Animal models and therapeutic molecules

Abstract

To provide rodent models have been developed to generate antibodies suitable for use in other species, such as companion animals.SOLUTION: Provided are rodents and cells that are engineered to contain the exogenous DNA of companion animals, their use in medicine and the study of disease, methods for production of rodents and cells, and antibodies and antibody chains produced by such animals and derivatives thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates, inter alia, to rodents and cells genetically engineered to contain exogenous DNA of a companion animal, their use in the study of medicine and disease, methods for producing the rodents and cells, and antibodies, antibody chains and derivatives thereof produced from such animals.

Background Art

[0002] The insertion of human DNA into rodents has been disclosed, for example, in Murphy et al., 111(14):5153 - 5158, doi:10.1073 / pnas.1324022111; MacDonald et al., 111(14):5147 - 5152, doi:10.1073 / pnas.1323896111; and Lee et al., Nature Biotechnology, 32:356 - 363, published in 2014, DOI: doi:10.1038 / nbt.2825. This approach is designed to create antibody products for therapeutic use in humans. However, rodent models for generating antibodies suitable for use in other species such as companion animals have not been developed.

[0003] The present invention relates to such rodents, cells, antibodies, and parts thereof produced therefrom, such as antibodies subsequently modified for use in companion animals, and methods for producing such rodents, cells, antibodies, and antibody chains.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non - Patent Documents

[0005]

Non - Patent Document 1

[0006] The present invention relates to the following. i) The IGH V - region gene of one or more companion animals, the IGH D - region gene of one or more companion animals, and the IGH J - region gene of one or more companion animals, and ii) Optionally, the IGL kappa V region gene of one or more companion animals, and the IGL kappa J region gene of one or more companion animals; and / or the IGL lambda V region gene of one or more companion animals, and the IGL lambda J region gene of one or more companion animals and a rodent or rodent cell having a genome comprising the rodent or rodent cell can express the variable region gene of the companion animal to form an antibody chain, a rodent or rodent cell, wherein the companion animal species is not a rodent.

[0007] i) The IGL kappa V region gene of one or more companion animals, and the IGL kappa J region gene of one or more companion animals; and / or the IGL lambda V region gene of one or more companion animals, and the IGL lambda J region gene of one or more companion animals, and ii) Optionally, the IGH V region gene of one or more companion animals, the IGH D region gene of one or more companion animals or the host, and the IGH J region gene of one or more companion animals or the host and a rodent or rodent cell having a genome comprising the rodent or rodent cell can express the variable region gene of the companion animal to form an antibody chain, a rodent or rodent cell, wherein the companion animal species is not a rodent.

[0008] A method for producing a rodent or rodent cell, wherein the rodent cell genome is subjected to i) The IGH V region gene of one or more companion animals, the IGH D region gene of one or more companion animals, and the IGH J region gene of one or more companion animals, and / or ii) The IGL kappa V region gene of one or more companion animals, and the IGL kappa J region gene of one or more companion animals, and / or The IGL lambda V region gene of one or more companion animals, and the IGL lambda J region gene of one or more companion animals comprising the step of inserting either of them, A method in which a rodent or rodent cell can express the gene of a companion animal to form an antibody chain in combination with the constant region of the rodent or companion animal.

[0009] A method for producing an antibody chain or antibody specific for a desired antigen, comprising the step of immunizing a rodent disclosed herein with the desired antigen, and the step of recovering the antibody chain alone or as part of a complete antibody, or recovering cells that produce the antibody chain alone or as part of a complete antibody.

[0010] A method for producing an antibody chain or antibody specific for a desired antigen and derived from a single species of companion animal, comprising the step of immunizing a rodent comprising the gene of a companion animal disclosed herein with the desired antigen, and then replacing the constant region of the antibody chain of the rodent with the constant region of a companion animal derived from the same companion animal by appropriately genetically engineering the nucleic acid encoding the antibody chain or antibody.

[0011] A method for producing an antibody, or a part thereof, comprising (i) a nucleic acid encoding an antibody or a part thereof obtained according to the present invention, or (ii) sequence information capable of expressing a nucleic acid encoding an antibody or a part thereof obtained according to the present invention to produce an antibody comprising the step of providing.

[0012] A method for producing an antibody chain or a part thereof having a variable region of a companion animal, comprising the step of expressing intracellularly a nucleic acid encoding the antibody chain or a part thereof, The DNA sequence encoding the variable region of the antibody chain is obtained or can be obtained by immunizing a rodent of the present invention with an antigen so that the antibody chain is produced, Optionally, the method is as follows: purifying and / or isolating an antigen receptor chain; optionally, then formulating the antigen receptor chain into a pharmaceutically acceptable formulation suitable for administration to a companion animal, preferably the same companion animal, as a variable region; A method comprising:

[0013] An antibody or antibody chain, or a part thereof, obtainable or obtained from a rodent or cell according to the invention.

[0014] An antibody or antibody chain, or a part thereof, obtainable or obtained according to the invention, for use in the treatment of companion animals.

[0015] A method for treating a companion animal, comprising delivering an antibody or antibody chain or a part thereof to a companion animal in need thereof, wherein the antibody or antibody chain or a part thereof is modified to be a fully companion animal antibody.

[0016] A pharmaceutical composition comprising an antibody of a canine, feline or equine animal having a lambda light chain, or a functional fragment or functional derivative thereof, for use in the prevention or treatment of a disease of a dog, cat or horse respectively, or suitable for such use, and a pharmaceutically acceptable excipient or carrier.

[0017] An antibody of a canine, feline or equine animal having a lambda light chain, or a functional fragment or functional derivative thereof, for use in the prevention or treatment of a disease of a dog, cat or horse respectively.

[0018] A lambda light chain of a canine, feline or equine animal, or a functional fragment or functional derivative thereof, for use in the prevention or treatment of a disease of a dog, cat or horse respectively.

[0019] A rodent, or a cell, such as a rodent cell, that expresses or encodes a lambda light chain of an animal of the Canidae family, the Felidae family, or the Equidae family.

[0020] A method for treating or preventing a disease of a companion animal, such as a dog, a horse, or a cat, the method comprising delivering to the companion animal an effective amount of an antibody, or a functional fragment or functional derivative thereof, or a composition disclosed herein that is suitable for the companion animal. BRIEF DESCRIPTION OF THE DRAWINGS

[0021]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0022] The present invention relates to i) the IGH V region gene of one or more companion animals, the IGH D region gene of one or more companion animals, and the IGH J region gene of one or more companion animals, and ii) optionally, the IGL kappa V region gene of one or more companion animals, and the IGL kappa J region gene of one or more companion animals; and / or the IGL lambda V region gene of one or more companion animals, and the IGL lambda J region gene of one or more companion animals and a rodent or rodent cell having a genome comprising the rodent or rodent cell is capable of expressing the variable region gene of the companion animal to form an antibody chain in combination with the constant region of the antibody, relates to one embodiment in a rodent or rodent cell where the companion animal species is not a rodent.

[0023] The present invention also relates to the following. i) the IGL kappa V region gene of one or more companion animals, and the IGL kappa J region gene of one or more companion animals; and / or the IGL lambda V region gene of one or more companion animals, and the IGL lambda J region gene of one or more companion animals, and ii) optionally, the IGH V region gene of one or more companion animals, the IGH D region gene of one or more companion animals, and the IGH J region gene of one or more companion animals and a rodent or rodent cell having a genome comprising the rodent or rodent cell is capable of expressing the variable region gene of the companion animal to form an antibody chain in combination with the constant region of the antibody, a rodent or rodent cell where the companion animal species is not a rodent.

[0024] As an illustrative example, without being bound by the present invention, the insertion of dog-derived immunoglobulin heavy (IGH) chain variable (V) region genes, IGH D region genes, and IGH J region genes into mice, in combination with a constant region, enables the production of antibody heavy chains containing variable antibody regions derived from the expression of DNA of canid animals in mice. The constant region may be a rodent immunoglobulin (IG) constant region, and as a result, chimeric heavy chains having a variable region of a canid animal and a constant region of a rodent are generated. Information regarding the variable region of such chimeric antibody chains, or nucleic acids containing the same, can be used, for example, for therapeutic use in dogs, to generate complete canid animal antibodies. Rodents containing DNA of canid animals can also serve as animal models for understanding diseases and testing pharmaceuticals.

[0025] All nucleotide coordinates of mice correspond to the December 2011 GRCm38 / mm10 assembly of mice (assembly accession GCA_000001635.2) unless otherwise specified.

[0026] To avoid doubt, the insertion points referred to in the mouse genome are the same as the insertion points detailed in Lee et al., Nature Biotechnology, Nature Biotechnology 32, pages 356 - 363 (2014).

[0027] The dog genome build is CanFam3.1 (assembly accession - GCA_000002285.2), created in September 2011 and last updated in May 2016.

[0028] The cat genome build is FelisCatus8.0 (assembly accession - GCA_000181335.3), created in November 2014.

[0029] The rodents of the present invention are preferably mice or rats, and more preferably mice.

[0030] The companion animal of the present invention is appropriately selected from dogs, cats, horses, birds, rabbits, goats, reptiles, fish, and amphibians. A dog is a preferred companion animal of the present invention. A cat is a preferred companion animal of the present invention. A horse is a preferred companion animal of the present invention. To avoid doubt, a human is not a companion animal.

[0031] In one aspect, the rodent is a mouse and the companion animal is a dog.

[0032] In one aspect, the rodent is a mouse and the companion animal is a cat.

[0033] In one aspect, the rodent is a mouse and the companion animal is a horse.

[0034] The immunoglobulin heavy chain (IGH) locus of a companion animal contains genes of a plurality of V, D, and J regions. When the genes of the V, D, and J regions are expressed together, a variable region of the heavy chain of an antibody is generated. The IGH V, D, and J genes are naturally expressed in combination with a heavy chain constant region. The immunoglobulin light chain locus (IGL), which can be lambda or kappa, contains a plurality of V and J gene segments that form a variable region of the light chain of an antibody when expressed together. The IGL V and J region genes are naturally expressed in combination with a kappa or lambda light chain constant region. The rodent or rodent cell of the present invention can express the VDJ or VJ region gene of a companion animal to form an antibody chain. The gene of a companion animal is operably linked to a constant region in the genome of a rodent in order to enable the expression of an antibody chain. The companion animal IG gene can be located in the genome of a rodent together with an exogenous constant region gene (derived from a species other than a rodent), or can be located in the genome of a rodent in a functional arrangement such as upstream, together with a rodent constant region that naturally exists in the genome of a rodent, and expression of a V region gene with a constant region can occur.

[0035] The genome of a rodent or rodent cell may contain the IGH V, IGH D, and IGH J region genes of one or more companion animals, but not the DNA of the light chain companion animal, or may contain the IGL V and IGL J region genes of one or more companion animals, but not the DNA of the heavy chain companion animal. The genome of a rodent or cell may contain genes of a companion animal derived from the heavy chain and kappa chain (not lambda), or the heavy chain and lambda chain (not kappa), or may contain genes of a companion animal derived from all three loci, the heavy chain, kappa, and lambda.

[0036] In one aspect, the inserted companion animal DNA contains at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, etc. of the heavy chain variable (V) gene of the companion animal, and in one aspect, contains all of the V genes of the companion animal.

[0037] In one aspect, the inserted companion animal DNA contains at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, etc. of the heavy chain diversity (D) gene of the companion animal, and in one aspect, contains all of the D genes of the companion animal.

[0038] In one aspect, the inserted companion animal DNA contains at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, etc. of the heavy chain joining (J) gene of the companion animal, and in one aspect, contains all of the J genes of the companion animal.

[0039] In one aspect, the inserted companion animal DNA contains at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, etc. of the light chain variable (V) gene of the companion animal, and in one aspect, contains all of the light chain V genes of the companion animal.

[0040] In one aspect, the inserted companion animal DNA includes at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, etc. of the light chain joining (J) gene of the companion animal, and in one aspect, includes all of the light chain J genes of the companion animal.

[0041] In one aspect, the rodent genome includes all of the IGH V, D, and J region genes and intervening sequences from the companion animal.

[0042] In one aspect, the rodent genome includes all of the IGL kappa V and J region genes and intervening sequences from the companion animal.

[0043] In one aspect, the rodent genome includes all of the IGL lambda V and J region genes and intervening sequences from the companion animal.

[0044] The genome of a rodent or rodent cell may include at least 4, 5, 10, 15, or 20 IGH V region genes of the companion animal, such as at least 30, 40, 50, 60, 70, 80 V region genes. In a preferred aspect, these are V region genes of canid animals. In a preferred aspect, the rodent genome includes at least 83 IGH V region genes of canid animals.

[0045] The genome of a rodent or rodent cell may include at least 1, 2, 3, 4, 5, or 6 IGHD region genes from the companion animal, preferably genes of canid animals.

[0046] The genome of a rodent or rodent cell may include at least 1, 2, 3, 4, 5, or 6 IGHJ region genes from the companion animal, preferably genes of canid animals.

[0047] The genome of a rodent or rodent cell may comprise at least 10, 15, 16, 17, 18, or 19 IGL kappa V region genes of a companion animal. In a preferred embodiment, these are kappa V region genes of a canine animal. In a preferred embodiment, the genome of the rodent comprises at least 19 light chain kappa V region genes of a canine animal.

[0048] The genome of a rodent or rodent cell may comprise at least 1, 2, 3, 4, or 5 IGL kappa J region genes from a companion animal, preferably a gene of a canine animal.

[0049] The genome of a rodent or rodent cell may comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or at least 160 IGL lambda V region genes of a companion animal. In a preferred embodiment, these are lambda V region genes of a canine animal. In a preferred embodiment, the genome of the rodent comprises at least 160 light chain lambda V region genes of a canine animal.

[0050] The genome of a rodent or rodent cell may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 IGL lambda J region genes from a companion animal, preferably a gene of a canine animal.

[0051] In another embodiment, the genome of a rodent or rodent cell may comprise at least 4, 5, 10, 15, or 20 IGH V region genes from a cat, such as at least 23 V region genes.

[0052] In another embodiment, the genome of a rodent or rodent cell may comprise at least 4, 5, 10, or 11 IGH D region genes of a companion animal from a cat.

[0053] In another aspect, the genome of a rodent or rodent cell may comprise at least 1, 2, 3, 4, or 5 IGH J region genes of a companion animal derived from a cat.

[0054] In another aspect, the genome of a rodent or rodent cell may comprise at least 4, 5, 10, 15 IGL kappa V region genes of a companion animal derived from a cat.

[0055] In another aspect, the genome of a rodent or rodent cell may comprise at least 1, 2, 3, 4, 5, or 6 IG kappa J region genes of a companion animal derived from a cat.

[0056] In another aspect, the genome of a rodent or rodent cell may comprise at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 100, or more, for example 113 IGL lambda V region genes of a companion animal derived from a cat.

[0057] In another aspect, the genome of a rodent or rodent cell may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 IG lambda J region genes of a companion animal derived from a cat.

[0058] The number of companion animal genes referred to in any of the above aspects may further increase and, in one aspect, will be doubled in the case of a homozygote having insertions in both alleles.

[0059] In one aspect, the inserted companion animal DNA comprises less than 100% of the heavy chain variable (V) region gene of the companion animal, for example less than 90%, 80%, 70%, or 60% of the V region gene of the companion animal. This applies to the heavy chain, kappa, and lambda loci. Reducing the size of the total inserted companion animal DNA reduces the number of required insertion steps. Insertion of less than 100% of the companion animal gene for any immunoglobulin locus can be based on a reasonable selection of V regions.

[0060] Preferred V-region genes are those that have the highest representation in the antibodies of naturally occurring companion animals. For example, in dogs, they would be as follows: IGHV: 4-1, 3-38, 3-9, 3-67, 3-41 IGLV: 1-136, 1-55, 1-138, 8-93, 1-149 IGKV: 2S16, 2-8, 2-11

[0061] In particular, the inventors choose that the above Ig kappa and / or Ig lambda segments are present in the genome of the rodent, preferably in the absence of the complete repertoire of the kappa and / or lambda gene segments of the animal of the Canidae family respectively.

[0062] In another aspect, when two different V-region genes encode the same amino acid sequence, the selection of the V-region gene is preferably based on the subsequent likelihood of change in the amino acid sequence encoded by the V-region gene, such as by AID activity. These sequences that are more susceptible to the effects of such amino acid changes, such as those nucleic acid sequences with a high likelihood of undergoing non-synonymous mutations, are preferred. For example, a single mutation in the genetic code results in a change in the encoded amino acid.

[0063] Preferably, the genes of the V, D, and J regions inserted into the genome are from the same companion animal. Preferably, all of the inserted IGH VDJ region genes, or IGL VJ region genes, are from animals of the Canidae family, or all from animals of the Felidae family, or all from animals of the Equidae family. Preferably, all the genes are from animals of the Canidae family.

[0064] In one aspect, all of the genes of the companion animal inserted are from the same breed of the companion animal, for example, the same dog breed.

[0065] In one aspect, the companion animal gene is located upstream of the genome of the constant region of the rodent, and for the inserted companion animal heavy chain variable region gene, it is appropriately located upstream of the heavy chain constant region(s), and / or for the inserted companion animal light chain variable region gene, it is appropriately located upstream of the light chain constant region, whereby the rodent or rodent cell can generate a chimeric antibody chain resulting from the expression of the inserted variable region gene and the constant region of the rodent.

[0066] Preferably, the heavy chain V, D, and J region genes derived from the companion animal are located in the rodent genome upstream of the heavy chain constant region of the rodent.

[0067] Preferably, the light chain kappa V, J region genes derived from the companion animal are located in the rodent genome upstream of the kappa light chain constant region of the rodent.

[0068] Any reference to the position of the variable region upstream of the constant region such as the constant region of the rodent means that there is an appropriate relative position of the two genomic parts encoding the variable region and the constant region of the antibody to enable the chimeric antibody chain to be expressed in vivo in the rodent. In this way, the inserted companion animal DNA and the rodent constant region are functionally arranged relative to each other for the production of an antibody or antibody chain.

[0069] In one aspect, the DNA of the companion animal into which a variable VDJ or VJ region gene, etc. is inserted is located at a site different from the site of the heavy or light chain constant region naturally present in the genome of the rodent, for example, on a different chromosome. In this case, the insertion of the VDJ or VJ region gene is accompanied by a constant region and preferably also by a 3' enhancer derived from the rodent or the companion animal. One preferred embodiment is the use of the constant region of a canid animal and the 3' enhancer of a canid animal, etc. in combination with the VDJ or VJ region of the companion animal and the constant region and 3' enhancer of the rodent. In one aspect, the gene of the companion animal is located in the genome in a functional arrangement with the constant region derived from the same companion animal, and the rodent can produce antibody chains resulting from the expression of the inserted VDJ or VJ region gene of the companion animal and the constant region of the companion animal. Alternatively, the gene of the companion animal is located in the genome in a functional arrangement with a constant region derived from another companion animal, such as a different companion animal, or a constant region derived from a rodent, etc.

[0070] When the gene of the companion animal is inserted into the genome of the rodent along with a constant region, since the endogenous constant region gene is not necessary for the production of antibody chains, the insertion can be at any suitable position within the genome of the rodent cell and may not target the IG locus of the rodent. It will be understood that the insertion can be made at a random location in the genome of the rodent.

[0071] The present invention also relates to a rodent comprising genomic DNA encoding a complete companion animal antibody. A complete companion animal antibody can be produced by inserting the constant region of the companion animal into the rodent genome in a functional arrangement together with the inserted V, (D) and J region genes of the companion animal, or by replacing the constant region of the rodent with the constant region of the companion animal if a suitable chimeric antibody having the variable region of the companion animal and the constant region of the rodent is identified. Preferably, the constant region of the companion animal is selected from IgG of the same companion animal species having the greatest homology, such as functional homology with human IgG1 or IgG4, from either the IgG constant gene and / or allele. For antibodies for use in dogs, preferably, IgGA or IgGD of canid animals is used.

[0072] Accordingly, the present invention particularly contemplates cells and rodents having an insertion of a companion animal gene (encoding a "fully" companion animal antibody chain) with the constant region of the companion animal in the endogenous rodent IG locus, such as DNA containing the lambda V, J and C genes of the companion animal, or DNA containing the V, J and C kappa genes of the companion animal, or companion animal DNA containing V, D, J and C heavy chain gene segments. Preferably, the light chain lambda V and J region genes from the companion animal are located in the genome in a functional arrangement, such as upstream, together with the lambda chain constant region from the same companion animal. In this way, a lambda antibody chain having a lambda constant region from the companion animal is produced. Accordingly, the present invention relates to a rodent or rodent cell whose genome comprises one or more companion animal IGL lambda V region genes, one or more companion animal IGL lambda J region genes, and one or more companion animal lambda constant regions, and which can express a lambda antibody chain having both the variable and constant regions of the companion animal.

[0073] In one aspect, the VJC lambda antibody chain of the companion animal described above is inserted into the lambda locus of a rodent, such as the mouse lambda locus, preferably between the last rodent C gene and the 3' enhancer.

[0074] In one aspect, a rodent or rodent cell contains one or more companion animal IGL lambda V region genes, one or more companion animal IGL lambda J region genes, and one or more companion animal lambda constant regions located within the kappa locus of the rodent cell, such as upstream or downstream of the kappa constant region of the rodent. The insertion is preferably upstream of the IGL kappa locus constant region. In one aspect, the genome of the rodent or rodent cell contains the insertion of the companion animal light chain lambda V and J region genes upstream of the rodent kappa constant region, whereby the companion animal IGL lambda V and J region genes are expressed together with the rodent IGL kappa constant region. Suitably, the insertion positions the companion animal genes at a location substantially the same as the native rodent kappa gene, potentially deleting or replacing them, such that, for example, from the last inserted 3' lambda J gene to the rodent kappa constant region gene, there is the same or substantially the same distance as from the last rodent kappa 3' J gene to the kappa constant region. In one aspect, the insertion of the companion animal lambda light chain DNA is within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb of the boundary (upstream or downstream) of the rodent immunoglobulin kappa locus. The mouse kappa light chain is naturally expressed at a higher level than the mouse lambda light chain, and inserting the companion animal DNA into this kappa locus can result in high-level expression of the companion lambda chain V region gene.

[0075] In one aspect, the genome of the rodent can be homozygous for the insertion of the companion animal gene at one, or both, or all three of the immunoglobulin loci.

[0076] In another aspect, the genome of the rodent can be heterozygous for the insertion of the companion animal gene at one, or two, or all three of the immunoglobulin loci.

[0077] In particular, the genome of the rodent is heterozygous for the insertion of the companion animal gene at the kappa locus.

[0078] In one aspect, the inserted DNA can be expressed with the constant regions of different rodents by isotype switching.

[0079] In one aspect, the inserted companion animal DNA can be expressed with the constant regions of different rodents by trans-switching.

[0080] In one aspect, the companion animal is a dog, the genome of the rodent contains the kappa variable region gene of a canid animal, and all of the kappa variable region genes of the canid animal in the genome of the rodent are upstream of the constant region with which the variable region gene is co-expressed, for example upstream of the kappa constant region of the rodent, or for example upstream of the kappa constant region of a canid animal.

[0081] In another preferred aspect, the companion animal is a horse, the genome of the rodent contains the kappa variable region gene of a equine animal, and all of the kappa variable region genes of the equine animal in the genome of the rodent are upstream of the constant region with which the variable region gene is co-expressed, for example upstream of the kappa constant region of the rodent, or for example upstream of the kappa constant region of an equine animal.

[0082] In one aspect, the companion animal DNA is inserted between the wild-type constant region of the rodent located at the wild-type locus, suitably the constant region of the rodent and the host VDJ or VJ region. In one aspect, the IGH variable region gene is inserted downstream of the heavy chain J region and upstream of the Emu enhancer.

[0083] In one aspect, the rodent is a mouse, and the IGH variable region gene is inserted downstream of the mouse heavy chain J region and upstream of the Emu enhancer. In one aspect, the insertion of the IGH V region gene is performed at position 114666435 of the mouse genome on mouse chromosome 12. In one aspect, the insertion of the IGL lambda V region gene is performed at position 19047551 of the mouse genome on mouse chromosome 16. In one aspect, the insertion of the IGL kappa V region gene(s) is performed at position 70674755 of the mouse genome on mouse chromosome 6.

[0084] In one aspect, the rodent is a mouse, and the genome comprises at least the IGH variable region genes of V4-1, V3-2, V3-3, and V3-4 of animals of the Canidae family.

[0085] In one aspect, the rodent is a mouse, and the genome comprises at least the IGL kappa variable region genes of V4-1, V7-2, V3-3, V2-4, V2-5, V2-6, V2-7, V2-8, V2-9, V2-10, and V2-11 of animals of the Canidae family.

[0086] In one aspect, the rodent or rodent cell is a mouse or mouse cell, and one or more, or all, of the kappa V genes of animals of the Canidae family, 4-S17, 2-S16, 3-S15, 2-S14, 2-S13, and 2-S12 are located upstream of the rodent kappa constant region.

[0087] In one aspect, the rodent is a mouse, and the genome comprises at least the IGL lambda variable region genes of V3-1, V3-2, V3-3, V3-4, V4-5, and V4-6 of animals of the Canidae family.

[0088] In one aspect, the rodent is a mouse, and the genome comprises a deletion of one or some or all of the mouse IGH V region genes, preferably V1-85 to V5-2.

[0089] In one aspect, the rodent is a mouse, and the genome comprises a deletion of one or some or all of the mouse IGL kappa V region genes, preferably V3-1 to V2-137.

[0090] In one aspect, the rodent is a mouse, and the mouse heavy chain D and J region genes are retained upstream of the genome of the heavy chain variable region gene of the companion animal to be inserted.

[0091] In one aspect, the genome of the rodent is modified to reduce or prevent the expression of a complete rodent antibody having both a variable region and a constant region derived from the rodent. This can be due to inversion of all or part of the VDJ region of the rodent, or deletion or insertion into the endogenous rodent VDJ or VJ region of the genome. In one aspect, all or part of the VDJ or VJ region of the rodent is deleted. In one aspect, all or some of the V region genes of the rodent are deleted, for example, at least 50%, preferably at least 75%, or at least 90%, or all of the rodent IGH gene and / or the rodent IGL kappa VJ region gene and / or the rodent lambda VJ gene. In one aspect, the IGL lambda gene of the rodent does not delete from the rodent genome.

[0092] In one aspect, insertion of the DNA of the companion animal at the kappa locus of the rodent causes deletion or inactivation, in whole or in part, of one or both alleles of the kappa locus of the rodent.

[0093] In one aspect, the kappa locus of the rodent is inactivated, in whole or in part, for example by insertion, or deletion, or inversion.

[0094] In one aspect, the lambda locus of the rodent is inactivated, in whole or in part, for example by insertion, or deletion, or inversion.

[0095] In one aspect, the heavy chain locus of the rodent is inactivated, in whole or in part, for example by insertion, or deletion, or inversion.

[0096] The variable region gene of the companion animal is appropriately inserted upstream of the constant region of the rodent, and the constant region of the rodent contains all of the DNA necessary to encode a complete constant region or a part of the constant region sufficient to allow the formation of a functional chimeric antibody that can specifically recognize an antigen. Thus, references herein to chimeric antibodies or antibody chains having a rodent constant region are not limited to antibodies or antibody chains having a complete constant region or a complete constant region locus, but also include chimeric antibodies or chimeric antibody chains having a constant region or a part of a constant region locus sufficient to provide one or more effector functions found in antibodies that naturally occur in rodents. Effector functions include the ability to interact with Fc receptors and / or the ability to bind complement. This teaching also applies to the rodents, cells, and methods of the invention in which the variable region DNA is located in the host genome to form a chimeric antibody chain with all or part of the rodent constant region to form an antibody chain or a part thereof.

[0097] Preferably, the rodent genome contains all of the lambda constant region DNA and intervening regions of the companion animal.

[0098] The constant region of the rodent that is expressed together with the variable region of the companion animal is preferably the wild-type constant region of the rodent located at the wild-type locus appropriate for the heavy or light chain VDJ or VJ of the companion animal.

[0099] In one aspect, one or more other regulatory sequences such as enhancer or switch regions of at least one rodent are maintained in a functional arrangement with the constant region of the rodent. In this way, the effect of the enhancer or other regulatory sequence can be exerted in whole or in part in a cell or transgenic rodent.

[0100] In one aspect, one or more regulatory sequences of the rodent, such as the Emu enhancer sequence, are maintained in their native position upstream of the Mu constant region of the rodent, appropriately with respect to the distance from the constant region.

[0101] In one aspect, one or more rodent control sequences, such as enhancer sequences, are maintained in their native position downstream of the rodent constant region, suitably with respect to the distance from the constant region.

[0102] In one aspect, the rodent Smu switch sequence is maintained in its native position upstream of the rodent Mu constant region, suitably with respect to the distance from the constant region.

[0103] In such a position, the rodent enhancer or switch sequence is suitably operable in vivo with the host (i.e., its own) constant region sequence.

[0104] In a further aspect, one or more promoter elements, or other control elements, of the V, D, or J region genes of the companion animal are genomically optimized to interact with the rodent transcriptional machinery.

[0105] In one aspect, the genome of a rodent or rodent cell comprises one or more promoters, or enhancers, of a companion animal and / or other control elements associated with the companion animal V, D, or J region. In one aspect, one or more control regions of the companion animal, such as a promoter or enhancer or switch region, replace one or more of the rodent promoter or enhancer or switch regions, respectively. The control sequences of the companion animal are suitably maintained in a functional arrangement with the constant region such that the effects of the control sequences are exerted, in whole or in part, in the cell or transgenic rodent.

[0106] In one aspect, at least one or more of the V, D, or J gene segments of the companion animal to be inserted are accompanied by control sequences such as recombination signal sequences (RSSs) from the same companion animal, and optionally the control sequences induce the success of the recombination of the V, D, or J gene segment(s).

[0107] In this context, the "same" companion animal is not limited to the exact companion animal from which the V, D, or J gene segment of the companion animal is obtained. In one aspect, the "same" companion animal refers to the same breed or species as the companion animal from which the V, D, or J gene segment of the companion animal is obtained. In one aspect, it is the exact same companion animal.

[0108] In one aspect, at least one or more of the companion animal V, D, or J gene segments to be inserted are directly accompanied by a control sequence either in cis or in trans, or the control sequence is adjacent to one or both sides, and optionally for one or more gene segments, the control sequence is directly adjacent.

[0109] In one aspect, the control sequence includes a promoter preceding the individual V gene segment, and / or a splice site within the individual V gene segment, and / or downstream of the V gene segment, a recombination signal sequence for V(D)J recombination adjacent to the D gene segment or upstream of the J gene segment.

[0110] In one aspect, the V, D, or J sequences of the companion animal to be inserted are flanked by RSS sequences from the same companion animal. For example, the RSS sequences of a canine animal can be used with the V, D, and / or J sequences of a canine animal. It will be appreciated that this can be provided by insertion of a genomic fragment from a companion animal into the genome of a rodent. In a further aspect, the invention provides a method of replacing all or part of the endogenous immunoglobulin variable region locus in a rodent cell with a locus from a companion animal, the method comprising obtaining a cloned genomic fragment or synthetic sequence that wholly or partly comprises a locus of a companion animal that includes at least one V or D (for the heavy chain) or J gene segment and at least one associated regulatory sequence, and appropriately inserting the DNA of the companion animal into the rodent genome at the endogenous mouse immunoglobulin locus, preferably at the locus of the heavy or light chain that naturally corresponds to the DNA of the companion animal to be inserted.

[0111] In one aspect, the DNA of the companion animal to be inserted comprises at least 5 kb, at least 10 kb, at least 15 kb, 20 kb or more of genomic DNA from the companion animal.

[0112] In another aspect, the reconstruction of the companion animal locus in the genome of a rodent can be designed to minimize the number of steps of genetic manipulation of the rodent while inserting a subset of the companion animal DNA suitable for obtaining an acceptable companion antibody profile. Individual V region genes can be inserted into the rodent genome from the germline context, for example, not in the germline configuration or, for example, representing only a subset of the genes of the companion animal genome. The V, D, or J region genes of the companion animal inserted into the genome of the rodent or rodent cell can be accompanied by 1 to 10 kb, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb of the naturally occurring adjacent companion animal DNA located appropriately upstream and / or downstream in the companion animal genome. This can include regulatory elements that occur in the companion animal genome and can be beneficial for the expression of the variable regions V, D, and J region genes.

[0113] In one aspect, the rodent cells of the present invention can be rodent ES cells, rodent hematopoietic stem cells, or other cells that can generate an antibody chain containing a variable region encoded by the DNA of a companion animal, such as a chimeric antibody heavy chain, or a chimeric antibody light chain, or a complete companion animal antibody chain or antibody repertoire encoded by the variable region of the companion animal having a variable region and a constant region.

[0114] In one aspect, the cells of the present invention are rodent ES cells or induced pluripotent stem cells (iPS cells).

[0115] In one aspect, the cells are isolated rodent cells.

[0116] In one aspect, the cells are isolated rodent B cells.

[0117] Preferably, the rodent cells are rodent ES cells or iPS cells. Such cells are suitable for the insertion of the DNA of the companion animal and generate a rodent that expresses the antibody chains described herein.

[0118] The ES cells may be cells having a hybrid genome containing genomic DNA of mouse cell line 129 or C57BL, such as C57BL / 6N, C57BL / 6J, 129S5 or 129Sv strain, or 129 or C57BL.

[0119] The present invention also relates to cell lines that grow from or are otherwise derived from the cells described herein, including immortalized cell lines.

[0120] The cells or cell lines or genomes of the present invention may contain the V, (D), or J genes of the companion animal after germline constitution or reconstitution after in vivo maturation.

[0121] The present invention also relates to cells or cell lines that express antibody chains such as chimeric antibody heavy chains, obtained by immunizing the rodents of the present invention with an antigen.

[0122] The present invention also relates to cells or cell lines that express an antibody or antibody chain having a companion animal variable region, preferably with a companion animal constant region, wherein the nucleic acid sequence of the variable region of the antibody or antibody chain is identified or has been identified by immunizing the rodents of the present invention with an antigen and obtaining the antibody or antibody chain, or the sequence of the antibody or antibody chain, from the rodent or rodent cells.

[0123] The expressed antibody chain is preferably a complete canine antibody or antibody chain, or a complete equine antibody or antibody chain, or a complete feline antibody or antibody chain, in which the variable region derived from the companion animal is expressed in the cells or cell lines or rodents of the present invention with the same constant region derived from the companion animal (not the constant region of the rodent).

[0124] The antibody chain, or the cell or cell line expressing the antibody, can be a CHO cell or other mammalian cell line suitable for the production of therapeutic agents for use in animals.

[0125] The cells can be immortalized by fusion with tumor cells to provide antibody-producing cells and cell lines, or can be produced by direct cell immortalization.

[0126] The present invention also relates to vectors for use in the present invention. In one aspect, such vectors are bacterial artificial chromosomes (BACs) containing all or part of the companion animal's IG locus. Other cloning vectors can be used in the present invention, and thus it will be understood that references herein to BACs can generally be construed to refer to any suitable vector. The vector may include one or more selectable markers and / or one or more site-specific recombination sites. In one aspect, the vector includes two or more, for example three, heterospecific and incompatible site-specific recombination sites. In one aspect, the site-specific recombination site can be a loxP site or a variant thereof, or an FRT site or a variant thereof. In one aspect, the vector includes one or more transposon ITR (terminal inverted repeat) sequences.

[0127] A suitable BAC containing the DNA of a canine animal is available as the CHORI-82 BAC library from the BACPAC Resource Center at the Oakland Institute for Children's Hospital.

[0128] A suitable BAC containing the DNA of a feline animal is available as the FCAB library from Amplicon Express.

[0129] A suitable BAC containing the DNA of an equine animal is available as the CHORI-241 BAC library from the BACPAC Resource Center at the Oakland Institute for Children's Hospital.

[0130] The present invention relates to a method for producing a rodent or rodent cell, the method comprising inserting into the rodent cell genome one or more IGH V region genes of a companion animal, one or more IGH D region genes of a companion animal, and one or more IGH J region genes of a companion animal, wherein the rodent or rodent cell is capable of expressing the variable region genes of the companion animal in combination with a constant region to form an antibody chain.

[0131] The present invention also relates to a method for producing a rodent or rodent cell, the method comprising inserting into the rodent cell genome one or more IGL V region genes of a companion animal, and one or more IGL region J genes of a companion animal, wherein the rodent or rodent cell is capable of expressing the variable region genes of the companion animal in combination with a constant region to form an antibody chain.

[0132] Preferably, the method relates to inserting the VDJ and VJ region genes of a companion animal for both the light chain and the heavy chain such that an antibody is produced that has variable regions derived from the expression of the DNA of the companion animal for both the light chain and the heavy chain.

[0133] The present invention also relates to a method for producing a rodent or rodent cell, the method comprising sequentially inserting DNA fragments of a plurality of companion animals into the rodent cell genome, wherein the inserted fragments form a continuous insertion in the rodent cell, i.e., they are directly joined without intervening sequences.

[0134] In one aspect, the insertion process begins at a site where an initiation cassette is inserted into the genome of a cell such as an ES cell. In one aspect, the initiation cassette is inserted into the heavy chain locus of a rodent for use in the insertion of the heavy chain DNA of a companion animal. Similarly, the initiation cassette may be inserted into the light chain locus of a rodent for use in the insertion of the light chain VJ DNA of a companion animal. The initiation cassette may be placed between the last J region and the C region of the heavy chain and kappa chain of the rodent. The initiation cassette may be placed downstream of the rodent kappa IGL locus on the same chromosome for use in the insertion of the genes of an IGL lambda companion animal.

[0135] The initiation cassette appropriately includes a continuous locus unique to the genome of a rodent that can effect the insertion of companion animal DNA.

[0136] In one aspect, after insertion of the first DNA fragment into the initiation cassette, insertion of the second DNA fragment into a portion of the first DNA fragment may follow. Insertion may be made into at least a portion of a previously inserted DNA fragment.

[0137] In one aspect, the method includes targeted insertion of the initiation cassette into the rodent genome by homologous recombination, insertion of a first DNA sequence into at least a portion of the initiation cassette by site-specific recombination, insertion of a second DNA sequence into a site of at least a portion of the first DNA sequence, and optionally, insertion of one or more additional DNA sequences into at least a portion of a preceding DNA sequence, to construct a continuous DNA fragment within a target that includes companion animal DNA. Insertion of a DNA fragment into at least a portion of an initial fragment can be by site-specific recombination, for example, by recombinase-mediated cassette exchange (RMCE). This method may include both homologous recombination (e.g., the first step of insertion of the initiation cassette) and site-specific recombination, e.g., RMCE (e.g., one or more subsequent insertion events). Site-specific recombinase systems are well known in the art and may include Cre-lox, and FLP / FRT, or combinations thereof.

[0138] In one aspect, the DNA of the companion animal to be inserted is stepwise constructed into the genome of cells such as ES cells using separate insertions for each of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more heavy or light chain regions. The DNA fragments of the companion animal are successively and appropriately inserted into the same or substantially the same locus of the cell, for example, the locus of the ES cell, to form a complete VDJ or VJ region, or a part thereof.

[0139] The present invention also relates to cells and rodents containing intermediates during the process, the genome of which may contain only a partial VDJ or VJ region of the companion animal, such as only the variable region gene DNA of the companion animal.

[0140] Methods for targeted insertion of exogenous DNA at endogenous mouse loci such that the inserted V, D, and J genes can be expressed in the constant region of the host are well known in the art. See Murphy et al., Vol. 111, No. 14, pp. 5153 - 5158, doi:10.1073 / pnas.1324022111; MacDonald et al., Vol. 111, No. 14, pp. 5147 - 5152, doi:10.1073 / pnas.1323896111; and Lee et al., Nature Biotechnology, Vol. 32, pp. 356 - 363, 2014 DOI:, doi:10.1038 / nbt.2825.

[0141] In particular, methods for producing transgenic rodents include insertion of the VDJ or VJ region gene of the companion animal disclosed herein upstream or downstream of the constant region of the corresponding rodent mammal by stepwise insertion of multiple DNA fragments by sequential recombinase-mediated cassette exchange (SRMCE).

[0142] In one aspect, the correct insertion event is confirmed before proceeding to the next step of the multi-step cloning process.

[0143] In one aspect, the companion animal is a dog, the rodent is a mouse, and the canine IGL kappa V region gene that is naturally located downstream of the canine kappa constant region of the Canidae family in the dog is inserted upstream in the same orientation as the canine IGL kappa V gene that is naturally found upstream of the rodent kappa constant region in the rodent, preferably upstream of the canine IGL kappa constant region.

[0144] In one aspect, the rodent can generate a diversity of combinations of at least 1×10 6 different functional chimeric immunoglobulin sequences.

[0145] In one aspect, a rodent and cells of the one aspect, for example, ES cells having one or more chimeric loci are used to generate a chimera, where a host embryo is generated from a RAG-1 deficient background or other suitable genetic background that prevents the production of mature host B and T lymphocytes. Thereby, all B and T cells can be made from the injected ES cells.

[0146] In one aspect, the preferred rodent is a mouse and the cells of the invention are mouse cells or ES cells. In another aspect, the preferred rodent is a rat and the cells of the invention are rat cells or ES cells.

[0147] The ES cells of the present invention can be used to generate animals using techniques well known in the art, including injecting the ES cells into a blastocyst, subsequently transplanting the chimeric blastocyst into a female to generate offspring, mating these to generate heterozygous offspring, and then mating to generate homozygous recombinants having the required insertions. In one aspect, the host blastocyst is Rag deficient.

[0148] The present invention relates to a chimeric rodent generated by injecting the ES cells of the present invention into a blastocyst and then transplanting the chimeric blastocystys into a female rodent to produce offspring.

[0149] In one aspect, the rodent or rodent cell is a mouse or mouse cell, the mouse ADAM6a and ADAM6b genes are present in the mouse genome and have not been previously deleted from and then reinserted into the IGH locus. For example, here, the mouse ADAM6 gene has not been previously deleted from the mouse IgH locus and then returned by introduction into the IGH locus to improve fertility.

[0150] In one aspect, the rodent ADAM6a and ADAM6b genes are located 5' to the V, D, and J genes of one or more inserted companion animals.

[0151] In one aspect, the rodent IGH D and J genes are present in the rodent genome. In one aspect, the rodent IGH D and J genes have not been deleted from the rodent genome. In one aspect, the rodent IGH D and J genes are located 5' to the V, D, and J genes of one or more inserted companion animals.

[0152] In one aspect, the alleles of the Canidae animals used in the present invention are the reference alleles of the genes of each Canidae animal. These are those of CanFam3.1. See the assembly accession - GCA_000002285.2, created in September 2011 and last updated in May 2016.

[0153] Surprisingly, from the genomic data of 107 dogs across 19 breeds, it was found by the inventors that the breed variation was minimal, the reference allele (from Boxer dogs) was found at a rate of 76% across the entire sample, and non - reference alleles were typically found as heterozygotes with the reference allele. This means that the antibody population generated from rodents containing the reference alleles of Canidae animals is widely applicable for use across different dog breeds.

[0154] Accordingly, the present invention relates to a rodent cell or rodent disclosed herein, wherein at least 90%, at least 95%, and preferably all of the inserted companion gene segments are reference alleles of Canidae animals from CanFam 3.1.

[0155] Preferably, the rodent or rodent cell contains alleles that are not reference alleles of one or more of the following gene segments (not "*01 alleles"): IGVK2-S13, IGLV1-57, IGLV1-68, IGLV1-72, IGLV1-88, IGLV1-96, IGLV8-60, IGLV8-90, IGLV8-120. Preferably, the genome of the rodent or rodent cell contains all 2, 3, 4, 5, 6, 7, 8, or 9 of these non-reference alleles.

[0156] The present invention also relates to an antibody chain or a part thereof having a variable region derived from the expression of a reference allele of a Canidae animal in the treatment of a disease, particularly in a dog breed other than the reference genome breed, as obtained or obtainable as disclosed herein. In one aspect, at least 50, 60, 70, 80, 90, or 100% of the V gene segments to be inserted are V gene reference alleles of Canidae animals, and / or at least 50, 60, 70, 80, 90, or 100% of the D gene segments to be inserted are D gene reference alleles of Canidae animals, and / or at least 50, 60, 70, 80, 90, or 100% of the J gene segments to be inserted are J gene reference alleles of Canidae animals, and combinations thereof. Preferably, at least 90%, such as 100%, of the Canidae animal gene segment alleles to be inserted are reference alleles of the gene segment.

[0157] The present invention also relates to the insertion of the V, D, and J segments of the Canidae animals disclosed herein into rodents for the production of antibodies or antibody chains, or portions thereof, for use in the prevention or treatment of diseases in different dog breeds, and to the use of rodents inserted with the Canine V, D, and J segments described herein for the production of antibody chains or portions thereof for use in the prevention or treatment of diseases in different dog breeds.

[0158] The present invention also relates to rodents having the V, D, and J segments of one or more or all of the Canidae animals derived from the Boxer dog breed, as described herein. The present invention further provides for the use of an antibody or fragment thereof obtainable or obtained from any rodent having the genetic segments of the Canidae animals of the genome described herein, wherein the antibody is at least partially expressed from the DNA of the Canidae animals in the prevention or treatment of diseases in dog breeds different from the breed in which the genetic segments of the Canidae animals are used.

[0159] For example, if the inserted Canidae animal DNA is from a Boxer, it is used in dog breeds other than Boxers.

[0160] The present invention also relates to the following. (i) A rodent having the V, D, J segments of a Canidae animal derived from the Boxer dog breed, preferably one or more Boxer gene segments that are reference alleles; (ii) A rodent having the V, D, and J segments of the Canidae animals described herein, preferably one or more V, D, and / or J gene segments that are identical to those of the Boxer dog (i.e., the dog has reference alleles), derived from a dog breed other than Boxer; (iii) Cells such as B cells, hybridomas, CHO, or other suitable cells that express an antibody or antibody chain derived from the rodent of (i) or (ii), wherein the antibody is expressed from the V, D, J DNA of the Canidae animal (or the V, and J DNA of the light chain of the Canidae animal), preferably containing at least some amino acids expressed from the reference allele; (iv) For example, a complete canine antibody or antibody chain comprising the antibody variable region of the antibody of (iii) above, which can be generated by expressing DNA encoding the variable region of a canine animal together with DNA encoding the constant region of a canine animal; (v) An expression cell such as a CHO cell comprising DNA encoding all or part of such a complete canine antibody.

[0161] Preferably, the V, D, and J gene segments of the boxer are from the CHORI-82 BAC library.

[0162] In another aspect, the present invention provides the use of an antibody or antibody chain having the variable region of a canine animal in the prevention or treatment of diseases in different dog breeds, and the antibody or antibody chain is obtained from or can be obtained from the rodents disclosed herein, and the V gene segment used to generate the variable region of the canine animal is a canine reference allele, preferably, the antibody or antibody chain is effective, for example, biologically effective in the treatment or prevention of diseases in at least 50%, such as 60% or at least 70% of different dog breeds. Preferably, the V gene segment of the canine animal derived from the heavy chain and / or light chain is from a boxer. Preferably, the D and J gene segments are also the gene segments of the boxer.

[0163] The present invention relates to the following. A method for producing an antibody chain specific for a desired antigen, comprising the steps of immunizing a rodent disclosed herein with the desired antigen, and recovering the antibody chain alone or as part of a complete antibody, or recovering cells that produce the antibody chain alone or as part of a complete antibody (see, for example, Harlow, E. and 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 Sciences (2004) 101: 257-259). Appropriately, an immunogenic amount of the antigen is delivered. The present invention also relates to a method for detecting a target antigen, comprising the step of detecting an antibody produced as described above with a secondary detection agent that recognizes a part of the antibody.

[0164] A method for producing an antibody chain or an antibody specific for a desired antigen and derived from a single species of companion animal, comprising the steps of immunizing a rodent comprising the gene of a companion animal disclosed herein, and then appropriately genetically engineering the nucleic acid encoding the antibody to replace the constant region of the antibody chain of the rodent with the constant region of a companion animal derived from the same companion animal. This can replace the constant region of a non-human mammal with an appropriate companion animal constant region DNA sequence by standard cloning techniques at the DNA level. See, for example, Sambrook, J and Russell, D. (2001, 3rd Edition) "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Lab. Press, Plainview, NY). Alternatively, it can be achieved by direct nucleic acid synthesis.

[0165] A method for producing an antibody chain or a part thereof having the variable region of a companion animal, comprising the step of expressing intracellularly DNA encoding the antibody chain or a part thereof, The sequence of DNA encoding the variable region of the antibody chain can be obtained from, or can be obtained by, immunizing the rodent of the invention with an antigen such that the antibody chain is produced. Optionally, the method is as follows: purifying and / or isolating the antigen receptor chain; optionally, then formulating the antigen receptor chain into a pharmaceutically acceptable formulation suitable for administration to a companion animal A method comprising.

[0166] An antibody or antibody chain, or a part thereof, obtainable or obtained from a rodent or cell according to the invention, or a nucleic acid encoding an antibody chain or a part thereof.

[0167] The invention also relates to part or all of an immunoglobulin molecule comprising a variable domain of a canine animal disclosed herein and a constant domain of a rodent derived from a rodent B cell. Similarly, hybridoma cells obtainable or obtained from such B cells, and part or all of an immunoglobulin molecule comprising a variable domain of a canine animal and a constant domain of a rodent obtainable or obtained from such hybridoma cells. The invention also relates to the identification of variable regions of canine animals by single cell sequencing and the generation of synthetic vectors expressing full antibody chains having corresponding constant regions of canine animals. The invention also relates to obtaining these sequences by PCR and ligating them to appropriate constant regions by suitable molecular biology techniques such as, but not limited to, bridge PCR and Gibson cloning.

[0168] Another aspect of the invention relates to the identification of antibodies of interest generated in a rodent of the invention using high-throughput cell screening as disclosed in International Publication No. WO 2015 / 040401, which is incorporated herein by reference, and to antibodies identified using the methods disclosed therein. For example, suitable antibodies may be obtained, or can be obtained, from a population of cells expressing a repertoire of antibodies and / or antibody chains (also referred to as the protein or POI of interest), and the antibodies and chains comprise variable regions of a companion animal and constant regions of a rodent, and the method for generating the cells is as follows: a) providing a population of cells expressing a repertoire of the POI; b) sorting the population of cells to generate a sorted population of single cells, each cell containing a nucleic acid encoding a respective POI; c) amplifying the nucleic acids contained in the sorted single cell population to generate a sorted repertoire of amplified nucleic acids encoding the POI; d) modifying the sorted amplified POI encoded by the nucleic acids from step (c) to generate a sorted repertoire of expression cassettes, each cassette containing a nucleic acid sequence encoding the POI and one or more control elements for expressing the POI; and e) transferring the POI expression cassette from the repertoire of the cassettes to a sorted population of host cells while maintaining the sorting of the POI expression cassettes and generating a sorted repertoire of host cells expressing the sorted repertoire of the POI, wherein the sorted repertoire of host cells is capable of stably expressing the repertoire of the POI wherein the POI is an immunoglobulin chain or a part thereof, and in step (a), the cells are cells isolated from one or more animals including B cells, germinal center cells, memory B cells, antibody-secreting cells, plasma cells or plasmablasts, and step (c) is performed using PCR.

[0169] The invention also relates to an antibody or chain, or a part thereof, obtainable or obtained according to the invention for use in the treatment of companion animals.

[0170] The invention also relates to a method for treating companion animals, the method comprising delivering to a companion animal in need thereof a suitable antibody or antibody chain or a part thereof obtainable or obtained according to the invention. In particular, the invention relates to a method of medical treatment comprising delivering to a companion animal in need thereof an antibody chain or antibody, or a part thereof, wherein at least the variable region of the antibody is obtained or otherwise identified by immunizing a rodent of the invention comprising the companion DNA V(D)J region gene with an antigen.

[0171] In one aspect, the present invention relates to chimeric companion animal antibodies and antibody chains having a rodent constant region and a companion animal variable region, and functional fragments and functional derivatives of said antibodies and chains, and to the use of said antibodies, chains and fragments in medicine including diagnosis and in in vitro or ex vivo research. Functional antibody fragments and derivatives may include fragments capable of specific binding to an antigen. Functional antibody fragments can be, for example, Fab, Fab', F(ab)2, Fv, scFv fragments, diabodies, linear antibodies or single-chain antibody molecules. In one aspect, the fragment includes at least CDR3, for example lambda CDR3. Functional derivatives can be, for example, antibodies or fragments thereof modified by attachment to another agent such as an immunoadhesion molecule, an imaging agent, a therapeutic agent and a cytotoxic agent. Suitable examples are well known in the art. See, for example, International Publication No. WO 2012 / 024650 A2, which is incorporated herein by reference. In a further aspect, the present invention relates to "fully" companion animal antibodies and antibody chains ("fully" reflects the fact that both the variable and constant regions of the antibody are expressed from genes of the same species of companion animal), and to fragments and functional derivatives of said antibodies and chains, and to the use of said antibodies, chains and fragments in medicine including diagnosis and in in vitro or ex vivo research.

[0172] Methods for generating both monoclonal and polyclonal antibodies are well known in the art, and the present invention relates to both polyclonal and monoclonal antibodies of chimeric or fully companion animal antibodies produced in response to antigen loading in the rodents of the present invention.

[0173] In a further aspect, the invention relates to the use of a rodent as described herein as a model for the testing of drugs and vaccines. Accordingly, the invention relates to a method for the identification or validation of a drug or vaccine, the method comprising the step of delivering a vaccine or drug to a mammal of the invention and the step of monitoring one or more of an immune response, a safety profile, an effect on a disease.

[0174] In yet another aspect, the chimeric antibodies or antibody chains generated in the present invention can be appropriately manipulated at the DNA level to generate molecules having antibody-like properties or structures, such as variable regions of companion animals from heavy or light chains without constant regions, such as domain antibodies, or variable regions of companion animals having any constant region from either a heavy or light chain of the same or different origin, or variable regions of companion animals having non-naturally occurring constant regions, or variable regions of companion animals having any other fusion partner. The invention relates to all such chimeric antibody derivatives derived from the chimeric antibodies identified according to the present invention.

[0175] The invention also relates to a kit comprising any of the antibodies or antibody derivatives disclosed herein and instructions for use of such antibodies or suitable laboratory reagents such as buffers, antibody detection reagents.

[0176] The invention also relates to a method for making an antibody, or a portion thereof, comprising (i) a nucleic acid encoding an antibody or a portion thereof obtained according to the invention, or (ii) sequence information capable of expressing a nucleic acid encoding an antibody or a portion thereof obtained according to the invention to produce an antibody and the step of providing.

[0177] The present invention also relates to chimeric antibodies comprising a variable region of a companion animal and a constant region of a rodent (optionally, C gamma or C mu), wherein the antibody is encoded by a nucleotide sequence corresponding to the nucleotide sequence of the chimeric heavy chain locus of a cell (optionally, a B cell, ES cell or hybridoma), the locus comprising the nucleotide sequence of the constant region of the rodent, and a rearranged VDJ nucleotide sequence generated by in vivo rearrangement of the V region, D region, and J region of the companion animal, and the V region of the companion animal is selected from the IGH variable region genes of IGH V4-1, V3-2, V3-3, V3-4, V3-5 of Canidae animals.

[0178] Optionally, the J region is any one of JH1, JH2, JH3, JH4, JH5, or JH6 of Canidae animals.

[0179] Optionally, the D region is any one of DH1, DH2, DH3, DH4, DH5, and DH6 of Canidae animals.

[0180] In one aspect, the antibody comprises any combination exemplified in the examples and drawings herein. Optionally, the in vivo rearrangement occurs in a cell (e.g., a B cell or ES cell) of the same rodent species (e.g., a mouse B cell or ES cell) as the constant region sequence. The present invention also relates to a non-human vertebrate or mammalian cell (e.g., a B cell, ES cell or hybridoma) whose genome comprises the chimeric heavy chain locus described above in this paragraph.

[0181] The present invention also relates to a non-human vertebrate or mammal (e.g., a mouse or rat) whose genome comprises the chimeric heavy chain locus described above in this paragraph.

[0182] The antibodies of the present invention may be isolated, and in one aspect, are isolated from the cells or organisms in which they are expressed.

[0183] The present invention also relates to a part of an antibody chain. In particular, this part includes at least the variable region of the antibody. This can be expressed from cells, particularly for antibody production. This part may include the Fab region of the antibody, or may include at least the CDR region.

[0184] The present invention relates to a method for producing an antibody or an antibody chain, which comprises the step of immunizing a rodent described herein with an antigen that can be obtained from a companion animal that is the same as the source of the companion animal DNA present in the genome of the rodent. For example, an antigen of a canine animal can be used to immunize a rodent containing the V, D, and J genes of the canine animal disclosed herein.

[0185] Accordingly, the present invention also relates to a rodent immunized with an antigen derived from a companion animal corresponding to the source of the companion animal DNA present in the genome of the rodent.

[0186] It is not necessary to use exactly the same companion animal. For example, an antigen of a canine animal collected from one breed can be used to immunize a rodent containing DNA of a canine animal from a different breed. Antigens from the same dog breed can also be used.

[0187] Alternatively, the antigen can be derived from a pathogen such as a bacterium or virus known to infect the companion animal. For example, an antigen derived from a pathogen that infects dogs and causes disease can be used to immunize a rodent containing the V, D, and J genes of the canine animal disclosed herein.

[0188] Accordingly, the present invention relates to a rodent immunized with an antigen that causes disease in a companion animal corresponding to the source of the companion animal DNA present in the genome of the rodent.

[0189] In a further aspect, the antigen may be an equivalent in a companion animal of a human antigen associated with a human disease, preferably an antigen that has been validated as a target for the prevention or treatment of a human disease.

[0190] In one aspect, the present invention provides an antibody chain or a fragment thereof obtainable or obtained by immunizing a rodent with an antigen as described herein.

[0191] The present invention also relates to nucleic acids such as DNA or RNA encoding said antibody, antibody chain, or a part thereof. In particular, a part thereof may be the variable part of the antibody chain, which is the part encoded by the DNA of the companion animal in the rodent.

[0192] The present invention also relates to a cell such as a B cell or a hybridoma expressing a partial or complete antibody chain of a canine animal, or a part of a partial or complete antibody chain of a canine animal, for example, a variable region, or an expression cell line (e.g., CHO cell), the DNA or protein sequence of which can be obtained from or has been obtained from the rodent described herein.

[0193] Once the antibody of interest has been identified from an immunized rodent, it is understood to be standard in the art to identify the DNA sequence encoding the antibody from the B cells and to be able to express the antibody or a part thereof from that sequence or, in fact, from a different DNA sequence that expresses the same protein as a result of the redundancy of the genetic code. In particular, the variable region of a canine animal can be expressed together with the constant region of a canine animal to produce a complete antibody chain or antibody of a canine animal.

[0194] Accordingly, the present invention also provides a method for obtaining a complete antibody of a canine animal, comprising Immunizing a rodent having at least one heavy chain canine immunoglobulin V gene segment, at least one canine heavy chain D gene segment, and at least one canine heavy chain J gene segment, and / or having at least one light chain canine immunoglobulin V gene segment and at least one canine heavy chain J gene segment with an antigen described herein, Selecting an antibody chain(s) produced by a rodent having a variable antibody region encoded by canine DNA, Expressing the variable antibody region from the DNA of an expression cell line, preferably that of a complete canine, to express, for example, an antibody or antibody chain or a part thereof comprising a variable region of a canine and a constant region of a canine, Optionally, purifying the antibody, antibody chain or a part thereof, Optionally, further formulating the antibody or chain or a part thereof with a pharmaceutically acceptable excipient suitable to enable its administration to a companion animal in need thereof and providing a method comprising the above.

[0195] The present invention also relates to the use of such an antibody or antibody chain or a fragment thereof in the treatment of companion animals.

[0196] The above approach is equally applicable to other preferred companion animals such as cats and horses. Antigens of feline animals can be used in an equivalent feline / rodent model, antigens of equine animals can be used in an equivalent equine / rodent model, and all other aspects of the present invention are equally applicable to cats and horses.

[0197] The present invention relates to a method for generating cross-reactive antibodies or antibody chains, which comprises immunizing a rodent containing the DNA of a companion animal disclosed herein with an antigen of interest derived from said rodent and a corresponding antigen derived from the companion animal, wherein the rodent comprises a gene knockout of the gene encoding said antigen. The antibody population generated from said immunized rodent can bind to both the antigens of the rodent and the companion animal, i.e., can comprise cross-reactive antibodies.

[0198] Accordingly, in a further aspect, the present invention relates to cross-reactive antibodies or antibody fragments derived from a rodent containing the DNA of a companion animal as described herein, immunized with an antigen of interest of the rodent and a corresponding antigen derived from the companion animal, wherein the rodent has a gene knockout of the gene encoding said antigen.

[0199] In one aspect, the rodent is immunized sequentially or simultaneously with the antigen of the rodent and the corresponding antigen derived from the companion animal, either directly with a protein or a peptide fragment thereof, or with a vector encoding a related antigen or a fragment thereof, or with a syngeneic cell line expressing the desired antigen.

[0200] In one aspect, the rodent is a mouse and the companion animal is a dog, a cat, or a horse.

[0201] Cross-reactive antibodies are highly important in the field of drug discovery. Cross-reactive antibodies can be rapidly validated as drug candidates for a species to which the antibody is reactive by using other species to which the antibody is reactive as models without the need for antibody modification.

[0202] The present invention also relates to the use of a canine antigen for immunizing a rodent as claimed herein, to a canine antigen for immunizing a rodent as claimed herein, wherein the canine antigen has a family gene or protein equivalent in humans, is preferably therapeutically validated, and antibodies against the human equivalent antigen have been shown to be effective in the treatment of diseases.

[0203] The present invention discloses for the first time a strong homology between the Ig variable regions of canines and humans. See Figure 16. [Strong homology of germline canine-human IG V, TCR V homology is less conserved]. This information suggests that a rodent model containing canine variable region DNA may be able to utilize canine control sequences rather than, for example, mouse control sequences, which is because humanized mice can use human control sequences within the mouse. Indeed, the inventors have experimentally verified that the control sequences and RSS sequences of the inserted canine genomic DNA are recognized by rodents and can be used to express chimeric antibodies in rodents.

[0204] These findings by the inventors are surprising and not expected based on the evolutionary differences between canines and humans.

[0205] Within the assigned phylogeny, it is well known that mice and humans are more closely related than, for example, carnivores including cats and dogs, and horses. This is likely the reason why the use of human control sequences in chimeric mouse models with human DNA V, D, and J gene segment insertions is successful in said models. Based on this, the same could not be predicted for more evolutionarily diverse animals such as humans and dogs. Indeed, in previous attempts to use canine gene segments in mice, murine regulatory control sequences were utilized (e.g., Trianni, US2017306352).

[0206] In one aspect, the invention relates to a rodent in which the inserted (companion animal) IGHJ4 and IGHJ6 are the major JH gene segments found in the mature B cell antibody repertoire. Preferably, the rodent contains V, D, and J gene segments of a canine animal, and J4 and J6 are the J4 and J6 gene segments of a canine animal. In one aspect of the invention, the rodent can use one, two, three, four, five, six, or more, or all of the different inserted IGH D segments, and / or one, two, three, four, five, six, or more, or all of the inserted IGH J gene segments, in the production of antibody chains within the rodent antibody repertoire.

[0207] The currently tested rodent mouse having the inserted heavy chain D1-6 and J1-6 gene segments of a canine animal can utilize all of the canine IGH J1-6 and D1-6 in antibody formation.

[0208] In one aspect of the invention, the rodent utilizes more IGLJ1 than other light chain J gene segments.

[0209] In one aspect of the invention, the rodent contains DNA of a canine-derived companion animal, and the rodent has one or more of the following characteristics: The rodent expresses a chimeric antibody heavy chain from IGHJ4 more, individually, than any of IGHJ1, 2, 3, 5, or 6, and the rodent expresses a chimeric antibody heavy chain from IGHJ6 more, individually, than any of IGHJ1, 2, 3, or 5; The rodent expresses a chimeric antibody heavy chain from IGHD5 more, individually, than any of IGHD1, 2, 3, 4, or 6; The rodent expresses a chimeric antibody heavy chain from IGHD2 more, individually, than any of IGHD1, 3, 4, or 6; The rodent expresses an antibody chain from IGHD2 together with IGHJ4; The rodent expresses an antibody chain from IGHD5 together with IGHJ4; Rodents express more antibody chains from either IGHD5 together with IGHJ4 or from other combinations of canine IGHD and IGHJ segments than from expression of other combinations of canine IGHD and IGHJ segments; Rodents express more chimeric antibody light chains from IGLJ1 than from any other IGL J gene segment.

[0210] In one aspect, the rodent comprises the following inserted canine heavy chain gene segments: J1-6, D1-6, V4-1, V3-2, V3-3, and V3-5, optionally together with V3-4 (which is a pseudogene).

[0211] In one aspect, the rodent comprises the following inserted canine lambda light chain gene segments: J1-9 with C1-9 (i.e., the complete canine lambda JC cluster), and V3-2, V3-3, V3-4, V4-5, V4-6, and optionally V3-1 and V3-7 (both of which are pseudogenes).

[0212] The rodents of the invention can be appropriately modified by N and / or P addition to the inserted companion animal V, D, and J gene segments, and / or can undergo somatic hypermutation of the inserted companion animal V, D, and J gene segments.

[0213] The invention also provides an HCDR3, VH domain, antibody heavy chain, or antibody, wherein the VH domain of the heavy chain or antibody comprises rodent AID pattern somatic hypermutation and / or mouse dTd pattern mutation. This pattern can be provided, for example, when the VH domain is produced in a rodent that comprises rodent AID and / or rodent TdT (e.g., endogenous AID or TdT). The mouse is a preferred rodent.

[0214] In one aspect of the invention, the variable region of the chimeric antibody chain is different from the amino acid sequence predicted from the germline sequences of the V, D, and J gene segments used to generate the antibody. Thus, there was a certain degree of somatic hypermutation and / or N / P addition.

[0215] Surprisingly, when both samples were surveyed at the same depth using the RNA sequencing approach of Example 4, the inventors found that the diversity of the chimeric antibody population generated using rodents containing the V, D, and J gene segments of canids, or the V and J segments described herein, was greater than the observed antibody diversity of the wild-type canids from which the gene segments were derived. FIG. 21 shows that the antibody nucleotide sequences found only once within the sequenced libraries of rodents containing the heavy chain V, D, and J gene segments, or the light chain V and J segments of canids, are far more numerous than those found in the canids themselves.

[0216] Accordingly, the present invention relates to any rodent described herein that expresses a population of chimeric antibody chains resulting from the expression of the gene segments of at least one companion animal, the population being more diverse or at least as diverse as the population of antibodies found in the corresponding companion animal. In other words, the diversity of the antibody population generated in the rodent is greater than or at least as great as that found in the antibody repertoire of the companion animal.

[0217] Preferably, the diversity of the antibody population generated in a mouse having the V, D, and J gene segments, or the light chain V and J segments, of the DNA of a canid is greater than or at least as great as that found in the antibody repertoire of the canid.

[0218] Suitably, the diversity of the population is evaluated by the number of unique antibody sequences present in the heavy chain population or the light chain population, or both.

[0219] The present invention also relates to a population of chimeric antibody chains that are at least 65%, or at least 70% unique, with respect to the sequences as determined by 5'RACE, for example in Example 4, for example a rodent comprising a population of chimeric antibody chains, such as a rodent in which 65 - 80% of the chimeric antibody sequences are unique, such as a rodent in which 65 - 75% are unique.

[0220] The rodent can be any rodent disclosed herein. For example, in one aspect, the rodent comprises inserted canine heavy chain gene segments such as J1 - 6, D1 - 6, V4 - 1, V3 - 2, V3 - 3, and V3 - 5, optionally together with V3 - 4 (a pseudogene). In one aspect, the rodent comprises inserted canine lambda light chain gene segments such as J1 - 9 (a complete canine lambda JC cluster) together with C1 - 9, and V3 - 2, V3 - 3, V3 - 4, V4 - 5, V4 - 6, and optionally V3 - 1 and V3 - 7 (both pseudogenes). In one aspect, the rodent comprises both a canine heavy chain insertion and a light chain insertion.

[0221] The present invention also relates to the use of any rodent (e.g., a mouse) comprising the DNA of a companion animal disclosed herein in the generation of a chimeric antibody chain or antibody repertoire that is more diverse than, or at least as diverse as, that found in the companion animal itself, and to a rodent such as a mouse comprising the DNA of a companion animal disclosed herein for generating a chimeric antibody chain or antibody repertoire that is more diverse than, or at least as diverse as, that found in the companion animal itself.

[0222] Accordingly, preferred embodiments of the present invention are as follows.

[0223] A method for generating an antibody or an antibody chain, comprising immunizing a rodent disclosed herein with an antigen obtained from or obtainable from a companion animal that is the same as the source of the companion animal DNA present in the rodent genome, wherein the antigen can be a protein antigen, a cell expressing the antigen, or a nucleic acid encoding the antigen.

[0224] A method for generating an antibody or an antibody chain, comprising immunizing a rodent disclosed herein with an antigen derived from a pathogen such as a bacterium or a virus that infects a companion animal species which is the source of the companion animal DNA present in the rodent genome.

[0225] A rodent disclosed herein that has been immunized with an antigen derived from a companion animal corresponding to the source of the companion animal DNA present in the rodent genome.

[0226] A rodent disclosed herein that has been immunized with an antigen that causes a disease in a companion animal corresponding to the source of the companion animal DNA present in the rodent genome.

[0227] A rodent disclosed herein that has been immunized with a companion animal antigen equivalent of a human antigen associated with a human disease.

[0228] A rodent disclosed herein, wherein the rodent contains companion animal DNA derived from a dog and has one or more or all of the following characteristics: The rodent expresses a chimeric antibody heavy chain from IGHJ4, individually, more than any of IGHJ1, 2, 3, 5, or 6; The rodent expresses a chimeric antibody heavy chain from IGHJ6, individually, more than any of IGHJ1, 2, 3, or 5; The rodent expresses a chimeric antibody heavy chain from IGHD5, individually, more than any of IGHD1, 2, 3, 4, or 6; The rodent expresses a chimeric antibody heavy chain from IGHD2, individually, more than from any of IGHD1, 3, 4, or 6; The rodent expresses an antibody chain from IGHD2 together with IGHJ4; The rodent expresses an antibody chain from IGHD5 together with IGHJ4; The rodent expresses more antibody chains from IGHD5 together with IGHJ4 than from any other combination of canine IGHD and IghJ segments; The rodent expresses a chimeric antibody light chain from IGLJ1 more than from any other IGL J gene segment.

[0229] A rodent disclosed herein, comprising the following inserted canine heavy chain gene segments: J1-6, D1-6, V4-1, V3-2, V3-3, and V3-5, optionally together with V3-4.

[0230] A rodent disclosed herein, comprising the following inserted canine lambda light chain gene segments: J1-9, C1-9, V3-2, V3-3, V3-4, V4-5, V4-6, and optionally, V3-1 and V3-7.

[0231] A rodent disclosed herein, wherein the inserted companion animal V, D, and J gene segments can be modified by N and / or P addition and / or exhibit somatic hypermutation.

[0232] A rodent disclosed herein, expressing a population of chimeric antibody chains, wherein each antibody chain results from the expression of at least one companion animal gene segment, and wherein the chimeric antibody population is more diverse than the antibody population found in the corresponding wild-type companion animal.

[0233] A rodent disclosed herein, comprising a population of chimeric antibody chains, at least 65%, or at least 70% of which are unique, and optionally, wherein the rodent is a dog.

[0234] A rodent disclosed herein, such as a rodent in which 65-75% is unique, and 65-80% of the chimeric antibody sequence is unique.

[0235] Use of any rodent (e.g., mouse) containing the DNA of a companion animal disclosed herein in the generation of a more diverse repertoire of chimeric antibody chains or antibodies than is found in the companion animal itself.

[0236] A rodent disclosed herein, such as a mouse, for generating a more diverse repertoire of chimeric antibody chains or antibodies than is found in the companion animal itself.

[0237] A rodent disclosed herein, wherein one or more of the inserted companion animal V, D, or J gene segments are accompanied by control sequences from the same companion animal, and optionally the control sequences include a promoter preceding each V gene segment, and / or a splice site, and / or a recombination signal sequence for V(D)J recombination.

[0238] A rodent disclosed herein, wherein an RSS sequence from the same companion animal is adjacent to the inserted V, D, or J sequence of the companion animal, and the RSS sequence of the host rodent is not used.

[0239] A rodent disclosed herein, containing the DNA of a certain breed of Canidae animal, for use in the production of an antibody or antibody chain, or a portion thereof, for use in the treatment or prevention of diseases in different dog breeds.

[0240] Use of a rodent disclosed herein in the production of an antibody chain or a portion thereof for use in the treatment of different dog breeds, containing the DNA of a certain breed of Canidae animal.

[0241] A rodent disclosed herein, wherein one or more or all of the V, D, and J segments of Canidae animals are from Boxer dogs.

[0242] In another aspect, the DNA of the companion animal inserted into the rodent genome does not contain a non-immunoglobulin gene or does not contain a functional non-immunoglobulin gene (the latter allowing for the inclusion of non-functional non-immunoglobulin genes). If non-immunoglobulin genes are located within the genome of the companion animal, they can be excised prior to insertion. Accordingly, the present invention relates to a rodent genome having the inserted companion animal DNA described herein, but not containing a non-immunoglobulin gene of the companion animal or not containing a functional non-immunoglobulin gene of the companion animal.

[0243] For example, the inventors have identified an Ig locus of a canine animal that has three genes located within the lambda gene segment of a canine animal that does not encode an immunoglobulin gene, and thus is preferably excised prior to insertion and not inserted into the rodent genome. Accordingly, in one aspect, the herein-disclosed canine-rodent contains none, or any one, or two, or three, or all of the following canine genes: ZNF280B, PRAME, RPIA, and PCBP2. Excision of non-immunoglobulin genes prior to insertion reduces the size of the insertion necessary to achieve insertion of the desired IGL lambda V gene. As will be understood by those skilled in the art, this facilitates the insertion and reduces the likelihood that non-IG genes will cause any unwanted interference in the rodent or rodent cells. Accordingly, in one aspect, the inserted companion animal DNA may include an "edited" genomic clone that is not a genomic clone but has one or more genes deleted. In another aspect, the present invention relates to any of the methods disclosed herein for generating a transgenic rodent, wherein the DNA inserted into the rodent does not contain a non-immunoglobulin gene, for example, does not contain any one or more of ZNF280B, PRAME, RPIA, and PCBP2.

[0244] In another aspect, the invention relates to a rodent or cell disclosed herein that contains companion animal DNA and whose genome further includes knockout of one or more rodent genes. The invention also relates to a rodent having the inserted companion animal DNA described herein, whose genome further includes a gene knockout, and wherein the rodent is immunized with an antigen encoded by the knocked-out gene or by a homolog of that gene in the companion animal. Suitable homologs have at least 60% homology at the nucleic acid or protein level, for example 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homology at the nucleic acid or protein level.

[0245] Accordingly, the invention also includes a method for producing antibodies against a specific antigen of interest, the method comprising immunizing a rodent that contains a genome having the inserted companion animal gene disclosed herein and that includes a knockout of the gene encoding the antigen of interest or a homolog of that gene in the companion animal.

[0246] The present invention also relates to a rodent or rodent cell disclosed herein that expresses a multispecific antibody or antibody chain, such as a bispecific antibody or antibody chain. Methods and various modes for making bispecific antibodies are incorporated herein by reference with respect to different methods for generating bispecific antibodies and functional portions thereof, "The making of bispecific antibodies", Brinkmann et al., MAbs. February - March 2017; 9(2):182 - 212. Multispecific antibodies may be in any of the modes described therein, such as in a form with low Fc - bispecificity, in the form of tandem single - chain variable fragments (scFv2, taFv) and triple - bodies, in the form of diabodies and diabody derivatives, in the form of Fab fusion proteins. Multispecific antibodies may include additional antigen - binding sites grafted onto scFv, may be asymmetric IgGs having heavy and light chains from two different antibodies, may be bispecific IgGs having an asymmetric Fc region, may be added IgGs or modified IgGs, or may be bispecific antibodies based on symmetric Fc and CH3 as also described by Brinkmann et al.

[0247] The rodent or rodent cell of the present invention may comprise a single unrearranged companion animal light - chain variable region gene segment (or two companion animal light - chain variable region hereditary segments) that is rearranged to form a rearranged companion animal light - chain variable region gene (or rearranged light - chain variable region genes) that expresses a single light chain (or either or both of two light chains). The rearranged companion animal light - chain variable domain can pair with a plurality of affinity - matured companion animal heavy chains selected by the companion animal, and the heavy - chain variable regions specifically bind to different epitopes. The light chain may be a kappa chain or a lambda chain.

[0248] For example, the present invention relates to the following. A rodent or rodent cell, wherein (a) Replacement by a single companion animal light chain VJ gene segment, or by two companion animal light chain VJ gene segments, at the endogenous rodent κ immunoglobulin light chain variable region locus of all or substantially all endogenous rodent κ immunoglobulin light chain variable region gene segments, wherein the gene segments of each companion animal are operably linked to the endogenous rodent light chain constant gene; and (b) Replacement of the locus of a plurality of companion animal heavy chain variable region gene segments of a part or all or substantially all endogenous rodent heavy chain variable region genes, wherein the companion animal heavy chain variable region gene segments are operably linked to the endogenous rodent heavy chain constant region, and the companion animal heavy chain variable region gene segments are capable of reconstructing and forming a reconstructed companion animal / rodent chimeric heavy chain gene A rodent or rodent cell comprising

[0249] Substantially all replacement preferably means 90% or more, for example, 95% or more of each rodent locus is replaced. The replacement can be a functional replacement in that the relevant rodent locus can be inactivated such that no rodent antibody chain, or less than 10% of the rodent chain, is expressed from the locus, preferably less than 1% expression from the inactivated locus.

[0250] In one aspect, the rodent lacks the endogenous rodent κ and / or lambda immunoglobulin light chain variable region locus capable of reconstructing and forming genes encoding the κ and / or lambda variable regions of the rodent, respectively, or lacks a portion therefor suitable for preventing the formation of the κ and / or lambda variable regions of the rodent, respectively, or contains a mutation (e.g., deletion or insertion or replacement) suitable for preventing the formation of the κ and / or lambda variable regions of the rodent, respectively.

[0251] The present invention relates to companion animals as well as livestock. Any disclosure or aspect of the present invention disclosed herein regarding the DNA of companion animals can be similarly construed to refer to the DNA of livestock. Livestock includes, for example, livestock animals raised in an agricultural environment for the production of labor and commodities such as meat, eggs, milk, fur, leather, and wool. Examples of such animals include, but are not limited to, cows, goats, pigs, deer, and sheep.

[0252] In addition, the present invention similarly relates to the use of DNA derived from poultry, including chickens, turkeys, and other domesticated birds. All aspects described herein regarding the DNA of companion animals are similarly applicable to such poultry.

[0253] Kappa CDR3 of Canidae animals In a further aspect of the present invention, the inventors have identified that the IgK CDR3 sequences of certain Canidae animals are expressed more strongly than others. The three most preferred sequences together constitute 27.55% of all sequences and are each 9 amino acids in length (excluding conserved amino acids adjacent to CDR3). For clarity and to avoid doubt, with reference to the unique numbering system of IMGT (Lefranc, M.-P., Immunology Today, Vol. 18, p. 509 (1997) PMID: 9386342), the first amino acid corresponds to position 105, and finally, in this case, the ninth amino acid corresponds to position 117.

[0254] Data on the expression of kappa light chain CDR3 of Canidae animals in dogs are as follows.

[0255] [Table 1]

[0256] Thus, it encodes or expresses 9 amino acids of the kappa CDR3 region of a canine animal (excluding conserved amino acids adjacent to CDR3), preferably in a rodent or rodent cell (e.g., a rodent B cell, or a cell expressing an antibody or antibody chain disclosed herein). For clarity and to avoid doubt, referring to the unique numbering system of IMGT (Lefranc, M.-P., Immunology Today, 18, 509 (1997) PMID: 9386342), the first amino acid corresponds to position 105, and finally, in this case, the 9th amino acid corresponds to position 117.

[0257] Preferably, the rodent or rodent cell (e.g., a rodent B cell, or an expression cell expressing an antibody or antibody chain) of the present invention containing canine IgK DNA encodes and / or expresses a kappa CDR3 region of a canine animal that comprises or consists of a sequence selected from one of the following:

[0258] [Table 2]

[0259] In one aspect, the CDR3 sequence encoded or expressed comprises or consists of QQSLHFPPT.

[0260] In one aspect, the CDR3 sequence encoded or expressed comprises or consists of QQSLHLPPT.

[0261] In one aspect, the CDR3 sequence encoded or expressed comprises or consists of GQGTHSPTT.

[0262] QQSLHFPPT and QQSLHLPPT of CDR3 are preferred.

[0263] In one aspect, the encoded or expressed CDR3 sequence does not contain QQSLHFPPT or does not consist of QQSLHFPPT. In particular, when in the form of an antibody of a complete canid animal within the natural repertoire or when purified from that repertoire, it is not QQSLHFPPT. In one aspect, the encoded or expressed CDR3 sequence is not in the form disclosed in WO 2012 / 024650. For the avoidance of doubt, the QQSLHFPPT of the encoded or expressed CDR3 may be in the kappa chain which is part of the pharmaceutical composition, or may be a fragment of the full-length kappa chain, or may be a combination such as a bispecific antibody format.

[0264] In another aspect, a rodent or rodent cell comprising canid animal IgK DNA encodes and / or expresses a canid animal kappa CDR3 region comprising a proline (P) residue at position 7. 96.68% of the CDR3 regions of the kappa light chain antibody chains of canid animals contain such proline.

[0265] In another aspect, a rodent or rodent cell comprising canid animal IgK DNA encodes and / or expresses a canid animal kappa CDR3 region comprising a glutamine (Q) residue at position 2. 97.75% of the CDR3 regions of the kappa light chain antibody chains of canid animals contain such glutamine.

[0266] In another aspect, a rodent or rodent cell comprising canid animal IgK DNA encodes and / or expresses a canid animal kappa CDR3 region comprising a threonine (T) residue at position 9. 97.10% of the CDR3 regions of the kappa light chain antibody chains of canid animals contain such threonine.

[0267] In one aspect, the rodent genome can be reconstructed to express only the light chain variable region gene of a single reconstructed companion animal, or either of two such light chains, with only the kappa light chain variable region gene segments of one or two canid animals, and the canid kappa DNA inserted into the genome contains, consists of, or encodes and / or expresses an antibody chain containing a CDR3 having one, two, or all of the preferred amino acids P, Q, or T mentioned above at specific positions.

[0268] In one aspect, the CDR3 is not any one or more of the following.

[0269]

Table 3A

[0270]

Table 3B

[0271] Lambda CDR3 In a further aspect of the invention, the inventors have identified that certain canid Ig lambda CDR3 sequences are expressed more strongly than others in the natural canid repertoire. These sequences are typically 11 amino acids long (excluding conserved amino acids adjacent to CDR3). For clarity and to avoid doubt, with reference to the unique numbering system of IMGT (Lefranc, M.-P., Immunology Today, 18, 509 (1997) PMID: 9386342), the first amino acid corresponds to position 105, and finally, in this case, the 11th amino acid corresponds to position 117.

[0272] Data on canid lambda CDR3 expression in dogs are shown below.

[0273]

Table 4

[0274] For preferred residues, they relate to the classes defined in Pommie, C. et al., J. Mol. Recognit., Vol. 17, pp. 17-32 (2004). PMID: 14872534. In one aspect, they are classified based on hydropathy, and the residues are hydrophobic (A, C, I, L, M, F, W, V), or neutral hydropathy (G, H, P, S, T, Y), or hydrophilic (R, N, D, Q, E, K). In another aspect, they are classified based on volume, and the residues are very small (A, G, S), small (N, D, C, P, T), intermediate (Q, E, H, V), large (R, I, L, K, M), or very large (F, W, Y). In another aspect, they are classified based on their chemical properties, and the residues are aliphatic (A, G, I, L, P, V), aromatic (F, W, Y), sulfur-containing (C, M), hydroxyl (S, T), basic (R, H, K), acidic (D, E), or amide (N, Q). In another aspect, they are classified based on their charge, and the residues are positively charged (R, H, K), negatively charged (D, E), or uncharged (A, N, C, Q, G, I, L, M, F, P, S, T, W, Y, V). In another aspect, they are classified based on whether they can donate and / or accept hydrogen bonds, and the residues are donors (R, K, W), acceptors (D, E), both donors and acceptors (N, Q, H, S, T, Y), or neither donors nor acceptors (A, C, G, I, L, M, F, P, V). In another aspect, they are classified based on their polarity, and they are polar (R, N, D, Q, E, H, K, S, T, Y), or nonpolar (A, C, G, I, L, M, F, P, W, V).

[0275] Position 1 In another aspect, a rodent or rodent cell containing canine lambda DNA encodes and / or expresses a canine lambda CDR3 region containing a polar residue at position 1.

[0276] In another aspect, a rodent or rodent cell comprising a lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains an uncharged residue at position 1.

[0277] In another aspect, a rodent or rodent cell comprising a lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a residue with a neutral hydrophobicity state at position 1.

[0278] In another aspect, a rodent or rodent cell comprising a lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a residue that can both donate and accept a hydrogen bond at position 1.

[0279] In another aspect, a rodent or rodent cell comprising a lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a very small residue at position 1.

[0280] In another aspect, a rodent or rodent cell comprising a lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a hydroxyl residue at position 1.

[0281] In one aspect, the residue at position 1 is one of the preferred classes listed above. In another aspect, the residue at position 1 is two or more of the preferred classes listed above, for example, 2, 3, 4, 5, or 6 of the preferred classes.

[0282] In another aspect, a rodent or rodent cell comprising a lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a serine (S) residue at position 1.

[0283] Position 4 In another aspect, a rodent or rodent cell comprising a lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a polar residue at position 4.

[0284] In another aspect, a rodent or rodent cell comprising a lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a negatively charged residue at position 4.

[0285] In another aspect, a rodent or rodent cell comprising a lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a hydrophilic residue at position 4.

[0286] In another aspect, a rodent or rodent cell comprising a lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a residue capable of accepting a hydrogen bond at position 4.

[0287] In another aspect, a rodent or rodent cell comprising a lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a small residue at position 4.

[0288] In another aspect, a rodent or rodent cell comprising a lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains an acidic residue at position 4.

[0289] In one aspect, the residue at position 4 is one of the preferred classes listed above. In another aspect, the residue at position 4 is two or more of the preferred classes listed above, e.g., 2, 3, 4, 5, or 6 of the preferred classes.

[0290] In another aspect, a rodent or rodent cell comprising a lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains an aspartic acid (D) residue at position 4.

[0291] Position 6 In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a polar residue at position 6.

[0292] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains an uncharged residue at position 6.

[0293] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a residue with a neutral hydropathy state at position 6.

[0294] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a residue that can both donate and accept a hydrogen bond at position 6.

[0295] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a very small residue at position 6.

[0296] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a hydroxyl residue at position 6.

[0297] In one aspect, the residue at position 6 is one of the preferred classes listed above. In another aspect, the residue at position 6 is two or more of the preferred classes listed above, e.g., 2, 3, 4, 5, or 6 of the preferred classes.

[0298] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a serine (S) residue at position 6.

[0299] Position 7 In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a nonpolar residue at position 7.

[0300] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains an uncharged residue at position 7.

[0301] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a hydrophobic residue at position 7.

[0302] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a residue that can neither donate nor accept a hydrogen bond at position 7.

[0303] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a large residue at position 7.

[0304] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains an aliphatic residue at position 7.

[0305] In one aspect, the residue at position 7 is one of the preferred classes listed above. In another aspect, the residue at position 7 is two or more of the preferred classes listed above, e.g., 2, 3, 4, 5, or 6 of the preferred classes.

[0306] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a leucine (L) residue at position 7.

[0307] Position 11 In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a nonpolar residue at position 11.

[0308] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains an uncharged residue at position 11.

[0309] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a hydrophobic residue at position 11.

[0310] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a residue that can neither donate nor accept a hydrogen bond at position 11.

[0311] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains a residue of intermediate size at position 11.

[0312] In another aspect, a rodent or rodent cell comprising lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal that contains an aliphatic residue at position 11.

[0313] In one aspect, the residue at position 11 is one of the preferred classes listed above. In another aspect, the residue at position 11 is two or more of the preferred classes listed above, e.g., 2, 3, 4, 5, or 6 of the preferred classes.

[0314] Preferred embodiments are as follows. A rodent or rodent cell containing lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal containing an aspartic acid (D) residue at position 4.

[0315] A rodent or rodent cell containing lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal containing a leucine (L) residue at position 7.

[0316] A rodent or rodent cell containing lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal containing a serine (S) at position 1.

[0317] A rodent or rodent cell containing lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal containing a serine (S) at position 6.

[0318] A rodent or rodent cell containing lambda DNA of a canine animal encodes and / or expresses a lambda CDR3 region of a canine animal containing a valine (V) at position 11.

[0319] In one embodiment, the rodent genome of the present invention contains only a single rearranged companion animal light chain variable region gene, or a single or two lambda light chain variable region gene segments of a canine animal rearranged to express either of the two such light chains, and the lambda DNA of the canine animal inserted into the genome encodes and / or expresses an antibody chain containing a CDR3 having one or more of the preferred amino acids mentioned above in the lambda CDR3 region, for example, 2, 3, 4, or 5 of all the preferred amino acids.

[0320] In one embodiment, the CDR3 is not any of the following.

[0321] [Table 5]

[0322] Canine lambda repertoire expressed in mice In another aspect, the inventors have identified that when expressed from the mouse genome, certain amino acids are expressed in the CDR3 of the canine lambda antibody chain.

[0323] In another aspect, a rodent or rodent cell containing canine lambda DNA encodes and / or expresses a canine lambda CDR3 region comprising one or more or all of the following A at position 7 or 9; D at position 4; Q at position 1; K at position 8; S at position 5 and / or 6; V at position 2 and / or 11; and W at position 3 encoding and / or expressing a canine lambda CDR3 region comprising one or more or all of the above.

[0324] Data on canine lambda CDR3 expression in mice are as follows.

[0325] [Table 6]

[0326] In one aspect, the rodent genome of the invention is reconstituted to express either a single reconstituted companion animal light chain variable region gene, or either of two such light chains, and contains only a single or two canine lambda light chain variable region gene segments, and the canine lambda DNA inserted into the genome encodes and / or expresses an antibody chain comprising a CDR3 having one or more of the preferred amino acids mentioned above in the lambda CDR3 region, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the preferred amino acids mentioned above.

[0327] Also, a rodent and rodent cells having a genome containing an amino acid having an occurrence frequency greater than 90% at a preferably defined position are preferred, and those having an occurrence frequency greater than 95%, for example V11, are more preferred.

[0328] In addition, in the antibody population of Canidae animals found in dogs and the antibody population of Canidae animals in mice, rodents and rodent cells having a genome containing the above-mentioned preferred amino acids, for example, serine at position 6, are preferred.

[0329] Regarding the lambda light chain of Canidae animals, the present invention relates to a lambda light chain or antibody of a Canidae animal containing such a chain; a pharmaceutical composition containing such a chain or antibody; an active antibody fragment or derivative such as a Fab or domain antibody or bispecific antibody containing any type of fragment or derivative described herein; any one of the above, wherein the CDR3 of the lambda light chain (or antibody or pharmaceutical composition or antibody fragment or derivative) of a Canidae animal contains any of the above-mentioned preferred amino acids or classes for rodents or rodent cells. The present invention also relates to any of these for use in medicine, for example, for the treatment or prevention of canine diseases, and the use of any of the above in the preparation of a medicament for the prevention or treatment of canine diseases. In another aspect, the CDR3 contains preferably 2, 3, 4 or 5 of the preferred positions (considering the preference of the repertoire of natural Canidae animals), or preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the preferred amino acid positions (when considering the preference of the CDR3 of Canidae animals as expressed in mice), of a preferred amino acid class or a preferred amino acid.

[0330] In one aspect, the CDR3 contains a preferred amino acid or amino acid class at two of the preferred positions. In one aspect, the CDR3 contains a preferred amino acid or amino acid class at three or more of the preferred positions.

[0331] In one aspect, the amino acid positions of each CDR3 include amino acids that are in at least two or at least three or more preferred classes for that amino acid position.

[0332] The observed very high degree of conservation of both the class of amino acids and / or specific amino acids at the positions of the lambda CDR3 of these canid animals indicates that the biological preference for antibodies having these sequences is very relevant to the production and use of a lambda light chain of a suitable canid animal for use as a canine agent. This has not been understood heretofore.

[0333] In particular, all canine agents heretofore have had a kappa light chain. The present invention enables the provision of a lambda light chain of a canid animal and an antibody having such a light chain as an agent.

[0334] In another aspect, the present invention relates to a rodent or rodent cell, such as a B cell, or any expression cell or cell line that expresses an antibody, and the antibody includes any of the above-described preferred CDR3 sequences, or a kappa light chain or a lambda light chain or both having the preferred CDR3 amino acids mentioned above. The light chain (either kappa or lambda or both) can be in the form of a complete canid animal antibody kappa or lambda light chain, or a chimeric kappa or lambda light chain having a reconstituted canid animal variable region and a rodent constant region derived from the host rodent cell. A complete canid animal kappa or lambda light chain may be accompanied by a chimeric (canid animal variable region, rodent constant region) heavy chain. The chimeric kappa or lambda light chain may be accompanied by a chimeric canid animal heavy chain, or a complete canid animal heavy chain having both a canid animal variable region and a canid animal constant region.

[0335] In another aspect, an antibody obtainable by or obtained by any of the methods described herein includes a light chain CDR3 having any of the preferred amino acids or amino acid sequences described above.

[0336] The numbering of the CDR3 regions in this specification is defined by referring to the IMGT system disclosed at http: / / www.imgt.org / IMGTScientificChart / Numbering / IMGTIGVLsuperfamily.html.

[0337] Additional aspects of the present invention include the following. An antibody of a Canidae animal having a heavy chain of a complete Canidae animal and a light chain of a complete Canidae animal, (i) the light chain is a lambda CDR3 region containing aspartic acid (D) at position 4, a lambda CDR3 region containing leucine (L) residue at position 7, a lambda CDR3 region containing serine (S) at position 1, a lambda CDR3 region containing serine (S) at position 6, a lambda CDR3 region containing valine (V) at position 11 and is a lambda light chain having one of them, or the lambda light chain contains 2, 3, 4 or 5 of the above-mentioned preferred amino acids at preferred positions, or the lambda light chain contains amino acids at the above-mentioned preferred positions having characteristics of two or more classes, or (ii) the light chain is a lambda CDR3 region containing An A at position 7 or 9, a lambda CDR3 region containing D at position 4, a lambda CDR3 region containing A K at position 8, a lambda CDR3 region containing An S at position 5 and / or 6, a lambda CDR3 region containing A V at position 2 and / or 11, and a lambda CDR3 region containing W at position 3, and is a lambda light chain having one of them, or the lambda light chain contains 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the above-mentioned preferred amino acids at preferred positions, In particular, the amino acid is S at position 6 of lambda of a Canidae animal, or (iii) the light chain is a kappa CDR3 region of a Canidae animal containing proline (P) at position 7, a kappa CDR3 region of a Canidae animal containing glutamine (Q) at position 2, a kappa CDR3 region of a Canidae animal containing threonine (T) at position 9, a kappa light chain having one of them, or the kappa light chain contains, for example, two or three of the above-mentioned preferred amino acids at preferred positions, or the light chain is a kappa light chain having a CDR3 selected from QQSLHFPPT, QQSLHLPPT or GQGTHSPTT, optionally, the antibody has a kappa CDR3 sequence

[0338]

Table 7A

[0339]

Table 7B

[0340] and does not contain any of antibodies of Canidae animals.

[0341] In addition, the present invention relates to the following.

[0342] A pharmaceutical composition comprising any of the antibodies of the Canidae animals in a suitable combination with a pharmaceutical diluent, carrier or excipient.

[0343] An antibody or pharmaceutical composition of a Canidae animal disclosed herein for use in the treatment or prevention of diseases in dogs, particularly in dogs, wherein in one aspect the dog is not a boxer.

[0344] Use of an antibody or pharmaceutical composition of a canid animal disclosed herein in the preparation of a medicament for the treatment or prevention of diseases in dogs.

[0345] A method for the treatment or prevention of a disease in a dog, the method comprising the step of delivering to the dog in need thereof an antibody comprising a lambda light chain, preferably the lambda CDR3 region described in the present invention.

[0346] The inventors have surprisingly determined that the above - mentioned preferred CDR3 amino acids and CDR3 sequences occur very highly in the population of antibodies of canid animals. This very strong preference for specific amino acid residues was not predictable.

[0347] In one aspect, the antibody does not comprise any of the following lambda CDR3 sequences

[0348]

Table 8

[0349] thereof.

[0350] A preferred method is a method for obtaining an antibody of a complete companion animal, for example, an antibody of a complete canid animal, the method comprising expressing an antibody comprising at least the variable region of the companion animal using any of the methods described herein, and as a final step, formulating the antibody of the complete companion animal with an acceptable pharmaceutical excipient to form a pharmaceutical composition, and then, optionally, packaging the antibody. The present invention also relates to the use of an antibody or composition in a suitable mammal, for example, a cognate companion animal.

[0351] References to antibodies herein may also refer to antibody chains or any biologically active antibody fragments disclosed herein.

[0352] References to "antibodies of canid animals" in this specification include, for example, the amino acid sequence of the constant region of a naturally occurring canid animal, or a functional portion thereof having one or more effector functions, linked to the amino acid sequence of the variable region of an antibody that can be naturally expressed in a dog or can be expressed by recombination of the VDJ or VJ region genes of a canid animal after insertion into the genome of a rodent such as a mouse. Thus, it will be understood that the antibodies of canid animals of the present invention can be obtained from the recombination of the V, (D) and J region genes of canid animals, but the origin of the antibodies is not limited.

[0353] Certain preferred embodiments of the present invention are reflected in the following description. 1. i) One or more companion animal IGH V region genes, one or more companion animal D region genes, and one or more companion animal J region genes, and ii) Optionally, one or more companion animal IGL kappa V region genes, and one or more companion animal IGL kappa J region genes; and / or one or more companion animal IGL lambda V region genes, and one or more companion animal IGL lambda J region genes and A rodent or rodent cell having a genome comprising The rodent or rodent cell is capable of expressing the variable region genes of the companion animal to form antibody chains, The companion animal species is not a rodent, The rodent or rodent cell. 2. i) One or more companion animal IGL kappa V region genes, and one or more companion animal IGL kappa J region genes; and / or one or more companion animal IGL lambda V region genes, and one or more companion animal IGL lambda J region genes, and ii) Optionally, one or more companion animal IGH V region genes, one or more companion animal or host D region genes, and one or more companion animal or host J region genes and A rodent or rodent cell having a genome comprising the rodent or rodent cell is capable of expressing a variable region gene of a companion animal to form an antibody chain, wherein the companion animal species is not a rodent, the rodent or rodent cell. 3. The rodent or rodent cell according to claim 1 or 2, wherein one or more of the inserted V, D, or J region genes of the companion animal are accompanied by a control sequence from the same companion animal. 4. The rodent or rodent cell according to any one of claims 1 to 3, wherein the rodent genome contains genes of a companion animal derived from both the heavy chain and at least one light chain. 5. The rodent or rodent cell according to any one of claims 1 to 4, comprising at least 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, or at least 80 IGH V region genes of a companion animal, and optionally the V region genes are those of a canine animal. 6. The rodent or rodent cell according to any one of claims 1 to 5, comprising at least 4, 5, 10, 15, 16, 17, 18, or 19 IGL kappa V region genes of a companion animal, and optionally the V region genes are those of a canine animal. 7. The rodent or rodent cell according to any one of claims 1 to 6, comprising at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or at least 160 IGL lambda V region genes of a companion animal, and optionally the V region genes are those of a canine animal. 8. The gene of the companion animal is appropriately located upstream of the genomic constant region of the rodent, appropriately upstream of the heavy chain constant region for the companion animal heavy chain variable region gene to be inserted, and appropriately upstream of the light chain constant region for the companion animal light chain variable region gene to be inserted, whereby the rodent or rodent cell can generate a chimeric antibody chain resulting from the expression of the variable region gene to be inserted and the host constant region. The rodent or rodent cell according to any one of Items 1 to 7. 9. The gene of the companion animal is located in the genome in a functional arrangement together with the constant region derived from the same companion animal, whereby the rodent can produce an antibody chain resulting from the expression of the VDJ or VJ region gene of the companion animal to be inserted and the constant region of the companion animal. Optionally, the gene of the companion animal is a lambda light chain V and J gene in a functional arrangement together with the lambda constant region derived from the same companion animal. The rodent or rodent cell according to any one of Items 1 to 7 having a genome. 10. One or more IGL lambda V region genes of the companion animal, one or more IGL lambda J region genes of the companion animal, and one or more companion animal lambda constant region genes located at the kappa locus of the rodent cell, such as downstream of the kappa constant region of the rodent. The rodent or rodent cell according to any one of Items 1 to 9. 11. Containing the kappa variable region gene of the Canidae animal, all of the kappa variable region genes of the Canidae animal in the genome of the rodent are located upstream of the constant region with which the variable region gene is expressed together, and optionally located upstream of the host kappa constant region. The rodent or rodent cell according to any one of Items 1 to 10. 12. Whether all of the companion's gene segments correspond to the reference alleles of Canidae animals in CanFam 3.1, or the companion's gene segments contain non-reference alleles from one or more of the following gene segments: IGKV2-S13, IGLV 1-57, IGLV 1-68, IGLV 1-72, IGLV 1-88, IGLV 1-96, IGLV 8-60, IGLV 8-90, and IGLV 8-120. A rodent or rodent cell according to any one of items 1 to 11. 13. The rodent is a mouse, and the companion animal is a dog, or a cat, or a horse, preferably a dog. A rodent or rodent cell according to any one of items 1 to 12. 14. A method for producing a rodent or rodent cell according to any one of items 1 to 13, comprising the step of inserting one or more IGH V region genes of a companion animal, one or more IGH D region genes of a companion animal, and one or more IGH J region genes of a companion animal into the rodent cell genome, wherein the rodent or rodent cell can express the variable region gene of the companion animal in combination with the constant region to form an antibody chain. 15. A method for producing a rodent or rodent cell according to item 14, comprising the step of inserting one or more IGL V region genes of a companion animal and one or more IGL region J genes of a companion animal into the rodent cell genome, wherein the rodent or rodent cell can express the variable region gene of the companion animal in combination with the constant region to form an antibody chain. 16. A method for producing an antibody chain specific to a desired antigen, comprising the step of immunizing a rodent according to any one of items 1 to 13 with the desired antigen, and the step of recovering the antibody chain alone or as part of a complete antibody, or recovering the cells that produce the antibody chain alone or as part of a complete antibody. 17. A method for producing an antibody chain or an antibody that is specific for a desired antigen and is derived from a single species of companion animal, the method comprising: immunizing a rodent comprising the gene of the companion as defined in any one of claims 1 to 13; and then appropriately manipulating the nucleic acid encoding the antibody so as to replace the constant region of the antibody chain of the rodent with the constant region of the companion animal derived from the same companion animal. 18. A method for producing an antibody chain or a part thereof having the variable region of a companion animal, the method comprising the step of expressing, in a cell, DNA encoding the antibody chain or a part thereof, wherein the sequence of the DNA encoding the variable region of the antibody chain is obtained from or can be obtained by immunizing the rodent as defined in any one of claims 1 to 13 with an antigen so that the antibody chain is produced, and optionally, the method further comprises the following: (i) a step of purifying and / or isolating the antigen receptor chain; and (ii) optionally, a step of formulating the antigen receptor chain into a pharmaceutically acceptable formulation suitable for administration to a companion animal. 19. An antibody or antibody chain, or a part thereof, or DNA encoding an antibody chain or a part thereof, which is obtained from or can be obtained from the rodent or rodent cell as defined in any one of claims 1 to 13, or from the method as defined in any one of claims 16 to 18. 20. An antibody or antibody chain, or a part thereof, which is obtained from or can be obtained from the rodent or cell as defined in any one of claims 1 to 13 for use in the treatment or prevention of a disease of a companion animal in need thereof. 21. A method for treating a companion animal, the method comprising delivering to a companion animal in need thereof an antibody or antibody chain, or a part thereof, which is obtained from or can be obtained from the rodent or cell as defined in any one of claims 1 to 13, or from the method as defined in any one of claims 16 to 18. 22. A method for replacing all or part of an endogenous immunoglobulin variable region locus in a rodent or rodent cell with the locus and control sequences of a homologous or orthologous companion animal, comprising: i) obtaining a cloned genomic fragment or synthetic sequence comprising all or part of the locus and control sequences of a homologous or orthologous companion animal; ii) using homologous recombination to genetically modify the cloned genomic fragment of (i) to create a large targeting vector for use in a rodent or rodent cell; iii) introducing the vector of (ii) into a rodent or rodent cell to replace all or part of the endogenous immunoglobulin variable locus. A method comprising the steps above.

[0354] It will be understood that the specific embodiments described herein are presented by way of example and not as limitations of the present invention. The main features of the present invention can be used in various embodiments without departing from the scope of the present invention. Those skilled in the art will recognize or be able to ascertain many equivalents to the specific procedures described herein using nothing more than routine experimentation. Such equivalents are considered to be within the scope of the present invention and are included in the claims. All publications and patent applications mentioned in this specification are indicative of the state of the art of those skilled in the art to which the present invention pertains. All publications and patent applications are hereby incorporated by reference into this specification 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 terms "a" or "an" in conjunction with the term "comprising" in the claims and / or in this specification may mean "one" in some cases, but it also conforms to the meanings of "one or more", "at least one", and "one or more than one". The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer only to alternatives, or to support a definition that refers only to alternatives, or unless the alternatives are mutually exclusive. Throughout this application, the term "about" is used to indicate that a value includes the inherent variability of error of the device, i.e., the method employed to determine the value, or the variability that exists between the subjects being studied.

[0355] As used in this specification and the claims, 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.

[0356] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed prior to 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, where order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations including repetitions of one or more of the items or terms, such as BB, AAA, ABAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are explicitly included. One of ordinary skill in the art will understand that, unless otherwise apparent from the context, typically there is no limit on the number of items or terms in any given combination.

[0357] Any portion of the present disclosure can be read in combination with any other portion of the present disclosure, unless it is clear from the context that the contrary is intended.

[0358] All compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be made in the compositions and / or methods and in the sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0359] The invention is illustrated in more detail in the following non-limiting examples.

Examples

[0360] (Example 1) Annotation of the IG locus of canid animals Dogs are excellent models of human diseases. For example, the treatment of lymphoma in canids often predicts the human response to that treatment. However, their use is limited because their antigen receptor (AR) loci are only incompletely understood. This study advanced the annotation of the AR locus in canids, developed ways to investigate its evolutionary stress, and examined breed-specific features of the locus. Using a bioinformatics approach together with unbiased RNA sequencing, the annotation of the AR genes in canids was completed, and 107 whole-genome sequences from 19 breeds were queried using these sequences. A combination of existing and novel methods was used to analyze the diversity and mutation rates across these genes. Over 5,500 novel alleles were identified across approximately 550 gene segments of the AR locus (326 of which were newly annotated), insights into AR evolution were gained, and it was confirmed that there is higher conservation between dogs and humans than between either of them and mice. This study brought the understanding of dog AR genetics and expression to the same high level as that of mice and humans, making it only the third species to have all annotated AR loci. The numerous genome sequences will be useful for future research and allowed for statistically powerful conclusions to be drawn about the stresses that shaped these loci.

[0361] 1. Introduction In this study, the IGK and IGL of canids were annotated and the IGH locus was updated (Figs. 1–3). Using whole-genome sequence data from over 100 dogs, 5,000 non-reference alleles were identified, which shed light on the evolutionary stress that shaped these loci. Inter-species comparisons provided further insights and confirmed that dogs are a more faithful immune model.

[0362] 2. Materials and Methods 2.1.1 Annotation by Bioinformatics The loci were initially annotated using a principle equivalent to the algorithm of Olivieri et al. following a method similar to that of Das et al. 1、2. Briefly stated, the reference genome of Canidae animals (CanFam3.1) was investigated using human and mouse sequences. Once a region was identified, the AR gene was searched locally. Initially, mouse and human AR genes and RSS consensus were used, but as more Canidae genes were identified, these were used instead. Next, annotations were verified and added based on the alignment of RNA sequencing data.

[0363] 2.1.2 Dog Peripheral blood samples were secured from 26 dogs. The samples were unused clinical excesses of blood collection mandated by veterinarians from affected animals seen at the University of Cambridge Veterinary Hospital. This study had prior approval from the Ethics Committee of the University of Cambridge's Veterinary Medicine Department.

[0364] 2.1.3 Sequencing Mononuclear cells were isolated from peripheral blood using Ficoll-Paque (GE Healthcare) according to the manufacturer's instructions. The cells were processed into mRNA using polyA pull-down by the core sequencing team at the Wellcome Trust Sanger Institute, fragmented, and sequenced on a HiSeq 2500 machine (Illumina) using 250bp paired-end reads.

[0365] 2.1.4 Gene Naming The AR gene was divided into families and assigned as functional, pseudogene, or ORF using the same criteria as Bao et al. 3 Family numbers were assigned based on homology to human families, and new numbers were given if no obvious match was found. All gene names were assigned according to the naming system of IMGT. 4

[0366] 2.1.5 Non-reference Alleles 10 7Mapping of the variant call file to the AR locus from the whole genome sequences of individual Canidae animals was kindly provided by Steven Friedenberg of the University of Minnesota.

[0367] 2.1.6 Inter- and intra-species locus alignment The sequences were masked using RepeatMasker and alignment plots were generated using PipMaker 5、6 。

[0368] 2.1.7 Phylogenetic tree analysis The sequences were aligned using Clustal Omega and the output tree was visualized using Interactive Tree of Life 7、8 。

[0369] 3. Results 3.1.1 Gene numbers Within previously annotated loci, three novel IGHJ genes were identified. 162 IGLV genes across seven gene families were identified, with IGLV1 being the largest with 86 members. Consistent with other IGL loci, J and C genes were found as a total of nine pairs. 19 IGKV genes were identified, 14 of which were IGKV2, and simultaneously five IGKJ genes and one IGKC gene were identified.

[0370] 3.1.2 Non-reference alleles Whole genome sequences from 107 dogs of 19 breeds, all aligned to the current reference build (CanFam3.1), were investigated for novel AR alleles, and 4,074 were identified across three loci. Regarding the allele distribution, the reference allele was found 53,311 times (77%) out of the 68,908 alleles called. No significant breed specificity in allele distribution was identified.

[0371] Functional, pseudogene, or ORF assignments were made for all novel alleles and compared back to the reference alleles. Most novel alleles (72.8%) had the same function as the reference alleles. When considering the function of the novel alleles as the function of the alleles under selection, there were more alleles that were "loss" (23.8%), i.e., the novel alleles were pseudogenes rather than "gain" (3.4%) where the novel alleles were functional. This is not surprising as there are many more ways to lose a function than to gain one, and thus mutations are likely to bring about that change in direction. Changes from a function to an ORF were classified as loss, and changes from a pseudogene to an ORF were classified as gain.

[0372] Non-reference V alleles were called 13,129 times across all samples and loci. Interestingly, IGKV2-S13, and eight IGLV genes were only detected as non-reference alleles, including in boxer samples. This is probably an error in the reference genome, but it is also possible that the reference dog has nine rare alleles that just happened not to be represented in this dataset. Considering non-reference alleles, when there is no selection stress, the distribution of change types within the dataset is the same as the distribution within the alleles themselves. For example, since 3.4% of the alleles are "gain", 446 gain alleles are expected to be detected within the dataset.

[0373] However, loss changes were detected at a lower frequency than expected, and changes and gains were not detected more frequently than expected (Figure 7). Furthermore, the differences between breeds were very low, and more weight was placed on the selection stress of AR loci with limited breed dependence. A z-test was performed, and the difference between the population and the expected mean was highly significant, ranging from p = 2.34×10 -25 to p = 0 (the software used to calculate the values could not display p-values less than 1×10 -250 ).

[0374] 3.1.3 Locus Structure The IGH locus of canids is located at the subtelomere of chromosome 8 on the antisense strand. This telomeric location is observed in all mammals except monotremes and marsupials. 1 However, the light chain locus does not show strong conservation of chromosomal location among humans, mice, and dogs.

[0375] Similar to the IGH loci of humans and mice, the IGH locus of canids has all the functional gene segments that are transcribed in the same sense as the constant region, and one pseudogene with reverse transcription (Figure 1). The structure of the IGH locus is similar to that published by Bao et al. 3 However, three novel IGHJ genes have been identified, and although there are slight differences in the positions and predicted functions of the V genes, the absolute ratios and numbers are consistent. This discrepancy may be due to the use of different builds of the reference genome, and this annotation uses the latest publicly available build (CanFam3.1).

[0376] The IGK locus of canids is small (about 400 kbp) and has an unusual structure (Figure 2). There are 11 V genes upstream of the J and C genes, and all 8 C-distal genes of the IGKV2 family are functional and, unlike the 3 C-proximal genes, are in the same transcriptional orientation as the J and C genes. However, there are also 8 V genes downstream of other genes, and most of them are inverted relative to the other genes. This is reminiscent of the IGL locus of equids and has been found not to affect V gene usage.

[0377] Inversions and block duplications appear to be characteristic of the light chain locus, particularly the IGK locus. The IGK loci of humans, pigs, mice, horses, and dogs not only contain V genes with a transcriptional orientation opposite to that of the C genes, but the loci of dogs, humans, and pigs have undergone inverted duplications of the entire block. 10、11 In the pig and human loci, since the genes of the two blocks have not diverged sufficiently, some or all are known by the same names as their counterparts in the other block. 10、12 ​

[0378] The IGLV genes of humans, pigs, and mice maintain their transcriptional orientation along the locus, while those of canids do not. 13 。The large (2.6 Mbp) canid IGL locus contains many V genes inverted relative to the J-C cluster (Figure 3). The inversion appears to be under some locus-specific selective stress, as only 3 out of 116 of the most C-proximal IGLV genes, which are in the opposite transcriptional orientation to the J-C genes, and 26 out of the last 46 are inverted V genes. This increase is partly due to block duplications that occurred in such a way that the regions of IGLV1 and IGLV8, which show a very high level of sequence identity, are positionally conserved within the member repeat blocks.

[0379] From a broader perspective of trends, the canid IG locus is consistent with other published loci. In previously annotated IGHV, the gene-to-pseudogene ratio was approximately 1:1, which was found to be consistent with the canid IgH locus 1. 1 。However, the two light chain loci appear to be close to 5:1, which may reflect different tolerances for pseudogenes between the heavy and light chains. Furthermore, the gene number has been found to correlate with the biased chain usage in the light chains of most species studied so far. 14、15 。For example, the mouse IGL locus, which contains only 9 functional genes, reflects its use in only 5% of the expressed antibodies. 14 。The canid repertoire shows a similar bias, but conversely, 91% IGK chain usage has been reported, depending on the relative sizes of the two light chain repertoires of the various species. 15 。

[0380] 3.1.4 Inter- and Intra-Species Locus Alignments Aligning genomic regions to themselves and to equivalent regions of other species is an established method for gaining insights into evolution, and PipMaker is a common tool used for this purpose. 16、17、6、18The IGK, IGL, and TRA / D loci of Canidae animals were used to perform alignments against themselves and against the mouse and human loci, respectively.

[0381] In the IGK self-alignment, three comparisons are of note: the self-alignment of the upstream and downstream blocks (Figure 8), and their mutual alignment (Figure 8). Within the percentage identity plot (PIP), the lines indicate regions of identity, and thus the sequences aligned against themselves always have a solid line running along the diagonal. Solid lines off the main diagonal indicate a high likelihood of overlapping events, and gaps represent indels and other mutations accumulated after the overlap.

[0382] The multiple dashed diagonals in the upstream self-alignment are characteristic of blocks, similar to the block duplications of the three gene cassettes within the TCRB locus of Canidae animals, where in this case a single V and its adjacent sequences are locally duplicated multiple times. 16 In the self-alignment of the downstream block, the lines are shorter and sometimes vertical, meaning local inverted homology. Finally, the homology comparison between the upstream and downstream blocks revealed a good degree of homology with the IgKV2 gene upstream of the C gene, particularly IGKV2-S18 and IGKV-S19. This pattern should be seen when a single gene is duplicated multiple times upstream of the C gene, followed by block inverted duplication in a manner reminiscent of human IGK and equine IGL. Thus, this downstream block is likely to have a lower selective stress and, except for the less divergent IGKV2-S18 and IGKV-S19 genes, accumulates mutations and local inversions at a higher rate. While this is not the only possible explanation, it is consistent with the proposed explanations for similar features across other species and AR loci.

[0383] In the comparison of the IGL loci between dogs and humans, two things are striking. One is an almost seamless diagonal near the midpoint of the human sequence, and the other is a very high degree of homology characterized by numerous lines in or near the coding sequence (Figure 9a). Further analysis of the diagonal in the human / dog PIP revealed that it extends into regions of the human locus containing the non-AR genes ZNF280A, ZNF280B, and PRAME (Figure 9b). The sequences of these genes and most of the regions surrounding them are highly conserved between dogs and humans, suggesting that they may have similar functional importance for dogs as for humans. Specific comparisons indicate that the ZNF280B and PRAME genes are likely to function in dogs, but ZNF280A could not be reliably identified at this locus (data not shown). Further investigation identified another non-IGL gene, PCBP2, near ZNF280B.

[0384] The problem loci and genes are not always conserved, but non-AR genes scattered in the AR locus are a common feature among species. For example, the ADAM6 gene is found in the IGH locus between the human and mouse IGHV and IGHD genes, but the human ortholog is non-functional. In dogs, the ortholog has not been characterized, but there are two candidates. One is between IGHV3-4 and IGHV3-5, and the other is upstream of the entire IGH locus. Due to the limited research on the ADAM gene family in dogs, this potential ortholog has not been added to the IGH annotation but is maintained as a candidate for future research. At present, no other non-AR genes have been identified in the IGK or IGL loci of canids.

[0385] 3.1.5 Allele Distribution One prominent feature of the use of dogs as a model organism is the high degree of heterogeneity between breeds, while the within-breed homogeneity is very high, and breed formation is estimated to be responsible for a 35% loss in nucleotide diversity. 19The selection has been very strict, and recent studies have identified 22 homozygous blocks longer than 1 megabase in specific breeds, which the authors attribute to the selection stress imposed by breeders. 20 This is in contrast to the diversity seen in humans. Even in geographically isolated populations, the variation due to this isolation is 5 - 10%, whereas more than a quarter of the genomic variation in dogs is due to breed variation rather than individual variation. 21 。

[0386] Given the level of breed specificity in the genomics of Canidae animals, it was perhaps surprising that non-reference alleles did not appear to follow strong breed-specific haplotypes. The two most represented breeds in this sample were Boxer and Standard Poodle (22 and 20 dogs respectively), and these followed a similar pattern, i.e., a pattern where a small number of non-reference alleles were found relatively frequently in both breeds, and rarer alleles were found on a single chromosome in one dog of one breed. Less represented breeds followed a similar distribution, and considering the number of alleles that only appeared once, non-reference alleles typically seemed to be found in heterozygotes.

[0387] Larger breed-specific cohort sizes may reveal trends not apparent in this dataset, but currently, the selection stress forming the AR locus appears to be breed-independent and to prevail over the breed itself. The possibility of exceptions is in the case of evolutionary bottlenecks that increase the abundance of less common alleles. For example, IGLC1*01 (the reference allele) is found in 19 out of 44 boxer chromosomes sequenced and 1 out of 6 toy poodle chromosomes, but not in any other breed. 194 out of 217 alleles called across all breeds are represented by the other allele, IGLC1*02. It is possible that toy poodles were previously crossed with boxers or that there were sequencing errors and this is actually a boxer-specific allele, but a larger dataset remains necessary to ultimately answer these questions in some way.

[0388] Considering the distribution of non-reference alleles, these all match what is expected. The fact that changes are not seen more frequently than expected is likely because the gene in question is under selection stress to maintain its current state and thus deviations from it in the future are unfavorable. This could be either the loss of a functional gene or the reactivation of a self-reactive gene, both of which would reduce the fitness of the organism and thus be selected against. Similarly, the AR gene is very important for the fitness of the organism and its loss incurs a selection cost.

[0389] The fact that gain of function is more common than expected lends weight to the theory proposed regarding the large amount of pseudogenes at the AR locus. The amount of pseudogenes is generally high at the AR locus, especially in dogs, and is often expressed 16、1、12、9 For example, if stop codons are lost due to SHM or recombination itself, they can gain functionality in recombination and act as substrates for gene conversion. 22Assuming that the reference allele is original along its most uses, the acquisition of a non-reference allele is an example of a pseudogene that is a mutational starting pool of new beneficial alleles and is under such selective stress. (Reference)

[0390] (Example 2) Construction of chimeric IG loci in mouse cells The IG locus of mouse ES cells was modified by BAC insertion to introduce canine heavy-chain DNA into the mouse IGH locus and canine light-chain immunoglobulin DNA into the mouse IGL kappa and lambda loci as follows:

[0391] Canine IGH insertion Insertion of canine DNA from chromosome 8 was performed on the mouse IGH locus by BAC insertion. The inserted DNA contains nucleotides 72,988,807 - 73,128,041 and includes IGHV4-1 to IGHV3-4, as well as IGHD1-6 and IGHJ1-6. See FIGS. 1 and 10.

[0392] Canine IGL lambda DNA insertion Insertion of canine DNA from chromosome 26 was performed on the IGL lambda locus on mouse chromosome 16. The inserted DNA contains nucleotides 27,509,860 - 27,646,373 and includes IGLV3-1 to IGLV4-6, as well as IGLJ1-9 and IGLC1-9. See FIGS. 2 and 13.

[0393] The coordinates are from the GRCm38 / mm10 assembly of December 2011 in mouse and from Canfam3.1 in dog.

[0394] Insertion of canine DNA was inserted into landing pads at the following positions in the mouse genome. Canine heavy-chain DNA: Inserted immediately upstream of position 114,666435 on mouse chromosome 12 Canine kappa DNA: inserted immediately upstream of mouse chromosome 6, position 70,674,7.55 Canine lambda DNA: inserted immediately upstream of mouse chromosome 16, position 19,047,551

[0395] Expression of the chimeric transcript has been confirmed by PCR analysis from the chimeric locus - see Figure 15.

[0396] The method was as follows.

[0397] BAC modification BACs of canine origin from the CHORI-82 library were obtained directly from the BAC library at the Research Institute, Children's Hospital Oakland.

[0398] All bacteria containing BACs were cultured at 32 °C on Luria Bertani (LB) medium or LB agar supplemented with 12.5 μg / ml chloramphenicol. BAC-containing cells were made recombinogenic by addition of the pSIM18 plasmid to the cells by the standard CaCl2 heat shock protocol, and maintenance of pSIM18 was selected by supplementing the medium with 75 μg / ml hygromycin.

[0399] Recombinant plasmids such as pRMCE38 were linearized and 1 kbp BAC-specific homology arms were introduced using Gibson assembly. The plasmids contain sequences necessary for downstream recombination such as cre-lox and PiggbyBac sequences, as well as selection markers for the BAC or ESC engineering steps. The completed plasmids were restriction digested and gel purified to obtain fragments spanning the BAC-specific homology arms, the introduced recombinant sequences, and the homology arms to the vector backbone of the BAC. These fragments were inserted into the BAC by electroporation and those with successful integration of the fragment into the BAC were selected.

[0400] The successful modification of the resistant clones was verified by PCR across the junction of the inserted DNA and the endogenous DNA. When both ends of the BAC were modified, the BAC DNA was purified and electroporated into Electromax™ DH10B cells (Life Technologies). Clones that lost hygromycin resistance and were thus pSIM18 negative were selected for further analysis. PCR reactions were performed on the DNA of these clones to ensure correct modification at both ends, as well as the presence of the exon to be inserted using the appropriate BAC. Clones that passed this quality control check were used for genetic manipulation of embryonic stem cells (ESCs).

[0401] ESC genetic manipulation The processes of ESC culture, electroporation, and drug selection were performed as described by Lee et al. in 2014. Mouse male AB2.1 cells that already contained the landing pads for SRMCE at each immunoglobulin locus were used, cultured in M15 medium (knockout DMEM supplemented with 15% FBS, 2 mM glutamine, and 100 μM β-mercaptoethanol), and maintained on irradiated SNL76 / 7 feeders. All cells used were tested to confirm the absence of contaminants such as mycoplasma.

[0402] For each transfection, 1×10 7 cells were used, and all transfections were performed at 500 μF and 230 V using a Bio-Rad electroporator (GenePulser Xcell). For the introduction of the BAC, 10 μg of BAC DNA and 25 μg of pCAGGS-iCRE were used for each transfection. After 24 hours, the cells were selected with 3 μg / ml puromycin for 1 week, and colonies were selected for expansion and testing. PCR using primers spanning the junction of the mouse and the BAC DNA was used to confirm successful integration of the BAC into the landing pad.

[0403] Next, the positive clones were subjected to excision of the 3' landing pad using PBase. The clones were grown and 1×10 5 cells per electroporation of 10 μg of the PiggyBac transposase plasmid were used. After a 3-day recovery in M15, the cells were split, seeded at low density, selected with FIAU the next day, and maintained for 10 days. Normal excision of the 3' end of the landing pad was confirmed by junction PCR. The positive clones were then subjected to the same exon test as the original BAC to confirm that no deletions had occurred.

[0404] Generation and analysis of mice Positive ES cell clones were injected into blastocysts of the C57BL / 6 Tyrc-Brd mouse strain by standard procedures. The injected blastocysts were transferred to the uterus of pseudopregnant female B6 / CBA F1 recipients. Approximately 40 blastocysts were injected for each clone. To enable identification of pups derived from the injected ESCs based on coat pigment, a high percentage of chimeric males were mated with albino C57BL / 6 Tyrc-Brd females. Mice derived from the ESC clones were subjected to PCR tests with primers spanning the junction between the mouse and the inserted DNA.

[0405] To verify the expression of the inserted gene, a reverse transcription-based approach was implemented. Mice confirmed to have the BAC and the blood, spleen, and femurs of the related wild-type controls were harvested. The femurs were flushed with PBS to obtain bone marrow samples. These three tissue types were converted to RNA using the NucleoSpin RNA-kit (Macherey-Nagel). Reverse transcription was performed using superscript II (Thermo Fisher) with primers against the 5' end of the constant region of the mouse related to the inserted BAC (e.g., IGLC of mice containing the IGL BAC) or the polyA tail of the mRNA. These transcripts were then amplified using barcoded primers nested in the murine C region and priming from the inserted leader region from the V gene of the canid. This confirmed that only chimeric transcripts, rather than all mouse immunoglobulin transcripts, were amplified. These PCR products were visualized on a gel, and it was confirmed that chimeric transcripts were present in mice with the BAC but not in wild-type mice. See Figure 15 - Confirmation of IGH chimeric transcripts. The first 8 lanes correspond to RNA from blood samples, the second 8 lanes to bone marrow samples, and the last 8 lanes to spleen samples. The first 4 out of a given 8 are primed in the C region, and the second 4 are primed at the polyA tail. Of the 4, the first 2 are controls, and the latter 2 are mice in which the production of chimeric transcripts is expected.

[0406] All animal experiments and breeding were carried out under the approval of the Wellcome Trust Sanger Institute AWERB (Animal Welfare and Ethical Review Body). Approval from the UK Home Office was provided under project license 80 / 2432.

[0407] (Example 3) Figures 4, 5, and 6 show the annotation of the feline Ig locus. The method of annotation is the same as the method outlined in 2.1.1 of Example 1, except that the feline genome was investigated instead of the canid genome and RNA sequencing data was not used.

[0408] The annotations provide tools and information for using cat DNA in the genomes of rodents using the methods described above.

[0409] (Example 4) 5' rapid amplification of cDNA ends (5'RACE) Materials and methods Blood samples Baseline samples: Peripheral blood was collected from 13 dogs at the University of Minnesota College of Veterinary Medicine. RNA was extracted on site and transported on dry ice. This study had prior approval from the Institutional Animal Care and Use Committee of the University of Minnesota College of Veterinary Medicine.

[0410] Library preparation First-strand cDNA was synthesized using a pooled mixture of C-specific primers and template switch oligo (TSO). Components included in the reaction: 666.7 μM dNTP (Sigma Aldrich) in a 30 μl volume, 666.7 nM TSO, 333.3 nM each of the heavy and light chain RT primer mixtures, 1 - 5 μg RNA, 2 mM DTT (Invitrogen), 3 mM MgCl2, 40 units of RNaseOUT (Invitrogen), and 100 units of SuperScript II reverse transcriptase (Invitrogen). Extension was for 60 minutes at 42 °C, after which 1 μl of RNase A / T1 mixture (Thermo Scientific) was added and the reaction was incubated at 37 °C for 15 minutes. The reaction was then cleaned up using the AMPure XP system (Agencourt) according to the manufacturer's recommendations (initial reaction: using an AMPure XP solution ratio of 5:4). The cDNA was resuspended in 21 μl of PCR-grade water and equally divided into one heavy chain reaction and one light chain reaction. PCR was performed in a 25 μl volume using Q5 high-fidelity polymerase (New England Biolabs), 10.5 μl of the resuspended cDNA, and 400 nM each of 5'RFWA and the PCR1 mixture of the heavy or light chain. The cycling conditions were as follows: 98 °C for 30 seconds, then 20 cycles of (98 °C for 10 seconds, 63 °C for 30 seconds, 72 °C for 20 seconds), followed by a final extension of 72 °C for 2 minutes. The PCR reaction was subjected to the same AMPure XP clean-up as before and resuspended in 10.5 μl of PCR-grade water. This was done in a 25 μL PCR reaction using 400 nM each of Q5 polymerase and the forward primer containing 5'RRVA and the sample-specific index hexamer. The PCR conditions were the same as before, and the product was cleaned up using the same AMPure XP system and resuspended in 20 μl of PCR-grade water. These libraries were then quantified, pooled into an equimolar mixture, and diluted to a final concentration of 10 nM for sequencing.

[0411] Sequencing and Data Analysis The library was sequenced on a MiSeq machine (Illumina) using 300 bp paired-end reads (including 10% PhiX spike-in) by the core sequencing team at the Wellcome Trust Sanger Institute. The quality of the demultiplexed data was then filtered before submission to the IMGT V-Quest software (using the "HighV-Quest" software and selecting "species" = "Canis lupus familiaris (dog)" and "receptor type or locus" = "IG"). 14 .

Claims

1. 1. A method of producing a canine antibody chain specific to a desired antigen, comprising the steps of immunizing a rodent with the desired antigen and recovering the antibody chain, alone or as part of a complete antibody, or recovering a cell that produces the antibody chain, alone or as part of a complete antibody, wherein the rodent has a gene encoding a canine CDR3 region of a canine lambda light chain that comprises a glutamine (Q) amino acid residue at position 1 of CDR3, a valine (V) amino acid residue at position 2 of CDR3, a tryptophan (W) amino acid residue at position 3 of CDR3, an aspartic acid (D) amino acid residue at position 4 of CDR3, and a valine (V) amino acid residue at position 11 of CDR3.

2. 1. Use of a rodent having a nucleic acid encoding a canine lambda light chain, or a gene encoding same, in the preparation of a medicament for the treatment or prevention of a canine disease or in the production of a canine antibody or antigen-binding fragment thereof, wherein the canine CDR3 region of the canine lambda light chain comprises a glutamine (Q) amino acid residue at position 1 of CDR3, a valine (V) amino acid residue at position 2 of CDR3, a tryptophan (W) amino acid residue at position 3 of CDR3, an aspartic acid (D) amino acid residue at position 4 of CDR3 and a valine (V) amino acid residue at position 11 of CDR3.

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