Transgenic mammals and methods of use thereof

Abstract

Described herein are transgenic mammals expressing immunoglobulins having equine variable domains, including transgenic rodents expressing immunoglobulins having equine variable domains, for the development of therapeutic equine antibodies.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the production of immunoglobulin molecules, including methods for producing transgenic mammals capable of producing antigen-specific antibody-secreting cells for the production of equine monoclonal antibodies. [Background technology]

[0002] 2. Background of the Invention In the following description, certain documents and methods are described for background and introductory purposes. Nothing contained herein should be construed as an "admission" of prior art. Applicants expressly reserve the right, at their discretion, to demonstrate that the documents and methods cited herein do not constitute prior art under applicable statutory provisions.

[0003] Antibodies have emerged as important biological medicines because (i) they exhibit extremely strong binding properties that allow them to target antigens in diverse molecular forms, (ii) they are physiological molecules with favorable pharmacokinetics that make them well tolerated in human and animal treatments, and (iii) they are associated with strong immunological properties that naturally fight off infectious agents. Furthermore, established techniques exist for the rapid isolation of antibodies from laboratory animals that can be readily mounted with a specific antibody response against virtually any foreign substance not naturally present in the body.

[0004] In its most basic form, an antibody comprises two identical heavy (H) chains, each paired with an identical light (L) chain. The N-terminus of both the H and L chains contains a variable domain (V H and V L ), which together provide a paired HL chain with unique antigen-binding specificity.

[0005] Antibody V H and V L The exons encoding the V domains are not present in germline DNA. Instead, H The exons are randomly selected V sequences present in the immunoglobulin heavy chain locus. H, D and J H produced by recombination of gene segments; L The exons are randomly selected V L and J. L They are produced by chromosomal rearrangements of gene segments.

[0006] In mammals, the genome typically contains two alleles capable of expressing heavy chains, two alleles capable of expressing kappa (κ) light chains, and two alleles capable of expressing lambda (λ) light chains (one allele from each parent). H , D and J H Multiple V gene segments and both the immunoglobulin kappa (IGK) and immunoglobulin lambda (IGL) light chain loci L and J. L There are gene segments (Collins and Watson (2018) Immunoglobulin Light Chain Gene Rearrangements, Receptor Editing and the Development of a Self-Tolerant Antibody Repertoire. Front. Immunol. 9: 2249. (doi: 10.3389 / fimmu.2018.02249)).

[0007] In the heavy chain locus, there are also exons for expressing various antibody classes (isotypes). For example, in horse animals, the encoded isotypes are IgM, IgD, IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgE and IgA. Polymorphic variants (also called allotypes) also exist within the encoded isotypes and can be useful as allelic markers. In horse animals, polymorphic variants exist in IgM, IgG3, IgG4, IgG7 and IgE allotypes.

[0008] During B cell development, gene rearrangement first occurs on one of the two homologous chromosomes that contain the heavy chain variable gene segments. In pre-B cells, the resulting VH The exon is then transduced into the C-terminal region at the RNA level for the expression of IgM H chain (μH chain). μ Most of the μ heavy chain synthesized by pre-B cells is retained in the endoplasmic reticulum (ER) and eventually forms a partially unfolded C exon of the μ heavy chain. H The μ heavy chain is degraded due to a non-covalent interaction between the μ heavy chain 1 domain 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 portion of the μ heavy chain binds to a surrogate light chain complex containing the invariant λ5 and V pre-B proteins. This binding displaces BiP, allowing the μ heavy chain / λ5 / V pre-B complex, together with the Igα / β signaling molecule heterodimer, to exit the ER as the pre-B cell receptor (pre-BCR) and be transported through the secretory pathway to the plasma membrane.

[0009] Then, V L -J L Rearrangement occurs in one L chain allele until a functional L chain is formed, at which point the L chain polypeptide can combine with an IgM H chain homodimer to form a fully functional antigen-specific B cell receptor (BCR) that is expressed on the surface of immature B cells.

[0010] Immature B cells migrate to secondary lymphoid organs where they differentiate into mature B cells that can respond to cognate antigens and differentiate into antibody-secreting plasma cells and memory B cells. With the help of T cells, B cells undergo isotype switching, which changes the antibody isotype from IgM to IgG, IgA, or IgE, and V cell differentiation. H and V L Exons can undergo somatic hypermutation that can change the amino acid sequence of the exon. These mutations are H and V LAlthough randomly introduced into exons, B cells with high affinity for the immunizing antigen are able to take up, process, and present more antigen to T follicular helper cells and are therefore preferentially activated compared to B cells with low or no affinity for the immunizing antigen. As a result, somatic mutations are enriched in complementarity determining regions (CDRs) 1, 2, and 3 because they are the most abundant V H and V L This is because it is the region that interacts with the domain's antigen.

[0011] Genes encoding various mouse immunoglobulins have been extensively characterized. For example, Blankenstein and Krawinkel describe the mouse variable heavy chain region in Eur. J. Immunol., 17: 1351-1357 (1987). Equine immunoglobulin genes (e.g., from the Thoroughbred, Equus caballus, Twilight breed) have been structurally characterized. Sun et al. (Dev. Comp. Immunol. 34: 109 (2010)) and Talmadge et al. (Dev. Comp. Immunol. 1: 33 (2013); Dev. Comp. Immunol. 46: 171 (2014)) describe the Ig heavy and lambda light chain genes in the horse genome, and Walther, et al. describe the molecular characterization of all the Ig loci (Igh, Igκ, and Igλ) (Dev. Comp. Immunol. 3: 303 (2015)).

[0012] The production of transgenic animals, such as mice with altered immunoglobulin loci, allows the use of such transgenic animals in various research and development applications, such as drug development and basic research in various biological systems. For example, the production of transgenic mice carrying human immunoglobulin genes is described in International Applications WO90 / 10077 and WO90 / 04036. WO90 / 04036 describes transgenic mice with integrated human immunoglobulin "mini" loci. WO90 / 10077 describes vectors containing immunoglobulin dominant control regions for use in the production of transgenic animals.

[0013] Numerous methods have been developed to modify mouse endogenous immunoglobulin variable region loci with, for example, human immunoglobulin sequences to generate partially or fully human antibodies for drug development purposes. Examples of such mice include those described in, for example, U.S. Patents 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.

[0014] The use of antibodies that function as drugs is not limited to the prevention or treatment of human diseases. Domestic animals, such as horses, suffer from similar afflictions as humans, such as cancer, atopic dermatitis and chronic pain. Monoclonal antibodies targeting CD20, IgE and nerve growth factor, respectively, have already been used in domestic animals for the treatment of some of these conditions. However, before clinical use, monoclonal antibodies made in mice must be equinized, i.e., the amino acid sequence must be changed from mouse to equine, to prevent adverse immune responses in the recipient horse. Summary of the Invention [Problem to be solved by the invention]

[0015] Based on the above, it is apparent that there is a need for efficient and cost-effective methods for producing equine antibodies for the treatment of disease in horses. More specifically, there is a need in the art for the production of small, rapidly reproducing, non-equine mammals capable of producing hybridomas capable of large-scale production of antigen-specific equine immunoglobulins, particularly equine monoclonal antibodies. Thus, there is a continuing need for improved methods of producing transgenic non-human animals capable of producing equine antibodies, e.g., antibodies having equine V regions. [Means for solving the problem]

[0016] Summary of the Invention This Summary is provided to introduce selected 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 for use in limiting 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 embodiments illustrated in the accompanying drawings and defined in the appended claims.

[0017] Described herein are methods for producing mouse antibodies having equine immunoglobulin variable regions. In one embodiment, antibodies having equine variable regions are provided that can be produced in transgenic mammals or in vitro cell culture.

[0018] In some embodiments, a non-equine mammalian cell or a non-equine mammal is provided having a genome that includes a heterologous partial equine immunoglobulin locus. In some embodiments, the heterologous locus includes coding sequences of an equine immunoglobulin variable region gene and non-coding sequences based on an endogenous immunoglobulin variable region locus of the non-equine mammalian host. In some embodiments, the non-equine mammalian cell or mammal is capable of expressing a chimeric B cell receptor (BCR) or antibody that includes equine heavy (H) and light (L) chain variable regions and a constant region that is endogenous to the non-equine mammalian host cell or mammal. In some embodiments, the transgenic mammalian host cell or mammal has a genome in which some or all of the endogenous immunoglobulin variable region locus has been removed.

[0019] To produce chimeric equine monoclonal antibodies in a non-equine mammalian host, the host genome must have a locus that expresses at least one chimeric equine immunoglobulin heavy or light chain. In certain embodiments, the host genome contains one heavy chain locus and two light chain loci that express chimeric equine immunoglobulin heavy and light chains, respectively.

[0020] In some embodiments, the partial equine immunoglobulin locus is an endogenous V H Horse V present at locus H In one embodiment, the partial equine immunoglobulin locus comprises the equine V H Coding sequences and endogenous V of non-equine mammalian hosts H In one embodiment, the partial equine immunoglobulin locus comprises the equine D H and J. H Gene segment coding sequences and endogenous D of non-equine mammalian host cell genomes H and J. H In one embodiment, the partial equine immunoglobulin locus comprises the equine D H and J. HGene segment coding sequences and endogenous D of non-equine mammalian host cell genomes H and J. H Includes non-coding regulatory or scaffolding sequences present in gene segments.

[0021] In other embodiments, the partial equine immunoglobulin locus is L Coding sequences and endogenous V of non-equine mammalian hosts L In another embodiment, the partial equine immunoglobulin locus comprises the equine V L Coding sequences and endogenous V of non-equine mammalian hosts L In one embodiment, the heterologous partial equine immunoglobulin locus comprises a non-coding regulatory or scaffold sequence present in the locus. L Coding sequence and horse J L Gene segment coding sequences and endogenous J of non-equine mammalian host cell genomes L In one embodiment, the heterologous partial equine immunoglobulin locus comprises the equine V L Coding sequence and horse J L Gene segment coding sequences and endogenous J of non-equine mammalian host cell genomes L Includes non-coding regulatory or scaffolding sequences present in gene segments.

[0022] In some embodiments, the non-equine mammal is a rodent, for example, a mouse or a rat.

[0023] In one embodiment, a method is provided for producing a non-equine mammalian cell that contains a partial equine immunoglobulin locus. In one embodiment, the method comprises: a) introducing two or more recombinase targeting sites into the genome of a non-equine mammalian host cell to selectively express an endogenous immunoglobulin V locus. H , D H and J. H Genetic or endogenous V L and J. L and b) integrating at least one site upstream and at least one site downstream of a genomic region containing a gene; H, D H and J. H Gene or Horse V L and J. L The method includes introducing a heterologous partial equine immunoglobulin variable locus comprising a gene coding sequence and a non-coding sequence based on a non-coding sequence present in an endogenous immunoglobulin variable region locus of the non-equine mammalian host into a non-equine mammalian host cell via recombinase-mediated cassette exchange (RMCE).

[0024] In other embodiments, the method comprises deleting an endogenous immunoglobulin variable region in the genome of the host animal that is flanked by two heterologous recombinase targeting sites prior to introducing the heterologous partial equine immunoglobulin variable locus via RMCE into the non-equine mammalian host cell.

[0025] In one embodiment, the heterologous partial equine immunoglobulin locus is H Gene segment coding sequence, horse D H and J. H Gene segment coding sequences and endogenous D in the genomes of non-equine mammalian hosts H Based on the sequence located upstream of the gene segment, H The gene segment includes a non-coding regulatory or scaffold sequence (pre-D sequence, FIG. 1) upstream. In some embodiments, the upstream scaffold sequence includes a non-immunoglobulin gene, such as Adam6 (FIG. 1), which is associated with male fertility (Nishimura et al. Developmental Biol. 233(1): 204-213 (2011)). In some embodiments, the partial equine immunoglobulin locus is located on the same chromosome as the endogenous immunoglobulin V gene. H Upstream of the locus and endogenous J H It is introduced into the host cell using a recombinase targeting site that has been previously introduced downstream of the gene locus.

[0026] In some embodiments, the scaffold sequence comprises a naturally occurring nucleic acid sequence from another species. In some embodiments, the scaffold sequence can be designed based on a naturally occurring nucleic acid sequence from another species, e.g., the scaffold sequence can comprise a naturally occurring nucleic acid sequence from another species that has been modified, e.g., by one or more nucleic acid substitutions, insertions, deletions or other modifications. In some embodiments, the scaffold sequence can comprise an artificial sequence. In some embodiments, the scaffold sequence comprises a sequence present in an immunoglobulin locus of the horse genome in combination with other sequences, e.g., a scaffold sequence from another species.

[0027] In other embodiments, the heterologous partial equine immunoglobulin locus is an equine immunoglobulin V L Gene segment coding sequence, horse J L The heterologous partial equine immunoglobulin locus comprises a gene segment coding sequence and a non-coding sequence based on a non-coding sequence present in an endogenous L chain locus of a non-equine mammalian host cell genome. In some embodiments, the non-coding sequence comprises a regulatory or scaffolding sequence. In some embodiments, the heterologous partial equine immunoglobulin locus comprises an endogenous immunoglobulin V locus of the same chromosome. L Upstream of the locus and endogenous J L It is introduced into the host cell using a recombinase targeting site that has been previously introduced downstream of the gene locus.

[0028] In some embodiments, a heterologous partial equine immunoglobulin locus is synthesized as a single nucleic acid and introduced into a non-equine mammalian host cell as a single nucleic acid region. A heterologous partial equine immunoglobulin locus can also be synthesized as two or more contiguous segments and introduced into a mammalian host cell as separate segments. A heterologous partial equine immunoglobulin locus can also be produced using recombinant methods and isolated prior to introduction into a non-equine mammalian host cell. In some embodiments, a partial equine immunoglobulin heavy chain variable region locus can be produced in silico as follows: H , D and J H Coding sequences are obtained, for example, from the National Center for Biotechnology Information. H , D and JH The coding sequence was analyzed in silico using commercially available software, e.g., H , D and J H Advantageously, V H , D and J H The coding sequence can be replaced with the intervening mouse non-coding sequences remaining intact. Similarly, a partial equine immunoglobulin light chain variable region locus can be generated in silico as follows: L and J. L Coding sequences are obtained, for example, from the National Center for Biotechnology Information. L and J. L The coding sequence was analyzed in silico using commercially available software, e.g., L and J. L Replace it with the code sequence. Similarly, V L and J. L The coding sequences can be replaced while leaving the intervening mouse non-coding sequences intact. Methods are known to synthesize DNA sequences containing partial equine immunoglobulin loci based on in silico sequences.

[0029] In other embodiments, methods are provided for producing a non-equine mammalian cell comprising a heterologous partial equine immunoglobulin locus. In one embodiment, the method comprises: a) introducing into the genome of the non-equine mammalian host cell two or more sequence-specific recombination sites that cannot recombine with each other, wherein at least one recombination site is introduced upstream of an endogenous immunoglobulin variable region locus and at least one recombination site is introduced downstream of the same endogenous immunoglobulin variable region locus; b) introducing into the genome of the non-equine mammalian host cell i) an equine immunoglobulin variable region gene coding sequence and ii) a non-coding regulatory or scaffold sequence based on an endogenous immunoglobulin variable region locus of the host cell genome (wherein the partial equine immunoglobulin locus the method comprises providing a vector comprising a heterologous partial equine immunoglobulin locus having an equine immunoglobulin variable region locus flanked by the same two sequence-specific recombination sites that are flanked by the endogenous immunoglobulin variable region locus of the host cell; c) introducing into the host cell the vector of step b) and a site-specific recombinase that recognizes the two recombinase sites; d) allowing a recombination event to occur between the genome of the cell and the heterologous partial equine immunoglobulin locus, resulting in replacement of the endogenous immunoglobulin variable region locus with the heterologous partial equine immunoglobulin variable region locus. In one embodiment, the partial equine immunoglobulin locus comprises an equine immunoglobulin variable region locus having an equine immunoglobulin variable region locus. H Immunoglobulin gene segment coding sequences and i) equine D H and J. H gene segment coding sequences, ii) individual Vs endogenously present in the genome of the non-equine mammalian host H , D H and J. H and iii) a pre-D sequence based on the endogenous genome of the non-equine mammalian host cell. In one embodiment, the recombinase targeting site is an endogenous immunoglobulin V H Upstream of the locus and endogenous J H It is introduced downstream of the gene locus.

[0030] In one embodiment, a transgenic rodent is provided having a genome in which a rodent endogenous immunoglobulin variable locus has been deleted and replaced with a heterologous partial equine immunoglobulin locus comprising equine immunoglobulin variable gene coding sequences and non-coding regulatory or scaffold sequences based on the rodent endogenous immunoglobulin variable locus. In one embodiment, the heterologous partial equine immunoglobulin locus of the transgenic rodent is functional and expresses immunoglobulin chains comprising equine variable domains and rodent constant domains. In one embodiment, the heterologous partial equine immunoglobulin locus comprises equine V immunoglobulin variable gene coding sequences and non-coding regulatory or scaffold sequences based on the rodent endogenous immunoglobulin variable locus. H , D H and J. H In one embodiment, the heterologous partial equine immunoglobulin locus comprises a coding sequence of the equine V L and J. L In one embodiment, the heterologous partial equine immunoglobulin locus comprises the equine kappa (κ) V L and J. L In one embodiment, the heterologous partial equine immunoglobulin locus comprises an equine lambda (λ) V L and J. L In some embodiments, a cell of B lymphocyte lineage from a transgenic rodent is provided. In some embodiments, a portion or all of an immunoglobulin molecule comprising an equine variable domain and a rodent constant domain sequence from a cell of B lymphocyte lineage is provided. In some embodiments, a hybridoma cell from a cell of B lymphocyte lineage is provided. In some embodiments, a portion or all of an immunoglobulin molecule comprising an equine variable domain and a rodent constant domain from a hybridoma cell is provided. In some embodiments, an immortalized cell from a cell of B lymphocyte lineage is provided. In some embodiments, a portion or all of an immunoglobulin molecule comprising an equine variable domain and a rodent constant domain from an immortalized cell is provided. In some embodiments, a heterologous partial equine immunoglobulin locus is provided comprising an equine variable domain and a rodent constant domain sequence from ... L and J. L In one embodiment, a transgenic rodent is provided comprising a coding sequence for a heterologous partial equine immunoglobulin locus. H , DH and J. H In one embodiment, the heterologous partial equine immunoglobulin locus is a coding sequence for equine kappa (κ) V. L and J. L In one embodiment, the heterologous partial equine immunoglobulin locus comprises an equine lambda (λ) V L and J. L In some embodiments, the rodent is a mouse. In some embodiments, the non-coding regulatory sequences include one or more of the following sequences of the endogenous host: a promoter in front of each V gene segment, a splice site, and a recombination signal sequence for V(D)J recombination. In some embodiments, the heterologous partial equine immunoglobulin locus further includes one or more of the following sequences of the endogenous host: an ADAM6 gene, a Pax-5 activating intergenic repeat (PAIR) sequence, and a CTCF binding site from the heavy chain intergenic control region 1 (IGCR1).

[0031] In some embodiments, the non-equine mammalian cell is a mammalian cell. In some embodiments, the non-equine mammalian cell is a mammalian embryonic stem (ES) cell.

[0032] In some embodiments, equine mammalian cells in which a non-endogenous immunoglobulin variable region locus has been replaced with a heterologous partial equine immunoglobulin variable region locus are selected and isolated. In some embodiments, the cells are non-equine mammalian ES cells, e.g., rodent ES cells. In some embodiments, at least one isolated non-equine mammalian cell is used to create a transgenic non-equine mammal expressing a heterologous partial equine immunoglobulin variable region locus. In some embodiments, at least one isolated non-equine mammalian ES cell is used to create a transgenic non-equine mammal expressing a heterologous partial equine immunoglobulin variable region locus.

[0033] In some embodiments, a method of producing a transgenic rodent is provided. In some embodiments, the method comprises: a) integrating at least one target site for a site-specific recombinase upstream of an endogenous immunoglobulin variable locus and at least one target site for the site-specific recombinase downstream of the endogenous immunoglobulin variable locus in the genome of a rodent cell. In some embodiments, the endogenous immunoglobulin variable locus is a V H , D H and J. HIn some embodiments, the endogenous immunoglobulin variable locus comprises a Vκ and a Jκ gene segment. In some embodiments, the endogenous immunoglobulin variable locus comprises a Vλ and a Jλ gene segment. In some embodiments, the endogenous immunoglobulin variable locus comprises a Vλ, a Jλ gene segment and a Cλ gene. In some embodiments, the method comprises b) providing a vector comprising a heterologous partial equine immunoglobulin locus. In some embodiments, the heterologous partial equine immunoglobulin locus comprises a chimeric equine immunoglobulin gene segment. In some embodiments, each of the partial equine immunoglobulin gene segments comprises an equine immunoglobulin variable gene coding sequence and a rodent non-coding regulatory or scaffold sequence. In some embodiments, the partial equine immunoglobulin variable locus is flanked by target sites for a site-specific recombinase. In some embodiments, the target sites are recombined with target sites introduced into the rodent cell. In some embodiments, the method includes c) introducing into the rodent cell the vector and a site-specific recombinase capable of recognizing the target site. In some embodiments, the method includes d) allowing a recombination event to occur between the genome of the cell and the heterologous partial equine immunoglobulin locus, where the endogenous immunoglobulin variable locus is replaced with the heterologous partial equine immunoglobulin locus. In some embodiments, the method includes e) selecting a cell comprising the heterologous partial equine immunoglobulin variable locus produced in step d); and using the cell to produce a transgenic rodent comprising the heterologous partial equine immunoglobulin variable locus. In some embodiments, the cell is a rodent embryonic stem (ES) cell. In some embodiments, the cell is a mouse embryonic stem (ES) cell.

[0034] In some embodiments, the method further comprises, prior to step a) and after step b), deleting the endogenous immunoglobulin variable loci by introduction of a recombinase that recognizes a first set of target sites, wherein the deleting step leaves in place at least one set of target sites that cannot recombine with each other in the genome of the rodent cell. H , D H and J.H In one embodiment, the vector comprises a horse V L and J. L In one embodiment, the heterologous partial equine immunoglobulin locus comprises the equine kappa (κ) V L and J. L In one embodiment, the heterologous partial equine immunoglobulin locus comprises a lambda (λ) V L and J. L In some embodiments, the vector further comprises one or more of the following: a promoter, a splice site, and a recombination signal sequence.

[0035] In some embodiments, methods are provided for producing a transgenic non-equine mammal comprising a heterologous partial equine immunoglobulin variable region locus. In some embodiments, the methods comprise a) introducing into the genome of the non-equine mammalian host cell one or more sequence-specific recombination sites that flank the endogenous immunoglobulin variable region locus and do not recombine with each other. In some embodiments, the methods comprise b) providing a vector comprising a partial equine immunoglobulin locus having i) an equine variable region gene coding sequence and ii) a non-coding regulatory or scaffold sequence based on the endogenous host immunoglobulin variable region locus. In some embodiments, the coding and non-coding regulatory or scaffold sequences are flanked by the same sequence-specific recombination sites introduced into the genome of the host cell of a). In some embodiments, the methods comprise c) introducing into the cell the vector of step b) and a site-specific recombinase capable of recognizing the set of recombinase sites. In some embodiments, the methods comprise d) allowing a recombination event to occur between the genome of the cell of a) and the heterologous partial equine immunoglobulin variable region locus. In some embodiments, the endogenous immunoglobulin variable region loci are replaced with partial equine immunoglobulin loci. In some embodiments, the method comprises: e) selecting cells that contain a partial equine immunoglobulin locus; and f) using the cells to generate a transgenic mammal that contains a partial equine immunoglobulin locus.

[0036] In some embodiments, the transgenic non-equine mammal is a rodent, for example, a mouse or a rat.

[0037] In some embodiments, at least 10 3 An immunoglobulin library (also referred to as a repertoire) is provided that contains a diversity of library members.

[0038] In some embodiments, a repertoire of antibodies is provided that includes the partial equine antibodies described herein. In some embodiments, the repertoire includes a variety of antibodies that each specifically recognize the same target antigen. Such a repertoire can be referred to as an antibody library of the same antibody type or structure, where the antibodies differ in antigen binding sites, for example, to generate antibody variants of a parent antibody that recognize the same epitope. In some embodiments, the antibody library includes affinity matured or otherwise optimized antibody variants. In some embodiments, the antibody library includes antibodies that specifically recognize a target antigen, but different epitopes of such target antigen.

[0039] In certain embodiments, antibody repertoires are screened and individual library members are selected according to desired structural or functional properties, for example, to generate antibody products.

[0040] In some embodiments, a repertoire of antibodies is provided that includes the partial equine antibodies described herein. In some embodiments, the repertoire includes a variety of antibodies that recognize different target antigens. In some embodiments, the repertoire is obtained by immunization of a non-equine mammal with a multicomponent antigen, including, but not limited to, a virus or a bacterium, which may have many different target antigens, each of which may contain multiple epitopes.

[0041] In some embodiments, the repertoire is a naive library of antibodies, which may also be referred to as a "pre-immune repertoire." In some embodiments, the pre-immune repertoire is expressed by mature, but antigen-naive, B cells that have recently exited the bone marrow.

[0042] In one embodiment, the repertoire of antibodies comprises at least about 10 3 Antibodies, e.g., at least about 10 4 Antibodies, about 10 5 Antibodies, about 10 6 antibody or about 10 7 It can be characterized by its diversity, including antibodies.

[0043] In some embodiments, a non-equine mammalian cell is provided that expresses a heterologous immunoglobulin variable region locus having equine variable region gene coding sequences and non-coding regulatory or scaffolding sequences based on an endogenous non-equine immunoglobulin locus of the host genome, hi some embodiments, the non-equine mammalian cell expresses a chimeric antibody comprising a complete equine heavy or light chain variable domain together with a complete equine heavy or light chain variable domain.

[0044] In some embodiments, a non-equine transgenic mammal is provided that expresses a heterologous immunoglobulin variable region locus having equine variable region gene coding sequences and non-coding regulatory or scaffolding sequences based on an endogenous non-equine immunoglobulin locus of the host genome, hi some embodiments, the non-equine transgenic mammal expresses a chimeric antibody comprising a complete equine heavy or light chain variable domain together with a complete equine heavy or light chain variable domain.

[0045] In some embodiments, B cells from a transgenic non-equine mammal capable of expressing a partial equine antibody having a complete equine variable sequence are provided. In some embodiments, immortalized B cells are provided as a source of monoclonal antibodies specific for a particular antigen.

[0046] In one embodiment, equine immunoglobulin variable region gene sequences cloned from B cells are provided for use in the production or optimization of antibodies for diagnostic, prophylactic and therapeutic uses.

[0047] In one embodiment, non-equine hybridoma cells capable of producing partial equine monoclonal antibodies having complete equine immunoglobulin variable region sequences are provided.

[0048] In one embodiment, the V H and V L Exons were removed from monoclonal antibody-producing hybridomas to produce V H and V L Methods are provided in which exons are modified to include equine constant regions, thereby producing fully equine antibodies that are not immunogenic when injected into horses.

[0049] In one embodiment, a method for producing an equine antibody for therapeutic or diagnostic use is provided. In one embodiment, the method comprises: (i) The genome is modified to have a deletion of the endogenous rodent immunoglobulin locus variable region and a chimeric V H , D H and J. H At least one of each of the immunoglobulin variable region gene segments and / or the immunoglobulin light chain locus contains a chimeric V L and J. L expressing antibodies having a cloned equine variable domain from an antibody-producing cell of the transgenic rodent in which the equine variable domain has been replaced with a heterologous immunoglobulin locus variable region comprising at least one of each of the variable gene segments, wherein each chimeric gene segment comprises an equine V, D or J immunoglobulin variable region coding sequence and a rodent immunoglobulin variable region non-coding gene segment sequence; and (ii) isolating an antibody having an equine variable domain, wherein said antibody is suitable for therapeutic or diagnostic use.

[0050] In some embodiments, the antibody is cloned from a B cell of a transgenic rodent. In some embodiments, the rodent is a mouse. In some embodiments, therapeutic or diagnostic antibodies produced by the methods described herein are provided.

[0051] In one embodiment, a method is provided for producing a therapeutic or diagnostic antibody having an equine variable domain. In one embodiment, the method comprises: (i) The genome is modified to have a deletion of the endogenous rodent immunoglobulin locus variable region and a chimeric V H , D H and J. H At least one of each of the immunoglobulin variable region gene segments and / or the immunoglobulin light chain locus contains a chimeric V L and J. L cloning equine variable domains of antibodies expressed by antibody-producing cells from the transgenic rodent, the equine variable domains being replaced with heterologous immunoglobulin locus variable regions comprising at least one of each of the variable gene segments, wherein each chimeric gene segment comprises an equine V, D or J immunoglobulin variable region coding sequence and a rodent immunoglobulin variable region non-coding gene segment sequence; and (ii) producing a therapeutic or diagnostic antibody comprising an equine variable domain of an antibody expressed by the transgenic rodent.

[0052] In some embodiments, the equine variable domain is cloned from an antibody expressed by a B cell from a transgenic rodent. In some embodiments, the rodent is a mouse. In some embodiments, therapeutic or diagnostic antibodies produced by the methods described herein are provided.

[0053] In one embodiment, a method for producing a monoclonal antibody comprising an equine variable domain is provided. In one embodiment, the method comprises: (i) The genome is modified to have a deletion of the endogenous rodent immunoglobulin locus variable region and a chimeric V H , D and J H At least one of each of the immunoglobulin variable region gene segments and / or the immunoglobulin light chain locus contains a chimeric V L and J. Lproviding B cells from the transgenic rodent, the B cells having a xenogeneic immunoglobulin locus variable region comprising at least one of each of the variable gene segments, wherein each chimeric gene segment comprises an equine V, D or J immunoglobulin variable region coding sequence embedded in a rodent immunoglobulin variable region non-coding gene segment sequence; (ii) immortalizing the B cells; and (iii) isolating a monoclonal antibody containing an equine variable domain expressed by the immortalized B cells, or a gene encoding said antibody.

[0054] In an embodiment, the method comprises: (iv) cloning the horse variable domains expressed by the B cells; and (v) producing therapeutic or diagnostic antibodies comprising the cloned equine variable domains from B cells of the transgenic rodent. This includes:

[0055] In one embodiment, a method is provided for producing an antibody comprising an equine variable domain. In one embodiment, the method comprises: generating an antibody comprising an equine variable domain ... H , D H and J. H At least one of each of the immunoglobulin variable region gene segments and / or the immunoglobulin light chain locus contains a chimeric V L and J. L The method includes providing a transgenic rodent in which an equine immunoglobulin locus variable region comprising at least one of each of the variable gene segments has been replaced with a xenogenous immunoglobulin locus variable region comprising an equine V, D or J immunoglobulin variable region coding sequence embedded in a rodent immunoglobulin variable region non-coding gene segment sequence, wherein the xenogenous immunoglobulin locus of the transgenic rodent expresses an antibody comprising an equine variable domain.

[0056] In one embodiment, the method comprises isolating an antibody having an equine variable region expressed by a transgenic rodent or a gene encoding said antibody. In one embodiment, the method comprises (i) obtaining B cells from the transgenic rodent that express an antibody specific for a target antigen; (ii) immortalizing the B cells; and (iii) isolating an antibody specific for the target antigen from the immortalized B cells.

[0057] In some embodiments, the method comprises cloning an equine variable region from a B cell specific for a particular antigen. In some embodiments, the rodent is a mouse. In some embodiments, the method comprises using the equine variable region cloned from the B cell to produce a therapeutic or diagnostic antibody. In some embodiments, a therapeutic or diagnostic antibody produced by the methods described herein is provided.

[0058] These and other aspects are described in further detail below. [Brief description of the drawings]

[0059] [Figure 1]FIG. 1 shows the mouse Igh locus (top) located at the telomeric end of chromosome 12 (containing the V (IghV), D (IghD), J (IghJ), and C (IghC) gene segments), the Igκ locus (middle) located on chromosome 6 (containing the V (IgkV), J (IgkJ), and C (IgkC) gene segments), and the Igλ locus (bottom) located on chromosome 16 (containing the IglV (V), IglJ (J), and IglC (C) gene segments). Also shown at the Igh locus are 1) the PAIR sequence, a cis-regulatory sequence important for Igh looping to ensure utilization of distal VH gene segments in VDJ rearrangement, 2) the gene enabling Adam6a male fertility, 3) the intergenic control region 1 (IGCR1), which contains sites controlling ordered, lineage-specific rearrangement at the Igh locus, 4) Eμ, a heavy chain intronic enhancer, 5) Sμ, a switch region, and 6) the 3'regulatory region (3'RR), a cis-acting region controlling isotype switching. Also shown at the Igκ locus are the 5' (E5') and 3' (E3') enhancers and at the Igλ locus are three enhancers, Eλ2-4, Eλ, and Eλ3-1.

[0060] [Diagram 2] FIG. 2 is a schematic diagram illustrating a homologous recombination targeting strategy for introducing a first set of sequence-specific recombination sites into a region upstream of the heavy chain variable region locus in the genome of a non-equine mammalian host cell.

[0061] [Diagram 3] 3 is a schematic illustrating the introduction of a second set of sequence-specific recombination sites into a region downstream of the heavy chain variable region locus in the genome of a non-equine mammalian cell via a homology targeting vector. The diagram also illustrates the deletion of an endogenous immunoglobulin heavy chain variable region locus and a selectable marker from the genome of the non-equine mammalian host cell.

[0062] [Figure 4]FIG. 4 is a schematic illustrating the RMCE strategy for introducing a heterologous partial equine immunoglobulin heavy chain locus into a non-equine mammalian host cell genome that has been previously modified to delete an endogenous immunoglobulin heavy chain variable region locus.

[0063] [Diagram 5] FIG. 5 is a schematic diagram illustrating the introduction of a heterologous partial equine immunoglobulin κ light chain variable region locus into an endogenous immunoglobulin κ light chain locus of the mouse genome.

[0064] [Figure 6] 6A and 6B are schematic diagrams illustrating the introduction of a heterologous partial equine immunoglobulin λ light chain variable region locus into an endogenous immunoglobulin λ light chain locus of the mouse genome. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] definition Terms used herein are intended to have their plain and ordinary meanings as understood by those of ordinary skill in the art. The following definitions are intended to aid the reader in understanding the invention, but are not intended to modify or otherwise limit such terms, unless specifically indicated.

[0066] As used herein, the term "locus" refers to a chromosomal segment or nucleic acid sequence that is endogenous to a genome or integrated (or about to be integrated) into a genome, respectively. For example, the immunoglobulin locus may contain some or all of the genes (i.e., H , D H and J. H Gene segment or V L and J. L The term "locus" (e.g., an immunoglobulin heavy chain variable region locus) can refer to a specific portion of a larger locus (e.g., a V H , DH and J. H A portion of an immunoglobulin heavy chain locus that comprises a gene segment. Similarly, an immunoglobulin light chain variable region locus can refer to a specific portion of a larger locus (e.g., a V L and J. L (a portion of the immunoglobulin light chain locus that contains a gene segment).

[0067] As used herein, the term "immunoglobulin variable region gene" refers to a gene encoding a portion of an immunoglobulin heavy chain variable domain or a portion of an immunoglobulin light chain variable domain, respectively. H , D H or J H Gene segment or V in immunoglobulin light chain variable region L or J L The term "immunoglobulin variable region locus" refers to a gene segment that includes a variable (V), diversity (D), or joining (J) gene segment. H , D H or J H Gene segment or V L or J L It refers to part or all of a chromosomal segment or nucleic acid strand, including a gene segment and any collection of intervening non-coding sequences, including, for example, non-coding regulatory or scaffolding sequences.

[0068] As used herein, the term "gene segment" refers to a nucleic acid sequence that encodes a portion of a heavy or light chain variable domain of an immunoglobulin molecule. A gene segment may include coding and non-coding sequences. The coding sequence of a gene segment is a nucleic acid sequence that can be translated into a polypeptide, such as a leader peptide and N-terminal portion of a heavy or light chain variable domain. The non-coding sequence of a gene segment is a sequence adjacent to the coding sequence, which may include a promoter, 5' untranslated sequences, introns intervening in the coding sequence for the leader peptide, recombination signal sequences (RSSs) and splice sites. Gene segments at the immunoglobulin heavy chain (IGH) locus include V H , D H and J. HThe light chain variable region gene segments in the immunoglobulin kappa and lambda light loci are V L and J. L In the κ light chain, V L and J. L The gene segment is V κ and J. κ Similarly, in the lambda light chain, V L and J. L The gene segment is V λ and J. λ They can be referred to as gene segments or IGLV and IGLJ.

[0069] The heavy chain constant region is C H or IGHC. C in horses that encodes IgM, IgD, IgG1-7, IgE, or IgA. H The exons in the region are C μ , C δ , C 1~7 , C ε or C. Similarly, the immunoglobulin kappa or lambda constant regions can be referred to as Cκ or Cλ and IGKC or IGLC, respectively.

[0070] As used herein, "partially equine" refers to a nucleic acid or its expressed protein and RNA products that includes a sequence that corresponds to a sequence found at a locus in both an equine and a non-equine mammalian host. As used herein, "partially equine" also refers to an immunoglobulin locus that includes a nucleic acid sequence from both an equine and a non-equine mammal. In some embodiments, "partially equine" refers to an immunoglobulin locus that includes a nucleic acid sequence from, for example, a rodent, such as a mouse. In some embodiments, a partial equine nucleic acid refers to a coding sequence for an equine immunoglobulin heavy or light chain variable region gene segment and sequence that is based on a non-coding regulatory or scaffold sequence of an endogenous immunoglobulin locus of a non-equine mammal.

[0071] The term "based on," when used in reference to endogenous non-coding regulatory or scaffold sequences of a non-equine mammalian host cell genome, refers to non-coding regulatory or scaffold sequences present at a corresponding endogenous locus of the mammalian host cell genome. In certain embodiments, the term "based on" means that the non-coding regulatory or scaffold sequences present at the partial equine immunoglobulin locus share a substantially high degree of homology with the non-coding regulatory or scaffold sequences at the endogenous locus of the host mammal. In certain embodiments, the non-coding sequences at the partial equine immunoglobulin locus share at least about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homology with the corresponding non-coding sequences found at the endogenous locus of the host mammal. In certain embodiments, the non-coding sequences at the partial equine immunoglobulin locus are the same as the corresponding non-coding sequences found at the endogenous locus of the host mammal. In some embodiments, the non-coding sequences at the partial equine immunoglobulin locus are retained from an immunoglobulin locus of the host mammal. In some embodiments, the non-coding sequences at the partial equine immunoglobulin locus are the same as the corresponding non-coding sequences present at an endogenous locus of the host mammal. In some embodiments, the equine coding sequences are embedded in non-regulatory or scaffolding sequences of the immunoglobulin locus of the host mammal. In some embodiments, the non-equine host animal is a rodent, such as a rat or mouse.

[0072] "Chimeric" refers to a nucleotide sequence that includes nucleotide sequences from more than one animal or a polypeptide, e.g., an antibody, encoded by a nucleotide sequence that includes nucleotide sequences from more than one animal. A "chimeric" immunoglobulin locus refers to an immunoglobulin locus that includes nucleic acid sequences from more than one animal. In some embodiments, a chimeric immunoglobulin locus includes an equine nucleic acid sequence and a murine nucleic acid sequence. In some embodiments, a chimeric immunoglobulin includes protein sequences from more than one animal. In some embodiments, a chimeric immunoglobulin includes an equine sequence and a murine sequence. In some embodiments, a chimeric immunoglobulin includes an equine variable domain and a murine constant domain. In some embodiments, a chimeric immunoglobulin variable region locus includes an equine V nucleic acid sequence and a murine V nucleic acid sequence. H , D H and J. H Coding sequence or horse V L and J. L In one embodiment, the chimeric immunoglobulin variable region locus comprises an equine V H , D H and J. H Coding sequence or horse V L and J. L Contains coding sequences and mouse non-coding sequences.

[0073] As used herein, "adjacent" refers to a sequence, e.g., a nucleotide sequence, that is upstream or downstream of a reference sequence. In some embodiments, an adjacent sequence is adjacent to a reference sequence. In some embodiments, a pair of sequences is adjacent to a reference sequence, such that the first sequence is upstream of the reference sequence and the second sequence is downstream of the reference sequence.

[0074] "Endogenous" refers to a nucleic acid sequence or polypeptide that is naturally present in an organism or cell.

[0075] "Heterologous" refers to a nucleic acid sequence or polypeptide that does not naturally occur in an organism or cell.

[0076] "Non-coding regulatory sequences" refer to sequences known to be essential for (i) V(D)J recombination, (ii) isotype switching, (iii) proper expression of full-length immunoglobulin H or L chains following V(D)J recombination, or (iv) alternative splicing, for example, to produce membrane and secreted forms of immunoglobulin H chains. "Non-coding regulatory sequences" may further include the following sequences: enhancer and locus control sequences, such as CTCF and PAIR sequences (Proudhon, et al., Adv. Immunol. 128: 123-182 (2015)); promoters in front of each endogenous V gene segment; splice sites; introns; or recombination signal sequences adjacent to each V, D, or J gene segment. In certain embodiments, the "non-coding regulatory sequences" of the partial equine immunoglobulin locus have at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% and up to about 100% homology to the corresponding non-coding sequences found in the non-equine mammalian endogenous immunoglobulin locus host cell. In certain embodiments, the "non-coding regulatory sequences" of the partial equine immunoglobulin locus have the same sequence as the corresponding non-coding sequences found in the non-equine mammalian endogenous immunoglobulin locus host cell.

[0077] "Scaffold sequence" refers to a sequence that intervenes between gene segments present at an endogenous immunoglobulin locus of a host cell genome. In some embodiments, the scaffold sequence is interspaced with sequences essential for expression of functional non-immunoglobulin genes, e.g., ADAM6A or ADAM6B. In some embodiments, the scaffold sequence may comprise a naturally occurring nucleic acid sequence from another species. In some embodiments, the scaffold sequence is heterologous, based on a naturally occurring nucleic acid sequence from another species. In some embodiments, the scaffold sequence may comprise an artificial sequence. In some embodiments, the scaffold sequence comprises a sequence present at an immunoglobulin locus of the horse genome in combination with other sequences, e.g., a scaffold sequence from another species. The term "non-coding regulatory or scaffold sequence" is inclusive and may refer to both non-coding regulatory sequences and scaffold sequences at an immunoglobulin locus.

[0078] "Specifically bind" refers to the ability of an antibody or immunoglobulin to bind to an epitope or antigenic determinant of a particular antigen with much greater affinity than the antibody or immunoglobulin binds to other antigens.

[0079] The term "homologous targeting vector" refers to a nucleic acid sequence used to modify the endogenous genome of a mammalian host cell by homologous recombination. A homologous targeting vector may contain a targeting sequence that has homology with a corresponding endogenous sequence adjacent to the locus to be modified, for example, present in the genome of a non-equine mammalian host. In some embodiments, a homologous targeting vector contains at least one sequence-specific recombination site. In some embodiments, a homologous targeting vector contains a non-coding regulatory or scaffold sequence. In some embodiments, a homologous targeting vector contains one or more selectable marker genes. In some embodiments, a homologous targeting vector may be used to introduce a sequence-specific recombination site into a specific region of a host cell genome.

[0080] "Site-specific recombination" or "sequence-specific recombination" refers to the process of DNA rearrangement between two compatible recombination sequences (also referred to as "sequence-specific recombination sites" or "site-specific recombination sequences"). Site-specific recombination can involve any of the following three events: a) the deletion of preselected nucleic acids flanked by the recombination sites; b) an inversion of the nucleotide sequence of preselected nucleic acids flanked by the recombination sites; and c) the reciprocal exchange of nucleic acid sequences adjacent to recombination sites that are located on different nucleic acid strands. It is understood that this reciprocal exchange of nucleic acid segments can be utilized as a targeting strategy for introducing heterologous nucleic acid sequences into the genome of a host cell.

[0081] The term "targeting sequence" refers to a sequence that is homologous to the DNA sequence in the genome of a cell adjacent or proximal to the region of the immunoglobulin locus to be modified.The adjacent or proximal sequence may be within the locus itself or upstream or downstream of the coding sequence in the genome of the host cell.The targeting sequence is inserted into a recombinant DNA vector that can be used to transfect host cells, such as ES cells, so that the sequence to be inserted into the host cell genome, such as the sequence of the recombination site, is adjacent to the targeting sequence of the vector.

[0082] As used herein, the term "site-specific targeting vector" refers to a vector that includes a sequence-specific recombination site, a heterologous partial equine locus, and optionally a nucleic acid encoding a selectable marker gene. In one embodiment, a "site-specific targeting vector" is used for modification of an endogenous immunoglobulin locus in a host using recombinase-mediated site-specific recombination. The recombination site of the targeting vector is suitable for site-specific recombination with other corresponding recombination sites that have been inserted (e.g., by a homology targeting vector) into the genomic sequence of the host cell that is adjacent to the immunoglobulin locus to be modified. Integration of the heterologous partial equine sequence into the recombination site of the immunoglobulin locus replaces the endogenous locus with the heterologous partial equine region.

[0083] The term "transgene" is used herein to refer to genetic material that has been or is about to be artificially inserted into the genome of a cell, and particularly a cell of a mammalian host animal. As used herein, the term "transgene" refers to partial equine nucleic acid, e.g., in the form of a heterologous expression construct or targeting vector.

[0084] "Transgenic animal" refers to a non-equine animal, usually a mammal, that has a heterologous nucleic acid sequence present as part of its cells or as an extrachromosomal element stably integrated into the germline DNA (i.e., into most or all of the genomic sequence of its cells). In the present invention, partial equine nucleic acid is introduced into the germline of such a transgenic animal, for example, by genetic manipulation of the host animal's embryos or embryonic stem cells by methods well known in the art.

[0085] "Vector" refers to plasmids and viruses and any DNA or RNA molecule, whether autonomously replicating or not, that can be used to transform or transfect cells.

[0086] Detailed Description of the Invention The implementation of the techniques described herein may use conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry and sequencing technology, which are within the skill of the person skilled in the art to implement the techniques, unless otherwise specified. Such conventional techniques include polymer array synthesis, hybridization and ligation of polynucleotides, and detection of hybridization using labels. Specific descriptions of suitable techniques may be obtained by referring to the examples herein. However, other equivalent conventional methods may of course be used.Such conventional techniques and descriptions can be found in Green, et al., Eds. (1999), Genome Analysis: A Laboratory Manual Series (Vols. I-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation: A Laboratory Manual; Dieffenbach and Veksler, Eds. (2007), PCR Primer: A Laboratory Manual; Bowtell and Sambrook (2003), DNA Microarrays: A Molecular Cloning Manual; Mount (2004), Bioinformatics: Sequence and Genome Analysis; Sambrook and Russell (2006), Condensed Protocols from Molecular Cloning: A Laboratory Manual; and Green and Sambrook (2012), Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) WH Freeman, New York NY; Gait, "Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press, London; Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3.sup.rd Ed., WH Freeman Pub., New York, NY; and Berg et al. (2002) Biochemistry, 5.sup.th Ed., WH Freeman Pub., New York, NY, all of which are incorporated herein by reference in their entireties for all purposes.

[0087] As used herein and in the appended claims, the singular terms include the plural unless the context indicates otherwise. Thus, for example, reference to a "locus" refers to one or more loci, reference to a "method" includes equivalent steps and methods known to those skilled in the art, and so forth.

[0088] As used herein, the term "or" may refer to "and / or" unless expressly states only alternatives or the alternatives are mutually exclusive. The terms "include", "including" and "including" are open-ended.

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications used herein are incorporated by reference for the purpose of describing and disclosing devices, formulations and methods that can be used in connection with the invention described herein.

[0090] When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also included in the invention, subject to any specifically excluded ranges in the stated range. When the stated range includes one or both limits, ranges excluding either or both are also included in the invention.

[0091] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and methods well known to those skilled in the art have not been described in order to avoid obscuring the present invention.

[0092] In the humoral immune system, a diverse antibody repertoire is produced by the combination and junctional diversity of IgH (Igh) and Igl chain loci by a process called V(D)J recombination. In developing B cells, the first recombination event to occur is between one D and one J gene segment of the heavy chain locus, resulting in the deletion of DNA between these two gene segments. After this DJ recombination, one V gene segment from the upstream region of the newly formed DJ complex is joined to form a rearranged VDJ exon. All other sequences between the recombined V and D gene segments of the newly created VDJ exon are deleted from the genome of the individual B cell. This rearranged exon is ultimately expressed on the B cell surface as the variable region of the H chain polypeptide combined with the L chain polypeptide to form the B cell receptor (BCR). The mouse and horse Ig loci are highly complex in the number of features they contain and how the coding regions are diversified by V(D)J rearrangement; however, this complexity does not extend to the fundamental details of the structure of each variable region gene segment. V, D and J gene segments are uniform in composition and organization. For example, V gene segments have the following features arranged in an essentially invariant sequential manner in the immunoglobulin locus: a short transcriptional promoter region (<600 bp long), exons encoding most of the signal peptides of the antibody chains; introns; exons encoding small portions of the signal peptides and most of the antibody variable domains of the antibody chains, and a 3' recombination signal sequence required for V(D)J rearrangement. Similarly, D gene segments have the following features: a 5' recombination signal sequence, a coding region and a 3' recombination signal sequence. J gene segments have the following features: a 5' recombination signal sequence, a coding region and a 3' splice donor sequence.

[0093] In one embodiment, a non-equine mammalian cell is provided that comprises a heterologous, partial equine nucleic acid sequence that includes an equine variable region coding sequence and a non-coding regulatory or scaffolding sequence present in an immunoglobulin locus of the mammalian host genome, e.g., a mouse genomic non-coding sequence when the host mammal is a mouse.

[0094] Horse Genome V H The region contains approximately 50 V sequences that map to a 510 kb region of horse chromosome 24. H , 40 D H and 8 J's H The lambda (λ) coding region maps to horse chromosome 8, spans approximately 1310 kb and contains approximately 144 Vλ, 7 Jλ and 7 Cλ genes, while the kappa (κ) coding region maps to horse chromosome 15, spans approximately 820 kb and contains approximately 60 Vκ, 4 functional Jκ and 1 Cκ gene. There are several properties of the equine Ig locus that are unusual, particularly the high frequency of apparently non-functional V gene segments. For example, there are 50 V HOnly 12 of the gene segments are functional; 33 are pseudogenes, and 5 are classified as open reading frames (ORFs), which are variable gene segments with open reading frames that have defects in splicing sites, recombination signal sequences, regulatory sequences, or highly conserved amino acid changes predicted to lead to misfolding of the V domain. Similarly, only 27 of 144 Vλ and 4 of 7 Jλ gene segments and 19 of 60 Vκ gene segments are functional. The genomic structure of the λ locus is also atypical. In humans and mice, for example, there is a cluster of Vλ gene segments (I) followed by a cluster of Jλ-Cλ genes. During B cell development, deletion rearrangements result in the combination of one of the Vλ gene segments with one of the Jλ-Cλ genes. In horses, on the other hand, there is a cluster of Vλ gene segments (I) followed by a cluster of Jλ-Cλ genes followed by another cluster of Vλ gene segments (II). Based on the orientation, Vλ gene segments in cluster II undergo inversion V→J gene rearrangements, which occur much less frequently than deletion gene rearrangements, creating Vλ exons containing recombined Vλ and Jλ gene segments. Furthermore, of the 34 Vλ gene segments in cluster II, 25 are pseudogenes and 2 are ORFs, but Walther et al. (Dev. Comp. Immunol. 3: 303 (2015)) have identified seven functional Vλ gene segments in this cluster. Analysis of the sequence of this contig (NW_001867428.1) indicates that none of the seven putatively functional Vλ gene segments in cluster II contain a conventional RSS, and thus are unlikely to be used in the equine λLC repertoire. However, as shown in Table 1, all seven of these Vλ gene segments are found as cDNAs in GenBank and are likely to be rearranged and expressed in B cells. In some embodiments, the partial equine Vλ locus described herein can include Vλ gene segments from both cluster I and cluster II. H , DH and J. H Segment and full V L and J. L The segments are flanked by murine RSSs to promote rearrangement during B cell development and contribution to the partial equine antibody repertoire of the transgenic mice.

[0095] Like humans and mice, horses express two types of Ig light chains (κ and λ). However, the κ to λ ratio differs significantly between these animals. In mice, approximately 96% of the light chains in serum antibodies are of the κ type, whereas in humans the κ type represents only 66% of the total population of Ig L chains. In contrast, the L chain repertoire in horses is λ dominated (95%).

[0096] A partial equine nucleic acid sequence integrated into an Igh, IgK, or Igλ locus enables the transgenic animal to produce antibodies that contain an equine heavy chain variable region paired with an equine K or λ variable region. The partial equine immunoglobulin variable region locus retains regulatory sequences and other elements within the intervening sequences of the host genome (e.g., rodent), thereby facilitating efficient antibody production and antigen recognition in the host.

[0097] In some embodiments, synthetic or recombinantly produced immunoglobulin V H Partial equine immunoglobulin loci are provided that include equine coding sequences from a Vλ, Vκ or Vκ locus and non-equine non-coding regulatory or scaffolding sequences.

[0098] In some embodiments, the synthetic heavy chain DNA segment comprises one or more of the following sequences: the ADAM6 gene required for male fertility, a Pax-5 activating intergenic repeat (PAIR) sequence involved in Igh locus contraction, a CTCF binding site from heavy chain intergenic control region 1 involved in controlling normal VDJ rearrangement ((Proudhon, et al., Adv. Immunol., 128: 123-182 (2015)), or a combination thereof. The location of these endogenous non-coding control and scaffolding sequences in the mouse Igh locus is set forth in FIG. 1, which includes, from left to right: approximately 100 functional heavy chain variable region gene segments (101); PAIR, a Pax-5 activating intergenic repeat involved in Igh locus contraction for VDJ recombination (102); Adam6a, a disintegrin and metallopeptidase domain 6A gene required for male fertility (103); pre-D region, the most distal D H gene segment, a 21,609-bp fragment upstream of Ighd-5 (104);V H Intergenic control region 1 (IGCR1) containing CTCF insulator sites that control gene segment utilization (106);D H , diverse gene segments (10–15 depending on mouse strain) (105); 4 concatenated J H gene segments (107); Eμ, an intronic enhancer involved in VDJ recombination (108); Sμ, a μ switch region for isotype switching (109); eight heavy chain constant region genes: Cμ, Cδ, Cγ3, Cγ1, Cγ2b, C2γa / c, Cε, and Cα (110); and a 3' regulatory region (3'RR) that controls isotype switching and somatic hypermutation (111). Figure 1 is a modification of a diagram taken from Proudhon, et al., Adv. Immunol., 128: 123-182 (2015).

[0099] In one embodiment, the heterologous partial equine immunoglobulin locus integrated into the mammalian host cell is a known equine V H In some instances, however, such a V HA subset of gene segments may be desirable. In some embodiments, only one horse V H The coding sequence may be contained in a partial equine immunoglobulin locus.

[0100] In one embodiment, the non-equine mammal or mammalian cell is an equine V. H , D H and J. H The heterologous partial equine immunoglobulin locus includes gene coding sequences. In some embodiments, the partial equine immunoglobulin locus is based on the endogenous Igh locus of a non-equine mammalian host and includes non-coding regulatory and scaffolding sequences, such as pre-D sequences. In some embodiments, the heterologous partial equine immunoglobulin locus includes a complete recombined V(D)J exon.

[0101] In one embodiment, the transgenic non-equine mammal is an equine V. H , D H and J. H A transgenic rodent, e.g., a mouse, that comprises a heterologous, partial equine immunoglobulin locus that includes intervening sequences, e.g., including the pre-D region, based on the gene and the intervening (non-coding regulatory or scaffolding) sequences in the rodent. In one embodiment, the transgenic rodent further comprises a partial equine Igl locus that includes an intervening sequence, such as an equine Vκ or Vλ coding sequence and an equine Jκ or Jλ coding sequence, respectively, and a non-coding regulatory or scaffolding sequence present in the Igl locus of the rodent.

[0102] In one embodiment, the endogenous V H The entire immunoglobulin locus was deleted, resulting in 12 functional equine V H J558 V in gene segments and the mouse genome H In one embodiment, the heterologous immunoglobulin locus is replaced by a non-coding sequence of the 40 equine D H and 8 J's H In one embodiment, the heterologous immunoglobulin locus comprises a mouse pre-D region. In one embodiment, the heterologous immunoglobulin locus comprises a horse V H , DH and J. H The coding sequence is embedded within rodent non-coding sequences.

[0103] In some embodiments, a combination of homologous and site-specific recombination is used to generate transgenic cells and animals. In some embodiments, a homology targeting vector is used to introduce a sequence-specific recombination site into the genome of a mammalian host cell at a desired position in the endogenous immunoglobulin locus. In some embodiments, the sequence-specific recombination site is introduced into the genome of a mammalian host cell by homologous recombination and does not affect the expression or coding sequence of any other genes in the mammalian host cell. In some embodiments, the ability of the immunoglobulin gene to be transcribed and translated to produce an antibody is maintained after the recombination site, and any additional sequence, such as a selectable marker gene, is inserted as desired. However, in some cases, other heterologous sequences can be inserted into the immunoglobulin locus sequence so that the amino acid sequence of the resulting antibody molecule is altered by the insertion, but the antibody retains sufficient functionality for the desired purpose. In some embodiments, one or more genetic polymorphisms are introduced into the endogenous locus in a constant region exon, thereby providing an allotype marker so that different Ig alleles can be distinguished.

[0104] In some embodiments, a homology targeting vector is used to replace sequences within an endogenous immunoglobulin locus and insert a sequence-specific recombination site and one or more selectable marker genes into the host cell genome. As used herein, it will be understood by those skilled in the art that selectable marker genes can be used to identify and eliminate cells that have not undergone homologous recombination or that carry random integration of the targeting vector.

[0105] Methods of homologous recombination are known, and include those described in US Pat. Nos. 6,689,610; 6,204,061; 5,631,153; 5,627,059; 5,487,992; and 5,464,764, each of which is incorporated herein by reference in its entirety.

[0106] Site / Sequence-Specific Recombination Site / sequence-specific recombination differs from homologous recombination in that the site where recombination occurs is only the site where a short, specific DNA sequence is required for recognition by the recombinase. Depending on the orientation of these sites on a particular DNA strand or chromosome, specialized recombinases that recognize these specific sequences catalyze i) DNA excision or ii) DNA inversion or rotation. Site-specific recombination can also occur between two DNA strands if these sites are not present on the same chromosome. Several bacteriophage and yeast-derived site-specific recombination systems, each containing a recombinase and its cognate recognition site, have been shown to work in eukaryotic cells, including, but not limited to, the bacteriophage P1 Cre / lox system, the yeast FLP-FRT system, and the Dre system of the tyrosine family of site-specific recombinases. Such systems and methods are described, for example, in U.S. Patents 7,422,889; 7,112,715; 6,956,146; 6,774,279; 5,677,177; 5,885,836; 5,654,182; and 4,959,317; each of which is incorporated herein by reference.

[0107] Other systems of the tyrosine family of site-specific recombinases can be used, including, but not limited to, bacteriophage lambda integrase, HK2022 integrase, and systems belonging to the serine family of recombinases, including, for example, bacteriophage phiC31 and R4Tp901 integrase.

[0108] Site-specific recombination can be used to introduce heterologous immunoglobulin loci into a host cell genome through a process called recombinase-mediated cassette exchange (RMCE) because the site-specific recombination is between two different DNA strands. The RMCE method is utilized using wild-type and mutant sequence-specific recombination sites for the recombinase protein. In some embodiments, RMCE involves negative selection. For example, the targeted chromosomal locus can be flanked on one end by a wild-type LoxP site and on the other by a mutant LoxP site. Similarly, a vector can contain a sequence to be inserted into a heterologous host cell genome flanked on one end by a wild-type LoxP site and on the other by a mutant LoxP site. When the vector is transfected into a host cell in the presence of Cre recombinase, Cre recombinase catalyzes RMCE between the endogenous DNA strand and the DNA of the vector, rather than catalyzing an excision reaction of the same DNA strand, because the wild-type and mutant LoxP sites of each DNA strand are incompatible for recombination with each other. That is, the LoxP site in one DNA strand can only recombine with a LoxP site in the other DNA strand; similarly, the mutated LoxP site in one DNA strand can only recombine with a mutated LoxP site in the other DNA strand.

[0109] In some embodiments, variants of sequence-specific recombination sites that are recognized by the same recombinase for RMCE are used. Examples of such sequence-specific recombination site variants include those that contain combinations of inverted repeats or contain recombination sites with mutant spacer sequences. For example, two classes of variant recombinase sites are available for designing stable Cre-loxP integration recombination. Both explore sequence mutations within the 8bp spacer region or 13bp inverted repeat in the Cre recognition sequence. Spacer mutants such as lox511 (Hoess, et al., Nucleic Acids Res, 14: 2287-2300 (1986)), lox5171 and lox2272 (Lee and Saito, Gene, 216: 55-65 (1998)), m2, m3, m7 and m11 (Langer, et al., Nucleic Acids Res, 30: 3067-3077 (2002)) readily recombine with themselves, but the rate of recombination with the wild-type site is significantly reduced. This class of mutants has been exploited for DNA insertion by RMCE using non-interacting Cre-Lox and FLP recombination sites (Baer and Bode, Curr Opin Biotechnol, 12: 473-480 (2001); Albert, et al., Plant J, 7: 649-659 (1995); Seibler and Bode, Biochemistry, 36: 1740-1747 (1997); Schlake and Bode, Biochemistry, 33: 12746-12751 (1994)).

[0110] Inverted repeat mutants are another class of variant recombinase sites. For example, LoxP sites can contain modified bases in the left inverted repeat (LE mutant) or right inverted repeat (RE mutant). The LE mutant, lox71, has 5 bp at the 5' end of the left inverted repeat where the wild type sequence is changed to TACCG (Araki, et al, Nucleic Acids Res, 25: 868-872 (1997)). Similarly, the RE mutant, lox66, has the 5 most 3' bases changed to CGGTA. Inverted repeat mutants are used to integrate plasmid inserts into chromosomal DNA with the LE mutant designated as the "target" chromosomal loxP site where the "donor" RE mutant recombines. After recombination, the loxP sites are located in cis adjacent to the inserted segment. The mechanism of recombination is such that, after recombination, one loxP site is double mutant (containing both LE and RE inverted repeat mutations) and the other is wild type (Lee and Sadowski, Prog Nucleic Acid Res Mol Biol, 80: 1-42 (2005); Lee and Sadowski, J Mol Biol, 326: 397-412 (2003)). The double mutant is sufficiently different from the wild-type site that it is not recognized by Cre recombinase and the inserted segment is not excised.

[0111] In some embodiments, the sequence-specific recombination sites are introduced into an intron, but not into the coding or regulatory sequences, to avoid interference with regulatory or coding sequences used in antibody expression.

[0112] Introduction of sequence-specific recombination sites can be achieved by conventional homologous recombination techniques, as described in references such as, for example, Green and Sambrook (2012) (Molecular cloning: a laboratory manual 4th ed. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press) and Nagy, A. (2003). (Manipulating the mouse embryo: a laboratory manual, 3rd ed. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).

[0113] Specific recombination into genome can be promoted by using vectors designed for positive or negative selection, as known in the art.To promote the identification of cells undergoing replacement reaction, suitable genetic marker systems can be used, and cells are selected, for example, by using selective tissue culture medium.In some embodiments, heterologous sequences, for example, marker systems or nucleic acid sequences at or adjacent to two ends of genes, can be removed after selection of cells containing heterologous nucleic acid.

[0114] In some embodiments, cells in which the endogenous immunoglobulin locus has been deleted can be positively selected using a marker gene, which can be optionally removed from the cells after or as a result of a recombination event. A positive selection system that can be used is based on the use of two non-functional portions of a marker gene, such as hypoxanthine-guanine phosphoribosyltransferase (HPRT), which are linked via a recombination event. In some embodiments, the two non-functional portions are functionally linked upon successful replacement of the endogenous immunoglobulin locus with the heterologous immunoglobulin locus. In some embodiments, the functionally rearranged marker gene is further flanked on either side by sequence-specific recombination sites (different from the sequence-specific recombination sites used in the replacement reaction) so that the marker gene can be excised from the genome using an appropriate site-specific recombinase. In other embodiments, cells are negatively selected by exposure to a toxin or drug. For example, cells in which the targeting construct has not integrated by homologous recombination, but has randomly integrated into the genome, will maintain expression of herpes simplex virus-thymidine kinase (HSV-TK) if the HSV-TK gene is located outside the region of homology, and such cells can be selected for using nucleoside analogs such as ganciclovir.

[0115] In some embodiments, the recombinase is provided as a purified protein. In some embodiments, the recombinase is provided as a protein expressed from a vector construct that is transiently transfected into a host cell or stably integrated into a host cell genome. Alternatively, transgenic animals containing heterologous immunoglobulin loci can be bred with animals expressing the recombinase.

[0116] In certain embodiments, two or more sets of sequence-specific recombination sites are contained within the engineered genome such that multiple rounds of RMCE can be utilized for insertion of a partial equine immunoglobulin variable region locus into the genome of a non-equine mammalian host cell.

[0117] In some embodiments, partial equine immunoglobulin loci are introduced using CRISPR technology. For example, the CRISPR / Cas9 genome editing system can be used for targeted recombination (He, et al., Nuc. Acids Res., 44: e85, (2016)).

[0118] Production of transgenic animals In one embodiment, a method of making a transgenic animal, eg, a rodent, eg, a mouse, that comprises a heterologous partial equine immunoglobulin locus is provided.

[0119] In one embodiment, the genome of the transgenic animal is modified such that the B cells of the transgenic animal express more than one functional VH domain per cell, i.e., such that the cells produce bispecific antibodies as described in WO20170 / 35252, filed August 24, 2016, entitled "Enhanced Production of Immunoglobulins," the disclosure of which is incorporated herein by reference.

[0120] In certain embodiments, the genome of the transgenic animal is modified such that the B cells of the transgenic animal express antibodies that contain heavy chains but no light chains, ie, the cells produce heavy chain-only antibodies.

[0121] In some embodiments, the host cells are embryonic stem (ES) cells, which can then be used to generate transgenic mammals. In some embodiments, the methods include isolating embryonic stem cells that contain heterologous partial equine immunoglobulin loci and using the ES cells to generate transgenic animals that contain heterologous partial equine immunoglobulin loci. EXAMPLES

[0122] The following examples are presented to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors perceive as their invention, nor are they intended to represent or imply that the experiments described below are all or the only experiments performed. Those of ordinary skill in the art will recognize that numerous changes or modifications can be made without departing from the spirit or scope of the invention described herein. Thus, the examples should be construed as illustrative, and not limiting.

[0123] Efforts have been made to ensure accuracy with respect to periods and numbers used (e.g., vectors, amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0124] The examples describe the introduction of synthetic DNA via targeting and RMCE with both 5' and 3' vectors adjacent to the site of recombination. By reading this specification, it is clear to those skilled in the art that 5' vector targeting can occur first, followed by 3', or 3' vector targeting can occur first, followed by 5' vector. In some situations, targeting can be performed simultaneously using a dual detection mechanism. Although a different strategy is used in each example for the selection of cells with properly integrated 5' or 3' vector, it is also clear that such a strategy is compatible with targeting Igh, Igκ or Igλ locus with minor modifications.

[0125] Example 1: Introduction of a heterologous partial equine immunoglobulin variable region locus into an immunoglobulin heavy chain variable region locus of a non-equine mammalian host cell genome Exemplary methods illustrating the introduction of a heterologous partial equine immunoglobulin locus into the genomic locus of a non-mammalian ES cell are shown in Figures 2-4. HA method is described for the introduction of site-specific recombination sequences upstream (5') of a gene segment. A 5' homology targeting vector (201) is provided that contains a puromycin phosphotransferase-thymidine kinase fusion protein (puro-TK) (203) flanked by two different recombinase recognition sites (e.g., FRT (207) and loxP (205) for Flp and Cre, respectively) and two different mutant sites (e.g., modified mutant FRT (209) and mutant loxP (211)) that lack the ability to recombine with their respective wild-type counterparts / sites (i.e., wild-type FRT (207) and wild-type loxP (205)). The targeting vector contains a diphtheria toxin receptor (DTR) cDNA (217) for use in negative selection of cells. The targeting vector also optionally contains a visible marker such as green fluorescent protein (GFP) (not shown). Regions 213 and 215 are flanked by endogenous non-equine VTR (216) and VTR (217) that lack the ability to recombine with their respective wild-type counterparts / sites (i.e., wild-type FRT (207) and wild-type loxP (205)). H The homology targeting vector (201) is homologous to the 5' and 3' portions of a contiguous region (229) in an endogenous non-equine locus that is 5' of the genomic region containing the gene segment (219). H Gene segment (219), pre-D region (221), D H Gene segment (223), J H The site-specific recombination sequence and DTR cDNA from the homology targeting vector (201) are introduced (202) into ES cells carrying the immunoglobulin locus (231) containing the gene segment (225) and the immunoglobulin constant gene region gene (227). H It integrates with the non-equine genome at site 5′ of the locus ( 204 ), resulting in the genome structure described in 233 .

[0126] Mouse embryonic stem (ES) cells (derived from C57B1 / 6NTac mice) are transfected by electroporation with the 5' vector (201) according to known methods. Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that cuts only the prokaryotic plasmid sequence or the polylinker associated with it. The transfected cells are plated and, after about 24 hours, are placed under selection for cells in which the 5' vector (201) has been integrated into their DNA. ES cells that do not have the 5' vector (201) integrated into their genome can be selected (dead) by including puromycin in the culture medium; only ES cells in which the 5' vector (201) has been stably integrated into their genome and which constitutively express the puro-TK gene are resistant to puromycin.

[0127] Colonies of drug-resistant ES cells are physically extracted from the plates after about a week when they become visible to the naked eye. These pooled colonies are disaggregated and replated in microwell plates and cultured for several days. Each clone of cells is then split so that some cells can be frozen as an archive and the rest can be used for DNA isolation for analytical purposes. Primary screening methods for 5' vector introduction can be performed by Southern blotting or PCR with confirmation by a secondary screening method such as Southern blotting.

[0128] DNA from ES cell clones is screened by PCR using a widely implemented gene targeting assay design. For this assay, one of the PCR oligonucleotide primer sequences maps outside the region of shared identity between the 5' vector (201) and genomic DNA, while the other maps within the 5' vector, e.g., to the Puro-TK gene (203). By standard design, these assays detect DNA present only in clones of ES cells that have undergone homologous recombination between the 5' targeting vector and the endogenous mouse Igh locus.

[0129] Southern blot assays are performed according to widely used methods using three probes and multiple restriction enzyme digested genomic DNA, selected such that the combination of probes and digests allows identification of the structure of the targeted locus in the clone as appropriately modified by homologous recombination. One of the probes maps to the DNA sequence adjacent to the 5' side of the region of shared identity between the 5' targeting vector and the genomic DNA; the second probe maps outside the region of identity but on the 3' side; and the third probe maps within the novel DNA between the two arms of genomic identity in the vector, e.g., to the Puro-TK gene (203). The Southern blot identifies the predicted restriction enzyme generated fragment portion of the DNA corresponding to the modified sequence of the Igh locus, i.e., due to homologous recombination with the 5' targeting vector, detected by one of the external probes and the Puro-TK probe. The external probe detects mutant fragments and also wild-type fragments from the non-mutant copy of the immunoglobulin Igh locus in the homologous chromosome.

[0130] The ES cell karyotypes of PCR and Southern blot positive clones are analyzed using an in situ fluorescent hybridization method designed to distinguish the most commonly occurring chromosomal abnormalities occurring in mouse ES cells. Clones with such abnormalities are excluded from further use. ES cell clones with the predicted genomic structure based on the Southern blot data and with no detectable chromosomal abnormalities based on karyotype analysis are selected for further use.

[0131] As shown in Figure 3, a 3' homology targeting vector (301) is provided that contains an optional hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene (335) that can be used for positive selection in HPRT-deficient ES cells; a neomycin resistance gene (337); and recombinase recognition sites FRT (307) and loxP (305) for Flp and Cre, respectively. Regions 329 and 339 are located in the cytoplasm of the endogenous J HThe homology targeting vector is homologous to the 5' and 3' portions of the contiguous region (341) of the endogenous mouse locus downstream of gene segment (325) and upstream of the constant region gene (327), respectively. H gene segment (319), pre-D region (321), D gene segment (323), J H The gene segment (325) and the constant region genes (327) are introduced (302) into a modified mouse immunoglobulin locus (331). The site-specific recombination sequences of the homology targeting vector (307, 305), the HPRT gene (335), and the neomycin resistance gene (337) are integrated (304) into the mouse genome upstream of the endogenous mouse constant region genes (327), resulting in the genome structure described in 333.

[0132] Acceptable clones modified with the 3' vector (301) are identified using methods and screening assays essentially identical in design to those used with the 5' vector (201), except that neomycin or HPRT selection is used instead of puromycin for selection. PCR assays, probes and digests are also tailored to be compatible with genomic region modifications with the 3' vector. The ES cell karyotypes of PCR and Southern blot positive clones are analyzed using an in situ fluorescent hybridization method designed to distinguish the most commonly occurring chromosomal abnormalities occurring in mouse ES cells. Clones with such abnormalities are excluded from further use.

[0133] Clones of ES cells that have been mutated by both the 3' and 5' vectors, i.e., doubly targeted cells carrying both engineered mutations, are isolated after vector targeting and analysis. Clones must be subjected to gene targeting on the same chromosome as opposed to the homologous chromosome (i.e., the engineered mutations made by the targeting vectors must be in cis on the same DNA strand, not in trans on separate homologous DNA strands). Clones in the cis configuration are distinguished from those in the trans configuration by analytical methods such as fluorescent in situ hybridization of metaphase spreads using a probe that hybridizes to novel DNA present on the two gene targeting vectors (303 and 337) between the arms of genomic identity. The two types of clones can also be distinguished from each other by transfecting them with a vector expressing Cre recombinase that deletes the HPRT (335) and neomycin resistance (337) genes if the targeting vector is integrated in cis, and then analyzing the drug resistance phenotype of the clones by a "sib selection" screen in which a portion of cells from each clone is tested for resistance to G418 / neomycin. The majority of the resulting cis-derived clones are also sensitive to G418 / neomycin, in contrast to the trans-derived clones that retain resistance to the drug. A doubly targeted clone of cells carrying the cis configuration of the engineered mutations in the heavy chain locus is selected for further use.

[0134] Once the two recombination sites have been integrated into the mammalian host cell genome, the endogenous immunoglobulin locus is then subjected to recombination by introduction of one of the recombinases, e.g., Flp or Cre, that corresponds to the sequence-specific recombination site integrated into the genome. In the presence of Flp or Cre (302), the entire intervening sequence between the DTR gene (317), the endogenous Igh variable region locus (319, 323, 325), the pre-D region (321) and the wild-type FRT or wild-type LoxP site containing the HPRT (335) and neomycin resistance (337) genes is deleted, resulting in the genome structure shown in 339. The method relies on the second targeting occurring on the same chromosome but not its homolog (i.e., in cis rather than trans). If targeting occurs in cis as intended, the cells will not be susceptible to negative selection by diphtheria toxin introduced into the culture medium because the DTR gene that confers sensitivity to diphtheria toxin (317) is absent (deleted) in the host cell genome. Similarly, ES cells carrying random integration of the first or second targeting vector will be sensitive to diphtheria toxin due to the presence of the non-deleted DTR gene.

[0135] ES cell clones carrying a sequence deletion in one of the two homologous copies of the immunoglobulin heavy chain locus were cloned using equine V-type genomic DNA embedded in a Cre recombinase expression vector and mouse non-coding sequences. H , D H and J. H The vector is retransfected with a vector containing a partial equine immunoglobulin heavy chain locus that contains gene segment coding sequences. FIG. 4 shows that a portion of the endogenous immunoglobulin heavy chain locus that encodes the heavy chain variable region domain is transfected with the endogenous V H and J. H The introduction of a heterologous partial equine immunoglobulin heavy chain locus into a mouse genome that is deleted, including the intervening sequences between the loci, is described. A site-specific targeting vector (441) containing the partial equine immunoglobulin locus to be inserted into a non-equine host genome is introduced (402) into the modified genome (439) of the host cell by RMCE. H locus (419), mouse pre-D region (421), partial equine DH Locus (423), partial equine J H A site-specific targeting vector (441) containing the locus (425) and flanking mutant FRT (409), mutant LoxP (411), wild-type FRT (407) and wild-type LoxP (405) sites is introduced into host cells by RMCE (402). H Locus (419) identifies 12 functional equine Vs present in endogenous non-equine genomes. H Contains gene segment coding sequence and 3' non-equine RSS and intervening sequences; pre-D region (421) contains 21.6 kb of non-equine sequence present upstream in the endogenous non-equine genome; H Region (423) is adjacent to the non-equine RSS and contains the endogenous non-equine D H 40 horse D embedded in intervening sequences present in the region H containing the codons of the gene segment; and J H Locus (425) contains eight horse J sequences with a 5' non-equine RSS embedded in intervening sequences present in the endogenous non-equine genome. H In one embodiment, the Igh locus of the host cell genome comprises the codons of all endogenous V, including the intervening sequences, as described in connection with FIG. H , D H and J. H The gene segment is modified to be deleted. As a result of this modification, the endogenous non-equine Igh locus (439) remains with a puro-TK fusion gene (403) flanked by mutant FRT sites (409) and mutant LoxP sites (411) upstream and wild-type FRT (407) and wild-type LoxP (405) downstream. By introduction of the appropriate recombinase (404), the partial equine immunoglobulin locus is integrated into the genome upstream of the endogenous mouse constant region gene (427) between the lox5171 (411) and loxP (405) sites, creating the DNA region described in 443.

[0136] ES cells that have not undergone RMCE and integration of the partial equine Igh locus retain the puro-TK fusion gene (403) and are eliminated by the addition of ganciclovir to the tissue culture medium.

[0137] Horse V H , D H and J. H The sequences of the gene segments are shown in SEQ ID NOs: 1 to 65.

[0138] The integration of the heterologous partial equine immunoglobulin region can be detected by Southern blotting or PCR with confirmation by a secondary screening method such as Southern blotting. H , D H or J H The ES cell karyotypes of PCR and Southern blot positive clones are analyzed using an in situ fluorescent hybridization method designed to distinguish the most commonly occurring chromosomal abnormalities occurring in mouse ES cells. Clones with such abnormalities are excluded from further use.

[0139] ES cell clones carrying a partial equine immunoglobulin heavy chain variable region (443) at the mouse heavy chain locus are microinjected into mouse blastocysts from strain DBA / 2 by standard methods to generate ES cell-derived chimeric mice. Male chimeric mice with the highest contribution to ES cell-derived hair are selected for mating with female mice. Progeny from these matings are analyzed for the presence of the partial equine immunoglobulin heavy chain locus and used to establish a colony of mice carrying the partial equine immunoglobulin heavy chain locus.

[0140] Example 2: Introduction of a heterologous partial equine immunoglobulin locus into the immunoglobulin kappa chain locus of the mouse genome The method for replacing a portion of the mouse Igκ locus with a partial equine Igκ locus is shown in Figure 5. This method replaces the endogenous V K (515) and J K(519) The introduction of a first site-specific recombinase recognition sequence into the mouse genome that can be introduced 5' or 3' to a cluster of constant region gene segments, followed by a first sequence-specific recombination site in combination with a V upstream of the constant region gene (521). K and J. K The method includes the introduction of a second site-specific recombinase recognition sequence into the mouse genome that flanks the entire locus including the gene segment, and using the associated site-specific recombinase, deletes the flanking regions and replaces them with a partial equine immunoglobulin light chain variable region locus.

[0141] V K (515) and J K (519) The targeting vector used to introduce the site-specific recombination sequences on either side of the gene segment also contains additional site-specific recombination sequences that are still efficiently recognized by the recombinase but are modified so that they cannot recombine with unmodified sites. K and J. K After gene segment cluster deletion, heterologous immunoglobulin light chain variable region loci are modified via RMCE K The heterologous immunoglobulin light chain variable region locus is placed in a targeting vector for use in a second site-specific recombination event to insert into the locus. In this example, the heterologous immunoglobulin light chain variable region locus is the equine V K and J. K It is a synthetic nucleic acid comprising gene segments and mouse Igκ variable region non-coding sequences.

[0142] Two gene targeting vectors are constructed to accomplish the steps outlined immediately above. One of the vectors (503) contains the most distal V K The other vector (505) contains mouse genomic DNA (525 and 541) taken from the 5' end of the gene locus, upstream of the gene segment. K It contains mouse genomic DNA (543 and 549) taken from within the locus downstream (3') of gene segment (519) and upstream of the constant region gene (521).

[0143] The key features of the 5' vector (503) are: a gene encoding the diphtheria toxin A subunit (DTA) under the transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter linked to two mutant transcriptional enhancers from polyomavirus (523); 6 Kb of mouse genomic DNA mapping upstream of the distal-most variable region gene in the kappa chain locus (525); an FRT recognition sequence for Flp recombinase (527); a piece of genomic DNA containing the mouse Polr2a gene promoter (529); a translation initiation sequence (535, a "Kozak" consensus sequence a methionine codon embedded in the cytosine codon; a mutated loxP recognition sequence for Cre recombinase (lox5171) (531); a transcription termination / polyadenylation sequence (533); a loxP recognition sequence for Cre recombinase (537); a gene encoding a fusion protein containing a protein conferring resistance to puromycin fused to a truncated form of thymidine kinase (pu-TK) under the transcriptional control of a promoter from the mouse phosphoglycerate kinase 1 gene (539); and 2.5 Kb of mouse genomic DNA at the 5' end of the vector, mapped to a 6 Kb sequence and arranged in a native-related orientation (541).

[0144] The key properties of the 3' vector (505) are: κ (519) and C κ(521) 6 Kb of mouse genomic DNA mapping within an intron between the loci (543); a gene encoding human hypoxanthine-guanine phosphoribosyltransferase (HPRT) under the transcriptional control of the mouse Polr2a gene promoter (545); a neomycin resistance gene under the control of the mouse phosphoglycerate kinase 1 gene promoter (547); a loxP recognition sequence for Cre recombinase (537); 3.6 Kb of mouse genomic DNA (549) mapping immediately downstream of the genome of a 6 Kb DNA fragment contained at the 5' end of the vector, with the two fragments oriented in the same relative manner as in the mouse genome; and a gene encoding diphtheria toxin A subunit (DTA) under the transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter linked to two mutant transcriptional enhancers from polyomavirus (523).

[0145] Mouse embryonic stem (ES) cells derived from C57B1 / 6NTac mice are transfected with the 3' vector (505) by electroporation using known methods. Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that cuts only the prokaryotic plasmid sequence or the polylinker associated with it. The transfected cells are plated and, after approximately 24 hours, are under positive selection for cells whose DNA has integrated the 3' vector by use of the neomycin analog drug G418. There is also negative selection for cells whose DNA has integrated the vector but not by homologous recombination. Non-homologous recombination results in retention of the DTA gene, which will kill the cell when the gene is expressed, whereas the DTA gene is deleted by homologous recombination because it is outside the region of the vector that is homologous to the mouse Igκ locus. Colonies of drug-resistant ES cells are physically extracted from the plates after about one week when they become visible to the naked eye. These collected colonies are disaggregated and replated into microwell plates and cultured for several days. Each of the cell clones is then split so that some cells are frozen as an archive and the rest can be used to isolate DNA for analytical purposes.

[0146] DNA from ES cell clones is screened by PCR using gene targeting assays. For this assay, one of the PCR oligonucleotide primer sequences maps outside the region of shared identity between the 3' vector (505) and the genomic DNA (501), while the other maps within the new DNA between the two arms of genomic identity of the vector, e.g., the HPRT (545) or neomycin resistance (547) genes. These assays detect pieces of DNA that are present only in clones of ES cells derived from transfected cells that have undergone homologous recombination between the 3' vector (505) and the endogenous mouse Igκ locus. PCR-positive clones are selected for expansion and further analysis using subsequent Southern blot assays.

[0147] A Southern blot assay is performed by known methods; it includes three probes and genomic DNA digested with multiple restriction enzymes, selected so that the combination of probes and digests can be used to draw conclusions about the structure of the targeted locus in the clone and whether it has been appropriately modified by homologous recombination. One of the probes maps to a DNA sequence adjacent to the 5' side of the region of shared identity between the 3' kappa targeting vector (505) and the genomic DNA; a second probe also maps outside the region of identity but on the 3' side; a third probe maps within the novel DNA between the two arms of genomic identity in the vector, e.g., the HPRT (545) or neomycin resistance (547) gene. The Southern blot identifies the expected restriction enzyme-generated fragment of DNA that corresponds to the portion of the kappa locus that has been correctly, i.e., mutated by homologous recombination with the 3' kappa targeting vector (505), as detected by one of the external probes and the neomycin resistance or HPRT gene probe. The external probe detects the mutant fragment and also the wild-type fragment from the non-mutant copy of the immunoglobulin kappa locus on the homologous chromosome.

[0148] The ES cell karyotypes of PCR and Southern blot positive clones are analyzed using an in situ fluorescent hybridization method designed to distinguish the most commonly occurring chromosomal abnormalities occurring in mouse ES cells. Clones with such abnormalities are excluded from further use. Karyotypically normal clones, judged to have the correct genomic structure predicted based on the Southern blot data, are selected for further use.

[0149] Acceptable clones are then modified with the 5' vector (503) using methods and screening assays essentially identical in design to those used with the 3' vector (505), except that puromycin selection is used instead of G418 / neomycin selection and the protocol is tailored to be compatible with the genomic region modified by the 5' vector (503). The goal of the 5' vector (503) transfection experiment is the isolation of clones of ES cells that are mutated in the predicted manner by both the 3' vector (505) and the 5' vector (503), i.e., doubly targeted cells carrying both engineered mutations. In these clones, Cre recombinase causes recombination (502) between the loxP sites introduced at the kappa locus by the two vectors, resulting in the genomic DNA configuration shown in 507.

[0150] Furthermore, clones must be subjected to gene targeting on the same chromosome, as opposed to the homologous chromosome; i.e., the engineered mutations made by the targeting vectors must be in cis on the same DNA strand, not in trans on separate homologous DNA strands. Clones in the cis configuration are distinguished from those in the trans configuration by analytical methods such as fluorescent in situ hybridization of metaphase spreads, using a probe that hybridizes to the novel DNA present in the two gene targeting vectors (503 and 505) between the arms of genomic identity. The two types of clones can also be distinguished from each other if the targeting vector is integrated in cis with a Cre recombinase expressing vector that deletes the pu-Tk (539), HPRT (545), and neomycin resistance (547) genes, by comparing the number of colonies that survive ganciclovir selection against the thymidine kinase gene introduced by the 5' vector (503), and the drug resistance phenotype of the surviving clones can be analyzed by a "sib selection" screening method in which a portion of cells from the clones are tested for resistance to puromycin or G418 / neomycin. Cells carrying the mutation in the cis configuration are approximately 10 times more likely to survive than those carrying the mutation in the trans configuration. 3 This is predicted to result in clones that are more resistant to ganciclovir. The majority of the resulting cis-derived ganciclovir-resistant clones are also sensitive to both puromycin and G418 / neomycin, in contrast to the trans-derived ganciclovir-resistant clones that retain resistance to both drugs. Clones of cells carrying the cis configuration of the engineered mutations in the kappa chain loci are selected for further use.

[0151] The double-targeted clones of cells are transiently transfected with a vector expressing Cre recombinase (502), and the transfected cells are then placed under ganciclovir selection as in the analytical experiments summarized above. Ganciclovir-resistant clones of cells are isolated and analyzed by PCR and Southern blot for the presence of the predicted deletion (507) between the two engineered mutations made by the 5' vector (503) and the 3' vector (505). In these clones, Cre recombinase causes recombination between the loxP sites (537) introduced into the kappa chain locus by the two vectors. Because the LoxP sites are positioned in the same relative orientation in the two vectors, recombination results in the excision of a circle of DNA that includes the entire genomic interval between the two loxP sites. The circle does not contain an origin of replication and therefore does not replicate during mitosis, and is therefore lost from the clone of cells as it is subjected to clonal expansion. The resulting clones have a deletion of the DNA that was originally between the two loxP sites. The ES cell karyotypes of PCR and Southern blot positive clones are analyzed using an in situ fluorescent hybridization method designed to distinguish the most commonly occurring chromosomal abnormalities occurring in mouse ES cells. Clones with such abnormalities are excluded from further use. Karyotypically normal clones, judged to have the correct genomic structure predicted based on the Southern blot data, are selected for further use.

[0152] ES cell clones carrying a deletion of sequences in one of the two homologous copies of the immunoglobulin kappa chain locus are retransfected with a Cre recombinase expression vector and a vector (509) containing a partial equine immunoglobulin kappa chain locus including the Vκ(551) and Jκ(555) gene segments (504). Key features of the vector include: a lox5171 site (531); a neomycin resistance gene open reading frame (547, lacking an initiator methionine codon but in frame and contiguous with the uninterrupted open reading frame at the lox5171 site (531) downstream of the methionine start codon (535); an FRT site (527); an array of 19 horse Vκ gene segments (551), each containing horse coding sequence flanked on the 3' side by a mouse RSS and embedded in mouse noncoding sequences; optionally, a 13.5 Kb piece of genomic DNA from just upstream of a cluster of J kappa region gene segments at the mouse kappa chain locus (not shown); DNA containing four horse Jκ region gene segments flanked on the 5' side by a mouse RSS and embedded in mouse noncoding DNA (555); and a loxP site (537) in the reverse relative orientation to the lox5171 site (531).

[0153] The sequences of the horse Vκ and Jκ gene coding regions are shown in SEQ ID NOs:66-86.

[0154] The transfected ES clones are placed under G418 selection, which enriches for clones of cells subjected to RMCE in which the donor DNA (509), containing a partial equine immunoglobulin kappa chain locus, is fully integrated into the deleted endogenous immunoglobulin kappa chain locus between the lox5171 (531) and loxP (537) sites placed by the 5' (503) and 3' (505) vectors, respectively. Only cells properly subjected to RMCE have the ability to express the neomycin resistance gene (547) because the promoter (529) and the initiator methionine codon (535) required for its expression are absent from the vector (509) and are already present in the modified host cell Igκ locus (507). The DNA region created using the 509 sequence is illustrated in 511. The remaining sequences from the 5' vector (503), located between the FRT sites (527), are removed by Flp-mediated recombination (506) in vitro or in vivo as described below, resulting in a partial equine immunoglobulin light chain locus shown at 513.

[0155] G418-resistant ES cell clones are analyzed by PCR and Southern blotting to determine whether they have undergone the expected RMCE process without unwanted rearrangements or deletions. The ES cell karyotypes of PCR- and Southern blot-positive clones are analyzed using an in situ fluorescent hybridization method designed to distinguish the most commonly occurring chromosomal abnormalities occurring in mouse ES cells. Clones with such abnormalities are excluded from further use. Karyotypically normal clones, judged to have the correct genomic structure predicted based on the Southern blot data, are selected for further use.

[0156] ES cell clones carrying a partial horse immunoglobulin kappa chain locus at the endogenous mouse immunoglobulin kappa chain locus (513) are microinjected into mouse blastocysts from strain DBA / 2 by standard methods to generate partial ES cell-derived chimeric mice. Male chimeric mice with the highest contribution to ES cell-derived hair are selected for mating with female mice. Female mice selected for use in offspring are of the C57B1 / 6NTac strain and also carry a transgene encoding Flp recombinase that is expressed in the germline and deletes the FRT-flanked neomycin resistance gene (520) and other elements from the 5' vector. Progeny from these matings are analyzed for the presence of the partial horse immunoglobulin kappa chain locus and for loss of the neomycin resistance gene. Mice carrying the partial horse immunoglobulin kappa chain locus are used to establish a mouse colony.

[0157] Mice bearing a partial equine immunoglobulin heavy chain locus, produced as described in Example 1, can be bred with mice bearing a partial equine immunoglobulin kappa chain locus. These offspring are then bred with each other in a scheme that ultimately produces mice that are homozygous for both partial equine Igh and partial equine Igκ. Such mice produce partial equine heavy chains that contain equine variable domains and mouse constant domains. They also produce partial equine Kappa proteins that contain equine Kappa variable domains and mouse Kappa constant domains. Monoclonal antibodies recovered from these mice contain equine heavy chain variable domains paired with equine Kappa variable domains.

[0158] In one embodiment, mice homozygous for both partial equine Igh and partial equine Igκ are bred with mice homozygous for the partial equine lambda locus generated in Example 3 to produce mice homozygous for all three loci.

[0159] Those skilled in the art will recognize that the 5' vector (503) and subsequent strategy used herein to target the Igκ locus can be used in place of the 5' vector (201) in Figure 2 as an alternative strategy to target the Igh locus, in which the 5' vector (503) is modified to replace the genomic DNA regions homologous to the Igκ locus (525 and 541) with genomic DNA regions homologous to the Igh locus (213 and 215 in Figure 2).

[0160] Example 3: Introduction of a heterologous partial equine immunoglobulin locus at the immunoglobulin lambda chain locus of the mouse genome A method for replacing a portion of the mouse Igλ locus with a partial equine Igλ locus is illustrated in Figures 6A and 6B. The method involves the deletion of approximately 200 Kb of DNA from the wild-type mouse immunoglobulin lambda locus (601 and Figure 1, bottom), including the Vλ2 / Vλ3 gene segments (613), the Jλ2 / Cλ2 gene cluster (615), and the Vλ1-Jλ3 / Cλ3-Jλ1 / Cλ1 gene cluster (617), by a homologous recombination method involving a targeting vector (603) that shares identity with the endogenous mouse immunoglobulin lambda locus both upstream of the Vλ2 / Vλ3 gene segments (613) and downstream of the Cλ1 gene segment (right-most box in 617) and both upstream or downstream of the Eλ enhancer (623). The vector replaces approximately 200 Kb of endogenous mouse genomic DNA with sequences designed to allow for subsequent site-specific recombination to replace the heterologous immunoglobulin lambda locus with a modified Vλ locus by RMCE (604). In this example, the heterologous immunoglobulin lambda locus is a synthetic nucleic acid that contains equine Igλ coding sequences and mouse Igλ non-coding sequences.

[0161] Key features of the gene targeting vector (603) for achieving the approximately 200 Kb deletion and site-specific recombination site insertion are: a negative selection gene (not shown), such as a gene encoding the A subunit of diphtheria toxin (DTA, 659) or the herpes simplex virus thymidine kinase gene; 4 Kb of genomic DNA from the 5' of the mouse Vλ2 / Vλ3 variable region gene segment at the immunoglobulin lambda locus (625); an FRT site (627); genomic DNA containing the mouse Polr2a gene promoter (629); a translation initiation sequence (a methionine codon embedded in a "Kozak" consensus sequence) (635); a mutated loxP recognition sequence for Cre recombinase (lox5171) (631); a transcription termination / polyadenylation sequence (633); an open reading frame encoding a protein that confers resistance to puromycin (637), while this open reading frame is encoded by the Polr a loxP recognition sequence for Cre recombinase (639); a translation initiation sequence on the same antisense strand as the puromycin resistance gene open reading frame (a methionine codon embedded in a "Kozak" consensus sequence) (635); a chicken beta-actin promoter and cytomegalovirus early enhancer element (641) oriented to direct transcription of the puromycin resistance open reading frame, with translation initiating at the start codon downstream of the loxP site (635) and leading back through the loxP site to the puromycin open reading frame, all antisense to the adjacent Polr2a promoter and translation initiation sequence; genomic DNA containing a mutant recognition site for Flp recombinase (643); and an Eλ enhancer element (623) (645).

[0162] Mouse embryonic stem (ES) cells derived from C57B1 / 6NTac mice are transfected (602) with the targeting vector (603) by electroporation using known methods. Homologous recombination replaces the endogenous mouse immunoglobulin lambda gene with the site-specific recombination site from the targeting vector (603) in an approximately 200 Kb region, resulting in the genomic DNA configuration described in (605).

[0163] Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that cuts only the prokaryotic plasmid sequence or the polylinker that joins it. The transfected cells are plated and, after approximately 24 hours, placed under positive drug selection using puromycin. There is also a negative selection for cells whose DNA has integrated the vector, but not by homologous recombination. Non-homologous recombination results in the retention of the DTA gene (659), which will kill the cell when the gene is expressed, whereas the DTA gene is deleted by homologous recombination, since it is outside the region of the vector that is homologous to the mouse Ig λ locus. Colonies of drug-resistant ES cells are physically extracted from the plates when they become visible to the naked eye after one week. These pooled colonies are disaggregated and replated in limiting dilution in microwell plates and cultured for several days. Each of the cell clones is then split so that some cells can be frozen as archives and the rest can be used for DNA isolation for analytical purposes.

[0164] DNA from ES cell clones is screened by PCR using known gene targeting assays. For these assays, one of the PCR oligonucleotide primer sequences maps outside the region of shared identity between the targeting vector and genomic DNA, while the other maps within the novel DNA between the two arms of genomic identity of the vector, e.g., the puro gene (637). These assays detect pieces of DNA that are present only in clones of cells derived from transfected cells that have undergone homologous recombination between the targeting vector (603) and endogenous DNA (601).

[0165] PCR positive clones from the transfections are selected for expansion and subsequent further analysis using a Southern blot assay, which contains genomic DNA from the clones digested with three probes and multiple restriction enzymes, such that the combination of probes and digests allows identification of whether the ES cell DNA has been appropriately modified by homologous recombination.

[0166] ES cell karyotypes of PCR- and Southern blot-positive clones are analyzed using an in situ fluorescent hybridization method designed to distinguish the most commonly occurring chromosomal abnormalities occurring in mouse ES cells. Clones showing evidence of abnormalities are excluded from further use. Karyotypically normal clones, judged to have the correct genomic structure predicted based on the Southern blot data, are selected for further use.

[0167] ES cell clones lacking one of the two homologous copies of the immunoglobulin lambda chain locus are retransfected with a Cre recombinase expression vector (604) together with a vector containing a partial equine immunoglobulin lambda chain locus including equine Vλ and Jλ region gene segment coding sequences (607). The key features of this vector (607) are: a lox5171 site (631); a neomycin resistance gene open reading frame (647) that lacks an initiator methionine codon but is in frame and contiguous with the uninterrupted open reading frame at the lox5171 site (631 in diagram 605); an FRT site (627); an array of 27 functional horse lambda variable region gene segments (651), each gene segment containing horse lambda coding sequence flanked on the 3' side by a mouse RSS and embedded in mouse lambda non-coding sequences; and an array of JC units, each unit containing a horse Jλ gene segment and a mouse lambda constant domain gene segment embedded within non-coding sequences from the mouse lambda locus (655), containing an Eλ 2-4 enhancer element (Figure 1). The horse Jλ gene segments are those encoding Jλ1, Jλ5, Jλ6 and Jλ7 (the other Jλ gene segments are pseudogenes), while the mouse lambda constant domain gene segments are Cλ1, Cλ2 or Cλ3 or combinations thereof; a mutant recognition site for Flp recombinase (643); an open reading frame conferring hygromycin resistance (657), located on the antisense strand to the immunoglobulin gene segment coding information in the construct; and a loxP site in the reverse relative orientation to the lox5171 site (639).

[0168] RCME inserts a partial equine immunoglobulin lambda chain locus (607) from the RCME vector into the modified endogenous mouse Igλ locus, resulting in the genomic DNA configuration shown in 609.

[0169] The sequences of the horse Vλ and Jλ gene coding regions are shown in SEQ ID NOs:87-122.

[0170] The transfected clones are placed under G418 or hygromycin selection, which enriches for clones of cells that have been subjected to the RMCE procedure in which the partial equine immunoglobulin lambda chain variable is integrated into the deleted endogenous mouse immunoglobulin lambda chain locus between the lox5171 and loxP sites placed by the gene targeting vector. The remaining sequences from the targeting vector (603) are removed by in vitro or in vivo FLP-mediated recombination (606) (see below) to obtain the final partial equine immunoglobulin lambda chain locus shown in 611.

[0171] A more detailed view of one configuration of a partial equine immunoglobulin lambda chain locus is at 613 and is provided as an example only. Other configurations and numbers of equine Vλ and Jλ gene segments and mouse Cλ gene segments as well as the location and number of enhancer sequences are possible.

[0172] G418 / hygromycin resistant ES cell clones are analyzed by PCR and Southern blotting to ensure that they have undergone the expected recombinase-mediated cassette exchange without unwanted rearrangements or deletions. ES cell PCR and Southern blot positive clones are analyzed for karyotype using an in situ fluorescent hybridization method designed to distinguish the most commonly occurring chromosomal abnormalities occurring in mouse ES cells. Clones that show evidence of abnormalities are excluded from further use. Karyotypically normal clones, judged to have the correct genomic structure predicted based on the Southern blot data, are selected for further use.

[0173] ES cell clones carrying a partial equine immunoglobulin lambda chain locus (611) at the mouse immunoglobulin lambda chain locus are microinjected into mouse blastocysts from strain DBA / 2 by known methods to generate partially ES cell-derived chimeric mice. Male chimeric mice with the highest contribution to ES cell-derived hair are selected for mating with female mice of the C57B1 / 6NTac strain carrying a transgene encoding Flp recombinase expressed in the germline and lacking an FRT-flanked selectable marker. Progeny from these matings are analyzed for the presence of the partial equine immunoglobulin lambda chain locus and for the loss of the FRT-flanked neomycin resistance gene and the mFRT-flanked hygromycin resistance gene created in the RMCE process. Mice carrying the partial equine immunoglobulin lambda chain locus are used to establish a mouse colony.

[0174] In one embodiment, mice homozygous for a partial equine immunoglobulin heavy chain locus and a partial equine immunoglobulin kappa light chain locus (as described in Examples 1 and 2) are bred with mice carrying a partial equine immunoglobulin lambda light chain locus. Mice produced from this type of breeding scheme are homozygous for a partial equine Igh locus and homozygous for partial equine Igκ and Igλ loci. Monoclonal antibodies recovered from these mice contain equine heavy chain variable domains paired in some cases with equine kappa variable domains and in other cases with equine lambda variable domains. [Table 1] 1 Nomenclature of Sun, et al. Dev. Comp. Immunol. 34: 1009(2010) 2 Nomenclature of Walther, et al. Dev. Comp. Immunol. 53: 303(2015) 3This cDNA is derived from E. asinus, the others from E. caballus

[0175] Sequence information Note: (RC) stands for reverse complement, indicating that the sequence is in the reverse orientation compared to another sequence. IGHV SEQ ID NO: 1: VH1 IGHV1-5*01 L1: ATGGACTGGAGCTGGAGCATCCTCTTCTTGGTGGCAGTGGCTGCAG L2: GTGTCTCCTCC VH: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCAGGGGCATCCGTGAAGGTCTCCTGCAAGGCTTCTGGAGACAGCTTCACTTATTACTCTATGAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGACGGGAT ATATCTATCCTGAATATGATGCTATGGGCTACCCGCAGAAGTTCCAGGGCAGAGTCACCATGACTGCGGACAAGTCCACGAGCACAGTCTACATGGAGCTGAGCAGTCTGGCATCTGAGGACACAGCCGTGTATTACTGTGCAACAGA SEQ ID NO:2: VH2 IGHV4-11*01 L1: ATGAATCACCTGTGGTTCTTCCTCTTTCTGGTGGCCGCTCCTGCAT L2: GTGTCCTGTCC VH: CAGGTGCAACTGAAGGAGTCAGGACCTGGCCTGGTGAAGCCCTCGCAGACCCTGTCCCTCACCTGCCCTGTCTCTAGATTCCCTTTAACCAACCATCATGTACACTGGACCCACCAGGCTCCAGGAAAAGGGCTGGAGTGGCTTGGTGATTCAAGGAGTGGTGAAAGCACATACTACAACTTAACTCTGAAGTCCCAACTCAGCATCCCCAGTGATACTTCCAAAAGCCAAATTTATTTAACGCTGAACAGGCTGAGAGGCGATGACATGGCCATGTACTACTGTGCCAGAGA Accession No. 3: VH3 IGHV4-17*02 L1: ATGAGACTCTTGTGTCTTCTCCTTTGCCTGGTGATGGCTCCCCAAG L2: GAGTCCTGTCC VH: CAGGTGAAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCCTCACAGACCCTCTCCCTCACCTGCTCTGTGTCTGGAGTCTCCATCACAAGCAGTGGTGACTGGTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAATGGATGGGGTACATAAGTTATAGTGGTAGCGCTTACTACACCACATCCCTCAAGAGCCGACTCTCCATCTCCAGAGACACGTCCAAGGACCAGTTCTCCCTGCAGCTGAGCTCCGTGACCACAGAGGACACGGCCGTTTATTACTGTGCAAGTGA Accession No. 4: VH4 IGHV4S1 L1: ATGAATCACCTGTGGTTCTTCCTCTTTCTGGTGGCCGCTCCTACAT L2: GTGTCCTGTCC VH: CAGGTGCAACTGAAGGAGTCGGGACCTGGCCTGGTGAAGCCCTCGCAGACCCTGTCCCTCACCTGCACTGTCTCTGGATTATCTTTGAGCAGTAATGCTGTAGGCTGGGTCCGCCAGGCTCCAGGAAAAGGGCTGGAGTGGGTTGGTGTTATATATGGTAGTGAAAGTACATACTACAACCCAGCCCTGAAGTCCCGAGCCAGCATCACCAAGGACACCTCAAAGAGCCAAGTTTATCTGACGCTGAACAGCCTGACAGGCGAAGACACGGCCGTCTATTACTGTGCAGGATG Accession No. 5: VH5 IGHV4-29*02 L1: ATGAGTCACCTGTGGTTCTTCCTCTTTCTGGTGGCCGCTCCTACAT L2: GTGTCCTGTCC VH: CAGGTGCAACTGAAGGAGTCAGGACCTGGCCTGGTGAAGCCCTCGCAGACCCTGTCCCTCACCTGCACTGTCTCTGGATTATCTTTGAGCAGTTATGCTGTAGGCTGGGTCCGCCAGGCTCCAGGAAAAGGGCTGGAATATGTTGGTGCTATATATGGTAGTGCAAGTGCAAACTACAACCCAGCCCTGAAGTCCCGAGCCAGCATCACCAAGGACACCTCAAAGAGCCAAGTTTATCTGACGCTGAACAGCCTGACAGGCGAGGACACGGCCGTCTATTACTGTGCGAGA Accession No. 6: VH6 IGHV1-41*01 L1: ATGGACTGGAGCTGGAGCATCCTCTTCTTGGTGGCAGTGGCTGCAG L2: GTGTCTCCTCC VH: GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAGGAAGCCAGGGGCATCCGTGAAGGTCTCCTGCAAGGCTTCTGGAGACAGCTTCACTTATTACTCTATGAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGACTGGATGGGAGGGATCTTGCCTATAGTTGATGATACAAGCTACACGCAGAAGTTCCAGGGCAGAGTCACCATGACTGCAGACAAGTCCACGAGCACAGTCTACATGGAGCTGAGCAGTCTGACATCCGAGGACACGGCCGTGTATTACTGTGCAAAAGA Accession number 7: VH7 IGHV1-70*01 L1: ATGGGCTGGAGCTGGAGAATCCTCTTCTTGGTGGCAGTAGCTTCAG L2: GTGTCTCCTCC VH: GAGGGTCAGCTGGAACAGTCGGGGCCGGAGTTGAAGAAGCCTGGGTCATCAGTGAAGATCTCCTGCAAGGCTTCTGGATACACCTTCAGTAGCTATGCTGTGCACTGGGTGCGACAGGCCAATGGAAAAGGGATTGAGTGGATGGGATCTATCTATGCTGAATATGATGATACAAGCTACGCACCGAAGTTCCAGGGCAGAGTCACCATGACTGCGGACAAGTCCACGAGCACAGTCTACATGGAGCTGAGCAGTCTGACATCTGAGGACATGGCCGTGTATTACTGTGCAACAGA Accession number 8: VH8 IGHV9-66*01 L1: ATGGCCCCTCTCCTGGTCATCTTCTGCCTGCTGGCTGCTCTCCACG L2: GTGTCGAGGCT VH: GAGGACCCTCTCGTGCAATGGGGAGGTGGAGTGGTGGTCTCCTCACAGACACTCAGCCTCACCTGTGCCGCCTACAAACGCAAAGTTTCAGAATATTCCCTGTGGTGGATTCGCCTTCTCCCAGGGAAGGGGTTGGAGTGCGTAGGTGTGATCTGGGCTAAGGGGGACACTCAGTGCAGCCCCCACCTGCAGTCTCGAGTCAGCATCTCCAGGGACGCCACCAAGAACCAAGTGTTCTTACAGCTGAGCAGTGTGATGCCTGAGGATTCAGGCGTGTATTACTGTGCTCAAGA Accession No. 9: VH9 IGHV4-65*02 L1: ATGAGACTCTTGTGTCTTCTCCTTTTCCTGGTGACGGCTCCCCAAG L2: GAGTCCTGTCC VH: CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGCAGCCCTCACAGACCCTGTCCCTCACCTGCACTGTCACTGGAGGCTCCATCACAAGCAGCTATTCTAGCTGGAGCTGGTTACGCCAGCCTCCAGGGAAGGGGCTGGAGTACATGGGGTACATATATTATGATGGTAGAACTTACTACAATCCTTCCTTCAAGAGCCGCACCTCCATCTCCAGAGACACCTCCAGGAACCAGTTCTCCCTGCAGCTGAGCTCCGTGACCACCGAGGACGCGGCCGTGTATTACTGTGCAAGAGA Accession No. 10: VH10 IGHV2-63*01 L1: ATGGACACACTGTATCCCACCCTCCTGCTGCTGACCATCCCTTCCT L2: GGGCTCTGTCC VH: CAGATCAGCCTGCAGGAGTCTGGTCCTGGGCTGCTGAAGCCCACCCAGACCCTTACGCTGACCTGCTCCTTCTCTGGGTTCTCACTGACTACTTCTGATATTGGTGTTGGTTGGATGCGTCAACCCCCTGGGAAGGCACTGGAGTGGCTCACCTATGTTTGGTGGACTGATGAAAAGCATTACAACCCATCTCTGAAGAGCCGGCTCACAATCTCCAAGGACACCTCCAAAAACCAGGTGATGCTGACAATGACCAGTTTGGACCCTCCAGACACAGCCACATATTACTGTGTAAAGAGGG Accession No. 11: VH11 IGHV4-59 L1: ATGAGGAGGCTGGGTCTTCTCCTTTTCCTGGTGACGGCTCCCCAAG L2: GTGTCCTCTCC VH: CAGGTGCAGCTGCAGGAGTCAGGACCAGGCCAGACGAATCCCTCACAGACCCTGTCCCTCACATGCACTGTCACTGGTTACTCCATCACCAGTGGTTATGGCTGGAACTGGATCCGCCAGCCACCAAACAAAGGGCTGGAGTGGATGGGGAGCATAAGCTATAGTGGTAGAACTAACTACAGCCCATCCCTCAGGAGCCGCATCACCATCTCCAGAGACACTTCCAAGAACCAGTTCTTGCTGCAGCTGAGCTCAGTAACCACTGAGGACACGGCCGTGTATTACTGTGCGACAGA Accession No. 12: VH12 IGHV4-55 L1: ATGAGGCTGTTGGGTCTTCTCCTTTGTCTTGTGACGGCTTACCAGG L2: GTGTCCTGTCC VH: CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCCTCACAGACCCTCTCCCTCACCTGCACTGTCACTGGTTACTCCATCACCAGTGGTTACTACTGGAGCTGGATCCGTCAGCCCCCAGGAAAGAGGCTGGAGTGGATGG GCTCCATATATTATAGTGGTAGCACTTACTACAGCCCATCCCTCAAGAGCCGCATCACCATCTCCACAGACACGTCCCAGAACCAGTCCTCCCTGCAGCTGAGCTCCGTGACCACCAAGGACACAGCTGTGTATTACTGTGCCAGAGA IGHD SEQ ID NO: 13: DH1 IGHD3-1 GTATTATTCTCCTGGATAGAGTAATTACAAC SEQ ID NO: 14: DH2 IGHD4-2 TTACTATGGCTGGGGTAAC SEQ ID NO: 15: DH3 IGHD2-3 ATGATGACTATGGTGATACTTTCTACTATACA SEQ ID NO: 16: DH4 IGHD3-4 GTATTACTCTTCTGCATATGATTGTATCAAC SEQ ID NO: 17: DH5 IGHD4-5 TTACTACAACTATAACTAC SEQ ID NO: 18: DH6 IGHD2-6 ATGGTTACTATAGTAGGAGTTGCTATACC SEQ ID NO: 19: DH7 IGHD3-7 GGATTTACTGTTCTGGGTGCAGATGCTCGTTACAACCACAGCAA SEQ ID NO: 20: DH8 IGHD4-8 TAACTACAGATATAGCTCC SEQ ID NO: 21: DH9 IGHD2-9 ATGGTTACTATGCTAGTGGTTATGACTACA SEQ ID NO: 22: DH10 IGHD3-10 GTATTACTCTTCTGCATATGCTTGTATCAAC SEQ ID NO: 23: DH11 IGHD4-11 TAACTACGGTTATGGTTATGCTAC SEQ ID NO: 24: DH12 IGHD2-12 ACTATAGTTATGGTAGTTACTATGCC SEQ ID NO: 25: DH13 IGHD3-13 GTATGACTGTACTGGTCATGGATGTGTCTACATC SEQ ID NO: 26: DH14 IGHD4-14 TAACTACTATGGTAGCAAC SEQ ID NO: 27: DH15 IGHD2-15 ATGGTTACTATGGTAGTTACTACAGTAGTTACTATGCC SEQ ID NO: 28: DH16 IGHD3-16 GTATTACTATTCTGGATATAATTATTACAAC SEQ ID NO: 29: DH17 IGHD4-17 GCCACTGATATAGCTCC SEQ ID NO: 30: DH18 IGHD2-18 ATGGTTACTATGCTGGTAGTTACTATGCC SEQ ID NO: 31: DH19 IGHD3-19 GTGTGAATGTCCTGGGCATGGATGTTATTACGAC SEQ ID NO: 32: DH20 IGHD4-20 TTCCTACCGATATAGCTCC SEQ ID NO: 33: DH21 IGHD2-21 ACGGTTCCTATGCTGGTAGTTACTTATACTACA SEQ ID NO: 34: DH22 IGHD3-22 GTATTACTATTCTGCATATGATTATTACAAC SEQ ID NO: 35: DH23 IGHD2-23 ATGATTACTATGGTATTAGTGACTCCTACA SEQ ID NO: 36: DH24 IGHD2-24 GTATTACTCTTTTGAATATGGTTATAACAAC SEQ ID NO: 37: DH25 IGHD4-25 CTGCTATAGCAGCTATGCTTACTAC SEQ ID NO: 38: DH26 IGHD2-26 ACTATGGTTATGGTGGTGCTTACTACTACA SEQ ID NO: 39: DH27 IGHD3-27 GTATTACTATTCTGCATTTCGTTATTACAAC SEQ ID NO: 40: DH28 IGHD4-28 CTGCTATAGCAGCTATGCTTACTAC SEQ ID NO: 41: DH29 IGHD2-29 ACAGTTACTATGGTGGTAGTTCCTGGTACTCC SEQ ID NO: 42: DH30 IGHD3-30 GTATTACTATTCTGGACATGATTATTACAACCTCAGCGT SEQ ID NO: 43: DH31 IGHD4-31 TTACGATGACGGATACTACAAC SEQ ID NO: 44: DH32 IGHD1-32 GGTCCTGGGTACAGCTCC SEQ ID NO: 45: DH33 IGHD7-33 AGATACTCCAGTGCTGGTTAC SEQ ID NO: 46: DH34 IGHD6-34 CTACGGTAGCGGTTGGCC SEQ ID NO: 47: DH35 IGHD4-35 TAACTATGGCTCCTATAATTACTAC SEQ ID NO: 48: DH36 IGHD2-36 ATGATTATTATGGTGCTATTGACTACATAAC SEQ ID NO: 49: DH37 IGHD3-37 TATGACAATTCTGTATATAGCTCTGACTACAGCAT SEQ ID NO: 50: DH38 IGHD4-38 GGAGAAGAGTTGGAGTAAC SEQ ID NO: 51: DH39 IGHD2-39 ACAGTTACTGGAGTAGTAGTTACTATGCC SEQ ID NO: 52: DH40 IGHD3-40 GAATAACTATGCTACATATGATTATATCAAC SEQ ID NO: 53: DH41 IGHD4-41 AACTGCTATGGTAACAAC SEQ ID NO: 54: DH42 IGHD1-42 GGTACTTGGGTACAGCTCC SEQ ID NO: 55: DH41 IGHD7-43 AGATACTCCAGTGTTGGTTAC SEQ ID NO: 56: DH41 IGHD6-44 ATACGGTAGTGGTTGGCC IGHJ SEQ ID NO:57: JH1 IGHJ1 CTTATGCTTACTTGCAGCACTGGGGCCACGGCACCCTGGTCACCGTCTCCTCAG SEQ ID NO:58: JH2 IGHJ2 GTCCTGGCACCTCGAGCACTGGGACCACGGCATCCTGGTCACCGTCTCCTCAG SEQ ID NO:59: JH3 IGHJ3 GTTATGGCTACGTGGATCACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG SEQ ID NO: 60: JH4 IGHJ4 ACTATTTTGGCTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG SEQ ID NO: 61: JH5 IGHJ5 ACAACGAGTTGGATTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG SEQ ID NO: 62: JH6 IGHJ6 ATTATTATGGTATAAACTACTGGGGCCAGGGCATCCTGGTCACCGTCTCCTCAG SEQ ID NO: 63: JH7 IGHJ6-2 ATTATTATAATGCTATGGACCCCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG SEQ ID NO:64: JH8 IGHJ6-3* ATTATTATGATATAGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG SEQ ID NO: 65: JH9 IGHJ6-4* ATTATTATGATATAGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG *JH8 and JH9 are separate gene segments but have the same sequence. IGKV SEQ ID NO: 66: IGKV2-48*01 L1: ATGAGGTTCTCTGCTCAGCTCCTGGGGTTGCTAATACTCTGGGTCCCAG L2: GATCCACTGGG VK: gacacagttttgacccagaccccactctctctgtctgtcatccctggagagtcggcctcc atctcttgcaagtctagtcagagcctcctacatggtaatggaaacacctatttgcattgg tacctgcagaagccaggccagtctcttcagcgcctgatctctatggtttccaatcgggca tctggggtcccagacaggttcagtggcagcgggtctgggacagatttcacccttataatc agcaagttggaagctgaggatgttggagtttattactgcatgcaagctacacaaagtccc cc Accession No. 67: IGKV2-46*01 L1: ATGAAATTCGCTAGTCAGCTCCTGGGGCTACTGATGCTCTGGATCCCAG L2: GATCCAGTGCG VK: gatgttgtgttgacccagactccactctccctgtctgtcgtccctggagagccggcctcc atctcctgcaagtctagtcagagcctcaaatatagtgatgggaaaacctatttgtattgg ttcctacagaagccaggccagtctccaaagctcctgatctatttggtttccacccggtac tctggggtctcagacaggttcagtggcagcggatcagaagcagatttcaccctgaaaatc agcagagtggagcctgaggatgttggagtctattactgctttcaagctctatatgcttct cc Accession No. 68: IGKV2-45*01 L1: ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTGATGCTCTGGATCACAG L2: GATCCAGTGGG VK: GATGTTGTGATGACCCAGACTCCACTCTCCCTGTCTGTCGTCCCTGGAGAGCCGGCCTCCATCTCCTGCAAGTCTAGTCAGAGCCTCAAACATAGTGATGGAAAAACCTATTTGTATTGGTTCCTACAGAAGCCAGGCCAGTCTCCAAAGTGCTTGATCTATTTGGTTTCCACCCGGGTCTCTGGAGTCTCAGACAGGTTCAGTGGGAGCGGGTCAGAAACAGATTTCACCCTGAAAATCAGCAGAGGGGAGCCTGAGGACGTTGGAGTCTATTACTGTGTGCAAGCTCTATATGCTTCTCC Accession number 69: IGKV5-43*01 L1: ATGGGCTCCCAGGCTCAGCTCCTCAGCTTCCTGCTCCTCTGGATTTTTG L2: ATACCAGGGCA VK: GAAATAACAGTCACACAGTCTCCGGAATCCATGTTAGTGATTCCAGGAGACAAAGTCATCATCACCTGCAAAGCCAGCCAAGACATTGGTGATGATGTGAACTGGTATCAATGGAAACCAGGAGAAGCTCCTAAGCTCATTATTAAAGAAGCTACTACTCTCTGGTCTGGGGTTCCCTCTCGGTTCAGTGGCACTGTGCATGGAGTAGATTTTACCCTGACAATTGAGGACGTAAAATCTGAGGATGCTGCATATTATTTCTGTCTACAACATGATCGTATACCTCT Accession number 70: IGKV2-39*01 L1: ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTGATGCTCTGGATCCCAG L2: GATCCAGTGGG VK: GATGTTGTGATGACCCAGACTCCACTCTCCCTGCCTGTCGTCCCTGGAGAGCCGGCCTCCATCTCCTGCAAGTCTAGTCAGAGCCTGCTGGATAGTGATGGAAAAACCTATTTGTATTGGTACCTGCAGAAGCCGGGCCAGTCTCCAAAGCTCCTGATCTATTCAGTTTCCAACCGGGACTCTGGGATCTCAGACAGGTTCAGTGGCAGCGGGTCAGGAACAGATTTCACCCTGAAAATCAGCAGAGTGGAGCCTGAGGATGTTGAAGTCTATTACTGTGTGCAAGCTACACATGCTCCTCC Accession number 71: IGKV1-36*01 L1: ATGAGGGTCCCTGCTCAGCTCCTCAGCCTTCTGCTGCTCTGGCTCCCAG L2: GTGCCAGGTGT VK: GAGATCCAGATGACCCAGTCTCCAGCCTCCCTGTCTGCATCTCTAGGAGACAGAGTCACCATCACTTGCCAGGCCACTCAGGGCATTAACACTTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCTTAAGTTCCTGATCAGTAAGGCAACCATTTTGCACACTGGCGTCTCTTCGAGGTTCAGTGGCAGTGGAACTTGGACAGATTTCACTCTCACCATCAGCAGCCTGGAGCCTGAAGATGCTGCAACTTATTACTGTCAGCAGTATAAGAGCAGCCCTCC Accession number 72: IGKV2-33*01 L1: ATGAAATTCCTTGCTCAGCTCCTGGGGCTGCTAATGCTCTGGATCCCAG L2: GATCCAGTGGA VK: GATATTGTGATGACCCAGACTCCACTCTCCCTGCCTGTCGTCCCTGGAGAGCTGGCCTCCAACTCATGCAGGTTTAGTCAGAGCCTCCTACATAGTAATGGAAACACCTATTTGCACTGGTTCCTGCAGAAGCCAGGCCAATCTCCAAGGCGTCTGACCTATAGGGTGTCCAACCGGAACTCTGGGGTCCCAGACAGGTTCATTGGCAGCGGGTCAGGGACAGATTTTACACTTAAAATCAGCAAGGTGGAGGCTGAAGATGGTGGAGTTTATTATTGCTCCCAAGGTACACAAAGTCCCCC Accession number 73: IGKV2-28*01 L1: ATGAGGTTCCCTGCTCAGCTCCTGGGGCTACTAATGCTCTGGATCCCAG L2: GATCCAGTGGA VK: GATATTGTGATGACCCAGACTCCCCTCTGCTTGGCCGTCACCTTGGGAGAGCCAGTTTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTCCGTAGTGATGACTACACCTATTTGGATTGGTACCTGCAGAAACCAGGCCAGTCTCCACGGCTGCTGATCTATGAGGTTTCCAAGCTGGTCTCTGGAGTCTCAGACAGGTTCAGTGGCAGTGGGTCAGGGACAGATTTCACCCTTCAAATCAGCAGAGTGGAGGCTGAGGATGTTGGAGTTTATTACTGCATGCAAGGTTCACAAAGACCTCC Accession number 74: IGKV4-18*01 (RC) L1: ATGATGTCACTGACAAAGGTGTTTATGTCTTTGTTGCTCTGGGTCTCAA L2: CTGCCTGTGGG VK: GACATCGTGATGACCCAGTCTCCAGGCTCCTTGGCAGTGTCTCCAGGACAGAGGGTCACCATTAGCTGCAAGGCCAGTCAGAGTGTTAGCAACTACTTAGACTGGTACCAGCAAAAACCAGGAGAGGCTCCTATGCTGCTTATCTATGCGGCATCCAGCAGAGCATCTGGGGTCCCCGACAGATTCAGTGGCGGTGGATCTGGGACAGATTTCGCTCTCACCATCAGCAGCCTCCAGGCTGAAGATGTGGCAGTTTTCACTGTCAGCAGCATTATACTAATCCTCCC Accession number 75: IGKV4-12*01 (RC) L1: ATGATGTGGGAGACACAGGTCCTTATGTCCTTATTGCTCGGGGTCTCAG L2: GTACCTTGGGG VK: GACATCATGATGACCCAGTCTCCAGACTCCTTGGCAGTGTCTCTAGGAGAGAGGGTCGACATGAAGTGCACGGCCAGTCAGAGTGTTTACCACTACTTAGCCTGGTACCAGCAAAAACCAGGACAGGCTCCTAAGCCCCTCATCTACTCAGCATCTACCAGACCATCTGGGATCCCTGACCGATTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAACTTCCAAGCTGAAGATGCGGCAGTTTATTGCTGTGAGCAGTATTATGGTAATCCTCC Accession number 76: IGKV4-9-1*01 (RC) L1: ATGATGTCACAGACACAGGTCCTCTTGTCGGTGTTTCTCTGGGTCTCAG L2: GTGCCTGTGGG VK: GACCTCGTGATGACGCAGTCTCCAGGCTCCTTGGCAGCGTCTCTAGGACAGAGAGTCGAGATGAAGTGCAAGGCCAGTCAGAGTGTTAGCAGCTACTTAGACTGGTACCAGCAGAAACCAGGACAGGCTCCTAAGCAGCTCATCTATGCTGCATCCAGCAGAGCGTCTGGGGTCCCCGACCGATTCAGTGGCAGTGGATCTGGGACAGATTTCACTATCACCATCAGCAGCCTCCAGGCTGAAGATCTGGCCATTTATTACTGTCAGCAGTATAATAGTGCTCCTCC Accession number 77: IGKV4-9*01 (RC) L1: ATGATGTCACAGACACAGGTCCTCTTGTCGGTGTTTCTCTGGGTCTCAG L2: GTGCCTGTGGG VK: GACCTCGTGATGACGCAGTCTCCAGGCTCCTTGGCAGCGTCTCTAGGACAGAGAGTCGAGATGAAGTGCAAGGCCAGTCAGAGTGTTAGCAGCTACTTAGACTGGTACCAGCAGAAACCAGGACAGGCTCCTAAGCAGCTCATCTATGCTGCATCCAGCAGAGCGTCTGGGGTCCCCGACCGATTCAGTGGCAGTGGATCTGGGACAGATTTCACTATCACCATCAGCAGCCTCCAGGCTGAAGATCTGGCCATTTATTACTGTCAGCAGTATAATAGTGCTCCTCC Accession number 78: IGKV4-8*01 (RC) L1: ATGATGTTGCAGACACAGGTCCTTATAACCTTGTTGCTCTGGGTCTCAG L2: GTGCCTGTGGG VK: GACATCGTGATGACCCAGTCTCCAGACTCCTTGTCTGTGTCTGCAGGACAAAGGGTCGACATGAAGTGCAGGGCCAGTCAGAGTGTTAGCAATGAGTTATCCTGGTACCAGCAAAAACCAGGACAGGCTCCTAAGCTGCTGATCTATGCAGCATCCAACAGAGCATCTGTGGTCCCTGACCGATTCAGTGGCGGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAGCCTCCAGGCTGAAGATGTGGCCGTTTATTACTGTCTGCAGCATTATAATAATCCTCC Accession No. 79: IGKV4-5-1*01 (RC) L1: ATGATGTCGCTGACAAAGGTCCTTATATCTGTGTTGCTCTGGGTCTCAG L2: GTGCCTGTGGG VK: GACATCGTGTTGACCCAGTCTCCAGAGTCCTTGGCAGTGTCTCTAGGACAGAGGGTCGAGATGAAGTGCAAGGCCAGTCAGAGTGCTAGCAGCAACTTGGACTGGCACCAGCACAAACCAGGACAGGCTCCTAAGCAGCTCATCTACAGAGCATCCAGCAGAGCGTCTGGGGTCCCTGACCGATTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTCCAGGCTGAAGATGTGGCCGTTTATTACTGTCAGCAGTATAATAGTGCTCCTCC Accession No. 80: IGKV5-5*01 (RC) L1: ATGATGTCATGGACTCAGATCCTTATGTCCTTGTTGCTCTGGGTCTCAG L2: GTGCCTGTGGG VK: GACATCGTGATGACCCAGTCTCCAGACTCCTTGGCAGTGTCTCTAGGACAGAGAGTCGAGATGAAGTGCAAGGCCAGTCAGAGTGTTAGCAACTACTTAGACTGGTACCAGCAAAAACCAGTAAAGGCTCCTAAGCTGCTCATCTATGCAGCATCCAGCAGAGCATCTGGGGTCCCCGACCGATTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTCCAGGCTGAAGATGTGGCAGTTTACTCCTGTCAGCAGCGTTATAGTTCTCCTCC Accession number 81: IGKV4-2*01 (RC) L1: ATGATGTCGCTGACACAGTTCCTTATATCTGTGTTGCTCTGGGTCTCAG L2: GTGCCTGTGGG VK: GACATCGTGATGACGCAGTCTCCAGACTCCTTGGCAGTGTCTCTAGGACAGAGAGTCGAGATGAAGTGCAAGGCCAGTCAGAGTGTTAGCAGCAGCTTGGACTGGCACCAGCACAAACCAGGACAGGCTCCTAAGCTGCTCATCTACAGAGCATCCAGCAGAGCGTCTGGGGTCCCTGACCGATTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTCCAGGTTGAAGATGTGGCAGTTTATTACTGTATGCAGTCTAATACTGCTCCTCC Accession number 82: IGKV4-1*01 (RC) L1: ATGCTGACGCAGACGCAGGTCCTTATATCTGTGTTGCTCTGGGTCTCAG L2: GAGCCTGTGGG VK: GACGTCATGATGACCCAGTCTCCAGACTCCTTGGCAGCGTCTCTAGGACAGAGAGTCGAGATGAAGTGCAAGGCCAGTCAGAGTGTTAGCAGCTACTTAGCTTGGTACCAGCACAAACCAGGACAGGCTCCTAAGCGGCTCAT CTATGCTGCATCCAGCAGAGCATCTGGGGTCCCTGACCGATTCAGTGGCAGTGGATCTGGGATGGATTTCACTCTCACCATCAGCAGCCTCCAGGCTGAAGATGTGGCCATTTATTACTGTATGCAGCATTATAATAATCCTCC IGKJ SEQ ID NO: 83: IGKJ1*01 GTGGACGTTCGGTGCCGGGACCAAGCTGGAAATCAAAC SEQ ID NO: 84: IGKJ2*01 TATACACGTTTGGCCAAGGGACCAAGCTGGAGATCAAAA SEQ ID NO: 85: IGKJ3*01 GTTCACTTTCGGCCAAGGGACCAAAACTGGAGATCAAAC SEQ ID NO: 86: IGKJ4*01 GCTTACGTTCGGCCAGGGGACCAAGCTGGAGATCAAAC Note: The sequence of the equine lambda locus is still incomplete. The sequences below do not necessarily represent the complete equine IGLV and IGLJ repertoire. IGLV SEQ ID NO: 87: IGLV28 (RC) L1: ATGGCCTGGTCCCCGCTCCTCCTCACCCTCATCGCTCTCTGCACAG VL: TATCCTGGGCCCAGTCTGTGCTGACTCAGCCGGCCTCAGTGTCTGGGAACCTGGGCCAGAGGGTCACCATCTCCTGCACTGGGAGCAGCTCCAACACCAGGGATAATTATGTGAACTGGTACCAGCAGCTCCCAGGAACCGCCCCCAAACTCATCATCTATGAAAATAGCAAAAGACCCTCCGGGACCCCAGATCGAATCTCTGGCTCCAAGTCTGGAAACCCGGCCTCCCTGACCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGATGACAACCTGAATGCTCG Accession number 88: IGLV27 L1: ATGGCCTGGTCCCCTCTCCTCCTCACCCTCATCGCTCTCTGCACAG VL: TCACTACTATCACTGCTGTAATAGGAACCACAGTAATAATCGGCCTCGTCCTCAGCCTGAAGCCCAGAGATGGTCAGGGTGGCTGTGTTGCCAGACTTGGAGCCAGAGAATCGATCTGGGACCCCTGAGGCTCGTTTGTTATTACCATAGATGAGGGTTTTGGGGGCTGTTCCTGGGATCTGTTGGTACCAGCCCACAGCACTATAACTATACCCGATGTTGGAGCTGCTTCCAGAGCAGGAGATGGTGACTGTCTGGCCCAGGGTCCCAGACACTGAGGCGGGCTGGGTCAGAGACTGGGCCCAGGATC Accession number 89: IGLV26 L1: ATGGCCTGGTCCCCTCTCCTCCTCACCCTCATCGCTCTCTGCACAG VL: GATCCTGGGCCCAGTCTCTGACTCAGCCCGCCTCAGTGTCTGGGACCCTGGGCCAGACAGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATCGGGTATAGTTATAGTGCTGTGGGCTGGTACCAACAGATCCCAGGAACAGCCCCCAAAACCCTCATCTATGGTAATAACAAACGAGCCTCAGGGGTCCCAGATCGATTCTCTGGCTCCAAGTCTGGCAACACAGCCACCCTGACCATCTCTGGGCTTCAGGCTGAGGACGAGGCCGATTATTACTGTGGTTCCTATTACAGCAGTGATAGTAGTGA Sequence number 90: IGLV25 L1: ATGGCCTGGTGCCCTCTCCTCCTCACCCTCATCGCTCTCTGCACAG VL: GATCCTGGGCCCAGTCTGTGACTCAGCCCGCCTCAGTGTCTGGGACCCTGGGCCAGACAGTCACCATCACCTGCACTGGAAGCAGCTCCAACATAGTTGCTTATGTGGGCTGGTACCAACAGATCCCAGGAACAGCCCCCAAAACCCTCATCTACGCTAATAACAAACGAGCCTCAGGGGTCCCAGATCGATTCTCTGGCTCCAAGTCTGGCAGCACAGCCACCCTGACCATCACTGGGCTCCAGGCTGAGGACGAGGCCGATTATTACTGTGGTACCTCTAGCAGCAGTGGTAGTAGTGA Sequence number 91: IGLV24 L1: ATGTCCTGTACTCCTCTCCTCCTCGTGCTCCTCTCTCACTGCACAG VL: GTTCCCTCTCCCAGCCTGTGCTGACCCAGCCTCCCTTCTTCTCTGCATCTCCTGGAGCATCAGCCAGACTCACCTGCACCCTGAGCAGTGACATCAGTGTTGACAGCTCTCTCATATTCTGGTGCCAGCAGAAGCCAGGGAGCCCTCCCCGGTATCTCCTGAGTTTCTACTCAGACTCAGTTAAGCACCAGGGCTCCGGGGTCCCCAGCCGCTTCTCTGGATCCAGAGACACCTCGGCCAATGCAGGGCTTCTGCTCATCTCTGGGCTCAAGGCTGAGGACGAGGCTGACTATTACTGTGCTACAGCTGATAGCAGTGGGAGCAGCTCTGGTTACT Accession number 92: IGLV23 L1: ATGGCCTGGTCCCCTCTCCTCCTCACCCTCATCGCTCTCTGCACAG VL: GATCCCGGGCCCAGTCTGTGACGCAGCCCGCCTCAGTGTCTGGGACCCTGGGCCAGACAGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATCGGGAGTGGTCATGTGTCCTGGTACCAACAGATCCCAGGAACAGCCCCCAAACGCCTCATCTATTCTTCCGCTAGCAGGGCTTCCAGGGTCCCCGACCGATTCTCTGGCTCCAGGTCTGGCAACACAGCCACCCTGACCATCTCTGGGCTCCAGGGTGAGGACGAGGCCGATTATTACTGTGGTACATTGTACAGCAGTTGGAGTAGTGA Accession number 93: IGLV22 L1: ATGGCCTGGTCCCCTCTCCTCCTCACCCTCATCGCTCTCTGCACAG VL: GATCCTGGGCCCAGTCTCTGACTCAGCCCGCCTCAGTGTCTGGGACCCTGGGCCAGACAGTCACCATCTCCTGCTCTGGAAGCAGCTCCAGCATAGGTTCTTATATGGGCTGGTACCAACAGATCCCAGGGACAGCCCCCAAAACCCTCATCTATGCTACTAACAAACGAGCCTCAGGGGTCCCAGATCGATTCTCTGGCTCCAAGTCTGGCAACACAGCCACCCTGACCATCTCTGGGCTTCAGGCTGAGGACGAGGCCGATTATTACTGTGGTTCCTATTACAGCAGTGATAGTAGTGA Accession number 94: IGLV21 L1: ATGGCCTGGACAGTGCTTCTTCTCTGGCTCCTCACTTACAGCTCAG VL: GGGCAGATTCTCAGGCTGTGGTGATCCAGGACCCATCATTCTCTGTGTCCCTAGGGGGGACGGTCATACTGACCTGTGGCCTTAGAACTGGGTCAGTCTCTACCAGTAACTATCCTAGATGGTACCAGCAGACACCAGGCAAGGCTCCCCGTACACTCACCTACAGCACAAACAACCGCCCCTCTGGGATCCCTGAACGCTTCTCTGGATCCATCTCAGGAAACAAAGCCGCCCTCACCATCACGGGGGCCCAGCCCGAGGACGAGGCCGACTATTACTGTGATCTGTATGTGGATCGTGGTGTTTC Accession number 95: IGLV20 (RC) L1: ATGGCCTGGATGGTGCTTCTTCTCGGGCTCCTTTCTTACAGCTCAG VL: GGGCGGATTCTCAGTCTGTGGTGACCCAGGAGCCATCACTCTCAGTGTCTTCAGGAGGGACAGTCACACTCACCTGTGGCCTTAACTCTGGGTCAGTCTCTTCCAGTAACCACCCCAGCTGGCACCAGCAAACCCCAGGCCAGGCTCCCCGCA CACTTATCTACTACACAAACACCCGTGCCTCTGGAGTCCCTAATCTCTTCTCTGGATCCATCTCCGGGAACAGAGCCACCCTCACCATCACGGGGGCCCAGCGTGAGGACGAGGCCGACTATTACTGCGCTCTGTATACGGGTAGTTACACTGA SEQ ID NO: 96: IGLV19 (RC) L1: ATGGCCTGGTCCCCTCTCCTCCTCACCCTCATCGCTCTCTGCACAG VL: GATCCTGGGCCCAGTCTGTGACTCAGCCCGCCTCAGTGTCTGGGACCCTGGGCCAGACAGTCACCATCTCCTGCACTGGAAGCATCTCCAACATAGGTGTTTATGTGGACTGGTACCAACAGATCCCAGGAACAGCCCCCAAAACCATCA TCTATGCTACTAACAAACAACCCTCAGGGGTCCCAGATCGATTCTCTGGCTCCAAGTCTGGCAACACAGCCACCCTGACCATCACTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGTGGTATCTATGACAGCAGCCTGAGTAGTGA IGLJ Note: The IGVL gene segment is present both upstream and downstream of the equine IGLJ gene segment and the IGLC gene (not shown). SEQ ID NO: 97: IGLJ7 J7: TGCGATGGTCAGGTGGGTGCCTCCGCCGAATGCACCA SEQ ID NO: 98: IGLJ6 J6: TGCGATGGTCAGGTGGGTGCCTCCGCCGAATGCACCA SEQ ID NO: 99: IGLJ5 J5: TGCGATGGTCAGGTGGGTGCCTCCGCCGAATGCACCA SEQ ID NO: 100: IGLJ4 J4: GCGATGGGTCAGGTGGGTGCCTCCGCCGAATACAGCACA SEQ ID NO: 101: IGLJ3 J3: AGGACTATCAGCTGGGTCCCTCAGCTGAGCACAGGA SEQ ID NO: 102: IGLJ2 J2: CTAGGACGGTCAGATGGGTACCTCCAGTGAACTATGAA SEQ ID NO: 103: IGLJ1 J1: CTAGGACGGTCAGATGGGTACCTCCAGTGAACTATGAA SEQ ID NO: 104: IGLV2 (RC) L1: ATGGCCTGGACCCCTCTCCTGCTCCTCCTCCTCACTCTCTGCACAG (RC) VL: GCTCTGTGGCTTCTTCTATGCTGACTCAGCCACTTACCTTGTCCGTGGCCTTTGGAAGCACAGTCACTATCACATGCCAGGGAGAGCTCCTAGACAGTTATTATGCTGAGTGGTACCAGCAGAAGCCAGACCAGGCTCCCGTGCTGGTCA TATATTATGGAAGCAAACGTCCTTCGGGGATTTCTACCCGATTCTCTGGCTCCTACTCAAGCAAGATGGCCACCCTGACCCTCAGTGGGGCCTTGGCCGAGGATGAGGCTGACTATTACTGTCAGGTGTGGGACAGCAGTGGTAACCAGCC SEQ ID NO: 105: IGLV3 (RC) L1: ATGGCCTGGACCCTTCTCCTGCTTCCCCTCCTCACTCTCTGCACAG VL: GTTCTGTGACCACCTATGACTTGACGCAACCACACTCAACTTCGGTGGCCCTAGGACAGACAGCGACAATCACCTGCTCTGGAGATAATCTCGAGGATGAATATGCTTACTGGTACCAGCAGAAGACAGGCCAGTCCCCTGCCCTGGTCATTTATAAGGATAGTGAGCACCCCTCAGGGATCCCTGACCGGTTCTCTGGCTCAAACTCAGGAAACACAGCCACGCTGACCATCAGAGGGGCCAAGACAGAGGACAAGGCTGACTATTACTGCCAATCGTGGAGCAGTGCTAATGCT Accession number 106: IGLV4 (RC) L1: ATGGCCTGGACCCCTCTCTTGTTTGCCTTCCTCACTCTCTGCACAG VL: GTCCTGTAGTCTCTTCTGAGGTGACTCAGCCAACTGCGGTGTCCGTGGCCTTGGGACAGACAGCCTCCATCACCTGCCAGGGAAGCGACTTTGAAAATTATTATGCTAGCTGGTACCAGCAGAAGCCAGGCCAGGCCCCAGTGCTGGTCATCAATGCTAATAATGAGCGGCCCTCAGGGATCCCTGAACGATTCTCTGGGTCCAGTTCAGGAGAGACAGCTACGCTGACCATCAGTGGAGCCCACGCTGAGGACGAGGCCGACTATTACTGTCTGGCAACAGATGCTTATGTTGCTGAAGCT Accession number 107: IGLV5 (RC) L1: CTGTGCAGAGAGTGAGGAAGGCTAACAAGAGAGGGGTCCAGGCCAT VL: AGCTTCATAATCAGAAGCATCTGCTGCCAGACAGTAATAGTCAGCCTCGTCCTCAGCCTGGGCCCCGCTGATGGTCAGCGTGGCTGTGTCTCCTGAGCTGGAGCCAGAGAATCGTTCAGGGATCCCTGAGGGCCGCTCATTACTAGCATCGATGACCAGCACAGGGGCCTGGCCTGGCTTCTGCTGGTACCAGCTACCAACAAAACTTTCAAAGTCGCCTCCCTTGCAGGTGATGGTGGCCGTCTGTCCCAAGGCCACAGACACTGAAGATGGCTGAGTCAGCTTAGAAGAGGCCACGGGAC Accession number 108: IGLV6 (RC) L1: ATGGCCTGGACCCCTCTCTTGTTAGCCTTCCTCACTCTCTGCACAG VL: GTCCTATGGCCTCTTCGGAGGTGACTCAGCCATCTGCGGTGTCTGTGGCCTTGGGACAGACAGCCACCCTCACCTGCCAGGGAGACTACTATGAAAGATATATTGTCAACTGGTACCAGCAGAAGCCAGGCCAGGCACCTGTGCTGGTCATCTATGCTAATAGTGAGCGGCCCTCAGGAATCCCTGAACGATTCTCTGGCTCCAGCTCATTAGGCACATCCACGCTGACCATCAGCGGGGCCCAGGCTGAGGATGAGGCTGACTATTACTGTCAGCCAGCAGATGCTCATCGTTCTGAATCTGTCCTATGGCCTCTTCGGAGGTGACTCAGCCATCTGCGGTGTCTGTGGCCTTGGGACAGACAGCCACCCTCACCTGCCAGGGAGACTACTATGAAAGATATATTGTCAACTGGTACCAGCAGAAGCCAGGCCAGGCACCTGTGCTGGTCATCTATGCTAATAGTGAGCGGCCCTCAGGAATCCCTGAACGATTCTCTGGCTCCAGCTCATTAGGCACATCCACGCTGACCATCAGCGGGGCCCAGGCTGAGGATGAGGCTGACTATTACTGTCAGCCAGCAGATGCTCATCGTTCTGAATCT Accession number 109: IGLV7 (RC) L1: ATGGCCTGGACCCCTCTCTTGTTAGCCTTCCTCTCTCTCTGCACAG VL: GTCCTGTTGTCTCTTCTGCAGTGACTCAGCCATCTGAGGTGTCCGTGGCCTTGGGACAGAGAGCCACCCTCACCTGCCAGGGAAGCAACTTTGAATTTTTTTCTCCTAGCTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTACTGCTCATCAATATTAATAATGAGCGCCACTCAGGGATCCCTGAACGATTCTCCGGCTCCAGCTCAGGAGACACGTCCACACTGACCATCAGTGGGGCCCAGGCTGAGGACGAGGCTGACTATTACTGTCTGGCAGTAGATGCTCTTAGTTCTGAAACT Accession number 110: IGLV8 (RC) L1: ATGGCCTGGACACTTCTCCTTCTCCCTCTCCTCACTCTCTGCACAG VL: GTTCTGTGGCCCCTTCTGAGCTGACTCAGTTAACTGTGGTGTCTGTGGCCTTGGCACAGACAGCCAGGGTCACCTGCCAGGGAGAGAGACCAAAAAGTGTCTATGCTGGCTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTATGGGTCATCTATAGTAAAAACAATTGGACCACAGGCACACCTGAACAATTCTCTGCCTCTGACTCAGGGGACACAGCCACCCTGACCATCAGTGGGGTCCAGGTTGAGGGCGAGACTGACTATTACTGTGGGGTAAGTGTTGGAAGTGGGAGCAGCTGGCAGTCACT Accession number 111: IGLV9 (RC) L1: ATGGCCTGGACCCCTCTCCTGCTCCCTCTCCTCACTCTCTGCACAG VL: GTTCTGTGTCCTCTTCTGAGCTTACTCAGTCTACTGCAGTGTCATTTTCCTTGGGACAGACAGCCACCATCACCTGCCAGGGAGAAACCCTAAGAAGCCACTATGCTAGCTGGTACCAGAAGAATCCAGGACAGGCCCCTGTATTGGTAATATATGGTAATAACAACCGGCCCTCAGGGATCCCTGCCCGATTTTCCAGCTCCTACTCAGAGGACACAGGCACCCTGACCATCAGTGGGGTCCAGATAGAGGATGAGGCTGACTATTACTGCCAATCATTGGGCAGTGATTATGCT Accession number 112: IGLV10 (RC) L1: ATGGCCTGGGCTCTGTTCCTCATCACCCTCCTCACTCAGGGCACAG VL: GGTCCTGGGGCCAGTCTGCCCTGGTTCAGCCTTCTTCGGTGTCCGTGGCTCTAGGACAGTCGGTCACCATCTCCTGTGCTGGAAGCAGCAGTGACATTGGGTATTATAACTCTATTTCCTGGTACCAACAGCACCCAGGCACAACCCCAAAGCTGCTGATTTACTATACCAATAAGAAGCACTCAGGGATCCCTGATCGCTTCTCTGGCTCCAAGTCTGGGAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGAGTATTACTGTTGCTCATATGCAGGCAGTGGCAATTTA Accession number 113: IGLV11 (RC) L1: ATGGCCTGGACTCTGCTCCTTCTCACCCTCCTCACTCAGGGTACAG VL: GGTCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCGTCAGTGTCCGGGGCTCTAGGACAGTCGGTCACCATCACCTGTGCTGGAAGCAGCAGTGACATTGGGGGTTATAATGCTGTCAGCTGGTTACAACAGCACCCGGGCACAGCCCCCAAAGTTCTGATTTATAGTGTGAATACTCGGGCCTCAGGGATCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGTTGAGGACGAGGCTGATTATTACTGTTACTCGCTTGTGAGTGGTTACACTTTC Accession number 114: IGLV12 (RC) L1: ATGGCCTGGGCTCTGCTCCTCATCAGCCTCTTCACTCAGGGCACAG VL: GGTCCTGGGCGCAGTCTGCCCTGACTCAGCCTGCGTCAGTGTCCGGGACTCTGGGACAGTCGGTCACCATCTCCTGTGCTGGAAGCAGCAGCAACATTGGGAGTTATAACTATGTTTCCTGGTACCAACAGCACCCGGGCACAGCCCCCAAACTCCTCATTTATAGTGCCAGTTCTCGAGCCTCAGGGATCCCTGATCGCTTCTCTGGCTCCAAGTCTGGGAACACGGCCTCTCTGACCATCTCGGGGCTCCAGGCTGAGGACGAGGCCGATTATTACTGTAGCTCATATATCAATGCTGATCCTTATC Accession number 115: IGLV13 (RC) L1: ATGGCCTGGGCTCTGCTCCTCATCACCCTCCTCACTCAGGGCACAG VL: GTAATTGTAACTGCCAACATACGCGTGACAGTAATAATCAGCCTCGTCCTCAGCCTGGAGCCCAGAGATGGTCAGGGACATCGTGTTGCCAGACGTGGAGCCGGAGAAGCGATCAGGGATCCCTGAGGCCCGATTATTCCCATTATAAATGAGGAGTTTGGGGGCTGTGCCTGGGTGCTGTTGGTACCAGGAAATATATTTATAAGATCCCGTGCTTCCAGCACAGGTGATGGTGACCGACTGTCCCAGAGTCCCGGAGACTGACGCAGGCTGAGTCAGGGCAGACTGCGCCCAGGACC Accession number 116: IGLV14 (RC) L1: ATGGCCTGGGTGCCACTCCTGCTCACACTTCTGGCTCACTGCACAG VL: GGTCCACTTCACAGGATGTGGTGATTCAGGAATCTTCACTGATCACAACTCCTGGGGGAACAGTCACACTCACCTGTGGCTCAAGTGCTGGGGCTGTCACCTCCAATAATTATGCCAACTGGGTCCAAGAGAAGCCCTATCAGGGACGCCAGGGTCTAATAGGTGGTACTAGCAACAGGGTCTCAGGGGGTCCCTGCCCGATTCTCTGGCTCCCTGCGCTTGGGAACAAGGCCGCCCTCACTATCATGGGGGCCCAGCCAGAGGACGAGGACGAGTGTTACTGTGCTCTGTGGTTCAGCAACCATTTC Accession number 117: IGLV15 (RC) L1: ATGGCCTGGTCCCCTCTCCTCCTCACCCTCATCGCTCTCTGCACAG VL: GATCCTGGGCCCAGTCTCTGACTCAGCCCGCCTCAGTGTCTGGGACCCTGGGCCAGACAGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATCGGGTATAGTTATAGTGCTGTGGGCTGGTACCAACAGATCCCAGGAACAGCCCCCAAAACCCTCATCTATGGTAATAACAAACGAGCCTCAGGGGTCCCAGATCGATTCTCTGGCTCCAAGTCTGGCAACACAGCCACCCTGACCATCTCTGGGGTCCAGGCTGAGGACGAGGCCGATTATTACTGCTCAGCAGGAGACAGCAGTGGTAGTAGTGA Accession number 118: IGLV16 (RC) L1: ATGGCCTGGACTCCTCTCATCCTCATGCTCCTGTCTCACTGCACAG VL: GTTCCCTCTCCCAGCCTGTGCTGACCCAGCCACCCTCCCTCTCTGCATCTCCTGGAACATCAGCCAGACTCACCTGCGCCCTGAGCAGTGATGTCAGTGTTAGCAGCTCTCTCATATTCTGGTACCAGCAGAAGCCAGGGAGCCCTCCGGGGTATCTTCTGAGTTTCTACTCAGACTCAGTTAAGCACCAGGGCTCCGGGGTCCCCAGCCACTTCTCTGGATCCAAAGACACCTCGTCCAATGCAGGGCTTCTGCTCATCTCTGGGCTCGAGGCTGAGGACGAGGCTGACTATTACTGTGCTACAGCTGATAGCAGTGGGATCAGCTCTGGTTACT Accession number 119: IGLV17 (RC) L1: ATGGCCTGGTCCCCTCTCCTCCTCACCCTCATCGCTCTCTGCACAG VL: GATCCTGGGCCCAGTCTGTGACTCAGCCCGCCTCAGTGTCTGGGACCCTGGGCCAGACAGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATCGGGTATAGTTATAGTTATGTGGGCTGGTTCCAACAGATCCCAGGAACAGCCCCCAAAACCCTCATCTATGGTAATAACAAACGAGCCTCAGGGGTCCCAGATCGATTCTCTGGCTCCAAGTCTGGCAACACAGCCACCCTGACCATCTCTGGGGTCCAGGCTGAGGACGAGGCCGATTATTACTGTGGTTCCTATGACAGCAGCAGTAGTAGTGA Accession number 120: IGLV18 (RC) L1: ATGGCCTGGTCCCCTCTCCTCCTCACCCTCATCGCTCTCTGCACAG L2: TCCCGGGCCCAGTCTGTGACTCAGCCCGCCTCAGTGTCTGGGACCCTGGGCCAGACAGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATCGGGAGTGGTCATGTGTCCTGGTACCAACAGATCCCAGGAACAGCCCCCAAACGCCTCATCTATTCTTCCACTAATAGGGCTTCTGGGGTCCCCGACCGATTCTCTGGCTCCAGGTCTGGCAACACAGCCACCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGTGGTACATTGTACAGCAGTTGGAGTAATGA Accession number 121: IGLV19 (RC) L1: ATGGCCTGGTCCCCTCTCCTCCTCACCCTCATCGCTCTCTGCACAG LV: CAGTTCTGTGACTCAGCCCGCCTCAGTGTCTGGGACCCTGGCCAGACAGTCACCATCTCCTGCACTGGAAGCATCTCCAACATAGGTGTTTATGTGGACTGGTACCAACAGATCCCAGGAACAGCCCCCAAAACCATCATCTATG CTACTAAACAAACAACCCTCAGGGGTCCCAGATCGATTCTCTGGCTCCAAGTCTGGCAACACAGCCACCCTGACCATCACTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGTGGTATCTATGACAGCAGCCTGAGTAGTGA SEQ ID NO: 122: IGLV20 (RC) L1: ATGGCCTGGATGGTGCTTCTTCTCGGGCTCCTTTCTTACAGCTCAG VL: GGGCGGATTCTCAGTCTGTGGTGACCCAGGAGCCATCACTCTCAGTGTCTTCAGGAGGGACAGTCACACTCACCTGTGGCCTTAACTCTGGGTCAGTCTCTTCCAGTAACCACCCCAGCTGGCACCAGCAAACCCCAGGCCAGGCTCCCCGCA CACTTATCTACTACACAAACACCCGTGCCTCTGGAGTCCCTAATCTCTTCTCTGGATCCATCTCCGGGAACAGAGCCACCCTCACCATCACGGGGGCCCAGCGTGAGGACGAGGCCGACTATTACTGCGCTCTGTATACGGGTAGTTACACTGA

[0176] Pre-DJ This is a 21609 bp fragment upstream of the Ighd-5 Dh gene. The pre-DJ sequence is found on Mus musculus strain C57BL / 6J chromosome 12, assembly: GRCm38.p4, annotation 106, sequence ID: NC_000078.6 The entire sequence is located between two 100 bp sequences: Upstream of the Ighd-5 Dh gene segment corresponding to positions 113526905-113527004 of NC_000078.6: ATTTCTGTACCTGATCTATGTCAATATCTGTACCATGGCTCTAGCAGAGATGAAATATGAGACAGTCTGATGTCATGTGGCCATGCCTGGTCCAGACTTG (SEQ ID NO: 123) 2 kb upstream of the Adam6a gene, corresponding to positions 113548415-113548514 of NC_000078.6: GTCAATCAGCAGAAATCCATCATACATGAGACAAAGTTATAATCAAGAAATGTTGCCCATAGGAAACAGAGGATATCTCTAGCACTCAGAGACTGAGCAC (SEQ ID NO: 124)

[0177] Adam6a Adam6a (a disintegrin and metallopeptidase domain 6A) is a gene involved in male fertility. The Adam6a sequence is found on Mus musculus strain C57BL / 6J chromosome 12, assembly: GRCm38.p4, annotation release 106, sequence IDs: 113543908-113546414 are NC_000078.6. Adam6a sequence ID: OTTMUSG00000051592 (VEGA)

Claims

1. A transgenic rodent in which an endogenous rodent immunoglobulin variable locus has been deleted and replaced with a partial equine immunoglobulin locus comprising equine immunoglobulin variable gene coding sequences and non-coding regulatory sequences based on the endogenous rodent immunoglobulin variable locus, wherein the partial equine immunoglobulin locus of the transgenic rodent is functional and expresses immunoglobulin chains comprising equine variable domains and rodent constant domains.

2. A partial equine immunoglobulin locus (i) Uma V Ｈ , D Ｈ and J. Ｈ Coding sequence; (ii) the Umakappa VL and JL coding sequences; (iii) the horse lambda VL and JL coding sequences; (iv) the ADAM6 gene; (v) Pax-5 activating intergenic repeat (PAIR) element; (vi) a CTCF binding site from heavy chain intergenic control region 1; or (vii) any combination thereof The transgenic rodent of claim 1, comprising:

3. The transgenic rodent of claim 1 , wherein the non-coding regulatory sequences include promoters in front of the individual V gene segments, splice sites and recombination signal sequences for V(D)J recombination.

4. A cell of the B lymphocyte lineage from the transgenic rodent of claim 1.

5. An immortalized cell or hybridoma cell derived from a cell of the B lymphocyte lineage of claim 4.

6. A partial or complete immunoglobulin molecule comprising an equine variable domain and a rodent constant domain derived from a cell of the B lymphocyte lineage of claim 4 or a hybridoma cell or immortalized cell of claim 5.

7. 12. A method for producing the transgenic rodent of claim 1, comprising: a) integrating into the genome of a rodent cell at least one site-specific recombinase target site upstream of an endogenous immunoglobulin variable locus and at least one site-specific recombinase target site downstream of an endogenous immunoglobulin variable locus, wherein the endogenous immunoglobulin variable locus is (i) a V Ｈ , D Ｈ and J. Ｈ (ii) a Vκ and a Jκ gene segment, (iii) a Vλ and a Jλ gene segment; or (iv) a Vλ and a Jλ gene segment and a Cλ gene; b) providing a vector comprising a partial equine immunoglobulin locus, the partial equine immunoglobulin locus comprising partial equine immunoglobulin variable region gene segments each comprising equine immunoglobulin variable region gene coding sequences and rodent non-coding regulatory sequences, and the partial equine immunoglobulin variable region locus flanked by target sites for a site-specific recombinase, wherein the target sites are capable of recombination of the target sites introduced into the rodent cell in step a); c) introducing into a cell the vector of step b) and a site-specific recombinase capable of recognizing the target site; d) causing a recombination event to occur in the genome of the cell and the partial equine immunoglobulin locus, resulting in replacement of the endogenous immunoglobulin variable locus with the partial equine immunoglobulin locus; e) selecting cells containing the partial equine immunoglobulin variable locus produced in step d); and f) Using the cells to generate transgenic rodents containing partial equine immunoglobulin variable loci. The method includes:

8. The method of claim 7, wherein the cell is a rodent embryonic stem (ES) cell.

9. The method of claim 7, further comprising, after the introducing step and before the providing step, a step of deleting endogenous immunoglobulin variable loci by introducing a recombinase that recognizes a first set of target sites, wherein the deleting step leaves at least two target sites in the genome of the rodent cell that cannot recombine with each other.

10. Vector (i) Uma V Ｈ , D Ｈ and J. Ｈ Coding sequence; (ii) the kappa or lambda V L and J L coding sequences; (iii) V gene promoters, splice sites and recombination signal sequences of endogenous host origin; (iv) the ADAM6 gene; (v) Pax-5 activating intergenic repeat (PAIR) element; (vi) a CTCF binding site from heavy chain intergenic control region 1; or (vii) any combination thereof The method of claim 7, comprising:

11. 1. A method for producing an antibody for therapeutic or diagnostic use comprising: (i) the genome is deleted of endogenous rodent immunoglobulin locus variable regions and contains a chimeric V Ｈ , D and J Ｈ At least one of each of the immunoglobulin variable region gene segments and / or the immunoglobulin light chain locus is provided with a chimeric V Ｌ and J. Ｌ expressing antibodies having the cloned equine variable domains from antibody-producing cells of the transgenic rodent replaced with an immunoglobulin locus variable region comprising at least one of each of the variable gene segments, wherein each chimeric gene segment comprises an equine V, D or J immunoglobulin variable region coding sequence and a rodent immunoglobulin variable region non-coding gene segment sequence; and (ii) isolating an antibody having an equine variable domain, wherein said antibody is suitable for therapeutic or diagnostic use.

12. The method of claim 11 , wherein the antibody is cloned from a B cell of a transgenic rodent.

13. 1. A method for producing a therapeutic or diagnostic antibody having an equine variable domain comprising: (i) The genome is modified to have a deletion of endogenous rodent immunoglobulin locus variable regions and a chimeric V Ｈ , D Ｈ and J. Ｈ At least one of each of the immunoglobulin variable region gene segments and / or the immunoglobulin light chain locus is provided with a chimeric V Ｌ and J. Ｌ cloning equine variable domains of antibodies expressed by antibody-producing cells from the transgenic rodent, the equine variable domains being replaced with immunoglobulin locus variable regions comprising at least one of each of the variable gene segments, wherein each chimeric gene segment comprises an equine V, D or J immunoglobulin variable region coding sequence and a rodent immunoglobulin variable region non-coding gene segment sequence; and (ii) producing a therapeutic or diagnostic antibody comprising an equine variable domain of an antibody expressed by the transgenic rodent; The method includes:

14. The method of claim 13 , wherein the equine variable domain is cloned from an antibody expressed by a B cell from a transgenic rodent.

15. 1. A method for producing a monoclonal antibody comprising an equine variable domain, comprising: (i) The genome is modified to have a deletion of endogenous rodent immunoglobulin locus variable regions and a chimeric V Ｈ , D Ｈ and J. Ｈ At least one of each of the immunoglobulin variable region gene segments and / or the immunoglobulin light chain locus is provided with a chimeric V Ｌ and J. Ｌ providing B cells from the transgenic rodent, wherein the B cells have been replaced with an immunoglobulin locus variable region comprising at least one of each of the variable gene segments, wherein each chimeric gene segment comprises an equine V, D or J immunoglobulin variable region coding sequence integrated into a rodent immunoglobulin variable region non-coding gene segment sequence; (ii) immortalizing the B cells; and (iii) isolating a monoclonal antibody comprising an equine variable domain expressed by the immortalized B cells or a gene encoding said antibody. The method includes:

16. moreover (iv) cloning the horse variable domains expressed by the B cells; and (v) Producing therapeutic or diagnostic antibodies comprising the cloned equine variable domains from B cells of the transgenic rodent.

16. The method of claim 15, comprising:

17. A method for producing an antibody comprising an equine variable domain, the genome being modified to have endogenous rodent immunoglobulin locus variable regions deleted and chimeric V domains at the immunoglobulin heavy chain loci. Ｈ , D Ｈ and J. Ｈ At least one of each of the immunoglobulin variable region gene segments and / or the immunoglobulin light chain locus is provided with a chimeric V Ｌ and J. Ｌ providing a transgenic rodent having an immunoglobulin locus variable region comprising at least one of each of the variable gene segments, wherein each chimeric gene segment comprises an equine V, D or J immunoglobulin variable region coding sequence integrated into a rodent immunoglobulin variable region non-coding gene segment sequence, wherein the immunoglobulin locus of the transgenic rodent expresses an antibody comprising an equine variable domain.

18. (i) obtaining B cells from the transgenic rodent that express an antibody specific for a target antigen; (ii) immortalizing the B cells; and (iii) isolating antibodies specific to the target antigen from the immortalized B cells.

20. The method of claim 17, further comprising:

19. 20. The method of claim 18, further comprising cloning the equine variable region from a B cell specific for a particular antigen.

20. 20. The method of claim 19, further comprising using the equine variable regions cloned from the B cells to produce therapeutic or diagnostic antibodies.

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