Heavy chain-only antibodies

Abstract

1. A method for producing a mouse whose B cells express a diverse repertoire of heavy chain-only antibodies (HCAbs) by incorporating a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment within an endogenous immunoglobulin heavy chain constant region locus, wherein the Cγ gene segment comprises a deletion of a nucleotide sequence encoding at least a portion of the CH1 domain.

Description

[Technical Field]

[0001] The present invention relates to heavy chain-only antibody (HCAb) constructs, transgenic mice expressing HCAbs, and methods for producing them in vitro and in vivo. [Background technology]

[0002] Antibodies have emerged as important biological pharmaceuticals because (i) they exhibit exquisite binding properties that allow them to target antigens in diverse molecular forms, (ii) they are physiological molecules that are well tolerated in treated humans and animals because of their favorable pharmacokinetics, and (iii) they are associated with potent immunological properties that allow them to naturally fight off infectious agents. Furthermore, established techniques exist for the rapid isolation of antibodies from laboratory animals that can readily mount specific antibody responses against virtually any foreign substance not naturally present in the body.

[0003] In their most basic form, antibodies are composed of two identical heavy (H) chains, each paired with an identical light (L) chain. The N-terminus of both the H and L chains consists of a variable domain (VH and VL, respectively), which combine to produce a paired H chain with unique antigen-binding specificity. The exons encoding the VH and VL domains of antibodies are not present in germline DNA. Instead, each VH exon is generated by recombination of randomly selected V, D, and J gene segments present in the H chain locus (Igh). Similarly, individual VL exons are produced by chromosomal rearrangements of randomly selected V and J gene segments in the light chain locus (Igl) (see schematic diagram of the mouse Igh locus, Igl kappa locus (Igk or Igκ), and Ig lambda locus (Igl or Igλ; Figure 1) (Tonegawa, Nature, 302:575, 1983; Bassing et al., Cell, 109 Suppl:S45, 2002). The mouse genome contains two alleles capable of expressing heavy chains (one allele from each parent), two alleles capable of expressing kappa (κ) light chains, and two alleles capable of expressing lambda (λ) light chains. Each heavy chain locus contains three alleles. The heavy chain locus contains multiple V, D, and J gene segments, and both light chain loci contain multiple V and J genes. Downstream of the J gene in each immunoglobulin (Ig) locus are one or more exons encoding the antibody constant region (C). The heavy chain locus also contains exons for the expression of different antibody classes (isotypes). In mice, the encoded isotypes are IgM, IgD, IgG1, IgG2a / c, IgG2b, IgG3, IgE, and IgA; in humans, they are IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgE, IgA1, and IgA2.

[0004] During B cell development in fetal liver and adult bone marrow, gene rearrangement first occurs on one of two homologous chromosomes containing the heavy chain V, D, and J gene segments. In pre-B cells, the resulting VH exon is then spliced ​​at the RNA level to exons encoding the constant region of the μ heavy chain. Most μ heavy chains synthesized by pre-B cells are retained in the endoplasmic reticulum (ER) and ultimately degraded through noncovalent interactions between the partially unfolded CH1 domain of the μ heavy chain and the resident ER chaperone BiP (Haas and Wabl, Nature, 306:387-9, 1983; Bole et al., J Cell Biol. 102:1558, 1986). However, a small proportion of μ chains associate with a surrogate light chain complex composed of the invariant λ5 and VpreB proteins, displacing BiP and enabling the μ heavy chain / λ5 / VpreB complex, together with Igα / β signaling molecules, to exit the ER as the preB cell receptor (preBCR) and traffic through the secretory pathway to the plasma membrane (Ubelhart et al., Curr. Top. Microbiol. Immunol. 393:3, 2016).

[0005] VJ rearrangement then occurs in one L chain allele over time to produce a functional L chain, after which the L chain polypeptide completely replaces BiP and can associate with the μ H chain to form a fully functional B cell receptor (BCR) for antigen, thereby forming an immature B cell.

[0006] The ER quality control mechanism, which prevents incompletely assembled Ig molecules from being expressed on the cell surface or secreted, is quite strict. Therefore, molecules such as HL, HHL, or HH are typically retained in the ER and degraded unless rescued by assembly into a complete H2L2 structure. (Because this system primarily focuses on the retention of Ig H chains, free L chains can often be secreted.) However, it has been known for decades that free monoclonal H chains can be secreted in a rare B-cell proliferative disorder called heavy chain disease (HCD) (Franklin et al., Am. J. Med., 37:332, 1964). The H chains in HCD are truncated, and subsequent structural studies have shown that the CH1 domain is deleted in most cases (Corcos et al., Blood, 117:6991, 2011). Mechanistically, the CH1 deletion frees the H chain from inhibitory interactions with BiP, thus allowing its secretion, and also prevents covalent disulfide association with the L chain; therefore, the HCD protein is an HH dimer. Heavy chain-only Abs (HCAbs) can also be found in non-disease contexts: i) Approximately 75% of normal camel serum IgG consists of HCAbs, which lack the CH1 domain and have a structurally altered VH domain that prevents effective association with the VL domain (de los Rios et al., Cur. Opin. Struct. Biol., 33:27, 2015). ii) Mice in which both the kappa and lambda light chain loci are inactivated still produce serum IgG, but production of this antibody requires an error in class switch recombination (CSR) that leads to the deletion of the exon encoding the CH1 domain in B cell DNA (Zou et al., J. Exp. Med., 204:3271, 2007).

[0007] HCAbs are attractive therapeutics due to their high stability and smaller size than conventional immunoglobulins. The VH antigen-binding portion of this molecule, unobstructed by the VL antigen-binding portion, can recognize epitopes within pockets in protein structures that are otherwise inaccessible to conventional antibodies, including enzyme active sites and epitopes on viruses and G-coupled protein receptors. For example, camel-based HCAbs derived from mice in which the endogenous VH gene has been replaced with a camel VH gene and the CH1-encoding exon has been deleted, are a potential source of such antibodies. However, such HCAbs have the disadvantage that the camel VH domain is immunogenic in humans and other animals in which they could be used as therapeutics. Mice exist in which the endogenous VH gene has been replaced with the corresponding human counterpart, and, combined with inactivation of the kappa and lambda light chain loci, could serve as a source of HCAbs. However, production of such antibodies relies on a relatively low frequency of errors in cross-reaction (CSR) during the immune response and is therefore inefficient.

[0008] The size of the binding site of a conventional antibody, consisting of a paired heavy (H) and light (L) chain, can be too large to fit into the groove or recess of the target epitope. The reduced width of the binding site of HCAbs can help access "difficult" epitopes, such as the active site of an enzyme. In another use, HCAbs facilitate the production of bispecific antibodies. The absence of L chains prevents "L chain scrambling," which results in loss of specificity. Transgenic mice producing (human) immunoglobulins consisting of H chains without L chains are useful for discovering such antibodies with desired antigen specificity.

[0009] However, as mentioned above, in the absence of light chains, native heavy chains are not secreted by plasma cells because the CH1 domain of the H chain is bound to the chaperone protein BiP and is therefore retained in the lumen of the endoplasmic reticulum. Deletion of CH1, as in heavy chain disease or by engineering, allows the secretion of the H chain as a disulfide-bonded dimer.

[0010] Immunoglobulin class switching, also known as isotype switching, isotype conversion, or class switch recombination (CSR), is a biological mechanism by which B cells change their immunoglobulin production from one isotype to another, such as from isotype IgM to isotype IgG. In this process, the constant region portion of the antibody heavy chain changes, but the variable region of the heavy chain remains the same. Because the variable region remains unchanged, class switching does not affect antigen specificity. Instead, the antibody retains affinity for the same antigen but can interact with different effector molecules via its Fc region.

[0011] Class switching occurs after activation of mature B cells via membrane-bound antibody molecules (B cell receptors, BCRs) to generate different classes of antibodies, all of which have the same variable domains as the original antibodies expressed by immature B cells as a result of the process of V(D)J recombination, but different constant domains in the heavy chains.

[0012] Naive wild-type mature B cells produce both IgM (Cμ) and IgD (Cδ), the first two heavy chain segments in the wild-type immunoglobulin locus. Following antigen activation, these B cells proliferate. When these activated B cells encounter specific signaling molecules via CD40 and cytokine receptors (both modulated by T helper cells), antibody class switching occurs, resulting in the production of IgG, IgA, or IgE antibodies. During class switching, the constant region of the immunoglobulin heavy chain changes, but the variable region, and therefore the antigen specificity, remains the same. Therefore, different daughter cells derived from the same activated B cell can produce antibodies of different isotypes or subtypes (e.g., IgG1, IgG2, etc.).

[0013] Various immunoglobulin modifications can be envisioned and engineered to produce HCAbs in cell culture. However, in the case of heavy chain-only (HCO) transgenic mice, B lymphocyte development must be kept in mind. B cells are generated daily through the differentiation of hematopoietic stem cells into pro-B, pre-B, and immature B cells. If the modified immunoglobulin does not support this differentiation sequence, B cells will not be generated.

[0014] Reports of HCO antibodies in llamas, transgenic mice, and rats have shown that B cells can develop in which the heavy chain is not paired with the light chain. However, CH1 must be deleted, and in llamas, the heavy chain without the light chain has a VH structure that differs from that of the canonical VH region. This latter finding indicates that not all human VH domains support the development of HCO B cells.

[0015] WO2019018770A1 discloses a single-chain VH antibody comprising a VH domain and an antigen-binding portion consisting of immunoglobulin constant domains CL and CH1. WO2014 / 141192A2 discloses the generation of heavy chain-only antibodies and transgenic non-human animals that produce them. Such antibodies lack the CH1 domain.

[0016] US8754287B2 discloses mice that produce heavy chain antibodies lacking the CH1 domain, and transgenic mice that contain a germline modification that deletes the nucleic acid encoding the CH1 domain.

[0017] VJC L The expression of heavy chain-only antibodies without associated light chains in knockout chickens is described by Schusser et al. (Eur. J. Immunol., 46:2137, 2016).

[0018] Klein et al. (Biochemistry, 18:1473, 1979) describe the interaction of isolated variable and constant domains of a light chain with the Fd' fragment of immunoglobulin G.

[0019] WO2011 / 072204A1 discloses transgenic mice containing a functional Cμ segment. As a result, mature B cells with canonical IgM as the antigen receptor develop normally. However, the downstream Cγ segment of the genome lacks the CH1 domain. When antibodies class switch to the (desired) γ chain during an immune response, these chains cannot pair with L chains. In H chain class switching, the VH is maintained but the CH is exchanged. This has two effects: (i) cells with antibodies whose specificity is defined by the combination of H and L chains are no longer stimulated and die, and (ii) cells whose specificity is primarily defined by the H chain may die because the unpaired VH is structurally inviable. Therefore, in these mice, VH selection is "deferred," i.e., occurs during the immune response.

[0020] WO2007096779A2 describes the use of transgenic mice for the production of class-specific heavy chain-only antibodies, the mice containing multiple heterologous VH heavy chain loci, each VH heavy chain locus containing one or more V gene segments, one or more D gene segments, one or more J gene segments, and a gene segment encoding a heavy chain constant region that does not contain a CH1 domain when expressed.

[0021] WO2009013620A2 describes the production of fully human soluble VH domains by incorporating human V segments into the mouse heavy chain locus, where the mouse V, D and J gene segments are replaced with V, D and J segments of human origin, and the immunoglobulin heavy chain effector constant region is replaced with an immunoglobulin heavy chain effector constant region lacking CH1.

[0022] WO2015143414A2 describes mice comprising a deletion in the immunoglobulin constant region CH1 gene of the heavy chain constant region gene sequence and replacement of one or all endogenous VH, DH and JH gene segments with at least one unrearranged VL gene segment and at least one unrearranged JL gene segment.

[0023] WO2019184014A1 describes mice containing a transgenic Cγ gene segment introduced into the immunoglobulin locus in place of the endogenous Cμ gene segment and containing a deletion of the CH1 domain (see Figure 1). EP2411408B1 and US9353179B2 describe transgenic mice containing randomly integrated VH and VHH loci. Problems with randomly integrated immunoglobulin (and other) loci, namely non-physiological levels of expression and instability (loss or silencing of function) of the inserted transgene, have been documented since the 1980s of the last century.

[0024] There is a need for efficient and cost-effective methods for the stable production of HCAbs, and more specifically, for transgenic non-human animals capable of producing antigen-specific HCAbs. Summary of the Invention [Problem to be solved by the invention]

[0025] This Summary is provided to introduce certain concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, applications, and advantages of the claimed subject matter will become apparent from the following Detailed Description, including the aspects illustrated in the accompanying drawings and defined in the appended claims.

[0026] The object of the present invention is to provide means, constructs, and methods for the efficient and facile production of heavy chain-only antibodies (HCAbs) and B cell repertoires expressing various such HCAbs, whether in transgenic animals or in vitro cell culture. [Means for solving the problem]

[0027] This object is solved by the subject matter of the present claims and as further described herein. In accordance with the present invention, methods are provided for producing mice whose B cells express a diverse repertoire of heavy chain-only antibodies (HCAbs) by incorporating a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment within an endogenous immunoglobulin heavy chain constant region locus, wherein the Cγ gene segment contains a deletion of a nucleotide sequence encoding at least a portion of the CH1 domain.

[0028] Specifically, when the mouse is immunized with an antigen, the mouse develops a B cell repertoire that secretes diverse antigen-specific HCAbs. Specifically, when the mouse is immunized with an antigen, the antigen-specific B cells differentiate into plasma cells that secrete antigen-specific HCAbs.

[0029] Specifically, upon immunization with an antigen, mice activate antigen-specific B cells and induce their differentiation into plasma cells that secrete a variety of antigen-specific HCAbs. Specifically, methods are provided for producing mice that express a B cell repertoire that secretes diverse antigen-specific HCAbs upon immunization with an antigen by incorporating a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment within an endogenous immunoglobulin heavy chain constant region locus, wherein the Cγ gene segment contains a deletion of a nucleotide sequence encoding at least a portion of the CH1 domain.

[0030] In wild-type B cells, IgM and IgD are coexpressed, but this occurs in mature B cells in the periphery (e.g., spleen) but not in B cells developing in the bone marrow. The Cγ gene segment, located downstream of an inactivated Cμ gene segment, may not be expressed during B cell development. Because Ig heavy chains are required for B cell development, mice containing a Cγ gene segment downstream of an inactivated Cμ gene segment may not produce B cells at all. If a Cγ gene segment is introduced into the Ig heavy chain locus instead of a Cμ gene segment, misfolded delta heavy chains will reside in the endoplasmic reticulum (ER), and coexpression of IgD, which is light chain-dependent, may be detrimental to B cells. This can lead to ER stress and activation of the unfolded protein response (UPR). The UPR aims to restore protein homeostasis; if not restored, B cells undergo apoptotic cell death. Specifically, by positioning a Cγ gene segment upstream of the endogenous location of the Cμ gene segment, pre-B cells express a Cγ-containing preBCR, which includes a V that can be stably expressed without light chains. H This has the significant advantage that positive selection of the -Cγ heavy chain occurs. The unstable version of Vγ does not dimerize with the light chain. H Pre-B cells expressing -Cγ heavy chains are unable to form preBCRs and are therefore excluded from selection, resulting in an improved B cell repertoire expressing a variety of HCAbs of the IgG type.

[0031] In particular, the mouse comprises cells comprising such recombinant immunoglobulin heavy chain loci that are capable of producing said B cell repertoire. Specifically, the deleted portion of at least the CH1 domain comprises all or a portion of the CH1 domain, particularly at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99%, or 100%, of the entire nucleotide sequence encoding the CH1 domain. Specifically, the Cγ gene segment comprises a deletion of at least a portion of the nucleotide sequence encoding CH1, wherein the portion is the chaperone binding domain, particularly wherein the portion is one or more amino acids bound to BiP.

[0032] In certain embodiments, the HCAbs described herein lack a CH1 domain, and in particular an IgG CH1 domain. According to certain embodiments, the mouse may be capable of expressing a portion of the CH1 domain, but the portion does not include a functional CH1 domain capable of pairing with a CL domain.

[0033] According to further specific embodiments, the HCAbs described herein comprise a CH1 domain lacking a chaperone-binding domain, particularly a BiP chaperone-binding domain. Specifically, the mouse is engineered to express an HCAb lacking at least such a chaperone-binding domain of the CH1 domain.

[0034] The HCAb secreted by the mouse cells described herein can be soluble or membrane-bound. By deleting all or at least part of the CH1 domain, the expressed HCAb is not retained in the endoplasmic reticulum (ER) by the ER quality control mechanism that typically prevents cell surface expression or secretion of heavy chain-only antibody constructs. Specifically, the ER chaperone BiP does not retain the HCAb of the present invention in the ER.

[0035] Specifically, the Cγ gene segment is positioned downstream of the Eμ major intronic enhancer within the endogenous immunoglobulin heavy chain locus, preferably downstream of the endogenous mouse Eμ major intronic enhancer located downstream of the J coding sequence within the endogenous mouse immunoglobulin heavy chain locus, specifically comprising SEQ ID NO:7.

[0036] As described herein, the Cγ gene segment is located upstream of the CM gene segment, which may or may not be mutated. Specifically, the Cγ gene segment is located upstream of the position where the CM gene segment is located in the wild-type heavy chain locus. Specifically, the Cγ gene segment is upstream of the CM gene segment and downstream of the JH gene segment in the immunoglobulin heavy chain locus of the mouse described herein.

[0037] Naive wild-type mature B cells produce both IgM (Cμ) and IgD (Cδ), the first two heavy chain segments in the wild-type immunoglobulin locus. As described herein, by positioning the Cγ gene segment upstream of the Cμ gene segment within the endogenous immunoglobulin heavy chain locus, naive mature B cells of the mice described herein produce IgG-type HCAbs, and these B cells proliferate after activation with antigen. Surprisingly, B cells containing the immunoglobulin locus described herein develop normally and can produce IgG-type membrane-bound or soluble HCAbs. Thus, the present invention provides an improved B cell repertoire that expresses a variety of IgG-type HCAbs.

[0038] Specifically, IgG-type HCAbs are characterized by a VH domain containing an IgG framework, particularly a human or mouse framework, and / or an antibody constant domain that is that of an IgG antibody. Specifically, IgG-type HCAbs are of any IgG subtype, for example, any of the mouse IgG1, IgG2a / c, IgG2b, or IgG3 subtypes, or any of the human IgG1, IgG2, IgG3, or IgG4 subtypes.

[0039] Specifically, the transgenic Cγ gene segment is integrated upstream of the endogenous Cμ gene segment within the endogenous mouse immunoglobulin heavy chain locus, and is therefore in a location that is different from its respective location in wild-type cells, where the Cγ gene segment is placed downstream of the Cμ gene segment within the immunoglobulin heavy chain locus.

[0040] In certain embodiments, the transgenic Cγ gene segment is derived from an endogenous nucleic acid sequence, specifically a mouse Cγ gene segment that has been modified by deletion of a nucleotide sequence encoding at least a portion of the CH1 domain and integrated into the endogenous mouse immunoglobulin heavy chain locus at a location where it is not naturally found, i.e., upstream of the endogenous Cμ gene segment.

[0041] According to different specific embodiments, the transgenic Cγ gene segment is an exogenous nucleic acid, such as a synthetic nucleic acid, that is integrated upstream of an endogenous Cμ gene segment within the endogenous mouse immunoglobulin heavy chain locus.

[0042] Specifically, the transgenic Cγ gene segment is of mammalian origin, preferably of rodent origin, and most preferably of murine origin. Specifically, the transgenic Cγ gene segments are of human origin.

[0043] Specifically, the transgenic Cγ gene segments comprise or consist of transgenic Cγ1, Cγ2a / c, Cγ2b, or Cγ3 gene segments.

[0044] Specifically, the transgenic Cγ gene segments comprise or consist of transgenic Cγ1, Cγ2, Cγ3, or Cγ4 gene segments. Specifically, the transgenic Cγ gene segment is a transgenic mouse or human Cγ1 gene segment.

[0045] Specifically, the endogenous immunoglobulin heavy chain loci of the mice described herein are endogenous mouse immunoglobulin heavy chain loci, particularly those that are in their endogenous locations within the mouse genome.

[0046] According to certain embodiments of the methods provided herein, the Cμ gene segment in the immunoglobulin heavy chain locus of the mouse or B cell described herein is inactive. Specifically, the Cμ gene segment is inactive due to a loss-of-function mutation, such as the introduction of a stop codon, or the deletion of a portion of the nucleotide sequence encoding the Cμ gene segment. Preferably, the Cμ gene segment is not deleted entirely, but only a portion thereof. Specifically, the Cμ gene segment is inactive due to the deletion of the CH1, CH2, CH3, and / or CH4 domains.

[0047] In certain cases, when the locus is transcribed, the VDJ exon is spliced ​​to the Cμ gene segment. This can result in the production of light chain-dependent μ heavy chains. Specifically, by inactivating the Cμ gene segment, the quality of the B cell repertoire described herein can be further improved.

[0048] Specifically, the Cμ gene segment is inactivated by deletion of the entire nucleotide sequence encoding the endogenous Cμ gene segment, or a portion thereof that is at least about any one of 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% of said sequence.

[0049] Specifically, the Cμ gene segment preferably contains a loss-of-function, deletion, or inactivating mutation, preferably the introduction of a stop codon, in any one or all of its CH1, CH2, CH3, and CH4 domains. Preferably, the Cμ gene segment contains a stop codon in the CH1, CH2, and CH3 domains.

[0050] According to certain embodiments of the methods provided herein, the μ switch (S) region in the immunoglobulin heavy chain locus of a mouse described herein or a B cell described herein is inactive. Specifically, the immunoglobulin heavy chain locus of a mouse described herein comprises a loss-of-function mutation, deletion, or inactivating mutation in the nucleotide sequence encoding the μ switch (S) region typically present in the endogenous Igh locus. Specifically, an Igh locus without a functional S region is unable to undergo isotype switching.

[0051] Specifically, when the B cells described herein undergo isotype switching, light chain-dependent heavy chain isotypes IgG, IgA, or IgE may be produced. This may result in the death of the B cells described herein. Therefore, preventing isotype switching can further improve the B cell repertoire.

[0052] In certain embodiments, the CH domains of additional heavy chain isotypes are inactivated, preferably by the introduction of loss-of-function, deletion, or inactivating mutations. Specifically, the constant region gene segments of the immunoglobulin heavy chain loci described herein preferably contain loss-of-function, deletion, or inactivating mutations, preferably the introduction of stop codons, in any one or all of their CH1, CH2, CH3, and CH4 domains.

[0053] According to certain embodiments, the mice produced according to the methods described herein comprise inactivated or deleted endogenous immunoglobulin light chain loci. Specifically, the transgenic mice described herein comprise loss-of-function mutations in either or both of the endogenous kappa or lambda light chain loci, or deletions in either or both of the endogenous kappa or lambda light chain loci. Such mice are characterized by expressing HCAbs that lack light chains.

[0054] In certain embodiments, the immunoglobulin heavy chain locus of a mouse produced according to the methods described herein is a human V H , D and J H Coding sequence, as well as V H , D and J H It includes an expression control sequence operably linked to control expression of the coding sequence, particularly a murine expression control sequence.

[0055] In certain embodiments of the methods provided herein, during B cell development, a V of the immunoglobulin heavy chain locus described herein is H , D and J H The coding sequences recombine to form a VDJ coding sequence that expresses a VH binding site. Each developing B cell produces one V for recombination and expression as an HCAb. H , one D, and one J HThe gene segment is randomly selected. Upon maturation, each of the millions of B cells in a mouse recognizes a specific antigen and becomes activated when it encounters that antigen.

[0056] Specifically, V H , D and J H The coding sequences are recombined to form a VDJ coding sequence that expresses a VH binding site that specifically recognizes an antigen, thereby generating a recombined VDJ coding sequence in a given B cell. H The coding sequence is obtained, and the B cells express the antigen-specific VH encoded by the recombined VH coding sequence. H It is possible to secrete IgG-type HCAbs containing the binding domain.

[0057] Specifically, V H , D and J H The coding sequences recombine to form a VDJ coding sequence that expresses a VH binding site that specifically recognizes an antigen, thereby generating a recombined VDJ coding sequence in a B cell. H The coding sequence is obtained, and the progeny of this B cell expresses the antigen-specific VH encoded by the recombined VH coding sequence. H It is possible to secrete IgG-type HCAbs containing the binding domain.

[0058] Specifically, V H , D and J H The coding sequences recombine to form a VDJ coding sequence that expresses a VH binding site that specifically recognizes an antigen, thereby generating a recombined VDJ coding sequence in a B cell. H The coding sequence is obtained, and the B cell differentiates into a plasma cell, specifically a plasma cell, which produces an antigen-specific VH encoded by the recombined VH coding sequence. H It is possible to secrete IgG-type HCAbs containing the binding domain.

[0059] In certain embodiments of the methods provided herein, after a mouse described herein is immunized with an antigen, B cells expressing a VH binding site that specifically recognizes that antigen are activated and produce antigen-specific VH binding sites.H They differentiate into plasma cells capable of secreting IgG-type HCAbs containing the binding domain.

[0060] Specifically, the HCAbs secreted by murine B cells described herein are antigen-specific V H The antibody comprises or consists of an IgG constant region lacking a CH1 domain or comprising or consisting of only a portion of the CH1 domain, wherein the portion of the CH1 domain lacks the chaperone-binding domain, preferably the CH1 domain lacks the BiP chaperone-binding domain.

[0061] Further provided herein is a B cell repertoire expressing various IgG-type HCAbs. Specifically, the B cell repertoire expresses various IgG-type HCAbs derived from a mouse obtained by the method described herein. The mouse described herein may contain the B cell repertoire, or the B cell repertoire may be isolated from the mouse. The B cell repertoire may be provided in the form of a library of B cells or a library of nucleic acid molecules derived from the B cell repertoire. Each library may be prepared using a display system that allows screening of molecules expressing antigen-binding properties. Specifically, such a library of nucleic acid molecules encodes various HCAbs or respective antigen-binding molecules comprising the VH-binding sites of such HCAbs. According to certain embodiments, such a library of nucleic acid molecules may be used in a method for producing a library of antigen-binding molecules comprising or consisting of VH-antigen-binding sites or VH domains with different binding properties.

[0062] Specifically, the B cell repertoires described herein can express a variety of HCAbs that differ in their (VH) antigen-binding sites, thereby enabling, for example, the production of a variety of antigen-binding molecules, such as antibodies, which recognize the same antigen or epitope, such as the production of affinity-matured or otherwise optimized antibody variants, or the production of antibodies that specifically recognize a target antigen (but different epitopes of such target antigen).

[0063] According to certain embodiments, the libraries described herein, e.g., libraries of B cells, or libraries of nucleic acid molecules or antigen-binding molecules, respectively, can be suitably screened, and library members selected according to desired structural or functional properties to identify and produce antigen-binding molecules comprising the VH binding site of the selected library members. Such libraries described herein can be suitably screened, and individual library members selected according to desired structural or functional properties to produce antigen-binding molecules, particularly antibody products.

[0064] Specifically, the B cell repertoire described herein is capable of expressing a variety of antigen-specific HCAbs with different VH domains. In certain embodiments, the B cell repertoire described herein expresses antibodies or antibody fragments, particularly various HCAbs described herein, which include various antibodies or antibody fragments that each specifically recognize the same target antigen or epitope. Specifically, the B cell repertoire expresses antigen-specific antibodies or antibody fragments after a mouse described herein is immunized with an antigen. According to certain embodiments, an antibody library is provided herein that includes or covers various antibodies that recognize the same target antigen or epitope.

[0065] In certain embodiments, the B cell repertoire described herein expresses antibodies or antibody fragments, particularly various HCAbs described herein, which include various antibodies that recognize different target antigens. Such a repertoire can be obtained by immunization with complex multicomponent antigens, such as viruses or bacteria, which have many different target antigens, each with multiple epitopes. According to certain embodiments, an antibody library is provided herein that includes or covers various antibodies that recognize different target antigens.

[0066] According to a specific embodiment, the present invention provides a repertoire of B cells expressing various antibodies or antibody fragments that lack any light chain. Specifically, the present invention provides a repertoire of B cells expressing various HCAbs described herein, each specifically recognizing the same target antigen or different target antigens. Specifically, the present invention provides a B cell repertoire that can be obtained or has been obtained by the methods provided herein. Specifically, the B cell repertoire is expressed by the transgenic mouse described herein.

[0067] Specifically, the various antigen-binding molecules referred to herein, for example, the various B cell repertoires or respective libraries described herein, are composed of various, at least 10 2 , 10 3 , 10 4 , 10 5 or 10 6 The antibody or antibody-specific molecule may comprise or cover any one of the antigen-specific molecules, such as an antibody or HCAb.

[0068] The diversity of the B cell repertoire can be determined, for example, by deep sequencing to determine the number of different VDJ configurations, indicating the diversity of HCAbs expressed by the B cell repertoire.

[0069] The B cell repertoire described herein can be provided as a repertoire of nucleic acid sequences, which can be obtained or obtained by sequencing the recombined VH sequence in the B cell produced by the mouse described herein.Such sequencing can be carried out using deep sequencing methods such as next-generation sequencing (NGS).Deep sequencing refers to sequencing a genome region multiple times, sometimes hundreds or even thousands of times.This NGS approach can provide the nucleic acid or amino acid sequences of various VH domains.

[0070] According to certain embodiments, an antibody library of HCAbs is provided, wherein: a) the genes encoding said antibodies are derived from a B cell repertoire as described herein, or b) The antibodies are secreted by mammalian plasma cells, preferably of rodent origin, especially of murine origin.

[0071] Specifically, this library can be obtained by cloning genes encoding said antibody repertoire from B cells of the B cell repertoire described herein, or by secreting antibodies from various mammalian plasma cells, wherein the antibodies secreted by mammalian plasma cells are characterized by a glycosylation pattern characteristic of the species from which the mammalian plasma cells originate.

[0072] Accordingly, the present invention further provides methods for producing the antibodies described herein, and in particular repertoires of the antibodies described herein, by engineering mammalian plasma cells that express and secrete such antibodies.

[0073] In particular, the mammalian plasma cells are derived from a rodent, preferably a mouse. It is appreciated that the antibodies described herein may be prepared using, for example, one or more sequences from a species other than mouse, including a non-mouse animal, or a human, or any combination thereof, including, for example, a human nucleic acid sequence encoding each human antibody domain.

[0074] Sequences of human antibody constant domains are well known in the art and can be obtained from various databases, including the National Center for Biotechnology Information (NCBI) and ImmunoGeneTics (IMGT). Exemplary sequences of the CH1, CH2, and CH3-S human IgG1 constant domain exons (S is the 3' portion of the CH3 exon encoding the secreted γ1 HC) are identified by SEQ ID NOs: 16-18.

[0075] It is fully understood that any of the sequences of human antibody domains are merely exemplary, and alternatively, the sequences of human antibody domains of each different allele may be used. In the structures of each antibody described herein, modified (artificial) nucleotide sequences and respective amino acid sequences, for example, respective sequences that contain any one of at least 80%, 85%, 90%, or at least 95% sequence identity, can be used in place of the animal- or human-derived nucleotide sequences encoding the antibody domains or the animal- or human-derived amino acid sequences, so long as the respective antibody domains are functional to pair and link.

[0076] According to particular embodiments, the antibodies are produced in host cells (in vitro) or in a non-human animal host, in particular a mouse (in vivo). The structure of an exemplary HCAb described herein is shown in Figure 3B.

[0077] A VH antigen-binding portion specifically comprises or consists of three CDR loops of the VH domain, i.e., VH-CDR1, VH-CDR2, and VH-CDR3. The antigen-binding portion can be affinity matured by mutating one or more of the CDR loops to optimize or increase the affinity of binding to the target antigen. Such mutations can be obtained, for example, by in vivo processes or by in vitro mutagenesis techniques, by one or more point mutations, e.g., one, two, three, or more point mutations, in any or each of the CDR sequences to obtain an affinity-matured antigen-binding site.

[0078] Specifically, HCAbs are molecules consisting of two identical heavy chains, each containing a VH domain fused to antibody constant domains CH2 and CH3. Specifically, the antibody constant domains are IgG constant domains. When the CH2 and CH3 domains of both heavy chains are paired, HCAbs contain two VH domains and an Fc region.

[0079] The Fc region of the HCAb described herein specifically includes the constant region of the antibody, excluding the first constant region immunoglobulin domain. "Fc region" typically refers to the last two constant region immunoglobulin domains of IgG, IgA, or IgD, the flexible hinge region N-terminal to these domains, and the last three constant region immunoglobulin domains of IgE and IgM. Specifically, the hinge is that of an IgG, IgA, or IgD antibody.

[0080] In the case of IgG-type HCAbs, the Fc specifically comprises or consists of the Cγ immunoglobulin domains CH2 and CH3, and optionally the hinge region between the Fc domain and the antibody domain of the VH arm or between VH and CH2. The Fc region of an IgG-type HCAb specifically does not comprise the CH1 domain, or comprises a portion of the CH1 domain lacking the chaperone binding sequence. The Fc region may comprise the CH2 or CH3 domain in the form of an artificial variant of the respective naturally occurring antibody domain, e.g., in the form of at least 90% sequence identity with said naturally occurring antibody domain.

[0081] In particular, the Fc region described herein comprises or consists of a dimer of CH2 and CH3 domains, which are part of an antibody heavy chain (HC), with the CH2 domain of one HC pairing with the CH2 of a second HC and the CH3 domain of the first HC pairing with the CH3 of the second HC. Such a dimer can be a homodimer, i.e., composed of two CH2-CH3 domain chains of the same amino acid sequence, or a heterodimer, i.e., composed of two CH2-CH3 domain chains, each with a different amino acid sequence, e.g., with a different CH3 amino acid sequence to stabilize the Fc.

[0082] In certain embodiments, HCAbs comprise a hinge region connecting the CH2-CH3 domain chain of the Fc region to the VH domain. Specifically, HCAbs comprise a hinge region connecting the VH domain and the antibody constant domain. Specifically, the hinge region is derived from a conventional antibody heavy chain hinge region linking the C-terminus of the CH1 domain to the N-terminus of the CH2 domain. Alternatively, any other natural or artificial linker of approximately the same length may be used. A suitable hinge region is a natural (naturally occurring, e.g., human or mouse) IgG or IgA heavy chain hinge region, or a functional variant thereof of the same length ±1 or 2 amino acids, optionally containing one or more, up to a maximum of 5 point mutations. The hinge region typically contains one or more cysteine ​​residues to create disulfide bridges in the HCAb, for example, to connect two HCs.

[0083] Specifically, the amino acid sequences of the two HCs (also referred to herein as the first and second HCs) contained in the HCAb are identical. Alternatively, the amino acid sequences of the two HCs are different, for example, because the antigen-binding sites of the VH domains are different.

[0084] For example, a first HC comprises a first VH, and a second HC comprises a second VH. The first and second VHs can comprise the same or different antigen-binding sites, e.g., those that specifically recognize two different target antigens. Thus, an HCAb can be monospecific, bivalent, or bispecific.

[0085] Antibodies produced by transgenic non-human animals, such as the mice described herein, are generally understood as natural or spontaneous antibodies. Such natural antibodies can be derived from a repertoire of antibodies that specifically recognize an antigen, such as those produced by the transgenic mice described herein.

[0086] According to a particular embodiment, the antibody or repertoire of antibodies is affinity matured in vivo, e.g., to a total of 10 -7 Less than M, e.g. 10-7 From 10 -10 K between M D This can result in high affinity antibodies that bind to specific target antigens.

[0087] Affinity matured antibodies produced by in vitro mutagenesis methods, such as those using random mutagenesis and / or library techniques, can be used to, for example, D is 10 -8 Less than M, e.g. 10 -11 This may result in even higher affinities below M.

[0088] Native antibodies are advantageously characterized by the native conformation of the VH-CDR sequences, which is characterized by the naturally occurring primary structure of the antigen-binding site and / or the naturally occurring primary structure of the full-length VH domain.

[0089] When producing HCAbs, the antibody domain and / or hinge region selected can be of human, artificial, or non-human animal origin. For example, HCAbs are produced in transgenic mice containing human and mouse sequences.

[0090] According to certain embodiments, the HCAb described herein is provided in a soluble form, for example, a water-soluble form, at a concentration suitable for use in pharmaceutical preparations.Specifically provided herein is a soluble preparation comprising the HCAb described herein in an isolated form, such as isolated from the serum or blood fraction of the animal that produces it, or isolated from a cell culture fraction.

[0091] Further provided herein is a mouse obtainable or obtained by the method described herein. Specifically provided herein is a mouse comprising the immunoglobulin heavy chain locus described herein. Specifically, the mouse described herein comprises a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment within its endogenous immunoglobulin heavy chain locus, wherein the Cγ gene segment comprises a deletion of a nucleotide sequence encoding at least a portion of the CH1 domain.

[0092] Specifically, the mice described herein comprise modified immunoglobulin alleles or other transgenes in their genomes. Specifically, the mice described herein comprise at least a transgenic Cγ gene segment in their endogenous immunoglobulin heavy chain loci and are thus transgenic animals.

[0093] In certain embodiments, the mice described herein further comprise human immunoglobulin regions. For example, numerous methods have been developed to replace endogenous mouse immunoglobulin regions with human immunoglobulin sequences to generate partially or fully human antibodies for drug discovery purposes. Examples of such mice include those described in, for example, U.S. Patent Nos. 7,145,056, 7,064,244, 7,041,871, 6,673,986, 6,596,541, 6,570,061, 6,162,963, 6,130,364, 6,091,001, 6,023,010, 5,593,598, 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,661,016, 5,612,205, and 5,591,669.

[0094] According to certain aspects, the immunoglobulin heavy chain loci described herein comprise chimeric gene segments. Specifically, the mice described herein comprise chimeric immunoglobulin segments, particularly human IGH (V), as described in US 2013 / 0219535, embedded in corresponding mouse non-coding regulatory sequences. H , D., J. H ) coding sequences. Immunoglobulin gene segments comprising human immunoglobulin coding sequences and mouse expression control sequences are also referred to herein as "chimeric immunoglobulin gene segments."

[0095] Specifically, such transgenic mice have a genome that includes an introduced exogenous immunoglobulin region that is partially human, the introduced region comprising human variable region coding sequences and mouse non-coding regulatory sequences that control expression of the human sequences, which are of mouse origin or endogenous to the mouse's genome and have been knocked into the mouse genome by the introduction of chimeric immunoglobulin gene segments or chimeric immunoglobulin loci. In particular, the mouse sequences, particularly the mouse non-coding regulatory sequences, are based on sequences identical to the corresponding mouse endogenous sequences, i.e., those of an endogenous wild-type immunoglobulin locus.

[0096] The mouse expression control sequences described herein for expressing human immunoglobulin gene sequences are specifically selected from the group consisting of promoters, transcriptional start and stop sequences, enhancer and activator sequences, and ribosome binding sites. Specific examples of such expression control sequences are promoters, 5' untranslated sequences, introns between the coding sequences for leader peptides, recombination signal sequences (RSSs), and sequences flanking the coding sequences, which may include splice sites.

[0097] Specifically, the mice described herein contain a human VV ... H , D and J H Coding sequence, as well as V H , D and J HV, which includes an expression control sequence operably linked to control expression of the coding sequence H Contains the heavy chain locus, specifically V H The heavy chain locus is chimeric and V H The expression control sequences of the heavy chain locus are murine, preferably endogenous expression control sequences.

[0098] Specifically, the VH heavy chain locus is H , D and J H Specifically, this includes a repertoire of coding sequences, which may include at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 V sequences. H coding sequence, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 D coding sequences, and at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 J coding sequences. H coding sequences, specifically human VH, D and JH coding sequences. Specifically, this includes up to 120 VH, D and JH coding sequences. H coding sequence, up to 40 D coding sequences, and up to 20 J coding sequences H Specifically, the VH heavy chain locus contains the complete repertoire of human VH, D and JH coding sequences.

[0099] Specifically, V H V of the heavy chain locus H , D and J H The coding sequences recombine to form a VDJ coding sequence that specifically recognizes an antigen, and the recombined V H B cells containing the heavy chain locus express antigen-specific V H An HCAb is expressed that comprises a binding domain and an IgG constant region lacking the CH1 domain or lacking at least a portion of the CH1 domain.

[0100] Specifically, the transgenic Cγ gene segments are included in the nucleic acid construct. In certain embodiments, the transgenic Cγ gene segment comprises at least one immunoglobulin gene segment coding sequence, particularly at least a Jγ gene segment coding sequence of an immunoglobulin heavy chain locus. H Gene segment coding sequences, preferably at least J H Included in the nucleic acid construct is a coding sequence for 2-6 gene segments.

[0101] Specifically, a nucleic acid construct comprising a transgenic Cγ gene segment and a chimeric immunoglobulin gene segment comprising a human IGH coding sequence, preferably at least a human IGH J coding sequence, embedded in corresponding mouse non-coding regulatory sequences can be introduced into the mouse genome as an exogenous nucleic acid construct or element upstream of the endogenous Cμ gene segment and replacing at least a portion of the mouse's endogenous immunoglobulin heavy chain locus. Specifically, the nucleic acid construct comprises a human IGH V coding sequence. H , D and J H Contains coding sequences.

[0102] Specifically, a nucleic acid construct comprising a transgenic Cγ gene segment, a chimeric immunoglobulin gene segment comprising a human IGH coding sequence, preferably at least a human IGH J coding sequence, embedded in corresponding mouse non-coding regulatory sequences, and an inactive Cμ gene segment, as described herein, can be introduced into the mouse genome upstream of the endogenous Cδ gene segment and as an exogenous nucleic acid construct or element that replaces at least a portion of the mouse's endogenous immunoglobulin heavy chain locus. Specifically, the nucleic acid construct comprises a human IGH V H , D and J H Contains coding sequences.

[0103] Alternatively, a transgenic Cγ gene segment, and optionally an inactive Cμ gene segment, is knocked into the immunoglobulin locus in the mouse genome.

[0104] In another preferred embodiment, the genomic content of the mice described herein is modified to enable their B cells to express multiple functional VH domains per cell, i.e., the cells produce bispecific antibodies as described in WO2017035252A1.

[0105] Specifically, the Cμ gene segment within the immunoglobulin heavy chain locus of the transgenic mice described herein is inactive, specifically, the Cμ gene segment is inactive due to a loss-of-function mutation, a deletion of the Cμ gene segment, or one or more mutations that introduce one or more stop codons.

[0106] Specifically, described herein is a mouse immunoglobulin heavy chain locus described herein, comprising a transgenic Cγ gene segment 5′ to the Cμ gene segment, the Cγ gene segment comprising a deletion of a nucleotide sequence encoding at least a portion of the CH1 domain.

[0107] In a specific embodiment, the Cμ gene segment is an endogenous gene segment of the immunoglobulin heavy chain locus of a mouse described herein. In certain embodiments, the Cμ gene segments of the immunoglobulin heavy chain loci described herein are transgenic gene segments that replace endogenous Cμ gene segments within an endogenous immunoglobulin heavy chain locus and contain one or more inactivating mutations.

[0108] Specifically, the transgenic Cμ gene segment is inactive by a loss-of-function mutation in the Cμ gene segment, preferably in any one or more of the CH1, CH2, CH3 and CH4 domains, or by a partial deletion of the Cμ gene segment, for example, at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or by the introduction of an exogenous stop codon.

[0109] Specifically, the Cμ gene segments described herein are located in their natural location in the wild-type mouse immunoglobulin heavy chain locus. Specifically, the mouse described herein comprises a modification within its endogenous immunoglobulin heavy chain locus described herein.

[0110] Specifically, the loci described herein are recombinant (e.g., "chimeric") loci that are derived from a mouse but include at least one exogenous element, e.g., one or more exogenous heavy chain regions, that are not naturally associated with the regulatory elements of the locus, such as a Cγ gene segment that includes a deletion in the nucleotide sequence encoding at least a portion of the CH1 domain.

[0111] Specifically, mice are engineered to integrate gene sequences into the immunoglobulin heavy chain locus by a suitable gene targeting technique, for example, by directed homologous recombination using site-specific recombinase techniques, or CRISPR / Cas9 techniques.

[0112] Specifically, the mice do not express the endogenous kappa and / or lambda loci because the endogenous kappa and / or lambda loci are deleted, silenced, or otherwise mutated to a loss of function.

[0113] According to certain embodiments, the method for producing the antibodies described herein further comprises immunizing a mouse described herein with an antigen to elicit an immune response against the antigen by antigen-specific B cells contained in the mouse's B cell repertoire, which ultimately differentiate into plasma cells that secrete HCAbs specific to the immunizing antigen.

[0114] The antigen, with or without an adjuvant, can be administered to the mice in any convenient manner and can be administered according to a predetermined schedule. Specifically provided herein is a method for producing an antigen-specific antibody, comprising: a) immunizing a transgenic mouse with an antigen, wherein the mouse comprises a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment within an immunoglobulin heavy chain locus, the Cγ gene segment comprising a deletion of a nucleotide sequence encoding at least a portion of a CH1 domain; b) expressing an HCAb comprising an antigen-specific VH and an IgG constant region; c) isolating one or more nucleic acid sequences encoding said antigen-specific VH; d) producing a monoclonal antibody comprising the antigen-specific VH; The method includes:

[0115] Specifically, provided herein is a method for producing an antibody comprising an antigen-specific VH domain, comprising: a) immunizing a mouse with an antigen, wherein the mouse comprises a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment within an immunoglobulin heavy chain locus, the Cγ gene segment comprising a deletion of a nucleotide sequence encoding at least a portion of a CH1 domain, thereby resulting in a repertoire of cells expressing antigen-specific HCAbs; b) selecting cells expressing IgG-type HCAbs containing antigen-specific VHs from the repertoire; c) determining the nucleic acid sequence encoding the antigen-specific VH from the HCAb; d) producing a monoclonal antibody comprising the antigen-specific VH; A method is provided which includes:

[0116] Specifically, monoclonal antibodies produced according to the methods described herein may be full-length immunoglobulins, antigen-binding antibody fragments thereof, or any other antibody construct comprising at least a variable heavy (VH) antibody domain, or antibodies comprising a single VH domain, e.g., one or more single-domain antibodies, Fab, F(ab'), (Fab)2, scFv, Fd, Fv, or full-length antibodies, e.g., IgG type (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies. Particular embodiments are heavy-chain-only antibodies, such as the HCAbs described herein.

[0117] Specifically, the nucleic acid sequence encoding the antigen-specific VH is determined by sequencing the recombined VH sequence in B cells produced by the mouse described herein. Such sequencing can be performed using deep sequencing methods such as next-generation sequencing (NGS).

[0118] To produce monoclonal antibodies, antibody-producing cells, such as cells from the spleen and / or lymph nodes, can be isolated from immunized transgenic mice and used for cell fusion with transformed cell lines to produce hybridomas, or antibody-encoding cDNAs can be cloned by standard molecular biology techniques and expressed in transfected cells. Procedures for producing monoclonal antibodies are well established in the art.

[0119] Specifically, this method further comprises the steps of preparing hybridomas and producing and screening antibody-producing cells, particularly those that specifically recognize the target antigen.

[0120] Specifically, provided herein is a method for producing an antibody comprising an antigen-specific VH domain, comprising: a) immunizing a mouse with an antigen, wherein the mouse comprises a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment within an immunoglobulin heavy chain locus, the Cγ gene segment comprising a deletion of a nucleotide sequence encoding at least a portion of a CH1 domain; b) isolating spleen and / or lymph node cells from said transgenic mice; c) generating hybridomas from said spleen and / or lymph node cells; d) selecting antigen-specific hybridomas; e) secreting IgG-type HCAbs containing antigen-specific VHs from the hybridomas; f) isolating the monoclonal HCAb from the culture supernatant; A method is provided which includes:

[0121] In certain embodiments, the method comprises determining the nucleic acid sequence encoding said antigen-specific VH from said hybridoma, and producing a recombinant monoclonal antibody comprising said antigen-specific VH.

[0122] Specifically, the method involves isolating nucleic acid sequences from immunized mice for producing in cell culture specific antibodies or fragments thereof, particularly antigen-binding fragments, such antibodies or antigen-binding fragments being understood herein as hyperimmune antibodies.

[0123] Further provided herein is the use of the transgenic mouse described herein in a method for producing a library of antigen-binding molecules comprising various VH binding sites derived from the B cell repertoire of the mouse described herein or each described herein. Specifically, provided herein is a repertoire of VH domains (e.g., a VH library) or a repertoire (or each library) of molecules comprising VH, particularly a repertoire of HCAbs (or HCAb libraries).

[0124] Provided herein is a method for producing a mouse as described herein, comprising: a) providing mouse embryonic stem cells; b) providing one or more vectors (also referred to as targeting vectors) comprising a nucleic acid sequence comprising a transgenic Cγ gene segment as described herein and, optionally, an inactive Cμ gene segment as described herein in one or more expression cassettes; c) introducing said one or more vectors into a cell; d) selecting transgenic cells, wherein the sequence of b) has been integrated into the cellular genome of said cells by targeted integration at the endogenous immunoglobulin heavy chain locus upstream of the endogenous Cμ gene segment (which may or may not be mutated), or at the Cμ upstream of the endogenous Cδ gene segment if the vector contains an inactive Cμ replacing the endogenous one; e) utilizing the transgenic cells to generate transgenic mice derived from the transgenic cells; Also provided is a method comprising:

[0125] Specifically, the endogenous Cμ gene segment is preferably inactivated by a loss-of-function mutation, a deletion of the Cμ gene segment, or a mutation that introduces a stop codon. Specifically, the mice express HCAbs described herein. Specifically, the mice express only heavy chain antibodies and / or do not express antibody constructs comprising a VL domain.

[0126] Specifically, a marker or markers are used that indicate successful integration of the one or more vectors into the cell genome, specifically, a selectable marker that is capable of expression in the host and allows for easy selection of hosts containing the introduced nucleic acid or vector.

[0127] Examples of selectable markers include, for example, proteins that confer resistance to antibacterial agents (e.g., puromycin, hygromycin, bleomycin, or chloramphenicol) or antiviral agents (e.g., gancyclovir), proteins that confer a metabolic advantage, such as a nutritional advantage, on the host cell, and proteins that confer a functional or phenotypic advantage on the cell (e.g., cell division).

[0128] In certain embodiments, the herpes simplex virus (HSV) thymidine kinase (TK) gene for negative selection with ganciclovir and / or the puromycin resistance gene for selection of cells in which the targeting vector has integrated into their genome are used.

[0129] Specifically, the vector is introduced so that the encoding nucleic acid sequence is inserted into the cell by means that allows the nucleic acid sequence to be integrated into the eukaryotic cell, where the nucleic acid sequence may be transiently present in the cell, but is preferably integrated or stably integrated into the genome (particularly chromosome) of the cell.

[0130] Specifically, the one or more vectors are integrated into the cell genome of the mouse cell at target site by any method of targeted recombination, for example, by homologous recombination or site-specific recombination technique.Specifically, for targeted recombination, CRISPR / Cas9 genome editing system can be used (He et al., Nuc.Acids Res., 44:e85, 2016).

[0131] According to certain embodiments, there is provided a method for generating a transgenic mouse as described herein, comprising: a) providing a mouse cell containing a target site located 5' to an endogenous Cδ gene segment of an endogenous immunoglobulin heavy chain locus; b) providing one or more vectors in one or more expression cassettes, the vectors comprising a nucleic acid sequence comprising a Cγ gene segment comprising a deletion of a nucleotide sequence encoding a CH1 domain and, optionally, a Cμ gene segment comprising at least one inactivating mutation, the nucleic acid sequence being flanked by DNA sequences homologous to the target site, and one or more markers for selecting for targeted homologous recombination of the vector into the cell genome; c) introducing the one or more vectors into the mouse cells; d) integrating the nucleic acid sequence into the genome of the mouse cells and selecting transgenic cells in which the nucleic acid sequence has been integrated into the cellular genome of the cells at the target site; e) using the transgenic cells to generate a transgenic mouse comprising the transgenic cells; A method is provided which includes:

[0132] Specifically, a homology targeting vector or "targeting vector" can be used, which is a vector that contains nucleic acid encoding a targeting sequence, a site-specific recombination site, and optionally a selectable marker gene, that is used to modify endogenous immunoglobulin regions in a host cell using homologous recombination, particularly homologous recombination. In specific examples, the homology targeting vector may further contain a site-specific recombination site and can be used to introduce the site-specific recombination site into a specific region of the host cell genome.

[0133] Specifically, a targeting vector is used that recombines with the host cell genome upon transfection of the host cell, and after productive VDJ rearrangement, the encoded antibody is expressed, inserted into the plasma membrane, and / or secreted by the host cell. Specifically, the vector comprises one or more exogenous or heterologous regulatory elements, such as an enhancer or promoter, operably linked to the antibody coding sequence, which regulatory elements are not naturally associated with said antibody coding sequence.

[0134] According to another specific embodiment, there is provided a method for generating a mouse as described herein, comprising: a) providing a mouse cell that incorporates two recombinase-mediated cassette exchange (RMCE) target sites flanked by recognition sequences for a site-specific recombinase at positions 5' and 3' of an endogenous Cμ gene segment of an endogenous immunoglobulin heavy chain locus; b) providing one or more vectors comprising a nucleic acid sequence comprising a Cγ gene segment comprising a deletion of a nucleotide sequence encoding at least part or all of the CH1 domain and, optionally, a Cμ gene segment comprising at least one inactivating mutation in one or more expression cassettes, the nucleic acid sequence being flanked by additional recognition sites for a site-specific recombinase, and one or more markers for selecting for targeted integration of the vector into the mouse genome, wherein the additional recognition sites are capable of recombining with the RMCE target site; c) introducing into the mouse cells the one or more vectors and a site-specific recombinase that recognizes the RCME target site and an additional recognition site; d) integrating the nucleic acid sequence into the genome of the mouse cells and selecting transgenic cells in which the nucleic acid sequence has been integrated into the cellular genome of the cells at the RMCE target site; e) using the transgenic cells to generate a transgenic mouse comprising the transgenic cells; A method is provided which includes:

[0135] Specifically, all of the recognition sites for a site-specific recombinase are recombinase recognition sites (e.g., Cre / lox, Flp-FRT, etc.) that allow the recombinase to excise the DNA sequence between two of its recognition sites.

[0136] According to a particular aspect, provided herein is a method for producing heavy chain-only antibodies, comprising: a) expressing a recombinant immunoglobulin heavy chain locus in a mouse, said locus comprising: i)V H , D and J H Each of the gene segments comprises one or more of the human V, preferably embedded in mouse regulatory sequences. H , D and J H a variable heavy chain region, including a coding sequence; ii) a constant heavy chain region comprising a transgenic Cγ gene segment upstream of a Cμ gene segment within an immunoglobulin heavy chain locus, wherein the Cγ gene segment comprises a deletion of a nucleotide sequence encoding at least part or all of a CH1 domain, and optionally, wherein the Cμ gene segment is inactive; and iii) a linking region, engineered and positioned to express the HCAbs described herein; Including, causing the mouse to not express endogenous kappa and / or lambda loci; b) producing an antibody that is the HCAb; A method is provided which includes:

[0137] According to certain embodiments, the antibodies described herein are produced in cell culture using a suitable production host cell line. Specifically, bacterial, yeast, plant, insect, or mammalian cell cultures are used for production. Specifically, host cells are used upon introduction of the respective nucleic acid molecules encoding the antibodies described herein. In particular, mammalian host cells such as BHK, CHO, HeLa, HEK293, MDCK, NIH3T3, NS0, PER.C6, SP2 / 0, or VERO cells are all advantageously used.

[0138] According to a particular aspect, the invention provides the use of a mouse as described herein to produce a library of antigen-binding molecules, such as antibodies, comprising at least a VH binding site or respective VH domains, e.g., HCAb antibodies or fragments thereof comprising at least a VH binding site, or a library of nucleic acid sequences encoding or expressing said library.

[0139] The transgenic cells described herein can be used to produce expression libraries for identifying antibodies of interest, for example, by cloning antibody-encoding genes from B cells or by selecting plasma cells with defined specificities in engineered mice that express antibodies on the plasma cell membrane, for example, as described in US20170226162A1. Thus, the present invention also includes antibody libraries produced using cell technologies for the identification of antigen-specific antibodies expressed by plasma cells.

[0140] Certain embodiments include part or all of an immunoglobulin protein transcribed from an immunoglobulin heavy chain gene derived from an engineered portion of a genetically modified mouse described herein, and part or all of an engineered immunoglobulin protein derived from a cell of the genetically modified mouse.

[0141] These and other aspects, objects, and features of the present invention are described in further detail below. [Brief explanation of the drawings]

[0142] [Figure 1] The mouse germline Igh locus (top) [containing the V (IghV), D (IghD), J (IghJ), and C (IghC) gene segments; there are multiple IghC exons to encode different Ig H chain isotypes], the Igκ locus (Igk, middle) [containing the V (IgkV), J (IgkJ), and C (IgkC) gene segments], and the Igλ locus (bottom) [containing the V (IglV), J (IglJ), and C (IglC) gene segments] are shown. Also shown are (1) the PAIR element, a cis-regulatory sequence important for Igh looping to ensure the utilization of distal VH gene segments in VDJ rearrangement; (2) the Adam6a male fertility enabler gene; (3) the intergenic control region 1 (IGCR1), which contains sites regulating the ordered lineage-specific rearrangement of the Igh locus; (4) the intronic enhancers Eμ, iEκ, and Eλ2-4 of the heavy, kappa, and lambda light chains; (5) the 3′ enhancers 3′Eκ, Eλ, and Eλ3-1 of the kappa and lambda light chains; (6) the μ switch region Sμ; and (7) the 3′ regulatory region (3′RR), a cis-acting element that controls isotype switching. [Figure 2] A targeting vector for introducing the mouse Ighg1ΔCH1 gene cassette into the endogenous mouse Igh locus upstream of Ighm by homologous recombination for the production of HCO antibodies. In this figure and Figure 3, the "endogenous mouse Igh locus" is in ES cells containing the partially human IGH locus described by Wabl and Killeen in US20130219535A1. [Figure 3](A) The endogenous mouse Igh locus is shown, targeted with the vector shown in Figure 2 to encode a heavy chain (HCO) antibody (ΔCH1 HCO IgG1). A mouse Cγ1 gene with a deletion of the CH1 exon was inserted into the mouse Igh locus between Eμ and Ighm, and stop codons (TGA, indicated by an asterisk) were inserted into each of the exons encoding the CH1, CH2, and CH3 domains of the μ heavy chain (HC). In wild-type mice, light chain (LC)-dependent μHC is produced after productive VDJH recombination in developing B cells. In the mutated Igh locus shown here, LC-independent ΔCH1γ1 HC is instead produced. Both transmembrane (M) and secreted (S) forms of IgG1 HCO antibodies are encoded by this modified Igh locus. Alternative splicing produces γ1m transcripts, which contain the M1 and M2 exons, and γ1s, which contains the S exon located 3' of the CH3 exon. The CH1 deletion allows the HC to translocate to the plasma membrane (γ1m) or be secreted (γ1s) without the need to associate with a surrogate or conventional (κ or λ) LC. The three stop codons in Cμ prevent LC-dependent μHC expression when the VDJH exon is spliced ​​directly to Cμ rather than Cγ1ΔCH1. (B) is a schematic diagram of a transmembrane IgG1 HCO antibody protein containing variable (VH), CH2, CH3, and M domains but lacking the CH1 domain. The HC exists as a dimer held together by noncovalent interactions and interchain disulfide bonds (indicated by the horizontal line between the VH and CH2 domains) and is inserted into the plasma membrane by its M domain. Secreted IgG1 HCO antibodies (not shown) lack the M domain and instead contain a short secretory (S) domain at the COOH terminus of the protein. This is expressed after B cell activation and differentiation into plasmablasts / plasmocytes. [Figure 4]Figure 3 shows B cell development in the bone marrow of TRN11 / 2 / 5 and TRN34 / 29 / 30 mice. In TRN11 / 2 / 5 mice, the endogenous VH, Vκ, and Vλ loci have been replaced with human VH, Vκ, and Vλ coding sequences flanked by mouse regulatory sequences, respectively. In TRN34 / 29 / 30 mice, the VH, D, and JH gene segments are identical to those in TRN11, but the remainder of the Igh locus has been modified to encode an IgG1 HCO antibody, as shown in Figure 3. The Vκ and Vλ loci are inactivated in the TRN29 and TRN30 alleles, respectively. Bone marrow cells were stained with fluorescently conjugated monoclonal antibodies (mAbs) specific for the indicated CD antigens and analyzed by flow cytometry. Numbers in the flow plots indicate the percentage of cells in the given gates. B cell developmental stages are also indicated. Mature recirculating B cells are B cells that are generated in the bone marrow, complete their maturation in the periphery, for example in the spleen, and recirculate back to the bone marrow (BM) via the bloodstream. [Figure 5] B cell differentiation in the spleens of TRN11 / 2 / 5 and TRN34 / 29 / 30 mice is shown. Spleen cells were stained with fluorescently conjugated mAbs specific for the indicated CD antigens and analyzed by flow cytometry. Numbers in the flow plots indicate the percentage of cells in the given gates. B cell developmental stages are also indicated. Fo.B, follicular B cells; MZ B, marginal zone B cells; T, transitional; and Mat, mature. [Figure 6] Surface IgG1 expression on splenic marginal zone and follicular B cells from TRN11 / 2 / 5 and TRN34 / 29 / 30 mice. Spleen cells gated on B cells from the MZ (top) and Fo. (bottom) were analyzed for cell surface expression of γ1 HC. [Figure 7] B cell differentiation and surface IgG1 expression in lymph nodes from TRN11 / 2 / 5 and TRN34 / 29 / 30 mice. Lymph node cells were stained with fluorescently conjugated mAbs specific for the indicated CD antigens and γ1 HC and analyzed by flow cytometry. Numbers in the flow plots indicate the percentage of cells in a given gate. [Figure 8] B cell differentiation and surface IgG1 expression in the peritoneal cavity of TRN11 / 2 / 5 and TRN34 / 29 / 30 mice were shown. Peritoneal cavity cells were stained with fluorescently conjugated mAbs specific for the indicated CD antigens and γ1 HC and analyzed by flow cytometry. Numbers in the flow plots indicate the percentage of cells in a given gate. [Figure 9] ELISA assays for detecting serum IgG1 and IgM in unimmunized TRN11 / 2 / 5 (open circles) and TRN34 / 29 / 30 HCO (filled circles) mice are shown. Optical density is shown on the Y-axis and serum dilutions on the X-axis. For normalization (open squares), 100 μg / ml monoclonal IgG1 (left) and IgM (right) were also serially diluted. [Figure 10] ELISA assays for detecting serum IgG2b, IgG2c, and IgG3 in unimmunized TRN11 / 2 / 5 (open circles) and TRN34 / 29 / 30 HCO (filled circles) mice are shown. Optical density is shown on the Y-axis and serum dilution is shown on the X-axis. For normalization (open squares), 100 μg / ml of monoclonal IgG2b (left), IgG2c (center), and IgG3 (right) were also serially diluted. [Figure 11-1] Nucleotide sequences referred to herein. [Figure 11-2] Nucleotide sequences referred to herein. [Figure 11-3] Nucleotide sequences referred to herein. [Figure 11-4] Nucleotide sequences referred to herein. [Figure 11-5] Nucleotide sequences referred to herein. [Figure 11-6] Nucleotide sequences referred to herein. [Figure 11-7] Nucleotide sequences referred to herein. [Figure 11-8] Nucleotide sequences referred to herein. [Figure 11-9] Nucleotide sequences referred to herein. DETAILED DESCRIPTION OF THE INVENTION

[0143] Unless otherwise stated or defined, all terms used herein have the usual meaning in the art, which is clear to those skilled in the art.For example, refer to standard textbooks such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd edition), vol. 1-3, Cold Spring Harbor Laboratory Press (1989), Lewin, "Genes IV", Oxford University Press, New York, (1990), and Janeway et al., "Immunobiology" (5th edition, or more recent edition), Garland Science, New York, 2001.

[0144] As used herein, the terms "comprise," "contain," "have," and "include" may be used interchangeably and shall be understood as an open-ended definition that allows for additional members, moieties, or elements. "Consisting of" is considered the closest definition that does not include the additional elements characteristic of the definition of "consisting of." Thus, "comprising" is broader and includes the definition of "consisting of."

[0145] As used herein, the term "about" refers to a value that is the same as the given value or that differs from it by ±5%. The present inventors have overcome the limitations of the prior art and demonstrated that generation of transgenic mice by targeted integration of a transgenic Cγ gene segment 5′ to the endogenous Cμ gene segment in the immunoglobulin heavy chain locus allows efficient production of heavy chain-only antibodies (HCAbs) expressed by B cells and secreted by plasma cells, which can then be used to generate a reliable source of HCAbs or antigen-binding fragments thereof, e.g., using established hybridoma technology.

[0146] The antibody constructs provided herein, referred to herein as HCAbs, and the repertoires and libraries described herein, are artificial constructs. It is well understood that the materials, methods, and uses of the present invention, which specifically refer to, for example, isolated nucleic acid sequences, amino acid sequences, expression constructs, transformed host cells, transgenic animals, and recombinant antibodies, are "artificial" or synthetic and therefore not considered to be "natural products" or the result of the "laws of nature."

[0147] As used herein, the term "antibody" refers to a polypeptide or protein consisting of or comprising antibody domains, understood as constant and / or variable domains of immunoglobulin heavy and / or light chains, in various combinations or configurations. A polypeptide is understood as an antibody domain if it comprises a beta-barrel structure consisting of at least two beta strands of the antibody domain structure connected by a loop sequence. The antibody domain may be in its native structure or may be modified by mutagenesis or derivatization to alter antigen-binding characteristics or any other property, such as stability or functional properties, such as binding to Fc receptors, Fcμ, Fcα / μ, Fcα, Fcε, and / or Fcγ receptors (e.g., murine FcRn, FcγRI, FcγRIIB, FcγRIII, or FcγRIV), or polymeric Ig receptor (pIgR).

[0148] As used herein, the terms "antibody" and "immunoglobulin" are used interchangeably. The term "antibody" as used herein is intended to specifically refer to an antibody construct comprising an antigen-binding site, in particular that of a VH domain. Particular embodiments refer to antibodies comprising or consisting of a VH as a single variable antibody domain, e.g., combined (or fused) with one or more other variable and / or constant antibody domains, and optionally comprising one or more linking sequences or hinge regions, e.g., heavy chain antibodies composed of one or two single chains, each single chain comprising or consisting of a variable heavy chain region (i.e., VH) linked to a constant domain.

[0149] The specific antibodies referred to herein may be full-length antibodies or antigen-binding antibody fragments thereof, or any other antibody constructs comprising or consisting of at least a variable heavy chain (VH) antibody domain, or a single VH domain, such as antibodies comprising or consisting of one or more single domain antibodies, Fab, F(ab'), (Fab)2, scFv, Fd, or Fv. Exemplary antibodies comprise or consist of any of the HCAbs further described herein. The antibodies described herein may be of a certain immunoglobulin type. Particular antibodies comprise antibody constant domains, particularly heavy chain constant domains, of the IgG type (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM type, or their corresponding murine IgG1, IgG2a / c, IgG2b, IgG3, IgA, IgD, IgE, or IgM. Preferably, the antibodies described herein comprise constant antibody domains that are of the IgG type (eg, IgG1, IgG2, IgG3, or IgG4 subtype).

[0150] Accordingly, antibodies are typically understood as proteins (or protein complexes) comprising one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the immunoglobulin variable region genes. Light chains (LC) are classified as either kappa or lambda. Heavy chains (HC) are classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively.

[0151] In a typical IgG antibody structure, the HC or LC each comprises at least two domains connected to each other to form a pair of binding site domains. In certain cases, a heavy chain may incorporate an LC constant domain but still be considered an HC, e.g., lacking a light chain variable domain or region.

[0152] The HC of an antibody may comprise a hinge region that connects one or two antigen-binding arms of the antibody to the Fc portion. The hinge region may be a naturally occurring heavy chain hinge region of an immunoglobulin, such as IgG1 or IgG3, or may be an artificial hinge region that comprises or consists of a number of consecutive amino acids that are approximately the same length as the naturally occurring one (± 20%, or ± 10%). A preferred hinge region comprises one or more, for example, two, three, or four, cysteine ​​residues, which can form disulfide bridges with another hinge region, thereby resulting in a dimeric construct.

[0153] The antibody described herein can comprise one or more antibody domains that are shortened or extended, for example, by using a linking sequence or linker.This linking can be specifically by recombinant fusion or chemical linking.Particular linking can be by linking the C-terminus of one domain to the N-terminus of another domain, for example, one or more amino acid residues in the terminal region can be deleted to shorten the domain size, or extended to increase the flexibility of the domain.

[0154] Specifically, shortened domain sequences include deletions of the C-terminal and / or N-terminal regions, resulting in the deletion of at least 1, 2, 3, 4, or 5, and up to 6, 7, 8, 9, or 10 amino acids.

[0155] The extension of the domain with a linker can be an amino acid sequence derived from the N- or C-terminal region of an immunoglobulin domain, which is naturally located adjacent to the domain, so as to include the natural junction between the domains. Alternatively, the linker can include an amino acid sequence derived from a hinge region. However, the linker can also be an artificial sequence, for example, rich in or consisting of multiple Gly and Ser amino acids.

[0156] The term "antigen-binding molecule" as used herein refers to any protein or protein complex (e.g., consisting of multiple polypeptide chains linked in a complex form) that comprises or consists of an antibody containing at least the antigen-binding site of the antibody. Furthermore, the antigen-binding molecule may further comprise one or more elements fused or conjugated to the antibody portion, thereby resulting in an antibody derivative such as a fusion protein.

[0157] The term "antigen-binding site" or "binding site" refers to the portion of an antibody that is involved in antigen binding. The antigen-binding site of a natural antibody is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and / or light ("L") chains, or their variable domains. Three highly variable sections within the V regions of the heavy chains (and sometimes the light chains) are called "hypervariable regions" and are interposed between more conserved flanking sections known as framework regions. An antigen-binding site provides a surface that is complementary to the three-dimensional surface of the epitope or antigen to which it binds, and the hypervariable regions are called "complementarity-determining regions" or "CDRs." Binding sites incorporated into CDRs are also referred to herein as "CDR-binding sites."

[0158] Specifically, the antigen-binding site of the HCAbs described herein is formed by amino acid residues of the N-terminal variable ("V") region of the heavy ("H") chain. The term "CDR region" or the respective sequence refers to the variable antigen-binding region of a variable antibody domain, such as a VH domain, which contains the variable structure capable of binding interactions with an antigen. An antibody domain containing the CDR region may be used by itself or may be incorporated into a larger proteinaceous construct, thereby forming a specific region of such construct that has a binding function. The variable structure may be derived from a natural repertoire of binding proteins, such as immunoglobulins, and in particular from antibodies or immunoglobulin-like molecules. The variable structure may also be produced by randomization techniques, particularly those described herein. These include mutagenized CDR loop regions of antibody variable domains, particularly immunoglobulin CDR loops.

[0159] Typically, an antibody having an antigen-binding site with a particular CDR structure can specifically bind to a target antigen, i.e., specifically recognize such a target antigen via the CDR loops of a pair of VH domains.

[0160] In HC antibodies, antigen-binding sites are characterized by specific CDR structures consisting only of VH-CDR1, VH-CDR2, and VH-CDR3 loops.When such antigen-binding sites are produced by animals such as transgenic mice described herein, they are understood to be in their natural state or in their natural structure and / or conformation.Antigen-binding sites can be artificially produced, for example, by recombinant techniques to synthesize new structures, but when the genes encoding each antibody are incorporated into transgenic non-human animals, new synthetic antibodies with natural conformation are produced.

[0161] Such native conformations can be further affinity matured by any in vivo or in vitro technique of affinity maturation to produce polyclonal and / or monoclonal antibodies comprising an artificial antigen binding site characterized by the native conformation and further characterized by high affinity for specifically binding to its target antigen.

[0162] The term "antibody" is intended to apply to antibodies of animal origin such as mammals, including humans, mice, rabbits, and rats, or birds such as chickens, and the term specifically includes recombinant antibodies based on animal-derived sequences, e.g., mouse sequences.

[0163] The term "antibody" also applies to fully human antibodies. The term "fully human" when used with respect to immunoglobulins is understood to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. Human antibodies include antibodies isolated from a human immunoglobulin or antibody library, or from animals transgenic for one or more human immunoglobulins.

[0164] The human immunoglobulin is preferably selected or derived from the group consisting of IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 and IgM. The mouse immunoglobulin is preferably selected or derived from the group consisting of IgA, IgD, IgE, IgG1, IgG2A, IgG2B, IgG2C, IgG3 and IgM.

[0165] The term "antibody" also applies to chimeric antibodies, which have mixed sequences from different species, such as sequences from a mouse and a human. Specifically, the term "antibody" applies to antibodies produced by a transgenic non-human animal, such as a mouse, that contain a human antigen-binding region and non-human, such as a mouse, constant region or framework sequences.

[0166] The term "chimeric" when used with respect to immunoglobulins or antibodies refers to a molecule in which one portion of the antibody chain is homologous to a corresponding sequence in immunoglobulins derived from a particular species or belonging to a particular class, while the remaining segment of the chain is homologous to a corresponding sequence in another species or class. Typically, the variable region mimics the variable region of an immunoglobulin derived from one mammalian species, while the constant portion is homologous to an immunoglobulin sequence derived from another. In one example, the variable region can be derived from currently known sources, for example, using readily available B cells from a human host organism in combination with constant regions derived from non-human cell preparations. Specifically, chimeric antibodies or antibody fragments can be produced by transgenic mice, such as those described herein, containing human variable region coding sequences (or respective variable antibody domains) and mouse constant region coding sequences (or respective constant region antibody domains).

[0167] The term "antibody" also applies to monoclonal antibodies, particularly recombinant antibodies, and includes all types of antibodies and antibody structures derived from animals, such as mammals, including humans, and prepared, expressed, created, or isolated by recombinant means, such as antibodies comprising genes or sequences of different origin, e.g., chimeric, humanized, or hybridoma-derived antibodies. Further examples refer to antibodies isolated from host cells transformed to express the antibody, or antibodies isolated from a recombinant combinatorial library of antibodies or antibody domains, or antibodies prepared, expressed, created, or isolated by any other means involving splicing of antibody gene sequences into other DNA sequences.

[0168] The term "antibody" is understood to include functionally active variants of new or existing (herein referred to as "parent") molecules, such as naturally occurring immunoglobulins. Furthermore, the term encompasses antibody variants, as well as derivatives of such molecules. Derivatives are any combination of one or more antibodies and / or fusion proteins, in which any domain of an antibody, e.g., an antibody domain comprising the antigen-binding site of a VH domain, or a VH domain, may be fused at any position to one or more other proteins, such as other antibodies, e.g., binding structures comprising CDR loops, receptor polypeptides, as well as other ligands, enzymes, toxins, etc. The antibodies described herein may be used specifically as isolated polypeptides or as composite molecules with other peptides or polypeptides, e.g., by recombinant, fusion, or conjugation techniques.

[0169] Derivatives of antibodies can also be obtained by association or binding with other substances through various chemical techniques, such as covalent coupling, electrostatic interaction, disulfide bonding, etc. Other substances bound to antibodies can be lipids, carbohydrates, nucleic acids, organic and inorganic molecules, or any combination thereof (e.g., PEG, prodrugs, or drugs). Derivatives can also include antibodies with the same amino acid sequence but made entirely or partially from non-natural or chemically modified amino acids. In certain embodiments, antibodies are derivatives containing an additional tag that enables specific interaction with a biologically acceptable compound. Tags that can be used in the present invention are not particularly limited, as long as they have no adverse effect on the binding of immunoglobulins to targets or are acceptable. Examples of suitable tags include His tags, Myc tags, FLAG tags, Strep tags, calmodulin tags, GST tags, MBP tags, and S tags. In another specific embodiment, the antibody is a derivative containing a label. As used herein, the term "label" refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody to generate a "labeled" antibody. The label may be one which is detectable by itself, eg, a radioisotope label or a fluorescent label, or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound which is detectable.

[0170] Antibody derivatives are those derived from a parent antibody or antibody sequence, such as a parent antigen-binding (e.g., CDR) or framework (FR) sequence, e.g., a mutant or variant obtained by in silico or recombinant engineering, or by chemical derivatization or synthesis.

[0171] The term "variant" is intended to specifically encompass functionally active variants. Functional variants of the antibodies described herein are particularly functional with respect to antigen-binding specificity.

[0172] The term "variant" specifically refers to antibodies, such as mutant antibodies or antibody fragments, obtained by mutagenesis, particularly to introduce deletions, replacements, insertions, or deletions in a particular antibody amino acid sequence or region, or to chemically derivatize the amino acid sequence of a constant domain to engineer, for example, improved antibody stability, increased effector function, or half-life, or to chemically derivatize the amino acid sequence of a variable domain to modulate antigen-binding properties, for example, by affinity maturation techniques available in the art. Any known mutagenesis method may be used, including point mutations at desired positions obtained by randomization techniques or domain deletions used in HCAb engineering. In some cases, positions are randomly selected, for example, by selecting any of the available amino acids or preferred amino acids to randomize the antibody sequence. The term "mutagenesis" refers to any art-recognized technique for modifying the sequence of a polynucleotide or polypeptide. Preferred types of mutagenesis include error-prone PCR mutagenesis, saturation mutagenesis, or other site-directed mutagenesis.

[0173] If the antigen is expressed on the cell surface, intracellularly, or on microbeads such as Luminex, the functional activity of the antibody in terms of antigen binding is typically determined in an ELISA assay, a BIAcore assay, an Octet BLI assay, or a flow cytometry-based assay.

[0174] Functionally active variants can be obtained, for example, by altering the sequence of a parent antibody, e.g., a monoclonal antibody having a particular native structure of an immunoglobulin, such as an IgG1 structure, to obtain a variant with the same specificity in recognizing the target antigen but a structure different from that of the parent, e.g., by modifying one of the antibody domains to introduce specific mutations, or by producing fragments of the parent molecule.

[0175] Particular functionally active variants include one or more functionally active CDR variants or parent antibodies, each of which contains at least one point mutation in the parent CDR sequence and comprises or consists of an amino acid sequence having at least 60% sequence identity with the parent CDR sequence, preferably at least 70%, at least 80%, at least 90% sequence identity.

[0176] Particular variants are functionally active variants of parent antibodies, for example, in which the parent CDR sequences are incorporated into human framework sequences and, optionally, one, two, three, or four amino acid residues of each of the parent CDR sequences are further mutated by introducing point mutations to improve the stability, specificity, and affinity of the parent or humanized antibody.

[0177] Specifically, the antibody can comprise a functionally active CDR variant of any of the CDR sequences of the parent antibody, where the functionally active CDR variant is a) 1, 2, or 3 point mutations in the parent CDR sequences (preferably the number of point mutations in each of the CDR sequences is either 0, 1, 2, or 3), and / or b) one or two point mutations in any of the four C-terminal amino acid positions or the four N-terminal amino acid positions or the four central amino acid positions of the parent CDR sequence, and / or c) at least 60% sequence identity with the parent CDR sequence Preferably, the functionally active variant antibody comprises at least one of the functionally active CDR variants described herein. Specifically, functionally active variant antibodies comprising one or more of the functionally active CDR variants have specificity for binding to the same epitope as the parent antibody.

[0178] According to a particular embodiment, the point mutation is either an amino acid substitution, a deletion and / or an insertion of one or more amino acids. "Percent (%) amino acid sequence identity," with respect to antibody sequences, is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in a particular polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, according to methods known in the art, such as CLUSTALW (Chenna et al., Nucleic Acids Res., 31:3497, 2003), to achieve the maximum percent sequence identity, and not considering conservative substitutions as part of the sequence identity. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0179] Antibody variants are specifically understood to include homologs, analogs, fragments, modified forms, or variants with specific glycosylation patterns, e.g., produced by glycoengineering, that are functional and can result in functional equivalents, e.g., that bind to a specific target and have different functional properties. Antibodies can be glycosylated or non-glycosylated. For example, the recombinant antibodies described herein can be expressed in suitable mammalian cells that allow for specific glycosylation of the molecule as determined by the host cell in which the antibody is expressed, or in prokaryotic cells lacking the glycosylation machinery, resulting in a non-glycosylated protein.

[0180] The "beta sheet" or "beta strand" of an antibody domain, particularly a constant antibody domain, is understood herein as follows: An antibody domain typically consists of at least two beta strands connected at their sides by at least two or three main-chain hydrogen bonds to form an overall twisted, pleated sheet. A beta strand is a continuous stretch of amino acids, typically 3 to 10 amino acids long, that adopts such an extended conformation that it participates in main-chain hydrogen bonds with at least one other strand, thereby forming a beta sheet. In a beta sheet, most beta strands are arranged adjacent to other strands, and together with their neighbors, form an extensive hydrogen-bonding network in which the NH group of the main chain of one strand establishes a hydrogen bond with the C=O group of the main chain of the adjacent strand.

[0181] The structure of antibody constant domains, such as the CL (Cκ, Cλ), CH1, CH2, or CH3 domain, is similar to that of variable domains, consisting of beta strands connected by loops, some of which contain short alpha-helical sections. As seen from the B-factors of X-ray crystallography of various Fc crystal structures, the framework is largely rigid, while the loops are relatively flexible. Antibody constant domains typically have seven beta strands (ABCDEFG) that form a beta sheet, connected by loops, with three loops located at the N-terminal end of the domain (AB, CD, EF) and three more loops located at the N-terminal end of the domain (BC, DE, FG). The "loop region" of a domain refers to the portion of the protein located between the beta strands (e.g., each CH3 domain contains seven beta sheets, A to G, from the N-terminus to the C-terminus).

[0182] In certain embodiments, antibody domains may comprise wild-type amino acid sequences, such as those derived from an animal (including a human), or may be artificial, containing mutations, e.g., at least a portion of one or more beta strands may be replaced with a heterologous sequence, such as to contain a mutation that promotes pairing with another domain, e.g., an interdomain disulfide bridge connecting the beta sheet regions of two antibody domains, a knob and / or hole mutation, or strand exchange.

[0183] Certain domain mutations involve the formation of additional interdomain or interchain disulfide bridges, a) stabilizing the antibody domains by additional intra-domain disulfide bonds, and / or c) stabilizing the two chains of the antibody domain by additional interchain disulfide bridges This may involve the incorporation of new (additional) amino acid residues, for example Cys residues, which may result in the formation of new (additional) amino acid residues.

[0184] Disulfide bonds are typically formed by oxidation of two cysteines or thiol groups of other amino acids to generate thiols, or by oxidation of thiol groups of amino acid analogs that link the S atoms of amino acid side chains to form artificial disulfide bridges. Specifically, cysteines (as additional amino acids or amino acid substitutions) can be inserted between a pair of domains to ensure additional cysteine ​​modifications, thereby creating domain pairs stabilized by disulfide bond formation.

[0185] Antibodies can be produced by first screening antigen-binding sites formed by folding CDR sequences at each binding site of an antibody library and selecting specific binders. As a next step, the selected library members can serve as a source of CDR sequences (or parent CDR sequences that can be further modified to modulate antigen binding and even phenotypic properties) that can be used to engineer any type of antibody construct, such as a full-length immunoglobulin or an antigen-binding fragment thereof.

[0186] A library of antibodies (e.g., one containing a repertoire of specific antibody constructs that recognize the same target antigen, or a naive library of antibodies produced by a particular animal or species, such as a transgenic mouse described herein, that contains a repertoire of antibodies that recognize different target antigens) refers to a set or collection of antibodies (e.g., HCAbs described herein) in which each antibody is appropriately displayed in a selected display system or storage container.

[0187] Certain display systems couple a predetermined protein, such as an antibody, such as the HCAb described herein, with its encoding nucleic acid, for example, its encoding mRNA, cDNA or gene.Therefore, each member of the library comprises the nucleic acid that encodes the antibody that it displays.Display systems include, but are not limited to, cells, viruses such as phage, ribosomes, eukaryotic cells such as yeast, DNA including plasmids, and mRNA.

[0188] Any antibody gene diversity library can be used for such purposes, e.g., containing a large number of individual library members to generate diverse antibody sequences, or using preselected libraries that are enriched or stabilized for functionally active library members. For example, a display system can be enriched for library members that bind to a particular target.

[0189] Libraries can be constructed by well-known techniques, including, for example, chain shuffling methods. In heavy chain shuffling, antibodies are cloned into vectors containing, for example, human VH gene repertoires to generate phage antibody library transformants. Additional methods include site-directed mutagenesis of antibody CDRs, or CDR randomization, in which part or all of the CDRs are randomized using either complete randomization of the targeted residues by applying mutagenic oligonucleotides containing NNK codons, or partial randomization of the targeted residues using parsimonious mutagenesis, in which the oligonucleotides at the positions encoding the targeted amino acid residues contain a mixture biased toward the original nucleotide base. Alternatively, in PCR reactions, the use of dNTP analogs, error-prone polymerases, or Mn 2+ Error-prone PCR with ion addition can be used to construct libraries.

[0190] A variety of techniques are available for producing genes encoding the design of a human antibody library construction. DNA can be produced by a completely synthetic approach, where the sequence is divided into overlapping fragments and then prepared as synthetic oligonucleotides. These oligonucleotides are mixed together and annealed to each other by first heating to approximately 100°C and then slowly cooling to ambient temperature. After this annealing step, the synthetically assembled genes can be cloned directly or amplified by PCR before cloning. This is particularly desirable when large, single-pot human libraries are desired and extensive resources are available for the construction process.

[0191] Certain methods use phage, phagemid, and / or yeast libraries for direct binder selection and internalizing phage antibody selection. Additional methods for site-directed mutagenesis can be used to generate library inserts, such as the Kunkel method (Kunkel, Proc. Natl. Acad. Sci. USA., 82:488, 1985) or the DpnI method (Weiner et al., Gene 151:119, 1994).

[0192] An "antigen-specific library" refers to a library of polynucleotides (or polypeptides encoded by such polynucleotides) that has been screened for the presence of antibodies with specificity for a particular antigen. Such libraries may be restricted to, or otherwise enriched for, antibody sequences with specificity for any given antigen group or particular antigen. An exemplary antigen-specific library is an antibody "optimized library" (e.g., a "maturation library" or "affinity maturation library").

[0193] Certain embodiments described herein are based on mice containing or expressing a preimmune library prior to immunization with an antigen. The preimmune library can be a naive library with sequence diversity similar to that of naturally occurring antibody sequences before such naturally occurring sequences undergo antigen selection. Preimmune libraries can be designed and prepared to reflect or mimic a preimmune repertoire and / or based on rational design informed by collections of V, D, and J genes and other large databases of heavy chain sequences (e.g., publicly known germline sequences). In certain embodiments of the invention, cassettes representing the V, D, and J diversity and junctional diversity (i.e., N1 and N2) likely to be found in human or non-human repertoires are synthesized de novo as single- or double-stranded DNA oligonucleotides.

[0194] An "optimized library" or "maturation library" refers to a library designed to enhance or improve at least one characteristic of antibody sequences identified by scanning a library, such as a naive library or a pre-immune library, for the presence of antibody sequences with specificity for an antigen. Such a maturation library can be generated by incorporating nucleic acid sequences corresponding to one or more CDRs, one or more antigen-binding regions, one or more VH regions, and / or one or more heavy chains obtained or identified from scanning a naive library into a library designed for further mutagenesis in vitro or in vivo to generate a library with introduced diversity relative to the initial (parent) antibody.

[0195] As another example of array technology, B cell cloning can be used to place genes encoding the antibody constructs described herein at manually or computer-addressable locations within an array of B cells. Robotics or manual methods can be used to manipulate the array to repopulate only cells that express a particular type of antibody and / or that specifically recognize a particular target.

[0196] In certain embodiments, B cell cloning from suitably immunized non-human transgenic animals, such as the mice described herein, genetically engineered to produce antibodies, or mammalian cell expression libraries are used, or large populations of stably transformed mammalian cells are generated by standard methods and robotic tools in antibody and protein engineering. Individual clones are kept viable in addressable wells arranged on plates in a suitable incubator and / or under long-term storage conditions, which may include, for example, freezing cell suspensions in liquid nitrogen and storing them at -135°C, or other acceptable conditions that allow for recovery of stored cell lines.

[0197] The term "repertoire," as used herein with respect to antibodies, is intended to refer to a collection of variants, such as variants characterized by diverse target epitope or antigen specificities. Typically, the structure (also called the "scaffold") of the antibody is the same in such a repertoire, but there are a variety of different CDR sequences.

[0198] As is well known in the art, there are a variety of display and selection techniques that can be used to identify and isolate proteins with certain binding properties and affinities, including, for example, cell-based and cell-free methods, particularly display technologies such as mobile display systems. Among cellular systems, phage display, viral display, yeast or other eukaryotic cell display, such as mammalian or insect cell display, can be used. Mobile systems refer to display systems in soluble formats, such as in vitro display systems, including ribosome display, mRNA display, or nucleic acid display.

[0199] Screening of the library for library members displaying antigen-binding structures capable of binding to the target can be carried out by any suitable method. The screening step can involve one or several rounds of selection.

[0200] Any screening method suitable for identifying antibodies capable of binding to a target antigen can be used. In particular, a round of selection may involve incubating the library in the presence of said target to select for antibodies that bind to said antigen or an epitope thereof.

[0201] Once an antibody with the desired structure is identified, such antibody can be produced by methods well known in the art, including, for example, hybridoma technology or recombinant DNA technology.

[0202] In the hybridoma method, a suitable non-human host animal, such as a mouse as described herein, is immunized to activate lymphocytes that produce or are capable of producing antibodies that specifically bind to the antigen used for immunization. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with plasmacytoma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells.

[0203] The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by an in vitro binding assay, such as flow cytometry, immunoprecipitation, or enzyme-linked immunosorbent assay (ELISA).

[0204] In another embodiment, recombinant monoclonal antibodies can be produced by isolating DNA encoding the desired antibody chains and transfecting the coding sequence for expression into a recombinant host cell using well-known recombinant expression vectors, such as plasmids or expression cassettes containing nucleotide sequences encoding the antibody sequences described herein. Recombinant host cells can be prokaryotic or eukaryotic.

[0205] According to certain embodiments, the coding nucleotide sequence can be used for genetic engineering to humanize the antibody or to improve the affinity or other characteristics of the antibody. For example, the constant region can be engineered to resemble a human constant region. It may be desirable to genetically engineer the antibody sequence to obtain higher affinity for the target antigen. It will be clear to those skilled in the art that one or more amino acid changes can be made to an antibody while maintaining its ability to bind to the target (epitope or antigen).

[0206] The production of antibody molecules by various means is generally well understood, and various techniques involved in the production of antibodies are described, for example, in Harlow et al., Antibodies: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2014).

[0207] Monoclonal antibodies can be produced using any method that provides for the production of antibody molecules by continuous cell lines in culture, for example. Examples of suitable methods for preparing monoclonal antibodies include the hybridoma method of Kohler et al. (Nature 256:495, 1975) and the human B-cell hybridoma method (Kozbor, J. Immunol. 133:3001, 1984; and Brodeur et al., 1987, Monoclonal Antibody Production Techniques and Applications, edited by L.B. Schook (Marcel Dekker, Inc., New York), pp. 51-63).

[0208] The term "B cell" refers to a type of lymphocyte that plays a major role in humoral immune responses (as opposed to cell-mediated immune responses, which are driven by T cells). The primary function of B cells is to produce antibodies against antigens; specifically, they express cell surface antibodies (forming the BCR complex) specific to a particular antigen, act as antigen-presenting cells (APCs), and ultimately develop into antibody-secreting plasma cells and memory B cells after activation by antigen interaction. B cells are essential components of the adaptive immune system. Specifically, upon exposure to antigen, B cells become activated and differentiate into immunoglobulin-expressing B cell variants, such as plasma cells, also known as plasma cells, and memory cells.

[0209] The term "repertoire" as used herein with respect to B cells, also referred to herein as "B cell repertoire," is intended to refer to a collection of variants, such as B cell variants, characterized by the expression of immunoglobulins comprising diverse target epitopes or antigen specificities.

[0210] As used herein, the term "target" is intended to refer to an epitope or antigen. The term "antigen" as used herein is intended to specifically include all antigens and target molecules that have been shown to be recognized by the binding site (at least one paratope) of an antibody as a result of exposure of the antigen to an animal's immune system or a library of antibodies. Specifically, preferred antigens targeted by the antibodies described herein are molecules that have already proven or can become immunologically or therapeutically important, particularly those whose clinical efficacy has been tested.

[0211] The term "antigen" is used to describe the entire target molecule, or a fragment of such a molecule, particularly a substructure such as a target polypeptide or carbohydrate structure. Such substructures (often referred to as "epitopes," such as B-cell epitopes, T-cell epitopes, etc.) may be immunologically significant, i.e., recognizable by natural or monoclonal antibodies.

[0212] As used herein, the term "epitope" specifically refers to a molecular structure present at the interface between an antigen and a specific antibody; the surface of the antibody that interacts with the epitope is called the "paratope." Chemically, an epitope can be composed of a carbohydrate sequence or structure, a peptide sequence or set of sequences within a discontinuous epitope, a fatty acid, or an oligonucleotide or polynucleotide. When an antigenic molecule is an organic, biochemical, or inorganic substance, it is called a "hapten." An epitope or hapten can consist of derivatives or any combination of the above substances. When an epitope is a polypeptide, the peptide typically contains at least 3 amino acids, preferably 8-50 amino acids, and more preferably about 10-20 amino acids. Epitopes can be either linear or discontinuous. A linear epitope consists of a single segment of the primary sequence of a polypeptide or carbohydrate chain. Linear epitopes can be continuous or overlapping. Discontinuous epitopes are composed of amino acids or carbohydrates that are grouped together by folding polypeptides to form tertiary structures, and the amino acids are not necessarily adjacent to each other in linear sequence.Specifically, epitopes are at least part of molecules of diagnostic importance, that is, the absence or presence of epitopes in samples correlates qualitatively or quantitatively with disease, or the health status of patients, or the process status in manufacturing, or the status of environment and food.Epitopes can also be at least part of molecules of therapeutic importance, that is, molecules that can be targeted by specific binding domains to change the course of disease.

[0213] As used herein, the term "specificity" or "specific binding" refers to a binding reaction that determines a cognate ligand of interest in a heterogeneous population of molecules. Thus, under certain conditions (e.g., immunoassay conditions), an antibody binds to its specific target and does not bind in significant amounts to other molecules present in the sample. Specific binding means that the binding is selective for the selected target identity, high, medium, or low binding affinity or avidity. Selective binding is usually achieved when the binding constant or binding kinetics differs from that of competing targets in the sample by at least 10-fold; preferably, this difference is at least 100-fold, more preferably at least 1000-fold.

[0214] Specific binding does not exclude certain cross-reactivity with similar antigens or the same antigen (analogs) in different species. For example, the binding entity preferably also cross-reacts with rodent or primate targets similar to the human target, to facilitate preclinical animal testing.

[0215] As used herein, the term "locus" refers to a DNA coding sequence or segment of DNA that encodes an expression product, i.e., a genomic sequence such as a portion of the genome of a host organism or a vector that is integrated at a target site, e.g., a defined restriction site or region of homology.

[0216] The restriction sites can be designed to ensure that the expression cassette is inserted in the proper reading frame. Typically, foreign (also referred to herein as exogenous) DNA is inserted at one or more restriction sites in the vector DNA and then carried by the vector into a host cell along with the transmissible vector DNA.

[0217] Typically, a locus includes at least one gene or one or more gene segments described herein. The term "locus" does not imply that the gene is actively transcribed or intact. Inactivated genes may be included.

[0218] The term "immunoglobulin heavy chain locus," as used herein, refers to a locus encoding a VH domain comprising one or more V gene segments, one or more D gene segments, and one or more J gene segments operably linked to one or more heavy chain constant regions. Preferably, the immunoglobulin heavy chain locus referred to herein is an endogenous mouse immunoglobulin heavy chain locus comprising at least one transgenic nucleic acid sequence, specifically a Cγ gene segment as described herein. The V, D, and J gene segments of the immunoglobulin heavy chain locus comprise VH, D, and JH coding sequences, respectively, and expression control sequences operably linked to control expression of the respective VH, D, and JH coding sequences. According to certain embodiments, the VH, D, and JH coding sequences are human, and the expression control sequences of the VH heavy chain locus are mouse, preferably endogenous, expression control sequences, thus resulting in chimeric V, D, and J gene segments.

[0219] The complexity of the V gene segments can be increased by increasing the number of V gene segments present at a locus or by using different loci, each containing a different V gene segment.

[0220] Preferably, the variable region of the immunoglobulin heavy chain locus comprises 5 to 120 (10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120) or more different V gene segments derived from any vertebrate species or chimera described herein.

[0221] Preferably, the variable region of the immunoglobulin heavy chain locus comprises 2 to 40 (2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, or 40) or more D gene segments. The D gene segments may be derived from any vertebrate species and may be chimeric as described herein.

[0222] Preferably, the variable region of the immunoglobulin heavy chain locus comprises 2 to 20 (2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20) or more J gene segments. The J gene segments may be derived from any vertebrate species and may be chimeric as described herein.

[0223] The V gene segment must be capable of recombining with the D and J gene segments at the DNA level to form a functional VDJ exon. After transcription and RNA splicing, the VDJ exon and heavy chain constant (effector) region (which may comprise several exons) are adjacent in the mRNA transcript and functional for translation, and, according to the present invention, when the nucleic acid is expressed, it produces a heavy chain-only antibody.

[0224] Operationally, the heavy chain constant region is encoded by a naturally occurring or engineered gene segment capable of being expressed together with VDJ exons in B cells. Antibodies can occur in a variety of different forms known as isotypes or classes. Placental mammals have five antibody isotypes known as IgA, IgD, IgE, IgG, and IgM. These are each named using the prefix "Ig" for immunoglobulin (a name sometimes used interchangeably with antibody) and differ in their biological properties, functional location, and ability to address various antigens. The different suffixes of antibody isotypes refer to the different types of heavy chains they contain, with each heavy chain class designated alphabetically as α (alpha), γ (gamma), δ (delta), ε (epsilon), and μ (mu). These are IgA, IgG, IgD, IgE, and IgM, respectively.

[0225] The heavy chain constant regions of the immunoglobulin heavy chain loci described herein contain at least the transgenic Cγ gene segment described herein expressed without a functional CH1 domain, so that IgG-type heavy chain-only antibodies can be generated. Each heavy chain constant region may contain one or more additional heavy chain constant region gene segments selected from the group consisting of Cδ, Cμ, Cε, and Cα gene segments. The heavy chain constant region gene segments are selected depending on the preferred class or desired mixture of antibody classes.

[0226] For example, expression of all or part of a heterologous immunoglobulin heavy chain locus containing a transgenic Cγ gene segment containing a deletion of all or part of the CH1 domain will result in the production of optionally some or all of the IgG isotypes, depending on the IgG1, IgG2, IgG3, and IgG4 isotypes present in the engineered Igh locus.

[0227] Alternatively, a mixture of selected antibodies can be obtained, for example, if the heavy chain constant region comprises Cα and Cμ gene segments, IgA and IgM can also be obtained. Specifically, the Cγ gene segments described herein include deletions of the nucleotide sequence encoding all or part of the CH1 domain, such as deletions of the CH1 exon. Such deletions may be partial deletions of the nucleotide sequence encoding the CH1 domain, as long as they deprive the CH1 domain of its functionality. Preferably, the partial deletions remove or inactivate the chaperone-binding domain, particularly the BiP chaperone-binding domain.

[0228] In some cases, the heterologous heavy chain locus is Cμ-deficient or contains an inactive Cμ gene segment. Specifically, the Cμ gene segment is inactive due to a loss-of-function mutation, such as the introduction of a stop codon, or deletion of at least a portion of the nucleotide sequence encoding the Cμ gene segment. Specifically, the Cμ gene segment is inactive due to deletion of the CH1, CH2, CH3, and / or CH4 domains.

[0229] Specifically, the Cμ gene segment is inactivated by deletion of any one of the entire nucleotide sequence encoding the Cμ gene segment, or at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% of said sequence.

[0230] Specifically, the Cμ gene segment comprises a loss-of-function, deletion, or inactivating mutation, preferably the introduction of a stop codon, in any one or all of its CH1, CH2, CH3, and CH4 domains. Preferably, the Cμ gene segment comprises a stop codon in the CH1, CH2, and CH3 domains.

[0231] In a specific example, for the derivation of HCAbs to be used for therapeutic applications in humans, the VDJ coding sequence present at this locus is derived from optionally modified human germline sequences, and the constant region may be of rodent origin when the host animal is a rodent, e.g., a mouse. Selected heavy chain-only antibodies comprising a soluble human VH binding domain and a rodent constant effector region may then be cloned, and the rodent effector region replaced with an optional human constant effector region, or a soluble VH domain that is used to derive alternative VH fusion proteins, VH domain-antibody complexes, and the like.

[0232] In another embodiment, when a heavy chain-only antibody is intended for veterinary or other use, the V, D, and J coding sequences are preferably derived from the vertebrate or mammal most suitable for the intended purpose. For example, the V and D and J gene segments may be derived from other mammals (e.g., mouse, rat, pig, cow, goat, sheep, camel, horse, etc.) depending on the intended use, such as veterinary, industrial, agricultural, diagnostic, or reagent use.

[0233] "Heavy chain constant region exon" ("CH exon") includes the sequence of a naturally occurring vertebrate, particularly a mammalian, CH exon, which differs in a class-specific manner. For example, IgG and IgA naturally lack a CH4 domain. The term "CH exon" also includes within its scope derivatives, homologs, and fragments thereof, so long as the CH exon, when a component of a heavy chain constant region, is capable of forming a functional HCAb as defined herein.

[0234] In certain embodiments described herein, the endogenous kappa and lambda light chain loci of the transgenic mice are non-functional due to one or more modifications, such as loss-of-function mutations or deletions of the endogenous κ and / or λ light chain loci or portions thereof.

[0235] An exemplary suitable modification is understood as follows: To inactivate the kappa chain locus, the entire 3.2 Mb genomic region between Vκ2-137, the Vκ gene segment furthest from Cκ, and Jκ5, the Jκ segment closest to Cκ, is deleted using the recombinase-mediated cassette exchange (RMCE) strategy. This is achieved by inserting appropriate targeting sequences upstream of Vκ2-137 and downstream of Jκ5, followed by in vitro Cre-mediated deletion of the intervening genomic region. A similar strategy is used to inactivate the lambda chain locus. The entire 194 Kb region containing the mouse lambda V gene segment (IglV) is deleted by RMCE. In this case, appropriate targeting sequences are inserted upstream of IglV2 and downstream of IglV1, followed by in vitro Cre-mediated deletion of the intervening genomic region.

[0236] The locus may be engineered to express or contain exons encoding antibodies, as further described herein. Recombinant loci can be created using a variety of conventional techniques for site-specific editing and / or recombination. Preferably, the modified locus is generated by inserting a piece of DNA containing a gene segment (referred to herein as "donor DNA") into a modified version of a non-human animal immunoglobulin locus, such as a heavy chain locus, of a host organism (referred to herein as "acceptor allele"). The acceptor allele may contain recognition sites for site-specific DNA recombinases such as Cre recombinase (loxP sites and mutated versions of loxP sites). The donor DNA may be flanked by the same Cre recombinase recognition sites (e.g., at both the 5' and 3' ends, such as a loxP site on one side and a mutated version of a loxP site on the other side). Cre recombinase may be used to catalyze the insertion of the donor DNA into the acceptor allele.

[0237] In alternative embodiments, gene segments are introduced into immunoglobulin loci primarily, if not exclusively, by homologous recombination. In such embodiments, targeting sequences or vectors are used that are composed of genomic targeting homology arms (i.e., nucleotide sequences at both the 5' and 3' ends that are homologous to and capable of hybridizing with the target sequence) flanking the nucleic acid sequence comprising the antibody-encoding gene segment. These genomic homology arms facilitate the insertion of DNA, such as DNA encoding an immunoglobulin heavy chain, into the immunoglobulin locus. A targeting sequence refers to a sequence that is homologous to DNA sequences that flank or occur adjacent to the region of the immunoglobulin locus to be modified in the genome of a cell. Flanking or adjacent sequences can be within the locus itself or upstream or downstream of the coding sequence in the genome of the host cell. The targeting sequence is inserted into a recombinant DNA vector used for cell transfection such that the sequence to be inserted into the cell genome, e.g., the sequence of a recombination site, is flanked by the vector's targeting sequences.

[0238] In many cases where homologous recombination is used to achieve genetic changes in genomes, such as insertions or deletions, further modification requires the use of engineered site-specific endonucleases to increase the likelihood of achieving the desired results. Such endonucleases are useful because they can be engineered to be highly specific to unique sequences in the target genome and cause double-stranded DNA breaks at the sites they recognize. The double-stranded breaks promote homologous recombination with a targeting vector that has targeting homology with the DNA in the immediate vicinity of the break. Therefore, the combination of a targeting vector and a site-specific endonuclease that cuts DNA within or near the region targeted by the vector typically results in significantly higher homologous recombination efficiency than the use of a targeting vector alone. Furthermore, the creation of genome deletions can be facilitated by using one or more site-specific endonucleases and a targeting vector composed of two targeting homology arms, one arm targeting one side of the region to be deleted and the other arm targeting the opposite side.

[0239] Site-specific recombination differs from general homologous recombination in that the only site at which recombination occurs is a short, specific DNA sequence required for recombinase recognition. Site-specific recombination requires specialized recombinases to recognize the sites and catalyze recombination at these sites. Several site-specific recombination systems derived from bacteriophage and yeast (each containing a recombinase and a specific cognate target site) have been shown to function in eukaryotic cells for DNA integration and are therefore applicable for use as described herein. These include the bacteriophage P1 Cre / lox, the yeast FLP-FRT system, and the Dre system of the tyrosine family of site-specific recombinases. Such systems and methods of use are well described in the prior art. The recombinase-mediated cassette exchange (RMCE) procedure is facilitated by using a combination of wild-type and mutant loxP (or FRT, etc.) sites in conjunction with appropriate recombinases (e.g., Cre or Flp) and negative and / or positive selection. RMCE occurs when the sites used are identical to each other and / or in the absence of selection, but the process is less efficient due to the preference for excision over insertion reactions, and (when positive selection is not employed) there is no enrichment for properly mutated cells.

[0240] Other tyrosine family systems, such as bacteriophage lambda Int integrase, HK2022 integrase, and additional systems belonging to distinct serine recombinase families, such as bacteriophage phiC31, R4Tp901 integrase, are known to function in mammalian cells using their respective recombination sites and are equally applicable for the uses described herein.

[0241] The method described herein specifically utilizes site-specific recombination sites that utilize the same recombinase but do not promote recombination between sites.For example, a loxP site and a mutant loxP site can be integrated into the genome of a host, but the introduction of Cre into the host does not cause the recombination of the two sites.Rather, a loxP site recombines with another loxP site, and a mutant site recombines only with another similar mutant loxP site.

[0242] To facilitate recombinase-mediated cassette exchange, two classes of variant recombinase sites are available: one harboring a mutation within the 8-bp spacer region of the site, and the other with a mutation in the 13-bp inverted repeat.

[0243] Spacer mutants such as lox511 (Hoess et al., Nucleic Acids Res., 14:2287, 1986), lox5171 and lox2272 (Lee and Saito, Gene, 216:55, 1998), m2, m3, m7, and mil (Langer et al., Nucleic Acids Res., 30:3067, 2002) readily recombine with themselves but exhibit significantly reduced recombination rates with the wild-type site. An example of the use of this type of mutant site for DNA insertion by recombinase-mediated cassette exchange can be found in Baer and Bode, Curr. Opin. Biotechnol., 12:473, 2001.

[0244] Inverted repeat mutants represent a second class of variant recombinase sites. For example, loxP sites can contain modified bases in the left inverted repeat (LE mutant) or the right inverted repeat (RE mutant). The LE mutant lox71 has 5 bases at the 5' end of the left inverted repeat changed from the wild-type sequence to TACCG (Araki, Nucleic Acids Res., 25:868, 1997). Similarly, the RE mutant lox66 has the 5 3'-most bases changed to CGGTA. Inverted repeat mutants can be used to integrate plasmid inserts into chromosomal DNA. For example, an LE mutant can be used as a "target" chromosomal loxP site into which a "donor" RE mutant recombines. After recombination, the donor portion of DNA that contained the RE site is found inserted into the genome flanked by a double mutant site (containing both the LE and RE inverted repeat mutations) on one side and a wild-type site on the other (Lee and Sadowski, Prog. Nucleic Acid Res. Mol. Biol., 80:1, 2005). The double mutant is sufficiently different from the wild-type site so that it is not recognized by Cre recombinase, and the inserted segment therefore cannot be excised by Cre-mediated recombination between the two sites.

[0245] In certain embodiments, the site-specific recombination site may be introduced into an intron or intergenic region rather than into a coding nucleic acid region or regulatory sequence, thereby avoiding the unintentional disruption of regulatory sequences or coding regions necessary for proper gene expression upon insertion of the site-specific recombination site into the genome of an animal cell.

[0246] Introduction of site-specific recombination sites can be achieved by conventional homologous recombination techniques, as described in references such as Sambrook and Russell (2001) Molecular cloning: a laboratory manual, 3rd ed. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press) and Nagy, (2003) Manipulating the mouse embryo: a laboratory manual, 3rd ed. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).

[0247] Specific recombination into genome can be facilitated by using vectors designed for positive or negative selection known in the art.In order to facilitate the identification of cells that have undergone replacement reaction, suitable gene marker systems can be used, for example, by using selective medium to select cells.However, in order to ensure that the genome sequence does not substantially contain exogenous nucleic acid sequences at or near the two end points of replacement interval, it is desirable that the marker system / gene can be removed after selecting cells that contain replaced nucleic acid.

[0248] The recombinase may be provided as a purified protein or may be expressed from a transiently expressed construct in cells to provide recombinase activity. Alternatively, the cells may be used to generate transgenic animals that can be bred with animals expressing the recombinase to produce offspring lacking the marker gene and associated recombination site.

[0249] As used herein, the term "endogenous" with respect to a gene indicates that the gene is native to the cell, i.e., that the gene resides at a particular locus within the genome of an unmodified cell. An endogenous gene may be a wild-type gene (as found in nature) present at that locus in a wild-type cell. An endogenous gene may also be a modified endogenous gene if it resides at the same locus in the genome as the wild-type gene. An example of such a modified endogenous gene is a gene that contains a deletion within its sequence or into which a foreign nucleic acid has been inserted. An endogenous gene may reside in the nuclear genome, mitochondrial genome, etc. Specifically, the immunoglobulin heavy chain locus described herein is an endogenous mouse immunoglobulin locus that contains the modifications described herein.

[0250] The immunoglobulin heavy chain loci described herein comprise a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment. In certain embodiments, the endogenous Cμ gene segment is modified to be inactive, but the Cμ gene segment is present in the same position within the locus as in a wild-type mouse.

[0251] In an alternative embodiment, gene segments are introduced into the immunoglobulin loci by CRISPR / Cas9 technology using a non-homologous end-joining approach (see, e.g., He et al., Nuc. Acids Res., 44:e85, 2016) rather than by homologous sequence-dependent repair, which is typically used in this system.

[0252] In the context of the present invention, the term "heterologous" means that the nucleotide sequence or locus as described herein is not endogenous to the mammal in which it is present, or that the endogenous locus has been modified by substitution or removal of endogenous sequences.

[0253] As used herein, the term "transgenic" with respect to a nucleic acid element, e.g., a nucleic acid construct such as an immunoglobulin gene segment, or a non-coding or coding gene sequence such as a gene, or a locus, indicates that the gene, gene segment, and locus, respectively, are not native to the cell (i.e., do not naturally occur at the same location in the cell's genome in wild-type cells of the same species) or are foreign to the cell to produce a recombinant cell, i.e., the nucleic acid element is present in the genome of a modified (recombinant) cell that is not a wild-type cell. A transgenic gene segment can be a wild-type gene segment that is present at a locus or location different from its respective locus or location in a wild-type cell (and therefore not found at the same locus in nature). A transgenic gene segment can include an endogenous coding sequence or gene (modified or unmodified) if it is present at a different locus in the genome than that found in the wild-type gene or organism. One example of such a transgenic nucleic acid element is a modified endogenous, e.g., Cγ gene segment described herein that contains a deletion or modification in the CH1 domain, which gene segment is integrated upstream of the endogenous Cμ gene segment within the endogenous mouse immunoglobulin heavy chain locus, thus placing the Cγ gene segment downstream of the Cμ gene segment within the immunoglobulin heavy chain locus in a location that is different from its respective location in a wild-type cell.

[0254] The term "transgene" is used herein to describe genetic material that has been artificially inserted into or is being inserted into the genome of a cell, particularly a cell of a host animal. As used herein, the term "transgene" refers to a nucleic acid molecule or element, such as a nucleic acid contained in an expression construct and / or targeting vector, that can be introduced and integrated into the genome of a host, such as, for example, the Cγ gene segment described herein that contains a deletion in the CH1 domain, thereby engineering the host organism (such as a mouse) as a "transgenic" host.

[0255] The term "recombinant" refers to a polynucleotide or polypeptide that does not naturally occur in a host cell. A recombinant or transgenic molecule can contain two or more naturally occurring sequences linked together in a way that does not occur in nature. A recombinant or transgenic cell contains a recombinant polynucleotide or polypeptide. When a cell receives a transgenic or recombinant nucleic acid, the nucleic acid is "exogenous" to the cell.

[0256] The term "recombinant" specifically means "prepared by or the result of genetic engineering." Alternatively, the term "engineered" is used. For example, antibodies or antibody domains can be modified to produce variants by engineering the respective parent sequences to result in engineered antibodies or domains. Recombinant hosts specifically include expression vectors or targeting vectors, or are genetically engineered to contain recombinant nucleic acid sequences, particularly using nucleotide sequences foreign to the host. Recombinant proteins are produced by expressing the respective recombinant nucleic acids in the host. As used herein, the term "recombinant antibody" includes immunoglobulins, particularly antibodies prepared, expressed, produced, or isolated by recombinant means, such as:

[0257] a) antibodies isolated from animals (e.g., non-human animals such as mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom; b) an antibody isolated from a host cell transformed to express the antibody, e.g., from a transfectoma; c) antibodies isolated from a recombinant combinatorial antibody library, and d) Antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences, or the like, into other DNA sequences. Such recombinant antibodies include antibodies engineered to contain rearrangements and mutations that occur, for example, during antibody maturation.

[0258] In certain aspects of the embodiments, the transgenic animals of the invention further comprise human immunoglobulin regions. For example, numerous methods have been developed to replace endogenous mouse immunoglobulin regions with human immunoglobulin sequences to generate partially or fully human antibodies for drug discovery purposes. Examples of such mice include those described in, for example, U.S. Patent Nos. 7,145,056, 7,064,244, 7,041,871, 6,673,986, 6,596,541, 6,570,061, 6,162,963, 6,130,364, 6,091,001, 6,023,010, 5,593,598, 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,661,016, 5,612,205, and 5,591,669.

[0259] As used herein, the term "chimeric immunoglobulin gene segment" refers to a human immunoglobulin gene segment coding sequence, particularly a human IGH (V H , D and J HThe term "chimeric immunoglobulin locus" is used to refer to immunoglobulin gene segments comprising human variable region coding sequences, mouse expression control sequences, and mouse expression regulatory sequences. Mice containing such chimeric gene segments have a genome comprising introduced exogenous immunoglobulin regions that are partially human, the introduced regions comprising human variable region coding sequences and mouse non-coding regulatory sequences that are of mouse origin or endogenous to the mouse's genome and control expression of the human sequences that have been knocked into the mouse genome by the introduction of the chimeric immunoglobulin gene segments or chimeric immunoglobulin loci. In particular, the mouse sequences, and particularly the mouse non-coding regulatory sequences, are based on sequences identical to the corresponding mouse endogenous sequences, i.e., those of the endogenous wild-type immunoglobulin loci.

[0260] The mouse expression control sequences described herein for expressing human immunoglobulin gene sequences are specifically selected from the group consisting of promoters, transcriptional start and stop sequences, enhancer and activator sequences, or ribosome binding sites. Specific examples of such expression control sequences are promoters, 5' untranslated sequences, introns between the coding sequences for leader peptides, recombination signal sequences (RSSs), and sequences flanking the coding sequences, which may include splice sites.

[0261] In particularly preferred embodiments, the transgenic mice described herein comprise chimeric immunoglobulin segments such as those described by Wabl and Killeen in U.S. Publication No. 2013 / 0219535. Such transgenic mice have genomes that include introduced, partially human, immunoglobulin regions, the introduced regions comprising human variable region coding sequences and non-coding variable sequences based on the mouse's endogenous genome. Specifically, the transgenic cells and mice of the invention have genomes in which some or all of the endogenous immunoglobulin regions have been removed.

[0262] In another preferred embodiment, the genomic content of the mice described herein is modified to enable their B cells to express multiple functional VH domains per cell, i.e., the cells produce bispecific antibodies as described in WO2017035252A1.

[0263] As used herein, a "vector" is defined as a DNA sequence required for the transcription of cloned recombinant nucleotide sequences, i.e., recombinant genes, and the translation of their mRNA in a suitable host organism. Vectors include plasmids and viruses, as well as any DNA or RNA molecule, whether autonomously replicating or not, that can be used to transform, transduce, or transfect cells. Vectors can contain autonomously replicating nucleotide sequences and nucleotide sequences that are integrated into a genome. An expression vector may further contain an origin of autonomous replication or a genome integration site in a host cell, one or more selectable markers (e.g., amino acid synthesis genes or genes that confer resistance to antibiotics such as puromycin, Zeocin™, kanamycin, G418, or hygromycin), multiple restriction enzyme cleavage sites, a suitable promoter sequence, and a transcription terminator, these components operably linked together.

[0264] A common type of vector is a "plasmid," which is usually a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and can be easily introduced into a suitable host cell. Plasmids often contain coding DNA and promoter DNA and have one or more restriction sites suitable for inserting foreign DNA. Specifically, the term "plasmid" refers to a vehicle capable of introducing a DNA or RNA sequence (e.g., a foreign gene) into a host cell in order to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.

[0265] As used herein, the term "host cell" refers to the primary subject cell transformed to produce a particular recombinant protein, such as an antibody, described herein, and any progeny thereof. While not all progeny may be exactly identical to the parent cell (due to deliberate or inadvertent mutations or differences in environment), it is understood that such modified progeny are included in these terms so long as the progeny retain the same functionality as the originally transformed cell. The term "host cell line" refers to a cell strain of host cells used to express a recombinant gene to produce a recombinant polypeptide, such as a recombinant antibody. As used herein, the term "cell line" refers to an established clone of a particular cell type that has acquired the ability to grow over long periods of time. Such host cells or host cell lines can be maintained and / or cultivated in cell culture to produce recombinant polypeptides.

[0266] The term "isolated" or "isolated" as used herein with respect to nucleic acids, antibodies, or other compounds is intended to refer to a compound that has been sufficiently separated from the environment with which it would be associated in nature, such that it exists in "substantially pure" form. "Isolated" does not necessarily imply artificial or synthetic mixtures with other compounds or substances, or the exclusion of the presence of impurities that do not interfere with the basic activity and that may be present, for example, due to incomplete purification. In particular, the isolated nucleic acid molecules described herein are also intended to include those that have been chemically synthesized.

[0267] The term "isolated nucleic acid" may be used in connection with nucleic acids described herein. When applied to DNA, this term refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism from which it originates. For example, an "isolated nucleic acid" may include a DNA molecule inserted into a vector, such as a plasmid or viral vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism. When applied to RNA, the term "isolated nucleic acid" primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that is sufficiently separated from other nucleic acids with which it would be associated in the natural state (i.e., in cells or tissues). An "isolated nucleic acid" (whether DNA or RNA) may also refer to a molecule produced directly by biological or synthetic means and separated from other components present during its production.

[0268] With respect to a polypeptide or protein, such as an isolated antibody, the term "isolated" is intended to specifically refer to compounds that are free or substantially free from materials with which they are naturally associated, such as other compounds found with them in their natural environment, or the environment in which they are prepared, e.g., in cell culture if such preparation is by recombinant DNA techniques in vitro or in vivo. Isolated compounds can be formulated with a diluent or adjuvant and still be isolated for practical purposes; for example, a polypeptide or polynucleotide can be mixed with a pharmaceutically acceptable carrier or excipient when used diagnostically or therapeutically.

[0269] The antibodies described herein are provided in an isolated form, substantially free of other antibodies, particularly those directed against different target antigens and / or containing different structural configurations of antibody domains. The isolated antibody may nevertheless be included in a composite preparation, comprising a combination of the isolated antibody with at least one other antibody, such as, for example, a monoclonal antibody or antibody fragment having a different specificity.

[0270] Specifically, the antibodies described herein are provided in substantially pure form. As used herein, the term "substantially pure" or "purified" refers to a preparation that contains at least 50% (w / w), preferably at least 60%, 70%, 80%, 90%, or 95% of a compound, such as a nucleic acid molecule or antibody. Purity is measured by a method appropriate for the compound (e.g., chromatography, polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0271] "Site-specific recombination" refers to the process of recombination between two compatible recombination sites, which involves one of the following three events: a) a deletion of a preselected nucleic acid flanked by recombination sites; b) an inversion of the nucleotide sequence of a preselected nucleic acid flanked by recombination sites; and c) Reciprocal exchange of nucleic acid regions adjacent to recombination sites on different nucleic acid molecules. It will be understood that this reciprocal exchange of nucleic acid segments results in an integration event when one or both of the nucleic acid molecules are circular.

[0272] The foregoing will be more fully understood with reference to the following examples, which, however, are merely representative of methods of practicing one or more embodiments of the invention and should not be construed as limiting the scope of the invention. [Example]

[0273] Example 1 Use of homologous recombination to introduce a mouse Ighg1ΔCH1 gene cassette into the endogenous mouse Igh locus upstream of Ighm for the production of heavy chain-only (HCO) antibodies.

[0274] An exemplary method for introducing the Ighg1ΔCH1 gene cassette for the production of HCO antibodies (or HCAbs) is shown in Figures 2 and 3. The two essential components of the homologous recombination targeting vector (Figure 2) are the short homology arm (SHA) and the long homology arm (LHA), which share sequence identity with homologous DNA segments flanking the region of the endogenous locus to be modified. In this case, the SHA consists of the human JH2-JH6 gene segment flanked by the corresponding mouse Jh noncoding sequence (SEQ ID NO: 2). The LHA consists of the entire Ighm gene, starting with the 5' intron and ending with the 3' intron (SEQ ID NO: 12). Other notable features of the targeting vector, starting from the 5' end, include: 1) Pgk_TK_pA (SEQ ID NO: 1), a herpes simplex virus (HSV) thymidine kinase (TK) gene driven by the phosphoglycerate kinase promoter (Pgk) and containing a polyA tract (pA). This element is used for negative selection with ganciclovir for cells that have integrated the targeting vector but not via homologous recombination. During homologous recombination, nonhomologous DNA 5' to the SHA (i.e., 3' to the LHA) is eliminated (Figure 2). In this example, if the vector integrates by homologous recombination, the HSV-TK gene is deleted. All cells that do not integrate the vector by homologous recombination retain the HSV-TK gene and are killed. 2) T3 promoter for T3 bacteriophage RNA polymerase (SEQ ID NO: 3). This DNA-dependent RNA polymerase is highly specific for the T3 phage promoter. This 99 KD enzyme catalyzes in vitro RNA synthesis, allowing rapid cloning of VDJ rearrangements from small numbers of B cells or hybridomas. 3) CAG_PuroR_pA (SEQ ID NO: 5), a puromycin resistance gene driven by the strong CAG promoter and containing a polyA site. This element is used for positive selection of cells that have integrated the targeting vector based on puromycin resistance. 4) Note that cells that have stably integrated the targeting vector into their genome by homologous recombination are resistant to both ganciclovir and puromycin.5) Note that the CAG_PuroR_pA element is flanked by FRT sites (SEQ ID NO: 4 and SEQ ID NO: 6), which can be used to remove this element in vitro or in vivo by supplying Flp recombinase after identification of a properly targeted ES cell clone. An Eμ enhancer (SEQ ID NO: 7) is included upstream of the Ighg1ΔCH1 gene cassette to promote transcription of the locus. In this targeting vector, this is followed by the sequence of a portion of the Ighg1 5' intron (SEQ ID NO: 8), the sequence of a portion of the Ighg1 hinge 5' intron (SEQ ID NO: 9), the Ighg1ΔCH1 gene (SEQ ID NO: 10), and the human growth hormone 1 polyadenylation signal sequence (hGH1 pA, SEQ ID NO: 11). Immediately downstream is LHA (SEQ ID NO: 12). It consists of the sequence of part of the Ighm 5' intron (SEQ ID NO: 13) and the Ighm gene including the 3' UTR (SEQ ID NO: 14), followed by the sequence of part of the Ighm 3' intron (SEQ ID NO: 15). VDJ. HTo prevent LC-dependent μHC expression when the exon is directly spliced ​​to the Cμ gene rather than the Ighg1ΔCH1 gene, a stop codon (TGA) was introduced into the CH1, CH2, and CH3 Ighm exons. Because this targeting vector lacks the μ switch (S) region present in the endogenous Igh locus, the targeted locus also lacks the S region and is therefore unable to undergo isotype switching. The targeting vector is introduced into ES cells by electroporation. The cells are grown in medium supplemented with ganciclovir and puromycin. Subsequently, surviving isolated ES cell clones are monitored for successful gene targeting by genomic PCR using primers located internally at the 5' and 3' ends of the newly introduced Ighg1ΔCH1 gene cassette, a widely used gene targeting strategy. Proper integration of the targeting cassette is further verified by genomic Southern blot using a probe mapping to the DNA sequence flanking the 5' side of SHA, a second probe mapping to the DNA sequence flanking the 3' side of LHA, and a third probe mapping within the novel DNA between the two arms of genomic identity within the vector (the structure of the correctly targeted locus is shown in Figure 3 and is hereafter referred to as the HCO locus).

[0275] The karyotypes of PCR- and Southern blot-verified ES cell clones are analyzed using an in situ fluorescent hybridization procedure designed to distinguish the most commonly occurring chromosomal abnormalities that occur in mouse ES cells. Clones with such abnormalities are excluded from further use. ES cell clones that are determined to have the correct predicted genomic structure based on the PCR and Southern blot data and that have no detectable chromosomal abnormalities based on karyotype analysis are selected for further use.

[0276] Using standard procedures, ES cell clones containing a properly targeted Ighg1ΔCH1 gene cassette within the mouse heavy chain locus are microinjected into mouse blastocysts from the DBA / 2 strain to generate chimeric mice derived in part from ES cells. Male chimeric mice with the highest levels of ES cell-derived contributions to the coat are selected for mating with female mice. The selected female mice are of the C57B1 / 6NTac strain, which carries a transgene encoding Flp recombinase in their germline. Offspring from these matings are analyzed for the presence of the Ighg1ΔCH1 gene cassette and loss of the FRT-flanked puromycin resistance gene (Figure 3). Correctly targeted mice, designated TRN0034 or TRN34, are used to establish a mouse colony.

[0277] Example 2 B cell development in the bone marrow (BM) of HCO mice. In the prior art (see, for example, WO2011 / 072204A1), IgG-type HCAbs are produced in mice lacking a functional light chain and having a functional Cμ gene segment upstream of a recombinant Cγ gene segment lacking the CH1 domain. The production of HCAbs in these mice has a major disadvantage. With a functional Cμ gene segment, mature B cells with canonical IgM as the antigen BCR develop normally. However, during the immune response, the antibody class switches to the (desired) γ chain, which cannot pair with it due to the lack of an L chain. This has two effects: (i) Cells bearing antibodies whose specificity is defined by the combination of heavy and light chains are no longer stimulated and die; (ii) Unpaired VH is structurally non-viable, which can lead to the death of cells whose specificity is primarily defined by their H chains.

[0278] Thus, in such mice of the prior art, VH selection is "deferred", ie occurs during the immune response. By incorporating a transgenic Cγ1 gene segment into the endogenous immunoglobulin locus upstream of a Cμ gene segment, VH selection is "front-loaded," ie, occurs during ontogeny.

[0279] To demonstrate normal B cell development in these mice, bone marrow cells were surface stained with mAbs specific for the CD antigens shown in Figure 4 and analyzed by flow cytometry. In control TRN11 / 2 / 5 mice, endogenous V cells were expressed as described in co-pending application U.S. Publication No. 20130219535 A1 by Wabl and Killeen. H , V κ and V λ The human V locus is flanked by mouse regulatory sequences H , V κ and V λ In TRN34 / 29 / 30 mice, V H , D and J H The gene segment is identical to TRN11, but the remainder of the Igh locus has been modified to encode an IgG1 HCO antibody, as shown in Figure 3. κ and V λ The loci are inactivated with the TRN29 and TRN30 alleles, respectively. Numbers in the flow plots indicate the percentage of cells in a given gate.

[0280] Genetic structure of TRN11 / 2 / 5 mice: - Human V flanked by mouse regulatory sequences H , V κ and V λ Endogenous V, each replaced with a locus coding sequence H , V κ and V λ locus Genetic structure of TRN34 / 29 / 30 mice: - HCO locus: Ighg1ΔCH1 and a stop codon (TGA) were introduced into the CH1, CH2 and CH3 Ighm exons; - Chimera VH Locus: endogenous V H Human V flanked by mouse regulatory sequences, replacing the locus H , V κ and V λ locus coding sequence, and - Inactivated V κ and V λ Locus.

[0281] Stages of B cell development are also shown: Mature recirculating Bs refer to B cells that are generated in the bone marrow, complete their maturation in the periphery, e.g., the spleen, and recirculate back to the bone marrow (BM) via the bloodstream. While the frequency of early B-lineage cells (B220+CD23-) is identical in both mouse strains, the frequency of pro-B cells is increased and the frequency of their progeny, pre-B cells, is decreased in HCO mice compared to controls (lower panel). This decrease leads to a corresponding decrease in transitional and mature B cells in the periphery (Figures 5, 7, and 8) and mature recirculating B cells in the BM (upper panel). There are several possible explanations for the altered frequencies of pro-B and pre-B cells. Normally, pre-B cells synthesize μHCs, which associate with surrogate light chains and CD79A / B and are expressed at low levels on the cell surface. This signals the cells to proliferate before V→J rearrangement and light chain gene expression, which indicate the immature B-cell stage. The γ1 HC in HCO mice does not associate with SLCs and is expressed on the cell surface together with CD79A / B. These pre-B cells may not proliferate to the same extent as wild-type (WT) cells and, because they lack the need for LC gene rearrangement, may differentiate more rapidly into immature B cells and leave the BM. The increase in pro-B cells suggests a possible defect in the pro-B to pre-B developmental step.

[0282] In any case, B lineage cells at all expected developmental stages are present in HCO mice. Example 3 B cell differentiation and cell surface IgG1 expression in the periphery of HCO mice.

[0283] Spleen cells from the same mice as in Example 3 were stained with fluorescently conjugated mAbs specific for the indicated CD antigens and analyzed by flow cytometry (Figure 5). Numbers in the flow plots indicate the percentage of cells in the given gates. As expected based on the reduced frequencies of pre-B cells and mature recirculating B cells in the BM (Figure 4), there was an overall reduction in all B cell subsets in the spleens of HCO mice. The analyzed subsets included total B, B1, B2, transitional stages 1 and 2 (T1 and T2), follicular (Fo.) B, and marginal zone (MZ) B. Cells at all expected stages of differentiation were present in HCO mice, albeit in reduced numbers.

[0284] A large number of splenic MZ (80%) and Fo.B (91%) cells expressed γ1 HC on the cell surface (Fig. 6 ), indicating that the HCO locus is functional in vivo. In lymph nodes (LN, Figure 7), total B cells (upper panel) and mature Fo. B cells (middle panel) were reduced. Similar to splenic B cells, 89% of LN B cells from HCO mice expressed γ1 HC on their cell surface (lower panel).

[0285] In the peritoneal cavity (Fig. 8), total B cells (upper panel), Fo. B cells, and B1 B cells (middle panel) were reduced. Similar to splenic and LN B cells, most peritoneal B cells from HCO mice expressed γ1 HC on their cell surface (lower panel).

[0286] In summary, although the frequencies of all B cell developmental stages and subsets were reduced in the BM and periphery of HCO mice (except for pro-B cells in the BM), all B cell developmental stages and subsets were present and the γ1 HCO locus functioned normally in vivo.

[0287] Example 4 Serum immunoglobulin (Ig) levels in HCO mice Figure 9 shows the results of ELISA assays to detect serum IgG1 and IgM in unimmunized TRN11 / 2 / 5 (open circles) and TRN34 / 29 / 30 HCO (filled circles) mice. Optical density is shown on the Y-axis, and serum dilutions are shown on the X-axis. For normalization (open squares), 100 μg / ml monoclonal IgG1 (left) and IgM (right) were also serially diluted. Significant levels of heavy chain-only IgG1 were detected in the serum of TRN34 / 29 / 30 HCO mice, slightly below the IgG1 (including heavy and light chains) in control TRN11 / 2 / 5 serum. In contrast, IgM in HCO mice was at background levels. A similar ELISA assay was used to detect serum IgG2b, IgG2c, and IgG3 (Figure 10). All three Ig isotypes were undetectable in TRN34 / 29 / 30 HCO mice.

[0288] These results indicate that B cells in TRN34 / 29 / 30 HCO mice cannot differentiate into IgM-secreting plasma cells because three stop codons are present in the μHC open reading frame. These results also indicate that isotype switching to IgG2b, IgG2c, or IgG3 is absent in HCO mice because the μ switch region is deleted from the Igh locus. In one aspect, the present invention may be as follows. [Embodiment 1] A method for producing a mouse whose B cells express a diverse repertoire of heavy chain-only antibodies (HCAbs) by incorporating a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment within an endogenous immunoglobulin heavy chain constant region locus, wherein the Cγ gene segment contains a deletion of a nucleotide sequence encoding at least a portion of the CH1 domain. [Embodiment 2] The method of embodiment 1, wherein when a mouse is immunized with an antigen, antigen-specific B cells are activated and their differentiation into plasma cells that secrete a variety of antigen-specific HCAbs is induced. [Embodiment 3] The method of embodiment 1 or 2, wherein the Cγ gene segment is positioned downstream of an Eμ major intronic enhancer, preferably downstream of an endogenous Eμ major intronic enhancer comprising SEQ ID NO:7. [Embodiment 4] The method according to any one of embodiments 1 to 3, wherein the Cγ gene segment comprises a Cγ1 gene segment. [Embodiment 5] The method of any one of embodiments 1 to 4, wherein at least a portion of the CH1 domain comprises a BiP chaperone binding domain. [Aspect 6] The method of any one of Aspects 1 to 5, wherein the mouse comprises an inactivated or deleted endogenous immunoglobulin light chain locus, preferably wherein the mouse comprises a loss-of-function mutation in either or both of the endogenous kappa or lambda light chain loci, or a deletion of either or both of the endogenous kappa or lambda light chain loci. [Embodiment 7] The method according to any one of embodiments 1 to 6, wherein the endogenous Cμ gene segment is inactive. [Embodiment 8] The method of any one of embodiments 1 to 7, wherein the endogenous Cμ gene segment is inactive due to a loss-of-function mutation, a deletion of a portion of the Cμ gene segment, or one or more mutations that introduce one or more stop codons. [Aspect 9] The immunoglobulin heavy chain locus is a human V H , D and J H coding sequence, and H , D and J H 9. The method according to any one of aspects 1 to 8, comprising an expression control sequence operably linked to the coding sequence, preferably wherein the expression control sequence is murine. [Aspect 10] V H , D and J H The coding sequences are recombined to form a VDJ coding sequence that expresses a VH binding site that specifically recognizes an antigen, thereby generating the recombined VDJ coding sequence in a given B cell. H The coding sequences are obtained, and the B cells, upon differentiation into plasma cells, express antigen-specific VH encoded by the recombined VH coding sequences. H10. The method of embodiment 9, wherein the HCAb of IgG type comprising the binding domain is secreted. [Aspect 11] a) providing mouse embryonic stem cells; b) providing one or more vectors comprising nucleic acid sequences comprising Cγ gene segments in one or more expression cassettes; c) introducing the one or more vectors into the cell; d) selecting transgenic cells in which the sequence of b) has been integrated into the cellular genome of the cells by targeted integration at the endogenous immunoglobulin heavy chain locus upstream of the endogenous Cμ gene segment, which Cμ gene segment is optionally inactive; e) utilizing the transgenic cells to generate transgenic mice derived from the transgenic cells; 11. The method according to any one of aspects 1 to 10, comprising: [Aspect 12] A mouse obtainable by the method of any one of Aspects 1 to 11, comprising, within its endogenous immunoglobulin heavy chain constant region locus, a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment, wherein the Cγ gene segment comprises a deletion of a nucleotide sequence encoding at least a portion of a CH1 domain. [Aspect 13] At least two V H , two Ds, and two Js H Human V H , D and J H 13. The mouse of embodiment 12, comprising a VH heavy chain locus comprising a repertoire of coding sequences. [Aspect 14] A mouse according to aspect 12 or 13, comprising a loss-of-function mutation in either or both of the endogenous kappa or lambda light chain loci, or a deletion of either or both of the endogenous kappa or lambda light chain loci. [Aspect 15] A B cell repertoire derived from a mouse and expressing various IgG-type HCAbs, obtainable by the method described in any one of Aspects 1 to 11. [Aspect 16] A B cell repertoire according to aspect 15, which expresses various antigen-specific HCAbs with different VH domains. [Aspect 17] A method for producing an antibody comprising an antigen-specific VH domain, comprising: a) immunizing a mouse with an antigen, wherein the mouse comprises a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment within an immunoglobulin heavy chain locus, the Cγ gene segment comprising a deletion of a nucleotide sequence encoding at least a portion of a CH1 domain, thereby resulting in a repertoire of cells expressing antigen-specific HCAbs; b) selecting cells expressing IgG-type HCAbs containing antigen-specific VHs from the repertoire; c) determining a nucleic acid sequence encoding an antigen-specific VH from the HCAb; d) producing a monoclonal antibody comprising the antigen-specific VH; A method comprising: [Aspect 18] Use of a mouse described in any one of aspects 12 to 14 in a method for producing a B cell repertoire or a repertoire of molecules comprising VH.

Claims

1. 1. A method for producing a mouse whose B cells express a diverse repertoire of heavy chain-only antibodies (HCAbs) by incorporating a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment within an endogenous immunoglobulin heavy chain constant region locus, wherein the Cγ gene segment comprises a deletion of a nucleotide sequence encoding at least a portion of a CH1 domain, and the endogenous Cμ gene segment is inactive due to a loss-of-function mutation, a deletion of a portion of the Cμ gene segment, or one or more mutations introducing one or more stop codons.

2. 2. The method of claim 1, wherein, when a mouse is immunized with an antigen, antigen-specific B cells are activated and their differentiation is induced into plasma cells that secrete a variety of antigen-specific HCAbs.

3. 3. The method of claim 1 or 2, wherein the Cγ gene segment is positioned downstream of an endogenous Eμ major intronic enhancer comprising SEQ ID NO:

7.

4. The method of any one of claims 1 to 3, wherein the Cγ gene segment comprises a Cγ1 gene segment.

5. The method of any one of claims 1 to 4, wherein at least a portion of the CH1 domain comprises a BiP chaperone binding domain.

6. 6. The method of any one of claims 1 to 5, wherein the mouse comprises a loss-of-function mutation in either or both of the endogenous kappa or lambda light chain loci, or a deletion of either or both of the endogenous kappa or lambda light chain loci.

7. The immunoglobulin heavy chain locus is human V Ｈ , D and J Ｈ coding sequence, and the V Ｈ , D and J Ｈ The method of any one of claims 1 to 6, comprising an expression control sequence operably linked to the coding sequence, wherein the expression control sequence is murine.

8. V Ｈ , D and J Ｈ The coding sequences are recombined to form a VDJ coding sequence that expresses a VH binding site that specifically recognizes an antigen, thereby generating the recombined VDJ coding sequence in a given B cell. Ｈ The coding sequence is obtained, and the B cells, upon differentiation into plasma cells, produce antigen-specific VH coding sequences encoded by the recombined VH coding sequences. Ｈ 8. The method of claim 7, wherein the method is capable of secreting an HCAb of the IgG type comprising the binding domain.

9. a) providing mouse embryonic stem cells; b) providing one or more vectors comprising nucleic acid sequences comprising Cγ gene segments in one or more expression cassettes; c) introducing the one or more vectors into the cell; d) selecting transgenic cells in which the sequence of b) has been integrated into the cellular genome of the cells by targeted integration at the endogenous immunoglobulin heavy chain locus upstream of the endogenous Cμ gene segment, which Cμ gene segment is optionally inactive; e) utilizing the transgenic cells to generate transgenic mice derived from the transgenic cells; The method according to any one of claims 1 to 8, comprising:

10. The method of any one of claims 1 to 9, wherein the transgenic mouse comprises, within its endogenous immunoglobulin heavy chain constant region locus, a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment, and the Cγ gene segment comprises a deletion of a nucleotide sequence encoding at least a portion of the CH1 domain.

11. A transgenic mouse having at least two V Ｈ , two Ds, and two Js Ｈ Human V Ｈ , D and J Ｈ 11. The method of claim 10, comprising a VH heavy chain locus comprising a repertoire of coding sequences.

12. The method of claim 10 or 11, wherein the transgenic mouse comprises a loss-of-function mutation in either or both of the endogenous kappa or lambda light chain loci, or a deletion of either or both of the endogenous kappa or lambda light chain loci.

13. A method for producing a B cell repertoire expressing various HCAbs of the IgG type, comprising the step of isolating B cells from a mouse obtainable by the method according to any one of claims 1 to 12.

14. The method described in claim 13, wherein the B cell repertoire expresses various antigen-specific HCAbs with different VH domains.

15. 1. A method for producing an antibody comprising an antigen-specific VH domain, comprising: a) immunizing a mouse with an antigen, wherein the mouse comprises a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment within an immunoglobulin heavy chain locus, the Cγ gene segment comprising a deletion of a nucleotide sequence encoding at least a portion of a CH1 domain, thereby resulting in a repertoire of cells expressing an antigen-specific HCAb; b) selecting from the repertoire cells expressing IgG-type HCAbs containing antigen-specific VHs; c) determining the nucleic acid sequence encoding the antigen-specific VH from the HCAb; d) producing a monoclonal antibody comprising the antigen-specific VH; A method comprising:

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