Pure heavy chain antibodies

Abstract

A method for generating mice in which B cells express a library of multiple pure heavy chain antibodies (HCAbs), the method comprising incorporating a transgenic Cγ gene fragment upstream of an endogenous Cμ gene fragment in an endogenous immunoglobulin heavy chain constant region locus, wherein the Cγ gene fragment contains a deletion of at least a portion of a nucleotide sequence encoding a CH1 domain.

Description

TECHNICAL FIELD

[0001] The present invention relates to pure heavy chain antibody (HCAb) constructs, transgenic mice expressing HCAbs, and methods of generating them in vitro and in vivo. BACKGROUND

[0002] Antibodies have become important biopharmaceuticals due to (i) their ability to target different molecular forms of antigens with exquisite binding properties, (ii) their physiological nature as molecules with desirable pharmacokinetics, making them well tolerated in humans and animals receiving treatment, and (iii) their association with strong immunological properties of natural defenses against infectious agents. Furthermore, established techniques exist for rapidly isolating antibodies from experimental animals that can readily generate specific antibody responses to virtually any non-naturally occurring foreign substance in vivo.

[0003] In their most basic form, antibodies are composed of two identical heavy chains (H) each paired with an identical light chain (L). The N-terminus of both H and L chains contain a variable domain (VH and VL, respectively) that together provide the paired H-L chain with a unique antigen-binding specificity. Exons encoding antibody VH and VL domains do not exist in germline DNA. Rather, each VH exon is generated by recombination of randomly selected V, D, and J gene segments found in the H chain locus (Igh); likewise, a single VL exon is generated by chromosomal rearrangement of randomly selected V and J gene segments in the light chain locus (Igl) (see mouse Igh locus, Igkappa locus (Igk or IgK), and Ig lambda locus (Igl or IgL, respectively, in Figure 1). Figure 1 (Tonegawa, Nature, 302:575, 1983; Bassing, et al., Cell, 109 Suppl: S45, 2002). The mouse genome contains two alleles that can express H chains (one allele from each parent), two alleles that can express kappa (K) L chains, and two alleles that can express lambda (L) L chains. There are multiple V, D, and J gene segments in the H chain locus, and multiple V and J genes in both L chain loci. One or more exons encoding antibody constant regions (C) are found downstream of the J genes in each immunoglobulin (Ig) locus. In the heavy chain locus, there are also exons that express different antibody classes (isotypes). In mice, the isotypes encoded are IgM, IgD, IgGl, IgG2a / c, IgG2b, IgG3, IgE, and IgA; in humans, IgM, IgD, IgGl, IgG2, IgG3, IgG4, IgE, IgAl, and IgA2.

[0004] During B cell development in the fetal liver and adult bone marrow, gene rearrangement first occurs on one of the two homologous chromosomes containing H chain V, D, and J gene segments. In pre-B cells, the resulting VH exon is spliced at the RNA level to an exon encoding the mu H chain constant region. The mu H chain synthesized by pre-B cells is largely retained in the endoplasmic reticulum (ER) and is ultimately degraded due to non-covalent interactions between the partially unfolded CH1 domain of the mu H 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 fraction of mu chains bind to an alternative light chain complex composed of invariant lambda 5 and VpreB proteins, displacing BiP and allowing the mu H chain / lambda 5 / VpreB complex to exit the ER with the Ig alpha / beta signaling molecules as the pre-B cell receptor (preBCR) and traffic through the secretory pathway to the plasma membrane (Ubelhart, et al., Curr. Top. Microbiol. Immunol. 393:3, 2016).

[0005] Subsequently, a VJ rearrangement occurs on one L chain allele until a functional L chain is produced, after which the L chain polypeptide can fully displace BiP and bind to the mu 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 that prevents cell surface expression or secretion of incompletely assembled Ig molecules is very stringent, so molecules such as HL, HHL or HH are usually retained in the ER, to be degraded if not rescued by assembly into intact H2L2 structures. (This system focuses primarily on retention of Ig H chains; thus, free L chains can often be secreted.) However, for decades it has been known that free monoclonal H chains can be secreted in a rare B cell proliferative disease 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 showed that the CH1 domain is almost always missing (Corcos, et al., Blood, 117:6991, 2011). Mechanistically, the CH1 deletion releases the H chain from inhibitory interactions with BiP, allowing its secretion, while also preventing disulfide-mediated covalent association with L chains, so HCD proteins are HH dimers. Pure heavy chain Abs (HCAbs) can also be found in non-disease settings. i) About 75% of serum IgG in normal camels consists of HCAbs, which lack CH1 domains and also have structurally altered VH domains that prevent efficient association with VL domains (de los Rios, et al., Cur. Opin. Struct. Biol., 33:27, 2015). ii) Mice in which both kappa and lambda L chain loci are inactivated still produce serum IgG, but this requires an error in class switch recombination (CSR) that results in deletion of the CH1 domain-encoding exon in B cell DNA (Zou, et al., J. Exp. Med., 204:3271, 2007).

[0007] HCAbs are attractive as therapeutic agents because they are highly stable and smaller than conventional immunoglobulins. The VH antigen-binding portion of the molecule is unobstructed by a VL antigen-binding portion and can recognize epitopes within protein structure pockets, including enzyme active sites and epitopes on viruses and G-coupled protein receptors that are inaccessible to conventional Abs. Camel-based HCAbs derived from, for example, mice in which the endogenous VH genes have been replaced by camel VH genes and the exon encoding CH1 has been deleted are a potential source of such Abs. However, their disadvantage is that the camel VH domains are immunogenic in humans and other animals in which they can be used as therapeutic agents. Mice exist in which the endogenous VH genes have been replaced by their human counterparts, combined with inactivation of the kappa and lambda L chain loci, which can be a source of HcAbs. However, production of such Abs is dependent on a relatively rare error in CSR during an immune response, so the efficiency is low.

[0008] The size of the binding site of a conventional antibody, which has a pair of heavy (H) and light (L) chains, can be too large to fit into the groove or cleft of a target epitope. Reduction in the width of the HCAb binding site can facilitate access to "difficult" epitopes, such as the active site of an enzyme. In another use, HCAb facilitates the production of bispecific antibodies. The absence of L chains prevents "L-chain shuffling" that leads to loss of specificity. Transgenic mice that produce (human) immunoglobulins consisting of H chains without L chains can be used to find antibodies with desired antigen specificity.

[0009] However, as noted above, in the absence of light chains, native heavy chains are not secreted by plasma cells. This is because the CH1 domain of the H chain is bound by the chaperone protein BiP, and thus remains in the lumen of the endoplasmic reticulum. Deletion of CH1, for example in heavy chain disease, or by engineering, allows the H chain to be secreted as a disulfide-linked dimer.

[0010] Immunoglobulin class switching, also known as isotype switching, isotypic commutation, or class switch recombination (CSR), is a biological mechanism that changes the isotype of an immunoglobulin produced by a B cell, for example from isotype IgM to isotype IgG. During this process, the constant region part of the antibody heavy chain changes, but the variable region of the heavy chain remains the same. Since the variable region is unchanged, class switching does not affect antigen specificity. Instead, the antibody retains affinity for the same antigen, but can interact with different effector molecules through its Fc region.

[0011] Class switching occurs after a mature B cell is activated by its membrane-bound antibody molecule (B cell receptor, BCR), producing antibodies of different classes as a result of the V(D)J recombination process, all with the same variable domain as the original antibody expressed by the immature B cell, but with different constant domains in their heavy chains.

[0012] Initial wild-type mature B cells produce IgM (Cμ) and IgD (Cδ), which are the first two heavy chain segments in the wild-type immunoglobulin locus. Upon antigen activation, these B cells proliferate. If these activated B cells encounter specific signaling molecules through their CD40 and cytokine receptors, both of which are regulated by helper T cells, they undergo antibody class switching, producing IgG, IgA, or IgE antibodies. During class switching, the constant region of the immunoglobulin heavy chain changes, but the variable region remains the same, so the antigen specificity remains the same. This allows different sub-cells from the same activated B cell to produce antibodies of different isotypes or subtypes (e.g., IgG1, IgG2, etc.).

[0013] While one can envision and design various immunoglobulin modifications to produce HCAb in cell culture, for pure heavy chain (HCO) transgenic mice, the development of B lymphocytes must be kept in mind. B cells are produced daily by differentiation of hematopoietic stem cells into pro-B cells, pre-B cells, and immature B cells. If the modified immunoglobulin does not support this sequence of differentiation, B cells will not be produced.

[0014] Reports on HCO antibodies in camels, transgenic mice, and rats show that B cells can be developed in which the H chain is not paired with an L chain. However, CH1 needs to be deleted, and in camels, the H chain without L chain has a different VH structure than the standard VH region. The latter shows that not all human VH domains support the development of HCO B cells.

[0015] WO2019018770 Al discloses single chain VH antibodies comprising an antigen binding portion consisting of a VH domain and immunoglobulin constant domains CL and CH1.

[0016] WO2014 / 141192 A2 discloses the production of pure heavy chain antibodies and transgenic non-human animals producing the same. Such antibodies lack a CH1 domain.

[0017] US8754287 B2 discloses mice producing heavy chain antibodies lacking a CH1 domain, and transgenic mice comprising a germline modification deleting nucleic acid encoding a CH1 domain.

[0018] Schusser et al. describe the development of VJC L Knockout chickens express antibodies of pure heavy chain without associated light chain. (Eur. J. Immunol., 46:2137, 2016).

[0019] Klein et al. (Biochemistry, 18:1473, 1979) describe the interaction of isolated light chain variable and constant regions with the Fd' fragment of immunoglobulin G.

[0020] WO2011 / 072204A1 discloses transgenic mice containing functional C mu segments. Thus, mature B cells with standard IgM as antigen receptor develop normally. However, the downstream C gamma segments of the genome lack a CH1 domain. When during the immune response, the antibody class switches to (desired) gamma chains, these cannot pair with L chains. In the H chain class switch, the V H is kept, while the CH is exchanged. This will have two effects: (i) cells with antibodies whose specificity is defined by the combination of H+L chains will no longer be stimulated and will die; and (ii) cells whose specificity is defined mainly by the H chain can die because unpaired V H cannot structurally survive. Thus, in this mouse, V H selection is "back-loaded", i.e. during the immune response.

[0021] WO2007096779A2 describes the use of transgenic mice for the production of class- specific pure heavy chain antibodies, the mice comprising one or more heterologous V H heavy chain loci, wherein each V H heavy chain locus comprises 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, when expressed, does not comprise a CH1 domain.

[0022] WO2009013620A2 describes the production of fully human soluble V H domains by incorporating human V segments into the mouse heavy chain locus, wherein the mouse V, D, and J gene segments are replaced by human V, D, and J segments, and the immunoglobulin heavy chain effector constant region is replaced by an immunoglobulin heavy chain effector constant region that lacks a CH1.

[0023] WO2015143414A2 describes a mouse 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 V H, D H, and J H gene segments with at least one unrearranged V L gene segment and at least one unrearranged J L gene segment.

[0024] WO2019184014A1 describes a mouse comprising a transgenic C gamma gene segment that has been introduced into the immunoglobulin locus in place of an endogenous C mu gene segment and comprises a deletion of the CH1 domain (see Figure 1 ). EP2411408B1 and US9353179B2 describe transgenic mice comprising randomly integrated V H and V HH loci. Since the 1980s, problems with randomly integrated immunoglobulin (and other) loci have been documented: non-physiological levels of expression and instability (loss or functional silencing) of the inserted transgenes.

[0025] There is a need for efficient and cost-effective methods to stably produce HCAbs. More specifically, there is a need for transgenic non-human animals that are capable of producing antigen-specific HCAbs. SUMMARY

[0026] This Summary is provided to introduce some 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, utilities, and advantages of the claimed subject matter will be apparent from the following DETAILED DESCRIPTION, including the drawings attached and defined aspects in the appended claims.

[0027] It is an object of the present invention to provide means, constructs and methods for efficient and easy production of pure heavy chain antibodies (HCAbs) and B cell repertoires expressing a plurality of such HCAbs in transgenic animals or in vitro cell cultures.

[0028] This object is solved by the subject matter of the present claims and is further described herein.

[0029] According to the present invention, a method of producing a mouse expressing a plurality of B cells secreting pure heavy chain antibodies (HCAbs) is provided 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 part of a CH1 domain.

[0030] Specifically, upon immunization of the mouse with an antigen, the mouse expresses a B cell repertoire secreting a plurality of antigen-specific HCAbs. Specifically, upon immunization of the mouse with an antigen, antigen-specific B cells differentiate into plasma cells secreting antigen-specific HCAbs.

[0031] Specifically, upon immunization with an antigen, the mouse activates antigen-specific B cells and induces their differentiation into plasma cells secreting a plurality of antigen-specific HCAbs.

[0032] Specifically, a method of producing a mouse expressing a B cell repertoire secreting a plurality of antigen-specific HCAbs upon immunization with an antigen is provided 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 part of a CH1 domain.

[0033] In wild-type B cells, IgM and IgD are co-expressed, but this occurs in mature B cells in the periphery (spleen, etc.) and not in B cells developing in the bone marrow. Cγ gene segments located downstream of the inactivated Cμ gene segment can not be expressed during B cell development. Since Ig heavy chains are essential for B cell development, mice containing Cγ gene segments downstream of the inactivated Cμ gene segment can not be able to produce any B cells at all. If the Cγ gene segments are introduced into the Ig heavy chain locus in place of the Cμ gene segments, light chain dependent co-expression of IgD can be detrimental to B cells because of misfolded δ heavy chains in the endoplasmic reticulum (ER). This can lead to ER stress, activation of the unfolded protein response (UPR) aimed at restoring protein homeostasis, and if not restored, B cells will undergo apoptotic cell death. Specifically, by positioning the Cγ gene segments upstream of the endogenous position of the Cμ gene segments, pre-B cells will express Cγ-containing preBCRs. This has the significant advantage that V H - Cγ heavy chains are positively selected. V H - pre-B cells of the Cγ heavy chain version are unstable, do not dimerize with light chains, cannot form preBCRs, and are thus selected, providing an improved B cell repertoire expressing a variety of IgG-type HCAbs.

[0034] Specifically, the mice comprise cells containing a recombinant immunoglobulin heavy chain locus capable of producing said B cell repertoire.

[0035] Specifically, said portion of at least the CH1 domain comprises all or part of the CH1 domain, in particular at least any one of 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 at least a portion of the nucleotide sequence encoding the CH1 that is the chaperone binding domain, in particular said portion is one or more amino acids bound by BiP.

[0036] In one specific embodiment, the HCAbs described herein lack a CH1 domain, in particular lack an IgG CH1 domain. Although the mice can be able to express a portion of the CH1 domain, according to one specific embodiment, the portion does not comprise a functional CH1 domain capable of pairing with a CL domain.

[0037] According to another specific embodiment, the HCAbs described herein comprise a CH1 domain that lacks a chaperone binding domain, in particular a BiP chaperone binding domain. Specifically, the mice are engineered to express HCAbs in which at least this type of chaperone binding domain of the CH1 domain is deleted.

[0038] The HCAb secreted by the mouse cells described herein can be soluble or membrane bound. By deleting all or at least part of the CHI domain, the expressed HCAb is not retained in the endoplasmic reticulum (ER) by the ER quality control mechanism that normally prevents cell surface expression or secretion of pure heavy chain antibody constructs. Specifically, the ER chaperone BiP does not retain the HCAb of the application in the ER.

[0039] Specifically, the Cγ gene segment is located downstream of the Eμ major intronic enhancer in the endogenous immunoglobulin heavy chain locus, preferably downstream of the endogenous Eμ major intronic enhancer in the mouse, and in particular, it comprises SEQ ID NO: 7, which is located downstream of the J-encoding sequence in the mouse endogenous immunoglobulin heavy chain locus.

[0040] As described herein, the Cγ gene segment is located upstream of a Cμ gene segment, which can be mutated or unmutated. Specifically, the Cγ gene segment is located upstream of the position in the wild type heavy chain locus where the Cμ gene segment is located. Specifically, the Cγ gene segment is located upstream of a Cμ gene segment and downstream of a JH gene segment in the immunoglobulin heavy chain locus of the mouse described herein.

[0041] The initial wild type mature B cells produce IgM (Cμ) and IgD (Cδ), which are the first two heavy chain segments in the wild type immunoglobulin locus. As described herein, by positioning the Cγ gene segment upstream of a Cμ gene segment in the endogenous immunoglobulin heavy chain locus, the initial mature B cells of the mouse described herein produce IgG type HCAb, and upon antigen activation, these B cells proliferate. Surprisingly, the B cells comprising the immunoglobulin locus described herein develop normally and are capable of producing IgG type membrane bound or soluble HCAb. Thus, the present application provides an improved B cell repertoire expressing a plurality of IgG type HCAb.

[0042] Specifically, the IgG type HCAb is characterized by a VH domain comprising an IgG framework, in particular a VH domain of a human or murine IgG framework, and / or an antibody constant domain of an IgG antibody. Specifically, the IgG type HCAb is of any IgG subtype, e.g., any mouse IgG1, IgG2a / c, IgG2b or IgG3 subtype or any human IgG1, IgG2, IgG3 or IgG4 subtype.

[0043] Specifically, the transgenic Cγ gene segment is integrated in the mouse endogenous immunoglobulin heavy chain locus upstream of the endogenous Cμ gene segment, thus at a position different from its corresponding position in a wild type cell, in which the Cγ gene segment is located downstream of the Cμ gene segment in the immunoglobulin heavy chain locus.

[0044] In one particular embodiment, the transgenic Cγ gene segment is derived from an endogenous nucleic acid sequence, in particular a mouse Cγ gene segment, which is modified by deletion of nucleotide sequences encoding at least part of the CH1 domain, and the transgenic Cγ gene segment is incorporated at a position in the mouse endogenous immunoglobulin heavy chain locus that does not naturally occur, i.e. upstream of the endogenous Cμ gene segment.

[0045] According to different particular aspects, the transgenic Cγ gene segment is an exogenous nucleic acid, e.g. a synthetic nucleic acid, and is integrated upstream of the endogenous Cμ gene segment in the mouse endogenous immunoglobulin heavy chain locus.

[0046] In particular, the transgenic Cγ gene segment is of mammalian origin, preferably of rodent origin, most preferably of mouse origin.

[0047] In particular, the transgenic Cγ gene segment is of human origin.

[0048] In particular, the transgenic Cγ gene segment comprises or consists of a transgenic Cγ1, Cγ2a / c, Cγ2b or Cγ3 gene segment.

[0049] In particular, the transgenic Cγ gene segment comprises or consists of a transgenic Cγ1, Cγ2, Cγ3 or Cγ4 gene segment.

[0050] In particular, the transgenic Cγ gene segment is a transgenic mouse or human Cγ1 gene segment.

[0051] In particular, the endogenous immunoglobulin heavy chain locus of the mouse described herein is the mouse endogenous immunoglobulin heavy chain locus, in particular at an endogenous position within the mouse genome.

[0052] According to one particular aspect of the methods provided herein, the Cμ gene segment in the immunoglobulin heavy chain locus of the mouse described herein or the B cell described herein is inactive. In particular, the Cμ gene segment is inactivated by a functional deletion mutation, e.g. by introduction of a stop codon, or by deletion of part of the nucleotide sequence encoding the Cμ gene segment. Preferably, the Cμ gene segment is not completely deleted, but only a part thereof is deleted. In particular, the Cμ gene segment is inactivated by deletion of the CH1, CH2, CH3 and / or CH4 domain.

[0053] In some cases, when the locus is transcribed, the VDJ exon is spliced onto the Cμ gene segment. This can lead to the production of light chain dependent μ heavy chains. In particular, by inactivating the Cμ gene segment, the quality of the B cell repertoire described herein is further improved.

[0054] Specifically, the Cm gene segment is inactivated by deletion of the entire nucleotide sequence encoding the endogenous Cm gene segment or a portion thereof that is at least any of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the sequence.

[0055] Specifically, the Cm gene segment comprises a loss-of-function mutation, a deletion, or an inactivating mutation, preferably introducing a stop codon, preferably in any one or all of its CHI, CH2, CH3, and CH4 domains. Preferably, the Cm gene segment comprises a stop codon in the CHI, CH2, and CH3 domains.

[0056] According to one specific aspect of the methods provided herein, the mu switch (S) region in the immunoglobulin heavy chain locus of the mouse described herein or the B cell described herein is inactive. Specifically, the immunoglobulin heavy chain locus of the mouse described herein comprises a loss-of-function mutation, a deletion, or an inactivating mutation of the nucleotide sequence encoding the mu switch (S) region, which is normally present in the endogenous Igh locus. Specifically, the Igh locus without a functional S region is unable to undergo isotype switching.

[0057] Specifically, if the B cell described herein undergoes isotype switching, it is possible to produce a light chain dependent heavy chain isotype IgG, IgA, or IgE. As described herein, this can lead to B cell death. Thus, by preventing isotype switching, the B cell repertoire can be further improved.

[0058] In one specific embodiment, the CH domains of the other heavy chain isotypes are inactivated, preferably by introducing a loss-of-function mutation, a deletion, or an inactivating mutation. Specifically, the constant region gene segments of the immunoglobulin heavy chain locus described herein comprise a loss-of-function mutation, a deletion, or an inactivating mutation, preferably introducing a stop codon, preferably in any one or all of its CHI, CH2, CH3, and CH4 domains.

[0059] According to one specific aspect, the mouse produced according to the methods described herein comprises an inactivated or deleted endogenous immunoglobulin light chain locus. Specifically, the transgenic mouse described herein comprises a loss-of-function mutation or a deletion in any of the endogenous kappa or lambda light chain loci or both. Such a mouse is characterized by the expression of HCAb without light chains.

[0060] In one specific embodiment, the mouse immunoglobulin heavy chain locus produced according to the methods described herein comprises human V H , D, and J H coding sequences and expression control sequences in operable linkage H , D, and J HThe representation of the encoded sequence. Specifically, the representation of the control sequence is mouse-based.

[0061] In one specific aspect of the method provided herein, during B cell development, the V gene at the immunoglobulin heavy chain locus described herein... H D and J H Coding sequence recombination forms expression V H The VDJ coding sequence for the binding site. Each developing B cell will randomly select a VDJ. H A D and a J H Gene fragments are used for recombination and expression as HCAb. Upon maturation, each of the millions of B cells in a mouse recognizes a specific antigen and is activated upon encountering that antigen.

[0062] Specifically, V H D and J H The coding sequence is recombined to form a VDJ coding sequence that expresses a VH binding site that specifically recognizes the antigen, thereby obtaining recombinant VDJ in a given B cell. H The coding sequence enables the secretion of antigen-specific V antibodies encoded by the recombinant VH coding sequence. H IgG-type HCAbs that bind to the binding domain.

[0063] Specifically, V H The coding sequences of D and J are recombined to form the coding sequence of VDJ, which expresses the VH binding site that specifically recognizes the antigen, thereby obtaining recombinant VDJ in B cells. H The coding sequence, whose progeny are able to secrete antigen-specific V containing the recombinant VH coding sequence. H IgG-type HCAbs that bind to the binding domain.

[0064] Specifically, V H D and J H The coding sequence is recombined to form the VDJ coding sequence that expresses the VH binding site that specifically recognizes the antigen, thereby obtaining recombinant VDJ in B cells. H The coding sequence enables this B cell, after differentiating into plasma cells (especially plasma cells), to secrete antigen-specific V antibodies encoded by the recombinant VH coding sequence. H IgG-type HCAbs that bind to the binding domain.

[0065] In one specific aspect of the method provided herein, after immunizing the mice described herein with the antigen, B cells expressing a VH binding site that specifically recognizes the antigen will undergo activation and differentiate into plasma cells capable of secreting antigen-specific VH. H IgG-type HCAbs that bind to the binding domain.

[0066] In particular, the HCAb secreted by the B cells of the mice described herein comprises or consists of antigen-specific V H The binding domain and the IgG constant region lacking the CH1 domain consist of, or comprise or consist of only, a part of the CH1 domain. In particular, said part of the CH1 domain lacks a partner binding domain, preferably the CH1 domain lacks the BiP partner binding domain.

[0067] Also provided herein are B cell repertoires expressing a plurality of IgG-type HCAbs. In particular, the B cell repertoire expresses a plurality of IgG-type HCAbs derived from a mouse obtained by the methods described herein. The mice described herein can comprise said B cell repertoire, or the B cell repertoire can be isolated from the mice. The B cell repertoire can be provided in the form of a B cell library or a library of nucleic acid molecules derived from said B cell repertoire. The respective libraries can be provided using a display system, thereby enabling the screening of molecules expressing antigen binding properties. In particular, such a library of nucleic acid molecules encodes a plurality of HCAbs, or respective antigen binding molecules comprising the VH binding site of such HCAbs. According to a particular aspect, such a library of nucleic acid molecules can be used in a method for generating a library of antigen binding molecules comprising or consisting of VH domains differing in their VH antigen binding site or binding properties.

[0068] In particular, the B cell repertoire described herein is capable of expressing a plurality of HCAbs differing in their (VH) antigen binding site, e.g. thereby allowing the generation of a plurality of antigen binding molecules, such as antibodies, recognizing the same antigen or epitope, e.g. generating affinity matured or optimized antibody variants; or generating antibodies specifically recognizing a target antigen but recognizing different epitopes of that target antigen.

[0069] According to a particular aspect, the libraries described herein, e.g. the B cell library or the respective libraries of nucleic acid molecules or antigen binding molecules, can be suitably screened, and library members can be selected according to desired structural or functional properties, to identify and generate 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 can be selected according to desired structural or functional properties, to generate antigen binding molecules, in particular antibody products.

[0070] In particular, the B cell repertoire described herein is capable of expressing a plurality of antigen-specific HCAbs differing in their VH domains.

[0071] In one particular aspect, the B cell repertoires described herein express a plurality of antibodies or antibody fragments, in particular HCAbs described herein, including a plurality of antibodies or antibody fragments each specifically recognizing the same target antigen or epitope. In particular, upon immunization of the mice described herein with an antigen, the B cell repertoire expresses antigen-specific antibodies or antibody fragments. According to one particular aspect, provided herein is an antibody library comprising or covering a plurality of antibodies recognizing the same target antigen or epitope.

[0072] In one particular aspect, the B cell repertoires described herein express a plurality of antibodies or antibody fragments, in particular HCAbs described herein, including a plurality of antibodies recognizing different target antigens. Such repertoires can be obtained by immunization with a complex multi-component antigen, e.g. a virus or bacterium, having a number of different target antigens, each target antigen having a plurality of epitopes. According to one particular aspect, provided herein is an antibody library comprising or covering a plurality of antibodies recognizing different target antigens.

[0073] According to one particular aspect, the present application provides a B cell repertoire expressing a plurality of antibodies or antibody fragments, these antibodies or antibody fragments lacking any light chain. In particular, the present application provides a B cell repertoire expressing a plurality of HCAbs described herein, each HCAb specifically recognizing the same target antigen or a different target antigen. In particular, provided herein is a B cell repertoire obtainable or obtained by the methods provided herein. In particular, the B cell repertoire is expressed by a transgenic mouse described herein.

[0074] In particular, the various antigen binding molecules referred to herein, e.g. the antigen binding molecules of the B cell repertoires or corresponding libraries described herein, comprise or cover at least any one of 10 2 , 10 3 , 10 4 , 10 5 or 10 6 antigen specific molecules, e.g. antibodies or HCAbs.

[0075] The diversity of a B cell repertoire can be determined, e.g. by deep sequencing, e.g. determining the number of different VDJ arrangements, indicating the diversity of HCAbs expressed by the B cell repertoire.

[0076] The B cell repertoires described herein can be provided as a library of nucleic acid sequences, obtainable by sequencing the recombinant VH sequences in B cells produced by the mice described herein. Such sequencing can be done using deep sequencing methods, e.g. next generation sequencing (NGS). Deep sequencing refers to sequencing a genomic region multiple times, sometimes hundreds or even thousands of times. Such NGS methods allow providing a plurality of VH domain nucleic acid or amino acid sequences.

[0077] According to a specific aspect, there is provided an antibody library of HCAbs, wherein

[0078] a) the genes encoding said antibodies are derived from a B cell library as described herein, or

[0079] b) the antibodies are secreted by mammalian plasma cells, preferably of rodent origin, in particular of mouse origin.

[0080] Specifically, the library can be obtained by cloning the genes encoding said antibody library from B cells of a B cell library as described herein or by secreting antibodies by a plurality of mammalian plasma cells. Specifically, the antibodies secreted by the mammalian plasma cells are characterized by a glycosylation pattern which is characteristic for mammalian plasma cell-derived species.

[0081] Accordingly, the present application further provides a method of producing a library of antibodies as described herein, in particular by engineering mammalian plasma cells expressing and secreting such antibodies.

[0082] Specifically, the mammalian plasma cells are derived from rodents, preferably mice.

[0083] It is well understood that the antibodies described herein can be prepared using one or more corresponding sequences of other species than mice, including for example non-mouse animals, or of human origin, or any combination thereof, for example human nucleic acid sequences encoding corresponding human antibody domains.

[0084] Sequences of human antibody constant domains are well known in the art and are available from various databases, including The National Center for Biotechnology Information (NCBI) and ImmunoGeneTics (IMGT). Exemplary sequences of human IgGl constant domains exon CHI, CH2 and CH3-S (S, 3' part of CH3 exon encoding the secreted form of g1 HC) are identified as SEQ ID NO: 16 - SEQ ID NO: 18.

[0085] It is well understood that any sequence of a human antibody domain is merely exemplary. Alternatively, a corresponding different allele of a human antibody domain sequence can be used.

[0086] As an alternative to an animal or human origin nucleotide sequence or animal or human amino acid sequence encoding an antibody domain, a modified (artificial) nucleotide sequence and corresponding amino acid sequence can be used, for example, a corresponding sequence comprising any of at least 80%, 85%, 90% or at least 95% sequence identity, as long as the corresponding antibody domain has the function of pairing and linking within the corresponding antibody structure as described herein.

[0087] According to a specific embodiment, the antibody is produced in a host cell (in vitro) or a non-human animal host, in particular a mouse (in vivo).

[0088] The structure of the exemplary HCAb described herein is shown in Figure 3B .

[0089] The VH antigen binding moiety specifically comprises or consists of three CDR loops of a VH domain, namely VH-CDR1, VH-CDR2 and VH-CDR3. The antigen binding moiety can be affinity matured by variation of one or more CDR loops, thereby optimizing or increasing the affinity for binding to the target antigen. Such variation can be obtained by one or more point mutations, for example, by in vivo processes or by in vitro mutagenesis techniques, 1, 2, 3 or more point mutations in any one or each CDR sequence to obtain an affinity matured antigen binding site.

[0090] Specifically, the HCAb is a molecule consisting of two identical heavy chains, each heavy chain comprising a VH domain fused to an antibody constant domain CH2 and CH3. Specifically, the antibody constant domain is an IgG constant domain. Upon pairing of the CH2 and CH3 domains of the two heavy chains, the HCAb comprises two VH domains and one Fc region.

[0091] The Fc region of the HCAb described herein specifically comprises the constant region of an antibody, excluding the first constant region immunoglobulin domain. The "Fc region" generally refers to the last two constant region immunoglobulin domains of IgG, IgA or IgD and the flexible hinge region N-terminal of these domains, and the last three constant region immunoglobulin domains of IgE and IgM. Specifically, the hinge is an IgG, IgA or IgD antibody.

[0092] For HCAbs of the IgG type, 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 VH arm of the antibody domain or between VH and CH2. The Fc region of the IgG type HCAb specifically does not comprise a CH1 domain or comprises a part of the CH1 domain lacking a partner binding sequence. The Fc region can also comprise a CH2 or CH3 domain in the form of an artificial variant of the corresponding naturally occurring antibody domain, for example, having at least 90% sequence identity to said naturally occurring antibody domain.

[0093] In particular, the Fc region described herein comprises or consists of a dimer of CH2 and CH3 domains that are part of antibody heavy chains (HC), wherein the CH2 domain of a first HC is paired with the CH2 of a second HC, and the CH3 domain of the first HC is paired with the CH3 of the second HC. Such a dimer can be a homodimer, i.e. composed of two CH2-CH3 domain chains of identical amino acid sequence, or a heterodimer, i.e. composed of two CH2-CH3 domain chains, wherein each chain has a different amino acid sequence, e.g. with different CH3 amino acid sequences to stabilize the Fc.

[0094] In one particular aspect, the HCAb comprises a hinge region connecting the CH2-CH3 domain chain of the Fc region to the VH domain. In particular, the HCAb comprises a hinge region connecting the VH domain and the constant domain of the antibody. In particular, the hinge region is derived from the conventional antibody heavy chain hinge region connecting the C-terminus of the CH1 domain to the N-terminus of the CH2 domain. Alternatively, any other natural or artificial linker of about the same length can be used. A suitable hinge region is a native (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, which optionally comprises one or more, up to 5 or fewer, point mutations. The hinge region typically comprises one or more cysteine residues to generate disulfide bonds in the HCAb, e.g. linking two HC.

[0095] In particular, the amino acid sequences of the two HC (also referred to herein as first and second HC) comprised in the HCAb are identical. Alternatively, the amino acid sequences of the two HC are different, e.g. such that the antigen binding sites of the VH domains are different.

[0096] For example, the first HC comprises a first VH and the second HC comprises a second VH. The first and second VH can comprise identical or different antigen binding sites, e.g. specifically recognizing two different target antigens. Thus, the HCAb can be monospecific, bivalent or bispecific.

[0097] Antibodies produced by transgenic non-human animals, e.g. mice as described herein, are generally understood to be natural or native antibodies. Such natural antibodies can be derived from an antibody library specifically recognizing an antigen, e.g. an antibody library produced by a transgenic mouse as described herein.

[0098] According to one particular aspect, the antibody or antibody library can be subjected to affinity maturation in vivo, resulting in high affinity antibodies binding to a specific target antigen, e.g. with a K -7 M, e.g. between 10 -7 and 10 -10 M. D .

[0099] Antibodies generated by in vitro mutagenesis methods, such as affinity matured antibodies generated using random mutagenesis and / or library techniques, can produce even higher affinities, e.g., with Kd of less than 10 -8 M, e.g., less than 10 -11 M of K D .

[0100] Natural antibodies advantageously feature the native conformation of the VH-CDR sequences. This native conformation is characterized by the natural primary structure of the antigen binding site, and / or the natural primary structure of the full-length VH domain.

[0101] When generating HCAbs, the selected antibody domains and / or hinge regions can be of human, artificial, or non-human animal origin. For example, HCAbs are generated in transgenic mice comprising human and mouse sequences.

[0102] According to a particular aspect, the HCAbs described herein are provided in soluble form, e.g., water-soluble form at a concentration suitable for use in pharmaceutical formulations. A soluble preparation is specifically provided herein, comprising an HCAb in isolated form as described herein, e.g., isolated from a serum or blood component of an animal producing the HCAb, or isolated from a cell culture component.

[0103] Mice obtainable or obtained by the methods described herein are also provided herein. Specifically, mice comprising an immunoglobulin heavy chain locus as described herein are provided herein. Specifically, the mice described herein comprise a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment in their endogenous immunoglobulin heavy chain locus, wherein the Cγ gene segment comprises a deletion of nucleotide sequences encoding at least part of a CH1 domain.

[0104] Specifically, the mice described herein comprise a modified immunoglobulin allele or other transgene in their genome. Specifically, the mice described herein comprise at least one transgenic Cγ gene segment in their endogenous immunoglobulin heavy chain locus, and are thus transgenic animals.

[0105] In one particular embodiment, the mice described herein further comprise human immunoglobulin regions. For example, a number of approaches have been developed to replace mouse endogenous immunoglobulin regions with human immunoglobulin sequences to produce partially or fully human antibodies for drug discovery purposes. Examples of such mice include those described in, e.g., 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.

[0106] According to one particular aspect, the immunoglobulin heavy chain loci described herein comprise a chimeric gene segment. In particular, the mice described herein comprise a chimeric immunoglobulin segment, e.g., a chimeric immunoglobulin segment as described in US 2013 / 0219535, in particular, a human IGH(V H , D, J H ) coding sequences are embedded in corresponding mouse non-coding and regulatory sequences. Immunoglobulin gene segments comprising human immunoglobulin coding sequences and murine expression control sequences are also referred to herein as "chimeric immunoglobulin gene segments".

[0107] In particular, the genome of such transgenic mice comprises an introduced exogenous immunoglobulin region that is partially human, wherein the introduced region comprises human variable region coding sequences and murine non-coding regulatory sequences that control expression of the human sequences, which regulatory sequences are derived from the mouse or the endogenous genome of the mouse and are knocked into the mouse genome by introduction of a chimeric immunoglobulin gene segment or a chimeric immunoglobulin locus. In particular, the murine sequences, in particular the murine non-coding and regulatory sequences, are based on the corresponding murine endogenous sequences, i.e., the same sequences as in the wild-type endogenous immunoglobulin loci.

[0108] The murine expression control sequences described herein for expression of human immunoglobulin gene sequences are in particular selected from the group consisting of promoters, transcriptional initiation and termination sequences, enhancer and activator sequences, ribosomal binding sites. Particular examples of such expression control sequences are sequences flanking the coding sequences, which can include promoters, 5' untranslated sequences, introns into which leader peptide coding sequences are inserted, recombination signal sequences (RSS) and splice sites.

[0109] In particular, the mice described herein comprise a V H heavy chain locus within the immunoglobulin heavy chain locus described herein, which locus comprises human V H , D and J HThe encoding sequence and the expression control sequence control V in an operationally connected manner. H D and J H The representation of the encoded sequence. Specifically, V H The heavy chain locus is chimeric, in which V H The expression control sequence of the heavy chain locus is mouse-derived, with an preferred endogenous expression control sequence.

[0110] Specifically, the VH heavy chain locus contains human V H D and J H A library of encoded sequences. Specifically, it is a library of at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 V's. H The encoding sequence consists of 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40 D-coded 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-coded sequences. H The coding sequence, particularly the human VH, D, and JH coding sequences. Specifically, it is a maximum of 120 V... H The encoding sequence can have a maximum of 40 D encoding sequences and a maximum of 20 J encoding sequences. H Coding sequences. Specifically, the VH heavy chain locus contains a complete library of the coding sequences for human VH, D, and JH.

[0111] Specifically, V H V of the heavy chain locus H D and J H The coding sequence is recombined to form a VDJ coding sequence that specifically recognizes the antigen, and contains recombinant V... H B-cell expression of heavy chain loci includes antigen-specific V H HCAbs that bind to the CH1 domain and lack the CH1 domain or at least a portion of the CH1 domain in the constant region of IgG.

[0112] Specifically, the transgenic Cγ gene fragment is contained in the nucleic acid construct.

[0113] In one specific implementation, the transgenic Cγ gene fragment is contained in a nucleic acid construct that includes at least one immunoglobulin gene fragment coding sequence, specifically at least the J locus of the immunoglobulin heavy chain. H Gene fragment coding sequence, preferably at least J H The coding sequences of gene segments 2-6.

[0114] In particular, a nucleic acid construct comprising a transgenic C gamma gene segment and a chimeric immunoglobulin gene segment comprising human IGH coding sequences, preferably at least human IGH J coding sequences, embedded in corresponding mouse non-coding and regulatory sequences, can be introduced into the mouse genome as an exogenous nucleic acid construct or element upstream of an endogenous C mu gene segment and replaces at least a portion of the mouse endogenous immunoglobulin heavy chain locus. In particular, the nucleic acid construct comprises a human IGH V H , D and J H coding sequences.

[0115] In particular, a nucleic acid construct comprising a transgenic C gamma gene segment, a chimeric immunoglobulin gene segment comprising human IGH coding sequences, preferably at least human IGH J coding sequences, embedded in corresponding mouse non-coding and regulatory sequences, and an inactivated C mu gene segment as described herein can be introduced into the mouse genome as an exogenous nucleic acid construct or element upstream of an endogenous C delta gene segment and replaces at least a portion of the mouse endogenous immunoglobulin heavy chain locus. In particular, the nucleic acid construct comprises a human IGH V H , D and J H coding sequences.

[0116] Alternatively, the transgenic C gamma gene segment and optionally the inactivated C mu gene segment are knocked into the immunoglobulin locus in the mouse genome.

[0117] In another advantageous aspect, the genome content of the mice described herein is modified such that their B cells are capable of expressing more than one functional VH domain per cell, i.e. the cells produce bispecific antibodies, as described in WO2017035252A1.

[0118] In particular, the C mu gene segment in the immunoglobulin heavy chain locus of the transgenic mice described herein is inactive. In particular, the C mu gene segment is inactivated by a loss-of-function mutation, a deletion of the C mu gene segment or one or more mutations introducing one or more stop codons.

[0119] In particular, it is described herein that the immunoglobulin heavy chain locus of the mice described herein comprises a transgenic C gamma gene segment 5' of a C mu gene segment, wherein the C gamma gene segment comprises a deletion of a nucleotide sequence encoding at least a portion of a CH1 domain.

[0120] In one particular embodiment, the C mu gene segment is an endogenous gene segment of the immunoglobulin heavy chain locus of the mice described herein.

[0121] In one particular embodiment, the Cμ gene segment of an immunoglobulin heavy chain locus described herein is a transgenic gene segment which replaces an endogenous Cμ gene segment in an endogenous immunoglobulin heavy chain locus and comprises one or more inactivating mutations.

[0122] In particular, the transgenic Cμ gene segment is inactivated by a loss-of-function mutation, or by partial deletion of the Cμ gene segment, e.g. by deletion of at least any of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, or by introduction of an exogenous stop codon in the Cμ gene segment, preferably in any one or more of the CH1, CH2, CH3 and CH4 domains.

[0123] In particular, the Cμ gene segment described herein is located in the natural position of a Cμ gene segment in a wild-type mouse immunoglobulin heavy chain locus.

[0124] In particular, the mouse described herein comprises a modification of its endogenous immunoglobulin heavy chain locus described herein.

[0125] In particular, the locus described herein is a recombinant (e.g. "chimeric") locus which is derived from a mouse, but comprises at least one exogenous element, e.g. one or more exogenous heavy chain regions which are not naturally associated with the regulatory elements of the locus, e.g. a Cγ gene segment comprising a deletion in the nucleotide sequence encoding at least part of a CH1 domain.

[0126] In particular, the mouse is treated by a suitable gene targeting technique, e.g. targeted homologous recombination, using a site-specific recombinase technology, or a CRISPR / Cas9 technology, to incorporate the genetic sequence into the immunoglobulin heavy chain locus.

[0127] In particular, the mouse does not express an endogenous κ and / or λ locus, either because the endogenous κ and / or λ locus is deleted or silenced, or is functionally mutated.

[0128] According to one particular embodiment, the method of producing an antibody as described herein further comprises the step of immunizing a mouse as described herein with an antigen, thereby eliciting an immune response against the antigen by antigen-specific B cells contained in the B cell repertoire of the mouse. The responding B cells will eventually differentiate into plasma cells which secrete HCAb specific for the immunizing antigen.

[0129] The antigen can be administered to the mouse in any convenient manner, with or without an adjuvant, and can be administered according to a predetermined schedule.

[0130] Particularly provided herein is a method of producing an antigen-specific antibody, comprising:

[0131] a) immunizing a transgenic mouse comprising a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment in an immunoglobulin heavy chain locus, wherein the Cγ gene segment comprises a deletion of nucleotide sequences encoding at least part of a CH1 domain, with an antigen,

[0132] b) expressing a HCAb comprising an antigen specific VH and an IgG constant region;

[0133] c) isolating one or more nucleic acid sequences encoding the antigen specific VH,

[0134] d) producing a monoclonal antibody comprising the antigen specific VH.

[0135] In particular, provided herein is a method of producing an antibody comprising an antigen specific VH domain, the method comprising:

[0136] a) immunizing a mouse comprising a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment in an immunoglobulin heavy chain locus, wherein the Cγ gene segment comprises a deletion of nucleotide sequences encoding at least part of a CH1 domain, with an antigen, thereby providing a cell pool expressing antigen specific HCAb;

[0137] b) selecting a cell from the pool expressing a IgG type HCAb comprising an antigen specific VH;

[0138] c) determining a nucleic acid sequence encoding the antigen specific VH from the HCAb,

[0139] d) producing a monoclonal antibody comprising the antigen specific VH.

[0140] In particular, the monoclonal antibody produced according to the methods described herein can be any full length immunoglobulin, an antigen binding antibody fragment thereof or any other antibody construct comprising at least one variable heavy chain (VH) antibody domain or a single VH domain, such as including one or more single domain antibodies, Fab, F(ab'), (Fab)2, scFv, Fd, Fv, or a full length antibody such as an IgG type (e.g. IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody. A particular embodiment is a pure heavy chain antibody such as a HCAb as described herein.

[0141] In particular, the nucleic acid sequence encoding the antigen specific VH is determined by sequencing the recombinant VH sequences in B cells produced by the mouse described herein. Such sequencing can be done using deep sequencing methods such as next generation sequencing (NGS).

[0142] To prepare monoclonal antibodies, antibody producing cells, such as spleen and / or lymph node cells, can be isolated from immunized transgenic mice and used to fuse with transformed cell lines to produce hybridomas or the cDNA encoding the antibodies can be cloned by standard molecular biology techniques and expressed in transfected cells. Procedures for preparing monoclonal antibodies are well established in the art.

[0143] In particular, the method further comprises the steps of preparing hybridomas and producing and screening antibody producing cells, in particular those specifically recognizing the target antigen.

[0144] In particular, provided herein is a method of producing an antibody comprising an antigen specific VH domain, the method comprising:

[0145] a) immunizing a mouse with an antigen, the mouse comprising a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment in an immunoglobulin heavy chain locus, wherein the Cγ gene segment comprises a deletion of nucleotide sequences encoding at least part of a CH1 domain,

[0146] b) isolating spleen and / or lymph node cells from the transgenic mouse;

[0147] c) producing hybridomas from the spleen and / or lymph node cells;

[0148] d) screening for antigen specific hybridomas;

[0149] e) secreting an IgG type HCAb comprising an antigen specific VH from the hybridomas; and

[0150] f) isolating the monoclonal HCAb from the culture supernatant.

[0151] In a particular embodiment, the method comprises the steps of determining a nucleic acid sequence encoding the antigen specific VH from the hybridomas and producing a recombinant monoclonal antibody comprising the antigen specific VH.

[0152] In particular, the method comprises the steps of isolating nucleic acid sequences from the immunized mouse for producing specific antibodies or fragments thereof, in particular antigen binding fragments, in cell culture. Such antibodies or antigen binding fragments thereof are understood herein as hyperimmune antibodies.

[0153] Also provided herein is the use of a transgenic mouse as described herein in a method of preparing a library of antigen binding molecules comprising a plurality of VH binding sites derived from the mouse described herein or the corresponding B cell repertoire described herein. In particular, provided herein is a library of VH domains (e.g. a VH library) or a library of molecules comprising VH (or a corresponding library), in particular a library of HCAbs (or a HCAb library).

[0154] Also provided herein are methods of producing the mice described herein, comprising:

[0155] a) providing murine embryonic stem cells;

[0156] b) providing one or more vectors (also referred to as targeting vectors) comprising nucleic acid sequences in one or more expression cassettes, said nucleic acid sequences comprising a transgenic Cγ gene segment as described herein and optionally a non-functional Cμ gene segment as described herein;

[0157] c) introducing the one or more vectors into the cells;

[0158] d) screening for transgenic cells in which the sequences of b) have been incorporated into the cellular genome of said cells by targeted integration upstream of an endogenous Cμ gene segment (which can be mutated or unmutated) in the endogenous immunoglobulin heavy chain locus, or, if the vector comprises a non-functional Cμ in place of the endogenous Cμ, into Cμ upstream of the endogenous Cδ gene segment; and

[0159] e) using said transgenic cells to produce a transgenic mouse derived from said transgenic cells.

[0160] In particular, the endogenous Cμ gene segment is inactivated, preferably by a functional deletion mutation, a Cμ gene segment deletion or by a mutation introducing a stop codon.

[0161] In particular, the mouse expresses an HCAb as described herein. In particular, the mouse expresses a pure heavy chain antibody, and / or does not express any antibody construct comprising a VL domain.

[0162] In particular, one or more markers are used to indicate successful integration of the one or more vectors into the cellular genome. In particular, a selectable marker is used that is capable of being expressed in the host, which allows for easy selection of those hosts that contain the introduced nucleic acid or vector.

[0163] Examples of selectable markers include, for example, proteins that confer resistance to antimicrobials (e.g., puromycin, hygromycin, bleomycin, or chloramphenicol) or antivirals (e.g., ganciclovir), proteins that confer a metabolic advantage (e.g., a nutritional advantage) to the host cell, and proteins that confer a functional or phenotypic advantage (e.g., cell division) to the cell.

[0164] In a particular embodiment, a herpes simplex virus (HSV) thymidine kinase (TK) gene is used for negative selection, and a ganciclovir and / or puromycin resistance gene is used to select for cells that have integrated the targeting vector into their genome.

[0165] In particular, the vector is introduced by a means capable of incorporating the nucleic acid sequence into the eukaryotic cell, such that the nucleic acid sequence is inserted into the cell, wherein the nucleic acid sequence can be present transiently in the cell, but is preferably incorporated or stably integrated into the genome (in particular the chromosome) of the cell.

[0166] In particular, the one or more vectors are integrated into the target site of the cellular genome of the mouse cell by any targeted recombination method, such as by homologous recombination or site-specific recombination technology. In particular, the CRISPR / Cas9 genome editing system can be used for targeted recombination (He, et al., Nuc. Acids Res., 44: e85, 2016).

[0167] According to one particular embodiment, a method of generating a transgenic mouse as described herein is provided, comprising:

[0168] a) providing a mouse cell comprising a target site located 5' of an endogenous Cδ gene segment of an endogenous immunoglobulin heavy chain locus;

[0169] b) providing one or more vectors comprising in one or more expression cassettes a nucleic acid sequence comprising a Cγ gene segment comprising a deletion of the nucleotide sequence encoding the CH1 domain, and optionally a Cμ gene segment comprising at least one inactivating mutation, said nucleic acid sequence being flanked by DNA sequences homologous to said target site, and one or more markers to select for targeted homologous recombination of the vector into the cellular genome;

[0170] c) introducing said one or more vectors into said mouse cell;

[0171] d) incorporating said nucleic acid sequence into the genome of said mouse cell, and selecting for transgenic cells wherein said nucleic acid sequence has been integrated into the cellular genome of said cell at said target site; and

[0172] e) using said transgenic cells to generate a transgenic mouse comprising said transgenic cells.

[0173] In particular, a homology targeting vector or "targeting vector" can be used, which is a vector comprising nucleic acid encoding a targeting sequence, a site-specific recombination site, and optionally a selectable marker gene, which is used to modify an endogenous immunoglobulin region in a host cell using homology- mediated recombination (in particular homologous recombination). In one particular embodiment, the homology targeting vector can further comprise 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.

[0174] Specifically, a targeting vector is used which, upon transfection of a host cell, recombines with the host cell genome, upon productive VDJ rearrangement, the encoded antibody is expressed and inserted into the plasma membrane and / or secreted by the host cell. Specifically, the vector comprises one or more exogenous or heterologous regulatory elements, e.g., enhancers or promoters, operably linked to the antibody-encoding sequence, which regulatory element is not naturally associated with the antibody-encoding sequence.

[0175] According to another specific embodiment, a method for producing a mouse as described herein is provided, comprising:

[0176] a) providing a mouse cell and integrating two recombinase-mediated cassette exchange (RMCE) target sites flanked by recognition sequences for a site-specific recombinase at 5' and 3' positions of an endogenous Cμ gene segment of an endogenous immunoglobulin heavy chain locus;

[0177] b) providing one or more vectors comprising in one or more expression cassettes nucleic acid sequences comprising a Cγ gene segment comprising a deletion of a nucleotide sequence encoding at least part or all of a CH1 domain and optionally a Cμ gene segment comprising at least one inactivating mutation, said nucleic acid sequences being flanked by further recognition sites for a site-specific recombinase, and one or more markers to select for targeted integration of the vectors into the mouse genome, wherein said further recognition sites are capable of recombining with said RMCE target sites;

[0178] c) introducing said one or more vectors and a site-specific recombinase recognizing said RCME target sites and further recognition sites into said mouse cell;

[0179] d) incorporating said nucleic acid sequences into the genome of said mouse cell and selecting transgenic cells, wherein said nucleic acid sequences have been integrated into the cellular genome of said cells at said RMCE target sites; and

[0180] e) using said transgenic cells to produce a transgenic mouse comprising said transgenic cells.

[0181] Specifically, any of said recognition sites for a site-specific recombinase are recombinase recognition sites (e.g., Cre / lox, Flp-FRT, etc.), wherein the recombinase is capable of excising a DNA sequence between its two recognition sites.

[0182] According to one specific aspect, a method of producing a pure heavy chain antibody is provided, said method comprising:

[0183] a) expressing in a mouse a recombinant immunoglobulin heavy chain locus, which locus comprises

[0184] i) a variable heavy chain region comprising one or more V H , D and J H gene segments, preferably comprising human V H , D and J H coding sequences embedded in mouse regulatory sequences,

[0185] ii) a constant heavy chain region comprising a transgenic Cg gene segment upstream of the Cm gene segment in the immunoglobulin heavy chain locus,

[0186] wherein the Cg gene segment comprises a deletion of nucleotide sequences encoding at least part or all of the CHI domain, and wherein, optionally, the Cm gene segment is inactive, and

[0187] iii) a joining region,

[0188] said regions are engineered and positioned to express the HCAb as described herein,

[0189] wherein the mouse does not express endogenous K and / or l gene loci; and

[0190] b) producing an antibody, i.e. the HCAb.

[0191] According to a specific embodiment, the antibody as described herein is produced in cell culture using a suitable production host cell line. Specifically, the production employs a bacterial, yeast, plant, insect or mammalian cell culture. Specifically, a host cell is used after introduction of a corresponding nucleic acid molecule encoding the antibody as described herein. In particular, advantageously any mammalian host cell is used: BHK, CHO, HeLa, HEK293, MDCK, NIH3T3, NSO, PER.C6, SP2 / 0 or VERO cells.

[0192] According to a specific aspect, the present application provides the use of a mouse as described herein for the production of a library of antigen binding molecules, such as antibodies, comprising at least a VH binding site or a corresponding VH domain, such as a HCAb antibody, or a fragment thereof comprising at least a VH binding site, or a library of nucleic acid sequences encoding or expressing said library.

[0193] The transgenic cells as described herein can be used to generate expression libraries for the identification of antibodies of interest, e.g. by cloning of the genes encoding the antibodies from B cells, or by selecting plasma cells with a defined specificity in engineered mice expressing antibodies on the plasma cell membrane, e.g. as described in US20170226162A1. Thus, the present application also encompasses the use of the antibody repertoires generated using the cell technology for the identification of antigen specific antibodies expressed by plasma cells.

[0194] Specific embodiments include partial or whole immunoglobulin proteins transcribed from immunoglobulin heavy chain genes of engineered portions from genetically modified mice described herein; and partial or whole engineered immunoglobulins derived from genetically modified mouse cells.

[0195] These and other aspects, objects, and features of the present application will be better understood from the following detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0196] Figure 1 : depicts the mouse germline Igh locus (top) [including V (IghV), D (IghD), J (IghJ), and C (IghC) gene segments; there are multiple IghC exons encoding different Ig H chain isotypes], Ig kappa locus (IgK, middle) [including V (IgkV), J (IgkJ), and C (IgkC) gene segments], and Ig lambda locus (bottom) [including V (IgIV), J (IglJ), and C (IgIC) gene segments]. Also shown are (1) the PAIR element, which is a key cis-regulatory sequence for Igh looping, to ensure utilization of distal VH gene segments in VDJ rearrangement, (2) Adam6a male fertility gene, (3) Intergenic Control Region 1 (IGCR1), which contains sites that regulate ordered, lineage-specific rearrangement of the Igh locus, (4) Eμ, IEK, and Eλ2-4, heavy chain, kappa, and lambda light chain intronic enhancers, (5) 3’EK, Eλ, and Eλ3-1, kappa and lambda light chain 3’ enhancers, (6) Sμ, the μ switch region, and (7) 3’ regulatory region (3’RR), a cis-acting element that controls isotype switching.

[0197] Figure 2 : Targeting vector for introducing mouse Ighg1 ΔCH1 gene cassette into mouse endogenous Igh locus upstream of Ighm to generate HCO Abs by homologous recombination. In this figure and Figure 3, the “mouse endogenous Igh locus” is in an ES cell that contains part of the human IGH locus, as described in Wabl and Killeen in US20130219535A1.

[0198] Figure 3: (A) depicts the mouse endogenous Igh locus targeted with the vector shown in Figure 2 to encode a pure heavy chain (HCO) antibody (ΔCH1 HCO IgG1). A C H 1 exon-deleted mouse Cγ1 gene was inserted between Eμ and Ighm of the mouse Igh locus, and a stop codon (TGA, indicated by an asterisk) was inserted into the C H 1, C H 2, and C H3 Each exon of the 3 domain. In wild-type mice, productive VDJ H Following recombination, a light chain (LC)-independent mu HC is produced. In the mutant Igh locus described herein, however, a LC-independent AC H 1 gamma 1 HC. Both transmembrane (M) and secreted (S) forms of the IgG1 HCO antibody are encoded by this modified Igh locus. The gamma 1 m transcript is produced by alternative splicing, including M1 and M2 exons, and the gamma 1 s includes the S exon located 3' to the C H 3 Exon 3' to the S exon. C H 1 Deletion allows HC trafficking to the plasma membrane (gamma 1 m) or secretion (gamma 1 s) without having to associate with surrogate or conventional (kappa or lambda) LC. When VDJ H The three stop codons in C mu prevent expression of LC-dependent mu HC when the Exon is spliced directly to C mu instead of C gamma 1 AC. (B) is a schematic of the transmembrane form of the IgG1 HCO antibody, which includes variable (V H ), C H 2, C H 3 and M domains, but lacks the C H 1 domain. The HC exists as a dimer, bound together by non-covalent interactions and inter-chain disulfide bonds (indicated by the horizontal lines between the V H and C H 2 domains), and inserted into the plasma membrane by its M domain. The secreted form of the IgG1 HCO antibody (not shown) lacks the M domain, instead having a short secreted (S) domain at the COOH end of the protein. It is expressed after B cell activation and differentiation into a plasmablast / plasma cell.

[0199] Figure 4 : B cell development in bone marrow of TRN11 / 2 / 5 and TRN34 / 29 / 30 mice is depicted. In the TRN11 / 2 / 5 mice, the endogenous V H , V κ , and V λ loci have been replaced by human V H , V κ , and V λ loci sequences flanked by mouse regulatory sequences, respectively. In the TRN34 / 29 / 30 mice, the V H , D, and J H gene segments are identical to those in the TRN11, but the remainder of the Igh locus has been modified, as shown in Figure 3, to encode an IgG1 HCO antibody. V κ , and V λLoci are inactivated in the TRN29 and TRN30 alleles, respectively. Bone marrow cells were stained with fluorescent conjugated monoclonal antibodies (mAbs) specific for the indicated CD antigens and analyzed by flow cytometry. Numbers in the flow diagrams represent the percentage of cells in a given gate. Developmental stages of B cells are also shown. Recirculating B (recirc. B), B cells are produced in the bone marrow and complete maturation in the periphery (e.g. spleen) and recirculate back to the bone marrow (BM) via the bloodstream.

[0200] Figure 5 B cell differentiation in the spleen of TRN11 / 2 / 5 and TRN34 / 29 / 30 mice. Spleen cells were stained with fluorescent conjugated mAbs specific for the indicated CD antigens and analyzed by flow cytometry. Numbers in the flow diagrams represent the percentage of cells in a given gate. Developmental stages of B cells are also shown. Fo. B, follicular B cells; MZ B, marginal zone B cells; T, transitional; Mat, mature.

[0201] Figure 6 Surface IgGl expression on marginal zone and follicular B cells of TRN11 / 2 / 5 and TRN34 / 29 / 30 mice. Cell surface expression of λ1 HC was analyzed on spleen cells gated on MZ (upper) and Fo. (lower) B cells.

[0202] Figure 7 B cell differentiation and surface IgGl expression in the lymph nodes of TRN11 / 2 / 5 and TRN34 / 29 / 30 mice. Lymph node cells were stained with fluorescent conjugated mAbs specific for the indicated CD antigens and λ1 HC and analyzed by flow cytometry. Numbers in the flow diagrams represent the percentage of cells in a given gate.

[0203] Figure 8 B cell differentiation and surface IgGl expression in the peritoneal cavity of TRN11 / 2 / 5 and TRN34 / 29 / 30 mice. Peritoneal cavity cells were stained with fluorescent conjugated mAbs specific for the indicated CD antigens and λ1 HC and analyzed by flow cytometry. Numbers in the flow diagrams represent the percentage of cells in a given gate.

[0204] Figure 9 ELISA assay to detect serum IgGl and IgM in unimmunized TRN11 / 2 / 5 (open circles) and TRN34 / 29 / 30 HCO (closed circles) mice. Optical density is shown on the Y axis and serum dilution on the X axis. For standardization (open boxes), monoclonal IgGl (left) and IgM (right) at 100 μg / ml were also serially diluted.

[0205] Figure 10ELISA assays to detect serum IgG2b, IgG2c and IgG3 in unimmunized TRN11 / 2 / 5 (open circles) and TRN34 / 29 / 30 HCO (closed circles) mice are depicted. The optical density is shown on the Y-axis and the serum dilution on the X-axis. For standardization (open boxes), monoclonal IgG2b (left), IgG2c (middle) and IgG3 (right) at 100 μg / ml were also serially diluted.

[0206] Figure 11 Nucleotide sequences referred to herein. DETAILED DESCRIPTION

[0207] Unless otherwise indicated or defined, all terms used herein have their ordinary meaning as is clear to one of skill in the art. Reference is made, for example, to standard manuals such as Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990) and Janeway et al., "Immunobiology" (5th Ed., or newer), Garland Science, New York, 2001.

[0208] The terms "comprise", "contain", "have" and "include" as used herein can be used as synonyms, and should be understood as open definitions, allowing more members or components or elements. "Consisting" is considered the most closed definition, without other elements not defining the characteristics of the definition. Thus, "comprising" is broader, encompassing the definition of "consisting".

[0209] The term "about" as used herein refers to the same value or a value that differs from the given value by + / - 5%.

[0210] The present inventors have overcome the limitations of the prior art and have shown that transgenic mice can be generated to efficiently produce pure heavy chain antibodies (HCAb) by 5' targeted integration of a transgenic C gamma gene fragment into the immunoglobulin heavy chain locus from an endogenous C mu gene fragment, which are expressed by B cells and secreted by plasma cells. These can then, for example, using established hybridoma technology, be used to generate a reliable supply of HCAb or antigen binding fragments thereof.

[0211] The antibody constructs referred to herein as HCAb and the libraries described herein and provided herein are artificial constructs. It is understood that the materials, methods and uses of the application, e.g., specifically the isolated nucleic acid sequences, amino acid sequences, expression constructs, transformed host cells, transgenic animals and recombinant antibodies, are "artificial" or synthetic and thus are not considered to be the result of "natural processes" or "natural laws".

[0212] The term "antibody" as used herein refers to a polypeptide or protein consisting of or comprising various combinations or arrangements of antibody domains, which are understood to be constant and / or variable domains of immunoglobulin heavy and / or light chains. A polypeptide is understood to be an antibody domain if it comprises a beta barrel structure consisting of at least two beta strands of antibody domain structures connected by loop sequences. Antibody domains can be native structures or modified by mutagenesis or derivatization, e.g., to modify antigen binding properties or any other properties, e.g., stability or functional properties, e.g., binding to Fc receptors, Fc mu, Fc alpha / mu, Fc alpha, Fc epsilon and / or Fc gamma receptors (e.g., FcRn, Fc gamma RI, Fc gamma RIIB, Fc gamma RIII or Fc gamma RIV in mice) or binding to polymeric Ig receptor (plgR).

[0213] In the present context, the terms "antibody" and "immunoglobulin" are used interchangeably.

[0214] The term "antibody" as used herein shall in particular refer to antibody constructs comprising an antigen binding site, in particular an antigen binding site of a VH domain. Specific embodiments relate to antibodies comprising or consisting of a VH as a single variable antibody domain, e.g., in combination (or fusion) with one or more further variable and / or constant antibody domains, optionally with one or more linker sequences or hinge regions, e.g., heavy chain antibodies, consisting of one or two single chains, wherein each single chain comprises or consists of a variable heavy chain region (or VH) connected to a constant domain.

[0215] The specific antibodies referred to herein can be full-length antibodies or antigen-binding antibody fragments thereof, or any other antibody construct comprising or consisting of at least one variable heavy chain (VH) antibody domain or a single VH domain, e.g. an antibody comprising or consisting of one or more single domain antibodies, Fab, F(ab'), (Fab)2, scFv, Fd, Fv. Exemplary antibodies comprise or consist of any HCAb described further herein. The antibodies described herein can be of some immunoglobulin type. The specific antibodies comprise antibody constant domains, in particular heavy chain constant domains, which are of the IgG type (e.g. IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM type, or their murine counterparts, IgG1, IgG2a / c, IgG2b, IgG3, IgA, IgD, IgE or IgM. Preferably, the antibodies described herein comprise constant antibody domains of the IgG type (e.g. IgG1, IgG2, IgG3 or IgG4 subtypes).

[0216] Accordingly, antibodies are typically understood to include proteins (or protein complexes) of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The 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 in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0217] In a typical IgG antibody structure, each of the HC or LC comprises at least two domains connected to each other to create a pair of binding site domains. In specific cases, a heavy chain can incorporate a LC constant domain, but is still considered a HC, e.g. lacking a light chain variable domain or light chain variable region.

[0218] The HC of an antibody can comprise a hinge region connecting one or both antigen binding arms of the antibody to the Fc portion. The hinge region can be a natural heavy chain hinge region of an immunoglobulin, e.g. IgG1 or IgG3, or an artificial hinge region comprising or consisting of a number of consecutive amino acids which is approximately the same length (+ / - 20%, or + / - 10%) as a natural hinge region. Preferred hinge regions comprise one or more, e.g. 2, 3 or 4 cysteine residues which can form disulfide bonds with another hinge region, thereby obtaining a dimeric construct.

[0219] The antibodies described herein can comprise one or more antibody domains which are shortened or extended, e.g. using a linker sequence or linker. Such linking is in particular by recombinant fusion or chemical linkage. The specific linkage can be by linking the C-terminus of one domain to the N-terminus of another domain, e.g. wherein one or more amino acid residues in the terminal regions are deleted to shorten the domain size or extended to increase the flexibility of the domain.

[0220] In particular, the shortened domain sequence comprises a deletion of the C-terminal and / or N-terminal region, e.g. a deletion of at least 1, 2, 3, 4 or 5, at most 6, 7, 8, 9 or 10 amino acids.

[0221] The domain extension by a linker can be by an amino acid sequence derived from the N- or C-terminal region of an immunoglobulin domain, which naturally is located in the vicinity of the domain, e.g. including the natural linkage between domains. Alternatively, the linker can contain an amino acid sequence derived from a hinge region. However, the linker can also be an artificial sequence, e.g. rich in or consisting of multiple Gly and Ser amino acids.

[0222] The term "antigen binding molecule" as used herein refers to any protein or protein complex (e.g. consisting of more than one polypeptide chain bound in complex form) which comprises or consists of an antibody comprising at least one antigen binding site of an antibody. Furthermore, the antigen binding molecule can also comprise one or more elements fused or complexed to the antibody portion, thereby providing an antibody derivative, e.g. a fusion protein.

[0223] The term "antigen binding site" or "binding site" refers to the portion of an antibody that is involved in antigen binding. In a natural antibody, the antigen binding site is formed by amino acid residues of the heavy ("H") and / or light ("L") chain N-terminal variable ("V") regions, or variable domains thereof. Within the V regions of the heavy (and optionally the light) chain are three hypervariable sequences referred to as "hypervariable regions" that are interposed between more conserved flanking sequences called framework regions. The antigen binding site provides a surface that is complementary to the three-dimensional surface of the bound epitope or antigen, the hypervariable regions being referred to as "complementarity determining regions" or "CDRs". The binding site incorporating the CDRs is also referred to herein as "CDR binding site".

[0224] In particular, the antigen binding site of the HCAb described herein is formed by amino acid residues of the heavy ("H") chain N-terminal variable ("V") region.

[0225] The term "CDR region" or corresponding sequence refers to the variable antigen binding region of a variable antibody domain, e.g. a VH domain, which comprises the variable structure capable of binding interaction with an antigen. An antibody domain having a CDR region can be used as such or integrated into a larger protein construct, thereby forming a specific region of such construct having binding function. Different structures can be derived from a natural repertoire of binding proteins, e.g. immunoglobulins, in particular antibodies or immunoglobulin-like molecules. Different structures can also be generated by randomization techniques, in particular those described herein. These include mutagenized CDR loop regions of antibody variable domains, in particular CDR loops of immunoglobulins.

[0226] Generally, an antibody having an antigen binding site of a specific CDR structure is capable of specifically binding to a target antigen, i.e. specifically recognizing such target antigen by the CDR loops of a pair of VH domains.

[0227] In an HC antibody, the antigen binding site is characterized by a specific CDR structure consisting of only VH-CDR1, VH-CDR2 and VH-CDR3 loops. If produced by an animal, e.g. a transgenic mouse as described herein, such antigen binding site is understood to be natural, or to have a natural structure and / or conformation. Although the antigen binding site can be artificially produced, e.g. because it is engineered by recombinant techniques synthesizing new structures, the incorporation of the corresponding gene encoding the respective antibody into a transgenic non-human animal leads to the production of a new synthetic antibody having a natural conformation.

[0228] Such natural conformation can be further affinity matured by any in vivo or in vitro affinity maturation technique, thereby leading to polyclonal and / or monoclonal antibodies comprising an artificial antigen binding site characterized by a natural conformation and further characterized by a high affinity for specifically binding to its target antigen.

[0229] The term "antibody" shall apply to antibodies of animal origin, e.g. mammals, including humans, mice, rabbits and rats, or birds, e.g. chickens, and shall in particular include recombinant antibodies based on sequences of animal origin, e.g. mouse sequences.

[0230] The term "antibody" further applies to fully human antibodies.

[0231] The term "fully human" used in connection with immunoglobulins is understood to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs. Human antibodies include antibodies isolated from a human immunoglobulin or antibody library or from a transgenic animal that is transgenic for human immunoglobulin genes.

[0232] The human immunoglobulin is preferably selected from or derived from the group consisting of IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4 and IgM.

[0233] The murine immunoglobulin is preferably selected from or derived from the group consisting of IgA, IgD, IgE, IgGl, IgG2A, IgG2B, IgG2C, IgG3 and IgM.

[0234] The term "antibody" further applies to chimeric antibodies, which have mixed sequences derived from different species, for example murine and human sequences.

[0235] In particular, the term "antibody" applies to antibodies produced by transgenic non-human animals, such as mice, which comprise human antigen binding regions and non-human, for example mouse, constant region or framework sequences.

[0236] The term "chimeric" in reference to an immunoglobulin or antibody refers to a molecule in which a portion of an antibody chain is homologous with the corresponding sequence in an immunoglobulin derived from a particular species or belonging to a particular class, while the remainder of that chain is homologous with a corresponding sequence in another species or class. Typically, the variable region mimics the variable region of an immunoglobulin derived from one mammal, while the constant region is homologous with an immunoglobulin sequence derived from another mammal. In one embodiment, the variable region can be derived from a currently known source, using readily available B cells from a human host organism, in combination with a constant region derived from, for example, a non-human cell preparation. In particular, chimeric antibodies or antibody fragments can be produced by transgenic mice, such as the transgenic mice described herein, which comprise human variable region encoding sequences (or corresponding variable antibody domains) and mouse constant region encoding sequences (or corresponding constant region antibody domains).

[0237] The term "antibody" also applies to monoclonal antibodies, in particular recombinant antibodies, which term includes all types of antibodies and antibody structures produced, expressed, created or isolated by recombinant means, such as antibodies derived from animals, such as mammals including humans, which comprise genes or sequences of different origin, such as chimeric antibodies, humanized antibodies or hybridoma-derived antibodies. Further examples relate to antibodies isolated from host cells transformed to express the antibodies, or antibodies isolated from recombinant combinatorial libraries of antibody or antibody domains, or antibodies prepared, expressed, created or isolated by any other means involving splicing of antibody gene sequences with other DNA sequences.

[0238] The term "antibody" is understood to include functionally active variants of new or existing (herein referred to as "parental") molecules, e.g., naturally occurring immunoglobulins. It is also understood that the term includes antibody variants, and should also include derivatives of these molecules. A derivative is any combination of one or more antibodies and / or fusion proteins, wherein any domain of an antibody, e.g., an antibody domain comprising a VH domain of an antigen binding site, or a VH domain, can be fused at any position to one or more other proteins, e.g., to other antibodies, such as to binding structures comprising CDR loops, receptor polypeptides, but also to other ligands, enzymes, toxins, etc. The antibodies described herein can be used in particular as isolated polypeptides or as combination molecules, e.g., by recombinant, fusion or conjugation techniques with other peptides or polypeptides.

[0239] Derivatives of antibodies can also be obtained by association or binding to other substances by various chemical techniques, e.g., covalent linkage, electrostatic interactions, disulfide bonds, etc. The other substances bound to the antibodies can be lipids, carbohydrates, nucleic acids, organic and inorganic molecules or any combination thereof (e.g., PEG, prodrugs or drugs). Derivatives can also comprise antibodies made of the same amino acid sequence but entirely or partially made of non-natural or chemically modified amino acids. In a particular embodiment, the antibodies are derivatives comprising additional tags allowing specific interaction with biologically acceptable compounds. There is no particular limitation as to the tags that can be used in the present application, provided that it has no negative or acceptable negative impact on the binding of the immunoglobulin to its target. Examples of suitable tags include His-tags, Myc-tags, FLAG-tags, Strep-tags, Calmodulin-tags, GST-tags, MBP-tags and S-tags. In another particular embodiment, the antibodies are derivatives comprising labels. The term "label" as used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody, thereby generating a "labeled" antibody. The label can itself be detectable, e.g., radioisotope labels or fluorescent labels, or, in the case of an enzyme label, it can catalyze chemical changes to a detectable substrate compound.

[0240] Derivatives of antibodies are, e.g., mutants or variants derived from the parental antibody or antibody sequences, e.g., parental antigen binding (e.g., CDR) sequences or framework (FR) sequences, such as obtained by, e.g., computational or recombinant engineering or chemical derivatization or synthesis.

[0241] The term "variant" shall include in particular functionally active variants. Functional variants of the antibodies described herein are in particular functional in terms of antigen binding specificity.

[0242] The term "variant" shall in particular refer to an antibody, e.g. a mutant antibody or antibody fragment, such as obtained by mutagenesis methods, in particular by deletion, exchange, introduction of insertions or deletions in a particular antibody amino acid sequence or region, or chemical derivatization of an amino acid sequence, such as in the constant domains to improve antibody stability, to enhance effector function or half-life, or in the variable domains to modulate antigen binding properties, such as by affinity maturation techniques available in the art. Any known mutagenesis method can be used, including point mutations at desired positions, e.g. obtained by randomization techniques, or domain deletions for HCAb engineering. In some cases, positions are randomly selected, e.g. to randomize an antibody sequence with any possible amino acid or to select preferred amino acids. The term "mutagenesis" refers to any art-recognized technique to alter a polynucleotide or polypeptide sequence. Preferred types of mutagenesis include error-prone PCR mutagenesis, saturation mutagenesis or other site-directed mutagenesis.

[0243] Functional activity of an antibody in terms of antigen binding is typically determined with an ELISA assay, a BIAcore assay, an Octet BLI assay or a flow cytometry-based assay when the antigen is expressed on the cell surface or intracellularly or on a microbead (e.g. Luminex).

[0244] Functional active variants can be obtained, e.g. by altering the sequence of a parent antibody, e.g. a monoclonal antibody having a particular native structure of an immunoglobulin such as an IgGl structure, to obtain a variant having the same specificity in recognizing a target antigen but having a structure different from the parent structure, e.g. modifying any antibody domain to introduce specific mutations or to generate fragments of the parent molecule.

[0245] Particular functional active variants comprise one or more functional active CDR variants or parent antibodies, each variant comprising at least one point mutation in the parent CDR sequence and comprising or consisting of an amino acid sequence having at least 60% sequence identity, preferably at least 70%, at least 80%, at least 90% sequence identity to the parent CDR sequence.

[0246] Particular variants are, e.g. functional active variants of a parent antibody, wherein the parent CDR sequences are incorporated into a human framework sequence, wherein optionally 1, 2, 3 or 4 amino acid residues of each parent CDR sequence can be further mutated by introducing point mutations to improve stability, specificity and affinity of the parent or humanized antibody.

[0247] In particular, the antibody can comprise a functional active CDR variant of any CDR sequence of a parent antibody, wherein the functional active CDR variant comprises at least one of:

[0248] a) 1, 2 or 3 point mutations in the parent CDR sequence, preferably wherein the number of point mutations in each CDR sequence is 0, 1, 2 or 3; and / or

[0249] b) 1 or 2 point mutations in any of the four C-terminal or four N-terminal or four central amino acid positions of the parent CDR sequence; and / or

[0250] c) at least 60% sequence identity to the parent CDR sequence;

[0251] Preferably, the functionally active variant antibody comprises at least one functionally active CDR variant as described herein. In particular, the functionally active variant antibody comprising one or more functionally active CDR variants has the specificity of binding to the same epitope as the parent antibody.

[0252] According to a particular aspect, the point mutation is any amino acid substitution, deletion and / or insertion of one or more amino acids.

[0253] The "percent (%) amino acid sequence identity" with respect to an antibody sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a specified polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity, according to methods well known in the art such as CLUSTALW (Chenna et al., Nucleic Acids Res., 31 :3497, 2003). A person skilled in the art can determine suitable parameters for the alignment, including any algorithm needed to achieve maximum alignment over the full length of the sequences being compared.

[0254] An antibody variant is to be specifically understood to include homologues, analogues, fragments, modifications or variants having a specific glycosylation pattern, e.g. resulting from glyco-engineering, which are functional and can act as functional equivalents, e.g. bind to a specific target and have different functional properties. The antibody can be glycosylated or non-glycosylated. For example, a recombinant antibody as described herein can be expressed in a suitable mammalian cell to allow specific glycosylation of the molecule as determined by the host cell expressing the antibody, or in a prokaryotic cell lacking glycosylation machinery, resulting in an unglycosylated protein.

[0255] The term "beta strand" or "beta strand" of an antibody domain, in particular a constant antibody domain, is understood herein as follows. An antibody domain typically consists of at least two beta strands that are connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted sheet of folding. A beta strand is a single contiguous amino acid sequence typically having a length of 3 to 10 amino acids, adopting this extended conformation and forming hydrogen bonds in the backbone with at least one other strand, such that they form a beta sheet. In a beta sheet, most beta strands are arranged next to each other with the other strands, and form a broad network of hydrogen bonds with the adjacent strands, in which the N-H group in the backbone of one strand makes a hydrogen bond with the C=0 group in the backbone of the adjacent strand.

[0256] The structure of an antibody constant domain (e.g. CL (Ck, C ), CHI, CH2 or CH3 domain) is similar to that of a variable domain, consisting of beta-strands connected by loops, some of which contain short alpha-helical stretches. As can be seen from the x-ray crystallographic B-factors of various Fc crystal structures, the framework is mostly rigid, while the loops are relatively more flexible. An antibody constant domain typically has seven beta strands (A-B-C-D-E-F-G) forming a beta sheet, with the beta strands being connected by loops, three of which are located at the N-terminus of the domain (A-B, C-D, E-F), and the other three of which are located at the N-terminus of the domain (B-C, D-E, F-G). The "loop regions" of a domain refer to the portions of the protein located between the beta strand regions (e.g. each CH3 domain contains 7 beta sheets, A to G, oriented from N- to C-terminus).

[0257] In certain embodiments, an antibody domain can comprise a wild-type amino acid sequence, e.g. a sequence derived from an animal (including a human), or an artificial sequence comprising mutations, e.g. one or more beta strands can be replaced at least in part by a heterologous sequence, e.g. comprising mutations that facilitate pairing with another domain, such as an interdomain disulfide bond, such as a beta-sheet region linking two antibody domains, "knob" and / or "hole" mutations or strand exchange.

[0258] The specific domain mutations can include the incorporation of new (additional) amino acid residues, e.g. Cys residues, which are capable of forming additional interdomain or interchain disulfide bonds, thereby

[0259] a) stabilizing the antibody domain by additional intra-domain disulfide bonds, and / or

[0260] c) stabilizing the two chains of the antibody domain by additional interchain disulfide bonds.

[0261] Disulfide bonds are typically formed by oxidation of the thiol groups of two cysteines or other thiol-forming amino acids, or of thiol groups of amino acid analogs, forming artificial disulfide bonds by linking the S atoms of the amino acid side chains. Specifically, a cysteine can be inserted (as an additional amino acid or amino acid substitution) between a pair of domains, which guarantees an additional cysteine modification, resulting in a stable domain pair by disulfide bond formation.

[0262] Antibodies can be generated by first screening for antigen binding sites that are formed by folding CDR sequences in each binding site of an antibody library to select specific binders. Next, the selected library members can serve as a source of CDR sequences (or parent CDR sequences, which can be further modified to modulate antigen binding or even phenotypic properties) that can be used to engineer any type of antibody construct, e.g., full-length immunoglobulins or antigen-binding fragments thereof.

[0263] An antibody library (e.g., a library comprising specific antibody constructs recognizing the same target antigen, or an initial library of antibodies produced by a certain animal or breed, e.g., a transgenic mouse as described herein, which library comprises a library of antibodies recognizing different target antigens) refers to a set or collection of antibodies (e.g., HCAbs as described herein), each antibody suitably displayed in a selected display system or vessel.

[0264] A particular display system links a given protein (e.g., an antibody, such as an HCAb as described herein) to its encoding nucleic acid (e.g., its encoding mRNA, cDNA, or gene). Thus, each member of a library comprises nucleic acid encoding the antibody displayed thereon. Display systems include, but are not limited to, cells, viruses (e.g., bacteriophage), ribosomes, eukaryotic cells (e.g., yeast), DNA (including plasmids), and mRNA.

[0265] Any antibody gene diversity library can be used for this purpose, e.g., including a large number of individual library members to generate diversity in antibody sequences, or using preselected libraries, which, e.g., are enriched for stable or functionally active library members. For example, the display system can be enriched for library members that bind a certain target.

[0266] Libraries can be constructed by well-known techniques, including, for example, chain-shuffling methods. For heavy chain shuffling, antibodies are cloned into vectors containing, for example, a human VHand library to generate phage antibody library transformants. Further methods include site-directed mutagenesis of antibody CDRs, or CDR randomization, in which some or all CDRs are randomized, either completely using mutagenic oligonucleotides containing NNK codons to target residues, or partially using parsimonious mutagenesis, in which oligonucleotides encoding the position of the targeted amino acid residue contain a mixture of nucleotide bases biased toward the original. Alternatively, libraries can be constructed using error-prone PCR, applying dNTP analogs, error-prone polymerases, or adding Mn 2+ ions.

[0267] Various techniques can be used to manufacture the genes encoding the human antibody library construction design. It is possible to produce DNA by a completely synthetic method, in which the sequence is divided into overlapping fragments, which are then made into synthetic oligonucleotides. These oligonucleotides are mixed together and annealed to one another by first heating to about 100°C and then slowly cooling to ambient temperature. After the annealing step, the synthetic assembled genes can be cloned directly or amplified by PCR before cloning. This is particularly desirable when large single-pot human libraries are required and large resources are available for the construction process.

[0268] Particular methods use phage, phagemid, and / or yeast libraries for direct binder selection and internalizing phage antibody selection. Other methods for site-directed mutagenesis can be used to generate library insertions, such as the Kunkel method (Kunkel, Proc. Natl. Acad. Sci. U S A., 82:488, 1985) or the Dpnl method [Weiner, et al., Gene 151:119, 1994].

[0269] An "antigen-specific library" refers to a library of polynucleotides (or polypeptides encoded by such polynucleotides) that has been used to interrogate whether antibodies specific for a particular antigen exist. Such a library can be limited to or biased toward or enriched for antibody sequences specific for any antigen group or a particular antigen. An exemplary antigen-specific library is an antibody "optimized library" (e.g., a "mature library" or "affinity matured library").

[0270] The specific embodiments described herein are based on mice that comprise or express a preimmune library prior to immunization with an antigen. The preimmune library can be a naive library that has similar sequence diversity to natural antibody sequences prior to the natural sequences undergoing antigen selection. The preimmune library can be designed and prepared to reflect or mimic a preimmune repertoire, and / or can be designed and prepared based on rational design provided by large databases of V, D, and J genes and other heavy chain sequences (e.g., known germline sequences). In certain embodiments of the application, expression cassettes representing the possible V, D, and J diversity, as well as joining diversity (i.e., N1 and N2) found within the human or non-human repertoire are recombined into single- or double-stranded DNA oligonucleotides.

[0271] An "optimized library" or "mature library" refers to a library designed to enhance or improve at least one characteristic of an antibody sequence identified when a library (e.g., a naive library or a preimmune library) is interrogated to determine whether an antibody sequence specific for an antigen is present. Such a mature 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 from or identified in an interrogation of a naive library designed to be further mutagenized in vitro or in vivo to generate a library with diversity introduced in the context of a naive (parental) antibody.

[0272] As a distinct example of array technology, B cell clones can be used to generate genes encoding antibody constructs described herein in artificial or computer addressable locations in a B cell array. The array can be manipulated using robotic or manual methods to rearrange cells that express only specific types of antibodies and / or specifically recognize particular targets.

[0273] In certain embodiments, B cell clones, e.g., from a suitably immunized non-human transgenic animal (such as a mouse described herein) that are genetically engineered to produce antibodies, or mammalian cell expression libraries, or large numbers of stably transformed mammalian cells generated by standard methods and robotic tools of antibody and protein engineering are used. Individual clones are maintained alive in addressable wells arranged on plates in suitable incubators and / or under long-term storage conditions, e.g., this can include freezing cell suspensions in liquid nitrogen, storage at -135°C, or storage under other acceptable conditions that allow recovery of the stored cell lines.

[0274] The term "repertoire" as used herein with respect to antibodies refers to a collection of variants, e.g., variants characterized by diversity in target epitope or antigen specificity. Typically, the structure of the antibody (also referred to as the "scaffold") is the same within such a repertoire, but with a variety of different CDR sequences.

[0275] As is well known in the art, a variety of display and selection techniques are available for identifying and isolating proteins having certain binding characteristics and affinities, including, for example, display techniques, such as cellular and non-cellular methods, particularly mobile display systems. In cellular systems, phage display, viral display, yeast or other eukaryotic cell display, such as mammalian or insect cell display, can be used. Mobile systems involve display systems in soluble form, such as in vitro display systems, including ribosome display, mRNA display, or nucleic acid display.

[0276] Library members displaying antigen binding structures capable of binding to a target can be screened from the library by any suitable method. The screening step can comprise one or several rounds of screening.

[0277] Any screening method suitable for identifying antibodies capable of binding to a target antigen can be used. In particular, a round of screening can comprise incubating the library in the presence of said target in order to select antibodies binding to said antigen or epitope thereof.

[0278] Once an antibody having the desired structure has been identified, this antibody can be produced by methods well known in the art, including, for example, hybridoma technology or recombinant DNA technology.

[0279] 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 can be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells.

[0280] The production of monoclonal antibodies directed against the antigen in the culture medium in which hybridoma cells are grown is determined. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by flow cytometry, immunoprecipitation, or by in vitro binding assays, such as enzyme-linked immunosorbent assay, ELISA.

[0281] According to another particular embodiment, recombinant monoclonal antibodies can be produced by isolating DNA encoding the desired antibody chains and transfecting a recombinant host cell with the coding sequences for expression using well-known recombinant expression vectors, such as plasmids or expression cassettes comprising nucleotide sequences encoding the antibody sequences described herein. The recombinant host cell can be prokaryotic and eukaryotic cells.

[0282] According to a specific aspect, the nucleotide sequences encoding can be used in genetic manipulation 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 that of a human. Genetic manipulation of the antibody sequence can be desirable to achieve greater affinity for the target antigen. It will be apparent to those skilled in the art that one or more amino acid changes can be made to the antibody and still maintain its ability to bind to the target (epitope or antigen).

[0283] The production of antibody molecules by various means is well known. Various techniques associated with antibody production are described, for example, in Harlow, et al., Antibodies: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2014).

[0284] Monoclonal antibodies can be produced, for example, using any method that produces antibody molecules by the use of a continuous cell line in culture. Examples of suitable methods for preparing monoclonal antibodies include the hybridoma method (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, in Monoclonal Antibody Production Techniques and Applications, LB Schook, ed., (Marcel Dekker, Inc., New York), pp. 51-63].

[0285] The term "B cell" refers to a class of lymphocytes that play an important role in the humoral immune response (as opposed to the cell-mediated immune response, which is controlled by T cells). The main function of B cells is to produce antibodies against antigens, in particular to express cell surface antibodies (forming BCR complexes) specific to a particular antigen, to function as antigen presenting cells (APCs), and ultimately to develop into antibody-secreting plasma cells and memory B cells upon activation by antigen interaction. B cells are an important component of the adaptive immune system. Specifically, when exposed to an antigen, B cells are activated and differentiate into immunoglobulin-expressing B cell variants, such as plasma cells, also known as plasmacytes, and memory cells.

[0286] The term "repertoire" as used herein with respect to B cells, also referred to herein as "B cell repertoire", refers to a collection of variants, such as B cell variants characterized by expression of immunoglobulins comprising a plurality of target epitope or antigen specificities.

[0287] The term "target" as used herein refers to an epitope or an antigen.

[0288] The term "antigen" as used herein shall particularly include all antigens and target molecules which are recognized by antibody binding sites (at least one antigenic determinant) due to the exposure of the antigen to the immune system of an animal or to an antibody library. In particular, preferred antigens targeted by the antibodies described herein are those molecules which have proven or can be able to be immunologically or therapeutically relevant, in particular those molecules which have been tested for clinical efficacy.

[0289] The term "antigen" is used to describe the complete target molecule or fragments of this molecule, in particular substructures, such as polypeptide or carbohydrate structures of the target. Such substructures are often referred to as "epitopes" (e.g. B cell epitopes, T cell epitopes), which can be immunologically relevant, i.e. can also be recognized by natural or monoclonal antibodies.

[0290] The term "epitope" as used herein refers in particular to a molecular structure present at the interface between an antigen and a specific antibody, wherein the antibody surface which interacts with the epitope is referred to as "paratope". Chemically, an epitope can be composed of a carbohydrate sequence or structure, a peptide sequence or a set of sequences in a discontinuous epitope, a fatty acid or an oligo- or polynucleotide. When the antigenic molecule is an organic, biochemical or inorganic substance, it is referred to as "hapten". An epitope or hapten can be composed of derivatives or any combination of the above. If the epitope is a polypeptide, it usually comprises at least 3 amino acids, preferably 8-50 amino acids, more preferably about 10-20 amino acids in the peptide. An epitope can be a linear or a discontinuous epitope. A linear epitope is composed of a single fragment of the primary sequence of a polypeptide or carbohydrate chain. Linear epitopes can be contiguous or overlapping. Discontinuous epitopes are composed of amino acids or carbohydrates which are brought together by folding the polypeptide to form a tertiary structure, and the amino acids are not necessarily adjacent to each other in the linear sequence. In particular, an epitope is at least a part of a diagnostically relevant molecule, i.e. the presence or absence of the epitope in a sample is correlated qualitatively or quantitatively with the health status of a patient or the state of a manufacturing process or the state of the environment and food. An epitope can also be at least a part of a therapeutically relevant molecule, i.e. a molecule which can be targeted by a specific binding domain which alters the disease progression.

[0291] The term "specific" or "bind specifically" as used herein refers to a binding reaction that is determinative of a cognate ligand of interest in a heterogeneous population of molecules. Thus, under designated conditions (e.g., immunoassay conditions), an antibody binds to a particular target and does not bind in a significant amount to other molecules present in the sample. Specific binding means selective binding in terms of target identity, high, medium or low binding affinity or avidity as chosen to be consistent. Selective binding can generally be achieved if the binding constant or binding kinetics differ by at least 10-fold, preferably by at least 100-fold, more preferably by at least 1000-fold, from competing targets in the sample.

[0292] Specific binding does not exclude cross-reactivity with similar antigens or the same antigen (analog) of a different species. For example, the binding entity can also preferably cross-react with rodent or primate targets analogous to the human target to facilitate preclinical animal studies.

[0293] The term "locus" as used herein refers to a DNA coding sequence or a DNA fragment encoding an expression product, i.e. a genomic sequence, e.g. a portion of the genome of a host organism, or a portion of a vector, such as integrated at a target site, e.g. a defined restriction site or a homologous region.

[0294] Restriction sites can be designed to ensure that the expression cassette is inserted into the correct reading frame. Typically, the foreign (also referred to herein as exogenous) DNA is inserted into one or more restriction sites of the vector DNA, which is then carried by the vector with the transferable vector DNA into the host cell.

[0295] Typically, a locus comprises at least one gene or one or more gene fragments as described herein. The term "locus" does not imply that the gene is actively transcribed or remains intact. Genes that have been inactivated can be included.

[0296] The term "immunoglobulin heavy chain locus" as used herein relates 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 a mouse endogenous immunoglobulin heavy chain locus comprising at least one transgenic nucleic acid sequence, in particular a Cy 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 and expression control sequences, respectively, which control the expression of the respective VH, D and JH coding sequences in operable linkage. According to a specific embodiment, the VH, D and JH coding sequences are human, the expression control sequences of the VH heavy chain locus are murine, preferably endogenous expression control sequences, thus providing a chimeric V, D and J gene segment.

[0297] The complexity of the V gene segments can be increased by increasing the number of V gene segments present in the locus or by using different loci, each locus comprising different V gene segments.

[0298] 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, which are derived from any vertebrate species or chimeras as described herein.

[0299] 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 can be derived from any vertebrate species or can be chimeras as described herein.

[0300] 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 can be derived from any vertebrate species or can be chimeras as described herein.

[0301] The V gene segments must be capable of recombining with the D gene segments, the J gene segments at the DNA level to form a functional VDJ exon. After transcription and RNA splicing, the VDJ exon and the heavy chain constant (effector) region, which can comprise several exons, become adjacent in the mRNA transcript and are functional for translation; upon expression of the nucleic acid according to the application, a pure heavy chain antibody is produced.

[0302] Operationally, the heavy chain constant region is encoded by a naturally occurring or engineered gene segment that is capable of being expressed with the VDJ exon in a B cell.

[0303] Antibodies can be of different classes, termed isotypes or subclasses. In placental mammals, there are five antibody isotypes, termed IgA, IgD, IgE, IgG, and IgM. They are all named with the “Ig” prefix that stands for immunoglobulin (sometimes used interchangeably with antibody), and they differ in biological properties, functional location, and ability to handle different antigens. The different suffixes of the antibody isotypes indicate different types of heavy chains that the antibodies comprise, each heavy chain class is named in alphabetical order: a (alpha), g (gamma), d (delta), e (epsilon), and m (mu). This results in IgA, IgG, IgD, IgE, and IgM, respectively.

[0304] The heavy chain constant region of the immunoglobulin heavy chain loci described herein comprises at least a transgenic Cγ gene segment described herein, which is expressed without a functional CH1 domain, such that a pure heavy chain antibody of the IgG type can be produced. Each heavy chain constant region can further comprise one or more additional heavy chain constant region gene segments selected from the group consisting of a Cδ, Cμ, Cε, and Cα gene segment. The selection of heavy chain constant region gene segments depends on the preferred class or mixed class of antibody class desired.

[0305] For example, depending on the IgG1, IgG2, IgG3, and IgG4 isotypes present in the engineered Igh locus, the whole or partial expression of the transgenic Cγ gene segment comprising a CH1 domain, all or in part deleted, of the heterologous immunoglobulin heavy chain locus will optionally produce some or all of the IgG isotypes.

[0306] Alternatively, a selected mixture of antibodies can be obtained. For example, when the heavy chain constant region comprises a Cα and a Cμ gene segment, IgA and IgM can additionally be obtained.

[0307] In particular, the Cγ gene segment described herein comprises a deletion of all or part of the nucleotide sequence encoding the CH1 domain, for example a deletion of the CH1 exon. This deletion can be a partial deletion of the nucleotide sequence encoding the CH1 domain, as long as it deprives the CH1 domain of its function. Preferably, the partial deletion is such that the partner binding domain, in particular the BiP partner binding domain, is removed or inactivated.

[0308] Optionally, the heterologous heavy chain locus is Cμ-deficient or comprises an inactive Cμ gene segment. In particular, the Cμ gene segment is inactivated by a functional loss mutation, for example the introduction of a stop codon, or a deletion of at least a portion of the nucleotide sequence encoding the Cμ gene segment. In particular, the Cμ gene segment is inactivated by a deletion of the CH1, CH2, CH3, and / or CH4 domains.

[0309] In particular, the Cμ gene segment is inactivated by a deletion of at least any one of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the entire nucleotide sequence encoding the Cμ gene segment or of said sequence.

[0310] In particular, the Cμ gene segment comprises a functional loss mutation, a deletion, or an inactivation 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.

[0311] In one specific embodiment, for the derivation of HCAbs for human therapeutic applications, the VDJ coding sequences present in the loci will be derived from human germline sequences, optionally modified, and if the host animal is a rodent, such as a mouse, the constant regions can be of rodent origin. Selected pure heavy chain antibodies comprising a soluble human VH binding domain and a rodent constant effector region can then be cloned and the rodent effector region replaced with a selected human constant effector region, or the soluble VH domain used to derive alternative VH fusion proteins, VH domain antibody complexes, etc.

[0312] According to another specific embodiment, when the pure heavy chain antibodies are for veterinary or replacement purposes, the V, D, and J coding sequences are preferably from a vertebrate or mammal best suited for the intended purpose. For example, the V gene segments and D and J gene segments can be derived from other mammals (e.g., mice, rats, pigs, cows, goats, sheep, camels, horses, etc.), depending on the intended use, such as veterinary, industrial, agricultural, diagnostic, or reagent applications.

[0313] A "heavy chain constant region exon" ("CH exon") includes naturally occurring CH exon sequences of vertebrates, especially mammals. This varies by class. For example, IgG and IgA naturally lack a CH4 domain. The term "CH exon" also includes within its scope derivatives, homologs, and fragments thereof, as long as the CH exon is capable of forming a functional HCAb when it is a component of a heavy chain constant region, as defined herein.

[0314] In specific embodiments described herein, the endogenous kappa and lambda light chain loci of the transgenic mouse are rendered non-functional due to one or more modifications, such as a loss-of-function mutation, or deletion of the endogenous kappa and / or lambda light chain loci or portions thereof.

[0315] Exemplary suitable modifications are understood as follows. To inactivate the kappa chain locus, the entire 3.2 Mb genomic region between VK2-137 (the most distal VK gene segment of CK) and JK5 (the most proximal VK of CK) is deleted by a recombination enzyme-mediated cassette exchange (RMCE) strategy. This is accomplished by inserting appropriate targeting sequences upstream of VK2-137 and downstream of JK5, 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 segments (IgIV) is deleted by RMCE. In this case, appropriate targeting sequences are inserted upstream of IgIV2 and downstream of IgIV1, followed by in vitro Cre-mediated deletion of the intervening genomic region.

[0316] The loci can be engineered to express or comprise exons encoding antibodies, such as further described herein.

[0317] The recombinant locus can be created using various site-specific editing and / or recombination techniques. Preferably, the modified locus is generated by inserting a piece of DNA containing the gene segment (herein referred to as "donor DNA") into a modified version of a non-human animal immunoglobulin locus (e.g., a heavy chain locus of the host organism, herein referred to as the "acceptor allele"). The acceptor allele can comprise recognition sites for a site-specific DNA recombination enzyme, such as Cre recombinase (loxP sites and mutant versions of loxP sites). The donor DNA can be flanked by identical Cre recombinase recognition sites (e.g., a loxP site on one side and a mutant version of a loxP site on the other side, at the 5' end and the 3' end). Cre recombinase can be used to catalyze the insertion of the donor DNA into the acceptor allele.

[0318] In another embodiment, the gene segment is introduced into the immunoglobulin locus primarily, if not exclusively, by homologous recombination. In this embodiment, a targeting sequence or vector comprising genomic targeting homology arms flanking the nucleic acid sequence comprising the gene segment of the antibody-encoding gene is used. These genomic homology arms facilitate the insertion of the DNA into the immunoglobulin locus, such as DNA encoding an immunoglobulin heavy chain. A targeting sequence refers to a sequence that is homologous to a DNA sequence that flanks or is proximal to the region of the immunoglobulin gene locus to be modified in the genome of a cell. The flanking or proximal sequence can be within the locus itself, or upstream or downstream of the coding sequence in the host cell genome. The targeting sequence is inserted into a recombinant DNA vector used for transfection of the cell such that the sequence to be inserted into the genome of the cell, such as the sequence of a recombination site, is flanked by the targeting sequence of the vector.

[0319] In many cases where homologous recombination is used to effect a genetic alteration (e.g., an insertion or deletion) in the genome, further modifications will involve the use of engineered site-specific endonucleases to increase the likelihood of achieving the desired result. Such endonucleases are valuable because they can be designed to have high specificity for unique sequences in the target genome, and because they cause double-stranded DNA breaks at the recognized site. The double-stranded break promotes homologous recombination with a targeting vector that has targeting homology to the DNA immediately proximal to the break. Thus, the combination of a targeting vector and a site-specific endonuclease that cuts DNA within or near the targeting region of the vector typically produces a much higher homologous recombination efficiency than the use of the targeting vector alone. Furthermore, it is possible to promote the creation of a genomic deletion by using one or more site-specific endonucleases and a targeting vector comprising two targeting homology arms, one targeting one side of the region to be deleted and the other targeting the other side.

[0320] Site-specific recombination differs from general homologous recombination in that the short specific DNA sequences that the recombinases recognize are the only sites where recombination occurs. Site-specific recombination requires specialized recombinases to recognize the sites and catalyze recombination at these sites. A number of site-specific recombination systems of phage and yeast origin (each comprising a recombinase and specific homologous target sites) have been shown to function in the context of eukaryotic cell DNA integration and are thus suitable for use in the uses described herein. These include the phage Pl 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 art. The Recombinase Mediated Cassette Exchange (RMCE) procedure is facilitated by the use of wild-type and mutant loxP (or FRT, etc.) sites in combination with the appropriate recombinase (e.g. Cre or Flp), and negative and / or positive selection. When the sites used are identical to each other and / or there is no selection, RMCE will occur, but the efficiency of the process is reduced because excision is favored over insertion, and (without incorporation of positive selection) there is no enrichment of properly mutated cells.

[0321] Other systems of the tyrosine family such as phage lambda Int integrase, HK2022 integrase, and others belonging to the independent serine family of recombinases such as phage phiC31, R4 Tp901 integrase are known to function in mammalian cells with their respective recombination sites and are also suitable for use in the uses described herein.

[0322] The methods described herein specifically utilize site-specific recombination sites that utilize the same recombinase, but do not promote recombination between the sites. For example, a loxP site and a mutated loxP site can be integrated into the genome of a host, but introduction of Cre into the host will not result in recombination of the two sites; instead, the loxP site will recombine with another loxP site, and the mutated site will only recombine with another similarly mutated loxP site.

[0323] Two classes of variant recombinase sites can be used to facilitate Recombinase Mediated Cassette Exchange. One has a mutation in the 8 bp spacer of the site, and the other has a mutation in the 13 bp inverted repeat.

[0324] Spacer mutants such as loxl 11 (Hoess, et al., Nucleic Acids Res., 14:2287, 1986), loxl 5171 and lox 2272 (Lee and Saito, Gene, 216:55, 1998), m2, m3, m7 and ml l (Langer, et al., Nucleic Acids Res., 30:3067, 2002) readily recombine with themselves, but have significantly reduced recombination rates with wild-type sites. Examples of the use of such mutant sites for DNA insertion by recombinase-mediated cassette exchange can be found in Baer and Bode, Curr. Opin. Biotechnol., 12:473, 2001.

[0325] Reverse repeat mutants represent a second class of altered recombinase sites. For example, loxP sites can contain altered bases in the left-hand repeat (LE mutants) or the right-hand repeat (RE mutants). LE mutant lox71 has a 5 bp change from the wild-type sequence at the 5' end of the left-hand repeat to TACCG (Araki, Nucleic Acids Res., 25:868, 1997). Similarly, the five most 3' end bases of RE mutant lox66 are changed to CGGTA. Reverse repeat mutants can be used to integrate plasmid inserts into chromosomal DNA. For example, LE mutants can be used as "target" chromosomal loxP sites for recombination by "donor" RE mutants. After recombination, a DNA donor fragment containing the RE site is found inserted into the genome, flanked on one side by a double mutant site (containing both le and RE reverse repeat mutations) and on the other side by a wild-type site (Lee and Sadowski, Prog. Nucleic Acid Res. Mol. Biol., 80:1, 2005). The double mutant is sufficiently different from the wild-type site that it cannot be recognized by Cre recombinase, so the inserted fragment cannot be excised by Cre-mediated recombination between the two sites.

[0326] In certain aspects, a site-specific recombination site can be introduced into an intron or an intergenic region, rather than a coding nucleic acid region or a regulatory sequence. This can avoid inadvertently disrupting any regulatory sequences or coding regions necessary for proper expression of a gene when inserting a site-specific recombination site into the genome of an animal cell.

[0327] Introduction of site-specific recombination sites can be achieved by conventional homologous recombination techniques. These techniques are described in the references, for example, Sambrook and Russell (2001) Molecular cloning: a laboratory manual, 3d ed. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press) and Nagy, (2003) Manipulating the mouse embryo: a laboratory manual, 3d ed. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press).

[0328] Use of vectors designed for positive or negative selection known in the art can facilitate the specific recombination into the genome. To facilitate the identification of cells that have undergone the replacement reaction, a suitable genetic marker system can be used and cells selected, for example, using selective media. However, to ensure that the genomic sequence is essentially free of extraneous nucleic acid sequences at or near the two termini of the replacement interval, it is desirable that the marker system / gene can be removed after selection of cells containing the replacement nucleic acid.

[0329] The recombinase can be provided as a purified protein or can be expressed from a construct that is transiently expressed in a cell in order to provide the recombinase activity. Alternatively, the cell can be used to generate a transgenic animal which can be crossed with an animal expressing the recombinase to generate offspring that lack the marker gene and associated recombination sites.

[0330] In this document, the term "endogenous" with respect to a gene means that the gene is native to the cell, i.e., the gene is present at a particular locus in the unmodified cell genome. An endogenous gene can be a wild-type gene that is present at that locus in a wild-type cell (as found in nature). If the endogenous gene is present at the same locus in the genome as the wild-type gene, the endogenous gene can be a modified endogenous gene. An example of such a modified endogenous gene is a gene that comprises a deletion or an insertion of an exogenous nucleic acid in its sequence. An endogenous gene can be present in the nuclear genome, mitochondrial genome, etc. In particular, the immunoglobulin heavy chain loci described herein are mouse endogenous immunoglobulin loci comprising the modifications described herein.

[0331] The immunoglobulin heavy chain loci described herein comprise a transgenic Cγ gene segment upstream of an endogenous Cμ gene segment. In one particular embodiment, the endogenous Cμ gene segment is modified to be inactive, but the Cμ gene segment is present in the locus at the same location as in a wild-type mouse.

[0332] In another embodiment, gene segments are introduced into the immunoglobulin loci by CRISPR / Cas9 technology using a non-homologous end joining method, e.g., see He, et al., Nuc. Acids Res., 44: e85, 2016, rather than the homology directed repair that is typically performed with this system.

[0333] In the context of the present application, the term "heterologous" refers to a nucleotide sequence or locus described herein that is not endogenous to the mammal in which it is located, or is a modified endogenous locus by replacement or removal of an endogenous sequence.

[0334] In the present context, the term "transgene" refers to a nucleic acid element (e.g., a nucleic acid construct such as an immunoglobulin gene segment, or a non-coding or coding genetic sequence such as a gene or locus) that is not native to the cell (i.e., not naturally occurring in the same location within the cellular genome of a wild-type cell of the same species), or is foreign to the cell from which the recombinant cell is generated, 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 in a different locus or location than in a wild-type cell (and thus the same locus does not exist in nature). A transgenic gene segment can comprise an (modified or unmodified) endogenous coding sequence or gene if it is present in a different locus in the genome than in a wild-type gene or organism. An example of such a transgenic nucleic acid element is a modified endogenous element, e.g., a Cγ gene segment described herein that comprises a deletion or modification in the CHI domain, and that is integrated in the mouse endogenous immunoglobulin heavy chain locus upstream of the endogenous Cμ gene segment, and thus in a different location than its corresponding location in a wild-type cell in which the Cγ gene segment is located downstream of the Cμ gene segment in the immunoglobulin heavy chain locus.

[0335] The term "transgene" is used herein to describe genetic material that has been or will be artificially inserted into the genome of a cell, particularly a host animal cell. The term "transgene" as used herein refers to a nucleic acid molecule or element, e.g., contained in an expression construct and / or targeting vector, that can be introduced and incorporated into a host genome, e.g., a Cγ gene segment described herein that comprises a deletion in its CHI domain. Thus, the host organism (e.g., mouse) is engineered to be a "transgenic" host.

[0336] The term "recombinant" refers to polynucleotides or polypeptides that are not naturally occurring in a host cell. Recombinant or transgenic molecules can comprise two or more naturally occurring sequences that are linked together in a non-natural manner. Recombinant or transgenic cells comprise recombinant polynucleotides or polypeptides. A cell is "foreign" to a cell if it receives a transgenic or recombinant nucleic acid.

[0337] The term "recombinant" refers to polynucleotides or polypeptides that are not naturally occurring in a host cell. Recombinant or transgenic molecules can comprise two or more naturally occurring sequences that are linked together in a non-natural manner. Recombinant or transgenic cells comprise recombinant polynucleotides or polypeptides. A cell is "foreign" to a cell if it receives a transgenic or recombinant nucleic acid.

[0338] a) antibodies isolated from an animal (e.g., a non-human animal, such as a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom,

[0339] b) antibodies isolated from a host cell transformed with (e.g., from a transfectoma),

[0340] c) antibodies isolated from a recombinant, combinatorial antibody library, and

[0341] d) antibodies prepared, expressed, created or isolated by any other means, including, for example, shuffling of human immunoglobulin gene sequences with other DNA sequences. Such recombinant antibodies include antibodies that have been engineered to include, for example, rearrangements and mutations that occur during antibody maturation.

[0342] In certain aspects of the embodiments, the transgenic animals of the application further comprise a human immunoglobulin region. For example, a number of approaches have been developed to replace mouse endogenous immunoglobulin regions with human immunoglobulin sequences to produce partially or fully human antibodies for drug discovery purposes. Examples of such mice include those described in, e.g., 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.

[0343] As used herein, the term "chimeric immunoglobulin gene segment" is used to refer to an immunoglobulin gene segment comprising human immunoglobulin gene segment coding sequences, particularly human IGH(V H , D and J H coding sequences and murine expression control sequences. Mice comprising such chimeric gene segments have a genome comprising an introduced exogenous immunoglobulin region that is partially human, wherein the introduced region comprises human variable region coding sequences and murine non-coding regulatory sequences that control expression of the human sequences from the mouse or mouse endogenous genome and are knocked into the mouse genome by introduction of the chimeric immunoglobulin gene segment or chimeric immunoglobulin locus. In particular, the murine sequences, particularly the murine non-coding and regulatory sequences, are based on the corresponding murine endogenous sequences, i.e., the same sequences as in the endogenous wild-type immunoglobulin loci.

[0344] The murine expression control sequences described herein for expression of human immunoglobulin gene sequences are particularly selected from the group consisting of promoters, transcription initiation and termination sequences, enhancer and activator sequences or ribosomal binding sites. Particular examples of such expression control sequences are sequences flanking the coding sequences, which can include promoters, 5' untranslated sequences, introns that insert leader peptide coding sequences, recombination signal sequences (RSS) and splice sites.

[0345] In a particularly advantageous aspect, the transgenic mice described herein comprise chimeric immunoglobulin segments as described in U.S. Patent 2013 / 0219535 to Wabl and Killeen. The genome of such transgenic mice comprises an introduced partially human immunoglobulin region, wherein the introduced region comprises human variable region coding sequences and non-coding variable region sequences based on the mouse endogenous genome. In particular, the transgenic cells and mice of the invention have a genome in which part or all of the endogenous immunoglobulin region is removed.

[0346] In another advantageous aspect, the genome content of the mice described herein is modified such that their B cells are capable of expressing more than one functional VH domain per cell, i.e., the cells produce bispecific antibodies, as described in WO2017035252A1.

[0347] As used herein, "vector" is defined as a DNA sequence that is required for transcription of a cloned recombinant nucleotide sequence (i.e., a recombinant gene) and translation of its mRNA in a suitable host organism. Vectors include plasmids and viruses, as well as any DNA or RNA molecule, whether or not self-replicating, that can be used to transform, transduce, or transfect a cell. Vectors can include autonomously replicating nucleotide sequences as well as genome-integrating nucleotide sequences. Expression vectors can additionally comprise an origin of replication in the host cell or a genome integration site, one or more selectable markers (e.g., an amino acid synthesis gene or a gene conferring resistance to an antibiotic such as puromycin, Zeocin TM G418, or hygromycin), a plurality of restriction enzyme cleavage sites, a suitable promoter sequence, and a transcription terminator, which components are operably linked together.

[0348] One common type of vector is a "plasmid," which is typically a self- contained molecule of double-stranded DNA that can readily accept additional (exogenous) DNA and can be readily introduced into a suitable host cell. Plasmids often contain DNA encoding a promoter and have one or more restriction sites suitable for insertion of exogenous DNA. In particular, the term "plasmid" refers to a vector by which a DNA or RNA sequence (e.g., an exogenous gene) can be introduced into a host cell, thereby transforming the host and facilitating expression (e.g., transcription and translation) of the introduced sequence.

[0349] As used herein, the term "host cell" refers to the original subject cell that is transformed to produce a particular recombinant protein (e.g., an antibody as described herein) and any progeny of that cell. It is understood that not all progeny will be identical to the parental cell (due to environmental production of mutations or differences), however, such progeny are included in the term "host cell" so long as the progeny retain the functional characteristics of the original transformed cell. The term "host cell line" refers to a cell line of host cells used to express a recombinant gene to produce a recombinant polypeptide, e.g., 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 proliferate indefinitely. Such host cells or host cell lines can be maintained in and / or cultured to produce the recombinant polypeptide.

[0350] As used herein, the term "isolated" or "isolation" with respect to a nucleic acid, antibody, or other compound means a compound that has been sufficiently separated from its natural environment such that it exists in "substantially pure" form. "Isolated" does not necessarily imply that the artificial or synthetic mixture of other compounds or materials is excluded, or that the presence of impurities that do not interfere with the basic activity is excluded, and can contain, for example, impurities that can be present due to incomplete purification. In particular, an isolated nucleic acid molecule as described herein also includes a chemically synthesized nucleic acid molecule.

[0351] The term "isolated nucleic acid" is sometimes used with respect to the nucleic acids described herein. When applied to DNA, the term refers to a DNA molecule that is separated from sequences that naturally flank the sequence in the genome of the organism from which the DNA molecule is derived. For example, an "isolated nucleic acid" can comprise a DNA molecule that is 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" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term can refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in a cell or tissue). An "isolated nucleic acid" (DNA or RNA) can further denote a molecule that is produced directly by biological or synthetic means and is separated from other components present during its production.

[0352] With respect to polypeptides or proteins, such as isolated antibodies, the term "isolated" shall specifically mean a compound free or substantially free of material with which it is naturally associated, such as other compounds found in its natural environment or in the environment in which it is produced (when such production is by recombinant DNA technology carried out in vitro or in vivo, such as cell culture). An isolated compound can be formulated with a diluent or adjuvant and still be isolated for purposes of utility, such as when used in diagnosis or therapy, a polypeptide or polynucleotide can be mixed with a pharmaceutically acceptable carrier or excipient.

[0353] The antibodies described herein are provided in particular in isolated form, substantially free of other antibodies to different target antigens and / or comprising different structural arrangements of antibody domains. However, the isolated antibodies can be included in combination preparations, including combinations of the isolated antibodies with, for example, at least one other antibody (e.g., a monoclonal antibody or antibody fragment having a different specificity).

[0354] In particular, the antibodies described herein are provided in substantially pure form. The term "substantially pure" or "purified" as used herein refers to a preparation of a compound (e.g., a nucleic acid molecule or an antibody) comprising at least 50% (w / w), preferably at least any of 60%, 70%, 80%, 90%, or 95%. Purity is measured by methods appropriate for the compound, e.g., chromatography, polyacrylamide gel electrophoresis, HPLC analysis, etc.

[0355] "Site-specific recombination" refers to the process of recombination between two compatible recombination sites, including any of the following three cases:

[0356] a) deletion of a preselected nucleic acid flanked by recombination sites;

[0357] b) inversion of a preselected nucleic acid sequence flanked by recombination sites, and

[0358] c) the reciprocal exchange of nucleic acid regions located near the recombination sites on different nucleic acid molecules. It will be appreciated that if one or both of the nucleic acid molecules is circular, such reciprocal exchange of nucleic acid fragments results in an integration event.

[0359] The foregoing description will be more fully understood with reference to the following Examples. These Examples, however, are merely representative of methods of practicing one or more embodiments of the application and should not be construed as limiting the scope of the application.

[0360] Examples

[0361] Example 1: Introduction of mouse Ighgl ΔCH1 gene cassette into mouse endogenous Igh locus Ighm upstream using homologous recombination to generate pure heavy chain (HCO) Abs. Figure 2

[0362] Figure 2 and FIG. 3 illustrate an exemplary method of introducing an IghglACHI gene cassette to generate HCO Abs (or HC Abs).

[0363] Two essential components of the homologous recombination targeting vector Figure 2 are short homology arms (SHA) and long homology arms (LHA) that have sequence identity to homologous DNA fragments flanking the region of the endogenous locus being modified. In this case, the SHA consists of the human JH2-JH6 gene fragment flanked by the corresponding mouse Jh non-coding sequences (SEQ ID NO: 2). The LHA consists of the entire Ighm gene starting at the 5' intron and ending at the 3' intron (SEQ ID NO: 12). Other notable features of the targeting vector starting at 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) including a polyadenylation site (pA). This element is used in conjunction with ganciclovir for negative selection of cells that have integrated (but not by homologous recombination) the targeting vector. During homologous recombination, the non-homologous DNA 5' of the SHA (or 3' of the LHA) will be eliminated B cell development in HCO mouse bone marrow (BM).In this case, if the vector integrates via homologous recombination, the HSV-TK gene is deleted. Any cells that do not integrate the vector via homologous recombination will retain the HSV-TK gene and be killed. 2) T3 promoter of T3 phage RNA polymerase (SEQ ID NO:3). This DNA-dependent RNA polymerase is highly specific to the T3 phage promoter. The 99KD enzyme catalyzes in vitro RNA synthesis, which allows for rapid cloning of VDJ rearrangements from a small number of B cells or hybridomas. 3) CAG_PuroR_pA, a puromycin resistance gene driven by a strong CAG promoter, including a polyA site (SEQ ID NO:5). This element is used for positive screening of cells that have integrated a puromycin-based target vector. 4) Note that cells that have stably integrated the target vector into their genome via homologous recombination will be resistant to both ganciclovir and puromycin. 5) Note that the CAG_PuroR_pA element with flanking FRT sites (SEQ ID NO:4 and SEQ ID NO:6) can be used. After identifying the correctly targeted ES cell clone, this element can be removed in vitro or in vivo by providing Flp recombinase. The Eμ enhancer (SEQ ID NO:7) is contained upstream of the Ighg1ΔCH1 gene cassette to promote transcription at this locus. In the targeting vector, the following sequences are partial sequences of the Ighg1 5' intron (SEQ ID NO:8), partial sequences 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 a partial sequence of the Ighm 5' intron (SEQ ID NO:13) and the Ighm gene (SEQ ID NO:14), the Ighm gene including a 3' UTR followed by a partial sequence of the Ighm 3' intron (SEQ ID NO:15). Stop codons (TGAs) are introduced in the CH1, CH2, and CH3 Ighm exons to prevent VDJ from being affected. HExpression of LC-dependent μHC when the exon is directly spliced onto the Cμ rather than the Ighg1 ΔCH1 gene. The targeting vector lacks the μ switch (S) regions present in the endogenous Igh locus, so the targeted locus will also lack the S regions and therefore will be unable to undergo isotype switching. The targeting vector is introduced into ES cells by electroporation. Cells are grown in media supplemented with ganciclovir and puromycin. Successful gene targeting of surviving isolated ES cell clones is then monitored by genomic PCR using primers located within 5' and 3' of the newly introduced Ighg1 ΔCH1 gene cassette. Correct integration of the targeting cassette is further confirmed by genomic Southern blotting using a probe mapping to the flanking 5' of the SHA DNA sequence; a second probe mapping to the flanking 3' of the LHA DNA sequence; and a third probe mapping within the new DNA between the two arms of the vector genomic identity. (The structure of the correctly targeted locus is shown in Figure 3, hereafter referred to as the HCO locus.)

[0364] Nucleotypes of the ES cell clones verified by PCR and Southern blotting are analyzed using an in situ fluorescence hybridization method designed to distinguish the most common chromosomal aberrations in mouse ES cells. Clones with such aberrations are excluded from further use. ES cell clones judged to have the expected correct genomic structure based on PCR and Southern blotting data and also free of detectable chromosomal aberrations based on nucleotype analysis are selected for further use.

[0365] ES cell clones carrying the correctly targeted Ighg1 ΔCH1 gene cassette in the mouse heavy chain locus are microinjected into mouse blastocysts from the DBA / 2 strain to generate partially ES cell-derived chimeric mice according to standard procedures. Male chimeric mice with the highest level of ES cell-derived contribution to their coat are selected and mated with female mice. The female mice selected here are of the C57B1 / 6NTac strain, carrying a transgene encoding the Flp recombinase in their germline. Offspring resulting from these matings are analyzed for the presence of the Ighg1 ΔCH1 gene cassette and the deletion of the FRT-flanked puromycin resistance gene. (Figure 3) Correctly targeted mice, referred to as TRN0034 or TRN34, are used to establish mouse populations.

[0366] Example 2: Figure 4

[0367] In the prior art (see e.g. WO 2011 / 072204 Al), IgG-type HCAb is produced in mice that lack functional light chains and that have a functional Cμ gene segment upstream of a recombinant Cγ gene segment that lacks a CH1 domain. The production of HCAb in these mice has clear disadvantages. The mature B cells with a functional Cμ gene segment, with standard IgM as antigen BCR, will develop normally. However, during the immune response, the antibody class switches to (desired) γ chains, which cannot pair with L chains because it is missing. This will have two effects:

[0368] (i) cells with antibodies, where the specificity is defined by the combination of H+L chains, will no longer be stimulated and die, and

[0369] (ii) cells with specificity defined primarily by H chains can die because unpaired VH cannot survive structurally.

[0370] Thus, in this mouse of the prior art, VH selection is "backloaded", i.e. it occurs during the immune response.

[0371] By integrating a transgenic Cγ1 gene segment upstream of the Cμ gene segment in the endogenous immunoglobulin locus, VH selection is "frontloaded", i.e. it occurs during ontogeny.

[0372] To show that B cells develop normally in these mice, bone marrow cells were surface stained for CD antigen specific monoclonal antibodies and analyzed by flow cytometry, as shown in Figure 5 In control TRN11 / 2 / 5 mice, the endogenous V H , VK and Vλ loci were replaced by human V H , VK and Vλ locus coding sequences flanked by mouse regulatory sequences, as described in the co-pending application US 20130219535 Al by Wabl and Killeen. In TRN34 / 29 / 30 mice, the V H , D and J H gene segments are identical to TRN11, but the rest of the Igh locus has been modified to encode IgGl HCO antibodies, as shown in Figure 3. The VK and Vλ loci are inactivated in the TRN29 and TRN30 alleles, respectively. Numbers in the flow diagrams represent the percentage of cells in a given gate.

[0373] Genetic composition of TRN11 / 2 / 5 mice:

[0374] The endogenous V H , VK and Vλ loci were replaced by human V H• Vκ and Vλ locus encoding sequences flanked by mouse regulatory sequences;

[0375] Genetic composition of TRN34 / 29 / 30 mice:

[0376] HCO locus: Ighg1 ΔCH1 and stop codon (TGA) introduced into CH1, CH2 and CH3 Ighm exons,

[0377] Chimeric V H Locus: human V H • Vκ and Vλ locus encoding sequences flanked by mouse regulatory sequences replacing endogenous V H loci, and

[0378] Inactivated Vκ and Vλ loci.

[0379] The developmental stage of B cells is also shown. Mature recirculating B indicates B cells that were generated in the bone marrow and completed maturation in the periphery (e.g. in the spleen) and recirculate back to the bone marrow (BM) through the blood stream.

[0380] In both mouse strains, the frequency of early B lineage cells (B220+CD23-) is identical, however, in HCO mice the frequency of pro-B cells is increased compared to control, while the frequency of their progeny pre-B cells is decreased (lower panel). This decrease results in a corresponding decrease of transitional and mature B cells in the periphery (lower panel) Peripheral B cell differentiation and cell surface IgGl expression in HCO mice. 、 7 , 8) and a corresponding decrease of mature recirculating B cells in the BM (upper panel). There are several possible explanations for the changes in pro-B and pre-B cell frequency. Normally, pre-B cells synthesize μHC associated with surrogate light chains and CD79A / B and express this at low levels on the cell surface. This marks the cell to proliferate before V→J rearrangement and light chain gene expression, marking the immature B cell stage. The γ1 HC of HCO mice does not associate with SLC and is expressed on the cell surface with CD79A / B. This pre-B cell can not proliferate to the same extent as WT and can differentiate into immature B cells and leave the BM faster because there is no need for LC gene rearrangement. The increase in pro-B cells indicates that there can be a bottleneck in the developmental stage from pro-B to pre-B.

[0381] In any case, all expected developmental stages of B lineage cells are present in HCO mice.

[0382] Example 3: Figure 5

[0383] Splenocytes from the same mice as in Example 3 were stained with fluorescent conjugated monoclonal antibodies specific for the indicated CD antigens and analyzed by flow cytometry (Figure 4 The numbers in the flow diagrams represent the percentage of cells in a given gate. Overall, there was a decrease in all B cell subpopulations in the spleen of HCO mice, in accordance with the prediction that the frequency of pre-B cells and mature recirculating B cells would decrease in the BM ( Figure 6 ), and that the frequency of pro-B cells would increase. The subpopulations analyzed included total B, B1, B2, transitional 1 and 2 (T1 and T2), follicular (Fo.) and marginal zone (MZ) B cells. Cells of all expected differentiation stages were present in HCO mice, although their numbers were reduced.

[0384] A large proportion of splenic MZ (80%) and Fo. B (91%) cells expressed γ1 HC ( Figure 7 ) on their cell surface, indicating that the HCO locus functions normally in vivo.

[0385] In the lymph nodes (LN, Figure 8 ), total B cells (top panel) and mature Fo. B cells (middle panel) were reduced. Similar to splenic B cells, 89% of LN B cells in HCO mice expressed γ1 HC (bottom panel) on their cell surface.

[0386] In the peritoneal cavity ( Serum immunoglobulin (lg) levels in HCO mice ), total B cells (top panel), Fo. B cells and B1 B cells (middle panel) were reduced. Similar to splenic and LN B cells, the majority of peritoneal B cells in HCO mice expressed γ1 HC (bottom panel) on their cell surface.

[0387] In summary, even though the frequency of all B cell developmental stages and subpopulations was reduced in the BM and periphery of HCO mice (except for pro-B cells in the BM), all B cell developmental stages and subpopulations were present, and the γ1 HCO locus functions normally in vivo.

[0388] Example 4: Figure 9

[0389] Figure 10 ELISA assays to detect serum IgG1 and IgM are shown for unimmunized TRN11 / 2 / 5 (open circles) and TRN34 / 29 / 30 HCO (closed circles) mice. The optical density is shown on the Y axis, and the serum dilution is shown on the X axis. For standardization (open boxes), 100 μg / ml of monoclonal IgG1 (left) and IgM (right) were also serially diluted. Significant levels of pure heavy chain IgG1 were detected in the serum of TRN34 / 29 / 30 HCO mice, slightly lower than the IgG1 (containing both heavy and light chains) in control TRN11 / 2 / 5 serum. In contrast, IgM in HCO mice was at background levels. Similar ELISA assays were used to detect serum IgG2b, IgG2c and IgG3 ( ​). None of the three Ig isotypes were detected in TRN34 / 29 / 30 HCO mice.

[0390] These results indicate that B cells in TRN34 / 29 / 30 HCO mice cannot differentiate into IgM-secreting plasma cells due to the presence of three stop codons in the μ HC open reading frame. The results also indicate that there is no isotype switching of IgG2b, IgG2c, or IgG3 in HCO mice due to the deletion of the μ switch region of the Igh locus in these mice. SEQUENCE LISTING <110> TrinamiX GmbH <120> Pure heavy chain antibodies <130> P23JM1WN00389AT <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 2182 <212> DNA <213> Artificial Sequence <220> <223> POLYNUCLEOTIDE <400> 1 agtcagcttc tgatggaatt agaacttggc aaaacaatac tgagaatgaa gtgtatgtgg 60 aacagaggct gctgatctcg ttcttcaggc tatgaaactg acacatttgg aaaccacagt 120 acttagaacc acaaagtggg aatcaagaga aaaacaatga tcccacgaga gatctataga 180 tctatagatc atgagtggga ggaatgagct ggcccttaat ttggttttgc ttgtttaaat 240 tatgatatcc aactatgaaa cattatcata aagcaatagt aaagagcctt cagtaaagag 300 caggcattta tctaatccca ccccaccccc acccccgtag ctccaatcct tccattcaaa 360 atgtaggtac tctgttctca cccttcttaa caaagtatga caggaaaaac ttccatttta 420 gtggacatct ttattgttta atagatcatc aatttctgca gacttacagg acggatcgat 480 cccctcagtt agcctccccc atctcccggg caaacgtgcg cgccaggtcg catatcgtcg 540 gtatggagcc gggggtggtg acgtgggtct ggaccatccc ggaggtaagt tgcagcaggg 600 cgtcccggca gccggcgggc gattggtcgt aatccaggat aaagacgtgc atggaacgga 660 ggcgtttggc caagacgtcc aaggcccagg caaacacgtt atacaggtcg ccgttggggg 720 ccagcaactc gggggcccga aacagggtaa ataacgtgtc cccgatatgg ggtcgtgggc 780 ccgcgttgct ctggggctcg gcaccctggg gcggcacggc cgtccccgaa agctgtcccc 840 agtcctcccg ccacgacccg ccgcactgca gataccgcac cgtattggca agtagcccgt 900 aaacgcggcg aatcgcagcc agcatagcca ggtccagccg ctcgccgggg cgctggcgtt 960 tggccaggcg gtcgatgtgt ctgtcctccg gaagggcccc aagcacgatg ttggtgccgg 1020 gcaaggtcgg cgggatgagg gccacgaacg ccagcacggc ctgggggtc atgctgccca 1080 taaggtaccg cgcggccggg tagcagga gggcggcgat gggatggcgg tcgaagatga 1140 gggtgagggc cggggcggg gcatgtgagc tcccagcctc ccccccgata tgaggagcca 1200 gaacggcgtc ggtcacggca taaggcatgc ccattgttat ctgggcgctt gtcattacca 1260 ccgccgcgtc cccggccgat atctcaccct ggtcgaggcg gtgttgtgtg gtgtagatgt 1320 tcgcgattgt ctcggaagcc cccagcaccc gccagtaagt catcggctcg ggtacgtaga 1380 cgatatcgtc gcgcgaaccc agggccacca gcagttgcgt ggtggtggtt ttccccatcc 1440 cgtgggggacc gtctatataa acccgcagta gcgtgggcat tttctgctcc gggcggactt 1500 ccgtggcttc ttgctgccgg cgagggcgca acgccgtacg tcggttgcta tggccgcgag 1560 aacgcgcagc ctggtcgaac gcagacgcgt gttgatggcc ggggtacgag gccatgatgg 1620 caaggacggt gcattggctg caggtcgaaa ggcccggaga tgaggaagag gagaacagcg 1680 cggcagacgt gcgcttttga agcgtgcaga atgccgggcc tccggaggac cttcgggcgc 1740 ccgccccgcc cctgagcccg cccctgagcc cgcccccgga cccacccctt cccagcctct 1800 gagcccagaa agcgaaggag caaagctgct attggccgct gccccaaagg cctacccgct 1860 tccattgctc agcggtgctg tccatctgca cgagactagt gagacgtgct acttccattt 1920 gtcacgtcct gcacgacgcg agctgcgggg cgggggggaa cttcctgact aggggaggag 1980 tagaaggtgg cgcgaagggg ccaccaaaga acggagccgg ttggcgccta ccggtggatg 2040 tggaatgtgt gcgaggccag aggccacttg tgtagcgcca agtgcccagc ggggctgcta 2100 aagcgcatgc tccagactgc cttgggaaaa gcgcctcccc tacccggtag aattgacctg 2160 ccggggccct cgaatcctgc ag 2182 <210> 2 <211> 2369 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide <400> 2 aggtgctcag caaaggaggt cggcaggagg gcggagggtg tgtttttgta tgggagaagc 60 aggagggcag aggctgtggg tttttgttat aggaacagag gcagaacaga cactgtgcta 120 ctggtacttc gatctctggg gccgtggcac cctggtcact gtctcctcag gtaagctggc 180 ttttttcttt ctgcacattc cattctgaaa cgggaaaaga tattctcaga tctccccatg 240 tcaggccatc tgccacactg tgagtcctgc atctggggac tgtggggttc aggtggccta 300 aggcaggatg tggagaggtt tttgttcggg gaacagaggg gtatccaaca ctgtgtgatg 360 cttttgatat ctggggccaa gggacaatgg tcaccgtctc ttcaggtaag atggctttcc 420 ttctgcctcc tttctctggg cccagcgtcc tctgtcctgg agctgggaga taatgtccgg 480 gggctccttg gtctgcgctg ggccatgtgg ggccctccgg ggctccttct ccggctgtct 540 gcatgctgca gaagcttttc tgtaaggata gggtcttcac tcccaggaaa agaggcagtc 600 agaggctagc tgcctgtgga acagtgacaa tcatggaaaa taggcattta cattgttagg 660 ctacatgggt agatgggttt ttgtacaccc actaaagggg tctatgacac tgtgactact 720 ttgactactg gggccaggga accctggtca ccgtctcctc aggtgagtcc ttacaacctc 780 tctcttctat tcagcttaaa tagattttac tgcatttgtt gggggggaaa tgtgtgtatc 840 tgaatttcag gtcatgaagg actagggaca ccttgggagt cagaaagggt cattgggagc 900 cctggctgac gcagacagac atcctcagct cccatacttc atggccagag atttataggg 960 atcctggcca gcattgccgc taggtccctc tcttctatgc tttctttgtc cctcactggc 1020 ctccatctga gatcatcctg gagccctagc caaggatcgg tttttgtcag gggtctaatc 1080 attgttgtca cactgtgaca actggttcga cccctggggc cagggaaccc tggtcaccgt 1140 ctcctcaggt gagtcctaac ttctcccatt ctaaatgcat gttgggggga ttctgggcct 1200 tcaggaccaa gattctctgc aaacgggaat caagattcaa cccctttgtc ccaaagttga 1260 gacatgggtc tgggtcaggg actctctgcc tgctggtctg tggtgacatt agaactgaag 1320 tatgatgaag gatctgccag aactgaagct tgaagtctga ggcagaatct tgtccagggt 1380 ctatcggact cttgtgagaa ttaggggctg acagttgatg gtgacaattt cagggtcagt 1440 gactgtctgg tttctctgag gtgaggctgg aatataggtc accttgaaga ctaaagaggg 1500 gtccaggggc ttctgcacag gcagggaaca gaatgtggaa caatgacttg aatggttgat 1560 tcttgtgtga caccaggaat tggcataatg tctgagttgc ccaggggtga ttctagtcag 1620 actctgggtt tttgttcggg tatagaggaa aaatccacca ctgtgattac tactactact 1680 actacatgga cgtctggggc aaagggacca cggtcaccgt ctcctcaggt aagaatggcc 1740 tctccaggtc tttattttta acctttgtta tggagttttc tgagcattgc agactaatct 1800 tggatatttg tccctgaggg agccggctga gagaagttgg gaaataaact gtctagggat 1860 ctcagagcct ttaggacaaa cggtggctgc cgtcctgaca ggggcttagg gaggctccag 1920 gacctcagtg ccttgaagct ggtttccaag agaaaaggat tgtttatctt aggaggcatg 1980 cttactgtta aaagacagga tatgtttgaa gtggcttctg agaaaaatgg ttaagaaaat 2040 tatgacttaa aaatgtgaga gattttcaag tatattaatt tttttaactg tccaagtatt 2100 tgaaattctt atcatttgat taacacccat gagtgatatg tgtctggaat tgaggccaaa 2160 gcaagctcag ctaagaaata ctagcacagt gctgtcggcc ccgatgcggg actgcgtttt 2220 gaccatcata aatcaagttt atttttttaa ttaattgagc gaagctggaa gcagatgatg 2280 aattagagtc aagatggctg catgggggtc tccggcaccc acagcaggtg gcaggaagca 2340 ggtcaccgcg agagtctatt ttaggaagc 2369 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> POLYNUCLEOTIDE <400> 3 cctttagtga gggttaatt 19 <210> 4 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> POLYNUCLEOTIDE <400> 4 gaagttccta ttccgaagtt cctattcttc aaaaggtata ggaacttc 48 <210> 5 <211> 2711 <212> DNA <213> Artificial Sequence <220> <223> POLYNUCLEOTIDE <400> 5 tcattctggc ctccccctcc cccaaggtca ttctggcctc cccctccccc aaggtcattc 60 tggcctcccc ctcccccttg gccgcgcctc attctggcct ccccctcccc caaggcacac 120 aaaaaaccaa cacacagatc tattgaaaat aatgatcttt tattgatccg cgcctggatc 180 TCGAACCGGT TCAGGCACCG GGCTTGCGGG Tcatgcacca ggtgcgcggt ccttcgggca 240 CCTCGACGTC GGCggtgacg gtgaagccga gccgctcgta gaaggggagg ttgcggggcg 300 Cggaggtctc caggaaggcg ggcaccccgg cgcgctcggc cgcctccact ccggggagca 360 CGACGGCGCT GCCCAGACCC TTGCCCTGgt ggtcgggcga gacgccgacg gtggccagga 420 ACCACGCGGG CTCCTTGGGC CGGTGCggcg ccaggaggcc ttccatctgt tgctgcgcgg 480 CCAGCCGGGA ACCGCTCAAC TCggccatgc gcgggccgat ctcggcgaac accgcccccg 540 CTTCGACGCT CTCCGGCgtg gtccagaccg ccaccgcggc gccgtcgtcc gcgacccaca 600 CCTTGCCGAT GTCGAGCCCG ACgcgcgtga ggaagagttc ttgcagctcg gtgacccgct 660 CGATGTGGCG GTCCGGGTCG ACGGTGTGGC GCgtggcggg gtagtcggcg aacgcggcgg 720 CGAGGgtcgt acggcccggg ggacgtcgtc gcgggtggcg aggcgcacc gtgggcttgt 780 ACTCGGTcat ggaaggctag gcggatcggc gatcgctccg gaggccgccc tgggagtgga 840 CACCTGTGGA GAGAAAGGCA AAGTGGATGT CAATGTCact caagtgtatg gccagatctc 900 aagcctgcca cacctcaagc ttgacaacaa aaagattgtc ttttctgacc agatggacgc 960 ggccaccctc aaaggcatca ccgcgggcca ggtgaatatc aaatcctcct cgtttttgga 1020 aactgacaat cttagcgcag aagtcatgcc cgcttttgag agggagtact caccccaacg 1080 cttctagagc cgccggtcac acgccagaag ccgaaccccg ccctgccccg tcccccccga 1140 aggcagccgt ccccccgcgg acagccccga ggctggagag ggagaagggg acggcggcgc 1200 ggcgacgcac gaaggccctc cccgcccatt tccttcctgc cggcgccgca ccgcttcgcc 1260 ccgcgcccgc tagagggggt gcggcggcgc ctcccagatt tcggctccgc acagatttgg 1320 gacaaaggaa gtccctgcgc cctctcgcac gattaccata aaaggcaatg gctgcggctc 1380 gccgcgcctc gacagccgcc ggcgctccgg gggccgccgc gcccctcccc cgagccctcc 1440 ccggcccgag gcggccccgc cccgcccggc acccccacct gccgccaccc cccgcccggc 1500 acggcgagcc ccgcgccacg ccccgtacgg agccccgcac ccgaagccgg gccgtgctca 1560 gcaactcggg gaggggggtg cagggggggt tgcagcccga ccgacgcgcc cacaccccct 1620 gctcaccccc ccacgcacac accccgcacg cagcctttgt tcccctcgca gccccccccg 1680 caccgcgggg caccgccccc ggccgcgctc ccctcgcgca cactgcggag cgcacaaagc 1740 cccgcgccgc gcccgcagcg ctcacagccg ccgggcagcg cggagccgca cgcggcgctc 1800 cccacgcaca cacacacgca cgcacccccc gagccgctcc ccccgcacaa agggccctcc 1860 cggagcccct caaggctttc acgcagccac agaaaagaaa caagccgtca ttaaaccaag 1920 cgctaattac agcccggagg agaagggccg tcccgcccgc tcacctgtgg gagtaacgcg 1980 gtcagtcaga gccggggcgg gcggcgcgag gcggcggcgg agcggggcac ggggcgaagg 2040 cagcgcgcag cgactcccgc ccgccgcgcg cttcgctttt tatagggccg ccgccgccgc 2100 cgcctcgcca taaaaggaaa ctttcggagc gcgccgctct gattggctgc cgccgcacct 2160 ctccgcctcg ccccgccccg cccctcgccc cgccccgccc cgcctggcgc gcgccccccc 2220 cccccccgcc cccatcgctg cacaaaataa ttaaaaaata aataaataca aaattggggg 2280 tggggagggg ggggagatgg ggagagtgaa gcagaacgtg gggctcacct cgaccatggt 2340 aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta 2400 ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg 2460 catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg 2520 acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa 2580 tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg 2640 gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt aataactagt 2700 caataatcaa t 2711 <210> 6 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide <400> 6 gaagttccta ttccgaagtt cctattcttc aaaaggtata ggaacttc 48 <210> 7 <211> 313 <212> DNA <213> Mus musculus <400> 7 ctgcagcagc tggcaggaag caggtcatgt ggcaaggcta tttggggaag ggaaaataaa 60 accactaggt aaacttgtag ctgtggtttg aagaagtggt tttgaaacac tctgtccagc 120 cccaccaaac cgaaagtcca ggctgagcaa aacaccacct gggtaatttg catttctaaa 180 ataagttgag gattcagccg aaactggaga ggtcctcttt taacttattg agttcaacct 240 tttaatttta gcttgagtag ttctagtttc cccaaactta agtttatcga cttctaaaat 300 gtatttagaa ttc 313 <210> 8 <211> 1000 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide <400> 8 ttaaaaaaaa aaaaaaagga aagggacttc tctgtgtttg gcaacacaag tgcgatgcac 60 aggcaggaag atcaaatctg tcccaacaat acaggggaca gagggtcaac ctacaaaagg 120 aaagaacctg gggcagtgtg aagacaacac tgtagaagcc aaggctgagt tcactgagct 180 ctcgttagtg agactacaca gcaaggaggt ggcgggcact gagcagtgag gccccgggaa 240 gtgggggtga tggtggtgac ggtggtaact gttaagaact gggggaaaga attgtggaga 300 accaagctaa atagttatgt caaaccacat gtttaggagc ctgggttgac ttcataggga 360 gtaggcatgg aggctaatct agaggtttgt gtataggcaa gaagtgaatc ctgacccaag 420 aatagagagt gctaaacgga cttagttcaa agacaactga aaaagacaat gcctgcaaaa 480 caaagctaag gccagagctc ttggactatg aagagttcag ggaacctaag aacagggacc 540 atctgtgtac aggccaaggc cggtagaagc agcctaggaa gtgtcaagag ccaacgtggc 600 tgggtgggca aagacaggaa gggactgtta ggctgcaggg atgtgccgac ttcaatgtgc 660 ttcagtattg tccagattgt gtgcagccat atggcccagg tataagaggt ttaacagtgg 720 aacacagatg cccacatcag acagctgggg ggcgggggtg aacacagata cccatactgg 780 aaagcaggtg gggcattttc ctaggaacgg gactgggctc aatggcctca ggtctcatct 840 ggtctggtga tcctgacatt gataggccca aatgttggat atcacctact ccatgtagag 900 agtcggggac atgggaaggg tgcaaaagag cggccttcta gaaggtttgg tcctgtcctg 960 tcctgtctga cagtgtaatc acatatactt tttcttgtag 1000 <210> 9 <211> 243 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide <400> 9 aagatcaaaa gttgttcttc tcccttctgg agatttctat gtcctttaca actcaattgg 60 ttaatatcct gggttggagt cccacacatc ttgacaaaca gagacaaatt tgagtatcac 120 cagccaaaag tcatacccaa aaacagcctg gcatgaccac acaccagact caaacttacc 180 ctacctttat cctggtggct tctcatctcc agaccccagt aacacatagc tttctctcca 240 cag 243 <210> 10 <211> 4252 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide <400> 10 tgcccaggga ttgtggttgt aagccttgca tatgtacagg taagtcagtg gccttcacct 60 gacccagatg caacaagtgg caatgttgga gggtggccag gtattgacct atttccacct 120 ttcttcttca tccttagtcc cagaagtatc atctgtcttc atcttccccc caaagcccaa 180 ggatgtgctc accattactc tgactcctaa ggtcacgtgt gttgtggtag acatcagcaa 240 ggatgatccc gaggtccagt tcagctggtt tgtagatgat gtggaggtgc acacagctca 300 gacgcaaccc cgggaggagc agttcaacag cactttccgc tcagtcagtg aacttcccat 360 catgcaccag gactggctca atggcaagga gttcaaatgc agggtcaaca gtgcagcttt 420 ccctgccccc atcgagaaaa ccatctccaa aaccaaaggt gagagctgca gtgtgtgaca 480 tagaagctgc aatagtcagt ccatagacag agcttggcat aacagacccc tgccctgttc 540 gtgacctctg tgctgaccaa tctctttacc cacccacagg cagaccgaag gctccacagg 600 tgtacaccat tccacctccc aaggagcaga tggccaagga taaagtcagt ctgacctgca 660 tgataacaga cttcttccct gaagacatta ctgtggagtg gcagtggaat gggcagccag 720 cggagaacta caagaacact cagcccatca tgaacacgaa tggctcttac ttcgtctaca 780 gcaagctcaa tgtgcagaag agcaactggg aggcaggaaa tactttcacc tgctctgtgt 840 tacatgaggg cctgcacaac caccatactg agaagagcct ctcccactct cctggtaaat 900 gatcccagtg tccttggagc cctctggtcc tacaggactc tgacacctac ctccacccct 960 ccctgtataa ataaagcacc cagcactgcc ttgggaccct gcaataacgt cctggtgatt 1020 tctgagatgt agagtctagc taggtcatgg aatgaggggt ctccatggtt tgagccctga 1080 gttgtgacta aggaaaaact cataggccta cactgccaca cccagcactt ttgaatttgc 1140 ctgacatgaa aagaatttac ctctccctgg aaagtggagc cttatcccta ggcagttccc 1200 ttaccagacc ttcctctagc ttgcactatg ttctgggcac agaatgtgtc taacccccca 1260 aagtaaggaa gacacaacct ctacttccct cactctgtcc ttaccccttt tcctggctaa 1320 gcatctcact gagtgcgctg aatagatgca tgtggccaca gcttgcagac agacctttgc 1380 catctctccg ctcagctttc cagaggctaa gtctagcccg tatggtgatg atgcagggag 1440 ctctatgcta tctcagtgtt atcagactcc taagtggagg atcaacatgg tcccattaaa 1500 accaacctgc tcagcaacac cctgccaata aggcccgtat gtgaaaatgt gcacacatct 1560 acacatgcac aggcacacac acacacacat gcatgggcac acacacatac agagagagag 1620 aatcacagaa actcccatga gcatcctata cagtactcaa agataaaaag gtaccaggtc 1680 tacccacatg atcatcctcg gcatttacaa gtgggccaac tgatacagat aaaacttttc 1740 tatgccaagg acgccaacaa ccttcctcat atacacaagt ccgctcatga caaatctgtc 1800 cctgaacctc agactggcgc ccgtgactca cacagtggac actcctccaa agctgtatag 1860 cttcctttac ttccctgtgt gtactttctc tgaagtacac tcatcacaca gaagaggccc 1920 tgtgattact ctggccctct gttcttggtc atcagagaat agacagaaga tcaggcaaac 1980 tacacagaca cttcccacaa tcatcacagg ccctgactct gctctccagt ctcaaaactg 2040 aaggctggag cacacagaaa taagctccta cacagcccag accagtatcg ggtccagtgt 2100 gtctgaatga gcccagggac aaaatggcag cactttgggg aactgagatt tctggtccaa 2160 gaaggagaga tggaggccca gggagggtct gctgacccag cccagcccag cccagctgca 2220 gctttctcct gggcctccat gcagcttcct gccacacagg gaatggccct agccccacct 2280 tattgggaca aacactgacc gccctctctg tccagggctg caactggacg agacctgtgc 2340 tgaggcccag gacggggagc tggacgggct ctggacgacc atcaccatct tcatcagcct 2400 cttcctgctc agcgtgtgct acagcgctgc tgtcacactc ttcaaggtca gccatactgt 2460 ccccacagtg tctacaatgt cctcatactc ttccccatac tgtccctgtg gtgacctata 2520 ccccacactg tcccatgcta atgaccacag tcttacatgc tatgtaatgc tgtctaccct 2580 tctgtatgca cagtctcaca atgtcccatg cagtctccac gatgctccat gctgcccctt 2640 gttccacgct atgctgtccc atgctattgt ctgtattttc atgctctttt cacactgtcc 2700 ctagtgtcac attctgccca tgttgtccac cacattgtcc ccactctgca cacagcctca 2760 cactgtaccc tgctacccga taatgttccc tgttgtcccc aactctctcc ctgcatcatt 2820 tgtcaactgt cccctgaatt cccatgttgt tcccacactg ttagtgtgta atgtgctctg 2880 tcccaggtgt accttgttcc gtgctgtctc acttcatcgc ccattctgtc cttttactaa 2940 ccccactcta tcaccacact gtccctatgc actgcccaca ttgtcctcat actgtcccat 3000 tttgtatctt catcctgtcc ccatagtgtc caatgatcta ccccacacta ttcccacttc 3060 atgcccctac aatttcccta ttccatccct ctctggtcac catgccatcc ttcccactcc 3120 tgcacagctg gagagggact cccgggatga gtccttgccc agatgagcta cctatctaga 3180 ggagtcttca ggtgggaagg gaatgcagtc ttgatcttgg tcttattcac cctgtctcac 3240 aggtaaagtg gatcttctcc tcggtggtgg agctgaagca gacactggtt cctgaataca 3300 agaacatgat tgggcaagcg ccctaggcca cctcttgtaa tggcagggga tttcccaggc 3360 cccaaaggac cctgtccaat atgccaagca gcacaactga gatcacactg tctgctcatc 3420 tcgctttcct ccgaccccga gactcagcta ctctcaaatt ttccctctct gaaggaccat 3480 gtggacatta cattgctcca ggccacagcc accaggacct aaaacaccat cacagcagca 3540 ccaaagacac tggatagacc cacaagagca atagcttcct caacagtata tccaaactgt 3600 tgggacaaac gagcaatcac tgaagaagtg acaagttccc acaatgtcag tgtccagctg 3660 agaaggggca aaaagtggta ccagccctgt ccacaccacc ttctaattca caggaatccg 3720 tgatagaaga ggcaggttgt agatccgaaa gatgagacag attttatcaa ctccagaaag 3780 agctgggccc aactgaatct aactgaatta ttctagcgac cttggcattg ccatgacctg 3840 ccatgacctt cctccttagc acttcgatga accctgggat atggaaaatg cctgtgtttc 3900 tcagggtttg ggaagaacca tccatgttgg gattcttgtg tagatcctcc tcctggtcac 3960 agatgcaata cactggattt tcaggcaaag gagcaaattc acagacaact ctggccctac 4020 agtcctcaga cctagacacc accatctcct tggaattatc aaatctaaca cccggcacac 4080 aacaaagaag gactgggact ttgaggcctt tgtgtagccc tagagggggc agaggccact 4140 gagcagggat tgggtgatca gcaaggacct cctggagagg gacctgagga gcaggttcca 4200 attgggccaa agaaagaaga agaacaatag aggtgaagga tgctggaaag ag 4252 <210> 11 <211> 623 <212> DNA <213> Homo sapiens <400> 11 gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca 60 gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc 120 ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca 180 acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc aatcttgg 240 ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg 300 ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg 360 ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct 420 tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg 480 attttaaaat aactatacca gcaggaggac gtccagacac agcataggct acctggccat 540 gcccaaccgg tgggacattt gagttgcttg cttggcactg tcctctcatg cgttgggtcc 600 actcagtaga tgcctgttga att 623 <210> 12 <211> 4565 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide <400> 12 gtgttcatgc ccctagagtt ggctgaaggg ccagatccac ctactctaga ggcatctctc 60 cctgtctgtg aaggcttcca aagtcacgtt cctgtggcta gaaggcagct ccatagccct 120 GCTGCAGTTT CTGCTGTATG CAGGTTCACC TACTACCATG TCTAGCCCTG CCTGCCTT 180 AAGAGTAGCA ACAAGGAAAT AGCAGGCTGT AGAGGGATCT CCTGTCTGAC AGGAGGCAAG 240 AAGACAGATT CTTACCCCTC CATTTCCTTT TTATCCCTCT CTGCTCCTCA GAGAGTCAGT 300 CCTTCCCAAA GTCTTCCCCC TCCTCTCCTG CGAGAGCCCC CTGTCTGATA AGAATCTGG 360 TGGCCATGGG CTGCCTGGCC CAGGACTTCC TGCCCAGCAC CATTTCCTTC CCTGAAACT 420 ACCAGAACAAC ACTGAAGTCA TCCAGGGTAT CAGAACCCTC CAACACTGAG GACAGGGG 480 GCAAGTACCT AGCCACCTCG CAGGTGTTGC TGTCTCCCAA GAGCATCCTT GAAGGTTCA 540 ATGAATACCT GGTATGCAAA ATCCACTACG GAGGCAAAAA CAAAGATCTG CATGTGCCC 600 TTCCAGGTAA GAACCAAACC CTCCCAGCAG GGCTGCCCAG GCCCAGGCAT GGCCCAGAGG 660 GAGCAGCGGG GTGGGGCTTA GGCCAAGCTG AGCTCACACC TTGACCTTTC ATTCCAGCTG 720 TCGCAGAGAT GAACCCCAAT GTAATGTGTT CGTCCCACCA CGGGATGGCT TCTCTGGCC 780 CTGCACCACG CAAGTCTAAA CTCACTTGCG AGGCCACGA ACTTCACCTC AAACCgATCA 840 cagtatcctg actaaaggat gggaagctcg tggaatctgg cttcaccaca gatccggtga 900 ccatcgagaa caaaggatcc acaccccaaa cctacaaggt cataagcaca cttaccatct 960 ctgaaatcga ctggctgaac ctgaatgtgt acacctgccg tgtggatcac aggggtctca 1020 ccttcttgaa gaacgtgtcc tccacatgtg ctgccagtga gtggcctggg ctaagcccaa 1080 tgcctagccc tcccagatta gggaagtcct cctacaatta tggccaatgc cacccagaca 1140 tggtcatttg ctccttgaac tttggctccc cagagtggcc aaggacaaga atgagcaata 1200 ggcagtagag gggtgagaat cagctggaag gaccagcatc ttcccttaag taggtttggg 1260 ggatggagac taagcttttt tccaacttca caactagata tgtcataacc tgacacagtg 1320 ttctcttgac tgcaggtccc tccacagaca tcctaacctt caccatcccc ccctcctttg 1380 ccgacatctt cctcagcaag tccgctaacc tgacctgtct ggtctcaaac ctggcaacct 1440 atgaaaccct gaatatctcc tgagcttctc aaagtggtga accactggaa accaaaatta 1500 aaatcatgga aagccatccc aatggcacct tcagtgctaa gggtgtggct agtgtttgtg 1560 TGGAAGACTG GAATAACAGG AAGGAATTTG TGTTGTACTG TGACTCACAGG GATCTGCCTT 1620 CACCACAGAA GAAATTCATC TCAAAACCCA ATGGTAGGTA TCCCCCCC TC CCTTCCCCTC 1680 CAATTGCAGG ACCCTTCCTG TACCTCATAG GGAGGGCAGG TCCTCTTCCA CCCTATCCTC 1740 ACTACTGTCT TCATTTACAG AGGTGCACAA ACATCCACCT GCTGTGTACC TGCTGCCACC 1800 AGCTCGTGAG CAACTGAACC TGAGGGAGTC AGCCACAGTC ACCCTGCCTG GTGAAGGGCT 1860 CTCTCCTGCA GACATCAGTG TGCAGTGGCT TCAGAGAGGG CAACCTCTTC CCCAAGAGAA 1920 GTATGTGACC AGTGCCCCGA TDGCCAGAGC CTGGGGCCCC AGGCTTCTAC TTTACCCACA G 1980 CATCCTGACT GTGACAGAGG AGGAATGGAA CTCCGGAGAG ACCTATACCT GTGTTAGG 2040 CCACGAGGCC CTGCCACACC TGGTGACCAG GAGGACCCTG GACAAGTCC ACTGGTAAAC 2100 CACACTGTAC AATGTCTCCC TGATCATGTC TGACACAGGC GGCACCTGCT ATTGACCATG 2160 CTAGCGCTCA ACCAGGCAGG CCCTGGGTGT CCAGTTGCTC GTGTATGC AAAC T AACC AT 2220 GTCAGAGTGA GATGTTGCAT TTTATAAAAA TTAGAAATAA AAAAATCCAT TCA AACGTC 2280 actggttttg attatacaat gctcatgcct gctgagacag ttgtgttttg cttgctctgc 2340 acacaccctg catacttgcc tccaccctgg cccttcctct accttgccag tttcctcctt 2400 gtgtgtgaac tcagtcaggc ttacaacaga cagagtatga acatgcgatt cctccagcta 2460 cttctagata tatggctgaa agcttgccta acctggtgca ggcagcattc aggcacatat 2520 atagacacac atgcatttat acatagatat ataggtacac atgtgtagac acatacatga 2580 atgtgtattc atggacacac agacaaaggt acacatatat acacatgagt tcatgcgcac 2640 acacatgcat ggacacttac aaacgccttc agagacaaat aggcatagac acacaaccac 2700 tcacagaaac agataccaat atgcatggtc ctgtgtacac agaaacagac tataggcaaa 2760 tatacacaaa taaactatat agatacaaag atatgcatat acacacatgt acagaaacat 2820 cttcacatgt gtacactaac atgtgaacag gtatagcaca cagatacacc tggactctga 2880 ccagggctgt aatctccaag gctcacggct cagagagcct acactaggct gggtcactga 2940 tactcctcag gagcccactc tatgattggg agagataacc ccaggtacaa agtatgccta 3000 tctgtctcaa caccatgggg cagaagatac tccactaacc acccatgaca gaaagttagc 3060 cttggctgtg tctccattaa tagaacacct cagaagacca atgtgaaatt gcctaaccca 3120 ctcacaccca ccctgatctc cagttcaaaa tgcagaaaac ataatgcagt tgtccaaaag 3180 atgccccaac cacacaca cacacaca cacacaca cacacaca cacacaca 3240 3300 gggctcatat cctggacatt cttcatattc atatccattt ggggcctagg ctttagatat 3360 ccccaagggc tcatctttac agggatcaga gatcccaata aatgccctgg tcccacagcc 3420 tccctcaggt atctgtctgt ttatctcttg gtaccaagac ccaacattgc tggcaggggt 3480 aggacaagca acgcacggga actctgatca aagaaagtca tgagatgcct gagtccttca 3540 ggaagtaagg agggacaacc tctggtatcc ctgttcttat tgctaaagcc caagacag 3600 ggagacctgc tctaaattct cagtctaaac agcaccgatg gcaccacctg ctcagggaaa 3660 gtccagagca caccaatatc attttgccac agttcctgag tctgccttta cccaggtcca 3720 tacattgcat ctgtcttgct tgctctgctg ccccagggct cctggaacaa aggctccaaa 3780 ttagtgtgtc ctacagcttg gcctgttctg tgcctccgtc tagcttgagc tattagggga 3840 ccagtcaata ctcgctaaga ttctccagaa ccatcagggc accccaaccc ttatgcaaat 3900 gctcagtcac cccaagactt ggcttgaccc tccctctctg tgtcccttca tagaggggga 3960 ggtgaatgct gaggaggaag gctttgagaa cctgtggacc actgcctcca ccttcatcgt 4020 cctcttcctc ctgagcctct tctacagcac caccgtcacc ctgttcaagg taggtggtt 4080 gtggggctga ggacacaggg ctgggacagg gagtcaccag tcctcactgc ctctacctct 4140 actccctaca agtggacagc aattcacact gtctctgtca cctgcaggtg aaatgactct 4200 cagcatggaa ggacagcaga gaccaagaga tcctcccaca gggacactac ctctgggcct 4260 gggatacctg actgtatgac tagtaaactt attctttacgt ctttcctgtg ttgccctcca 4320 gcttttatct ctgagatggt cttctttcta gactgaccaa agactttttg tcaacttgta 4380 caatctgaag caatgtctgg cccacagaca gctgagctgt aaacaaatgt cacatggaaa 4440 taaatacttt atcttgtgaa ctcactttat tgtgaaggaa tttgttttgt ttttcaaacc 4500 tttcctgcgg tgttgacagc ccaaggatta tctgaataga gcttaggaac tggaaatgga 4560 acagt 4565 <210> 13 <211> 291 <212> DNA <213> Artificial Sequence <220> <223> Polynuclotide <400> 13 gtgttcatgc ccctagagtt ggctgaaggg ccagatccac ctactctaga ggcatctctc 60 cctgtctgtg aaggcttcca aagtcacgtt cctgtggcta gaaggcagct ccatagccct 120 gctgcagttt cgtcctgtat accaggttca cctactacca tatctagccc tgcctgcctt 180 aagagtagca acaaggaaat agcagggtgt agagggatct cctgtctgac aggaggcaag 240 aagacagatt cttacccctc catttctctt ttatccctct ctggtcctca g 291 <210> 14 <211> 4227 <212> DNA <213> Mus musculus <400> 14 agagtcagtc cttcccaaat gtcttccccc tcgtctcctg cgagagcccc ctgtctgata 60 agaatctggt ggccatgggc tgcctggccc gggacttcct gcccagcacc atttccttca 120 cctgaaacta ccagaacaac actgaagtca tccagggtat cagaaccttc ccaacactga 180 ggacaggggg caagtaccta gccacctcgc aggtgttgct gtctcccaag agcatccttg 240 aaggttcaga tgaatacctg gtatgcaaaa tccactacgg aggcaaaaac aaagatctgc 300 atgtgcccat tccaggtaag aaccaaaccc tcccagcagg ggtgcccagg cccaggcatg 360 gcccagaggg agcagcgggg tggggcttag gccaagctga gctcacacct tgacctttca 420 ttccagctgt cgcagagatg aaccccaatg taaatgtgtt cgtcccacca cgggatggct 480 tctctggccc tgcaccacgc aagtctaaac tcatctgcga ggccacgaac ttcactccaa 540 aaccgatcac agtatcctga ctaaaggatg ggaagctcgt ggaatctggc ttcaccacag 600 atccggtgac catcgagaac aaaggatcca caccccaaac ctacaaggtc ataagcacac 660 ttaccatctc tgaaatcgac tggctgaacc tgaatgtgta cacctgccgt gtggatcaca 720 ggggtctcac cttcttgaag aacgtgtcct ccacatgtgc tgccagtgag tggcctgggc 780 taagcccaat gcctagccct cccagattag ggaagtcctc ctacaattat ggccaatgcc 840 acccagacat ggtcatttgc tccttgaact ttggctcccc agagtggcca aggacaagaa 900 tgagcaatag gcagtagagg ggtgagaatc agctggaagg accagcatct tcccttaagt 960 aggtttgggg gatggagact aagctttttt ccaacttcac aactagatt gtcataacct 1020 gacacagtgt tctcttgact gcaggtccct ccacagacat cctaaccttc accatcccc 1080 cctcctttgc cgacatcttc ctcagcaagt ccgctaacct gacctgtctg gtctcaaacc 1140 tggcaaccta tgaaaccctg aatatctcct gagcttctca aagtggtgaa ccactggaaa 1200 ccaaaattaa aatcatggaa agccatccca atggcacctt cagtgctaag ggtgtggcta 1260 gtgtttgtgt ggaagactgg aataacagga aggaatttgt gtgtactgtg actcacaggg 1320 atctgcctt accacagaag aaattcatct caaaacccaa tggtaggtat ccccccttcc 1380 cttcccctcc aattgcagga cccttcctgt acctcatagg gagggcaggt cctcttccac 1440 cctatcctca ctactgtctt catttacaga ggtgcacaaa catccacctg ctgtgtacct 1500 gctgccacca gctcgtgagc aactgaacct gagggagtca gccacagtca cctgcctggt 1560 gaagggcttc tctcctgcag acatcagtgt gcagtggctt cagagaggc aactcttgcc 1620 ccaagagaag tatgtgacca gtgccccgat gccagagcct ggggccccag gcttctactt 1680 tacccacagc atcctgactg tgacagagga ggaatggaac tccggagaga cctatacctg 1740 tgttgtaggc cacgaggccc tgccacacct ggtgaccgag aggaccgtgg acaagtccac 1800 tggtaaaccc acactgtaca atgtctccct gatcatgtct gacacaggcg gcacctgcta 1860 ttgaccatgc tagcgctcaa ccaggcaggc cctgggtgtc cagttgctct gtgtatgcaa 1920 actaaccatg tcagagtgag atgttgcatt ttataaaaaat tagaaataaa aaaaatccat 1980 tcaaacgtca ctggttttga ttatacaatg ctcatgcctg ctgagacagt tgtgttttgc 2040 ttgctctgca cacaccctgc atacttgcct ccaccctggc ccttctctcta ccttgccagt 2100 ttcctccttg tgtgtgaact cagtcaggct tacaacagac agagtatgaa catgcgattc 2160 ctccagctac ttctagatat atggctgaaa gcttgcctaa cctggtgcag gcagcattca 2220 ggcacatata tagacacaca tgcatttata catagatata taggtacaca tgtgtagaca 2280 catacatgaa tgtgtattca tggacacaca gacaaaggta cacatatata cacatgagtt 2340 catgcgcaca cacatgcatg gacacttaca aacgccttca gagacaaata ggcatagaca 2400 cacaaccact cacagaaaca gataccaata tgcatggtcc tgtgtacaca gaaacagact 2460 ataggcaaat atacacaaat aaactatata gatacaaaga tatgcatata cacacatgta 2520 cagaaacatc ttcacatgtg tacactaaca tgtgaacagg tatagcacac agatacacct 2580 ggactctgac cagggctgta atctccaagg ctcacggctc agagagccta cactaggctg 2640 ggtcactgat actcctcagg agcccactct atgattggga gagataaccc caggtacaaa 2700 gtatgcctat ctgtctcaac accatggggc agaagatact ccactaacca cccatgacag 2760 aaagttagcc ttggctgtgt ctccattaat agaacacctc agaagaccaa tgtgaaattg 2820 cctaacccac tcacacccac cctgatctcc agttcaaaat gcagaaaaca taatgcagtt 2880 gtccaaaaga tgccccaacc acacacacac acacacacac acacacacac acacacacac 2940 acacacacac acacacac catcaaggag cctctgtaag gagtcaccac ccaataacac 3000 tgcctctttg ggctcatatc ctggacattc ttcatattca tatccatttg gggcctaggc 3060 tttagatatc cccaagggct catctttaca gggatcagag atcccataa atgccctggt 3120 cccacagcct ccctcaggta tctgtctgtt tatctcttgg taccaagacc caacattgct 3180 ggcaggggta ggacaagcaa cgcacgggaa ctctgatcaa agaaagtcat gagatgcctg 3240 agtccttcag ggatagag gggacaacct ctggtatccc tgttcttatt gctaaagccc 3300 aagagacagg gagacctgct ctaaattctc agtctaaaca gcaccgatgg caccacctgc 3360 tcagggaaag tccagagcac accaatatca ttttgccaca gttcctgagt ctgcctttac 3420 ccaggtccat acattgcatc tgtcttgctt gctctgctgc cccagggctc ctggaaaaa 3480 ggctccaaat tagtgtgtcc tacagcttgg cctgttctgt gcctccgtct agcttgagct 3540 attaggggac cagtcaatac tcgctaagat tctccagaac catcagggca ccccaaccct 3600 tatgcaaatg ctcagtcacc ccaagacttg gcttgaccct ccctctctgt gtcccttcat 3660 agagggggag gtgaatgctg aggaggaagg ctttgagaac ctgtggacca ctgcctccac 3720 cttcatcgtc ctcttcctcc tgagcctctt ctacagcacc accgtcaccc tgttcaaggt 3780 agtgtggttg tggggctgag gacacagggc tgggacaggg agtcaccagt cctcactgcc 3840 tctacctcta ctccctacaa gtggacagca attcacactg tctctgtcac ctgcaggtga 3900 aatgactctc agcatggaag gacagcagag accaagagat cctcccacag ggacactacc 3960 tctgggcctg ggatacctga ctgtatgact agtaaactta ttcttacgtc tttcctgtgt 4020 tgccctccag cttttatctc tgagatggtc ttctttctag actgaccaaa gactttttgt 4080 caacttgtac aatctgaagc aatgtctggc ccacagacag ctgagctgta aacaaatgtc 4140 acatggaaat aaatacttta tcttgtgaac tcactttatt gtgaaggaat ttgttttgtt 4200 tttcaaacct ttcctgcggt gttgaca 4227 <210> 15 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide <400> 15 gcccaaggat tatctgaata gagcttagga actggaaatg gaacag 46 <210> 16 <211> 294 <212> DNA <213> Homo sapiens <400> 16 cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc acctctgggg 60 gcacagcagc cctgggctgc ctggtcaagg actacttccc cgaaccggtg acggtgtcgt 120 ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta cagtcctcag 180 gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc acccagacct 240 acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa gttg 294 <210> 17 <211> 330 <212> DNA <213> Homo sapiens <400> 17 cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc 60 tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc 120 ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc aagacaaagc 180 cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc 240 aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc ctcccagccc 300 ccatcgagaa aaccatctcc aaagccaaag 330 <210> 18 <211> 320 <212> DNA <213> Homo sapiens <400> 18 ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga 60 accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc gccgtggagt 120 gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg ctggactccg 180 acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg cagcagggga 240 acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca cagaagagcc 300 tctccctgtc tccgggtaaa 320

Claims

1. A method for producing mice, wherein B cells express a library of multiple pure heavy chain antibodies (HCAbs), the method comprising incorporating a transgenic Cγ gene fragment upstream of an endogenous Cμ gene fragment in an endogenous immunoglobulin heavy chain constant region locus, wherein the Cγ gene fragment contains a deletion of at least a portion of a nucleotide sequence encoding a CH1 domain. The endogenous Cμ gene fragment is inactivated due to loss-of-function mutation, partial deletion of the Cμ gene fragment, or one or more mutations that introduce one or more stop codons; The Cγ gene fragment is located downstream of the Eμ major intron enhancer.

2. The method according to claim 1, wherein the mouse, after being immunized with the antigen, activates antigen-specific B cells and induces them to differentiate into plasma cells that secrete multiple antigen-specific HCAbs.

3. The method according to claim 1, wherein the Cγ gene fragment is located downstream of the endogenous Eμ major intron enhancer containing SEQ ID NO:

7.

4. The method according to claim 1, wherein the Cγ gene fragment comprises the Cγ1 gene fragment.

5. The method of claim 1, wherein at least a portion of the CH1 domain comprises a BiP chaperone-binding domain.

6. The method of claim 1, wherein the mouse comprises an inactivated or missing endogenous immunoglobulin light chain locus.

7. The method of claim 6, wherein the mouse comprises a loss-of-function mutation or deletion in either or both of the endogenous κ or λ light chain loci.

8. The method of claim 1, wherein the immunoglobulin heavy chain locus comprises human V H D and J H Encoded sequence and related to the V H D and J H The encoded sequence is an operable linker of the expression control sequence.

9. The method of claim 8, wherein the expression control sequence is mouse-derived.

10. The method of claim 8, wherein the V H D and J H The coding sequence is recombined to form a VDJ coding sequence that expresses a VH binding site that specifically recognizes the antigen, thereby obtaining recombinant VDJ in a given B cell. H The coding sequence indicates that the B cells, after differentiating into plasma cells, can secrete IgG-type HCAbs, wherein the HCAbs contain the recombinant V... H The coding sequence encodes antigen-specific V H Combined structural domains.

11. The method according to claim 1, comprising: a) Provide mouse embryonic stem cells; b) Provide one or more vectors containing a nucleic acid sequence in one or more expression cassettes, the nucleic acid sequence containing the Cγ gene fragment; c) Introduce the one or more of the vectors into the cells; d) Screening for transgenic cells, wherein the sequence of b) has been incorporated into the cellular genome of the cells via targeted integration upstream of an endogenous Cμ gene fragment at an endogenous immunoglobulin heavy chain locus, the Cμ gene fragment optionally being inactive; and e) Using the transgenic cells to produce transgenic mice derived from the transgenic cells.

12. A B cell library expressing multiple IgG-type HCAbs, derived from mice obtained by the method of any one of claims 1 to 11.

13. The B cell library according to claim 12, wherein multiple antigen-specific HCAbs are expressed on different VH domains.

14. A method for generating an antibody comprising an antigen-specific VH domain, the method comprising: a) Immune mice with an antigen, the mice containing a transgenic Cγ gene fragment located upstream of an endogenous Cμ gene fragment in an immunoglobulin heavy chain locus, wherein the Cγ gene fragment contains a deletion of at least a portion of a nucleotide sequence encoding a CH1 domain, thereby providing a cell bank expressing antigen-specific HCAbs. b) Screen from the library for cells expressing IgG-type HCAb containing antigen-specific VH; c) Determine the nucleic acid sequence encoding the antigen-specific VH from the HCAb; and d) Generate a monoclonal antibody containing the antigen-specific VH.

15. A method for generating a B cell library or a molecular library containing VH, using mice obtained by the method of any one of claims 1 to 11.

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