Rodents expressing common light chains

Abstract

The present invention relates to genetically modified rodents whose genomes contain only a single canine light chain V gene segment, methods for using said rodents to obtain and identify antibodies, such as bispecific antibodies, that share a common light chain, and the bispecific antibodies resulting from said methods.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to UK Patent Application No. 2217978.2, filed November 30, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to genetically modified rodents whose genomes contain only a single canine light chain V gene segment. The present invention also relates to genetically modified rodents that express antibodies containing a common canine light chain. The present invention also provides methods for producing bispecific antibodies using genetically modified rodents, and antibodies produced from the genetically modified rodents. [Background technology]

[0003] Antibodies typically contain two identical heavy chains (homodimers), with each heavy chain monomer associated with an identical light chain. Antibodies with heterodimeric heavy chains, such as bispecific antibodies, can be used as effective therapeutic antibodies.

[0004] The challenge in creating bispecific antibodies stems from the fact that four chains (two heavy chains and two light chains) can form several undesired products. The problem of heavy chain mispairing can be solved by introducing mutations into the CH3 domain of the Fc to promote heterodimer formation. However, light chains can mispair with unmatched heavy chains, resulting in undesired impurities. One solution is to use a common light chain, where a single light chain pairs with both heavy chains of the bispecific antibody. This reduces the number of undesired products.

[0005] A discussion of the common light chain approach to bispecific antibodies can be found in WO2013 / 134263 and WO2019 / 008123, both of which are incorporated by reference.

[0006] Antibodies containing human DNA can be obtained from rodents. The insertion of human DNA into rodents is disclosed, for example, in Murphy et al., Vol. 111 no. 14, 5153-5158, doi:10.1073 / pnas.1324022111, MacDonald et al., vol. 111 no. 14, 5147-5152, doi:10.1073 / pnas.1323896111, and Lee et al., Nature Biotechnology Volume: 32, Pages: 356-363 2014 doi:10.1038 / nbt.2825. This approach is designed to generate antibody products for human therapeutic use. The insertion of canine DNA into rodents is disclosed in WO2018189520. All of these documents are incorporated herein by reference. Summary of the Invention

[0007] The present invention relates to the following:

[0008] A rodent or rodent embryonic stem (ES) cell whose genome contains only a single canine light chain V gene segment.

[0009] a rodent expressing a population of antibodies, wherein all canine immunoglobulin light chains of the population of antibodies comprise a canine light chain variable region derived from the same single canine light chain V gene segment.

[0010] 1. A method for producing antibodies with multiple specificities, the method comprising: (a) immunizing a rodent as disclosed herein that contains only a single canine light chain V gene segment with a first antigen; (b) identifying a first canine nucleic acid encoding a first canine heavy chain variable region that binds to a first antigen; (c) immunizing the rodent with a second antigen; (d) identifying a second canine nucleic acid encoding a second canine heavy chain variable region that binds to a second antigen; (e) expressing a first canine heavy chain variable region, a second canine heavy chain variable region, and one single canine light chain variable region obtained from a rearranged single canine light chain V gene segment; (f) thereby obtaining an antibody comprising the first canine heavy chain variable region, the second canine heavy chain variable region, and a single canine light chain variable region; (g) optionally formulating the antibody as a pharmaceutical composition. [Brief explanation of the drawings]

[0011] [Figure 1A] 1 shows the construction of a canine common light chain targeting vector. [Figure 1B] Figure 1 shows mouse immunoglobulin kappa V gene usage in naive mouse PBMCs (peripheral blood mononuclear cells), characterized using single cell VDJ sequencing. [Figure 1C] 1 shows an ES cell targeting strategy to replace endogenous mouse immunoglobulin kappa light chain variable region gene segments with a rearranged canine lambda common light chain comprising canine VL3-3, lambda J1, and lambda C5 constant regions. [Figure 2] 1 shows characterization of common light chain mouse whole blood and splenic B cells by flow cytometry. [Figure 3A] Figure 1 shows the sorting strategy for a population enriched in common light chains.Figure 2 shows a schematic diagram of the gating strategy for isolating splenic B cells that do not express mouse kappa or lambda light chains. [Figure 3B] 1 shows the sorting strategy for the common light chain enriched population. A representative density plot of common light chain mice is shown showing the gates used for cell sorting. [Figure 4A] Characterization of the consensus light chain transcript is shown. Using the mouse IKGV14-111 promoter, characterization of the 5'UTR at base pair resolution is shown. [Figure 4B] 1 shows characterization of the consensus light chain transcript. The annotated consensus light chain amino acid sequence is shown. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention relates to platforms for antibody sequence discovery. These platforms are genetically modified rodents that contain only a single canine light chain V gene segment in their genome. The rodents may also contain a canine light chain J gene segment that can optionally be expressed with a canine constant region, allowing the rodents to produce complete canine antibody light chains. The rodents may also contain canine heavy chain V, D, and J gene segments that can be expressed with rodent or canine constant regions, allowing the rodents to produce complete canine antibody chains or chimeric canine antibodies, for example, in which a canine V gene segment is expressed with a rodent constant region.

[0013] All canine antibodies produced from such rodents contain the same canine light chain V gene segments but produce a repertoire of heavy chain variable regions, and the rodents preferably contain antibodies that contain a repertoire of canine heavy chain V gene segments. Thus, separate immunizations with two different antigens (either from the same animal or from different animals with the same genetic makeup) allow for the discovery of antibody chain sequences suitable for use in bispecific antibodies with a common light chain.

[0014] The invention also relates to, inter alia, rodents and cells engineered to contain exogenous canine DNA, their uses in medical treatment and disease research, methods for producing the rodents and cells, and antibodies and antibody chains produced by such rodents, and derivatives thereof.

[0015] The invention also relates to antibody repertoires, antibodies and antibody portions (including complete canine antibodies, such as those produced from rodents containing canine immunoglobulin DNA), and the use of such antibodies and portions thereof in dogs for the prevention and treatment of disease, as well as methods for producing such rodents, cells, antibodies, antibody chains, and repertoires.

[0016] The rodent of the present invention has a genome containing only a single canine light chain V gene segment. This rodent produces antibodies with a light chain derived from a single canine light chain V gene segment. Preferably, the common light chain is a rearranged light chain in the genome, and rearrangement of the light chain VJ gene segment is not required to generate the common light chain. As disclosed herein, such rodents can be used as a platform for discovering therapeutic canine antibodies. This approach is referred to as the common light chain approach.

[0017] In one aspect, the present invention relates to a rodent or rodent embryonic stem (ES) cell whose genome comprises only a single canine light chain V gene segment.

[0018] Although only a single canine light chain V gene segment is present in the genome of a rodent, the resulting population of antibodies derived from that single V gene segment may have slightly different V regions due to somatic hypermutation. Thus, although canine light chain antibodies are all derived from a single canine light chain V region, in one embodiment, they are not all necessarily identical within a rodent.

[0019] If there is somatic hypermutation, it will be reflected in the different sequences expressed by different B cells within the mouse. Thus, the somatic hypermutated genomes of B cells may differ from each other, although for the avoidance of doubt, this is consistent with the disclosure that ES cells or rodent genomes contain only a single canine light chain V gene segment.

[0020] In one embodiment, the rodent genome is a germline genome.

[0021] In one embodiment, the rodent genome is the genome of a cell that has not undergone somatic hypermutation.

[0022] In one embodiment, the rodent genome is the genome of a cell that is not a B cell.

[0023] In one embodiment, somatic hypermutation is minimized or prevented, for example, by placing part or all of the canine light chain V gene sequence downstream of an intronic enhancer at the light chain locus, e.g., downstream of an intronic enhancer at the kappa locus, as further described herein. Approaches to preventing somatic hypermutation are described, for example, in EP 3648587.

[0024] In one embodiment, the rodent ES cell or genomic cell comprises an antibody light chain locus, the locus comprising (in a 5' to 3' direction): (a) light chain intronic enhancer, (b) all or part of a canine light chain V gene segment; (c) a canine light chain J gene segment, and (d) a light chain constant region of a canine antibody; This locus is operable to express an antibody light chain.

[0025] In one embodiment, (b) is a portion of a canine light chain V gene segment, and the locus further comprises a remaining portion of the canine light chain V gene segment located 5' of the light chain intronic enhancer, wherein the two portions together form a single contiguous canine light chain V gene segment after expression and splicing, and preferably the effect of somatic hypermutation on the canine light chain is reduced or eliminated compared to wild-type levels of somatic hypermutation.

[0026] In one embodiment, the canine light chain locus comprises a rearranged canine VJ region expressed in combination with a constant region. The constant region can be a canine constant region or a rodent constant region. Preferably, the constant region is a canine constant region.

[0027] In one embodiment, the canine light chain locus comprises a rearranged canine VJ region expressed in combination with a constant region, and the CDR3 of the expressed light chain is as disclosed herein, e.g., SEQ ID NO: 3.

[0028] The insertion of the rearranged canine light chain locus may prevent further editing of the light chain, such as by modifying the sequence encoding the light chain CDR3.

[0029] The specific single canine light chain V gene segment selected for use in the common light chain may be a canine light chain V gene segment that is highly expressed in naive (unimmunized) dogs or that pairs with many different canine heavy chain V gene segments in naive dogs, for example, it may be the most highly expressed, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth most highly expressed in unimmunized dogs.

[0030] In another embodiment, a single canine light chain V gene segment is selected by immunizing a dog with an antigen of interest and then selecting a canine light chain V gene segment that is highly expressed in the immunized dog, that is, the most highly expressed one, for example, the second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth most highly expressed one.

[0031] In one embodiment, the single canine light chain V gene segment is a canine V gene segment, e.g., selected from IGKV2-5, IGKV2-11, IGKV2S13, IGKV3-8, IGLV3-18, IGLV3-11, IGLV3-21, IGKV2-9, IGKV2-10, IGKV2-12, IGKV2-16, IGLV3-14, IGLV3-24, IGKV2-8, and IGLV3-3.

[0032] In one embodiment, the single canine light chain V gene segment is a canine kappa V gene segment.

[0033] Gene families, such as IGKV family 3, are terms well known in the art. In one aspect, references to gene families refer to the IMGT repertoire (ImMunoGeneTics) notation, which is available (as of May 19, 2022) at https: / / www.imgt.org / IMGTrepertoire / index.php?section=LocusGenes&repertoire=genetable&species=dog&group=IGHK.

[0034] In one embodiment, the single canine light chain V gene segment is a canine lambda V gene segment. In one embodiment, the single canine light chain V gene segment is a canine lambda V gene segment of the canine lambda V3 family, e.g., selected from IGLV3-18, IGLV3-11, IGLV3-21, IGLV3-14, IGLV3-24, and IGLV3-3.

[0035] In one embodiment, the single canine light chain V gene segment is IGLV3-3.

[0036] In one embodiment, the single canine light chain V gene segment comprises the amino acid sequence of SEQ ID NO:2, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO:2.

[0037] In one embodiment, the sequence encoding the CDR3 of the canine light chain comprises or consists of 33 nucleotides, particularly when the resulting antibody is capable of binding to an NK cell antigen or a T cell antigen, such as a CD16 polypeptide or a CD3 polypeptide, respectively, and the light chain is combined with an antibody heavy chain.

[0038] The NK cell antigens or T cell antigens disclosed herein, such as CD16 or CD3, are in one embodiment canine antigens.

[0039] In one embodiment, the canine light chain rodent or rodent ES cell CDR3-encoding sequences disclosed herein include canine light chain CDR3-encoding sequences that can result in a CDR3 of 11 amino acids.

[0040] In one embodiment, the canine light chain rodent or rodent ES cell CDR3-encoding sequences disclosed herein include canine light chain CDR3-encoding sequences that can result in a CDR3 of exactly 11 amino acids.

[0041] In one embodiment, the rodent or rodent ES cell disclosed herein comprises the sequence of SEQ ID NO:4, or a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO:4.

[0042] In one embodiment, the antigen of interest used to immunize a rodent and determine a single canine light chain V gene segment is a cancer-associated antigen. In one embodiment, the antigen of interest is an NK cell antigen or a T cell antigen, such as CD16 or CD3, respectively. In one embodiment, the light chain, when combined with a suitable canine heavy chain, is capable of recognizing a cancer-associated antigen, or an NK cell antigen or a T cell antigen, such as CD16 or CD3.

[0043] In one embodiment, in addition to the insertion of the single canine light chain V gene segment, a canine J gene segment is inserted into the genome of the rodent. In this manner, the rodent expresses an antibody having a canine V gene segment and a J gene segment. In one embodiment, the single canine light chain V gene segment is operably linked to a canine lambda J gene segment in the genome of the rodent or rodent ES cell disclosed herein. In one embodiment, the single canine light chain V gene segment is operably linked to a canine lambda J1 gene segment.

[0044] In one embodiment, the single canine light chain V gene segment is operably linked to a canine light chain J gene segment comprising the sequence of SEQ ID NO:8, or a sequence having at least 75%, 80%, 85%, 90%, or 95% sequence similarity to SEQ ID NO:8.

[0045] In one embodiment, in addition to inserting a single canine light chain V gene segment (and, in one embodiment, a canine light chain J gene segment), a canine light chain constant region can be inserted into the genome of the rodent. Inserting a canine light chain V gene segment, a canine light chain J gene segment, and a canine light chain constant region allows the rodent to produce a complete canine (common) light chain.

[0046] Insertion of a canine light chain V gene segment and a canine light chain J gene segment upstream of a rodent constant region allows rodents to produce chimeric (common) light chains.

[0047] In one embodiment, a single canine light chain V gene segment is operably linked to a canine light chain constant region in a rodent or rodent ES cell disclosed herein. In one embodiment, a single canine light chain V gene segment is operably linked to a canine lambda constant region in a rodent or rodent ES cell disclosed herein. In one embodiment, a single canine light chain V gene segment is operably linked to a canine λ5 lambda constant region in a rodent or rodent ES cell disclosed herein.

[0048] In one embodiment, a single canine light chain V gene segment is operably linked to a canine λ5 lambda constant region in a rodent or rodent ES cell disclosed herein, wherein the constant region comprises the nucleotide sequence of SEQ ID NO:6, or a sequence having at least 75%, 80%, 85%, 90%, or 95% sequence similarity to SEQ ID NO:6.

[0049] In one embodiment, only a single canine light chain J gene is inserted into the genome of the rodent. Thus, in combination with a single canine light chain V gene segment, the rodent produces an antibody light chain expressed from a single light chain V gene segment and a single light chain J gene segment, which may be a single canine common light chain.

[0050] Thus, the present invention relates to rodents or rodent ES cells whose genomes comprise only a single canine light chain V gene segment and only a single canine light chain J gene segment. The present invention also relates to methods for producing multispecific antibodies, comprising, inter alia, immunizing a rodent as disclosed herein that comprises only a single canine light chain V gene segment and only a single canine light chain J gene segment.

[0051] The present invention relates to rodents or rodent ES cells whose genome encodes only a single canine light chain variable region. The present invention also relates to a method for producing multispecific antibodies, which method comprises, inter alia, immunizing a rodent as disclosed herein that contains only a single canine light chain variable region.

[0052] It is observed that the common light chain expressed from the rodents of the present invention may be of a particular length and sequence, and the inventors note that a CDR3 length of 11 amino acids may be of particular value, particularly when combined with a suitable heavy chain as described herein, where the light chain is capable of binding to a CD3 polypeptide.

[0053] In one embodiment, the light chain CDR3 resulting from expression of a canine V gene segment, in combination with an antibody heavy chain, is capable of binding to a CD3 polypeptide, such as a canine CD3 polypeptide.

[0054] In one embodiment, a single canine light chain V gene segment is inserted into the genome of the rodent as a single contiguous sequence.

[0055] An alternative to inserting a single canine light chain V gene segment is to insert it into the rodent genome in two (or more) (discontinuous) parts, in which embodiment a regulatory region may be present between the two parts.

[0056] Thus, as disclosed herein, a single V gene segment need not be a contiguous piece of DNA in the genome, but may be composed of two or more DNA pieces that are spliced ​​together after expression to form a single V gene segment that can be expressed along with a J gene segment and constant region to form a canine light chain.

[0057] In one embodiment, the single canine light chain V gene segment is non-contiguous in the genome. In one embodiment, the single canine light chain V gene segment is separated into two parts in the genome. In one embodiment, the single canine light chain V gene segment is separated into two parts in the genome, and these parts can be spliced ​​to form a continuous nucleic acid sequence.

[0058] It may be preferable to insert a single canine light chain V gene segment into a location in the host rodent where it can be expressed at sufficient levels, for example, because rodents naturally express kappa light chains at higher levels than lambda light chains, insertion at the rodent kappa locus may be advantageous.

[0059] In one embodiment, the insertion of the single canine light chain V gene segment is made into the immunoglobulin (Ig) locus of the host rodent. In one embodiment, the single canine light chain V gene segment is located at the mouse immunoglobulin kappa locus on chromosome 6, e.g., between positions 70,500,000 and 71,000,000 on chromosome 6, e.g., between positions 70,698,946 and 70,726,755 on chromosome 6, e.g., the single canine light chain V gene segment is located between positions 70,698,946 and 70,726,755 on chromosome 6, optionally in two portions that can be spliced ​​to form a contiguous sequence.

[0060] In one embodiment, at least a portion of a single canine light chain V gene segment is downstream of a rodent intronic enhancer, for example, downstream of the mouse kappa intronic enhancer at the mouse immunoglobulin kappa locus in a mouse.

[0061] In one embodiment, the single canine light chain V gene segment is present upstream of the canine kappa constant region at the endogenous rodent kappa locus.

[0062] Alternatively, a single canine V gene segment, which may be kappa or lambda, may be located at the lambda locus in combination with any suitable J gene segment and constant region.

[0063] Expression of the canine common light chain is driven by a promoter. In one embodiment, the promoter is inserted into the genome of the rodent. The promoter operably linked to a single canine light chain V gene segment allows for the production of the canine common light chain.

[0064] In one embodiment, the promoter is any promoter effective to drive expression of a light chain V gene segment in a host rodent, and may be a host promoter, such as, for example, a mouse promoter in a mouse.

[0065] In one embodiment, the single canine light chain V gene segment is operably linked to a canine promoter, which may be the naturally occurring canine promoter for that gene segment.

[0066] In one embodiment, the single canine light chain V gene segment is operably linked to a mouse promoter.

[0067] In one embodiment, the single canine light chain V gene segment is operably linked to a mouse kappa IGK promoter or a mouse lambda IGL promoter.

[0068] In one embodiment, the single canine light chain V gene segment is operably linked to a mouse promoter selected from the IGKV1 family, the IGKV14 family, the IGKV4 family, the IGKV6 family, the IGKV8 family, or the IGKV3 family, e.g., the IGKV14-111 promoter, the mouse IGKV6-15 promoter, or the mouse IGKV6-23 promoter.

[0069] In one embodiment, the single canine light chain V gene segment is operably linked to a mouse promoter of the mouse IGK14 family.

[0070] In one embodiment, the single canine light chain V gene segment is operably linked to a mouse IGKV14-111 promoter. In one embodiment, the single canine light chain V gene segment is operably linked to a mouse IGKV6-15 promoter. In one embodiment, the single canine light chain V gene segment is operably linked to a mouse IGKV6-23 promoter.

[0071] In one embodiment, the inserted promoter for expression of at least a single canine V gene segment is located upstream of a rodent intronic enhancer, for example, upstream of the mouse kappa intronic enhancer at the mouse immunoglobulin kappa locus in a mouse.

[0072] In one embodiment, the genome of the rodent or rodent ES cell comprises an endogenous rodent 3' enhancer operably linked to the expression of a single canine light chain V gene segment. The enhancer may be a naturally occurring rodent enhancer, for example, the 3' enhancer located at the kappa locus for insertion of a canine V gene segment that occurs at the rodent kappa locus.

[0073] In one embodiment, an enhancer may be inserted into the genome of the rodent, and the enhancer may be an intronic enhancer, such as a kappa intronic enhancer.

[0074] In one embodiment, the rodent comprises a rodent splice donor sequence operably associated with the inserted canine sequence and mediating correct splicing of the canine sequence in the ES cell or the mouse. In one embodiment, the mouse IGK J5 exon splice donor is located immediately after the first portion of the single light chain variable gene segment and operates to splice this first exon to the second canine light chain sequence.

[0075] To discover suitable heavy chain sequences for therapeutic canine (bispecific) antibodies, rodents can be genetically engineered to contain canine heavy chain V gene segments, preferably along with canine heavy chain D and / or J gene segments, optionally along with a canine heavy chain constant region, and optionally along with a canine heavy chain regulatory region.

[0076] In one embodiment, all of the canine heavy chain V genes, or substantially all of the V gene segments, e.g., at least 50%, 60%, 70%, 80%, 90% or more of the canine V gene segments, are inserted into the genome of the host rodent.

[0077] In one embodiment, only some of the canine heavy chain V gene segments are inserted into the genome of the host rodent, e.g., to reduce the amount of genetic recombination required. To generate a diverse repertoire, it may be beneficial to introduce only specific canine heavy chain V gene segments. This may be based on the natural frequency of heavy chain V gene segments in naive dogs or the number of different light chains in each heavy chain pair within naive dogs.

[0078] In one embodiment, the rodent cell or ES cell is selected from the group consisting of IGHV4-1cf, IGHV3-4cf, IGHV3-5-1cf, IGHV3-5cf, IGHV1-4-1cf, IGHV3-2cf, IGHV3-7cf, IGHV3-8cf, IGHV(II)-1cf, IGHV(II)-2cf, IGHV3-6cf, IGHV3-12cf, IGHV3-11cf, IGHV3-29cf, IGHV3-23cf, IGHV3-21-1cf , IGHV3-18cf, IGHV3-19cf, IGHV3-20cf, IGHV3-21cf, IGHV3-22cf, IGHV3-31cf, IGHV3-24cf, IGHV1-30cf, IGHV3-25cf , IGHV3-26cf, IGHV3-27cf, IGHV3-28cf, IGHV3-42cf, IGHV3-33cf, IGHV3-32cf, IGHV3-46cf, IGHV3-47cf, IGHV3-40cf, IGHV3-45cf, IGHV3-39cf, IGHV3-38cf, IGHV3-37cf, IGHV3-43cf, IGHV3-41cf, IGHV3-34cf, IGHV3-35cf, IGHV3-44cf, IGHV3-36cf, IGHV3-70cf, IGHV3-62cf, IGHV3-69cf, IGHV3-74cf, IGHV3-60cf, IGHV3-59cf, IGHV3-67cf, IGHV3-68cf, I The canine heavy chain V gene segments comprise one or more or all of the canine heavy chain V gene segments selected from the group consisting of GHV3-66cf, IGHV3-65cf, IGHV3-64cf, IGHV3-63cf, IGHV3-61cf, IGHV3-81cf, IGHV3-54cf, IGHV3-10cf, IGHV3-58cf, IGHV3-3cf, IGHV3-9cf, IGHV3-75cf, IGHV3-80cf, IGHV3-16cf, and IGHV3-50cf.

[0079] In one embodiment, the canine heavy chain V gene segments found to be most frequently used in naive or immunized dogs are included in the genome of the rodent or rodent cell, e.g., at least the most frequently used 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 canine heavy chain V gene segments are included in the rodent genome.

[0080] Depending on how the canine DNA is inserted into the host rodent genome, the host rodent may still be able to make rodent antibodies.

[0081] In one embodiment, it is preferable to prevent the host rodent from making complete rodent antibodies. This can be achieved by various methods, such as excising the host DNA or inverting the endogenous locus of the variable region so that there is no production of host antibody chain expression. In one embodiment, inserting canine DNA into the Ig locus upstream of the rodent constant region (between the naturally occurring rodent gene segments and the constant region) moves the host rodent V(D) and J gene segments away from the rodent constant region, thereby reducing or inactivating expression of the host rodent antibody from that locus.

[0082] In one embodiment, the insertion of canine DNA results in or is associated with the removal of an endogenous rodent light chain J gene segment, resulting in an inability to produce a complete rodent antibody. Thus, in one embodiment, the rodent cell or rodent lacks a rodent J gene segment at one or both light chain loci.

[0083] In one embodiment, expression of some or all of the endogenous V gene segments is prevented, optionally by inversion or excision of the V gene segments relative to the native constant region.

[0084] In one embodiment, all or some of the rodent V region genes are deleted, e.g., at least 50%, preferably at least 75% or at least 90%, or all of the IGH V gene segments and / or D gene segments and / or J gene segments of the rodent; and / or At least 50%, preferably at least 75%, or at least 90%, or all of the rodent IGL V and / or J gene segments from kappa and / or lambda are deleted.

[0085] In one embodiment, the rodent is a mouse and the genome preferably comprises a deletion of one, some, or all of the mouse IGH V-region genes V1-85 through V5-2.

[0086] In one aspect, the rodent is a mouse and the genome preferably comprises a deletion of one, some, or all of the mouse IGL kappa V-region genes V3-1 through V2-137.

[0087] In one embodiment, the rodent is a mouse, and the mouse heavy chain D and J region genes are retained in the genome upstream of the inserted canine heavy chain variable region gene.

[0088] In one aspect, no rodent IGL lambda genes are deleted from the rodent genome. In an alternative embodiment, all of the lambda genes are deleted.

[0089] In one embodiment, one or both alleles of the rodent kappa locus are deleted or inactivated in whole or in part by inserting canine DNA into the rodent kappa locus.

[0090] In one aspect, the rodent kappa locus is inactivated in whole or in part, for example, by insertion, deletion, or inversion.

[0091] In one aspect, the rodent lambda locus is inactivated in whole or in part, for example, by insertion, deletion, or inversion.

[0092] In one aspect, the rodent heavy chain locus is wholly or partially inactivated, for example, by insertion, deletion, or inversion.

[0093] In one embodiment, the genetic modification is performed in host rodent ES cells, which are then developed into rodents using well-known techniques. Thus, the present invention relates to both rodents and ES cells.

[0094] The present invention also relates to cells that are not embryonic stem cells but that can be obtained from rodents as disclosed herein.

[0095] In one embodiment, the rodent or rodent ES cell is a mouse or mouse ES cell. In one aspect, the rodent cell of the invention is a rodent ES cell, rodent hematopoietic stem cell, or other cell that can develop into a rodent capable of producing a repertoire of antibody chains comprising variable regions encoded by canine DNA (such as chimeric antibody heavy chains or chimeric antibody light chains), or complete canine antibody chains, or antibodies encoded by canine variable regions with variable and constant regions.

[0096] In one aspect, the cells of the invention are rodent ES cells or induced pluripotent stem cells (iPS cells). Such cells are suitable for inserting canine DNA to generate rodents that express antibody chains as described herein.

[0097] In one embodiment, the cell is an isolated rodent cell.

[0098] In one embodiment, the cell is an isolated rodent B cell.

[0099] The rodent or rodent cell of the present invention is preferably a mouse or rat, or a mouse or rat cell (such as a mouse or rat ES cell), preferably a mouse or mouse ES cell. The ES cell may be of the 129 or C57BL strain of mouse cells, e.g., C57BL / 6N, C57BL / 6J, 129S5, or 129Sv strain group, or may be in a cell with a hybrid genome comprising 129 and / or C57BL genomic DNA.

[0100] The present invention also relates to cell lines propagated or otherwise derived from cells as described herein, including immortalized cell lines.

[0101] The cells or cell lines of the invention may contain canine V, D, or J genes in an unrearranged configuration or after rearrangement following in vivo maturation.

[0102] The present invention also relates to cells or cell lines, such as CHO cell lines, that express antibody chains having variable regions obtainable by immunizing a rodent of the present invention with an antigen, or for which the nucleic acid sequences of the variable regions can or have been identified from a rodent or rodent cell described herein, or from the antibody repertoire described herein. The antibody chains may be chimeric antibody heavy chains, preferably complete canine antibody chains. The cells or cell lines expressing the antibody chains or antibodies may be CHO cells or other mammalian cell lines suitable for producing animal therapeutics. Cells may be immortalized by fusion to tumor cells to provide antibody-producing cells and cell lines, or may be generated by direct cell immortalization.

[0103] Various dog breeds can be used as sources of canine DNA. Preferred canine DNA is boxer DNA, and therefore preferred cells and rodents contain boxer DNA gene segments as disclosed herein. The use of boxer regulatory sequences is also preferred. Boxer DNA may be provided as genomic DNA.

[0104] In one embodiment, the canine DNA is beagle DNA, and thus the cells and rodents contain beagle DNA gene segments as disclosed herein. Use of beagle regulatory sequences may also be used. The beagle DNA may be provided as genomic DNA.

[0105] Although the invention relates to canine DNA, it will be understood that the same teachings apply equally to other canids, and therefore the invention more generally relates to the use of DNA from such other non-canine canids, and all teachings relating to dogs can be read as teachings relating to other non-canine canids.

[0106] It will be appreciated that, in a related aspect, the present invention provides a rodent expressing an antibody population, wherein all of the canine immunoglobulin light chains of the antibody population comprise a canine light chain variable region derived from the same single canine light chain V gene segment. Preferably, all or substantially all of the canine immunoglobulin light chains of the antibody population comprise the same single canine light chain V gene segment. In one embodiment, the antibody population does not contain, or is substantially free of, mouse V gene segments (e.g., less than 10%, or less than 5%, or less than 1% of the light chains have mouse V gene segments). All features disclosed herein apply equally to such aspects, e.g., with respect to selection of canine V and J gene segments, selection of constant regions, selection of canine heavy chain gene segments, inactivation of host antibody expression, and regulatory control elements. Preferably, the rodent is a mouse. In a related aspect, the present invention relates to cells, such as ES cells, that can be developed into rodents as disclosed herein.

[0107] In one embodiment, the invention relates to a rodent as disclosed herein, wherein the antibody light chain repertoire is at least 95% pure for a single canine light chain V domain species.

[0108] In one embodiment, the invention relates to a rodent as disclosed herein, wherein the antibody light chain repertoire is at least 99% pure for a single canine light chain V domain species.

[0109] In one embodiment, the remaining light chains of the light chain repertoire comprise variants of the canine light chain V domain species.

[0110] In one embodiment, the invention relates to a rodent as disclosed herein, wherein the antibody light chain repertoire is substantially 100% pure for a single canine light chain V domain species.

[0111] In one embodiment, the invention relates to a rodent as disclosed herein, wherein at least 95% of all VL domains of the rodent's antibody light chain repertoire are encoded by the same canine light chain variable region sequence contained by the rodent's genome.

[0112] In one embodiment, the invention relates to a rodent as disclosed herein, wherein the light chain V domain species is canine lambda V3-3.

[0113] In one embodiment, the invention relates to rodent ES cells as disclosed herein that can be developed into rodents as disclosed herein.

[0114] As disclosed herein, the present invention can be used as a platform for the discovery of canine heavy chains for use in canine therapeutic antibodies, such as bispecific antibodies.

[0115] Thus, the present invention relates to a method for producing antibodies with multiple specificities, the method comprising: (a) immunizing a rodent as disclosed herein that contains only a single canine light chain V gene segment with a first antigen; (b) identifying a first canine nucleic acid encoding a first canine heavy chain variable region that binds to a first antigen; (c) immunizing the rodent with a second antigen; (d) identifying a second canine nucleic acid encoding a second canine heavy chain variable region that binds to a second antigen; (e) expressing in the cell (i) a first canine heavy chain variable region, (ii) a second canine heavy chain variable region, and (iii) one single canine light chain variable region obtained from expression of a single canine light chain V gene segment, preferably together as first and second antibody heavy chains and a common antibody light chain; (f) thereby obtaining an antibody comprising the first canine heavy chain variable region, the second canine heavy chain variable region, and a single canine light chain variable region; (g) optionally formulating the antibody as a pharmaceutical composition.

[0116] Step (e) optionally and preferably includes expressing in the cell (i) a first heavy chain comprising a first canine heavy chain variable region, (ii) a second heavy chain comprising a second canine heavy chain variable region, and (iii) only one common canine light chain comprising a single canine light chain variable region obtained from the expression of a single canine light chain V gene segment, thereby obtaining an antibody having a heavy chain and a light chain. The light chain is preferably entirely canine. The rodent may contain only one light chain canine J gene segment that is expressed together with one canine V gene segment to form the light chain variable region of the antibody chain. The heavy chain is preferably expressed in the rodent as a chimeric heavy chain (canine-variable rodent constant region).

[0117] Optionally, between step (d) and step (e), the method includes optimizing the binding of one or both of the first canine heavy chain variable region and the second canine heavy chain variable region to the first antigen and the second antigen, respectively.

[0118] The single light chain expressed in (e) is capable of pairing with both heavy chains and is preferably identified as paired with a heavy chain after immunization in steps (a) and (c).

[0119] In one embodiment, the first antigen or second antigen is an NK cell antigen or a T cell antigen such as CD16 or CD3, for example, mouse CD3 or canine CD3, or is a cancer-associated antigen.

[0120] WO2004 / 106375, WO2009 / 1577771, EP2147594, and WO2014 / 160179 discuss technology related to the production of bispecific antibodies and common light chain mice, all of which are incorporated by reference.

[0121] It should be noted that although the disclosure herein may refer to bispecific antibodies, the disclosure applies equally to multispecific antibody formats capable of binding to three or more antigens.

[0122] Disclosed herein is an in vivo approach for identifying common light chains for bispecific antibodies using chimeric rodents, in which B cells from the rodents are derived from ES cells with a light chain locus that expresses an immunoglobulin light chain repertoire with a limited number (e.g., only one) of light chain protein variable domains. Chimeric mice can be generated from wild-type, RAG knockout, or IgL knockout mouse blastocysts injected with mouse ES cells carrying knocked-in canine light chain coding sequences and an unrearranged canine or endogenous IgH locus. Methods for generating such chimeric rodents expressing common light chains are provided. Methods for identifying antigen-specific common light chain antibodies useful as bispecific antibodies, as well as the antibodies themselves and cells expressing these antibodies, are also provided.

[0123] Disclosed herein are genetically modified rodents that express canine heavy and light chain variable domains and have a limited light chain variable repertoire.

[0124] Disclosed herein is a biological system for generating canine light chain variable domains that are expressed in association with a diverse repertoire of affinity-matured canine heavy chain variable domains.

[0125] Disclosed herein is a method for producing binding proteins comprising an Ig variable domain, which method comprises immunizing a rodent having a limited Ig light chain repertoire with an antigen of interest and employing immunoglobulin variable region gene sequences from the rodent into a binding protein that specifically binds to the antigen of interest.

[0126] The term "bispecific antibody" refers to an antibody with specificity for two target molecules, and is exemplified by DVD-Ig (DiGiammarino et al., "Design and generation of DVD-Ig™ molecules for dual-specific targeting", Meth. Molecular Biology, 2012, 889, 145 156), mAb2 (see WO2008 / 003103), FIT-Ig (see WO2015 / 103072), mAb-dAb, Dock and Lock, Fab arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ-body, orthogonal Fab, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, scDiabody, scDiabody-CH3, diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 These formats include KIH, scFv-CH-CL-scFv, F(ab')2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knobs-in-hole, knobs-in-hole with a common light chain, knobs-in-hole with a common light chain and charge pair, charge pair, charge pair with a common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, and zybody. For a review of bispecific formats, see Spiess, C., et al., Mol. Immunol. (2015).In another embodiment, the bispecific molecule is an antibody fused to another non-Ig format, such as an antibody fused to a T-cell receptor binding domain, i.e., an immunoglobulin superfamily domain, a jawless variable lymphocyte receptor, a fibronectin domain (e.g., Adnectin™), an antibody constant domain (e.g., a CH3 domain, e.g., CH2 and / or CH3 of an Fcab™) that is not a functional CH1 domain, an scFv, (scFv)2, sc-diabody, scFab, a centrinin and epitope binding domain from a scaffold selected from CTLA-4 (Evibody™), a lipocalin domain, Protein A, such as the Z domain of protein A (e.g., Affibody™ or SpA), A domains (e.g., Avimer™ or Maxibody™), heat shock proteins (such as epitope binding domains derived from GroEI and GroES), transferrin domains (e.g., transbodies), ankyrin repeat proteins (e.g., DARPin™), peptide aptamers, C-type lectin domains (e.g., Tetranectin™), human gamma-crystallin or human ubiquitin (affilin), PDZ domains, scorpion toxins, and Kunitz-type domains of human protease inhibitors.

[0127] Antibody structure has been exploited to engineer a variety of different antibody formats to target human diseases. An example of such an engineered antibody format is the bispecific antibody. Bispecific antibodies bind to two different targets, allowing them to simultaneously bind to two different epitopes. One area of ​​interest is T cell-directed bispecific antibodies for efficient tumor killing. Bispecific antibodies have a "dual target" function, binding to two different surface receptors or ligands, thereby affecting multiple disease pathways. Bispecific antibodies can also place two targets in close proximity to support the formation of a protein complex on a single cell or to induce cell-to-cell contact. Bispecific antibody formats differ in many respects, including their molecular weight, the number of antigen-binding sites, the spatial relationship between the different binding sites, the valency of each antigen, the ability to support secondary immune functions, and their pharmacokinetic half-lives. These diverse formats offer great opportunities to customize the design of bispecific antibodies to fit the proposed mechanism of action and intended clinical use (Kontermann and Brinkmann Bispecific Antibodies Drug Discovery Today Volume 20, Number 7, 2015).

[0128] Producing IgG-type bispecific antibodies by coexpressing two light chains and two heavy chains in a single host cell can be extremely challenging due to low yields of the desired bispecific IgG and the difficulty of removing closely related mismatched IgG impurities. This reflects the fact that heavy chains form homodimers as well as the desired heterodimers, a problem known as the heavy chain pairing problem. Furthermore, light chains can mispair with non-matching heavy chains, a problem known as the light chain pairing problem. As a result, coexpression of two antibodies can produce up to nine undesired IgG species in addition to the desired bispecific antibody.

[0129] The art describes various approaches to promote heterodimerization, i.e., the formation of certain bispecific antibodies intended for human therapy, thereby reducing the content of undesired homodimers in the resulting mixture. Suitable methods for companion animal therapy are described, for example, in WO2021214460, the contents of which are incorporated herein by reference.

[0130] In one embodiment, the antibody is a multispecific antibody or fragment thereof. Multispecific proteins, such as multispecific antibodies, bind to at least two different targets, i.e., are at least bispecific. Thus, in one embodiment, the antibody is a bispecific antibody or fragment thereof that binds to at least two different targets. In other embodiments, the multispecific antibody or fragment thereof binds to three, four, or more targets.

[0131] A bispecific antibody is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In another embodiment, a bispecific antibody comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope and heavy and light chain variable domain sequences that have binding specificity for a second epitope. In further embodiments, a bispecific antibody molecule comprises an antibody that has binding specificity for a first epitope and an antibody that has binding specificity for a second epitope. In one embodiment, a bispecific antibody molecule comprises an antibody or fragment thereof that has binding specificity for a first epitope and a half antibody or fragment thereof that has binding specificity for a second epitope. In one embodiment, a bispecific antibody or fragment thereof comprises a Fab that has binding specificity for a first epitope and a Fab that has binding specificity for a second epitope.

[0132] Bispecific antibodies of the present invention, based on the IgG format consisting of two heavy chains and two light chains, can be produced by various methods known in the art. For example, bispecific antibodies can be produced by fusing two antibody-secreting cell lines to create a new cell line or by expressing two antibodies in a single cell using recombinant DNA technology. These approaches result in multiple antibody species because the heavy chains from each antibody form monospecific dimers (also called homodimers) containing two identical heavy chain pairs with the same specificity and bispecific dimers (also called heterodimers) containing two different heavy chain pairs with different specificities. Furthermore, the light and heavy chains from each antibody may pair randomly, forming inappropriate and non-functional combinations. This problem, known as heavy-light chain mispairing, can be resolved by selecting antibodies that share a common light chain for expression as a bispecific antibody. Methods to address the problem of light-heavy chain mispairing include generating bispecific antibodies using a single light chain. This requires the development of novel antibody libraries that utilize either heavy and light chain recombinations or a single light chain to limit diversity. Furthermore, antibodies with a common light chain have been identified from transgenic mice with a single light chain. Another approach is to swap the CH1 domain of one heavy chain with the CL domain of its corresponding light chain (Crossmab technology). The scFv format is also encompassed.

[0133] Methods for making bispecific antibodies are described in WO2019008123 and WO2014160179, which are incorporated by reference.

[0134] Other embodiments of the present invention are outlined below.

[0135] A method for producing an antibody or antibody chain specific to a desired antigen, comprising immunizing a rodent as disclosed herein with the desired antigen and recovering the antibody chain or antibody, or recovering a cell producing the antibody chain or antibody.

[0136] A method for producing an antibody chain or antibody specific to a desired antigen comprises immunizing a rodent as disclosed herein and then replacing any rodent constant regions of the antibody chain or antibody with canine constant regions, preferably by recombining nucleic acids encoding the antibody.

[0137] As used herein, a reference to substituting any rodent constant region can mean that if the constant region is a rodent constant region, it is substituted with, for example, a canine constant region, or that if the constant region is already, for example, a canine constant region, no such substitution is necessary.

[0138] 1. A method of producing an antibody, antibody chain, or portion thereof, wherein the antibody chain has a canine variable region, the method comprising expressing in a cell a nucleic acid, such as a DNA, encoding the antibody, antibody chain, or portion thereof; The nucleic acid sequences encoding the variable regions of the antibody chains are obtained by immunizing a rodent disclosed herein with an antigen; Optionally, the subsequent steps: purifying and / or isolating the antibody or antibody chain; and Optionally, formulating the antibody or antibody chain into a pharmaceutically acceptable formulation suitable for administration to a dog.

[0139] A method of making a pharmaceutical composition, the method comprising producing an antibody, preferably a whole canine antibody, according to the methods disclosed herein, and further comprising combining the antibody with a pharmaceutically acceptable carrier or other excipient to produce a composition.

[0140] An isolated antibody or antibody chain or portion thereof, such as a complete canine antibody chain or a complete canine antibody, obtained or obtainable from a rodent or rodent cell described herein or from a repertoire described herein, or a nucleic acid, such as DNA, encoding an antibody chain or portion thereof, or a pharmaceutical composition comprising same.

[0141] An antibody or antibody chain or portion thereof, such as a complete canine antibody chain or a complete canine antibody, obtained or obtainable from a rodent or rodent cell described herein or from a repertoire described herein, or a nucleic acid such as DNA encoding an antibody chain or portion thereof, or a pharmaceutical composition comprising same for use in treating or preventing a disease in a dog in need of such treatment or prevention.

[0142] A method of treating a dog, the method comprising delivering an antibody or antibody chain or portion thereof to a dog in need of treatment, wherein the antibody is obtained or is obtainable from a rodent or rodent cell or repertoire as described herein.

[0143] A further embodiment disclosed herein is a rodent or rodent cell that has only one canine IGH V gene segment, only one canine IGH D gene segment, and only one canine IGH J gene segment, as listed above, to form a "common heavy chain." In one aspect, the common heavy chain approach can also be used in methods for generating bispecific antibodies.

[0144] In one embodiment, the inserted canine gene segment may be inserted as part of an array or cluster of canine gene segments, optionally embedded in rodent or canine non-coding regulatory or scaffold sequences.

[0145] In one embodiment, the inserted canine gene segment may be inserted as part of a minilocus.

[0146] An alternative method for inserting gene segments into rodents is described in US20170306352, which is incorporated by reference.

[0147] Reference herein to a gene may, where appropriate, be a reference to a gene segment. Use of the term "gene" is not intended to exclude that such a feature is equally disclosed with respect to a gene segment, unless otherwise dictated by context or necessity. Similarly, reference herein to a gene segment may, where appropriate, be a reference to a gene. Use of the term "gene segment" is not intended to exclude that such a feature is equally disclosed with respect to a gene, unless otherwise dictated by context or necessity.

[0148] In all embodiments and aspects of the invention, the canine gene segment may be located in the rodent genome upstream of a rodent constant region, preferably upstream of the heavy chain constant region of the inserted canine heavy chain variable region gene segment, such that the rodent or rodent cell can produce the resulting chimeric antibody heavy chain from expression of the inserted variable region gene segment and the host constant region.

[0149] References to the position of a variable region upstream of a constant region, such as a rodent constant region, mean that the two genomic portions encoding the antibody variable and constant regions are in suitable relative positions to allow for the expression of a chimeric antibody chain in vivo in a rodent. In this manner, the inserted canine DNA and the constant region are functionally aligned with each other for the production of an antibody or antibody chain.

[0150] Information about, or nucleic acids comprising, the variable regions of the chimeric antibody chains can be obtained from cells expressing the chimeric antibody using standard techniques. These sequences can be used to generate complete canine antibodies, for example, for use in canine therapy, by expressing nucleic acids encoding the antibody variable regions together with canine constant regions to produce the canine antibody.

[0151] The canine DNA can be inserted at a rodent wild-type constant region located at the wild-type locus, preferably between the rodent constant region and the host VDJ or VJ region. The rodent constant region expressed with the canine heavy chain variable region is preferably a rodent wild-type constant region located at the wild-type locus of the canine heavy chain VDJ.

[0152] In one embodiment, the canine IGH variable region gene is inserted downstream of the heavy chain J region and upstream of the Emu enhancer.

[0153] In one embodiment, the canine IGH variable region gene is inserted downstream of the mouse heavy chain J region and upstream of the Emu enhancer. In one embodiment, the rodent is a mouse, and insertion of the IGH V region gene is at position 114666435 of the mouse genome on mouse chromosome 12.

[0154] Preferably, the inserted canine VDJ gene segments are capable of undergoing VDJ rearrangement to form antibody chains in a rodent.

[0155] In one embodiment, the rodent is a mouse.

[0156] Alternatively, the inserted canine VDJ gene segments are positioned within the genome (inserted into the genome) in functional alignment with a canine constant region, such that the rodent is able to produce antibody chains resulting from expression of the inserted canine VDJ gene segments with the canine constant region, which antibody chains can be expressed and paired with antibody chains resulting from expression of the inserted canine VJ gene segments with the canine constant region.

[0157] One possibility is in rodents where a complete canine antibody light chain is expressed with a chimeric heavy chain having a canine VDJ and a mouse heavy chain constant region.

[0158] As used herein, references such as "inserted canine gene segment" and "inserted canine DNA" can refer to a canine sequence that is present in the genome of a rodent or rodent cell and thus inserted into the genome of a rodent or rodent cell.

[0159] In one embodiment, the inserted canine DNA comprises at least 50%, e.g., at least 60%, at least 70%, at least 80%, at least 90%, and in one embodiment, all of the canine heavy chain diversity (D) genes.

[0160] In one embodiment, the inserted canine DNA comprises at least 50%, e.g., at least 60%, at least 70%, at least 80%, at least 90%, and in one embodiment, all of the canine heavy chain joining (J) genes.

[0161] The genome of the rodent or rodent cell can include at least 1, 2, 3, 4, 5, or 6 IGHD region gene segments from a dog.

[0162] The genome of the rodent or rodent cell can include at least 1, 2, 3, 4, 5, or 6 IGHJ region genes from a dog.

[0163] In either embodiment, the number of dog genes mentioned above can be further increased, and in one embodiment doubled in the case of homozygotes with insertions in both alleles.

[0164] In one embodiment, the rodent genome is homozygous for the inserted canine gene. Such homozygous rodents are preferably used for producing canine antibodies by immunization. The insertion can be made at the native rodent Ig locus.

[0165] In another embodiment, the rodent genome can be heterozygous for insertion of canine genes at one, two, or three loci, for example, at immunoglobulin loci.

[0166] In one embodiment, the rodent genome may be heterozygous for an insertion of the canine gene at the kappa locus.

[0167] In one embodiment, the inserted canine DNA is located at a different site in the rodent genome from the naturally occurring heavy or light chain constant region, e.g., on a different chromosome. Insertion can be at a random location in the rodent genome. In this case, the insertion of the VDJ region gene or VJ region gene is accompanied by a constant region from a rodent or dog, preferably a 3' enhancer. One preferred embodiment is to use a rodent constant region and a rodent 3' enhancer together with a canine VDJ region, or a canine constant region and a canine 3' enhancer together with a canine VJ region. In one aspect, the canine gene segment(s) are positioned in the genome in functional alignment with the constant region so that the rodent can produce the antibody chain.

[0168] In one aspect, the rodent or rodent cell comprises one canine IGL lambda V region gene, one canine IGL lambda J region gene, and one canine constant region located within, or upstream or downstream of, the kappa locus of the rodent cell, e.g., the kappa constant region locus of the rodent.

[0169] In one embodiment, the canine lambda V and / or J gene segments are associated with canine regulatory or scaffold sequences, hi another embodiment, the canine lambda V and / or J gene segments are associated with regulatory or scaffold sequences from a rodent, such as regulatory or scaffold sequences native to the rodent cell used to insert the canine DNA.

[0170] As used herein, references to a gene segment being associated with a regulatory or scaffold sequence, and similar such descriptions, may be references to the gene segment being operably linked to the regulatory or scaffold sequence. The term "associated" in this context means, and may be used synonymously with, "operably linked."

[0171] The canine heavy chain variable region gene segment(s) is / are preferably inserted upstream of a constant region that includes all of the DNA necessary to encode a complete constant region or a sufficient portion of the constant region to allow the formation of an effective antibody capable of specifically recognizing an antigen. Thus, reference herein to an antibody chain having a constant region is not limited to antibody chains having a complete constant region, but also includes antibodies or chains having a sufficient portion of the constant region to provide one or more effector functions found in naturally occurring antibodies in rodents. Effector functions include the ability to interact with Fc receptors and / or the ability to fix complement.

[0172] In one embodiment, any inserted heavy chain canine DNA can be expressed with different rodent constant regions via isotype switching. In one embodiment, any inserted heavy chain canine DNA can be expressed with different rodent constant regions via trans-switching.

[0173] In one embodiment, one, more, or all of the inserted canine V region gene segments, (D) region gene segments, or J region gene segments are associated with canine regulatory sequences, preferably from the same dog breed.

[0174] Even if the use of human regulatory sequences had been considered when inserting the human VDJ, based on the current state of the art it may not have been expected that the use of canine regulatory sequences rather than host regulatory sequences would work.

[0175] Das et al. (2008) - Immunogenetics, 60(1), 2008, pp. 47-55. and Flajnik (2002) - Nature Reviews Immunology, Vol. 2, September 2002, pp. 688-698, both of which are incorporated by reference, teach that even within mammals, key aspects of antibody production and regulation are highly variable. For example, Flajnik shows that different organs are used to form B cell repertoires in different species, which immediately raises the question of how predictable regulation and expression are given these different biological contexts. This suggests different mechanisms for generating diversity across species, such as gene conversion. Gene conversion relies on V gene recombination without the use of RSSs and utilizes cryptic regions of sequence homology within V genes.

[0176] Both documents also teach that the human / mouse paradigm is exceptional compared to other species that have been studied and therefore should not be presumed to be typical.

[0177] Matsushita et al. (2015) - PloS ONE, 10(6) (2015), e0130699, specifically teaches that bovine / human transgenic combinations do not work due to incompatibilities in both protein / protein and protein / DNA interactions between species, which is incorporated by reference. With regard to dogs and cats in particular, there is evidence showing how different they are from humans and mice, as described in Steiniger et al. (2014) - Molecular Immunology 59 (2014), p. 71-78, and Das et al. (2008), both of which are incorporated by reference.

[0178] Bolland et al. (2016) - Cell Reports 15 (2016), pp. 2475-2487, which is incorporated by reference, show that mouse non-coding sequences and other aspects such as chromatin structure can have a significant impact on VDJ recombination efficiency. Bolland et al. show that it is not clear how elements of the two different systems can be combined in an effective way to achieve successful expression.

[0179] Surprisingly, in association with canine regulatory elements, canine VDJ genes can be successfully expressed in mouse models to generate chimeric antibody expression. See, e.g., WO2018189520 and WO2020074874, both of which are incorporated herein by reference.

[0180] In one embodiment, at least one rodent enhancer or other regulatory sequence, such as a switch region, is maintained in functional alignment with the rodent constant region, so that the effect of the enhancer or other regulatory sequence can be exerted in whole or in part in the cell or transgenic rodent.

[0181] In one embodiment, one or more rodent regulatory sequences, such as the Emu enhancer sequence, are maintained upstream of the rodent Mu constant region, preferably in their native position with respect to distance from the constant region.

[0182] In one aspect, one or more rodent regulatory sequences, such as enhancer sequence(s), are maintained downstream of the rodent constant region, preferably in their native position with respect to distance from the constant region.

[0183] In one embodiment, the rodent Smu switch sequence is maintained upstream of the rodent Mu constant region, preferably in its native position with respect to distance from the constant region.

[0184] In such a location, the rodent enhancer or switch sequence preferably operates in vivo in conjunction with the host constant region sequence(s).

[0185] In a further embodiment, one or more promoter or other regulatory elements of the canine V region, D region, or J region genes are optimized within the genome to interact with rodent transcription machinery.

[0186] In one aspect, the genome of the rodent or rodent cell comprises one or more canine promoters, enhancers, and / or other regulatory elements associated with a canine V, D, or J gene segment. In one aspect, one or more canine regulatory regions, such as promoters, enhancers, or switch regions, replace one or more rodent promoters, enhancers, or switch regions, respectively. The canine regulatory sequences are maintained in functional alignment with the constant regions such that the effect of the regulatory sequence(s) can be exerted, in whole or in part, in the cell or transgenic rodent.

[0187] In one aspect, at least one or more of the inserted canine V, D, or J gene segments are associated with a regulatory sequence, e.g., a recombination signal sequence (RSS), from the same dog, which optionally directs successful recombination of the V, D, or J gene segment or segments.

[0188] In one aspect, the "same" dog refers to a dog of the same breed. In one aspect, the same dog can be the exact same animal.

[0189] In one embodiment, at least one or more of the inserted canine V, D, or J gene segments is directly associated with or flanked on one or both sides by regulatory sequences in cis or trans, and optionally, one or more gene segments is directly adjacent to a regulatory sequence.

[0190] In one aspect, the regulatory sequences include promoters preceding individual V gene segments, and / or splice sites within individual V gene segments, and / or recombination signal sequences for V(D)J recombination downstream of a V gene segment, adjacent to a D gene segment, or upstream of a J gene segment.

[0191] In one aspect, the inserted canine V, D, or J sequence is flanked by RSS sequences from the same dog. For example, canine RSS sequences can be used together with canine V, D, and / or J sequences. It will be appreciated that this can be provided by inserting a canine-derived genomic fragment into a rodent genome. In a further aspect, the present invention provides a method for replacing, in whole or in part, an endogenous immunoglobulin variable region locus with a canine locus in a rodent cell, comprising obtaining a cloned genomic fragment or synthetic sequence comprising, in whole or in part, a companion locus comprising at least one V gene segment, a D gene segment (for a heavy chain), or a J gene segment, and at least one associated regulatory sequence, and inserting the canine DNA into the rodent genome, suitably into an endogenous mouse immunoglobulin locus, preferably into a heavy or light chain rodent locus corresponding to the nature of the inserted canine DNA.

[0192] In one embodiment, the inserted canine DNA is associated with rodent regulatory sequences that allow V(D)J recombination in rodents. Such an approach is disclosed, for example, in US20170306352, the contents of which are incorporated herein by reference.

[0193] In one aspect, the regulatory sequences are non-coding regulatory sequences, which include the following sequences derived from the endogenous host: a promoter preceding each V gene segment coding sequence, introns, splice sites, and recombination signal sequences for V(D)J recombination. In another aspect, the regulatory sequences are one or more of a promoter preceding each V gene segment coding sequence, introns, splice sites, and recombination signal sequences for V(D)J recombination, all of which are derived from the endogenous host.

[0194] In another aspect, the partial canine immunoglobulin locus generated by the present invention comprises one or more of the following endogenous host-derived sequences: an ADAM6A or ADAM6B gene, a Pax-5 activator intergenic repeat (PAIR) element, or a CTCF binding site from heavy chain intergenic control region 1.

[0195] In one aspect, the invention relates to a transgenic mouse having a genome in which the entire endogenous immunoglobulin variable locus has been deleted and replaced with a recombined, partially canine-derived immunoglobulin locus comprising the coding sequences of canine immunoglobulin variable genes VH, D and JH, and canine VL and JL, as described herein, and the non-coding regulatory sequences of a mouse immunoglobulin variable locus, wherein the recombined, partially canine-derived immunoglobulin locus of the transgenic mouse is functional and expresses immunoglobulin chains comprising canine variable domains.

[0196] The inserted canine DNA can be, for example, a minigene having multiple different V gene segments.

[0197] The inserted canine V gene segments can be arranged so that the most desired gene segment or segments are located closest to the constant region. Gene segments located closer to the constant region are usually expressed at higher levels. Alternatively, the gene segments most naturally occurring in the canine antibody population can be located farther from the constant region to provide a balanced repertoire.

[0198] As disclosed herein, the present invention relates to a method for producing bispecific antibodies, comprising immunizing a rodent as disclosed herein with an antigen, wherein the rodent contains only a single type of canine light chain variable region (a single canine V and J gene segment, canine VJ) in the rodent genome, and selecting for bispecific antibodies capable of binding to the antigen and, preferably, also to a second preferred antigen target.

[0199] The present invention also relates to bispecific antibodies obtained or obtainable from the methods of the invention, wherein the antibody light chains are obtained from expression of preferred V gene segments as disclosed herein, preferably from a rodent of the invention. Preferably, the bispecific has preferred light and heavy chain pairings as disclosed herein.

[0200] Any bispecific antibody format can be used, for example, any of those disclosed in Brinkmann U and Kontermann RE, MAbs. 2017 Feb-March, 9(2):182-212.

[0201] The present invention also relates to a method for producing a bispecific antibody, comprising: immunizing a first rodent as disclosed herein with a first antigen, wherein the rodent comprises only a single canine lambda light chain variable region (a single canine V and J gene segment, canine VJ) in the rodent genome; and selecting an antibody capable of binding to the first antigen; immunizing a second rodent as disclosed herein with a second antigen, wherein the rodent comprises the same single canine lambda light chain variable region (a single canine V and J gene segment, canine VJ) in the rodent genome; and selecting an antibody capable of binding to the second antigen.

[0202] Optionally, the single canine lambda light chain V gene segment is selected from the list including canine lambda V1-138, V1-136, V1-141, V1-48, V1-55, V1-103, V1-41, V8-153, V1-75, V1-147, V8-128, V1-58, V1-100, V1-125, V1-84, V1-46, V2-8, V3-21, and V1-149.

[0203] In one embodiment, the single canine lambda light chain V gene segment is V1-138.

[0204] In one embodiment, the single canine lambda light chain V gene segment is V3-3.

[0205] The present invention also relates to bispecific antibodies obtained or obtainable from the methods of the invention, wherein the canine antibody light chain is obtained from expression of a single preferred lambda V gene segment as disclosed herein, preferably from a rodent of the invention. Preferably, the bispecific has a preferred light chain and heavy chain pairing as disclosed herein. In one embodiment, the single lambda V gene segment is selected from the list comprising canine lambda V1-138, V1-136, V1-141, V1-48, V1-55, V1-103, V1-41, V8-153, V1-75, V1-147, V8-128, V1-58, V1-100, V1-125, V1-84, V1-46, V2-8, V3-21, and V1-149.

[0206] The present invention provides rodents or rodent cells that contain a single canine lambda V and J gene for the light chain variable region in their genome. In this way, the rodent produces only a single type of variable region for the light chain of the canine antibody chains that it produces. In one embodiment, the single canine lambda light chain V gene segment is V1-138. In one embodiment, the single canine lambda light chain V gene segment is V3-3.

[0207] The present invention also relates to a method for producing a bispecific antibody, the method comprising: immunizing a first rodent as disclosed herein with a first antigen, wherein the rodent comprises only a single canine kappa light chain variable region (a single canine V and J gene segment, canine VJ) in the rodent genome, and selecting an antibody capable of binding to the first antigen; immunizing a second rodent as disclosed herein with a second antigen, wherein the rodent comprises the same single canine kappa light chain variable region (a single canine V and J gene segment, canine VJ) in the rodent genome, and selecting an antibody capable of binding to the second antigen, optionally wherein the single canine kappa light chain V gene segment is selected from the list comprising canine kappa V2-8, V2-7, V2-4, V2-5, and V2-11.

[0208] In one embodiment, the single Kappa Inu light chain V gene segment is V2-8.

[0209] In one embodiment, the single Kappa Inu light chain V gene segment is V2-7.

[0210] The present invention also relates to bispecific antibodies obtained or obtainable from the methods of the present invention, wherein the canine antibody light chain is obtained from expression of a single preferred kappa V gene segment as disclosed herein, preferably from a rodent of the present invention. Preferably, the bispecific has a preferred light and heavy chain pairing as disclosed herein. In one embodiment, the single kappa V gene segment is selected from the list comprising canine kappa V2-8, V2-7, V2-4, V2-5, and V2-11.

[0211] The present invention provides rodents or rodent cells that contain a single canine kappa V and J gene segment for a light chain variable region in their genome. In this manner, the rodent produces only a single type of variable region for the light chain of the canine antibody chains that it produces. In one embodiment, the single canine kappa light chain V gene segment is V2-8. In one embodiment, the single lambda canine light chain V gene segment is V2-7.

[0212] The present invention also relates to specific antibodies in which both light chains are identical and selected from the list comprising canine lambda V1-138, V1-136, V1-141, V1-48, V1-55, V1-103, V1-41, V8-153, V1-75, V1-147, V8-128, V1-58, V1-100, V1-125, V1-84, V1-46, V2-8, V3-21, and V1-149.

[0213] In one embodiment, both light chains are canine lambda V1-138.

[0214] In one embodiment, both light chains are canine lambda V3-3.

[0215] In one embodiment, both light chains are canine kappa V2-8.

[0216] The present invention also relates to vectors for use in the present invention. In one aspect, such a vector is a bacterial artificial chromosome (BAC) containing all or part of the canine IG locus suitable for insertion into ES cells. It is understood that other cloning vectors may also be used in the present invention, and therefore, reference to a BAC herein may be generally interpreted as referring to any suitable vector. A vector may contain one or more selectable markers and / or one or more site-specific recombination sites. In one aspect, a vector contains two or more, e.g., three, heterospecific and incompatible site-specific recombination sites. In one aspect, the site-specific recombination sites may be loxP sites or mutants thereof, or FRT sites or mutants thereof. In one aspect, a vector contains one or more transposon ITR (inverted terminal repeat) sequences.

[0217] A suitable BAC containing canine DNA is available from the BACPAC Resource Center at Children's Hospital Oakland Research Institute as the CHORI-82 BAC library.

[0218] In one embodiment, some or all of the inserted DNA is from a Boxer dog.

[0219] Preferably, the cell or rodent contains boxer dog V, D, and J gene segments from the CHORI-82 BAC library.

[0220] In one embodiment, the one or more canine gene segment alleles used in the present invention are reference alleles for the respective canine gene. These are the reference alleles of CanFam3.1, see assembly accession number GCA_000002285.2 (created September 2011, last updated May 2016). In one embodiment, at least 90%, at least 95%, and preferably all of the inserted canine gene segments are canine reference alleles from CanFam3.1. In one embodiment, at least 50, 60, 70, 80, 90, or 100% of the inserted V gene segments are the reference allele of a canine V gene, and / or at least 50, 60, 70, 80, 90, or 100% of the inserted D gene segments are the reference allele of a canine D gene, and / or at least 50, 60, 70, 80, 90, or 100% of the inserted J gene segments are the reference allele of a canine J gene, or combinations thereof.

[0221] Additional canine reference alleles may be available at canFam4 (UU_CFam_GSD_1.0 / canFam4, https: / / www.nature.com / articles / s42003-021-01698-x#MOESM1).

[0222] In one aspect, a reference to a canine gene segment (such as IGHV3-38) is a reference to the equivalent gene segment in CanFam3.1, however, it is understood that such sequences used in the present invention can differ from the exact sequence of CanFam3.1 and can be, for example, 99% identical, 98% identical, 97% identical, 96% identical, or 95% identical.

[0223] In one embodiment, the rodent or rodent cell comprises one or more reference alleles of a canine gene segment.

[0224] Preferably, the V region gene segments, (D) region gene segments, and J region gene segments inserted into the genome are all from the same breed or the same dog.

[0225] In one embodiment, the rodent comprises at least 1 x 10 6 A combinatorial diversity of different functional chimeric immunoglobulin sequences can be generated.

[0226] It should be noted that, in this specification, reference to a "dog" can refer to any individual belonging to Canis familiaris (domestic dog) / Canis lupus familiaris (domestic dog). A reference to a dog can also separately be a reference to any other canid that is not a dog.

[0227] The invention also relates to methods for producing rodents containing inserted canine DNA, methods for producing antibodies and antibody chains from these rodents, and methods for producing pharmaceutical compositions containing canine antibody chains or antibodies.

[0228] In one embodiment, rodent ES cells carrying one or more chimeric loci are used to generate chimeras in which host embryos are generated from a RAG-1 deficient background or other suitable genetic background that prevents the production of mature host B and T lymphocytes, allowing for the derivation of all B and T cells from the injected ES cells.

[0229] The ES cells of the present invention can be used to generate animals using techniques well known in the art, which may include, for example, injecting the ES cells into blastocysts and then implanting the chimeric blastocysts into females to produce offspring, which can be mated to produce heterozygous offspring, which can then be mated to produce homozygous recombinants having the required insertion. In one embodiment, the host blastocyst is Rag-deficient, such as RAG-1-deficient.

[0230] The present invention relates to chimeric rodents produced by injecting ES cells of the present invention into blastocysts, followed by implantation of the chimeric blastocyst into a female rodent to produce offspring.

[0231] In one embodiment, the rodent or rodent cell is a mouse or mouse cell, and the mouse ADAM6a and ADAM6b genes are present in the mouse genome and have not been previously deleted from the IGH locus and subsequently reinserted.

[0232] In one embodiment, the rodent ADAM6a and ADAM6b genes are located 5' to one or more inserted canine V, D, and J genes.

[0233] In one embodiment, the rodent IGH D and J genes are present in the rodent genome. In one embodiment, the rodent IGH D and J genes have not been deleted from the rodent genome. In one embodiment, the rodent IGH D and J genes are located 5' to one or more inserted canine V, D, and J gene segments.

[0234] The present invention also relates to a method for producing an antibody or antibody chain specific to a desired antigen, comprising immunizing a rodent as disclosed herein with the desired antigen and recovering the antibody or antibody chain, alone or as part of an intact antibody, or recovering a cell that produces the antibody or antibody chain, alone or as part of an intact antibody (see, e.g., Harlow, E. & Lane, D. 1998, 5th edition, Antibodies: A Laboratory Manual, Cold Spring Harbor Lab. Press, Plainview, NY, and Pasqualini and Arap, Proceedings of the National Academy of Science (2004) 101:257-259).

[0235] Preferably, an immunogenic amount of the antigen is delivered. The present invention also relates to a method for detecting a target antigen, which comprises detecting the antibody produced as described above with a secondary detection agent that recognizes a portion of the antibody.

[0236] The present invention also relates to a method for producing an antibody chain or antibody specific to a desired antigen, comprising immunizing a rodent containing a canine gene segment located upstream of a non-canine constant region, as disclosed herein, and then preferably recombining the nucleic acid encoding the antibody to replace the antibody chain or antibody constant region with that of a canine constant region. Preferably, the constant region is from the same breed of dog. Standard cloning techniques are known for replacing rodent constant regions with appropriate canine constant region DNA sequences; see, e.g., Sambrook, J. and Russell, D. (2001, 3rd edition) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY). Alternatively, direct nucleic acid synthesis can be used to generate the entire canine sequence using sequence information from DNA encoding the chimeric antibody.

[0237] The present invention also relates to methods that involve identifying canine variable regions by single-cell sequencing and creating vectors that express complete antibody chains with the corresponding canine constant regions. The present invention also relates to obtaining these canine sequences by PCR and connecting them to appropriate constant regions, such as canine constant regions, by suitable molecular biology techniques, including but not limited to bridge PCR and Gibson cloning. DNA may be synthesized for incorporation into expression vectors.

[0238] In yet another aspect, the chimeric antibody or antibody chain produced by the present invention is preferably engineered at the DNA level: a canine variable region from a heavy or light chain lacking a constant region; domain antibodies, Canine variable regions with any constant region from either the heavy or light chain of the same or different species; Molecules with antibody-like properties or structures can be generated, such as canine variable regions with non-naturally occurring constant regions, or canine variable regions combined with any other fusion partner. The present invention relates to all such chimeric antibody derivatives derived from the chimeric antibodies identified according to the present invention.

[0239] The present invention also relates to a method for producing an antibody or a portion thereof, the method comprising: (i) a nucleic acid encoding an antibody or part thereof obtained or obtainable according to the present invention; or (ii) providing sequence information to enable expression of an antibody or a portion thereof obtained or obtainable according to the present invention to produce the antibody; and expressing the antibody chain.

[0240] Also disclosed is a method for producing an antibody chain or portion thereof, wherein the antibody chain has a canine variable region, the method comprising expressing in a cell a nucleic acid, such as a DNA, encoding the antibody chain or portion thereof; the nucleic acid sequences encoding the variable regions of the antibody chains are or can be obtained by immunizing a rodent as disclosed herein with an antigen to produce the antibody chains, or are obtained from antibodies of the repertoire as described herein; Optionally, the subsequent steps: purifying and / or isolating the antibody chains; and Optionally, then formulating the antibody into a pharmaceutically acceptable formulation suitable for administration to a dog.

[0241] Also disclosed are methods of making pharmaceutical compositions, the methods comprising producing an antibody according to the methods disclosed herein and further comprising combining the antibody with a pharmaceutically acceptable carrier or other excipient to produce a composition.

[0242] The present invention further relates to chimeric antibodies or antibody chains expressed from the gene segments identified as important for canine antibody production, and nucleic acids encoding same.

[0243] The antibody, antibody chain, or nucleic acid may be or may be derived from a rodent or rodent cell, or may be derived from the repertoire of antibodies disclosed herein.

[0244] The present invention also relates to part or all of an immunoglobulin molecule comprising a canine variable domain and a rodent constant domain derived from the restricted insertion of a B cell of rodent origin, and hybridoma cells obtainable or obtained from the B cell, as well as part or all of an immunoglobulin molecule comprising a canine variable domain and a rodent constant domain obtainable or obtained from the hybridoma cells.

[0245] In another aspect, the present invention relates to fragments and functional derivatives of the antibodies and chains disclosed herein, also referred to as portions of antibody chains, and the use of such antibodies, chains, and fragments in diagnostics and medical treatment, including in vitro or ex vivo studies. Functional antibody fragments / portions can include fragments capable of specifically binding to an antigen. Functional antibody fragments can be, for example, FAB or single-chain variable fragments (scFv). The fragments can include at least the variable regions of the antibody. The fragments can include at least the CDR regions. Preferably, the portion is functional in that it can bind to the desired antigen, preferably the same antigen used to immunize the rodent to stimulate antibody production.

[0246] The present invention also relates to nucleic acids, such as DNA or RNA, that encode said antibodies, antibody chains, or portions thereof. In particular, the portions may be variable portions of antibody chains that are encoded by canine DNA in rodents.

[0247] In one aspect, the antibodies or fragments include any combination exemplified in the examples and figures herein, or any derivative thereof that is an intact canine antibody or fragment thereof capable of antigen binding.

[0248] The antibodies of the present invention may be isolated, and in one aspect are isolated from the cell or organism in which they are expressed.

[0249] The present invention relates to both polyclonal and monoclonal antibodies, either chimeric or whole canine, which may be produced in response to antigen challenge in the rodents of the invention or may be derived therefrom as described herein and / or may comprise V gene segments identified herein as highly utilized, such as IGH3-38. Methods for producing both monoclonal and polyclonal antibodies are well known in the art.

[0250] Also disclosed herein are pharmaceutical compositions comprising the antibodies and antibody chains, or nucleic acids encoding the antibodies or chains.

[0251] Suitable excipients and carriers are well known in the art and include water, surfactants, carbohydrates (eg, cyclodextrin derivatives), and amino acids.

[0252] The present invention also relates to pharmaceutical compositions comprising whole canine antibodies packaged in a delivery vehicle such as an IV bag or injection device.

[0253] The antibody chain may be a chimeric antibody chain or a complete canine antibody chain.

[0254] The present invention also relates to antibody repertoires, regardless of how they are produced. For example, the repertoire may be derived from a transgenic mouse, or alternatively, it may be a synthetic antibody repertoire, such as a phage display system, that utilizes the same limited set of immunoglobulin V gene segments as disclosed herein for the inserted heavy and / or light chain canine DNA. Suitable methods for generating synthetic antibody repertoires are described, for example, in WO2018234438, the contents of which are incorporated herein by reference.

[0255] In one embodiment, the repertoire comprises any one of those VH and VL regions described in WO2018234438, see in particular SEQ ID NOs: 1 to 36 described on pages 59 to 62 of that document.

[0256] The repertoire may include any one of the following germline VH1 regions as disclosed in WO2018234438: Vs618 (SEQ ID NO: 4 therein), Vs624 (SEQ ID NO: 1 therein), Vs628 (SEQ ID NO: 5 therein), and Vs635 (SEQ ID NO: 2 therein).

[0257] The repertoire may comprise any one of the following germline VL regions as disclosed in WO2018234438: Vs236 (kappa) (SEQ ID NO: 12 therein), Vs321 (lambda) (SEQ ID NO: 14 therein), Vs323 (lambda) (SEQ ID NO: 16 therein), Vs365 (lambda) (SEQ ID NO: 13 therein), and Vs843 (lambda) (SEQ ID NO: 15 therein).

[0258] The antibodies and antibody chains disclosed herein can be used in methods for preventing or treating diseases in dogs. In particular, complete dog antibodies with dog constant regions can be used.

[0259] Thus, the present invention relates to antibodies or antibody chains or portions thereof as disclosed herein, including, for example, antibodies that have been or can be obtained from a rodent or rodent cell disclosed herein or from a repertoire disclosed herein, including complete dog antibodies made using sequence information from a rodent or rodent cell, for use in the treatment or prevention of disease in a dog in need thereof.

[0260] The present invention also relates to methods of treating dogs, comprising delivering to a dog in need thereof an antibody or antibody chain or portion thereof disclosed herein, where the antibody is, for example, an antibody obtained or obtainable from a rodent or rodent cell disclosed herein, or from a repertoire as disclosed herein, or a complete dog antibody generated using sequence information from a rodent or rodent cell.

[0261] In a further aspect, the invention relates to the use of rodents as described herein as models for testing drugs and vaccines. Accordingly, the invention relates to a method of drug or vaccine identification or validation, comprising delivering the vaccine or drug to a rodent of the invention and monitoring one or more of the immune response, safety profile, and effect on disease.

[0262] The present invention also relates to kits comprising an antibody or antibody derivative disclosed herein and either instructions for use of such an antibody or suitable laboratory reagents (such as buffers, antibody detection reagents, or excipients for formulation with the antibody).

[0263] It will be understood that the specific embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are encompassed by the claims. All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the words "a" or "an," when used in conjunction with the word "comprising" in the claims and / or this specification, may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." Use of the term "or" in the claims is used to mean "and / or," unless expressly stated to refer only to alternatives or where the alternatives are mutually exclusive, but the present disclosure supports the definition and "and / or" to refer only to alternatives. Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method employed to determine the value, or the variation that exists among study subjects.

[0264] As used in this specification and claim(s), the terms "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0265] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and also includes BA, CA, CB, CBA, BCA, ACB, BAC, or CAB, if order is important in the particular context. Continuing with this example, combinations including repeats of one or more items or terms are expressly included, such as BB, AAA, ABAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those of skill in the art will understand that there is typically no limit to the number of items or terms in any combination, unless otherwise apparent from the context.

[0266] Any part of this disclosure may be read in combination with any other part of this disclosure, unless otherwise clear from the context.

[0267] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the compositions and / or methods, and in the steps or in the sequence of steps of the methods, described herein without departing from the concept, spirit, and scope of the invention.

[0268] Any part of this disclosure may be read in combination with any other part of this disclosure, unless otherwise clear from the context.

[0269] The present invention is described in further detail in the following non-limiting examples. [Example]

[0270] The following examples will serve to demonstrate the invention.

[0271] Example 1: Construction of a targeting vector The common light chain vector was constructed in three steps as illustrated in Figure 1A.

[0272] As shown in Figure 1A, four DNA fragments were synthesized (at Genscript) and sequentially assembled using NEB HiFi Assembly (New England Biolabs). Fragment 1 encompasses the 5' targeting arm, which contains a series of unique DNA sequences surrounding the IgVK3-2 gene. Due to the repetitive nature of the IGH locus, it is important to use unique DNA sequences as the targeting arm to ensure efficient homologous recombination at the desired genomic location. Furthermore, because IgVK3-2 expression is relatively high, it is plausible that the sequences surrounding IgKV3-2 provide a permissive regulatory environment for transcription (Figure 1B).

[0273] The 5' targeting arm is followed by 1 kb of the mouse IGKV14-111 promoter region, including the 5' UTR, located immediately upstream of the canine IgLV3-3 part 1 exon. The mouse IGK J5 exon splice donor is located immediately after the IgLV3-3 part 1 exon. This design ensures that the IGLV3-3 L part 1 exon is spliced ​​to the remainder of the canine IGLV3-3 common light chain using the endogenous mouse J5 splice donor and the downstream mouse kappa constant exon splice acceptor. Furthermore, this strategy also ensures the deletion of all mouse J genes during targeted replacement.

[0274] Fragment 1 ends at the natural TTAA site between the J5 exon and the mouse iεk enhancer. Fragment 2 begins at the natural TTAA site and encompasses a unique genomic DNA sequence downstream of the mouse kappa constant exon, forming the 3' targeting arm. In this fragment, the kappa constant exon is replaced with an engineered AscI site for subsequent modifications. Fragments 1 and 2 were assembled into a low-copy-number plasmid backbone to ensure cloning stability. The integrity of the intermediate plasmid was confirmed by diagnostic restriction enzyme digestion, and sequence identity was confirmed by Sanger sequencing. The second step in the construction was the introduction of a piggyBac transposon carrying the mammalian positive and negative drug selection cassette PuroDeltaTK.

[0275] This cassette was introduced into the native TTAA site within fragment 1 to ensure seamless excision upon removal of the piggyBac transposon by transient transfection with a PiggyBac transposase-containing plasmid. The third step involved linearizing the vector with AscI digestion. NEB HiFI assembly was then used to introduce the canine IGLV3-3-based common light chain into the AscI site. Correct assembly removes the AscI site and retains the endogenous mouse kappa constant exon splice acceptor before introducing the common light chain exon.

[0276] The mouse IgKV14-111 gene promoter is used to drive expression of the common light chain. Because expression regulation of the light chain lambda and kappa loci differs, matching species to cis-regulatory elements such as the V gene promoter and 5'UTR may be important in providing optimal expression in the correct regulatory environment. Therefore, the mouse IgK V gene promoter is preferable to the mouse and canine IgL V gene promoters. Furthermore, mouse IGKV gene expression varies among different V genes, which may be due to promoter strength. Therefore, selecting a promoter with a high expression level may improve expression of the common light chain. To this end, we examined mouse IGK V gene expression in single-cell 5'VDJ NGS sequencing data generated from naive mouse peripheral blood B cells. IgKV14-111 is identified as the most highly expressed IGKV gene (Figure 1B).

[0277] Figure 1C illustrates an ES cell targeting strategy for replacing endogenous mouse immunoglobulin kappa light chain variable region gene segments with a canine VL3-3-based common light chain. Targeting events following selection cassette removal by piggyBac transposition (CLC-modified IgK locus) result in the removal of all mouse J genes and a portion of the mouse VK gene 3′ to VK3-2. The targeting event also results in the replacement of mouse kappa constant exons with common light chain cDNA exons. The canine IGLV3-3 L part 1 exon, encoding most of the IGLV3-3 leader peptide, is positioned adjacent to the endogenous mouse kappa V gene. The mouse VK14-111 promoter is used to drive CLC expression. PB-PuTK is a PuroDeltaTK positive and negative selection cassette flanked by piggyBac transposon ITRs. iεk is an enhancer of the mouse kappa locus. Ck is a kappa constant region exon. The dotted line between the wild-type and target loci represents the genomic region replaced after modification. The genomic sequence from JK5 to the splice acceptor of Ck (including the splice donor after JK5, iεk, and the splice acceptor of Ck) remains intact. The mouse genomic location where the modification occurs is indicated in the CLC-modified IgK locus. The GRCm38 / mm10 mouse genome annotation is used.

[0278] Example 2: Generation of a consensus light chain mouse ES cell line AB2.1 mouse ES cells containing canine IgH BAC KI (WO2018 / 189520) were cultured on a monolayer of γ-irradiated STO feeders at 37°C in a humidified incubator with 5% CO2. ES cell culture medium (M15) consisted of knockout DMEM, 15% fetal bovine serum, 0.1 mM β-mercaptoethanol, 2 mM L-glutamine, 50 U / ml penicillin, and 39 μg / ml streptomycin. Cells were typically split 1:3 or 1:4 onto fresh feeder plates every 2–3 days and were ready for electroporation when they reached 70–80% confluency.

[0279] The procedure for gene targeting of ES cells was as follows: 2 hours before electroporation with the targeting vector (Figure 1C), the cell culture medium was refreshed. ES cells were trypsinized, resuspended to a single cell suspension, washed with PBS, and counted before mixing with the targeting vector. Electroporation was performed using a Biorad gene pulser. Electroporated ES cells were then plated onto feeder plates and treated with 1 μg / ml puromycin for 7 days starting the following day. Puromycin-resistant colonies were manually picked using a P20 pipette under a light microscope. ES cell colonies were expanded by splitting into multiple 96-well plates. Cells from one sister plate were lysed overnight at 55°C in sarkosyl-containing cell lysis buffer (2.5 g sarkosyl per 500 ml of buffer, 10 mM Tris-HCl, 10 mM EDTA, 10 mM NaCl) supplemented with proteinase K (1 mg / ml). Genomic DNA was extracted using ethanol precipitation. Correctly targeted ES cells were identified by PCR for precise replacement in both the 5' and 3' targeting arms. After correctly targeted clones were identified, excision of the piggyBac transposon was performed by electroporation of a piggyBac transposase-containing plasmid. To select for ES cells in the absence of the PuroTK selection cassette, negative selection was applied using 200 nM FIAU (1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil). FIAU-resistant colonies were picked, and excision of the piggyBac transposon was further confirmed by PCR across the junction.

[0280] The resulting recombined mouse IGK locus is shown in Figure 1C. After genotyping, selected ES cell lines were further tested by quantitative PCR for the absence of chromosomal abnormalities in chromosomes 8, 11, and Y. Chromosomally stable ES cell lines were advanced to mouse generations.

[0281] Example 3: Generation of common light chain mice from targeted mouse ES cells The engineered ESCs were injected into E3.5 blastocyst-stage embryos harvested from C57BL6c- / c- or C57BL6c- / c-Rag- / - mice. After microinjection, the blastocysts were transferred to the uterus of 2.5-day-post-coitum (dpc) B6CBAF1 female mice that were pseudopregnant by mating with vasectomized sterile male mice. After delivery, the resulting chimeric pups were identifiable by the presence of agouti coat color contributions from 10 days of age onward. Chimeric male mice derived from C57BL6c- / c-host embryos were mated with C57BL6c- / c-female mice at 6 weeks of age to obtain germline transmission of the common light chain knock-in mice. Germline transmission was initially identified by coat color transmission, and genotypes were further confirmed by PCR using genomic DNA extracted from ear tags. Mice homozygous for the common light chain IGK allele and carrying at least one allele of the canine IGH BAC KI are used for phenotypic and molecular characterization.

[0282] Example 4: Characterization of splenic and peripheral blood CD19+ B cells by flow cytometry Four mice from the same litter homozygous for the common light chain allele were used for phenotypic characterization by flow cytometry, along with a wild-type mouse as a control. For B cell analysis from whole blood, approximately 200 μl was collected from each mouse into a tube with anticoagulant (EDTA) and washed with FACS buffer (3% FCS in PBS) to remove serum antibody impurities. After blocking nonspecific binding with TruStain FcX™ PLUS (anti-mouse CD16 / 32) antibody, the blood was stained with the following panel for 30 minutes at room temperature, followed by VersaLyse (Beckman Coulter) treatment according to the manufacturer's recommendations to remove red blood cells. The remaining blood cells were washed with FACS buffer and analyzed using a BD FACSAria™ Fusion (BD Biosciences) and FlowJo™ v10.8.1 software (BD Life Sciences).

[0283] For spleen samples, a single-cell suspension of splenocytes was prepared by repeatedly puncturing the tissue with a syringe needle and flushing it with collection medium (RPMI / 10% FCS / 20 mM HEPES). Final mechanical dissociation of the spleen was performed by transferring the tissue to a 40 μm cell strainer and squashing it with a gentle circular motion. After passing through the cell strainer, the cells were ready to be stained with the same panel used for whole blood immunophenotyping. After 30 minutes, the stained cells were washed and acquired using a BD FACSAria™ Fusion (BD Biosciences) and analyzed using FlowJo™ v10.8.1 software (BD Life Sciences). [Table 1]

[0284] Flow cytometry profiles of B cell surface light chain usage in blood- and spleen-derived B cells are shown in Figure 2. Wild-type mice predominantly use mouse kappa light chains, with approximately 90% of B cell receptors (BCRs) preferentially pairing with mouse kappa light chains, while less than 10% of BCRs use mouse lambda light chains. In contrast to the wild-type scenario, no staining for mouse kappa light chains was observed, suggesting successful targeted replacement. The constant region of the common light chain allele is canine lambda, which cannot be detected by this staining panel due to the lack of commercially available antibodies. BCRs with a common light chain should be represented within the mouse lambda and kappa double-negative population.

[0285] As expected, in PBMC samples, we observed an increase in the lambda and kappa double-negative population in all four CLC mice (10.6%, 21.9%, 24.9%, and 27.6%) compared with wild-type controls (2.77%) (Figure 5, left panel). This result indicates that the recombined common light chain is used by the BCR, allowing B cells with the common light chain BCR to progress through B cell development. Mouse lambda light chain usage is also increased in CLC mice (89.3%, 61.1%, 63%, and 58%) compared with wild-type controls (2.99%). This increase is also expected given that CLC alleles are limited in variable region diversity; mouse lambda alleles are likely activated to compensate for the lack of diversity. Analysis of spleen-derived B cells yielded similar results ( Figure 5 , right panel), with a significantly increased double-negative population in CLC mice (26.5%, 31.4%, 37.3%, and 24.9%) compared with wild-type (1.07%).

[0286] Figure 2 shows the characterization of whole blood and spleen from common light chain mice by flow cytometry. The whole blood and spleen of four homozygous common light chain mice were derived from the same litter. Animal IDs are indicated on the left. Single live CD19+ / CD90.2- cells from the blood (left panel) and single live CD19+ cells from the spleen (right panel) were analyzed for surface expression of mouse lambda light chain and mouse kappa light chain.

[0287] Example 5: Sorting of splenic double-negative B cells and generation of a 10x VDJ library To further characterize the common light chain BCR at the molecular level, viable splenic CD19+ mouse lambda and kappa double negative cells were enriched by FACS sorting and subjected to single-cell VDJ sequencing (Figure 3).

[0288] After spleens were harvested and dissociated into single-cell suspensions as described above, splenocytes from each mouse were individually barcoded using TotalSeq™ anti-mouse hashtag antibody (BioLegend). After blocking nonspecific binding, splenocytes were stained with antibodies against mouse cell surface markers CD90.2, CD19, kappa, and lambda light chains, including a viability dye, as described above. After a 30-minute incubation at 4°C, cells were washed and resuspended in PBS / 2% FCS / 1 mM EDTA / 20 mM HEPES.

[0289] Cells were then subjected to fluorescence-activated cell sorting (FACS) using a BD FACSAria™ Fusion (BD Biosciences) to isolate a population of single, live, CD19+ / lambda and kappa double-negative B cells, as well as a control population of single, live, CD19+ / lambda+ B cells. 25,000 cells were sorted from each mouse and pooled into a single collection tube per population. The resulting cells were pelleted by centrifugation, and approximately 35,000 cells were loaded per sample, processed using the microfluidic encapsulation protocol from the 10X Genomics Chromium Next Gem Single Cell 5' v2.0 Kit (10X Genomics) according to the manufacturer's protocol. The resulting cDNA was quality-controlled by quantification using the highly sensitive Qubit and Bioanaylser assays (ThermoFisher and Agilent, respectively). The cDNA was then processed according to the 10X Genomics protocol to generate 5'VDJ NGS sequencing libraries.

[0290] The generated 5'VDJ libraries were sequenced on an Illumina NGS platform using 150 bp paired-end sequencing. At this read length, a minimum of 2000 read pairs per cell barcode is recommended. The resulting sequencing data was demultiplexed, and each library contains a pair of .fastq.gz files for Read1 and Read2.

[0291] The resulting FASTQ file sets were processed using Cell Ranger software (10X Genomics), and for the combination of VDJ and 5'GEX libraries, the "cellranger multi" command was used, taking into account the list of heavy and light chain V+D+J+C reference sequences within the canine repertoire provided as a reference set to the CanFam3.1 canine genome reference and annotation, which includes a list of validated Ig genes for the IgH locus within the canine genome and common light chains from internal curation, as well as a list of internal primers designed as parameters for this purpose.

[0292] The results of the VDJ library after the "cellranger multi" step were QC'd and compared with the cell count estimated in the cell counting step. The chains reconstructed from Cellranger (all_contig.fasta) were light chains corresponding to the CLC chains in their encapsulation (barcodes) and paired with the heavy chain counterpart with the highest UMI (unique molecular identifier) ​​in their encapsulation. These pairings were analyzed for frequency analysis of the heavy chain V gene frequency table.

[0293] Figure 3 shows the sorting strategy for populations enriched for common light chains. Figure 3A is a schematic diagram of the gating strategy for isolating splenic B cells that do not express mouse kappa or lambda light chains and are likely enriched for surface expression of common light chains. The sorting strategy involves selection of lymphocyte populations by FSC and SSC, followed by single cell selection, live cell selection, CD19+ B cell selection, and sorting for double-negative mouse kappa and lambda light chains. FSC-A is forward scatter area, SSC-A is side scatter area, FSC-H is forward scatter height, and SSC-H is side scatter height. Figure 3B shows a representative density plot from a common light chain mouse showing the gates used for cell sorting.

[0294] Example 6: Single-cell VDJ analysis In total, 450 common light chain cells were identified that passed QC. To confirm the integrity of the common light chain transcripts, all common light chain transcripts were aligned. The majority of CLC transcripts (>80% of CLC transcripts) utilize the mouse VK14-111 5'UTR encoded by the recombined IGKV14-111 fragment (Figure 1). 5'UTR usage is shown at nucleotide resolution in Figure 4A. The predominant form of the transcript (>80% of CLC transcripts) has a short 5'UTR, 15 nucleotides long, immediately upstream of the translation start. Longer alternative forms can range up to 35 nucleotides in length (11.4%–38.3% of CLC transcripts). This nucleotide-level examination confirms successful recombination of the CLC locus and transcription of the common light chain allele. Furthermore, this analysis demonstrates that CLC transcription is primarily driven by the IGKV14-111 promoter, resulting in the intended transcripts from the chimeric format.

[0295] The coding regions of all CLC transcripts were also analyzed. The mismatch rate of the transcripts relative to the reference sequence was 0.0635%, which is consistent with the expected NGS sequencing error rate (the Q30 threshold for NGS is 0.1%). This suggests that the common light chain has not undergone somatic hypermutation or editing, at least in naive mice. Furthermore, the modified transcriptional splice sites function as intended. The amino acid sequence of the common light chain, including the leader, is shown in Figure 4B. Part of the leader sequence is encoded by the canine IGLV3-3 L part 1 exon (Figure 1C). CLC is encoded by the canine IGLV3-3 gene recombined with the canine J1 gene. The canine lambda C5 gene is used as the constant region. CDRs 1, 2, and 3 are also highlighted in Figure 4B.

[0296] Single-cell VDJ sequencing allows the possibility to investigate heavy chain V gene usage and VH-VL common light chain pairing.

[0297] The functional canine IGHV gene introduced into the mouse IGH locus may be any of IGHV4-1, IGHV3-2, IGHV3-3, IGHV3-5, IGHV3-6, IGHV3-7, IGHV3-8, IGHV3-9, IGHV3-10, IGHV3-16, IGHV3-18, IGHV3-19, IGHV3-23, IGHV4-1, IGHV3-2, IGHV3-3, IGHV3-5, IGHV3-6, IGHV3-7, IGHV3-8, IGHV3-9, IGHV3-10, IGHV3-16, IGHV3-18, IGHV3-19, IGHV3-23, IGHV4-1, IGHV3-2, IGHV3-3, IGHV3-4, IGHV3-5, IGHV3-6, IGHV3-7, IGHV3-8, IGHV3-9, IGHV3-10, IGHV3-16, IGHV3-18, IGHV3-19, IGHV3-23, IGHV4-1 ...4-1, IGHV4-1, IGHV4-2, IGHV4-3, IGHV4-4, IGHV4-5, IGHV4-6, IGHV4-7, IGHV4-8, IGHV4-9, IGH The CLC VL and canine VH genes are listed as follows: IGHV1-30, IGHV3-35, IGHV3-37, IGHV3-38, IGHV3-39, IGHV3-41, IGHV3-46, IGHV3-47, IGHV3-50, IGHV3-54, IGHV3-58, IGHV3-61, IGHV3-67, IGHV3-69, IGHV3-70, IGHV3-75, IGHV3-80, and IGHV3-81. The present inventors have observed pairing of recombinant CLC VL with canine VH, demonstrating that the CLC alleles are not only expressed but also can form functional B cell receptors with the canine heavy chain genes.

[0298] Figure 4 shows the characterization of the consensus light chain transcript. Figure 4A shows the use of the IGKV14-111 promoter and characterization of the 5'UTR at base pair resolution. All CLC transcripts were aligned, and the frequency of each nucleotide position in the predicted transcript sequence was calculated and displayed above the consensus. Figure 4B shows the complete amino acid sequence of the consensus light chain with annotated features. [Table 2-1] [Table 2-2]

Claims

1. A rodent or rodent embryonic stem (ES) cell whose genome contains only a single canine light chain V gene segment.

2. 2. The rodent or rodent ES cell of claim 1, wherein the rodent genome is the genome of a cell that has not undergone somatic hypermutation.

3. 2. The rodent or rodent ES cell of claim 1, wherein the rodent genome is the genome of a cell that is not a B cell.

4. The rodent or rodent ES cell of claim 1 , wherein the rodent genome is a germline genome.

5. 5. The rodent or rodent ES cell of any one of claims 1 to 4, wherein the single canine light chain V gene segment is a canine light chain V gene segment selected from IGKV2-5, IGKV2-11, IGKV2S13, IGKV3-8, IGLV3-18, IGLV3-11, IGLV3-21, IGKV2-9, IGKV2-10, IGKV2-12, IGKV2-16, IGLV3-14, IGLV3-24, IGKV2-8, and IGLV3-3.

6. The rodent or rodent ES cell of any one of claims 1 to 4, wherein the single canine light chain V gene segment is a canine lambda V gene segment.

7. 7. The rodent or rodent ES cell of claim 6, wherein the single canine light chain V gene segment is a canine lambda V gene segment of the canine lambda V3 family, optionally selected from IGLV3-18, IGLV3-11, IGLV3-21, IGLV3-14, IGLV3-24, and IGLV3-3.

8. 8. The rodent or rodent ES cell of claim 7, wherein the single canine light chain V gene segment comprises the nucleic acid sequence of SEQ ID NO:2 or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO:

2.

9. 9. The rodent or rodent ES cell of any one of claims 1 to 8, wherein the sequence encoding the canine light chain CDR3 comprises 33 nucleotides.

10. The rodent or rodent ES cell of any one of claims 1 to 9, wherein the sequence encoding the canine light chain CDR3 consists of 33 nucleotides.

11. 11. The rodent or rodent ES cell of any one of claims 1 to 10, wherein the genome comprises a sequence encoding a canine light chain CDR3 that can result in a CDR3 of 11 amino acids.

12. 12. The rodent or rodent ES cell of any one of claims 1 to 11, wherein the genome comprises the sequence of SEQ ID NO:4 or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO:

4.

13. 13. The rodent or rodent ES cell of any one of claims 9 to 12, wherein the CDR3, in combination with an antibody heavy chain, is capable of suitably binding to an NK cell antigen or a T cell antigen, such as a CD16 polypeptide or a CD3 polypeptide.

14. The rodent or rodent ES cell of any one of claims 1 to 13, wherein the single canine light chain V gene segment is operably linked to a canine light chain constant region.

15. 15. The rodent or rodent ES cell of any one of claims 1 to 14, wherein the single canine light chain V gene segment is operably linked to a canine lambda constant region.

16. 16. The rodent or rodent ES cell of any one of claims 1 to 15, wherein the single canine light chain V gene segment is operably linked to a canine λ5 constant region.

17. 17. The rodent or rodent ES cell of any one of claims 1 to 16, wherein the single canine light chain V gene segment is operably linked to a canine light chain constant region comprising the sequence of SEQ ID NO:6, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO:

6.

18. 18. The rodent or rodent ES cell of any one of claims 1 to 17, wherein the single canine light chain V gene segment is operably linked to a canine light chain J gene segment.

19. 19. The rodent or rodent ES cell of any one of claims 1 to 18, wherein the single canine light chain V gene segment is operably linked to a canine lambda J gene segment.

20. 20. The rodent or rodent ES cell of any one of claims 1 to 19, wherein the single canine light chain V gene segment is operably linked to a canine lambda J1 gene segment.

21. 21. The rodent or rodent ES cell of any one of claims 1 to 20, wherein the single canine light chain V gene segment is operably linked to a canine light chain J gene segment comprising the sequence of SEQ ID NO:8, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence similarity to SEQ ID NO:

8.

22. 22. The rodent or rodent ES cell of any one of claims 1 to 21, wherein the single canine light chain V gene segment is non-contiguous in the genome.

23. 23. The rodent or rodent ES cell of any one of claims 1 to 22, wherein the single canine light chain V gene segment is divided into two parts in the genome.

24. 24. The rodent or rodent ES cell of any one of claims 1 to 23, wherein the single canine light chain V gene segment is split into two parts in the genome at the mouse kappa locus on chromosome 6.

25. 25. The rodent or rodent ES cell of any one of claims 1 to 24, wherein the single canine light chain V gene segment is divided into two parts in the genome, and the parts can be spliced ​​to form a contiguous sequence.

26. 26. The rodent or rodent ES cell of any one of claims 1 to 25, wherein the single canine light chain V gene segment is split into two parts in the genome, and the splice donor and splice acceptor are native rodent splice donors and acceptors, such as the splice donor after JK5 and the splice acceptor of Ck.

27. 27. The rodent or rodent ES cell of any one of claims 1 to 26, wherein some or all of the endogenous V, D, or J gene segments are inactivated, optionally by inversion or excision.

28. 28. The rodent or rodent ES cell of any one of claims 1 to 27, wherein the single canine light chain V gene segment is present on chromosome 6.

29. 29. The rodent or rodent ES cell of any one of claims 1 to 28, wherein the single canine light chain V gene segment is located at the mouse immunoglobulin kappa locus on chromosome 6.

30. 30. The rodent or rodent ES cell of any one of claims 1 to 29, wherein at least a portion of the single canine light chain V gene segment is downstream of a rodent intronic enhancer in the mouse immunoglobulin kappa locus.

31. 31. The rodent or rodent ES cell of any one of claims 1 to 30, wherein the single canine light chain V gene segment is upstream of a rodent kappa constant region at the endogenous rodent kappa locus.

32. 32. The rodent or rodent ES cell of any one of claims 1 to 31, wherein the single canine light chain V gene segment is operably linked to a mouse promoter.

33. 33. The rodent or rodent ES cell of any one of claims 1 to 32, wherein the single canine light chain V gene segment is operably linked to a mouse IGK promoter.

34. 34. The rodent or rodent ES cell of any one of claims 1 to 33, wherein the single canine light chain V gene segment is operably linked to a mouse promoter selected from the IGKV1 family, IGKV14 family, IGKV4 family, IGKV6 family, IGKV8 family, or IGKV3 family, such as an IGKV14-111 promoter, a mouse IGKV6-15 promoter, or a mouse IGKV6-23 promoter.

35. 35. The rodent or rodent ES cell of any one of claims 1 to 34, wherein the single canine light chain V gene segment is operably linked to a mouse promoter of the mouse IGKV14 family.

36. 36. The rodent or rodent ES cell of any one of claims 1 to 35, wherein the single canine light chain V gene segment is operably linked to a mouse IGKV14-111 promoter.

37. 37. The rodent or rodent ES cell of any one of claims 1 to 36, wherein the single canine light chain V gene segment is operably linked to a mouse IGKV6-15 promoter.

38. 38. The rodent or rodent ES cell of any one of claims 1 to 37, wherein the single canine light chain V gene segment is operably linked to a mouse IGKV6-23 promoter.

39. 39. The rodent or rodent ES cell of any one of claims 1 to 38, wherein the genome comprises an endogenous rodent 3' enhancer.

40. 40. The rodent or rodent ES cell of any one of claims 1 to 39, wherein the rodent or rodent ES cell is a mouse or mouse ES cell.

41. 1. A method for producing antibodies with multiple specificities, comprising: (a) immunizing a rodent according to any one of claims 1 to 40, which comprises only a single canine light chain V gene segment, with a first antigen; (b) identifying a first canine nucleic acid encoding a first canine heavy chain variable region that binds to the first antigen; (c) immunizing the rodent with a second antigen; (d) identifying a second canine nucleic acid encoding a second canine heavy chain variable region that binds to the second antigen; and (e) expressing the first canine heavy chain variable region, the second canine heavy chain variable region, and one single canine light chain variable region derived from the rearranged single canine light chain V gene segment; (f) thereby obtaining an antibody comprising the first canine heavy chain variable region, the second canine heavy chain variable region, and the single canine light chain variable region; (g) optionally formulating the antibody as a pharmaceutical composition.

Images