptable carrier, and an emulsifier. In some cases, a pharmaceutical composition provided herein can comprise an antibody (e.g., a nanobody or a heavy chain antibody) provided herein, a pharmaceutically acceptable carrier, and an adjuvant. In some cases, a pharmaceutical composition provided herein can comprise an antibody (e.g., a nanobody or a heavy chain antibody) provided herein, a pharmaceutically acceptable carrier, a buffer, and an adjuvant. In some cases, a pharmaceutical composition provided herein can comprise an antibody (e.g., a nanobody or a heavy chain antibody) provided herein, a pharmaceutically acceptable carrier, an emulsifier, and an adjuvant. In some cases, a pharmaceutical composition provided herein can comprise an antibody (e.g., a nanobody or a heavy chain antibody) provided herein, a pharmaceutically acceptable carrier, an emulsifying agent, a buffering agent, and an adjuvant.

Any suitable method may be used to design and construct the nucleic acid and polypeptide reagents described herein. In general, recombinant methods may be used. In general, see Smales and James (editors), "therapeutic proteins: methods and protocols (methods in molecular biology) (Therapeutic Proteins: methods and Protocols (Methods in Molecular Biology)), humana Press (2005); and Crommelin, sindelar and Meibohm (editions), "pharmaceutical biotechnology: basic knowledge and applications (Pharmaceutical Biotechnology: fundamentals and Applications), springer Press (2013). Methods for designing, preparing, evaluating, purifying, and manipulating nucleic acid compositions are described in Green and Sambrook (editions), molecular cloning: laboratory Manual (Fourth Edition) (Molecular Cloning: A Laboratory Manual (Fourth Edition)), cold spring harbor laboratory Press (Cold Spring Harbor Laboratory Press) (2012).

Also provided herein are methods of administering a composition (e.g., a pharmaceutical composition provided herein) comprising an antibody (e.g., a heavy chain antibody or a nanobody) provided herein to a mammal (e.g., a human) to treat a disease or disorder. For example, a composition (e.g., a pharmaceutical composition provided herein) comprising one or more antibodies provided herein can be administered to a mammal (e.g., a human) having an inflammatory disease to treat the mammal. In some cases, a composition (e.g., a pharmaceutical composition provided herein) comprising one or more antibodies provided herein can be administered to a mammal (e.g., a human) to reduce the severity of an inflammatory disease in the mammal and/or to increase the survival rate of a mammal having an inflammatory disease as compared to a mammal (e.g., a human) without the composition.

The compositions described herein may be administered to a subject by any mode of delivery, including, for example, by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or interstitial space), or by rectal, oral (e.g., tablet, spray), vaginal, topical, transdermal (see, e.g., international PCT patent application publication No. WO 99/27961) or transdermal (see, e.g., international PCT patent application publication nos. WO02/074244 and WO 02/064162), intranasal (see, e.g., international PCT patent application publication No. WO 03/028760), ocular, aural, pulmonary, or other mucosal administration. Multiple doses may be administered by the same or different routes.

The compositions provided herein (e.g., compositions containing heavy chain antibodies and/or nanobodies produced by non-human animals provided herein) can be administered prior to, concurrently with, or after delivery of other therapeutic agents. Moreover, the site of administration may be the same as or different from the other therapeutic agents being administered.

Dosage treatments using the compositions provided herein may be single dose regimens or multiple dose regimens. A multi-dose regimen is one in which the primary vaccination course may be 1-10 separate doses, with additional doses administered in subsequent time intervals, selected to maintain and/or enhance the immune response, e.g. a second dose at 1-4 months, and if desired, subsequent doses several months later. The dosage regimen will also be determined at least in part by the efficacy of the treatment regimen, the delivery employed, the needs of the subject, and will depend on the discretion of the practitioner.

The above-described functional characteristics may be achieved by combining the above-described embodiments. This is also illustrated by the following examples showing exemplary combinations and functional features implemented.

Examples

EXAMPLE 1 genetic engineering of an odd mice

Manipulation of genetic engineering

Blastocysts isolated from the 129S6 and C57BL/6N mice crosses were used to establish the LVGN-YF ES cell line (40, XY) and used for all genetic engineering. The F1 hybrid ES cell line exhibits robust germline capability after multiple rounds of genetic modification, and the use of the F1 hybrid ES cell line also allows for the strain-specific SNP to recognize consecutive genetic modifications occurring on the same chromosome. ES cells are supplemented with 20% ES cell-qualified fetal bovine serumFBS), 0.1mM MEM nonessential amino acids, 0.1mM 2-mercaptoethanol, 1mM sodium pyruvate, 2mM GlutaMAX-I supplement, 100 units/mL penicillin-streptomycin, 25nM MEK inhibitor PD98059 (Sigma)), 3nM GSK-3 inhibitor CHIR99021 (Sigma), and knockout DMEM (Knockout DMEM) of 1,000 units/mL mouse leukemia inhibitory factor (LIF, sigma) were cultured on feeder cells. hygro ^R /neo ^R /puro ^R Triple resistant feeder line LVGN-SHNPL was engineered from the SNL76/7 feeder line and cultured in knock-out DMEM supplemented with 10% ES cell-conforming FBS, 0.1mM MEM nonessential amino acids, 0.1mM 2-mercaptoethanol, 1mM sodium pyruvate, 2mM Glutamax-I supplement, and 100 units/mL penicillin-streptomycin. Feeder cells were prepared by treating the proliferating cells with 10mg/mL mitomycin C (Sigma Co.) for 3 hours.

All transfection was done by lipofection using Lipofectamine LTX (thermo fisher) or electroporation using a Bio-Rad gene pulser II device. For lipofection, the instructions provided by the manufacturer will be 0.1-1X10 ⁶ The dissociated ES cells were mixed with 0.5-2.5. Mu.g of plasmid DNA-lipofectamine complex in Opti-MEM and cultured overnight in growth medium, 24 hours later for antibiotic selection as desired. For electroporation, 0.5-1.5x10 will be ⁷ The individual ES cells were mixed with 10-30. Mu.g of plasmid and/or BAC DNA in PBS and electroporated at 250V/500. Mu.F in 4mm gap cuvettes and incubated overnight in growth medium, after 24 hours antibiotic selection was performed as required. The antibiotic concentrations used for selection were: geneticin (G418) was 250. Mu.g/mL, hygromycin B was 200. Mu.g/mL, puromycin was 5. Mu.g/mL. For CRISPR-mediated gene editing, cas9 and sgrnas are delivered on different plasmids, co-transfected with PGK-puro or PGK-hygro genes, and selected with the corresponding antibiotics for 2 days to enrich for transfectants, or co-transfected with HDR templates and selected with the corresponding antibiotics for 10-14 days to obtain stably transfected clones. To remove the selection marker cassette flanked by recombination sites (lox-lox, frt-frt or attB-attP), ES cells are transiently transfected with plasmids expressing the corresponding recombinase or integrase (Cre, flp or phiC31, respectively) and at low density Plates were used to isolate individual clones. For recombinase-mediated cassette exchange (RMCE), ES cells were co-transfected with RMCE construct and Cre expression plasmid and selected with the corresponding antibiotic for 10-14 days. ES cell colonies were picked into 96-well plates for amplification and genotyped by PCR to screen for the desired mutation, followed by Mulberry sequencing (Sanger sequencing). Positive clones that were sequence verified were amplified from 96-well plates to 24-well plates, then to 6-well plates and cryopreserved.

Positive ES cell lines carrying engineered mutations were used to generate chimeras according to standard procedures. Briefly, blastula were isolated from superovulated C57BL/6N females at 3.5dpc, microinjected with ES cells, and then transferred to the uterus of 2.5dpc pseudopregnant Swiss Webster females for implantation. A high percentage of chimeric males mate with C57BL/6N females to achieve germline transmission of the engineered mutation. Heterozygous F1 mice were identified by ligation PCR on genomic DNA isolated from biopsies. The F1 mice are then crossed to produce homozygous F2 mice with the same mutation, or crossed with other strains as desired. All engineered mice were cultured in a 129S6 and C57BL/6N mixed background.

Generation of the Extra murine allele

Igh is one of the largest loci in the mouse genome, spanning several megabases (Mb) near the right end of the q-arm of chromosome 12. It encodes a number of different elements that are involved in the generation of almost endless antibody diversity. This locus contains variable regions of more than 2.5Mb encoding hundreds of gene segments responsible for most antibody diversity, and a much smaller 220kb constant CH region encoding expression of multiple antibody classes and subtypes (fig. 1A). The CH region has 8 CH genes encoding different Ig isoforms: mu (Ighm), delta (Ighd), gamma 3 (Ighg 3), gamma 1 (Ighg 1), gamma 2b (Ighg 2 b), gamma 2a/2C (Ighg 2 a/2C), epsilon (Ighe) and alpha (Igha). Regulatory elements flanking this region and located throughout this region are involved in type switching recombination (CSR) and in the timely expression of isoforms (fig. 2).

In addition to the elements described above, located upstream of each Ig isotype (except IgD) are the I promoter/exon and S switch regions. The latter is involved in CSR, which is involved in germline transcription of its corresponding Ig isotype. Transcription of Ig isoforms is highly regulated. In resting B cells, germ Line Transcription (GLT) is limited to cμ transcription, driven by the E μ enhancer and the constitutive I μ promoter. In activated B cells in response to antigen encounter and cytokines, transcription of the downstream Ig isotype is activated at its I promoter containing the corresponding response element. Simultaneous transcription at the I.mu.promoter and the activated Ig isotype downstream I promoter results in AID-mediated CSR.

The step-wise procedure was used to generate the odd murine allele (fig. 1B). Unlike normal tetrameric antibodies generated by wild-type (WT) mice (fig. 1C), mice homozygous for this allele only produced hcabs (fig. 1D), whose genetic composition and diversity were entirely derived from the mouse's natural immune repertoire.

The generation of the odd murine allele was accomplished by 3 rounds of genetic engineering in LVGN-YF ES cells (FIG. 3). The Igh loci of these ES cells encode a Cγ2c similar to that present in C57BL/6N mice, but not to Cγ2a in the BALB/C strain (FIG. 3A). The first round of engineering was by CRISPR mediated non-homologous end joining (NHEJ) which removed the 92.6Kb genomic DNA fragment spanning the first exons of cμ, cδ, cγ3 and cγ1, which encode the CH1 domain of IgG1 (fig. 3B). The sgrnas were designed to cleave directly downstream of the cμ switch region (sμ) and upstream of the cγ1 second exon (which encodes the hinge domain of IgG 1), placing the truncated cγ1 gene under direct control of the I μ promoter and sμ, making the original cytokine-induced IgG1 transcription constitutive, as is the case for IgM in the WT allele. The second round of engineering was by CRISPR mediated Homology Directed Repair (HDR), which removes the 63.2Kb genomic DNA fragment spanning the first three exons of cγ2b, cγ2c, cε and cα, while introducing a selectable marker cassette (PGK/Em 7-neo) flanked by frt sites (fig. 3C). The CRISPR cleavage site was selected to avoid removal of the 3'γ1e element downstream of cγ1 and the 5' hsr1 element within cα intron 3. The third round of engineering utilized transient expression of Flp recombinase, removing the selectable marker cassette, leaving a single frt site for the co-linear validation of subsequent modifications (fig. 3D). Regulatory elements (including eμ, iμ, sμ, 3'γ1e, 5' hsr1, 3'rr, and 3' cbe) were kept intact to allow for high level constitutive transcription of CH1 truncated IgG1 (igg1Δch1) of the endogenous Igh allele (fig. 2 and 3E). The thus generated mice of the idiopathic murine species only produce hcabs of the IgG1 subtype; all other antibody types (IgM, igD, igE and IgA) and IgG subtypes (IgG 2b, igG2c and IgG 3) were eliminated to avoid any potential mechanism that would impair HCAb production and facilitate nanobody discovery, expression and purification.

Generation of odd superposition alleles

To extend the versatility of the odd platform and generate hcabs from other species, the odd hyper-berth allele was generated by removing 2.58Mb genomic DNA fragments containing all mouse VH, DH and JH genes (from Ighv86-1 upstream to Ighj4 downstream) and inserting a consecutive RMCE docking cassette upstream of E μ through CRISPR mediated HDR (fig. 4 and 5). The HDR template contains a left homology arm, a frt site, an attB site, a PGK promoter, a loxP site, an Em7-neo cassette, an attP site and a lox2272 site, followed by a right homology arm. The frt site was included to verify that the introduced RMCE docking cassette was located on the same chromosome (C57 BL/6N) as the odd murine allele after Flp recombinase expression. The wild-type loxP site (and not the other iso-specific lox sites) was selected to be placed between the PGK promoter and the Em7-neo cassette to achieve efficient RMCE events by selection marker exchange. These modifications produced a mouse VDJ null odd-superposition allele that contained RMCE docking sites for sequential introduction of BACs, cloning constructs, or synthesis of fragments containing any combination of fragments of V, D or J genes of human or other species heavy or light chain alleles (fig. 5).

Generation of an odd-numbered human allele series

Engineered BACs containing human VH, DH, and JH genes were introduced into the odd superpositioned alleles to generate an odd series of human alleles (fig. 6-8). Overlapping IGH BAC clones (FIG. 9 and Table 4) from the CH17 BAC library and RPCI-11 library (BACPAC resource) were modified at both ends by bacterial homologous recombination (recombinant engineering) to incorporate Em7-hyg or Em7-neo cassettes to allow for selectable marker exchange while introducing a DNA flanking the genomic sheetThe corresponding iso-specific lox sites of the segments (table 1) were used for the sequential RMCE. Briefly, synthetic gBlock (IDT or Twist) containing two 75-150 base pair (bp) homology arms flanked by appropriate lox sites and an antibiotic resistance cassette was electroporated into E.coli strains containing heat-inducible Red recombinase at 1.75kV, 25. Mu.F and 200 ohm using a Bio-Rad Gene pulser II device in a 1-mm gap electroporation cuvette. Next, 1.0mL of SOC medium was added to each cuvette, transferred to a microcentrifuge tube, and then incubated for 1 hour with shaking (200 rpm) at 32 ℃. Cells were then plated onto LB agar plates containing the corresponding antibiotics. The resulting colonies were screened by PCR using the ligation primers and then verified by sanger sequencing. FIG. 10 illustrates the recombinant engineering process of the first introduced BAC (hIGH-BAC 1) containing 3 human VH genes (two functional; IGHV1-2 and IGHV 6-1), 27 human D _H Gene and 9 human J _H And (3) a gene. A loxP-Em7-hyg-attP-lox5171 cassette is introduced immediately upstream of the human IGHV1-2 gene at the 5 'end of the original BAC clone by recombinant engineering, and then a lox2272-aadA cassette is introduced immediately downstream of the human IGHJ6 gene at the 3' end. The engineered BACs were then used in the first round of RMCE (between loxP and lox 2272) to introduce three human VH genes, all human DH genes and all human JH genes immediately upstream of the mouse Igh intron enhancer eμ, while introducing a different, iso-specific lox site (lox 5171) for the next round of RMCE (fig. 7A-7B). Subsequent overlapping BACs were modified in a similar manner, but using alternate selection markers (Em 7-neo and Em 7-hyg) and different iso-specific lox sites, with overlapping fragments trimmed away to gradually assemble the human VDJ genomic region (fig. 7C-7D and 8). The original BAC clones and the bispecific lox sites used to reconstruct the fully human VDJ region are shown in table 4 and table 1, respectively. Wild-type loxP sites exhibiting high recombination efficiency were paired with different hetero-specific lox sites for each round of RMCE. All engineered BACs were confirmed by PacBio SMRT sequencing prior to transfection into ES cells. No significant mutations were found except for several SNPs and small indels in the intergenic region. The sequential RMCE process resulted in the generation of a series of humanized odd alleles (SSVs 1-5) with increased VH diversity. By means of After PCR analysis and sanger sequencing of SSV4 mice, VH-specific primers confirmed the integration of all 37 functional VH elements (fig. 11). hIGH-BAC5 was engineered to contain sequences from three source BACs (FIG. 12) and SSV4 ES was introduced by RMCE to complete the construction of SSV5, where SSV5 was designed to contain the complete human VH repertoire (126 VH, 27 DH and 9 JH genes).

TABLE 4 human genomic coordinates (GRCh 38/hg 38) of engineered BACs and source BACs used in the S.SSV 1-SSV5 engineering of the idiosyncratic human allele series.

Generation of Igk and Igl knockouts for the production of light chain-free odd mice

To prevent the light chain from causing unnecessary interference to the production of hcabs by odd mice, the mouse light chain was removed by removing V and J gene fragments from the IgK and IgL loci.

To remove kappa light chains, the 3.17Mb genomic DNA fragment containing the entire mouse VK and JK gene fragments was removed by CRISPR/Cas9 mediated HDR and replaced with a docking cassette (fig. 13A). Similar to the odd superpositions at the Igh allele, the engineered IgK superpositions/KO alleles containing attB sites upstream of the 5 'enhancer element, PGK promoter, loxP sites, em7-neo cassette, attP sites and lox2272 sites are located 5' to the mouse CK gene, allowing for continuous RMCE at the IgK alleles. Engineered Igk superposition/KO mice were generated and confirmed by PCR and sequencing (fig. 13B).

To remove the lambda light chain, an about 200kb genomic DNA fragment between Olfr164 and Gm10086 was removed by CRISPR/Cas9 mediated NHEJ, which contained the entire lambda locus, including VL1, VL2, VL3 and all JL and CL gene segments (fig. 14A). Engineered Igl KO ES cells were generated and confirmed by PCR and sequencing (fig. 14B).

Example 2 characterization of an odd mouse

Odd mouse constitutively expresses CH1 truncated IgG1 heavy chain-only antibodies

To confirm this, in contrast to WT mice that can express all Ig isotypes (fig. 15A), the idiosyncratic murine mice expressed only IgG1- Δch1 (fig. 15B), and transcripts of different Ig classes and subtypes were analyzed. Total RNA was isolated from spleens of WT and odd murine mice using Trizol reagent and RNA concentration and quality were determined using a Bioanalyzer (Bioanalyzer). Reverse transcription was performed using Superscript IV reverse transcriptase (sameiser) and oligo (dT) 20 primers according to manufacturer's instructions. Transcription of all Ig types and subtypes was analyzed by RT-PCR according to standard procedures, with the mouse B cell marker Cd19 as an internal control. Although Ighm, ighd, ighg, ighg1, ighg2b, ighg2C, ighe and Igha were all detected in WT mice, the idiosyncratic murine mice only expressed Ighg1 transcripts of reduced size (FIG. 15C) and were sequencing-verified to lack CH1 sequence.

To examine the transcription of Ighg1 in the idiopathic human mice, RT-PCR was performed on cDNA reverse transcribed from total spleen RNA of the idiopathic human (SSV 1) mice. SSV1 (FIG. 16A) from an curious murine platform was designed to contain a complete human D _H And D _J Group and two functional human V _H (IGHV 6-1, IGHV1-2) (FIG. 16B). The PCR primers were designed with a set of forward primers specific for human IGHV6-1, IGHV1-2 and IGHJ3 and reverse primers specific for mouse Ighg1 CH 2. Chimeric transcripts with human VDJ-mouse Ighg 1-. DELTA.CH1 were detected in the odd human (SSV 1) but not in the odd murine mice (FIG. 16C), which were further verified for correct splicing by sequencing (FIG. 16D).

To detect protein expression of different Ig types and subtypes, immunoglobulins in plasma samples of immunized wild-type and odd-line mice were purified using protein A/G magnetic beads, separated by reducing SDS-PAGE, and transferred to electrophoresis according to standard proceduresP on membrane (Millipore Sigma). Immunodetection using HRP-conjugated secondary antibodies and enhanced chemiluminescence and autoradiography using ECL Western blotting reagentsAnd (5) shadow. Truncated IgG1 of about 40kDa was detected in the odd murine mice (compared to the full length IgG1 of about 50kDa in wild type mice), whereas IgM and IgG2b were not detected (fig. 15D). Similarly, truncated IgG1 of about 40kDa (corresponding to human VDJ mouse IgG1- Δch1) was detected in immunized, odd human mice (SSV 1) that did not express IgM or full length IgG1 seen in wild-type mice (fig. 17).

Up-regulation of IgG expression in odd mouse B cells

The shape and size of the spleens of the mice of the odd-numbered mice were similar to those of the wild-type mice (fig. 18A). To examine IgM and IgG expression on B cell membranes, single cell suspensions were prepared with spleen, treated with ACK lysis buffer to remove erythrocytes, blocked with Fc blocker, and stained in FACS buffer (PBS with 1% FBS), rat anti-mouse IgM (PE-Cy 7), rat anti-mouse IgG (BV 421), and rat anti-mouse CD19 (AF 700). After staining, the cells were analyzed by flow cytometry (BD LSR II). Although IgM was not detected in the odd murine mice ⁺ B cell, detected IgG ⁺ The proportion of B cells is significantly higher than that of IgG in wild-type mice ⁺ B cell proportion, which is consistent with increased expression levels resulting from IgG1 constitutive high-level germ line transcription (fig. 18B). Similarly, splenocytes from both the odd human line (SSV 2) and wild type mice were FACS analyzed (FITC) using the procedure described above with rat anti-mouse IgM (APC), rat anti-mouse IgG1 (APC), rat anti-mouse IgD, and rat anti-mouse B220 (PerCP-Cy5.5). Analysis IgM in SSV2 mice was not detected ⁺ Or IgD ⁺ B cells, but a large amount of IgM was found in wild type mice ⁺ Or IgD ⁺ B cells (fig. 19A). IgG1 detected in SSV2 mice ⁺ The cell fraction was significantly higher (fig. 19B).

Robust humoral immune response in odd mice

Protein antigens (Table 3) were prepared in Phosphate Buffered Saline (PBS) and freshly mixed with either complete Freund's adjuvant (Sigma catalog number 5881, for start of injection) or incomplete Freund's adjuvant (Sigma catalog number 5506, for boost injection) at 1:1 (v/v) by repeated passes through two connected syringes until a smooth emulsion was formed. Male or female mice of 4-12 weeks of age were injected using a 1mL syringe and 27 gauge needle. Priming and boosting injections were performed at intervals of 2 weeks at 10-25 μg antigen protein/mouse, with subcutaneous injections into the left and right groins (50 μl) and/or intraperitoneal injections of 100 μl, respectively. Tail venous blood was collected prior to each injection. The last boost was performed intraperitoneally with adjuvant-free antigen protein at week 4 or week 6. Animals were sacrificed 3-4 days later for terminal blood collection and tissue harvest. Blood samples were processed into plasma according to standard procedures.

To determine antibody titers in plasma, ELISA plates were coated with 1 μg/mL antigen protein diluted in PBS, overnight at 4 ℃. After repeated washing with PBST (pbs+0.05% Tween-20) and blocking with SuperBlock (Thermo Fisher), plasma samples were serially diluted in dilution buffer and applied to the plates. After removing unbound protein by multiple washes, bound protein was detected using the corresponding HRP-conjugated secondary antibody, developed with 3,3', 5' Tetramethylbenzidine (TMB) substrate (BM blue, sigma Co.) and used 50. Mu.L 1M H ₂ SO ₄ The reaction was terminated. Absorbance was read at 450 nm. After SAT immunization for 4 weeks (D28), a robust humoral immune response comparable to that of wild-type mice was observed in the odd murine mice (fig. 20A and 20B), and significantly higher titers (SSV 1) were obtained after 6 weeks (D51) for both the odd murine and the odd human (fig. 21A). Similar results were obtained with other immunogens, such as PD-L1 (FIG. 21B).

NGS analysis of the odd mouse VH pool

Total RNA was isolated from spleens of wild-type or odd-line mice immunized with different antigens using Trizol reagent and RNA quality and concentration were determined using a bioanalyzer. The recombinant variable region sequences (VH) of wild-type or odd mice were amplified by 5'cdna end rapid amplification (5' race) for Next Generation Sequencing (NGS). Briefly, reverse transcription was performed using Superscript IV reverse transcriptase, oligo (dT) 20 primer and template switching primer comprising 5' RACE adaptor and Unique Molecular Identifier (UMI) sequence (5 ' -CTACA-CTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNr GrGrGrGrG-3'; SEQ ID NO: 37). The template switched reverse transcribed product was then amplified in a first round of PCR reaction using a 5' -RACE adaptor forward primer (5 ' -CTACCTC-TTTCCCTACACGACCGATCTTCCGATCT-3 '; SEQ ID NO: 38) and a reverse primer specific for the IgGl CH2 domain (5'-GGTGGTTGTGCAGGCCCTCATG-3'; SEQ ID NO: 39). The purified products were further amplified in a second round of PCR using 5' RACE adaptor forward and reverse primers specific for IgGl CH1 domain (5'-CCATGGAGTTAGTTTGGCAGCA-3' for wild type IgG1 transcript; SEQ ID NO: 40) or IgG1 hinge domain (5'-CAAGGCTTACAACCACAATCCCT-3' for odd IgG1 transcript; SEQ ID NO: 41) to ensure that both mouse lines produced about 600bp amplicon (FIG. 22). The resulting nested PCR products were further amplified in a third round of PCR reaction to incorporate Illumina P5 and P7 adaptor sequences for NGS and barcodes to enable sample multiplex analysis. The final 5' race library was purified and sequenced using a 2x 300bp double-ended run on Illumina MiSeq.

Paired-end sequence reads in fastq format were processed and aligned using a KAligner (a proprietary version of the K-mer linking algorithm) (Liao et al, nucleic Acids res.,41 (10): e108 (2013)) to reference germline genes for VH, DH, and JH based on the annotation of the international ImMunoGeneTics information system (IMGT, world Wide Web): IMGT. Org), and identified CDR regions. When CDR3 is identical and there are no more than 2 mismatched nucleotide residues in the sequence, the full-length in-frame sequence is further assembled into a clonotype. Low quality sequences are excluded from assembly. Clonotypes are ordered for each animal based on abundance and further analysis does not include clonotypes with counts less than 5.

A total of 18 samples from wild-type and odd murine mice immunized with SARS-CoV2 spike-active trimer SAT (R & D Systems; catalog No. 10549-CV), PD-L1 (R & D Systems; catalog No. 156-B7), rabbit IgG (Siemens, catalog No. 02-6102), rat IgG (Siemens; catalog No. 31933) or goat IgG (Siemens; catalog No. 31245) were processed for NGS to determine their corresponding VH libraries. Each of all samples recovered more than 100 ten thousand reads, approximately half of which were successfully aligned with the Igh locus (table 3). Notably, the number of clonotypes for all antigens tested in the odd murine mice was significantly higher than in the WT mice, from several fold to over 20 fold. Furthermore, while 79-99 IGHV gene segments against these antigens were utilized in WT mice, significantly higher numbers of IGHVs (103-122) were utilized in the odd murine mice, nearly approaching the theoretical limit (125 functional IGHVs annotated based on the mouse genome GRCm38/mm 10) (fig. 23A and 24 and table 3). Among the various test antigens, the ability of the odd murine mice to acquire a greater number of IGHV segments than WT was very pronounced (fig. 24), probably due to the high levels of IgG1 GLTs in the odd murine mice, while IgG1 GLT in the WT mice was inducible, cytokine dependent expressed, and all other Ig types and subtypes were removed, so that IgG1 was expressed alone, regardless of the immunogen.

Analysis of VH sequences showed that the odd mice exhibited similar diversified use of IGHV gene segments compared to WT mice (fig. 23A and 24A-24B). IGHV gene segments were generated in wild-type mice in higher abundance or in lower abundance clonotypes, as well as in the odd murine mice (FIGS. 24A-24B). Although all 4 IghJ segments were used, ighJ3 was different in the odd murine mice, while IghJ4 exhibited advantages, possibly due to the structural preference of HCAb formation (fig. 23B and 25). No significant differences in CDR3 size distribution were observed between clonotypes, with wild-type and odd murine mice having an average size of about 14 (including unchanged C and W residues at the CDR3 boundaries) (fig. 23C and 26).

To confirm somatic hypermutation, top-ranked 100 clonotype sequences from each naive or SAT-immunized, odd murine mice were aligned with the corresponding germline IGHV sequences using Igblast (world Wide Web: ncbi.nlm.gov/IgBlast /). Mutation rates for each residue position were calculated and plotted according to IMGT numbering scheme (fig. 27). Although low levels of mutation rates were observed in naive mice, immunized mice exhibited significantly higher levels of somatic hypermutation, which was highly enriched in CDR regions (fig. 27). This was further confirmed in a later analysis of the complete VH sequence of the validated nanobody conjugate (fig. 37).

EXAMPLE 3 nanobody discovery with odd mice

Sequence driven, high throughput screening of nanobody conjugates

Next Generation Sequencing (NGS) and bioinformatics analysis were performed to analyze and select VH sequences (clonotypes) from immunized odd mice, followed by gene synthesis, cloning, expression, and ELISA screening to confirm nanobody binders (fig. 28). Alternatively, nanobody binders can be confirmed by other methods, including but not limited to hybridomas, single B cell clones, single B cell sequencing, and various display methods (such as bacterial display, yeast display, mammalian cell display, and phage display).

To select candidate clonotypes for nanobody expression and binder screening, each animal is ranked according to clonotype abundance and somatic hypermutation rate. Phylogenetic analysis of clonotype sequences was performed using Clustal Omega (Web ebi.ac. uk/Tools/msa/clustalo) to select representative sequences from different branches (FIG. 29). Candidate clonotype sequences (VH) from odd mice immunized with SAT were first human codon optimized, flanked by cloning adaptors, and synthesized as eBlock gene fragments (IDT). The synthesized eBlock was then cloned into pFuse-hIgG1-Fc2 vector (invitrogen) by NEBuilder HiFi DNA assembly to generate an in-frame fusion of the IL-2 signal peptide, VH and Fc domains (hinge-CH 2-CH 3) of human IgG1 (fig. 30A-30B). The sequence-verified expression constructs were then transfected into 96-well format of Expi293F cells (sameifer) to produce secreted nanobody-Fc fusions according to the manufacturer's instructions, and culture supernatants were collected 6 days post-transfection and used in ELISA screening to confirm antigen-specific binders.

Screening of 92 clonotypes selected from the odd murine mice immunized with SAT found 21 (23%) with binding (ELISA OD > 0.5), 11 (52%) with high levels of binding (ELISA OD > 3.0) (fig. 31-32). These VH sequences were then used to query the original clonotype sequence library by phylogenetic analysis to confirm homologous VH sequences, which were then used for secondary screening for hit expansion. Of the 30 clonotypes screened, 15 (50%) were binders (most were low abundance and therefore not listed as prescreening), 11 (73%) exhibited high affinity (fig. 31-32). In contrast, control screening using clonotypes screened from wild-type mice according to the same criteria (high abundance and high mutation rate) failed to find any binders (0/29), indicating that functional nanobodies could only be obtained from hcabs produced by odd mice, but not from traditional H2L2 antibodies produced by wild-type mice (fig. 31-32). SAT nanobody conjugate screening using an odd human mouse (SSV 2) confirmed the 14/41 (34%) Nb-Fc conjugate of the human VH sequence, indicating that functional HCAbs were produced by the same mechanism in humanized mice (FIGS. 31-32).

ELISA screening was performed on nanobodies against PD-L1, goat IgG, rabbit IgG and rat IgG, confirming 33% -61% of the corresponding antigen conjugates, further demonstrating the high efficiency of the NGS-driven screening method, particularly suitable for nanobody discovery (FIGS. 31-32). Because of the single-chain nature of hcabs, the various clonotypes identified represent unique antibodies, which are identified by a batch RNA-seq, without the use of any single-cell method.

Biophysical and biochemical properties of purified Nb-Fc

30mL of an expi293F cell culture was transfected with a selected set of ELISA-positive SAT Nb-Fc constructs (from mouse and human VH sequences; table 5) to generate nanobody-Fc fusions, which were then purified using protein A affinity chromatography according to standard procedures. Briefly, six days post-transfection, cell culture supernatants were collected, filtered with a 0.22 μm filter and loaded onto a 0.5mL protein a column (MabSelect SuRe, sitova (cytova)) pre-equilibrated with PBS. After washing with 2mL of PBS, the binding protein was eluted from the column with 4mL of lemon buffer (25 mM citric acid, 150mM sodium chloride, pH 3.5) and neutralized with additional 1M Tris-HCl (pH 8.8). The final buffer was replaced with PBS using Vivospin Turbo (30,000 MWCO PES). SDS-PAGE analysis showed that the purified Nb-Fc fusion migrated at about 80kDa under non-reducing conditions and at about 40kDa under reducing conditions, consistent with the expected size of VH-based Nb-Fc as homodimer, compared to a conventional antibody (about 150 kDa) with two heavy chains (about 50 kDa) and two light chains (about 25 kDa) (FIGS. 33-34).

Table 5 Biochemical and biophysical properties of mouse and human SAT Nb.

To check the quality of purified Nb-Fc, size Exclusion Chromatography (SEC) analysis was performed. Briefly, 2-10. Mu.L of purified Nb-Fc sample was injected into ACQUITY UPLC (Waters) protein BEH SEC 200,1.7 μm, 4.6X150 mm column, flow rate of 0.3 mL/min for 10 min. The mobile phase of 50mM sodium phosphate, 500mM NaCl,pH 6.2, was used. The high percentage Nb-Fc produced did not show aggregation propensity (representative SEC graph in fig. 35).

To assess the binding affinity of purified SAT Nb-Fc fusions, ELISA assays were performed using serial dilutions of protein samples against SAT antigen. All tested Nb-fcs showed ELISA ECs in the nanomolar and subnanomolar range ₅₀ In order to understand the neutralization potency, competitive ELISA assays were performed using the COVID-19 spike-ACE 2 binding assay kit (Raybiotech) according to the manufacturer' S instructions, in contrast to human SAT Nb-Fc control VH-Ab-8 (HAb 8-S) (Li et al, cell,183:429 (2020) (FIG. 36A and Table 5), many Nb-Fc showed potent neutralization potency against spike-ACE 2 binding, IC thereof ₅₀ Corresponding to HAb8-S (FIG. 36B; table 5). Interestingly, many of the nanobodies were determined by secondary screening using two potent SAT neutralizing nanobodies (LVGN-S3205 and LGVN-S52135) found in primary screening, further demonstrating the powerful function of the sequence driven nanobody discovery procedure (fig. 31 and 37).

The kinetics of Nb-Fc binding to SAT was analyzed using Surface Plasmon Resonance (SPR) and/or Biological Layer Interferometry (BLI) (table 5). For BLI, the binding experiments were performed on an Octet HTX at 25 ℃. Antibodies were loaded onto an anti-human Fc capture (AHC) sensor and then immersed in serial dilutions of antigen (1:3 dilution, 5 spots starting at 333 nM). The reference sample wells (buffers) were used for data analysis. Kinetic constants were calculated using a monovalent (1:1) binding model. Representative kinetic and sensory maps are shown in FIGS. 38-39As shown. Most Nb-fcs exhibit one or two bit nanomoles (10 ^-8 -10 ^-9 M) KD, which corresponds to HAb8-S (Table 5).

To evaluate thermal stability, the thermal denaturation midpoint temperature (Tm) of the purified Nb-Fc was measured using Differential Scanning Fluorescence (DSF). Briefly, nb-Fc was combined with Thermal Shift ^TM The dyes (zemoeimerter) were mixed to a final concentration of 1 μg/mL and 10 μl/well mixture was transferred into 384 well plates. For the boardOptical adhesive was sealed and loaded into Roche480 on the instrument. Fluorescence signals were collected when the temperature was increased from 20 ℃ to 85 ℃ at a rate of 0.06 ℃/sec. Most purified Nb-Fc showed high thermal stability, average tm1= 64.28 ±0.64 ℃ (PBS, ph 7.4) (table 5). Representative melting curves are shown in FIG. 40.

FACS-based cell binding assays were performed to examine the binding properties of Nb-Fc to spike proteins on the cell surface (fig. 41-42). HEK293 parent cells and HEK 293-spike cells expressing SARS-CoV-2 spike (S) protein with an inactivated furin site (293-SARS 2-S-dfur, injetty #293-CoV 2-sdf) were incubated with individual Nb-Fc at 1 μg/mL at 4℃for 1 hour, washed, and then incubated with DyLight 594 (Simerfeverish) conjugated goat anti-human IgG-Fc at 4℃for 1 hour. FACS analysis was performed on these samples on BD LSR II and Geometric Mean Fluorescence Intensity (GMFI) was calculated using FlowJo V10. 12 of the 18 Nb-fcs (bound to SAT in ELISA) also showed cell surface SAT binding above background, GMFI ratios between 4.0-42.9 (fig. 41-42).

Example 4 other odd-based non-human animals

Generation of the odd human-L and-K allele series

Variable light chain (V) _L ) The gene segments promote immune diversity of conventional tetrameric antibodies. Human tetrameric antibodies contain kappa (kappa) or lambda (lamda) light chains. Human immunoglobulinThe light chain is derived from two different sites: IGK and IGL located on chromosome 2 and chromosome 22, respectively. Similar to the IGH loci, each locus encodes a large number of V' s _L A gene segment. However, unlike the IGH locus, the light chain locus lacks a diverse D gene segment; recombination of the light chain locus requires RAG1/RAG2 proteins, but involves direct ligation of the VL fragment to the JL fragment.

To obtain V _L The unique nature of the gene segments and expanding the immune repertoire of the idiopathic human platform, two different approaches have been used to exploit the diversity provided by the light chain variable gene segments. First, since IGLV gene segments are flanked by 23RSS signals similar to IGHV gene segments, they can be correctly paired with 12RSS signals immediately upstream of IGHD gene segments, satisfying the "12/23 rule" of RAG1/RAG2 preference (fig. 43A). For the lambda light chain gene, the human IGHV fragment was first removed by editing using the CRISPR-Cas9 gene, leaving only the human IGHD and IGHJ fragments, generating the odd human DJ-dock allele from the SSV1 allele. Next, a set of IGLV gene fragments from a series of human BACs (CH 17-262M19, CH17-329P5, CH17-238D3, CH17-261A15, CH17-264L24, CH17-117C7, RP11-1040J16, CH17-320F 4) were modified (hLGLV-BAC) and sequentially integrated by RMCE in a similar manner as previously described (FIG. 43B).

Second, since IGKV gene segments flank 12RSS signals that are incompatible with IGHD segments, a different approach is required to introduce kappa light chain genes into the odd alleles. First, the odd superposition allele was used to integrate a sequence comprising VK and JK fragments (CH 17-272M2, CH17-405H5, CH17-140P2, CH17-13E7, CH17-84J8, CH17-53L 15) (FIG. 44A). Alternatively, the odd human J dock allele is generated from the SSV1 allele by removing the human IGHV and IGHD segments using CRISPR-Cas9 gene editing, leaving only the human IGHJ segment. Next, a set of IGKV gene segments from the engineered human BAC (hIGKV-BAC) of CH17-272M2, CH17-405H5, CH17-140P2, CH17-13E7, CH17-84J8, CH17-53L15 were integrated into the IGHJ gene segments by sequential RMCE, allowing individual IGKV to recombine with each IGHJ gene segment flanked upstream by 23RSS signals (FIG. 44B).

Generation of the odd horned cattle and Mi Nuotao alleles

Bovine antibodies are characterized by the presence of ultralong Complementarity Determining Regions (CDRs) (Berens et al, int. Immunol.,9 (1): 189-199 (1997)). These ultralong CDRs have antibody knob domains that can bind their antigens tightly as autonomous entities, thereby allowing the generation of ultra-small nanobodies of about 3-5kDa in size (MacPherson et al, PLoS biol.,18 (9): e3000821 (2020)). In humans and mice, CDR-H3 ranges from 6 to 20 amino acids in length, whereas in cattle CDR-H3 can reach 50 to 70 residues in length. The ultralong CDR-H3 portion is derived from abnormally long heavy diversity encoded in bovine germline genomes (D _H ) 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, the longest D known _H One, contributing at least 50 amino acid residues to bovine CDR-H3, the combination of IGHV1-7, IGHD8-2 and IGHJ2-4 is found predominantly in isolated ultralong CDR3 bovine antibodies (MacPherson et al, PLoS biol.,18 (9): e3000821 (2020)).

A synthetic 3252bp gene fragment was constructed on the odd-numbered platform, which included the approximately 2.5kb promoter and the 5' UTR region upstream of bovine (Bos Taurus) IGHV1-7, the entire IGHV1-7 leader exons, introns and coding sequences, followed by IGHD8-2, IGHJ2-4 and the 250bp sequence immediately downstream of the IGHJ2-4 region containing the splice donor sequence (FIG. 45A). This construct was integrated into the odd superphage allele by RMCE, resulting in mice designated as odd long horns (fig. 45B). PCR genotyping and sequencing confirmed the correct integration and transmission of the odd-horned bovine allele in F1 mice (fig. 45C), from which bovine VDJ-mouse igg1Δch1 transcripts were detected.

Sequences of bovine VDJ at long angles (bovine IGHV1-7 leader sequence and intron 1 underlined, bovine IGHV1-7 bold, bovine IGHD8-2 italics, bovine IGHJ2-4 bold and underlined)

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Based on the demonstration that the odd long horn bovine mice are capable of expressing D with the known longest _H Successful results of the bovine-mouse chimeric heavy chain antibody of the gene segment, construction of bovine D containing 8 longest species _H Synthetic arrays of gene segments (IGHD 4-1, IGHD5-3, IGHD8-2, IGHD1-3, IGHD7-4, IGHD6-3, and IGHD 3-3) to replace the human IGHD component of an exotic human mouse. The sequence of the human framework is based on a genomic region spanning 550bp upstream of IGHD4-4 to 550bp downstream of IGHD4-17, wherein human D _H The coding sequence of the gene was replaced with the bovine counterpart while retaining all the genetic elements including the 12RSS signal (fig. 46). Synthetic human-bovine D _H The array comprises the following sequences for substitution of human IGHD gene fragments in the odd human allele by CRISPR/Cas9 mediated HDR. Mice generated by these genetic engineering, known as the idiosyncratic Mi Nuotao of human-dairy hybrid, were intended to produce human nanobodies with ultralong CDR-H3 derived from cattle (Bos Taurus).

Sequence of the odd Mi Nuotao array (bovine IGHD in bold and underlined)

1) People: IGHD4-4; bovine surrogate: IGHD4-1

2) People: IGHD5-5; bovine surrogate: IGHD5-3

3) People: IGHD6-6; bovine surrogate: IGHD8-2

4) People: IGHD1-7; bovine surrogate: IGHD1-3

5) People: IGHD4-11; bovine surrogate: IGHD7-3

6) People: IGHD5-12; bovine surrogate: IGHD7-4

7) People: IGHD6-13; bovine surrogate: IGHD6-3

8) People: IGHD4-17; bovine surrogate: IGHD3-3

Generation of the sequence of the Qimake alleles

By constructing a synthetic array containing 5 known alpaca (Vicugna pacos) VHHs (sapaca VHHs), the function of the odd platform was extended (Achour et al, j.immunol.,181 (3): 2001-2009 (2008)). Individual VHH elements were grafted onto the framework of selected human VH components, including their approximately 250bp upstream human promoter, human leader exons 1 and 2, human introns and human Recombination Signal Sequences (RSS) containing regulatory elements involved in VH transcription (e.g. TATA box, octamer and heptamer) (fig. 47A). Human VH was selected based on evidence of its high availability in human and humanized rodent models (e.g., rats and mice). The saparaceae VHH array was designed to contain flanking different lox elements to facilitate its targeted integration into the odd human IgH locus by RMCE (fig. 47B). Syn sapataceae arrays (see sequence below) were inserted into the odd human DJ dock allele by RMCE (FIG. 47B). Mice produced by such genetic engineering are referred to as aspacae 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 antibodies).

Alpaca-human-mouse chimeric heavy chain antibodies produced by the amateur sapacae mice may have the naturally optimized nanobody properties of alpaca and may acquire other immune diversity of human D and J elements, enabling rapid humanization for human therapeutic applications. The saparaceae VHH array may be an RMCE mediated array (containing other V from alpaca and other camelids) _H H) Is easily expanded by repeated round integration.

The sequence of the Azimuth array (alpaca VHH sequence bold and underlined)

1) Human VH: IGHV6-1; alpaca V _H H substitute: VHH3-1

2) Human VH: IGHV1-2; alpaca V _H H substitute: VHH3-S1

3) Human VH: IGHV4-4; alpaca V _H H substitute: VHH3-S2

/>

4) Human VH: IGHV2-5; alpaca V _H H substitute: VHH3-S9

5) Human VH: IGHV3-7; alpaca V _H H substitute: VHH3-S10

Generation of the sequence of the odd-sex salva alleles

Heavy chain antibodies produced by cartilaginous fish (e.g., shark, ray, and ray) are derived from a special class of immunoglobulins called variable neoantigen receptors (VNAR) (Greenberg et al, nature,374 (6518): 168-73 (1995)).

By constructing a synthetic shark VNAR array, the capabilities of the odd platform are extended. Individual VNAR elements selected from the germline sequence of nurse shark (Ginglymostoma cirratum) were transplanted onto the framework of a selected human VH component comprising an approximately 250bp upstream human promoter, human leader exons 1 and 2, human introns and human Recombination Signal Sequences (RSS) containing regulatory elements involved in VH transcription (e.g. TATA box, octamer and heptamer) (fig. 48A). The VNAR array called savland was synthesized and inserted into the odd human DJ allele by RMCE (fig. 48B). Mice produced by such genetic engineering are referred to as odd-numbered savland mice, and are evaluated for the ability to produce shark-human-mouse chimeric heavy chain antibodies (e.g., shark-human-mouse chimeric heavy chain IgG1- Δch1 antibodies).

Shark-human-mouse chimeric heavy chain antibodies produced by the odd savland mice may have excellent biophysical properties of VNAR and may acquire other immune diversity of human D and J elements, enabling rapid humanization for human therapeutic applications. The immune repertoire of the odd savland mice can be easily expanded by repeated rounds of RMCE-mediated integration of arrays (containing other VNARs from other shark species).

Odd savland array sequence (alpaca V) _H The H sequence is bold and underlined

1) Human VH IGHV6-1; nurse shark VNAR substitute: l38968

2) Human VH IGHV3-23; nurse shark VNAR substitute: l38967

Example 5 exemplary embodiment

Embodiment 1A. A genetically engineered mouse comprising a germline modification comprising a deletion of a nucleic acid sequence comprising one or more heavy chain C-region genes; wherein the mouse expresses and secretes IgG heavy chain antibodies into its serum.

Embodiment 2A. The genetically engineered mouse of embodiment 1, wherein the one or more heavy chain C region groups are IgM C region genes (cμ), igD C region genes (cδ), igE C region genes (cε), igG 3C region genes (cγ3), igG2b C region genes (cγ2b), igG2C C region genes (cγ2c), or a combination thereof.

Embodiment 3A. The genetically engineered mouse of any one of embodiments 1A-2A, further comprising a deletion of a nucleic acid sequence encoding a CH1 domain of an IgG 1C region gene (cγ1).

Embodiment 4A. The genetically engineered mouse of embodiment 3A, wherein the deletion of the nucleic acid sequence encoding the CH1 domain of the IgG 1C region gene comprises exon 1.

Embodiment 5A. The genetically engineered mouse of any one of embodiments 1A-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. The genetically engineered mouse of any one of embodiments 1A-5A, wherein the germline modification further comprises a native nucleic acid sequence comprising an endogenous enhancer.

Embodiment 7A. The genetically engineered mouse of embodiment 6A, wherein the enhancer is E μ, 3' rr, 3' γ1e, 5' hsri, or a combination thereof.

Embodiment 8A. The genetically engineered mouse of any one of embodiments 1A-7A, wherein the germline modification further comprises a native nucleic acid sequence comprising a switch tandem repeat element (sμ), wherein sμ drives IgGl expression.

Embodiment 9A. The genetically engineered mouse of any one of embodiments 1A-8A, wherein the IgG heavy chain antibody comprises an IgG1 heavy chain antibody.

Embodiment 10A. The genetically engineered mouse of embodiment 9A, wherein the IgG1 heavy chain antibody is an igg1Δch1 protein.

Embodiment 11A. The genetically engineered mouse of any one of embodiments 1A-10A, wherein the IgG heavy chain antibody lacks a light chain.

Embodiment 12A. The genetically engineered mouse of any one of embodiments 1A-11A, wherein the IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

Embodiment 13A. The genetically engineered mouse of any one of embodiments 1A-12A, wherein the mouse does not express a wild-type IgM protein, a wild-type IgD protein, a wild-type IgE protein, a wild-type IgG3 protein, or a combination thereof.

Embodiment 14A. The genetically engineered mouse of any one of embodiments 1A-14A, wherein the mouse does not express a wild-type IgA protein, a wild-type IgG2b protein, a wild-type IgG2c protein, or a combination thereof.

Embodiment 15A. An engineered non-human animal comprising a germline genome comprising an engineered immunoglobulin heavy chain (IgH) allele at an endogenous IgH locus; wherein the engineered IgH allele lacks an endogenous heavy chain C region gene; and wherein the endogenous heavy chain C region gene comprises cμ, cδ, cε, cγ3, cγ2b, cγ2c, or a combination thereof.

Embodiment 16A. The genetically engineered mouse of embodiment 15A, wherein the IgH allele comprises a deletion of a nucleic acid sequence encoding a CH1 domain of an IgG 1C region gene (cγ1).

Embodiment 17A. The engineered non-human animal of embodiment 16A, wherein the CH1 domain of the IgGl C domain gene comprises exon 1.

Embodiment 18A the engineered non-human animal of any one of embodiments 15A-17A, 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.

Embodiment 19A. The engineered non-human animal of any one of embodiments 15A-18A, wherein the IgH locus comprises a native nucleic acid sequence comprising an endogenous enhancer.

Embodiment 20A. The engineered non-human animal of embodiment 19A, wherein the enhancer is E μ, 3' rr, 3' γ1e, 5' hsri, or a combination thereof.

Embodiment 21A the engineered non-human animal of any one of embodiments 15A-20A, wherein the IgH locus comprises a native nucleic acid sequence comprising a switch tandem repeat element (sμ), wherein the sμ drives IgGl expression.

Embodiment 22A the engineered non-human animal of any one of embodiments 15A-21A, wherein the non-human animal expresses an IgG heavy chain antibody.

Embodiment 23A. The engineered non-human animal of embodiment 22A, wherein the IgG heavy chain antibody comprises an IgG1 heavy chain antibody.

Embodiment 24A. The engineered non-human animal of any one of embodiments 22A-23A, wherein the IgG1 heavy chain antibody is an igg1Δch1 protein.

Embodiment 25A the engineered non-human animal of any one of embodiments 22A-24A, wherein the IgG heavy chain antibody lacks a light chain.

Embodiment 26A. The engineered non-human animal of any one of embodiments 22A-25A, wherein the IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

Embodiment 27A the engineered non-human animal of any one of embodiments 15A-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. The engineered non-human animal of any one of embodiments 15A-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 the engineered non-human animal of any one of embodiments 15A-28A, wherein the IgH locus comprises an endogenous V, D or J gene.

Embodiment 30A. The engineered non-human animal of any one of embodiments 15A-29A, wherein the engineered non-human animal is homozygous for the engineered IgH allele.

Embodiment 31A the engineered non-human animal of any one of embodiments 15A-30A, wherein the endogenous IgH locus does not comprise an exogenous nucleic acid sequence.

Embodiment 32A the engineered non-human animal of any one of embodiments 15A-30A, wherein the endogenous IgH locus does not comprise an exogenous nucleic acid sequence.

Embodiment 33A. The engineered non-human animal of embodiment 32A, wherein the exogenous nucleic acid sequence comprises a barcode.

Embodiment 34A. The engineered non-human animal of any one of embodiments 15A-33A, wherein the non-human animal is a mammal.

Embodiment 35A. The engineered non-human animal of embodiment 34A, wherein the mammal is a mouse or a rat.

Embodiment 36A. A method of producing a genetically modified non-human animal capable of producing heavy chain antibodies, comprising (a) deleting an endogenous nucleic acid sequence comprising one or more heavy chain C region genes from an endogenous immunoglobulin heavy chain locus in a stem cell of the non-human animal; (b) implanting stem cells into the blastula; (c) Implanting blastula into pseudopregnant mice to obtain chimeric mice; (d) Crossing the chimeric mice with wild-type mice to produce offspring; (e) screening the offspring for heterozygosity; and (f) identifying a first-established mouse carrying one or more heavy chain C region gene deletions; and wherein the non-human animal is capable of producing heavy chain antibodies.

Embodiment 37A. The method of embodiment 36A, wherein the stem cells are embryonic stem cells.

The method of any one of embodiments 36A-37A, wherein the one or more heavy chain C region genes comprise cμ, cδ, cγ3, cγ2b, cγ2c, cε, or a combination thereof.

Embodiment 39A. The method of any one of embodiments 36A-38A, further comprising deleting the nucleic acid sequence encoding the CH1 domain of the IgG 1C 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 IgG 1C region gene comprises exon 1.

Embodiment 41A. The method of any of embodiments 36A-40A, further comprising retaining 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. The method of any one of embodiments 36A-41A, further comprising retaining the native nucleic acid sequence comprising the 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 the method of any one of embodiments 36A-43A, further comprising retaining a native nucleic acid sequence comprising a switch tandem repeat element (sμ), wherein sμ drives IgGl expression.

Embodiment 45A. The method of any one of embodiments 36A-44A, wherein the heavy chain antibody is an IgG heavy chain antibody.

Embodiment 46A the method of any one of embodiments 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 an igg1Δch1 protein.

Embodiment 48A the method of any one of embodiments 45A-47A, wherein the IgG heavy chain antibody lacks a light chain.

Embodiment 49A the method of any one of embodiments 45A-48A, wherein the IgG heavy chain antibody comprises a hinge domain, CH2 domain, CH3 domain, or combination thereof.

Embodiment 50A. The method of any one of embodiments 36A-49A, 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 51A the method of any one of embodiments 36A-50A, 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 52A the method of any of embodiments 36A-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 the method of any one of embodiments 36A-53A, wherein deleting the endogenous nucleic acid sequence comprising one or more heavy chain C region genes comprises CRISPR/Cas9 genome editing.

Embodiment 55A. The method of any one of embodiments 36A-54A, wherein the genetically modified non-human animal is fertility.

Embodiment 56A. The method of any one of embodiments 36A-55A, wherein the genetically modified non-human animal has substantially normal B cell development and maturation.

Embodiment 57A the method of any one of embodiments 36A-56A, wherein the genetically modified non-human animal does not express a wild-type IgM protein, a wild-type IgD protein, a wild-type IgE protein, a wild-type IgG3 protein, a wild-type IgA protein, a wild-type IgG2b protein, a wild-type IgG2c protein, or a combination thereof.

Embodiment 58A. A method of making a soluble heavy chain antibody in the engineered non-human animal of any one of embodiments 36A-57A, comprising (a) administering an antigen to the 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) confirming clonotype based on mRNA sequence, and (f) performing a phylogenetic analysis of the clonotype; thereby producing soluble heavy chain antibodies.

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 of producing a single domain antibody (sdAb) that recognizes an engineered non-human animal from any of embodiments 36A-57A, comprising (a) expressing in a cell a polypeptide encoding a heavy chain variable (V) comprising V, D and J _H ) A nucleic acid sequence of a domain, wherein the cell produces a heavy chain variable domain; (b) The heavy chain variable domain is isolated from the sample, thereby producing a single domain antibody.

Embodiment 62A. The method of embodiment 61A, wherein the single domain antibody is a murine single domain 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 the method of any of embodiments 61A-64A, wherein the single domain antibody lacks a light chain.

Embodiment 66A the method of any one of embodiments 61A-65A, wherein the single domain antibody lacks a hinge domain, CH2 domain, CH3 domain, or a combination thereof.

Embodiment 1B. A genetically engineered mouse comprising a germline modification comprising a deletion of a nucleic acid sequence comprising one or more heavy chain C-region genes; wherein the mouse expresses the humanized IgG heavy chain antibody and secretes the humanized IgG heavy chain antibody into its serum.

Embodiment 2B. The genetically engineered mouse of embodiment 1B, wherein the one or more heavy chain C region groups are IgM C region genes (cμ), igD C region genes (cδ), igE C region genes (cε), igG 3C region genes (cγ3), igG2B C region genes (cγ2b), igG2C C region genes (cγ2c), or a combination thereof.

Embodiment 3B the genetically engineered mouse of any one of embodiments 1B-2B, further comprising a deletion of a nucleic acid sequence encoding a CH1 domain of an IgG 1C region gene (cγ1).

Embodiment 4B. The genetically engineered mouse according to embodiment 3B, wherein the deletion of the nucleic acid sequence encoding the CH1 domain of the IgG 1C region gene comprises exon 1.

Embodiment 5B the genetically engineered mouse of any one of embodiments 1B-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 the genetically engineered mouse of any one of embodiments 1B-5B, wherein the germline modification further comprises a native nucleic acid sequence comprising an endogenous enhancer.

Embodiment 7B. The genetically engineered mouse of embodiment 6B, wherein the enhancer is E μ, 3' rr, 3' γ1e, 5' hsr1, or a combination thereof.

Embodiment 8B the genetically engineered mouse of any one of embodiments 1B-7B, wherein the germline modification further comprises a native nucleic acid sequence comprising a switch tandem repeat element (sμ), wherein sμ drives IgGl expression.

Embodiment 9B the genetically engineered mouse of any one of embodiments 1B-8B, wherein the humanized IgG heavy chain antibody comprises a humanized IgG1 heavy chain antibody.

Embodiment 10B. The genetically engineered mouse of embodiment 9B, wherein the humanized IgG1 heavy chain antibody is an igg1Δch1 protein.

Embodiment 11B the genetically engineered mouse of any one of embodiments 1B-10B, wherein the humanized IgG heavy chain antibody lacks a light chain.

Embodiment 12B the genetically engineered mouse of any one of embodiments 1B-11B, wherein the humanized IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

Embodiment 13B the genetically engineered mouse of any one of embodiments 1B-12B, wherein the mouse does not express a wild-type IgM protein, a wild-type IgD protein, a wild-type IgE protein, a wild-type IgG3 protein, or a combination thereof.

Embodiment 14B the genetically engineered mouse of any one of embodiments 1B-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. An engineered non-human animal comprising a germline genome comprising an engineered immunoglobulin heavy chain (IgH) allele at an endogenous IgH locus; wherein the engineered IgH allele lacks an endogenous heavy chain C region gene; and wherein the endogenous heavy chain C region gene comprises cμ, cδ, cε, cγ3, cγ2b, cγ2c, or a combination thereof.

Embodiment 16B. The genetically engineered mouse of embodiment 15B, wherein the IgH allele comprises a deletion of a nucleic acid sequence encoding a CH1 domain of an IgG 1C region gene (cγ1).

Embodiment 17B. The engineered non-human animal of embodiment 16B, wherein the CH1 domain of the IgGl C region gene comprises exon 1.

Embodiment 18B the engineered non-human animal of any one of embodiments 15B-17B, 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.

Embodiment 19B the engineered non-human animal of any one of embodiments 15B-18B, wherein the IgH locus comprises a native nucleic acid sequence comprising an endogenous enhancer.

Embodiment 20B. The engineered non-human animal of embodiment 19B, wherein the enhancer is E μ, 3' rr, 3' γ1e, 5' hsri, or a combination thereof.

Embodiment 21B the engineered non-human animal of any one of embodiments 15B-20B, wherein the IgH locus comprises a native nucleic acid sequence comprising a switch tandem repeat element (sμ), wherein the sμ drives IgGl expression.

Embodiment 22B the engineered non-human animal of any one of embodiments 15B-21B, wherein the non-human animal expresses a humanized IgG heavy chain antibody.

Embodiment 23B the engineered non-human animal of any one of embodiment 22B, wherein the humanized IgG heavy chain antibody comprises a humanized IgG1 heavy chain antibody.

Embodiment 24B the engineered non-human animal of any one of embodiments 22B-23B, wherein the humanized IgG1 heavy chain antibody is an igg1Δch1 protein.

Embodiment 25B the engineered non-human animal of any one of embodiments 22B-24B, wherein the humanized IgG heavy chain antibody lacks a light chain.

Embodiment 26B the engineered non-human animal of any one of embodiments 22B-25B, wherein the humanized IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

Embodiment 27B the engineered non-human animal of any one of embodiments 15B-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 the engineered non-human animal of any one of embodiments 15B-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 the engineered non-human animal of any one of embodiments 15B-28B, wherein the IgH locus comprises a human V, D or J gene.

Embodiment 30B the engineered non-human animal of any one of embodiments 15B-29B, wherein the engineered non-human animal is homozygous for the engineered IgH allele.

Embodiment 31B the engineered non-human animal of any one of embodiments 15B-30B, wherein the endogenous IgH locus does not comprise an exogenous nucleic acid sequence.

Embodiment 32B the engineered non-human animal of embodiments 15B-31B, wherein the exogenous nucleic acid sequence comprises one or more human V _H Gene segment, one or more humans D _H Gene segments and one or more J _H A gene segment.

Embodiment 33B the engineered non-human animal of any one of embodiments 15B-32B, wherein the exogenous nucleic acid sequence comprises 65 human V _H A gene segment.

Embodiment 34B the method according to any one of embodiments 15B-32BThe engineered non-human animal wherein the exogenous nucleic acid sequence comprises 27 human D _H A gene segment.

Embodiment 35B the engineered non-human animal of any one of embodiments 15B-32B, wherein the exogenous nucleic acid sequence comprises 6J _H A gene segment.

Embodiment 36B the engineered non-human animal of any one of embodiments 15B-35B, wherein the exogenous nucleic acid sequence comprises 65 human V _H Gene segment, 27 person D _H Gene segment and 6J _H A gene segment.

Embodiment 37B the engineered non-human animal of embodiment 31B, wherein the exogenous nucleic acid sequence comprises a barcode.

Embodiment 38B the engineered non-human animal of any one of embodiments 15B-37B, wherein the non-human animal is a mammal.

Embodiment 39B the engineered non-human animal of embodiment 38B, wherein the mammal is a mouse or a rat.

Embodiment 40B. A method of producing a genetically modified non-human animal capable of producing heavy chain antibodies, comprising (a) deleting an endogenous nucleic acid sequence comprising one or more heavy chain C region genes from an endogenous immunoglobulin heavy chain locus in a stem cell of the non-human animal; (b) implanting stem cells into the blastula; (c) Implanting blastula into pseudopregnant mice to obtain chimeric mice; (d) Crossing the chimeric mice with wild-type mice to produce offspring; (e) screening the offspring for heterozygosity; and (f) identifying a first-established mouse carrying one or more heavy chain C region gene deletions; and 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 the method of any one of embodiments 40B-41B, wherein the one or more heavy chain C region genes comprise cμ, cδ, cγ3, cγ2b, cγ2c, cε, or a combination thereof.

Embodiment 43B the method of any one of embodiments 40B-42B, further comprising deleting a nucleic acid sequence encoding a CH1 domain of an IgG 1C region gene (Cγ1).

Embodiment 44B. The method of embodiment 43B, wherein the deletion of the nucleic acid sequence encoding the CH1 domain of the IgG 1C region gene comprises exon 1.

Embodiment 45B the method of any one of embodiments 40B-44B, further comprising retaining 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 46B the method of any one of embodiments 40B-45B, further comprising retaining the native nucleic acid sequence comprising the endogenous enhancer.

Embodiment 47B. The method of embodiment 46B, wherein the enhancer is E μ, 3' rr, 3' γ1e, 5' hsri, or a combination thereof.

Embodiment 48B the method of any one of embodiments 40B-47B, further comprising retaining a native nucleic acid sequence comprising a switch tandem repeat element (sμ), wherein sμ drives IgGl expression.

Embodiment 49B the method of any one of embodiments 40B-48B, wherein the humanized heavy chain antibody is a humanized IgG heavy chain antibody.

Embodiment 50B the method of any one 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 an igg1Δch1 protein.

Embodiment 52B the method of any of embodiments 50B-51B, wherein the humanized IgG heavy chain antibody lacks a light chain.

Embodiment 53B the method of any one of embodiments 49B-52B, wherein the humanized IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

Embodiment 54B the method of any one of embodiments 40B-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 the method of any one of embodiments 40B-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 the method of any one of embodiments 40B-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 the method of any one of embodiments 40B-57B, wherein deleting the endogenous nucleic acid sequence comprising one or more heavy chain C region genes comprises CRISPR/Cas9 genome editing.

Embodiment 59B the method of any one of embodiments 40B-58B, wherein the genetically modified non-human animal is fertility.

Embodiment 60B the method of any one of embodiments 40B-59B, wherein the genetically modified non-human animal has substantially normal B cell development and maturation.

Embodiment 61B the method of any one of embodiments 40B-60B, wherein the genetically modified non-human animal does not express a wild-type IgM protein, a wild-type IgD protein, a wild-type IgE protein, a wild-type IgG3 protein, a wild-type IgA protein, a wild-type IgG2B protein, a wild-type IgG2c protein, or a combination thereof.

Embodiment 62B. A method of producing a soluble humanized heavy chain antibody in the engineered non-human animal of any one of embodiments 40B-61B, comprising (a) administering an antigen to the 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) confirming clonotype based on mRNA sequence, and (f) performing phylogenetic analysis of the clonotype; thereby producing soluble humanized heavy chain antibodies.

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 of producing a humanized single domain antibody (sdAb) that recognizes an engineered non-human animal from any of embodiments 40-61, comprising (a) expressing in a cell a polypeptide encoding a human heavy chain variable (V) comprising V, D and J _H ) A nucleic acid sequence of a domain, wherein the cell produces a human heavy chain variable domain; and (b) isolating the human heavy chain variable domain from the 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 the method of any one of embodiments 65B-68B, wherein the single domain antibody lacks a light chain.

Embodiment 70B the method of any one of embodiments 65B-69B, wherein the single domain antibody lacks a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

Embodiment 71B the method of any one of embodiments 65B-70B, wherein the cell is a bacterial cell or a human cell.

Embodiment 1℃ A 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 comprising a CH1 constant domain, an IgM constant domain, an IgD constant domain, an IgE constant domain, an IgA constant domain, or any combination thereof of an IgG subclass.

Embodiment 2C the DNA according to embodiment 2C wherein the DNA is germline genomic DNA.

Embodiment 3C the DNA of any one of embodiments 1C-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 comprising a CH1 constant domain of an IgG subclass.

Embodiment 4C the DNA according to embodiment 3C wherein the IgG subclass comprises IgGl, igG2a, igG2b, igG2C, igG3, or IgG4 subclass.

Embodiment 5C the DNA of embodiment 3C wherein the IgG subclass is the IgG1 subclass.

Embodiment 6C the DNA according to any one of embodiments 1C-5C wherein the genetically modified non-human IgH allele comprises a nucleic acid sequence (Cγ1-. DELTA.CH1) encoding a CH1 truncated IgG1 constant domain (IgG 1-. DELTA.CH1).

Embodiment 7C the DNA of any one of embodiments 1C-6C, wherein the genetically modified non-human IgH allele comprises a nucleic acid sequence encoding a hinge (H) domain, a CH2 domain, a CH3 domain of an IgG subclass, or any combination thereof.

The DNA of any one of embodiments 1C-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 comprising an IgG2 constant domain, an IgG3 constant domain, an IgG4 constant domain, or any combination thereof.

Embodiment 9C the DNA of any one of embodiments 1C-8C, wherein the genetically modified non-human IgH allele comprises one or more endogenous enhancers comprising E μ, 3' γ1e, 5' hsr1, 3' rr, or any combination thereof.

Embodiment 10C the DNA of any one of embodiments 1C-9C, wherein the genetically modified non-human IgH allele comprises an I μ promoter, an I μ exon, or both.

Embodiment 11C the DNA of any one of embodiments 1C-10C, wherein the genetically modified non-human IgH allele comprises a switch tandem repeat element (sμ).

Embodiment 12C the DNA of any one of embodiments 1C-11C wherein IgG1 expression is driven by E.mu.I.mu.promoter, S.mu.or any combination thereof.

The DNA of any one of embodiments 1C-12C, wherein the genetically modified non-human IgH allele lacks one or more endogenous switching regions comprising sγ3, sγ1, sγ2b, sγ2c, sε, sα, or any combination thereof.

Embodiment 14C the DNA of any one of embodiments 1C-13C, wherein the genetically modified non-human IgH allele comprises the following components (from 5 'to 3'): eμ, Iμ promoter, Iμ exon, Sμ, Cγ1-. DELTA.CH1, 3' γ1E, 5' hsR1 and 3' RR.

Embodiment 15C the DNA of any one of embodiments 1C-14C, wherein the genetically modified non-human IgH allele comprises a invertase recognition target (frt) site.

The DNA of any one of embodiments 1C-15C, wherein the genetically modified non-human IgH allele comprises an endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof.

The DNA of any one of embodiments 1C-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.

The DNA of any one of embodiments 1C-17C, wherein the genetically modified non-human IgH allele comprises a docking box.

Embodiment 19C the 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 selectable marker, or any combination thereof.

Embodiment 20C the DNA of embodiment 18C, wherein said docking cassette comprises a nucleic acid sequence encoding a selectable marker.

Embodiment 21C the DNA of embodiment 20C, wherein the selectable marker comprises geneticin, hydromycin, puromycin, or any combination thereof.

The DNA of any one of embodiments 1C-21C, wherein the genetically modified non-human IgH allele encodes an IgG heavy chain antibody.

The DNA of any one of embodiments 1C-22C, wherein the genetically modified non-human IgH allele comprises an exogenous V gene segment, an exogenous D gene segment, an exogenous J gene segment, or any combination thereof.

The DNA of any one of embodiments 23C, wherein the exogenous gene segment is selected from the group consisting of: human, mouse, rat, bovine, alpaca, and shark gene segments.

Embodiment 25C the DNA of embodiment 23C wherein the exogenous gene segment comprises a human gene segment.

Embodiment 26C the DNA according to any one of embodiments 1C-25C wherein the genetically modified non-human IgH allele comprises one or more human V _H Gene segment, one or more humans D _H Gene segments and one or more J _H A gene segment.

The DNA of any one of embodiments 1C-26C, wherein the genetically modified non-human IgH allele comprises at least 10, 20, 30, 40, 50, 60, 80, 100, 120, or 126 human VH gene segments.

The DNA of any one of embodiments 1C-27C, wherein the genetically modified non-human IgH allele comprises at least 10, 15, 20, 25, or 27 human DH gene segments.

The DNA of any one of embodiments 1C-28C, wherein the genetically modified non-human IgH allele comprises at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 human JH gene segments.

Embodiment 30C the DNA of any one of embodiments 1C-29C, wherein the genetic modificationThe ornamental non-human IgH allele comprises 126 human V _H Gene segment, 27 person D _H Gene segment and 9 individuals J _H A gene segment.

The DNA of any one of embodiments 1C-30C, wherein the genetically modified non-human IgH allele comprises one or more bovine gene segments.

Embodiment 32C the DNA of embodiment 31C wherein the one or more bovine gene segments comprise the L1 exon, the L2 exon of IGHV1-7, the coding segment of IGHD8-2, the coding sequence of IGHJ2-4, the IGH2-4 splice donor, or any combination thereof.

Embodiment 33C the DNA of any one of embodiments 31C-32C wherein the one or more bovine gene segments comprise IGHD4-1, IGHD5-3, IGHD8-2, IGHD1-3, IGHD7-4, IGHD6-3, IGHD3-3, or any combination thereof.

The DNA of any one of embodiments 31C-33C wherein the one or more bovine gene segments comprise a nucleic acid sequence selected from the group consisting of SEQ ID NOs:42-49 and 57.

The DNA of any one of embodiments 32C-34C, wherein the DNA comprises one or more human VH gene segments.

The DNA of any one of embodiments 32C-35C, wherein the DNA comprises one or more human JH gene segments.

The DNA of any one of embodiments 1C-36C, wherein the genetically modified non-human IgH allele comprises one or more alpaca gene segments.

Embodiment 38C the DNA according to embodiment 37C wherein the one or more alpaca gene segments comprise VHH3-1, VHH3-S2, VHH3-S9, VHH3-S10 or any combination thereof.

The DNA of any one of embodiments 37C-38C wherein the alpaca gene segment comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 50-54.

Embodiment 40C the DNA of any one of embodiments 37C-39C, wherein the DNA comprises one or more human VH gene segments.

Embodiment 41C the DNA of any one of embodiments 37C-40C, wherein the DNA comprises one or more human JH gene segments.

Embodiment 42C the DNA of any one of embodiments 1C-41C, wherein the genetically modified non-human IgH allele comprises one or more shark gene segments.

Embodiment 43C the DNA of embodiment 42C, wherein the one or more shark gene segments comprise VNAR-L38968, VNAR-L38967, or both

Embodiment 44C the DNA according to any one of embodiments 42C-43C wherein the shark gene segment comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs 55-56.

Embodiment 45C the DNA of any one of embodiments 42C-44C, wherein the DNA comprises one or more human VH gene segments.

Embodiment 46C the DNA of any one of embodiments 42C-45C, wherein the DNA comprises one or more human JH gene segments.

The DNA of any one of embodiments 1C-46C, wherein the genetically modified non-human IgH allele encodes an IgG heavy chain antibody, and wherein the IgG heavy chain antibody comprises a kappa light chain variable domain, a lambda light chain variable domain, or both.

Embodiment 48C the DNA of embodiment 47C wherein the genetically modified non-human IgH allele comprises one or more exogenous human lambda light chain (LV) gene segments.

Embodiment 49℃ The DNA of embodiment 48C wherein the one or more human LV gene segments comprise CH17-262M19, CH17-329P5, CH17-238D3, CH17-261A15, CH17-264L24, CH17-117C7, RP11-1040J16, CH17-320F4 or any combination thereof.

Embodiment 50C the DNA of any one of embodiments 47C-49C, wherein the genetically modified non-human IgH allele comprises one or more exogenous human kappa light chain (KV) gene segments.

Embodiment 51C the DNA of embodiment 50C wherein the one or more human KV gene segments comprise CH17-272M2, CH17-405H5, CH17-140P2, CH17-13E7, CH17-84J8, CH17-53L15, or any combination thereof.

The DNA of any one of embodiments 47C-51C, wherein the DNA comprises one or more human VH gene segments.

The DNA of any one of embodiments 47C-52C, wherein the DNA comprises one or more human JH gene segments.

Embodiment 54C a genetically modified cell comprising the DNA of any one of embodiments 1C-53C.

Embodiment 55℃ The cell of embodiment 54C, wherein the cell is a non-human animal cell.

Embodiment 56C the cell of embodiment 54C, wherein the cell is a mammalian cell.

Embodiment 57℃ The cell of embodiment 56C, wherein the mammalian cell is a mouse, rat, bovine, alpaca, cat, dog, rabbit, pig, monkey, or chimpanzee cell.

Embodiment 58C the cell of embodiment 54C, wherein the cell is a mouse cell.

Embodiment 59℃ The cell of embodiment 54C, wherein the cell is a shark cell.

Embodiment 60℃ The cell of embodiment 54C, wherein the cell is a human cell.

Embodiment 61C the cell of any one of embodiments 54C-60C, wherein the cell is a stem cell.

Embodiment 62℃ The cell of embodiment 61C, wherein the stem cell is an Embryonic Stem Cell (ESC) or an Induced Pluripotent Stem Cell (iPSC).

Embodiment 63C the cell of any one of embodiments 54C-60C, wherein the cell is a B cell.

The genetically modified non-human animal of embodiment 64C, wherein the genetically modified non-human animal comprises the cell of any one of embodiments 54C-63C.

Embodiment 65C the genetically modified non-human animal of embodiment 64C, wherein the non-human animal is a mammal.

Embodiment 66C the genetically modified non-human animal of embodiment 65C, wherein the mammalian cell is a mouse, rat, bovine, alpaca, cat, dog, rabbit, pig, monkey, or chimpanzee cell.

Embodiment 67C the genetically modified non-human animal of embodiment 64C, wherein the non-human animal is a mouse.

Embodiment 68C the genetically modified non-human animal of any one of embodiments 64C-67C, wherein the genetically modified non-human animal comprises cells that express an IgG heavy chain antibody.

Embodiment 69C the genetically modified non-human animal of embodiment 68C, wherein the IgG heavy chain antibody is secreted into the serum of the genetically modified non-human animal.

Embodiment 70C the genetically modified non-human animal of any one of embodiments 68C-69C, wherein the IgG heavy chain antibody is a CH1 truncated IgG1 heavy chain antibody (igg1Δch1).

Embodiment 71C the engineered non-human animal of any one of embodiments 68C-70C, wherein the IgG heavy chain antibody lacks a light chain.

The engineered non-human animal of any one of embodiments 68C-71C, wherein the IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

Embodiment 73C the genetically modified non-human animal of any one of embodiments 68C-72C, wherein the 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 the genetically modified non-human animal of any one of embodiments 68C-73C, wherein the IgG heavy chain antibody is a human IgG1 heavy chain antibody.

Embodiment 75C the genetically modified non-human animal of any one of embodiments 68C-74C, wherein the IgG heavy chain antibody comprises an exogenous variable domain selected from the group consisting of: human, mouse, rat, bovine, alpaca, and shark variable domains.

The genetically modified non-human animal of any one of embodiments 68C-75C, wherein the IgG heavy chain antibody comprises a kappa light chain variable domain, a lambda light chain variable domain, or both.

Embodiment 77℃ A method for preparing germline genomic DNA, wherein the method comprises deleting one or more nucleic acid sequences from a non-human immunoglobulin heavy chain (IgH) allele, wherein the deleted one or more nucleic acid sequences encode at least a portion of one or more endogenous constant domains (comprising a CH1 constant domain, igM constant domain, igD constant domain, igE constant domain, igA constant domain, or any combination thereof of IgG subclasses) thereby producing a genetic constant domain.

Embodiment 78C the method of embodiment 77C, wherein the germline genomic DNA comprises the DNA of any one of embodiments 1C-53C.

Embodiment 79C the method of any one of embodiments 77C-78C, wherein said IgG constant domain comprises a constant domain of the IgG subclass.

Embodiment 80℃ The method of embodiment 79C, wherein the IgG subclass comprises IgGl, igG2a, igG2b, igG2C, igG3, or IgG4 subclass.

Embodiment 81c. a method for preparing a genetically modified non-human animal, wherein the method comprises:

(a) Removing one or more nucleic acid sequences from a non-human immunoglobulin heavy chain (IgH) allele, wherein the deleted one or more nucleic acid sequences encode at least a portion of one or more endogenous constant domains (comprising a CH1 constant domain, igM constant domain, igD constant domain, igE constant domain, igA constant domain, or any combination thereof of an IgG subclass) to thereby produce a genetic constant domain.

(b) Implanting cells comprising germline genomic DNA into a blastocyst;

(c) Implanting the blastocyst into a pseudo-pregnant non-human animal to obtain a chimeric non-human animal;

(d) Crossing the chimeric non-human animal with a wild-type non-human animal to produce offspring;

(e) Screening heterozygosity of offspring; and

(f) A genetically modified non-human animal carrying a deletion of one or more nucleic acid sequences and capable of producing heavy chain antibodies is identified.

Embodiment 82C the method of embodiment 81C wherein the genetically modified non-human animal is a genetically modified non-human animal of any one of embodiments 64C-76C.

Embodiment 83C the method of embodiments 81C-82C, wherein the one or more nucleic acid sequence deletions comprises use of 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) and a Cas protein that targets an endogenous heavy chain C region gene.

Embodiment 85C the method of embodiments 83C-84C, wherein the Cas protein comprises a Cas9 protein.

Embodiment 86C the method of any one of embodiments 81C-85C, wherein the deleted one or more nucleic acid sequences encodes a CH1 constant domain, an IgG3 constant domain, an IgM constant domain, and an IgD constant domain of IgG 1.

Embodiment 87C the method of any one of embodiments 81C-86C, wherein the deleted one or more nucleic acid sequences encode an IgG2 constant domain and an IgA constant domain.

Embodiment 88C the method of any one of embodiments 81C-87C, wherein said removing the nucleic acid sequence comprises removing a selectable marker from the non-human IgH allele using transient expression of Flp recombinase.

Embodiment 89C the method of any one of embodiments 81C-88C, wherein the deleted one or more nucleic acid sequences encodes a CH1 constant domain, an IgM constant domain, an IgD constant domain, and IgE constant domain, and an IgA constant domain of the IgG1 subclass.

The method of any one of embodiments 81C-89C, wherein the method comprises removing a nucleic acid sequence from the non-human IgH allele, wherein the nucleic acid sequence comprises an endogenous V gene segment, a D gene segment, a J gene segment, or any combination thereof.

Embodiment 91C the method of any of embodiments 81C-90C, wherein the method comprises inserting a docking box.

Embodiment 92C the method of embodiment 91C, wherein the method comprises contacting the docking cassette with a Bacterial Artificial Chromosome (BAC), wherein the BAC comprises a polypeptide comprising an exogenous V _H 、D _H And J _H Nucleic acid sequence of the gene segment.

Embodiment 93C the method of embodiment 92C, wherein the method comprises inserting the exogenous gene segment into a docking box.

The method of any one of embodiments 92C-93C, wherein the exogenous gene segment is a human gene segment.

Embodiment 95℃ A genetically modified non-human animal, wherein said genetically modified non-human animal is prepared using the method of any one of embodiments 81C-94C.

The embodiment 96℃ A method of producing an IgG heavy chain antibody in a genetically modified non-human animal, wherein the method comprises:

(a) Administering an antigen to the genetically modified non-human animal of any one of embodiments 64C-76C;

(b) Isolating one or more B cells from the genetically modified non-human animal;

(c) Isolating mRNA from one or more B cells; and

(d) IgG heavy chain antibodies were produced.

Embodiment 97C the method of embodiment 96C, wherein the genetically modified non-human animal comprises the DNA of any one of embodiments 1C-53C.

The method of any one of embodiments 96C-97C, wherein the method comprises sequencing mRNA isolated from one or more B cells.

The method of any one of embodiments 96C-98C, wherein the method comprises confirming clonotype based on mRNA sequence.

Embodiment 100C the method of embodiment 99C, wherein the method comprises performing a phylogenetic analysis of the clonotypes.

Embodiment 101C the method of any one of embodiments 96C-100C, wherein the IgG heavy chain antibody is a humanized IgG heavy chain antibody.

Embodiment 102C the method of any one of embodiments 96C-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 103℃ An IgG heavy chain antibody, wherein said IgG heavy chain antibody is produced by the method of any one of embodiments 96C-102C.

Embodiment 104℃ 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 capable of deleting one or more nucleic acid sequences from a non-human immunoglobulin heavy chain (IgH) allele, wherein the deleted one or more nucleic acid sequences encode at least a portion of one or more endogenous constant domains comprising CHl constant domains, igM constant domains, igD constant domains, igE constant domains, igA constant domains of the IgG subclass, or any combination thereof.

Other embodiments

It is to be understood that while the invention has been described in conjunction with the specific embodiments, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and improvements are within the scope of the claims.

All references, publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

While the present disclosure has been particularly shown and described with reference to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

1. A genetically engineered mouse comprising a germline modification comprising a deletion of a nucleic acid sequence comprising one or more heavy chain C-region genes; wherein the mouse expresses and secretes IgG heavy chain antibodies into its serum.

2. The genetically engineered mouse of claim 1, wherein the one or more heavy chain C region groups are IgM C region gene (cμ), igD C region gene (cδ), igE C region gene (cε), igG 3C region gene (cγ3), igG2b C region gene (cγ2b), igG2C C region gene (cγ2c), or a combination thereof.

3. The genetically engineered mouse of any one of claims 1-2, further comprising a deletion of a nucleic acid sequence encoding a CH1 domain of an IgG 1C region gene (cγ1).

4. The genetically engineered mouse of claim 3, wherein the deletion of the nucleic acid sequence encoding the CH1 domain of the IgG 1C region gene comprises exon 1.

5. The genetically engineered mouse of any one of claims 1-4, 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 engineered mouse of any one of claims 1-5, wherein the germline modification further comprises a native nucleic acid sequence comprising an endogenous enhancer.

7. The genetically engineered mouse of claim 6, wherein the enhancer is E μ, 3' rr, 3' γ1e, 5' hsr1, or a combination thereof.

8. The genetically engineered mouse of any one of claims 1-7, wherein the germline modification further comprises a native nucleic acid sequence comprising a switch tandem repeat element (sμ), wherein the sμ drives IgGl expression.

9. The genetically engineered mouse of any one of claims 1-8, wherein the IgG heavy chain antibody comprises an IgG1 heavy chain antibody.

10. The genetically engineered mouse of claim 9, wherein the IgG1 heavy chain antibody is an igg1Δch1 protein.

11. The genetically engineered mouse of any one of claims 1-10, wherein the IgG heavy chain antibody lacks a light chain.

12. The genetically engineered mouse of any one of claims 1-11, wherein the IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

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

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

15. A genetically engineered mouse comprising a germline modification comprising a deletion of a nucleic acid sequence comprising one or more heavy chain C-region genes; wherein the mouse expresses the humanized IgG heavy chain antibody and secretes the humanized IgG heavy chain antibody into its serum.

16. The genetically engineered mouse of claim 15, wherein the one or more heavy chain C region groups are IgM C region gene (cμ), igD C region gene (cδ), igE C region gene (cε), igG 3C region gene (cγ3), igG2b C region gene (cγ2b), igG2C C region gene (cγ2c), or a combination thereof.

17. The genetically engineered mouse of any one of claims 15-16, further comprising a deletion of a nucleic acid sequence encoding a CH1 domain of an IgG 1C region gene (cγ1).

18. The genetically engineered mouse of claim 17, wherein the deletion of the nucleic acid sequence encoding the CH1 domain of an IgG 1C region gene comprises exon 1.

19. The genetically engineered mouse of any one of claims 15-18, 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 engineered mouse of any one of claims 15-19, wherein the germline modification further comprises a native nucleic acid sequence comprising an endogenous enhancer.

21. The genetically engineered mouse of claim 20, wherein the enhancer is E μ, 3' rr, 3' γ1e, 5' hsr1, or a combination thereof.

22. The genetically engineered mouse of any one of claims 15-21, wherein the germline modification further comprises a native nucleic acid sequence comprising a switch tandem repeat element (sμ), wherein the sμ drives IgGl expression.

23. The genetically engineered mouse of any one of claims 15-22, wherein the humanized IgG heavy chain antibody comprises a humanized IgG1 heavy chain antibody.

24. The genetically engineered mouse of claim 23, wherein the humanized IgG1 heavy chain antibody is an igg1Δch1 protein.

25. The genetically engineered mouse of any one of claims 15-24, wherein the humanized IgG heavy chain antibody lacks a light chain.

26. The genetically engineered mouse of any one of claims 15-25, wherein the humanized IgG heavy chain antibody comprises a hinge domain, a CH2 domain, a CH3 domain, or a combination thereof.

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

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

29. An engineered non-human animal comprising a germline genome comprising an engineered immunoglobulin heavy chain (IgH) allele at an endogenous IgH locus; wherein the engineered IgH allele lacks an endogenous heavy chain C region gene; and wherein the endogenous heavy chain C region gene comprises cμ, cδ, cε, cγ3, cγ2b, cγ2c, or a combination thereof.

30. The engineered non-human animal of claim 29, wherein the IgH allele comprises a deletion of a nucleic acid sequence encoding a CH1 domain of an IgG 1C region gene (cγ1).

31. The engineered non-human animal of claim 30, wherein the CH1 domain of the IgGlC region gene comprises exon 1.

32. The engineered non-human animal of any one of claims 29-31, 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. The engineered non-human animal of any one of claims 29-32, wherein the IgH locus comprises a native nucleic acid sequence that comprises an endogenous enhancer.

34. The engineered non-human animal of claim 33, wherein the enhancer is E μ, 3' rr, 3' γ1e, 5' hsr1, or a combination thereof.

35. The engineered non-human animal of any one of claims 29-34, wherein the IgH locus comprises a native nucleic acid sequence comprising a switch tandem repeat element (sμ), wherein the sμ drives IgGl expression.

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

37. The engineered non-human animal of any one of claims 29-35, wherein the non-human animal expresses a humanized IgG heavy chain antibody.

38. The engineered non-human animal of any one of claims 37-22, wherein the humanized IgG heavy chain antibody comprises a humanized IgG1 heavy chain antibody.

39. The engineered non-human animal of any one of claims 37-38, wherein the humanized IgG1 heavy chain antibody is an igg1Δch1 protein.

40. The engineered non-human animal of any one of claims 37-39, wherein the humanized IgG heavy chain antibody lacks a light chain.

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

42. The engineered non-human animal of any one of claims 37-41, wherein the IgH locus comprises a human V, D or J gene.

43. The engineered non-human animal of any one of claims 37-42, wherein the endogenous IgH locus comprises an exogenous nucleic acid sequence.

44. The engineered non-human animal of claim 43, wherein the exogenous nucleic acid sequence comprises one or more human V _H Gene segment, one or more humans D _H Gene segments and one or more J _H A gene segment.

45. The engineered non-human animal of any one of claims 43-44, wherein the exogenous nucleic acid sequence comprises 65 human V _H A gene segment.

46. The engineered non-human animal of any one of claims 43-45, wherein the exogenous nucleic acid sequence comprises 27 human D _H A gene segment.

47. The engineered non-human animal of any one of claims 43-46, wherein the exogenous nucleic acid sequence comprises 6J _H A gene segment.

48. The engineered non-human animal of any one of claims 43-47, wherein the exogenous nucleic acid sequence comprises 65 human V _H Gene segment, 27 person D _H Gene segment and 6J _H A gene segment.

49. The engineered non-human animal of any one of claims 43-48, wherein the exogenous nucleic acid sequence comprises a barcode.

50. The engineered non-human animal of any one of claims 29-49, 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. The engineered non-human animal of any one of claims 29-50, 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 engineered non-human animal of any one of claims 29-51, wherein the engineered non-human animal is homozygous for an engineered IgH allele.

53. The engineered non-human animal of any one of claims 29-52, wherein the non-human animal is a mammal.

54. The engineered non-human animal of claim 53, wherein the mammal is a mouse or a rat.

55. A method of making a genetically modified non-human animal capable of producing heavy chain antibodies, comprising (a) deleting an endogenous nucleic acid sequence comprising one or more heavy chain C region genes from an endogenous immunoglobulin heavy chain locus in a stem cell of the non-human animal; (b) implanting stem cells into the blastula; (c) Implanting blastula into pseudopregnant mice to obtain chimeric mice; (d) Crossing the chimeric mice with wild-type mice to produce offspring; (e) screening the offspring for heterozygosity; and (f) identifying a first-established mouse carrying one or more heavy chain C region gene deletions; and wherein the non-human animal is capable of producing heavy chain antibodies.

56. A method of making a genetically modified non-human animal capable of producing a humanized heavy chain antibody comprising (a) deleting an endogenous nucleic acid sequence comprising one or more heavy chain C region genes from an endogenous immunoglobulin heavy chain locus in a non-human animal stem cell; (b) implanting stem cells into the blastula; (c) Implanting blastula into pseudopregnant mice to obtain chimeric mice; (d) Crossing the chimeric mice with wild-type mice to produce offspring; (e) screening the offspring for heterozygosity; and (f) identifying a first-established mouse carrying one or more heavy chain C region gene deletions; and wherein the non-human animal is capable of producing humanized heavy chain antibodies.

57. The method of any one of claims 55-56, wherein the stem cell is an embryonic stem cell.

58. A method of producing a soluble heavy chain antibody in the engineered non-human animal of claim 55, comprising (a) administering an antigen to the non-human animal; (B) isolating one or more B cells from the non-human animal; (c) isolating mRNA from one or more B cells; (d) sequencing the mRNA; (e) confirming clonotype based on mRNA sequence; and (f) performing phylogenetic analysis of the clonotypes; thereby producing soluble heavy chain antibodies.

59. A method of producing a single domain antibody (sdAb) that is validated from the engineered non-human animal of claim 55, comprising(a) Expression in cells of a heavy chain variable (V) encoding a polypeptide comprising V, D and J _H ) A nucleic acid sequence of a domain, wherein the cell produces a heavy chain variable domain; and (b) isolating the heavy chain variable domain from the sample, thereby producing a single domain antibody.

60. The method of claim 59, wherein the single domain antibody is a murine single domain antibody.

61. A method of making a soluble humanized heavy chain antibody in the engineered non-human animal of any one of claims 56-57, comprising (a) administering an antigen to the non-human animal; (B) isolating one or more B cells from the non-human animal; (c) isolating mRNA from one or more B cells; (d) sequencing the mRNA; (e) confirming clonotype based on mRNA sequence; and (f) performing phylogenetic analysis of the clonotypes; thereby producing soluble humanized heavy chain antibodies.

62. A method of producing a single domain antibody (sdAb) identified as derived from the engineered non-human animal of any one of claims 56-57, comprising (a) expressing in a cell a polypeptide encoding a human heavy chain variable (V) comprising V, D and J _H ) A nucleic acid sequence of a domain, wherein the cell produces a human heavy chain variable domain; and (b) isolating the human heavy chain variable domain from the sample, thereby producing a single domain antibody.

63. The method of claim 62, wherein the single domain antibody is a human single domain antibody.

64. The method of claim 62, wherein the cell is a bacterial cell or a human cell.

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 or CH3 domain of an IgG subclass, wherein the IgH allele lacks a nucleic acid encoding at least a portion of an endogenous CH1 domain of the IgG subclass, and wherein the IgH allele lacks an endogenous nucleic acid encoding at least a portion of an IgM constant domain, an endogenous nucleic acid encoding at least a portion of an IgD constant domain, an endogenous nucleic acid encoding at least a portion of an IgE constant domain, or an endogenous nucleic acid encoding at least a portion of an IgA constant domain.

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

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

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

69. The non-human animal of any one of claims 65-67, wherein the IgG subclass is an IgG2a, igG2b, igG2c, igG3, or IgG4 subclass.

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

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

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

73. The non-human animal of claim 70, wherein the IgH allele lacks an endogenous nucleic acid encoding each IgG2 constant domain, an endogenous nucleic acid encoding each IgG3 constant domain, or an endogenous nucleic acid encoding each IgG4 constant domain.

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

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

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

77. The non-human animal of any one of claims 65-74, wherein the IgH allele lacks an endogenous nucleic acid encoding each IgM constant domain.

78. The non-human animal of any one of claims 65-77, wherein the IgH allele lacks an endogenous nucleic acid encoding each IgD constant domain.

79. The non-human animal of any one of claims 65-78, wherein the IgH allele lacks an endogenous nucleic acid encoding each IgE constant domain.

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

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

82. The non-human animal of any one of claims 65-81, wherein the IgH allele comprises endogenous E μ.

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

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

85. The non-human animal of any one of claims 65-84, wherein the IgH allele comprises an endogenous sμ, an endogenous I μ promoter, an endogenous I μ exon, or a combination thereof.

86. The non-human animal of any one of claims 65-85, 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 of any one of claims 65-86, 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 an IgG1 CH2 domain.

88. The non-human animal of any one of claims 65-87, wherein the IgH allele comprises endogenous 3' γ1e.

89. The non-human animal of any one of claims 65-88, wherein the IgH allele lacks an endogenous nucleic acid encoding a downstream full length CH2 domain of the endogenous 3' γ1e.

90. The non-human animal of any one of claims 65-89, wherein the IgH allele comprises endogenous 5' hsr1.

91. The non-human animal of any one of claims 65-90, wherein the first nucleic acid sequence encoding the upstream full length CH2 domain of the endogenous 5' hsr1 is a nucleic acid encoding an IgG CH2 domain.

92. The non-human animal of any one of claims 65-91, wherein the first nucleic acid sequence encoding the upstream full length CH2 domain of the endogenous 5' hsr1 is a nucleic acid encoding an IgG1 CH2 domain.

93. The non-human animal of any one of claims 65-92, wherein the IgH allele comprises an endogenous 3' rr.

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

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

96. The non-human animal of any one of claims 65-95, wherein the IgH allele comprises an endogenous 3' cbe.

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

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

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

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

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

102. The non-human animal of any one of claims 65-100, wherein neither allele of the genome comprises an exogenous exon of an Ig heavy chain variable region.

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

104. The non-human animal of any one of claims 65-101, 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 of claim 104, wherein the IgH allele comprises one or more exogenous human Ig VH gene segments.

106. The non-human animal of claim 104, wherein the IgH allele comprises three or more human Ig VH gene segments.

107. The non-human animal of claim 104, wherein the IgH allele comprises 26 or more human Ig VH gene segments.

108. The non-human animal of claim 104, wherein the IgH allele comprises 65 or more human Ig VH gene segments.

109. The non-human animal of claim 104, wherein the IgH allele comprises 126 human Ig VH gene segments.

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

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

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

113. The non-human animal of any one of claims 104-112, wherein the IgH allele comprises 9 human Ig VJ gene segments.

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

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

117. The non-human animal of any one of claims 65-101 and 104-116, 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 of claim 117, wherein the IgH allele comprises one or more exogenous human igκ variable gene segments.

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

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

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

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

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

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

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

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

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

128. The non-human animal of any one of claims 117-127, wherein the IgH allele comprises 40 human igκ variable gene segments and five human igκvj gene segments.

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

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

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

132. The non-human animal of any one of claims 65-101, 104-115, and 117-130, wherein the non-human animal belongs to a first non-human species, and wherein the IgH allele comprises 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 of claim 132, wherein the IgH allele comprises one or more Ig VH gene segments of the second non-human species.

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

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

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

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

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

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

140. The non-human animal of claim 132, wherein the IgH allele comprises all of an Ig VH gene segment, an Ig VD gene segment, and an Ig VJ gene segment of the second non-human species.

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

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

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

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

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

147. The non-human animal of any one of claims 145-146, wherein the IgH allele comprises one, two, three, four, five, six, seven, eight, nine, or ten different exogenous recombinase site-recognition nucleic acid sequences.

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

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

150. The non-human animal of any one of claims 147-149, wherein the different exogenous recombinase site recognition nucleic acid sequences are each located less than 2.5Mb upstream of endogenous E.

151. The non-human animal of any one of claims 147-149, wherein the different exogenous recombinase site recognition nucleic acid sequences are each located less than 2.0Mb, less than 1.5Mb, less than 1.0Mb, less than 500kb, or less than 250kb upstream of the endogenous E.

152. The non-human animal of any one of claims 147-149, wherein the different exogenous recombinase site recognition nucleic acid sequences are each located less than 200Mb, less than 100Mb, less than 50Mb, less than 25kb, or less than 10kb upstream of the endogenous E.

153. An antibody comprising a variable region, the variable region comprising:

(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.

154. The antibody of claim 153, wherein the antibody binds to a SARS-CoV2 spike polypeptide.

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

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