Animal models and therapeutic molecules

Abstract

To provide, for example, rodents, cells, antibodies and parts produced therefrom, antibodies modified afterward for use in companion animals, as well as methods for preparing such rodents, cells, antibodies and antibody chains.SOLUTION: The invention relates to, inter alia, to a rodent or rodent cell having a genome comprising: 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, where the rodent or rodent cell is capable of expressing the companion animal variable region gene(s) to form an antibody chain and where the companion animal species is not a rodent.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 diseases, 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 is disclosed, for example, in 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, Publication Year: 2014 DOI:, doi:10.1038 / nbt.2825. This approach is designed to produce antibody products for human therapeutic use. However, rodent models for generating antibodies suitable for use in other species such as companion animals have not been developed.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non - Patent Document 4

Non - Patent Document 5

Non - Patent Document 6

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention relates to such rodents, cells, antibodies, and parts thereof produced therefrom, for example, antibodies modified thereafter for use in companion animals, and methods for producing such rodents, cells, antibodies, and antibody chains.

Means for Solving the Problems

[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) one or more IGL kappa V region genes of a companion animal, and one or more IGL kappa J region genes of a companion animal; and / or one or more IGL lambda V region genes of a companion animal, and one or more IGL lambda J region genes of a companion animal 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, a rodent or rodent cell, wherein the companion animal species is not a rodent.

[0007] i) one or more IGL kappa V region genes of a companion animal, and one or more IGL kappa J region genes of a companion animal; and / or one or more IGL lambda V region genes of a companion animal, and one or more IGL lambda J region genes of a companion animal, and ii) optionally, one or more IGH V region genes of a companion animal, one or more IGH D region genes of a companion animal or a host, and one or more IGH J region genes of a companion animal or a host 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, a rodent or rodent cell, wherein the companion animal species is not a rodent.

[0008] A method for producing a rodent or rodent cell, comprising introducing into the rodent cell genome i) 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, and / or ii) one or more IGL kappa V region genes of a companion animal, and one or more IGL kappa J region genes of a companion animal, 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 one of them, A method in which a rodent or rodent cell can express the gene of a companion animal and 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 to 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 the cells that produce the antibody chain alone or as part of a complete antibody.

[0010] A method for producing an antibody chain or antibody that is specific to 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 the 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 comprises the following: 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, or a human or other mammal in need thereof, as a variable region; A method comprising:

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

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

[0015] A method of treating a companion animal, comprising delivering an antibody or antibody chain, or a portion thereof, to a companion animal in need thereof, wherein the antibody or antibody chain, or a portion thereof, is modified to be fully a companion animal antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0016]

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Embodiments for Carrying out the Invention

[0017] 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, relating to a rodent or rodent cell, wherein the companion animal species is not a rodent.

[0018] 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, wherein the companion animal species is not a rodent.

[0019] 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 a mouse, in combination with a constant region, enables the production of an antibody heavy chain containing a variable antibody region derived from the expression of DNA of a canine animal in the mouse. The constant region may be a rodent immunoglobulin (IG) constant region, and as a result, a chimeric heavy chain having a variable region of a canine animal and a constant region of a rodent is generated. Information regarding the variable region of such a chimeric antibody chain, or a nucleic acid containing the same, can be used, for example, for therapeutic use in dogs, to generate a complete canine animal antibody. A rodent containing canine animal DNA can also serve as an animal model for understanding diseases and testing pharmaceuticals.

[0020] All nucleotide coordinates of the mouse correspond to the December 2011 GRCm38 / mm10 assembly of the mouse (assembly accession GCA_000001635.2) unless otherwise specified.

[0021] 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).

[0022] The canine genome build is CanFam3.1 (assembly accession - GCA_000002285.2), created in September 2011 and last updated in May 2016.

[0023] The feline genome build is FelisCatus8.0 (assembly accession - GCA_000181335.3), created in November 2014.

[0024] The rodent of the present invention is preferably a mouse or a rat, more preferably a mouse.

[0025] 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, humans are not companion animals.

[0026] In one aspect, the rodent is a mouse and the companion animal is a dog.

[0027] In one aspect, the rodent is a mouse and the companion animal is a cat.

[0028] In one aspect, the rodent is a mouse and the companion animal is a horse.

[0029] The IG heavy chain (IGH) locus of the 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, the variable region of the heavy chain of the antibody is generated. The IGH V, D, and J genes are naturally expressed in combination with the heavy chain constant region. The IG light chain locus (IGL), which can be lambda or kappa, contains a plurality of V and J gene segments that form the variable region of the light chain of the antibody when expressed together. The IGL V and J region genes are naturally expressed in combination with the kappa or lambda light chain constant region of the light chain. The rodent or rodent cell of the present invention can express the VDJ or VJ region gene of the companion animal to form an antibody chain. The gene of the companion animal is operably linked to the constant region in the genome of the rodent in order to enable the expression of the antibody chain. The companion animal IG gene can be located in the genome of the rodent together with an exogenous constant region gene (derived from a species other than the rodent), or together with the constant region of the rodent that naturally exists in the genome of the rodent, for example, in a functional arrangement such as upstream in the genome of the rodent, and the expression of the V region gene with the constant region can occur.

[0030] 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 animals, 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 animals. The genome of a rodent or cell may contain the genes of companion animals derived from the heavy chain and kappa chain (not lambda), or the heavy chain and lambda chain (not kappa), or may contain the genes of companion animals derived from all three loci, the heavy chain, kappa, and lambda.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] In one aspect, the inserted companion animal DNA comprises 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, comprises all of the light chain J genes of the companion animal.

[0036] In one aspect, the rodent genome comprises all of the IGH V, D, and J region genes and intervening sequences from the companion animal.

[0037] In one aspect, the rodent genome comprises all of the IGL kappa V and J region genes and intervening sequences from the companion animal.

[0038] In one aspect, the rodent genome comprises all of the IGL lambda V and J region genes and intervening sequences from the companion animal.

[0039] The genome of a rodent or rodent cell may comprise 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 canines. In a preferred aspect, the rodent genome comprises at least 83 IGH V region genes of canines.

[0040] The genome of a rodent or rodent cell may comprise at least 1, 2, 3, 4, 5, or 6 IGHD region genes from the companion animal, preferably genes of canines.

[0041] The genome of a rodent or rodent cell may comprise at least 1, 2, 3, 4, 5, or 6 IGHJ region genes from the companion animal, preferably genes of canines.

[0042] 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 a rodent comprises at least 19 kappa light chain V region genes of a canine animal.

[0043] 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 genes of a canine animal.

[0044] 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 a rodent comprises at least 160 lambda light chain V region genes of a canine animal.

[0045] 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 genes of a canine animal.

[0046] 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 feline, such as at least 23 V region genes.

[0047] 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 feline.

[0048] 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.

[0049] 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.

[0050] In another aspect, the genome of a rodent or rodent cell may comprise at least 1, 2, 3, 4, 5, or 6 IGH kappa J region genes of a companion animal derived from a cat.

[0051] 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.

[0052] 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 IGH lambda J region genes of a companion animal derived from a cat.

[0053] The number of companion animal genes mentioned in any of the above aspects may further increase and, in one aspect, is doubled in the case of a homozygote having insertions in both alleles.

[0054] Preferably, the V, D, and J region genes inserted into the genome are from the same companion animal. Preferably, the inserted IGH VDJ region genes, or IGL VJ region genes, are all from animals of the Canidae family, all from animals of the Felidae family, or all from animals of the Equidae family. Preferably, the genes are all from animals of the Canidae family.

[0055] In one aspect, all of the genes of the companion animal to be inserted are from the same breed of companion animal, for example, the same dog breed.

[0056] In one aspect, the gene of the companion animal is located upstream of the genome of the constant region of the rodent, and for the heavy chain variable region gene of the companion animal to be inserted, it is appropriately located upstream of the heavy chain constant region(s), and / or for the light chain variable region gene of the companion animal to be inserted, 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 variable region gene to be inserted and the constant region of the rodent.

[0057] Preferably, the heavy chain V, D, and J region genes derived from the companion animal are located in the genome of the rodent upstream of the heavy chain constant region of the rodent.

[0058] Preferably, the light chain kappa V, J region genes derived from the companion animal are located in the genome of the rodent upstream of the kappa light chain constant region of the rodent.

[0059] 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 DNA of the companion animal and the constant region of the rodent are functionally arranged relative to each other for the production of an antibody or antibody chain.

[0060] In one aspect, the DNA of the companion animal to be inserted, such as a variable VDJ or VJ region gene, 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, preferably also accompanied by a 3' enhancer derived from the rodent or the companion animal. One preferred embodiment is the use of the constant region of a canine animal and the 3' enhancer of a canine animal, such as the constant region of a rodent and the 3' enhancer of a rodent with the VDJ or VJ region of a companion animal. 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.

[0061] 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.

[0062] The present invention also particularly contemplates cells and rodents having an insertion of a gene of a companion animal (encoding a "fully" antibody chain of the companion animal) accompanied by a constant region of the companion animal in the endogenous IG locus of the rodent, such as DNA encoding the lambda V, J, and C genes of the companion animal.

[0063] Preferably, the light chain lambda V and J region genes derived from a companion animal are located in the genome in a functional arrangement such as upstream, together with the lambda chain constant region derived from the same companion animal. In this way, a lambda antibody chain having a lambda constant region derived from a companion animal is produced. Accordingly, the present invention relates to a rodent or rodent cell whose genome contains the IGL lambda V region gene of one or more companion animals, the IGL lambda J region gene of one or more companion animals, and the lambda constant region of one or more companion animals, and which can express a lambda antibody chain having both the variable region and the constant region of the companion animal.

[0064] In one aspect, the above-described VJC lambda antibody chain of a companion animal is inserted between the lambda locus of a mouse, preferably between the last rodent C gene and the 3' enhancer.

[0065] In one aspect, a rodent or rodent cell 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 located within the kappa locus of the rodent cell, e.g., at or upstream or downstream of the rodent kappa constant region locus. The insertion is preferably upstream of the IGL kappa locus constant region such that the IGL lambda V and J region genes are expressed in the IGL kappa constant region. Suitably, the insertion positions the companion animal genes at approximately the same position as the native rodent kappa genes, potentially deleting or replacing them, e.g., 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 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 companion animal DNA into this kappa locus can result in high-level expression of the companion lambda chain V region gene.

[0066] In one aspect, the rodent genome can be homozygous for the insertion of companion animal genes at one, or both, or all three of the immunoglobulin loci.

[0067] In another aspect, the rodent genome can be heterozygous for the insertion of companion animal genes at one, or two, or all three of the immunoglobulin loci.

[0068] In particular, the rodent genome is heterozygous for the insertion of companion animal genes at the kappa locus.

[0069] In one aspect, the inserted DNA can be expressed with different rodent constant regions by isotype switching.

[0070] In one aspect, the DNA of the inserted companion animal can be expressed together with the constant regions of different rodents by trans-switching.

[0071] In one aspect, the companion animal is a dog, the rodent genome contains the kappa variable region gene of a canine animal, and all of the kappa variable region genes of the canine animals in the rodent genome are upstream of the constant region with which the variable region gene is expressed together, for example, upstream of the rodent kappa constant region, or for example, upstream of the kappa constant region of a canine animal.

[0072] In another preferred aspect, the companion animal is a horse, the rodent genome contains the kappa variable region gene of a horse family animal, and all of the kappa variable region genes of the horse family animals in the rodent genome are upstream of the constant region with which the variable region gene is expressed together, for example, upstream of the rodent kappa constant region, or for example, upstream of the kappa constant region of a horse family animal.

[0073] In one aspect, the DNA of the companion animal 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.

[0074] 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.

[0075] 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 an animal of the Canidae family.

[0076] 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 an animal of the Canidae family.

[0077] In one aspect, the rodent or the cell of the rodent is a mouse or a cell of a mouse, and one or more, or all, of the kappa V genes of an animal of the Canidae family, 4-S17, 2-S16, 3-S15, 2-S14, 2-S13, and 2-S12 are located upstream of the kappa constant region of the rodent.

[0078] 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 an animal of the Canidae family.

[0079] In one aspect, the rodent is a mouse and the genome comprises one or several or all of the mouse IGH V region genes, preferably including the deletion of V1-85 to V5-2.

[0080] In one aspect, the rodent is a mouse and the genome comprises one or several or all of the mouse IGL kappa V region genes, preferably including the deletion of V3-1 to V2-137.

[0081] 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.

[0082] In one aspect, the genome of a rodent is modified to reduce or prevent the expression of a full-length rodent antibody having both a variable region and a constant region derived from the rodent. This can be by 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 rodent IGL lambda gene is not deleted from the rodent genome.

[0083] In one aspect, insertion of companion animal DNA at the kappa locus of the rodent results in deletion or inactivation, in whole or in part, of one or both alleles of the rodent kappa locus.

[0084] In one aspect, the kappa locus of the rodent is inactivated, in whole or in part, for example by insertion, deletion, or inversion.

[0085] In one aspect, the lambda locus of the rodent is inactivated, in whole or in part, for example by insertion, deletion, or inversion.

[0086] In one aspect, the heavy chain locus of the rodent is inactivated, in whole or in part, for example by insertion, deletion, or inversion.

[0087] 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 either the complete constant region or a portion of the constant region sufficient to enable the formation of a functional chimeric antibody that can specifically recognize an antigen. Accordingly, references herein to chimeric antibodies or antibody chains having a rodent constant region are not limited to 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 portion 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 a portion of the rodent constant region to form an antibody chain or a portion thereof.

[0088] Preferably, the rodent genome contains all of the lambda constant region DNA and intervening regions of the companion animal.

[0089] The constant region of the rodent that is expressed 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.

[0090] In one aspect, at least one other regulatory sequence such as a rodent enhancer or switch region is maintained in a functional arrangement with the rodent constant region. In this way, the effect of the enhancer or other regulatory sequence can be exerted, in whole or in part, in the cell or transgenic rodent.

[0091] In one aspect, one or more rodent regulatory sequences, such as the Emu enhancer sequence, are maintained in their native position upstream of the rodent Mu constant region, appropriately with respect to the distance from the constant region.

[0092] 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.

[0093] 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.

[0094] In such a position, the rodent enhancer or switch sequence is operable in vivo appropriately with the host constant region sequence.

[0095] 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.

[0096] In one aspect, the genome of a rodent or rodent cell comprises one or more promoters, or enhancers, and / or other control elements associated with the V, D, or J regions of a companion animal. In one aspect, one or more control regions of a companion animal, such as a promoter or enhancer or switch region, replace one or more rodent promoters or enhancers 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 effect of the control sequences is exerted, in whole or in part, in the cell or transgenic rodent.

[0097] 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 recombination of the V, D, or J gene segment(s).

[0098] In this context, a "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, a "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.

[0099] 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.

[0100] In one aspect, the control sequence includes a promoter preceding an individual V gene segment, and / or a splice site within an individual V gene segment, and / or a recombination signal sequence for V(D)J recombination downstream of the V gene segment, adjacent to the D gene segment, or upstream of the J gene segment.

[0101] In one aspect, the V, D, or J sequences of the companion animal to be inserted are adjacent to RSS sequences from the same companion animal. For example, the RSS sequences of canines can be used with the V, D, and / or J sequences of canines. It will be understood 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, in a rodent cell, the locus of an endogenous immunoglobulin variable region gene, in whole or in part, with the locus of a gene of a companion animal, the method comprising obtaining a cloned genomic fragment or synthetic sequence that wholly or partly comprises the locus of a companion gene comprising at least one V or D (for heavy chain) or J gene segment, and at least one regulatory sequence associated therewith, 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 naturally corresponding to the DNA of the companion animal to be inserted.

[0102] In one aspect, the DNA of the companion animal to be inserted comprises genomic DNA of at least 5 kb, at least 10 kb, at least 15 kb, 20 kb or more from the companion animal.

[0103] In one aspect, the rodent cells of the invention can be rodent ES cells, rodent hematopoietic stem cells, or other cells that can generate in a rodent an antibody chain comprising a variable region encoded by the DNA of a companion animal, such as a chimeric heavy antibody chain, or a chimeric antibody light chain, or a complete antibody chain or antibody repertoire of a companion animal having a variable region and a constant region encoded by the variable region of the companion animal.

[0104] In one aspect, the cells of the invention are rodent ES cells or induced pluripotent stem cells (iPS cells).

[0105] In one aspect, the cells are isolated rodent cells.

[0106] In one aspect, the cell is an isolated rodent B cell.

[0107] Preferably, the rodent cell is a rodent ES cell or iPS cell. Such cells are suitable for insertion of companion animal DNA and generate a rodent that expresses the antibody chains described herein.

[0108] The ES cell may be a mouse cell line 129 or C57BL, such as C57BL / 6N, C57BL / 6J, 129S5 or 129Sv strain, or a cell having a hybrid genome containing genomic DNA of 129 or C57BL.

[0109] The present invention also relates to cell lines that grow from or are otherwise derived from the cells described herein, including immortalized cell lines.

[0110] The cells or cell lines of the present invention may contain the V, (D), or J genes of the companion animal after germline composition or reconstitution after in vivo maturation.

[0111] The present invention also relates to cells or cell lines that express antibody chains such as chimeric antibody heavy chains, obtained by immunizing the rodent of the present invention with an antigen.

[0112] The present invention also relates to a cell or cell line that expresses an 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 may be identified or has been identified by immunizing a rodent of the present invention with an antigen and obtaining an antibody chain or the sequence of an antibody chain from the rodent or rodent cells. The antibody chain to be expressed is preferably an antibody chain of a complete canine animal, or a complete equine animal, or a complete feline animal, in which the variable region derived from the companion animal is expressed in the rodent of the present invention with a constant region derived from the same companion animal (not a rodent constant region). The cell or cell line that expresses the antibody chain or antibody can be a CHO cell or another mammalian cell line suitable for the production of therapeutic agents for use in humans or animals.

[0113] 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.

[0114] The present invention also relates to a vector for use in the present invention. In one aspect, such a vector is a bacterial artificial chromosome (BAC) that contains all or part of the IG locus of a companion animal. It will be understood that other cloning vectors can be used in the present invention, and thus the reference to BAC herein can generally be interpreted to refer to any suitable vector. The vector may contain one or more selectable markers and / or one or more site-specific recombination sites. In one aspect, the vector contains 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 contains one or more transposon ITR (terminal inverted repeat) sequences.

[0115] A suitable BAC containing canine animal DNA is available from the BACPAC Resource Center at the Children's Hospital Oakland Research Institute as the CHORI-82 BAC library.

[0116] Appropriate BACs containing DNA of feline animals are available from Amplicon Express as the FCAB library.

[0117] Appropriate BACs containing DNA of equine animals are available from the BACPAC Resource Center of the Children's Hospital Oakland Research Institute as the CHORI-241 BAC library.

[0118] The present invention relates to a method for producing a rodent or rodent cell, comprising the step of 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 expresses the variable region genes of the companion animal in combination with a constant region and can form an antibody chain.

[0119] The present invention also relates to a method for producing a rodent or rodent cell, comprising the step of 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 expresses the variable region genes of the companion animal in combination with a constant region and can form an antibody chain.

[0120] Preferably, the method relates to inserting the VDJ and VJ region genes of companion animals for both the light chain and the heavy chain so that an antibody having variable regions derived from the expression of the DNA of the companion animal is produced for both the light chain and the heavy chain.

[0121] The present invention also relates to a method for producing a rodent or rodent cell, comprising the step of 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 containing intervening sequences.

[0122] In one aspect, the insertion process begins at the site where the initiation cassette is inserted into the genome of cells such as ES cells. In one aspect, the initiation cassette is inserted into the heavy chain locus of a rodent for use in the insertion of human heavy chain DNA. Similarly, the initiation cassette may be inserted into the light chain locus of a rodent for use in the insertion of human light chain VJ DNA. The initiation cassette may be placed between the last J region and the C region of the rodent heavy chain and kappa chain. 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 the IGL lambda companion animal.

[0123] The initiation cassette appropriately includes a continuous locus unique to the rodent genome that can effect the insertion of companion animal DNA.

[0124] In one aspect, after the insertion of the first DNA fragment into the initiation cassette, the insertion of the second DNA fragment into a portion of the first DNA fragment may follow. Subsequent insertions may be made into at least a portion of the previously inserted DNA fragment.

[0125] In one aspect, the method includes targeted insertion of the initiation cassette into the rodent genome by homologous recombination, insertion of the first DNA sequence into at least a portion of the initiation cassette by site-specific recombination, insertion of the second DNA sequence into the 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 the preceding DNA sequence, to construct a continuous DNA fragment within the target that includes the DNA of the companion animal. The insertion of the DNA fragment into at least a portion of the 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, for example, 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.

[0126] 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 cellular locus, for example, the locus of ES cells, to form a complete VDJ or VJ region, or a portion thereof.

[0127] 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.

[0128] 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 host's constant region are well known in the art. See 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.

[0129] 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).

[0130] In one aspect, correct insertion events are confirmed before proceeding to the next step of the multi - step cloning process.

[0131] 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 constant region 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, preferably upstream of the canine IGL kappa constant region.

[0132] In one aspect, the rodent can generate a diversity of combinations of at least 1×10 6 different functional chimeric immunoglobulin sequences.

[0133] In one aspect of the rodent, chimeras are generated using ES cells having one or more chimeric loci, where the 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.

[0134] 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.

[0135] The ES cells of the invention can be used to generate animals using techniques well known in the art, including injecting the ES cells into blastocysts, subsequently transplanting the chimeric blastocysts into females to generate progeny, mating these to generate heterozygous progeny, and then mating to generate homozygous recombinants having the desired insertions. In one aspect, the host blastocyst is Rag deficient.

[0136] The present invention relates to chimeric rodents generated by injecting the ES cells of the invention into blastocysts and then transplanting the chimeric blastocysts into female rodents to produce progeny.

[0137] 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 and reinserted from the IGH locus.

[0138] 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.

[0139] 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 are not 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.

[0140] 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.

[0141] Surprisingly, from the genomic data of 107 dogs across 19 breeds, it has been found by the inventors that the breed variation is minimal, the reference allele (from Boxer dogs) is found at a rate of 76% across the entire sample, and non - reference alleles are 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.

[0142] 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.

[0143] Preferably, the rodent or rodent cell contains alleles that are not one or more reference alleles of the following gene segments (not "allele *01"): IGKV2-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.

[0144] In one aspect, the rodent or rodent cell contains one or more reference alleles of the companion animal's gene segments, such as 2, 3, 4, 5, 6, 7, 8, or 9 reference alleles, etc.

[0145] 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 inserted V gene segment is a V gene reference allele of a Canidae animal, and / or at least 50, 60, 70, 80, 90, or 100% of the inserted D gene segment is a D gene reference allele of a Canidae animal, and / or at least 50, 60, 70, 80, 90, or 100% of the inserted J gene segment is a J gene reference allele of a Canidae animal, and combinations thereof. Preferably, at least 90%, such as 100%, of the inserted Canidae animal gene segment alleles are reference alleles of that gene segment.

[0146] The present invention also relates to the insertion of V, D, and J segments of Canidae animals disclosed herein in 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 a rodent into which the Canidae animal V, D, and J segments described herein are inserted in the production of antibody chains or portions thereof for use in the prevention or treatment of diseases in different dog breeds.

[0147] The present invention also relates to a rodent having one or more or all of the V, D, and J segments of 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 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.

[0148] For example, if the inserted Canidae animal DNA is from a Boxer, it is used in dog breeds other than Boxers.

[0149] The present invention also relates to the following. (i) A rodent having V, D, J segments of Canidae animals 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 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 Canidae animal V, D, J DNA (or V, and J DNA of the light chain of Canidae animals), preferably including at least some amino acids expressed from reference alleles; (iv) For example, a complete antibody or antibody chain of a Canidae animal comprising the antibody variable region of the antibody of (iii) above, which can be generated by expressing DNA encoding the constant region of a Canidae animal together with DNA encoding the variable region of a Canidae animal; (v) An expression cell such as a CHO cell containing DNA encoding all or part of such a complete antibody of a Canidae animal.

[0150] Preferably, the V, D, and J gene segments of the boxer are from the CHORI-82 BAC library.

[0151] In another aspect, the present invention provides the use of an antibody or antibody chain having a variable region of a Canidae 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 Canidae animal is a reference allele of the Canidae animal. Preferably, the antibody or antibody chain is effective in at least 50%, for example, 60% or at least 70% of different dog breeds. Preferably, the V gene segment of the Canidae animal derived from the heavy chain and / or light chain is from a boxer. Preferably, the D and J gene segments are also gene segments of the boxer.

[0152] The present invention relates to the following. A method of producing an antibody chain specific for a desired antigen, comprising immunizing a rodent disclosed herein with the desired antigen, and 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 (see, e.g., Harlow, E. and Lane, D., 1998, 5th ed., "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 of detecting a target antigen, comprising detecting the antibody produced as described above with a secondary detection agent that recognizes a part of the antibody.

[0153] A method of producing an antibody chain or an antibody that is specific for a desired antigen and is derived from a single species of companion animal, comprising immunizing a rodent that contains the gene of the 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 the companion animal derived from the same companion animal. This can replace the constant region of a non-human mammal with an appropriate human constant region DNA sequence by standard cloning techniques at the DNA level. See, e.g., Sambrook, J and Russell, D. (2001, 3rd ed.) "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Lab. Press, Plainview, NY). Or it can be achieved by direct nucleic acid synthesis.

[0154] A method of producing an antibody chain or a part thereof having the variable region of a companion animal, comprising expressing in a cell the DNA encoding the antibody chain or a part thereof, The sequence of the DNA encoding the variable region of the antibody chain can be obtained from, or can be obtained by, immunizing the rodents of the present invention with an antigen so 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.

[0155] An antibody or antibody chain, or a part thereof, or a nucleic acid encoding an antibody chain or a part thereof, obtainable or obtained from a rodent or cell according to the present invention.

[0156] The present invention also relates to a part or the whole 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 a part or the whole 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 present invention also relates to the identification of variable regions of canine animals by single cell sequencing and the generation of synthetic vectors expressing complete antibody chains having corresponding constant regions of canine animals. The present 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.

[0157] An antibody or antibody chain, or a part thereof, obtainable or obtained from a rodent or cell according to the present invention for use in the treatment of companion animals.

[0158] A method of treating a companion animal, the method comprising delivering to a companion animal in need thereof a suitable antibody or antibody chain or a portion thereof obtainable or obtainable according to the present invention. In particular, the present invention relates to a method of medical treatment comprising delivering to a companion animal in need thereof an antibody chain or antibody, or a portion thereof, wherein at least the variable region of the antibody is obtained or otherwise identified by immunizing a rodent of the present invention containing a companion DNA V(D)J region gene with an antigen.

[0159] 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 fragments and functional derivatives of said antibodies and chains, and the use of said antibodies, chains and fragments in medicine including diagnosis and in in vitro or ex vivo studies. Functional antibody fragments can include fragments capable of specific binding to an antigen. The functional antibody fragment may be, for example, a FAB or a single chain variable fragment (scFv). 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 fragments and functional derivatives of said antibodies and chains, and the use of said antibodies, chains and fragments in medicine including diagnosis and in in vitro or ex vivo studies.

[0160] 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.

[0161] In a further aspect, the present invention relates to the use of a rodent as described herein as a model for the testing of drugs and vaccines. Accordingly, the present 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 present invention and the step of monitoring one or more of an immune response, a safety profile, an effect on a disease.

[0162] 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 present invention relates to all such chimeric antibody derivatives derived from the chimeric antibodies identified according to the present invention.

[0163] The present invention also relates to a kit comprising any of the antibodies or antibody derivatives disclosed herein and instructions for use of suitable laboratory reagents such as such antibodies or buffers, antibody detection reagents.

[0164] The present invention also relates to a method of making an antibody, or a part thereof, (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 and comprising the step of providing.

[0165] 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), the antibody being 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 a nucleotide sequence of the constant region of the rodent, and a rearranged VDJ nucleotide sequence generated by in vivo rearrangement of the V region of the companion animal, the D region of the companion animal, and the J region of the companion animal, the V region of the companion animal being selected from the IGH variable region genes of IGH V4-1, V3-2, V3-3, V3-4, V3-5 of Canidae animals.

[0166] Optionally, the J region is any one of JH1, JH2, JH3, JH4, JH5, or JH6 of Canidae animals.

[0167] Optionally, the D region is any one of DH1, DH2, DH3, DH4, DH5, and DH6 of Canidae animals.

[0168] In one aspect, the antibody comprises any combination exemplified in the examples and figures 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 or ES cell or hybridoma) whose genome comprises the chimeric heavy chain locus described above in this paragraph.

[0169] 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.

[0170] 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.

[0171] 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 at least the CDR region.

[0172] The present invention relates to a method for generating an antibody or an antibody chain, which method comprises immunizing a rodent described herein with an antigen obtainable from the same companion animal 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.

[0173] 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.

[0174] 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.

[0175] Alternatively, the antigen can be derived from a pathogen such as a bacterium or a virus known to infect the companion animal. For example, an antigen derived from a pathogen that infects a dog and causes a disease can be used to immunize a rodent containing the V, D, and J genes of the canine animal disclosed herein.

[0176] Accordingly, the present invention relates to a rodent immunized with an antigen that causes a disease in a companion animal corresponding to the source of the companion animal DNA present in the genome of the rodent.

[0177] In a further aspect, the antigen may be an equivalent in a companion animal of a human antigen associated with a human disease, preferably a human antigen that has been validated as a target for the prevention or treatment of a human disease.

[0178] In one aspect, the present invention provides an antibody chain or a fragment thereof that can be obtained or is obtained by immunizing a rodent with an antigen described herein.

[0179] The present invention also relates to nucleic acids such as DNA or RNA encoding the 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.

[0180] The present invention also relates to a cell such as a B cell or a hybridoma that partially or completely expresses an antibody chain of a canine animal, or a part of an antibody chain of a canine animal, or an expression cell line (e.g., CHO cell), and the DNA or protein sequence thereof can be obtained from or has been obtained from the rodent described herein.

[0181] 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 express the antibody or a part thereof from that sequence or, in fact, from a different DNA sequence that can express 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 generate a complete antibody chain or antibody of a canine animal.

[0182] 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 animal immunoglobulin V gene segment, at least one canine animal heavy chain D gene segment, and at least one canine animal heavy chain J gene segment, and / or having at least one light chain canine animal immunoglobulin V gene segment and at least one canine animal 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 animal DNA, Preferably that of a complete canine animal, for example, expressing the variable antibody region from the DNA of an expression cell line to express an antibody or antibody chain or a part thereof comprising a variable region of a canine animal and a constant region of a canine animal, 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 administration to a companion animal in need thereof, A method comprising is provided.

[0183] The invention also relates to the use of such an antibody or antibody chain or a fragment thereof in the treatment of companion animals.

[0184] The approaches described above are 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 invention are equally applicable to cats and horses.

[0185] The present invention relates to a method for generating cross-reactive antibodies or antibody chains, comprising 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 resulting from said immunized rodent can bind to both the antigens of the rodent and the companion animal, i.e., can comprise cross-reactive antibodies.

[0186] 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 described herein, immunized with an antigen of interest of a rodent and a corresponding antigen derived from a companion animal, wherein the rodent has a gene knockout of the gene encoding said antigen.

[0187] In one aspect, the rodent is immunized sequentially or simultaneously with the antigen of the rodent directly as a protein or peptide fragment thereof, or by any of a vector encoding a related antigen or fragment thereof, or a syngeneic cell line expressing the desired antigen, and with the corresponding antigen derived from the companion animal.

[0188] In one aspect, the rodent is a mouse and the companion animal is a dog, a cat, or a horse.

[0189] 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 a model without the need for antibody modification.

[0190] 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, preferably is therapeutically validated, and it is meant that antibodies against the human equivalent antigen have been shown to be effective in the treatment of diseases.

[0191] 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 not well 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 mice. In fact, 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.

[0192] These findings by the inventors are surprising and not expected based on the evolutionary differences between canines and humans.

[0193] Within the assigned phylogeny, it is well known that mice and humans are more closely related than either carnivores including (for example) cats and dogs, or horses. This is likely the reason for the success of the use of human control sequences in chimeric mouse models having V, D, and J gene segment insertions of human DNA in said models. Based on this, the same could not be predicted for more evolutionarily diverse animals such as humans and dogs. In fact, in previous attempts to use canine gene segments in mice, murine regulatory control sequences were utilized (for example, Trianni, US2017306352).

[0194] 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.

[0195] 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.

[0196] In one aspect of the invention, the rodent utilizes more IGLJ1 than other light chain J gene segments.

[0197] 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 from any of IGHJ1, 2, 3, 5, or 6, and the rodent expresses a chimeric antibody heavy chain from IGHJ6 more, individually, than from any of IGHJ1, 2, 3, or 5; The rodent expresses a chimeric antibody heavy chain from IGHD5 more, individually, than from any of IGHD1, 2, 3, 4, or 6; The rodent expresses a chimeric antibody heavy chain from IGHD2 more, individually, 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; Rodents express more antibody chains from either IGHD5 together with IGHJ4 or any other combination of canine IGHD and IGHJ segments than from the others; Rodents express more chimeric antibody light chains from IGLJ1 than from any other IGL J gene segment.

[0198] 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).

[0199] In one aspect, the rodent comprises inserted canine lambda light chain gene segments such as 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 pseudogenes).

[0200] The rodents of the invention can be appropriately modified by N and / or P addition of 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.

[0201] 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 comprising rodent AID and / or rodent TdT (e.g., endogenous AID or TdT). The mouse is a preferred rodent.

[0202] In one aspect of the invention, the variable region of the chimeric antibody chain differs 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.

[0203] 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, is greater than the observed antibody diversity of the wild-type canids from which the gene segments are 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.

[0204] Accordingly, the 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.

[0205] 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 canid DNA is greater than or at least as great as that found in the antibody repertoire of the canid.

[0206] 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.

[0207] The present invention also relates to a rodent, for example a rodent comprising a population of chimeric antibody chains, at least 65% or at least 70% of which are unique, with respect to the sequences as determined by 5'RACE, for example in Example 4, for example a rodent in which 65 - 80% of the chimeric antibody sequences are unique, for example a rodent in which 65 - 75% are unique.

[0208] The rodent can be any rodent disclosed herein. For example, 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). In one aspect, the rodent comprises the following inserted canine lambda light chain gene segments: J1-9 (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 of which are pseudogenes). In one aspect, the rodent comprises both the canine heavy chain insertion and the light chain insertion.

[0209] 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.

[0210] Accordingly, preferred embodiments of the present invention are as follows.

[0211] A method for producing an antibody or an antibody chain, comprising immunizing the rodent according to any one of claims 1 to 13 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 genome of the rodent, wherein the antigen can be a protein antigen, a cell expressing the antigen, or a nucleic acid encoding the antigen.

[0212] A method for producing an antibody or an antibody chain, comprising immunizing the rodent according to any one of claims 1 to 13 with an antigen derived from a pathogen such as a bacterium or a virus that infects a companion animal species that is the source of the companion animal DNA present in the genome of the rodent.

[0213] A rodent according to any one of claims 1 to 13, 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.

[0214] A rodent according to any one of claims 1 to 13, immunized with an antigen that causes a disease in a companion animal corresponding to the source of the companion animal DNA present in the genome of the rodent.

[0215] A rodent according to any one of claims 1 to 13, immunized with a companion animal antigen equivalent of a human antigen associated with a human disease.

[0216] The rodent according to any one of claims 1 to 13, wherein the rodent contains companion animal DNA derived from a dog and the rodent has one or more or all of the following characteristics: The rodent expresses a chimeric antibody heavy chain expressed from IGHJ4, individually, more than any of IGHJ1, 2, 3, 5, or 6; The rodent expresses a chimeric antibody heavy chain expressed from IGHJ6, individually, more than any of IGHJ1, 2, 3, or 5; The rodent expresses a chimeric antibody heavy chain expressed 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.

[0217] The rodent according to any one of claims 1 to 13, 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.

[0218] The rodent according to any one of claims 1 to 13, 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.

[0219] The rodent according to any one of claims 1 to 13, 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.

[0220] The rodent according to any one of claims 1 to 13, expressing a population of chimeric antibody chains, each antibody chain resulting from the expression of at least one companion animal gene segment, and the chimeric antibody population being more diverse than the antibody population found in the corresponding wild-type companion animal.

[0221] The rodent according to any one of claims 1 to 13, comprising a population of chimeric antibody chains, at least 65% or at least 70% of which are unique, and optionally the rodent is a dog.

[0222] The rodent according to any one of claims 1 to 13, wherein 65 to 80% of the chimeric antibody sequence, such as 65 to 75% in rodents, is unique.

[0223] Use of any rodent (e.g., mouse) comprising the DNA of a companion animal as defined in claims 1 to 13 in the generation of a more diverse repertoire of chimeric antibody chains or antibodies than is found in the companion animal itself.

[0224] A rodent such as a mouse for generating a more diverse repertoire of chimeric antibody chains or antibodies than is found in the companion animal itself, according to any one of claims 1 to 13.

[0225] The rodent according to any one of claims 1 to 13, 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 individual V gene segment, and / or a splice site, and / or a recombination signal sequence for V(D)J recombination.

[0226] The rodent according to any one of claims 1 to 13, wherein an RSS sequence from the same companion animal is adjacent to the inserted companion animal V, D, or J sequence, and the RSS sequence of the host rodent is not used.

[0227] The rodent according to any one of claims 1 to 13, comprising the DNA of an animal of a certain breed of Canidae and used in the generation of an antibody or antibody chain, or a part thereof, for use in the treatment or prevention of diseases of different dog breeds.

[0228] Use of the rodent according to any one of claims 1 to 13 in the generation of an antibody chain or a part thereof for use in the treatment of different dog breeds, comprising the DNA of an animal of a certain breed of Canidae.

[0229] The rodent according to any one of claims 1 to 13, wherein the V, D, and J segments of one or more or all of the Canidae animals are from a boxer dog.

[0230] It will be understood that the specific embodiments described herein are presented by way of example and not limitation 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. One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures described herein. 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 level of skill of those of ordinary skill 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" when used in conjunction with the term "comprising" in the claims and / or this specification may mean "one" in some cases, but it also conforms to the meaning 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 the alternatives are mutually exclusive, even if the present disclosure supports a definition that refers only to alternatives and "and / or". Throughout this application, the term "about" is used to indicate that a value includes the inherent variability of error for the device, i.e., the method employed to determine the value, or the variability that exists between the subjects of study.

[0231] As used in this specification and the claims, the term "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") is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0232] 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 that include 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 to the number of items or terms in any given combination.

[0233] Any part of this disclosure can be read in combination with any other part of this disclosure, unless the context clearly indicates otherwise.

[0234] All compositions and / or methods disclosed and claimed in this specification can be made and executed without undue experimentation in light of this 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 steps or the order of the 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.

[0235] The invention is illustrated in more detail in the following non-limiting examples.

Examples

[0236] (Example 1) Annotation of the IG locus of Canidae 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 the loci of their antigen receptor (AR) genes are only incompletely understood. This study advanced the annotation of the AR loci in canids, developed a method to investigate their evolutionary stress, and examined breed-specific features of the loci. 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. Using a combination of existing and novel methods, the diversity and mutation rates across these genes were analyzed. 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 or either of them and mice than between mice and humans. 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 AR loci annotated. The numerous genome sequences will be useful for future research and were able to draw statistically powerful conclusions about the stresses that shaped these loci.

[0237] 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.

[0238] 2. Materials and Methods 2.1.1 Annotation by Bioinformatics Seats were initially annotated using a principle equivalent to the algorithm of Olivieri et al. according to a method similar to that of Das et al. 1、2 . Briefly, the reference genome of Canidae (CanFam3.1) was investigated using human and mouse sequences. When regions were identified, the AR gene was searched locally. Initially, the AR genes and RSS consensus of mouse and human 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.

[0239] 2.1.2 Dog Peripheral blood samples were obtained from 26 dogs. The samples were unused clinical excesses of blood collection mandated by veterinarians from affected animals seen at the veterinary hospital of the University of Cambridge. This study had been pre-approved by the Ethics Committee of the University of Cambridge's Veterinary School.

[0240] 2.1.3 Sequencing According to the manufacturer's instructions, Ficoll-Paque (GE Healthcare) was used to isolate mononuclear cells from peripheral blood. 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.

[0241] 2.1.4 Gene Naming The AR genes were divided into families and assigned as functional, pseudogenes, or ORFs using the same criteria as Bao et al. 3 . Family numbers were assigned based on homology to human families, and new numbers were given when no obvious match was found. All gene names were assigned according to the naming system of IMGT. 4

[0242] 2.1.5 Non-reference Alleles 10 7 Mapping of the variant call file to the AR locus from the whole genome sequences of 10 individual canids was kindly provided by Steven Friedenberg of the University of Minnesota.

[0243] 2.1.6 Inter- and Intra-species Locus Alignments The sequences were masked using RepeatMasker and alignment plots were generated using PipMaker. 5、6 。

[0244] 2.1.7 Phylogenetic Tree Analysis The sequences were aligned using Clustal Omega and the output tree was visualized using the Interactive Tree of Life. 7、8 。

[0245] 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.

[0246] 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 out of the 68,908 alleles called (77%). No significant breed specificity in allele distribution was identified.

[0247] Functional, pseudogene, or ORF assignments were made for all novel alleles and compared back to the reference alleles. Most (72.8%) of the novel alleles have the same function as the reference alleles. When considering the function of the novel alleles as the function of the alleles under selection, there are more alleles that are "loss" (23.8%), i.e., the novel alleles are pseudogenes rather than being "gain" (3.4%) where the novel alleles are functional. This is not surprising as there are far 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 a loss, and changes from a pseudogene to an ORF were classified as a gain.

[0248] 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 happens to have nine rare alleles that do not appear in this dataset. Considering non-reference alleles, when there is no selection pressure, 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.

[0249] 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 pressure 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 cannot display p-values less than 1×10 -250 .

[0250] 3.1.3 Structure of the Locus 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.

[0251] 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 location and predicted function 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).

[0252] 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.

[0253] 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 gene, but the loci of dogs, humans, and pigs have undergone an inverted duplication of the entire block. 10、11 In the pig and human loci, the genes of the two blocks have not diverged sufficiently, so some or all are known by the same names as their counterparts in the other block. 10、12 ​

[0254] 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 selection pressure, with only 3 out of 116 of the most C-proximal IGLV genes in the opposite transcriptional orientation to the J-C genes, and 26 out of the last 46 inverted V genes. This increase is partly due to duplication of blocks in which segments of IGLV1 and IGLV8, showing very high levels of sequence identity, are positionally conserved within the member repeat blocks.

[0255] From a broader perspective of trends, the canid IGL 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, potentially reflecting different tolerances for pseudogenes between the heavy and light chains. Furthermore, the gene number has been found to correlate with the skewed chain usage in the light chains of most species studied to date. 14、15 For example, the mouse IGL locus, containing 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% usage of the IGK chain has been reported, depending on the relative sizes of the two light chain repertoires of the various species. 15 。

[0256] 3.1.4 Inter- and Intra-Species Locus Alignments Aligning genomic regions against themselves and against 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、18Alignments were performed using the IGK, IGL, and TRA / D loci of Canidae animals against themselves and against the mouse and human loci, respectively.

[0257] In the IGK self-alignment, three comparisons are of note: the self-alignments of the upstream and downstream blocks (the green and red boxes in Figure 8, respectively), and their mutual alignment (the blue box in Figure 8). Within the percent 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 the gaps represent indels and other mutations accumulated after the overlap.

[0258] 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 sequence are locally duplicated multiple times. 16 In the self-alignment of the downstream block, the lines are shorter and sometimes vertical, indicating 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 would be expected if a single gene was 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 subject to reduced selective pressure and has accumulated mutations and local inversions at a higher rate, excluding the less divergent IGKV2-S18 and IGKV-S19 genes. While this is not the only possible explanation, it is consistent with the proposed explanations for similar features across other species and AR loci.

[0259] In the comparison of the IGL loci between dog and human, two things are striking. A nearly seamless diagonal near the midpoint of the human sequence, and 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 dog and human, hinting that they may have a similar functional importance for the dog as for humans. Specific comparisons suggest that the ZNF280B and PRAME genes are likely to function in the dog, but ZNF280A could not be reliably identified at this locus (data not shown). Further investigation identified another non-IGL gene, PCBP2, near ZNF280B.

[0260] 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, no ortholog has 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.

[0261] 3.1.5 Allele Distribution One prominent feature of the use of dogs as model organisms is that while there is high heterogeneity between breeds, there is very high breed homogeneity, 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 certain breeds, which the authors attribute to the selection pressure imposed by breeders. 20 This is in contrast to the diversity seen in humans. Even in geographically isolated populations, the variation resulting from this separation is 5 - 10%, whereas more than a quarter of the genomic variation in dogs results from breed variation rather than individual variation. 21 。

[0262] 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 the Boxer and the Standard Poodle (22 and 20 dogs respectively), and they 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.

[0263] Larger breed-specific cohort sizes may reveal trends not apparent in this dataset, but currently, the selection pressure shaping the AR gene locus appears to be independent of breed and stronger than the breed itself. The potential for 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 sequenced boxer chromosomes and 1 out of 6 toy poodle chromosomes, but not in any other breeds. 194 out of 217 alleles called across all breeds are represented by the other allele, IGLC1*02. Whether the toy poodle was previously crossed with a boxer or there were sequencing errors and this is actually a boxer-specific allele remains an open question that may require a larger dataset to ultimately answer in some way.

[0264] Considering the distribution of non-reference alleles, these all match what would be expected. The lack of more frequent changes than expected is likely because the gene in question is under selection pressure 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.

[0265] The fact that gain of function is more common than expected lends weight to theories proposed regarding the high 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, a mutational starting pool of new beneficial alleles, and is under such selection pressure.

[0266] (References) 1. Das, S., Nozawa, M., Klein, J. & Nei, M. Evolutionary dynamics of the immunoglobulin heavy chain variable region genes in vertebrates. Immunogenetics 60, 47-55 (2008). 2. Olivieri, D., Faro, J., Von Haeften, B., Sanchez-Espinel, C. & Gambon-Deza, F. An automated algorithm for extracting functional immunologic V-genes from genomes in jawed vertebrates. Immunogenetics 65, 691-702 (2013). 3. Bao, Y., Guo, Y., Xiao, S. & Zhao, Z. Molecular characterization of the VH repertoire in Canis familiaris. Vet. Immunol. Immunopathol. 137, 64-75 (2010). 4. Lefranc, M. P. et al. IMGT(R), the international ImMunoGeneTics information system(R). Nucleic Acids Res. 37, 1006-1012 (2009). 5. Smit, A. F. A., Hubley, R. & Green, P. RepeatMasker. unpublished data Available at: http: / / www.repeatmasker.org / cgi-bin / WEBRepeatMasker. 6. Schwartz, S. PipMaker---A Web Server for Aligning Two Genomic DNA Sequences. Genome Res. 10, 577-586 (2000). 7. McWilliam, H. et al. Analysis Tool Web Services from the EMBL-EBI. Nucleic Acids Res. 41, W597-W600 (2013) 8. Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics 23, 127-128 (2007). 9. Hara, S., Diesterbeck, U. S., Konig, S. & Czerny, C. P. Transcriptional analysis of equine λ-light chains in the horse breeds Rhenish-German Coldblood and Hanoverian Warmblood. Vet. Immunol. Immunopathol. 145, 50-65 (2012). 10. Schwartz, J. C., Lefranc, M.-P. & Murtaguh, M. P. Evolution of the porcine (Sus scrofa domestica) immunoglobulin kappa locus through germline gene conversion. Immunogenetics 64, 303-311 (2012) 11. Walther, S., Rusitzka, T. V, Diesterbeck, U. S. & Czerny, C. Equine immunoglobulins and organization of immunoglobulin genes. Dev. Comp. Immunol. 53, 303-319 (2015). 12. Kawasaki, K. et al. Evolutionary dynamics of the human immunoglobulin kappa locus and the germline repertoire of the Vkappa genes. Eur. J. Immunol. 31, 1017-28 (2001). 13. Schwartz, J. C., Lefranc, M.-P. & Murtaguh, M. P. Organization, complexity and allelic diversity of the porcine (Sus scrofa domestica) immunoglobulin lambda locus. Immunogenetics 64, 399-407 (2012) 14. Sun, Y., Wei, Z., Li, N. & Zhao, Y. A comparative overview of immunoglobulin genes and the generation of their diversity in tetrapods. Dev. Comp. Immunol. 39, 103-9 (2013). 15. Arun, S. S., Breuer, W. & Hermanns, W. Immunohistochemical examination of light-chain expression (lambda / kappa ratio) in canine, feline, equine, bovine and porcine plasma cells. Zentralbl. Veterinarmed. A 43, 573-6 (1996). 16. Mineccia, M. et al. New insight into the genomic structure of dog T cell receptor beta (TRB) locus inferred from expression analysis. Dev. Comp. Immunol. 37, 279-293 (2012). 17. Massari, S. et al. The deduced structure of the T cell receptor gamma locus in Canis lupus familiaris. Mol. Immunol. 46, 2728-2736 (2009). 18. Koop, B. F. & Hood, L. Striking sequence similarity over almost 100 kilobases of human and mouse T-cell receptor DNA. Nat Genet 7, 48-53 (1994). 19. Dobson, J. M. Breed-Predispositions to Cancer in Pedigree Dogs. ISRN Vet. Sci. 2013, 1-23 (2013). 20. Vaysse, A. et al. Identification of Genomic Regions Associated with Phenotypic Variation between Dog Breeds using Selection Mapping. PLoS Genet. 7, e1002316 (2011). 21. Parker, H. G. Genomic analyses of modern dog breeds. Mamm. Genome 23, 19-27 (2012). 22. Sun, Y. et al. Immunoglobulin genes and diversity: what we have learned from domestic animals. J. Anim. Sci. Biotechnol. 3, 18 (2012).

[0267] (Example 2) Construction of chimeric IG locus 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:

[0268] 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.

[0269] Canine IGL lambda DNA insertion Insertion of Canidae 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 through IGLV4-6, as well as IGLJ1-9 and IGLC1-9. See Figures 2 and 13.

[0270] Coordinates are from the GRCm38 / mm10 assembly in December 2011 for mouse and Canfam3.1 for dog.

[0271] Insertion of Canidae DNA was made into landing pads at the following locations in the mouse genome. Canidae heavy chain DNA: inserted immediately upstream of mouse chromosome 12, position 114,666435 Canidae kappa DNA: inserted immediately upstream of mouse chromosome 6, position 70,674,7.55 Canidae lambda DNA: inserted immediately upstream of mouse chromosome 16, position 19,047,551

[0272] Expression of chimeric transcripts has been confirmed by PCR analysis from chimeric heavy loci - see Figure 15.

[0273] The method was as follows.

[0274] BAC modification BACs of Canidae from the CHORI-82 library were obtained directly from the BAC library at the Children's Hospital Oakland Research Institute.

[0275] 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.

[0276] The 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 selectable markers for the BAC or ESC engineering process. The completed plasmids were digested with restriction enzymes and gel-purified to obtain fragments spanning the BAC-specific homology arms, the introduced recombination sequences, and the homology arms for the vector backbone of the BAC. These fragments were inserted into the BAC by electroporation, and those with successful integration of the fragments into the BAC were selected.

[0277] The successful modification of the resistant clones was verified by PCR across the junctions between the inserted DNA and the endogenous DNA. When both ends of the BAC were modified, the BAC DNA was purified and electroporated into Electromax (trademark) DH10B cells (Life Technologies). Clones that lost hygromycin resistance and were thus negative for pSIM18 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).

[0278] ESC Genetic Manipulation The processes of ESC culture, electroporation, and drug selection were carried out 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.

[0279] For each transfection, 1×10 7Individual cells were used and all transfections were performed using a Bio-Rad electroporator (GenePulser Xcell) at 500 μF and 230 V. For the introduction of BAC, 10 μg of BAC DNA and 25 μg of pCAGGS-iCRE were used per transfection. After 24 hours, the cells were selected with 3 μg / ml puromycin for one week and colonies were selected for growth and testing. PCR using primers spanning the junction between the mouse and the BAC DNA was used to confirm successful integration of the BAC into the landing pad.

[0280] The positive clones were then subjected to excision of the 3' landing pad using PBase. The clones were grown and 1 × 10 5 cells were used per electroporation of 10 μg of PiggyBac transposase plasmid. After a 3-day recovery in M15, the cells were split, seeded at low density, and then 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.

[0281] Generation and analysis of mice Positive ES cell clones were injected into blastocysts of the C57BL / 6 Tyrc-Brd mouse strain according to standard procedures. The injected blastocysts were transferred into the uterus of pseudopregnant female B6 / CBA F1 recipients. Approximately 40 blastocysts were injected for each clone. To enable identification of puppies 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 testing using primers spanning the junction between the mouse and the inserted DNA.

[0282] 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 aspirated 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 the mouse 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 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.

[0283] All animal experiments and breeding were carried out under the approval of the Wellcome Trust Sanger Institute AWERB (Animal Welfare and Ethical Review Body). Home Office approval in the UK was provided under project license 80 / 2432.

[0284] (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.

[0285] Annotations provide tools and information for using feline DNA in the genomes of rodents using the methods described above.

[0286] (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 Veterinary Medicine. RNA was extracted on site and transported on dry ice. This study had prior approval from the University of Minnesota Veterinary Medicine Institutional Review Board.

[0287] Library Preparation First-strand cDNA was synthesized using a mixed pool of C-specific primers and template switch oligos (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 cleanup as before and resuspended in 10.5 μl of PCR-grade water. This was done in a 25 μL PCR reaction using Q5 polymerase and 400 nM each of the forward primer containing 5'RRVA and the sample-specific index hexamer. The PCR conditions were the same as before, and the products were 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.

[0288] 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 of the Wellcome Trust Sanger Institute. The quality of the demultiplexed data was then filtered (using the "HighV-Quest" software and selecting "species" = "Canis lupus familiaris (dog)" and "receptor type or locus" = "IG") before being sent to the IMGT V-Quest software 14 。

Claims

【Claim 1】 i) comprising the IGH V region gene of one or more companion animals, the D region gene of one or more companion animals, and the J region gene of one or more companion animals, 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 may be included A rodent or rodent cell having a genome, wherein the rodent or rodent cell can express the variable region gene of the companion animal to form an antibody chain, the companion animal species is not a rodent, a rodent or rodent cell.

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