Animal models and therapeutic molecules

Abstract

The present invention relates 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, wherein the rodent or rodent cell is capable of expressing the companion animal variable region genes to form antibody chains, and wherein the species of the companion animal is not rodent.

Description

[Technical Field]

[0001] The present invention relates, in particular, to rodents and cells genetically engineered to contain the exogenous DNA of companion animals, their use in medical and disease research, methods for producing rodents and cells, and antibodies and antibody chains and derivatives thereof produced from such animals. [Background technology]

[0002] 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, 2014 DOI:, doi:10.1038 / nbt.2825. This approach is designed to produce antibody products for therapeutic use in humans. However, rodent models suitable for generating antibodies for use in other species, such as companion animals, have not been developed. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] US2017306352 [Non-patent literature]

[0004] [Non-Patent Document 1] Murphy et al., Volume 111, Issue 14, Pages 5153-5158, doi:10.1073 / pnas.1324022111 [Non-Patent Document 2] MacDonald et al., Volume 111, Issue 14, Pages 5147-5152, doi:10.1073 / pnas.1323896111 [Non-Patent Document 3] Lee et al., Nature Biotechnology, Vol. 32, pp. 356-363, Publication Year: 2014, DOI:, doi:10.1038 / nbt.2825 [Non-Patent Document 4] Harlow, E. and Lane, D., 1998, 5th edition, "Antibodies: A Laboratory Manual," Cold Spring Harbor Lab. Press, Plainview, NY. [Non-Patent Document 5] Pasqualini and Arap, Proceedings of the National Academy of Sciences (2004) 101: pp. 257-259 [Non-Patent Document 6] Sambrook, J. and Russell, D. (2001, 3rd edition) "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Lab. Press, Plainview, NY) [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The present invention relates to such rodents, cells, antibodies, and portions thereof produced therefrom, for example, subsequently modified antibodies for use in companion animals, as well as methods for producing such rodents, cells, antibodies, and antibody chains. [Means for solving the problem]

[0006] This invention relates to the following: i) IGH V region genes of one or more companion animals, IGH D region genes of one or more companion animals, and IGH J region genes of one or more companion animals, 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 A rodent or rodent cell having a genome containing 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 A rodent or rodent cell having a genome containing 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 a gene of a companion animal and form an antibody chain in combination with a constant region of the rodent or companion animal.

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

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

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

[0012] A method for producing an antibody chain or a part thereof having a variable region of a companion animal, comprising the step of expressing, in a cell, a nucleic acid encoding the antibody chain or a part thereof, The sequence of the DNA encoding the variable region of the antibody chain is obtained or can be obtained by immunizing the rodent of the present invention with an antigen so that the antibody chain is produced, If necessary, the method is as follows: A step of purifying and / or isolating antigen receptor chains, If necessary, the process then includes a variable region, which involves formulating the antigen receptor chain into a pharmaceutically acceptable formulation suitable for administration to a companion animal, preferably the same companion animal, or to a human or other mammal that requires it. Methods that include...

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

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

[0015] A method for treating a companion animal, comprising the step of 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 a complete companion animal antibody. [Brief explanation of the drawing]

[0016] [Figure 1A] This figure shows an immunoglobulin heavy chain locus of a canid animal, covered by a bacterial artificial chromosome spanning the locus. The BAC is shown to further illustrate both that a group of genes from canids and felids are located in publicly available BACs, and that such genes can be incorporated into rodent loci according to the present invention. [Figure 1B] The continuation of Figure 1A is shown. [Figure 2A] This figure shows the immunoglobulin kappa locus of a canid animal, covered by a bacterial artificial chromosome spanning the locus. The BAC is shown to further illustrate both that a group of genes from canids and felids are located in the publicly available BAC, and that such genes can be incorporated into the rodent locus according to the present invention. [Figure 2B] The continuation of Figure 2A is shown. [Figure 2C] Figure 2B continues. [Figure 3] This figure shows the immunoglobulin lambda locus of a canid animal, covered by a bacterial artificial chromosome spanning the locus. The BAC is shown to further illustrate both that a group of genes from canids and felids are located in the publicly available BAC, and that such genes can be incorporated into the rodent locus according to the present invention. [Figure 4] This figure shows a feline immunoglobulin heavy chain locus covered with a bacterial artificial chromosome spanning the locus. The BAC is shown to further illustrate both that a group of canine and feline genes are located in publicly available BACs, and that such genes can be incorporated into rodent loci according to the present invention. [Figure 5] This figure shows the immunoglobulin kappa locus of a feline animal, covered by a bacterial artificial chromosome spanning the locus. The BAC is shown to further illustrate both that a group of genes for canids and felines are located in the publicly available BAC, and that such genes can be incorporated into the rodent locus according to the present invention. [Figure 6] This figure shows the immunoglobulin lambda locus of a feline animal, covered by a bacterial artificial chromosome spanning the locus. The BAC is shown to further illustrate both that a group of genes for canids and felines are located in the publicly available BAC, and that such genes can be incorporated into the rodent locus according to the present invention. [Figure 7]This diagram shows allele changes. The abundance of non-reference V gene alleles is plotted for each variety based on three classes. Alleles that functioned when the reference allele was absent, as well as alleles whose reference was a pseudogene (ORF), are classified as "gain." Alleles that were pseudogenes without a reference allele, as well as functional alleles that became ORFs, are classified as "loss." Alleles with no change in function are classified as "None." The black crosshairs represent the expected value for each type of change. [Figure 8] This diagram shows the self-alignment of the immunoglobulin kappa locus in canids. The green box (A) represents the upstream V gene aligned to itself. The red box (C) represents the downstream V gene aligned to itself. The blue box (B) represents the alignment of the upstream and downstream V genes. [Figure 9] This figure shows the alignment of immunoglobulin loci in canids and humans. (A) Complete loci. (B) Amplified section of the alignment corresponding to the red box in (A). [Figure 10] This figure shows the insertion of BAC 1 in a canid animal into the mouse IGH locus. [Figure 11] This figure shows the deletion site for mouse IGH. [Figure 12] This figure shows the results of deleting IGH in mice. [Figure 13] This figure shows the insertion of BAC 1 in a canid animal into the mouse IGL lambda locus. [Figure 14] This figure shows the deletion site for mouse IGK. [Figure 15] This figure shows the expression of chimeric transcripts confirmed by PCR analysis of chimeric heavy chain loci. [Figure 16] This figure shows strong germline homology between canids and humans, but TCR homology is not well conserved. [Figure 17] This figure shows that a comparison of germline IG V homology reveals a strong correlation between dogs and cats. [Figure 18] This diagram shows the BAC recombination process. A: Modified vector with unmodified 5' (top) and 3' (bottom) ends, with the linearization site shown in red. B: Linearized vector with BAC homology arms, ready for Gibson assembly. C: Assembled vector ready for digestion to release vector fragments. The digestion site is shown in red. D: Purified vector fragment ready for recombination. E: Recombination occurs along the dashed line between the fragment and the unmodified BAC. F: Recombinated BAC ready for S-RMCE. [Figure 19] This figure shows the S-RMCE process and screening. A: Recombinant BAC is inserted into the landing pad by the action of Cre recombinase. B: Successful BAC insertion can be screened using primers P1+P2 and P3+P4. C: 3' modified DNA is excised by the action of PB-ase (excision points are indicated by dashed lines). Successful excision can be screened using primers P5+P6. [Figure 20] This figure shows the seating positions of chimeric IGH and IGL in Ky9 mouse V0.5. [Figure 21] This figure shows that Ky9 mouse V0.5 generates a very diverse antibody repertoire. [Figure 22] This figure shows the chronotype richness in Ky9 V0.5 compared to healthy dogs. [Figure 23] This figure shows the use of IGHD and IGHJ in Ky9 V0.5 on baseline (healthy dogs). [Figure 24] This diagram shows the use of IGLJ in Ky9 V0.5. [Figure 25] This figure shows the addition of N and P nucleotides observed in Ky9 V0.5 mice. [Figure 26] The figure shows the mutation rates for each region, providing evidence of somatic hypermutation in Ky9 V0.5 mice. [Modes for carrying out the invention]

[0017] The present invention i) IGH V region genes of one or more companion animals, IGH D region genes of one or more companion animals, and IGH J region genes of one or more companion animals, ii) If necessary, the IGL kappa V region gene and the IGL kappa J region gene of one or more companion animals; and / or the IGL lambda V region gene and the IGL lambda J region gene of one or more companion animals A rodent or rodent cell having a genome containing, Rodents or rodent cells can express variable region genes of companion animals and form antibody chains in combination with the constant region of an antibody. This relates to rodents or rodent cells whose companion animal species are not rodents.

[0018] The present invention also relates to the following: i) IGL kappa V region genes of one or more companion animals and IGL kappa J region genes of one or more companion animals; and / or IGL lambda V region genes of one or more companion animals and IGL lambda J region genes of one or more companion animals, ii) If necessary, 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 A rodent or rodent cell having a genome containing, Rodents or rodent cells can express variable region genes of companion animals and form antibody chains in combination with the constant region of an antibody. A companion animal species that is not a rodent, but a rodent or rodent cell.

[0019] As an exemplary example, and not limited to the present invention, insertion of canine immunoglobulin heavy (IGH) chain variable (V) region genes, IGH D region genes, and IGH J region genes into mice, in combination with a constant region, enables the production of antibody heavy chains containing variable antibody regions derived from the expression of canine DNA in mice. The constant region may be a rodent immunoglobulin (IG) constant region, resulting in the production of a chimeric heavy chain having a canine variable region and a rodent constant region. Information on the variable region of such a chimeric antibody chain, or nucleic acids containing it, can be used, for example, to produce complete canine antibodies for therapeutic use in dogs. Rodents containing canine DNA can also serve as animal models for understanding diseases and testing pharmaceuticals.

[0020] Unless otherwise specified, all mouse nucleotide coordinates correspond to the mouse GRCm38 / mm10 assembly from December 2011 (assembly contract GCA_000001635.2).

[0021] To avoid any doubt, the insertions referenced in the mouse genome are identical to those detailed in Lee et al., Nature Biotechnology, Nature Biotechnology 32, pp. 356-363 (2014).

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

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

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

[0025] The companion animals of the present invention are appropriately selected from dogs, cats, horses, birds, rabbits, goats, reptiles, fish, and amphibians. Dogs are preferred companion animals of the present invention. Cats are preferred companion animals of the present invention. Horses are preferred companion animals of the present invention. To avoid doubt, humans are not companion animals.

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

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

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

[0029] The IG heavy chain (IGH) locus of the companion animal contains multiple V, D, and J region genes. When the V, D, and J region genes are expressed together, a variable region of the antibody heavy chain is generated. The IGH V, D, and J genes are spontaneously expressed in combination with the heavy chain constant region. The IG light chain locus (IGL), which may be lambda or kappa, contains multiple V and J gene segments that, when expressed together, form a variable region of the antibody light chain. The IGL V and J region genes are spontaneously expressed in combination with the light chain constant region of the kappa or lambda light chain. The rodent or rodent cell of the present invention can express the VDJ or VJ region genes of the companion animal to form an antibody chain. The companion animal genes are operably ligated to a constant region in the rodent genome to enable antibody chain expression. Companion animal IG genes can be located in the rodent genome along with exogenous constant region genes (derived from non-rodent species), or they can be located in the rodent genome in a functional arrangement, such as upstream, along with naturally occurring rodent constant regions, and expression of V region genes associated with constant regions can occur.

[0030] The genome of a rodent or rodent cell may contain IGH V, IGH D, and IGH J region genes of one or more companion animals but not light chain companion animal DNA, or may contain IGL V and IGL J region genes of one or more companion animals but not heavy chain companion animal DNA. The genome of a rodent or cell may contain companion animal genes derived from heavy chain and kappa chain (not lambda), or heavy chain and lambda chain (not kappa), or companion animal genes derived from all three loci, heavy chain, kappa, and lambda.

[0031] In one embodiment, the inserted companion animal DNA includes at least 50% of the companion animal's heavy chain variable (V) genes, for example, at least 60%, at least 70%, at least 80%, at least 90%, etc., and in one embodiment, it includes all of the companion animal's V genes.

[0032] In one embodiment, the inserted companion animal DNA includes at least 50% of the companion animal's heavy chain diversity (D) genes, for example, at least 60%, at least 70%, at least 80%, at least 90%, etc., and in one embodiment, it includes all of the companion animal's D genes.

[0033] In one embodiment, the inserted companion animal DNA includes at least 50% of the companion animal's heavy chain (J) genes, for example, at least 60%, at least 70%, at least 80%, at least 90%, etc., and in one embodiment, it includes all of the companion animal's J genes.

[0034] In one embodiment, the inserted companion animal DNA includes at least 50% of the companion animal's light chain variable (V) genes, for example, at least 60%, at least 70%, at least 80%, at least 90%, etc., and in another embodiment, it includes all of the companion animal's light chain V genes.

[0035] In one embodiment, the inserted companion animal DNA includes at least 50% of the companion animal's light chain ligature (J) genes, for example, at least 60%, at least 70%, at least 80%, at least 90%, etc., and in another embodiment, it includes all of the companion animal's light chain J genes.

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

[0037] In one embodiment, the rodent genome includes all of the IGL kappa V and J region genes, as well as intervening sequences, derived from the companion animal.

[0038] In one embodiment, the rodent genome includes all of the IGL-lambda V and J region genes, as well as intervening sequences, derived from the companion animal.

[0039] The genome of a rodent or rodent cell may contain at least 4, 5, 10, 15, or 20 companion animal IGH V region genes, for example, at least 30, 40, 50, 60, 70, or 80 V region genes. In one preferred embodiment, these are canid V region genes. In one preferred embodiment, the rodent genome contains at least 83 canid IG heavy chain V region genes.

[0040] The genome of a rodent or rodent cell may contain at least one, two, three, four, five, or six IGHD region genes derived from a companion animal, preferably from a canid.

[0041] The genome of a rodent or rodent cell may contain at least one, two, three, four, five, or six IGHJ region genes derived from a companion animal, preferably from a canid.

[0042] The genome of a rodent or rodent cell may contain at least 10, 15, 16, 17, 18, or 19 companion animal IGL kappa V region genes. In a preferred embodiment, these are kappa V region genes of canids. In a preferred embodiment, the genome of a rodent contains at least 19 light chain kappa V region genes of canids.

[0043] The genome of a rodent or rodent cell may contain at least one, two, three, four, or five IGL kappa J region genes derived from a companion animal, preferably from a canid.

[0044] The genome of a rodent or rodent cell may contain at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or at least 160 companion animal IGL lambda V region genes. In a preferred embodiment, these are lambda V region genes of canids. In a preferred embodiment, the rodent genome contains at least 160 canid light chain lambda V region genes.

[0045] The genome of a rodent or rodent cell may contain at least one, two, three, four, five, six, seven, eight, or nine IGL lambda J region genes derived from a companion animal, preferably from a canid.

[0046] In another embodiment, the genome of a rodent or rodent cell may include at least 4, 5, 10, 15, or 20 IGH V region genes derived from cat, for example, at least 23 V region genes.

[0047] In another embodiment, the genome of a rodent or rodent cell may include at least 4, 5, 10, or 11 companion animal IGH D region genes of cat origin.

[0048] In another embodiment, the genome of a rodent or rodent cell may include at least one, two, three, four, or five companion animal IGH J region genes of cat origin.

[0049] In another embodiment, the genome of a rodent or rodent cell may contain at least 4, 5, 10, or 15 IGL kappa V region genes derived from cats.

[0050] In another embodiment, the genome of a rodent or rodent cell may include at least one, two, three, four, five, or six companion animal IGH kappa J region genes of cat origin.

[0051] In another embodiment, the genome of a rodent or rodent cell may contain 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 feline origin.

[0052] In another embodiment, the genome of a rodent or rodent cell may include at least one, two, three, four, five, six, seven, eight, or nine companion animal IGH-lambda J region genes of feline origin.

[0053] The number of genes in the companion animal mentioned in any of the above embodiments may increase further, and in one embodiment, it may double in the case of a homozygous individual with insertions in both alleles.

[0054] Preferably, the V, D, and J region genes inserted into the genome are derived from the same companion animal. Preferably, the inserted IGH VDJ region genes or IGL VJ region genes are all from canids, all from felines, or all from equids. Preferably, all genes are from canids.

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

[0056] In one embodiment, the companion animal genes are located upstream of the rodent's constant region genome, and for the inserted companion animal heavy chain variable region genes, they are appropriately located upstream of the heavy chain constant region(s), and / or for the inserted companion animal light chain variable region genes, they are appropriately located upstream of the light chain constant region, thereby enabling the rodent or rodent cell to produce a chimeric antibody chain resulting from the expression of the inserted variable region gene and the rodent's constant region.

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

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

[0059] Any reference to the location of a variable region upstream of a constant region, such as the constant region of a rodent, implies the existence of appropriate relative positions between the two genomic regions encoding the variable and constant regions of the antibody, enabling the expression of a chimeric antibody chain in vivo in the rodent. In this way, the inserted companion animal DNA and the constant region of the rodent are functionally positioned relative to each other for the production of the antibody or antibody chain.

[0060] In one embodiment, the companion animal DNA into which a variable VDJ or VJ region gene is inserted is located at a different site from the naturally occurring heavy or light chain constant region of the rodent genome, 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 a 3' enhancer derived from the rodent or companion animal. One preferred embodiment is the use of a rodent constant region and a rodent 3' enhancer in conjunction with the companion animal's VDJ or VJ region, such as a canid constant region and a canid 3' enhancer. In one embodiment, the companion animal gene is located in the genome in a functional arrangement with a constant region derived from the same companion animal, and the rodent can produce antibody chains resulting from the expression of the inserted companion animal's VDJ or VJ region gene and the companion animal's constant region. Alternatively, companion animal genes are located in the genome in a functional arrangement with constant regions derived from other companion animals, such as different companion animals, or constant regions derived from rodents.

[0061] When a companion animal gene is inserted into the rodent genome along with a constant region, it is understood that the insertion may occur at any suitable location within the rodent cell genome, and may not target the rodent IG locus, since the endogenous constant region gene is not required for antibody chain production. The insertion may occur at a random location within the rodent genome.

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

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

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

[0065] In one embodiment, a rodent or rodent cell contains one or more companion animal IGL lambda V region genes, one or more companion animal IGL lambda J region genes, and one or more companion animal lambda constant regions located within the kappa locus of the rodent cell, for example, at or upstream of the kappa constant region of the rodent. Preferably, the insertion is located upstream of the IGL kappa locus constant region so that the IGL lambda V and J region genes are expressed in the IGL kappa constant region. Appropriately, the insertion places the companion animal genes at substantially the same position as the kappa genes of the native rodent, potentially deleting or substituting them, for example, the distance from the last inserted 3' lambda J gene to the rodent kappa constant region gene is the same or substantially the same distance as from the last rodent kappa 3' J gene to the kappa constant region. In one embodiment, 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. Mouse kappa light chains are naturally expressed at higher levels than mouse lambda light chains, and insertion of companion animal DNA into this kappa locus can result in high levels of expression of companion lambda V region genes.

[0066] In one embodiment, the genome of a rodent may be homozygous for the insertion of a companion animal gene at one, both, or all three immunoglobulin loci.

[0067] In another embodiment, the genome of a rodent may be heterozygous for the insertion of a companion animal gene at one, two, or all three immunoglobulin loci.

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

[0069] In one embodiment, the inserted DNA can be expressed along with the constant regions of different rodents by isotype switching.

[0070] In one embodiment, the inserted companion animal DNA can be expressed together with the constant region of a different rodent by trans-switching.

[0071] In one embodiment, the companion animal is a dog, the rodent genome contains kappa variable region genes of canids, and all kappa variable region genes of canids in the rodent genome are located upstream of the constant region in which the variable region genes are expressed together, for example upstream of the kappa constant region of rodents, or for example upstream of the kappa constant region of canids.

[0072] In another preferred embodiment, the companion animal is a horse, the rodent genome contains kappa variable region genes of an equid, and all kappa variable region genes of an equid in the rodent genome are located upstream of the constant region in which the variable region genes are expressed together, for example upstream of the kappa constant region of the rodent, or for example upstream of the kappa constant region of an equid.

[0073] In one embodiment, the companion animal's DNA is inserted between the wild-type constant region of the rodent located at the wild-type locus, and preferably between the constant region of the rodent and the host VDJ or VJ region. In another embodiment, the IGH variable region gene is inserted downstream of the heavy chain J region and upstream of the Emu enhancer.

[0074] In one embodiment, 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 embodiment, the insertion of the IGH V region gene occurs at position 114666435 of the mouse genome on mouse chromosome 12. In one embodiment, the insertion of the IGL lambda V region gene occurs at position 19047551 of the mouse genome on mouse chromosome 16. In one embodiment, the insertion of the IGL kappa V region gene(s) occurs at position 70674755 of the mouse genome on mouse chromosome 6.

[0075] In one embodiment, the rodent is a mouse, and its genome includes at least the IGH variable region genes V4-1, V3-2, V3-3, and V3-4 of canids.

[0076] In one embodiment, the rodent is a mouse, and the genome includes at least the IGL kappa variable region genes V4-1, V7-2, V3-3, V2-4, V2-5, V2-6, V2-7, V2-8, V2-9, V2-10, and V2-11 of canids.

[0077] In one embodiment, the rodent or rodent cell is a mouse or a mouse cell, and one or more or all of the kappa V genes of the canid, 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 embodiment, the rodent is a mouse, and the genome includes at least the IGL lambda variable region genes V3-1, V3-2, V3-3, V3-4, V4-5, and V4-6 of canids.

[0079] In one embodiment, the rodent is a mouse, and the genome contains deletions of one, some, or all of the mouse IGH V region genes, preferably V1-85 to V5-2.

[0080] In one embodiment, the rodent is a mouse, and the genome contains deletions of one, some, or all of the mouse IGL kappa V region genes, preferably V3-1 to V2-137.

[0081] In one embodiment, the rodent is a mouse, and the mouse heavy chain D and J region genes are retained upstream of the heavy chain variable region genes of the companion animal into which the insertion occurs.

[0082] In one embodiment, the genome of a rodent is modified to reduce or prevent the expression of a fully rodent antibody that has both variable and constant regions derived from the rodent. This may be by inversion of all or part of the rodent's VDJ region, or by deletion or insertion of an endogenous rodent VDJ or VJ region in the genome. In one embodiment, the rodent's VDJ or VJ region or part thereof is deleted. In one embodiment, all or some of the rodent's V region genes, for example, at least 50%, preferably at least 75%, or at least 90%, or all of the rodent's IGH gene and / or the rodent's IGL kappa VJ region gene and / or the rodent's lambda VJ gene are deleted. In one embodiment, the rodent's IGL lambda gene is not deleted from the rodent's genome.

[0083] In one embodiment, insertion of companion animal DNA into the kappa locus of a rodent results in the complete or partial deletion or inactivation of one or both alleles in the kappa locus of the rodent.

[0084] In one embodiment, the kappa position of a rodent is deactivated entirely or partially by, for example, insertion, deletion, or inversion.

[0085] In one embodiment, the lambda locus of a rodent is deactivated entirely or partially by, for example, insertion, deletion, or inversion.

[0086] In one embodiment, the heavy chain locus of a rodent is deactivated entirely or partially by, for example, insertion, deletion, or inversion.

[0087] The companion animal's variable region gene is appropriately inserted upstream of the rodent's constant region, and the rodent's constant region contains all the DNA necessary to encode the complete constant region or a portion of the constant region sufficient to enable the formation of an effective chimeric antibody capable of specifically recognizing an antigen. Accordingly, references herein to chimeric antibodies or antibody chains having the rodent's constant region are not limited to antibody chains having the complete constant region or the complete constant region locus, but also include chimeric antibodies or chimeric antibody chains having the constant region or a portion of the constant region locus sufficient to provide one or more effector functions found in antibodies naturally occurring in rodents. Effector functions include the ability to interact with Fc receptors and / or the ability to bind to complement. This teaching also applies to the rodents and cells and methods of the present invention in which the variable region DNA is located in the host genome so as to form an antibody chain or a portion thereof by forming a chimeric antibody chain with all or a portion of the rodent's constant region.

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

[0089] The rodent constant region expressed along with the companion animal's variable region is preferably a rodent wild-type constant region located in a wild-type locus appropriate for the companion animal's heavy chain or light chain VDJ or VJ.

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

[0091] In one embodiment, one or more rodent regulatory sequences, such as Emu enhancer sequences, are maintained upstream of the rodent's Mu constant region, and appropriately at their natural location relative to the distance from the constant region.

[0092] In one embodiment, one or more rodent regulatory sequences, such as enhancer sequences, are maintained downstream of the rodent's steady region, and appropriately at their natural location relative to the distance from the steady region.

[0093] In one embodiment, the Smu switch sequence of a rodent is maintained upstream of the rodent's Mu constant region, and appropriately at its natural position relative to the distance from the constant region.

[0094] In such locations, enhancer or switch sequences in rodents can be properly activated in vivo with host constant region sequences.

[0095] In a further embodiment, one or more promoter elements or other regulatory elements of the V, D, or J region genes of a companion animal are optimized in the genome to interact with the transcription mechanism of the rodent.

[0096] In one embodiment, the genome of a rodent or rodent cell includes a promoter or enhancer of one or more companion animals, and / or other regulatory elements associated with companion animal V, D, or J regions. In one embodiment, one or more companion animal regulatory regions, such as promoters, enhancers, or switch regions, each replace one or more rodent promoters, enhancers, or switch regions. The companion animal regulatory sequences are appropriately maintained in a functional arrangement with constant regions so that the effects of the regulatory sequences are exerted holistically or partially in the cell or transgenic rodent.

[0097] In one embodiment, at least one or more of the V, D, or J gene segments of the companion animal to be inserted are accompanied by a regulatory sequence, such as a recombinant signaling sequence (RSS), derived from the same companion animal, which, if necessary, induces successful recombination of the V, D, or J gene segment(s).

[0098] In this context, the “same” companion animal is not limited to the exact companion animal from which the V, D, or J gene segments of the companion animal are obtained. In one embodiment, the “same” companion animal refers to the same breed or species as the companion animal from which the V, D, or J gene segments of the companion animal are obtained. In one embodiment, it is the exact same companion animal.

[0099] In one embodiment, at least one or more of the inserted companion animal V, D, or J gene segments are directly accompanied by a control sequence in cis or trans, or the control sequence is adjacent to one or both sides, and if necessary, one or more gene segments are directly adjacent to the control sequence.

[0100] In one embodiment, 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 embodiment, the V, D, or J sequence of the companion animal to be inserted is adjacent to an RSS sequence derived from the same companion animal. For example, an RSS sequence of a canid can be used together with the V, D, and / or J sequences of a canid. This can be achieved by inserting a companion animal-derived genome fragment into the rodent genome. In a further embodiment, the present invention provides a method for whole- or partially replacing the locus of an endogenous immunoglobulin variable region gene in a rodent cell with the locus of a companion animal gene, comprising the steps of: obtaining a cloned genome fragment or synthetic sequence that whole- or partially comprises the locus of a companion gene including at least one V or D (for heavy chain) or J gene segment and at least one associated regulatory sequence; and appropriately inserting the companion animal DNA into the rodent genome at the endogenous mouse immunoglobulin locus, preferably at the rodent locus of the heavy chain or light chain corresponding to the natural heavy chain of the companion animal DNA to be inserted.

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

[0103] In one embodiment, the rodent cells of the present invention are rodent ES cells, rodent hematopoietic stem cells, or other cells that can develop in a rodent capable of producing antibody chains containing a variable region encoded by the DNA of a companion animal, such as a chimeric antibody heavy chain or a chimeric antibody light chain, or a complete companion animal antibody chain or antibody repertoire encoded by the variable region of the companion animal having a variable region and a constant region.

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

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

[0106] In one embodiment, the cells are isolated rodent B cells.

[0107] Preferably, the rodent cells are rodent ES cells or iPS cells. Such cells are suitable for inserting the DNA of a companion animal and generate a rodent expressing the antibody chain described herein.

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

[0109] The present invention also relates to cell lines that are grown from or 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 after reconstitution following 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 rodents of the present invention with an antigen.

[0112] The present invention also relates to cells or cell lines expressing antibody chains having a companion animal variable region accompanied by a companion animal constant region, wherein the nucleic acid sequence of the antibody variable region may or has been identified by immunizing the rodent of the present invention with an antigen and obtaining the antibody chain or antibody chain sequence from the rodent or rodent cells. The expressed antibody chain is preferably a complete canid antibody chain, a complete equine antibody chain, or a complete feline antibody chain, in which the companion animal-derived variable region is expressed in the rodent of the present invention accompanied by a constant region (not the rodent constant region) also derived from the same companion animal. The cells or cell lines expressing the antibody chain or antibody may be CHO cells, or other mammalian cell lines suitable for the production of therapeutic agents for human or animal use.

[0113] Cells can be immortalized by fusion with tumor cells to provide antibody-producing cells and cell lines, or they can be produced by direct cell immortalization.

[0114] The present invention also relates to vectors for use in the present invention. In one embodiment, such a vector is a bacterial artificial chromosome (BAC) containing all or part of the IG locus of a companion animal. Other cloning vectors may be used in the present invention, and it will be understood that references to BACs herein can generally be interpreted as referring to any suitable vector. The vector may contain one or more selection markers and / or one or more site-specific recombination sites. In one embodiment, the vector contains two or more, for example, three heterospecific and incompatible site-specific recombination sites. In one embodiment, the site-specific recombination sites may be loxP sites or variants thereof, or FRT sites or variants thereof. In one embodiment, the vector contains one or more transposon ITR (terminal inversion sequence) sequences.

[0115] A suitable BAC containing canid DNA is available as the CHORI-82 BAC Library from the BACPAC Resource Center at the Children's Hospital Oakland Laboratory.

[0116] A suitable BAC containing feline DNA is available as an FCAB library from Amplicon Express.

[0117] A suitable BAC containing DNA from equid animals is available as the CHORI-241 BAC Library from the BACPAC Resource Center at the Children's Hospital Oakland Laboratory.

[0118] The present invention relates to a method for producing a rodent or rodent cell, comprising the step of inserting one or more companion animal IGH V region genes, one or more companion animal IGH D region genes, and one or more companion animal IGH J region genes into the genome of a rodent cell, wherein the rodent or rodent cell can express the variable region genes of the companion animal in combination with a constant region and 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 one or more companion animal IGL V region genes and one or more companion animal IGL J gene genes into the rodent cell genome, wherein the rodent or rodent cell can express the companion animal's variable region genes in combination with a constant region and form an antibody chain.

[0120] Preferably, the method relates to inserting the VDJ and VJ region genes of both the light and heavy chains of the companion animal, respectively, so that an antibody is produced in which both the light and heavy chains have variable regions derived from the expression of the companion animal's DNA.

[0121] The present invention also includes 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 genome of a rodent cell, wherein the inserted fragments form a serial insertion in the rodent cell, that is, they are directly bound without intervening sequences.

[0122] In one embodiment, the insertion process begins at the site where the start cassette is inserted into the genome of a cell such as an ES cell. In one embodiment, the start cassette is inserted into a rodent heavy chain locus for use in the insertion of human heavy chain DNA. Similarly, the start cassette may be inserted into a rodent light chain locus for use in the insertion of human light chain VJ DNA. The start cassette may be positioned between the last J and C regions of the rodent heavy chain and kappa chain. The start cassette may be positioned downstream of the rodent kappa IGL locus on the same chromosome for use in the insertion of a gene in an IGL lambda companion animal.

[0123] The start cassette appropriately contains contiguous loci specific to the rodent genome, allowing for the insertion of companion animal DNA.

[0124] In one embodiment, the insertion of a first DNA fragment into the start cassette may be followed by the insertion of a second DNA fragment into a portion of the first DNA fragment. Subsequent insertions may be performed on at least a portion of the DNA fragments that are to be inserted beforehand.

[0125] In one embodiment, the method comprises targeted insertion of an initiation cassette into a rodent genome by homologous recombination, insertion of a first DNA sequence into at least a portion of the initiation cassette by site-directed recombination, insertion of a second DNA sequence into at least a portion of the first DNA sequence, and, optionally, insertion of one or more further DNA sequences into at least a portion of the preceding dNA sequence, thereby constructing a contiguous DNA fragment within a target containing the DNA of a companion animal. The insertion of the DNA fragment into at least a portion of the initial fragment may be by site-directed recombination, for example, by recombinase-mediated cassette exchange (RMCE). The method may include both homologous recombination (e.g., the initial step of inserting the initiation cassette) and site-directed recombination, such as RMCE (e.g., one or more subsequent insertion events). Site-directed recombinase systems are well known in the art and may include Cre-lox, FLP / FRT, or combinations thereof.

[0126] In one embodiment, the companion animal DNA to be inserted is constructed stepwise into the genome of a cell such as an ES cell using separate insertions of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more heavy or light chain regions. The companion animal DNA fragments are successively and appropriately inserted into the same or substantially the same cellular locus, for example, the locus of an ES cell, to form a complete VDJ or VJ region, or a portion thereof.

[0127] The present invention also relates to cells and rodents that include intermediates during the process, wherein the genome may consist only of a partial companion animal VDJ or VJ region, such as only the variable region gene DNA of the companion animal.

[0128] Methods for targeted insertion of exogenous DNA at endogenous mouse loci, enabling the insertion of V, D, and J genes in a constant region of the host, are well known in the art. See Murphy et al., Vol. 111, No. 14, pp. 5153-5158, doi:10.1073 / pnas.1324022111; MacDonald et al., Vol. 111, No. 14, pp. 5147-5152, doi:10.1073 / pnas.1323896111; and Lee et al., Nature Biotechnology, Vol. 32, pp. 356-363, 2014 DOI:, doi:10.1038 / nbt.2825.

[0129] In particular, the method for producing transgenic rodents includes the insertion of a VDJ or VJ region gene of a companion animal disclosed herein into the upstream or downstream of a corresponding constant region of a rodent mammal by sequential recombinase-mediated cassette exchange (SRMCE) through the stepwise insertion of multiple DNA fragments.

[0130] In one embodiment, the correct insertion event is confirmed before proceeding to the next step of the multi-step cloning process.

[0131] In one embodiment, the companion animal is a dog, the rodent is a mouse, and the canid IGL kappa V region gene, which is naturally located downstream of the kappa constant region of the canid, is inserted upstream of the rodent kappa constant region, preferably upstream of the canid IGL kappa V gene, which is naturally found upstream of the canid IGL kappa constant region.

[0132] In one embodiment, rodents have at least 1 × 10 6 This can generate a diversity of combinations of different functional chimeric immunoglobulin sequences.

[0133] In one embodiment, a chimera is created using ES cells having one or more chimeric loci, from which a host embryo is generated from a RAG-1-deficient background or another suitable genetic background that inhibits the production of mature host B and T lymphocytes. This allows all B and T cells to be derived from the injected ES cells.

[0134] In one embodiment, the preferred rodent is a mouse, and the cells of the present invention are mouse cells or ES cells. In another embodiment, the preferred rodent is a rat, and the cells of the present invention are rat cells or ES cells.

[0135] The ES cells of the present invention can be used to generate animals using techniques well known in the art, which include injecting the ES cells into a blastocyst, subsequently transplanting the chimeric blastocyst into a female to produce offspring, mating these offspring to produce heterozygous offspring, and then mating them to produce homozygous recombinants having the necessary insertions. In one embodiment, the host blastocyst is Rag-deficient.

[0136] This invention relates to a chimeric rodent produced by injecting the ES cells of the present invention into a blastocyst, and then transplanting the chimeric blastocyst into a female rodent to produce offspring.

[0137] In one embodiment, the rodent or rodent cell is a mouse or mouse cell, and the mouse ADAM6a and ADAM6b genes are present in the mouse genome and have not been previously deleted and reinserted from the IGH locus.

[0138] In one embodiment, the rodent ADAM6a and ADAM6b genes are located at the 5' position of the inserted V, D, and J genes of one or more companion animals.

[0139] In one embodiment, the rodent IGH D and J genes are present in the rodent genome. In another embodiment, the rodent IGH D and J genes are not deleted from the rodent genome. In another embodiment, the rodent IGH D and J genes are located at the 5' position of the inserted V, D, and J genes of one or more companion animals.

[0140] In one embodiment, the canid alleles used in the present invention are the reference alleles of the genes of each canid. These are from CanFam 3.1. See assembly contract GCA_000002285.2, created in September 2011 and last updated in May 2016.

[0141] Remarkably, the inventors found minimal breed variability in genomic data from 107 dogs across 19 breeds, with the reference allele (derived from Boxer dogs) found in 76% of the samples, and non-reference alleles typically found as heterozygotes to the reference allele. This means that antibody populations generated from rodents containing reference alleles of canids are widely applicable for use across different dog breeds.

[0142] Accordingly, the present invention relates to rodent cells or rodents disclosed herein, wherein at least 90%, at least 95%, and preferably all, of the inserted companion gene segments are reference alleles of canid animals from CanFam 3.1.

[0143] Preferably, the rodent or rodent cell contains one or more non-reference alleles (not "*01 alleles") from the following gene segments: 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 embodiment, the rodent or rodent cell includes one or more reference alleles of the companion animal's gene segment, such as 2, 3, 4, 5, 6, 7, 8, or 9 reference alleles.

[0145] The present invention also relates to antibody chains or portions thereof that can be obtained or obtained as disclosed herein, particularly for the treatment of diseases in canine breeds other than the reference genome breed, having variable regions derived from the expression of reference alleles of canids. In one embodiment, at least 50, 60, 70, 80, 90, or 100% of the inserted V gene segment is a reference allele of the V gene of a canid, and / or at least 50, 60, 70, 80, 90, or 100% of the inserted D gene segment is a reference allele of the D gene of a canid, and / or at least 50, 60, 70, 80, 90, or 100% of the inserted J gene segment is a reference allele of the J gene of a canid, as well as combinations thereof. Preferably, at least 90%, for example 100%, of the alleles of the inserted canine gene segment are reference alleles of that gene segment.

[0146] The present invention also relates to rodents with V, D, and J segments of canid animals disclosed herein inserted in the production of antibodies or antibody chains or parts thereof for use in the prevention or treatment of diseases in different canine breeds, and to the use of rodents with canine V, D, and J segments as described herein inserted in the production of antibody chains or parts thereof for use in the prevention or treatment of diseases in different canine breeds.

[0147] The present invention also relates to a rodent having one or more or all of the V, D, and J segments of a canid animal derived from the Boxer breed, as described herein. The present invention further provides the use of an antibody or fragment thereof obtained or obtainable from any rodent having the canid gene segment of the genome described herein, the antibody being at least partially expressed from the canid DNA in the prevention or treatment of a disease in a dog breed different from the breed in which the canid gene segment is used.

[0148] For example, if the canid DNA to be inserted is derived from a Boxer, it will be used in dog breeds other than Boxers.

[0149] The present invention also relates to the following: (i) A rodent having the V, D, and J segments of a canid animal derived from the Boxer breed, preferably one or more Boxer gene segments that are reference alleles; (ii) A rodent having one or more V, D and / or J gene segments that are identical to those of a canid described herein, preferably a boxer dog, derived from a dog breed other than a boxer (i.e., the dog has a reference allele); (iii) Cells such as B cells, hybridomas, CHOs, or other suitable cells expressing antibodies or antibody chains derived from rodents of (i) or (ii), wherein the antibodies express at least several amino acids expressed from a reference allele, preferably from the V, D, and J DNA (or the V and J DNA of the light chain of a canid); (iv) A complete canine antibody or antibody chain containing the antibody variable region of the antibody described in (iii) above, which may be produced by expressing DNA encoding the constant region of a canine animal along with DNA encoding the variable region of a canine animal; (v) Expression cells, such as CHO cells, containing DNA encoding all or part of the antibodies of such a complete canid animal.

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

[0151] In another aspect, the present invention provides antibodies or antibody chains having a variable region of a canid animal, and the use of antibodies or antibody chains in the prevention or treatment of diseases in different dog breeds, wherein the antibodies or chains are obtained or can be obtained from rodents disclosed herein, the V gene segment used to generate the variable region of a canid animal is a reference allele of a canid animal, and preferably the antibodies or antibody chains are effective in at least 50%, e.g., 60% or at least 70% of different dog breeds. Preferably, the V gene segment of a canid animal derived from the heavy chain and / or light chain is derived from a boxer. Preferably, the D and J gene segments are also boxer gene segments.

[0152] This invention relates to the following: A method for producing an antibody chain specific to a desired antigen, comprising the steps of immunizing a rodent disclosed herein with the desired antigen, and recovering the antibody chain alone or as part of a complete antibody, or recovering cells that produce the antibody chain alone or as part of a complete antibody (see, for example, Harlow, E. and Lane, D., 1998, 5th edition, "Antibodies: A Laboratory Manual," Cold Spring Harbor Lab. Press, Plainview, NY; and Pasqualini and Arap, Proceedings of the National Academy of Sciences (2004) 101: pp. 257-259). Appropriately, an immunogenic amount of antigen is delivered. The present invention also relates to a method for detecting a target antigen, comprising the step of detecting an antibody produced as described above with a secondary detection agent that recognizes a portion of the antibody.

[0153] A method for producing an antibody chain or antibody that is specific to a desired antigen and derived from a single companion animal, comprising the steps of: immunizing a rodent containing the genes of the companion animal disclosed herein; and subsequently replacing the constant region of the rodent antibody chain with the constant region of the companion animal derived from the same companion animal by appropriately genetically manipulating the nucleic acid encoding the antibody. This can be achieved by replacing the constant region of a non-human mammal with an appropriate human constant region DNA sequence using standard cloning techniques at the DNA level. See, for example, Sambrook, J and Russell, D. (2001, 3rd edition) "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Lab. Press, Plainview, NY). Alternatively, it can be achieved by direct nucleic acid synthesis.

[0154] A method for producing an antibody chain or a part thereof having a variable region of a companion animal, comprising the step of expressing DNA encoding the antibody chain or a part thereof in a cell, The DNA sequence encoding the variable region of the antibody chain is obtained, or can be obtained, by immunizing the rodent of the present invention with the antigen so that the antibody chain is produced. If necessary, the method is as follows: A step of purifying and / or isolating antigen receptor chains, If necessary, the process then involves formulating the antigen receptor chain into a pharmaceutically acceptable formulation suitable for administration to a companion animal. Methods that include...

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

[0156] The present invention also relates to some or all immunoglobulin molecules comprising a variable domain of a canid animal and a constant domain of a rodent derived from a rodent B cell, as disclosed herein, and similarly to hybridoma cells that can be obtained from or obtained from such B cells, and some or all immunoglobulin molecules comprising a variable domain of a canid animal and a constant domain of a rodent that can be obtained from or obtained from such hybridoma cells. The present invention also relates to the identification of a variable region of a canid animal by single-cell sequencing and the preparation of synthetic vectors that express complete antibody chains having the corresponding constant region of a canid animal. The present invention also relates to obtaining these sequences by PCR and binding them to appropriate constant regions by appropriate molecular biology techniques, such as but not limited to bridge PCR and Gibson cloning.

[0157] Antibodies or antibody chains, or parts thereof, obtained or obtainable from rodents or cells according to the present invention, for use in the treatment of companion animals.

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

[0159] In one embodiment, the present invention relates to chimeric companion animal antibodies and antibody chains having a constant region of a rodent and a variable region of a companion animal, as well as fragments and functional derivatives of the antibodies and chains, and the use of the antibodies, chains and fragments in medicine, including diagnostics, and in vitro or ex vivo research. Functional antibody fragments may include fragments capable of specific binding to antigens. Functional antibody fragments may be, for example, FABs or single-chain variable fragments (scFvs). In a further embodiment, the present invention relates to "fully" companion animal antibodies and antibody chains ("fully" reflecting the fact that both the variable and constant regions of the antibody are expressed from genes of the same species of companion animal), as well as fragments and functional derivatives of the antibodies and chains, and the use of the antibodies, chains and fragments in medicine, including diagnostics, and in vitro or ex vivo research.

[0160] Methods for producing 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 complete companion animal antibodies produced in response to antigen loading in rodents according to the present invention.

[0161] In a further embodiment, the present invention relates to the use of rodents, as described herein, as models for testing drugs and vaccines. Accordingly, the present invention relates to a method for identifying or validating a drug or vaccine, comprising the steps of delivering the vaccine or drug to a mammal of the present invention and monitoring one or more of the immune response, safety profile, and disease effects.

[0162] In yet another embodiment, the chimeric antibody or antibody chain generated in the present invention can be appropriately manipulated at the DNA level to generate a variable region of a companion animal from a heavy or light chain that lacks a constant region, such as a heavy or light chain of an antibody having antibody-like properties or structure, or a variable region of a companion animal having any constant region from either a heavy or light chain of the same or different species, or a variable region of a companion animal having a constant region that does not exist in nature, or a variable region of a companion animal having any other arbitrary fusion partner. The present invention relates to all such chimeric antibody derivatives derived from the chimeric antibody identified in accordance with the present invention.

[0163] The present invention also relates to a kit comprising an antibody or antibody derivative disclosed herein, and instructions for use of such antibody or buffer, antibody detection reagent, or other suitable experimental reagent.

[0164] The present invention also relates to a method for producing an antibody, or a part thereof, (i) nucleic acids encoding an antibody or a part thereof obtained in accordance with the present invention, or (ii) Sequence information that allows the expression of nucleic acids encoding an antibody or a part thereof obtained in accordance with the present invention to produce an antibody The present invention relates to a method that includes a step of providing

[0165] The present invention also relates to a chimeric antibody comprising a variable region of a companion animal and a constant region of a rodent (optionally, C-gamma or C-mu), wherein the antibody is encoded by a nucleotide sequence corresponding to the nucleotide sequence of a locus of a chimeric heavy chain of a cell (optionally, a B cell, ES cell, or hybridoma), the locus comprising the nucleotide sequence of the constant region of the rodent, as well as a reconstituted VDJ nucleotide sequence produced by in vivo reconstitution of the V region, D region, and J region of the companion animal, the V region of the companion animal being selected from the IGH variable region genes of canids IGH V4-1, V3-2, V3-3, V3-4, and V3-5.

[0166] If necessary, the J region is one of the following from canid animals: JH1, JH2, JH3, JH4, JH5, or JH6.

[0167] If necessary, region D is one of DH1, DH2, DH3, DH4, DH5, and DH6 in canids.

[0168] In one embodiment, the antibodies include any combination illustrated in the examples and drawings herein. If necessary, in vivo rearrangement occurs within cells of the same rodent species (e.g., mouse B cells or ES cells) as the constant region sequence. The present invention also relates to non-human vertebrate or mammalian cells (e.g., B cells or ES cells or hybridomas) whose genome contains the chimeric heavy chain loci described above.

[0169] The present invention also relates to a non-human vertebrate or mammal (e.g., mouse or rat) whose genome contains the chimeric heavy chain loci described above in this paragraph.

[0170] The antibodies of the present invention may be isolated, and in one embodiment, they are isolated from cells or organisms that express them.

[0171] The present invention also relates to a portion of an antibody chain. In particular, this portion includes at least the variable region of the antibody. This can be expressed from a cell, especially for antibody production. This portion may include the Fab region of the antibody, or at least the CDR region.

[0172] The present invention relates to a method for producing antibodies or antibody chains, comprising the step of immunizing a rodent described herein with an antigen that can be obtained from the same companion animal as the source of companion animal DNA present in the rodent's genome. For example, antigens from canids can be used to immunize a rodent containing the V, D, and J genes of a canid disclosed herein.

[0173] Therefore, the present invention also relates to rodents immunized with companion animal antigens corresponding to the sources of companion animal DNA present in the rodent genome.

[0174] It is not necessary to use exactly the same companion animal. For example, antigens from a canid animal taken from one breed can be used to immunize rodents containing DNA from canid animals of different breeds. Antigens from the same dog breed can also be used.

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

[0176] Therefore, the present invention relates to rodents immunized with antigens that cause disease in companion animals, corresponding to the sources of companion animal DNA present in the genome of the rodents.

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

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

[0179] The present invention also relates to nucleic acids such as DNA or RNA that encode the antibody, antibody chain, or a portion thereof. In particular, the portion may be a variable portion of the antibody chain, which is a portion encoded by the DNA of a companion animal within a rodent.

[0180] The present invention also relates to cells such as B cells or hybridomas that partially or completely express antibody chains of canid animals, or to expressing cell lines (e.g., CHO cells) whose DNA or protein sequences can or have been obtained from rodents described herein.

[0181] It will be understood that, once a target antibody is identified from an immunized rodent, it is standard practice in this art to identify the DNA sequence encoding that antibody from B cells and to express the antibody or a portion thereof from that sequence, or from a different DNA sequence that can actually express the same protein as a result of genetic code redundancy. In particular, the variable region of a canid can be expressed together with the constant region of a canid to generate a complete canid antibody chain or antibody.

[0182] Therefore, the present invention also provides a method for obtaining complete canine antibodies, A step of immunizing a rodent having at least one heavy chain canine immunoglobulin V gene segment, at least one canine heavy chain D gene segment, and at least one canine heavy chain J gene segment, and / or at least one light chain canine immunoglobulin V gene segment and at least one canine heavy chain J gene segment, with the antigens described herein. A step of selecting antibody chains (or more) produced by rodents that have a variable antibody region encoded by the DNA of canids, Preferably, the method involves expressing an antibody or antibody chain, or a part thereof, that is a complete canine animal, and for example, an antibody or antibody chain containing the variable region and the constant region of a canine animal, from the DNA of an expressing cell line. If necessary, a step to purify the antibody, antibody chain, or a part thereof, The process of further formulating the antibody or chain or a portion thereof with pharmaceutically acceptable excipients suitable for administration to companion animals that require it, if necessary. This provides a method that includes this.

[0183] The present invention also relates to the use of such antibodies, antibody chains, or fragments thereof in the treatment of companion animals.

[0184] The approach described above is similarly applicable to other preferred companion animals such as cats and horses. Antigens of felines can be used in equivalent feline / rodent models, antigens of equids can be used in equivalent equid / rodent models, and all other aspects of the present invention are similarly applicable to cats and horses.

[0185] The present invention relates to a method for producing cross-reactive antibodies or antibody chains, comprising the steps of immunizing a rodent containing the DNA of a companion animal disclosed herein with a target antigen derived from the rodent and a corresponding antigen derived from the companion animal, the method comprising gene knockout of the gene encoding the antigen in the rodent. The antibody population resulting from the immunized rodent may include antibodies that can bind to both the antigens of the rodent and the companion animal, i.e., cross-reactive antibodies.

[0186] Accordingly, in a further embodiment, the present invention relates to a cross-reactive antibody or antibody fragment derived from a rodent comprising the DNA of the companion animal described herein, immunized with the antigen of the rodent of interest and the corresponding antigen derived from the companion animal, wherein the rodent has a gene knockout of the gene encoding the antigen.

[0187] In one embodiment, rodents are immunized sequentially or simultaneously by rodent antigens and corresponding antigens derived from a companion animal, either directly with proteins or peptide fragments thereof, or by vectors encoding relevant antigens or fragments thereof, or by syngeneic cell lines expressing the desired antigens.

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

[0189] Cross-reactive antibodies are extremely important in the field of drug discovery. Cross-reactive antibodies can be rapidly validated as drug candidates for the species to which the antibody is reactive, by using another 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 canine antigens for the immunity of rodents as claimed herein, meaning that the canine antigens have family genes or protein equivalents in humans, are preferably therapeutically validated, and antibodies against human equivalent antigens have been shown to be effective in treating diseases.

[0191] This invention is the first to disclose strong homology between the Ig variable regions of canids and humans. See Figure 16. [Strong homology between germline canids and humans in IG V, while TCR V homology is not well conserved.] This information suggests that rodent models containing DNA from the variable regions of canids may be able to utilize canid regulatory sequences rather than, for example, mouse regulatory sequences, because humanized mice can utilize human regulatory sequences within mice. In fact, we experimentally verified that the regulatory and RSS sequences of inserted canid genomic DNA are recognized by rodents and can be used to express chimeric antibodies in rodents.

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

[0193] Within their assigned phylogenetic ranges, mice and humans are well known to be more closely related than any of the carnivores, including cats and dogs (e.g.), or horses. This is likely why the use of human regulatory sequences in chimeric mouse models with insertions of human V, D, and J gene segments has been successful in those models. Based on this, the same could not have been predicted for evolutionarily more diverse animals such as humans and dogs. In fact, previous attempts to use canid gene segments in mice have utilized mouse regulatory sequences (e.g., Trianni, US2017306352).

[0194] In one embodiment, the present invention relates to a rodent in which inserted (companion animal) IGHJ4 and IGHJ6 are major JH gene segments found in a mature B cell antibody population. Preferably, the rodent comprises the V, D, and J gene segments of a canid, and J4 and J6 are the J4 and J6 gene segments of a canid. In one embodiment of the present invention, the rodent can use one, two, three, four, five, six or more, or all, of different inserted IGH D segments, and / or one, two, three, four, five, six or more, or all, of inserted IGH J gene segments in the generation of antibody chains within the rodent antibody repertoire.

[0195] Currently tested rodent mice possessing inserted heavy chain D1-6 and J1-6 gene segments from canids can utilize all of the canid IGH J1-6 and D1-6 in antibody formation.

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

[0197] In one aspect of the present invention, the rodent contains the DNA of a companion animal derived from dogs, and the rodent has one or more of the following characteristics: Rodents express chimeric antibody heavy chains from IGHJ4 in greater quantities than any of IGHJ1, 2, 3, 5, or 6, and rodents express chimeric antibody heavy chains from IGHJ6 in greater quantities than any of IGHJ1, 2, 3, or 5; Rodents individually express chimeric antibody heavy chains derived from IGHD5 in greater amounts than any of IGHD1, 2, 3, 4, or 6; Rodents individually express chimeric antibody heavy chains derived from IGHD2 in greater amounts than any of IGHD1, 3, 4, or 6; Rodents express antibody chains from IGHD2 along with IGHJ4; Rodents express antibody chains from IGHD5 along with IGHJ4; Rodents express more antibody chains from IGHD5 along with IGHJ4 than canids express from any other combination of IGHD and IGHJ segments; Rodents express more chimeric antibody light chains from IGLJ1 than any other IGL J gene segment.

[0198] In one embodiment, the rodent contains the following inserted heavy chain gene segments of a canid: J1-6, D1-6, V4-1, V3-2, V3-3, and V3-5, along with V3-4 (which is a pseudogene) as needed.

[0199] In one embodiment, the rodent contains the following inserted canine lambda light chain gene segments: J1-9 with C1-9 (i.e., the complete canine lambda JC cluster), as well as V3-2, V3-3, V3-4, V4-5, V4-6, and optionally V3-1 and V3-7 (both pseudogenes).

[0200] The rodent of the present invention can appropriately modify the V, D, and J gene segments of the inserted companion animal by N and / or P addition, and / or can undergo somatic hypermutation of the V, D, and J gene segments of the inserted companion animal.

[0201] The present invention also provides HCDR3, a VH domain, an antibody heavy chain, or an antibody, wherein the VH domain of the heavy chain or antibody comprises a somatic hypermutation of the AID pattern in rodents and / or a 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). Mice are preferred rodents.

[0202] In one aspect of the present 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. Therefore, some degree of somatic hypermutation and / or N / P addition was present.

[0203] Surprisingly, when both samples were investigated at the same depth using the RNA sequencing approach of Example 4, the inventors found that the diversity of the chimeric antibody population generated using rodents containing the V, D, and J gene segments of canids, or the V and J segments described herein, was greater than the observed antibody diversity in wild-type dogs from which the gene segments originated. Figure 21 shows that antibody nucleotide sequences found only once in sequenced libraries of rodents containing the heavy chain V, D, and J gene segments of canids, or the light chain V and J segments, are far more numerous than those found in dogs themselves.

[0204] Therefore, the present invention relates to any rodent described herein in which each antibody expresses a population of chimeric antibody chains resulting from the expression of at least one companion animal gene segment, wherein the population is more diverse than, 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 produced 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 produced in mice having the V, D, and J gene segments of canid DNA, or the light chain V and J segments, is greater than, or at least as great as, that seen in the canine antibody repertoire.

[0206] Appropriately, population diversity is assessed by the number of unique antibody sequences present in the heavy chain population, the light chain population, or both.

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

[0208] The rodent may be any rodent disclosed herein, for example, in one embodiment the rodent includes the inserted heavy chain gene segments of a canid, such as: J1-6, D1-6, V4-1, V3-2, V3-3, and V3-5, along with V3-4 (which is a pseudogene) as an option. In one embodiment the rodent includes the inserted lambda light chain gene segments of a canid, such as: J1-9 together with C1-9 (a complete lambda JC cluster of a canid), as well as V3-2, V3-3, V3-4, V4-5, V4-6, and V3-1 and V3-7 (both pseudogenes) as an option. In one embodiment the rodent includes both heavy chain and light chain insertions of a canid.

[0209] The present invention also relates to the use of any rodent (e.g., mouse) containing the DNA of a companion animal disclosed herein in generating a repertoire of chimeric antibody chains or antibodies that is more diverse than or at least as diverse as that found in the companion animal itself, and to rodents such as mice containing the DNA of a companion animal disclosed herein for generating a repertoire of chimeric antibody chains or antibodies that is more diverse than or at least as diverse as that found in the companion animal itself.

[0210] Therefore, preferred embodiments of the present invention are as follows:

[0211] A method for producing an antibody or antibody chain, comprising the step of immunizing a rodent according to claims 1 to 13 with an antigen obtained from or obtainable from a companion animal which is the same as the source of companion animal DNA present in the genome of the rodent, wherein the antigen may be a protein antigen, a cell expressing the antigen, or a nucleic acid encoding the antigen.

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

[0213] A rodent according to claims 1 to 13, immunized with a companion animal antigen corresponding to a source of companion animal DNA present in the genome of the rodent.

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

[0215] A rodent according to 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 claims 1 to 13, wherein the rodent contains DNA of a companion animal of canine origin, and the rodent has one or more or all of the following characteristics: Rodents individually express chimeric antibody heavy chains derived from IGHJ4 in greater amounts than any of IGHJ1, 2, 3, 5, or 6; Rodents individually express chimeric antibody heavy chains derived from IGHJ6 in greater amounts than any one of IGHJ1, 2, 3, or 5; Rodents individually express chimeric antibody heavy chains derived from IGHD5 in greater amounts than any of IGHD1, 2, 3, 4, or 6; Rodents individually express chimeric antibody heavy chains derived from IGHD2 in greater amounts than any of IGHD1, 3, 4, or 6; Rodents express antibody chains from IGHD2 along with IGHJ4; Rodents express antibody chains from IGHD5 along with IGHJ4; Rodents express more antibody chains from IGHD5 along with IGHJ4 than canids express from any other combination of IGHD and IghJ segments; Rodents express more chimeric antibody light chains from IGLJ1 than any other IGL J gene segment.

[0217] A rodent according to claims 1 to 13, comprising the following inserted heavy chain gene segments of a canid: J1-6, D1-6, V4-1, V3-2, V3-3, and V3-5, along with V3-4 as appropriate.

[0218] A rodent according to claims 1 to 13, comprising the following inserted lambda light chain gene segments of a canid: 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 claims 1 to 13, wherein the V, D, and J gene segments of the inserted companion animal can be modified by N and / or P addition, and / or exhibit somatic hypermutation.

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

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

[0222] A rodent according to claims 1 to 13, wherein 65-75% of the chimeric antibody sequences are unique, such as a rodent.

[0223] The use of any rodent (e.g., mouse) containing the DNA of a companion animal as defined in claims 1 to 13 in generating 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, according to claims 1 to 13, for generating a more diverse repertoire of chimeric antibody chains or antibodies than those found in the companion animal itself.

[0225] The rodent according to claims 1 to 13, wherein one or more inserted companion animal V, D, or J gene segments include a promoter and / or splice site and / or a recombination signal sequence for V(D)J recombination, wherein the control sequence is accompanied by a control sequence derived from the same companion animal, and optionally the control sequence precedes the individual V gene segment.

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

[0227] A rodent according to claims 1 to 13, comprising DNA of a certain breed of canid, used for producing an antibody or antibody chain, or a part thereof, for use in the treatment or prevention of diseases of different dog breeds.

[0228] The use of the rodent according to claims 1 to 13 in the production of an antibody chain or a portion thereof, which contains DNA of a certain breed of canid, for use in treating different dog breeds.

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

[0230] It will be understood that the specific embodiments described herein are provided as examples, not as limitations of the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention. Those skilled in the art will be able to recognize or confirm, by mere routine study, many equivalents of the specific procedures described herein. Such equivalents are deemed to be within the scope of the invention and are included in the claims. All publications and patent applications referenced herein represent the state of the art to which the invention relates. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually incorporated by reference. The use of the terms “a” or “an” in the claims and / or herein, when used in conjunction with the term “comprising,” may mean “one,” but it also coincides with the meanings of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and / or” unless it is explicitly indicated that it refers only to options, or where the options are mutually exclusive, even if this disclosure supports a definition that refers only to options and “and / or.” Throughout this application, the term “about” is used to indicate that a value includes inherent variations in the error of the apparatus, i.e., the method employed to determine the value, or variations that exist between the subjects of study.

[0231] When used herein and in the claims, the terms “comprising” (and any form of “comprise,” such as “comprise,” and “comprises”), “having” (and any form of “have,” such as “have,” and “has”), “including” (and any form of “includes,” such as “includes,” and “include”), or “containing” (and any form of “contains,” such as “contain”) are compatible or open-ended and do not preclude any additional, unmentioned elements or steps of the method.

[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 also includes BA, CA, CB, CBA, BCA, ACB, BAC, or CAB, where the order is important in the particular context. Continuing this example, combinations containing repetitions of one or more items or terms, such as BB, AAA, ABAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. A person skilled in the art will understand that, unless otherwise evident from the context, there is typically no limit to the number of items or terms in any combination.

[0233] Any part of this disclosure may be read in conjunction with any other part of this disclosure unless it is evident from the context otherwise.

[0234] All compositions and / or methods disclosed and claimed herein can be prepared and performed without undue experimentation in light of this disclosure. While the compositions and methods of the present invention have been described in relation to preferred embodiments, it will be apparent to those skilled in the art that the compositions and / or methods, as well as the steps or order of steps of the methods, may be modified without departing from the concept, spirit and scope of the invention. All such similar substitutions and modifications that are apparent to those skilled in the art shall be deemed to fall within the spirit, scope and concept of the invention as defined by the appended claims.

[0235] The present invention will be described in more detail in the following non-limiting embodiments. [Examples]

[0236] (Example 1) Annotation of IG loci in canids 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. A combination of existing and novel methods was used to analyze the diversity and mutation rate across these genes. Over 5,500 novel alleles were identified across approximately 550 gene segments of the AR locus (326 of which were newly annotated), insights into AR evolution were obtained, 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 canine AR genetics and expression to the same high level as that of mice and humans, making it the only third species with all annotated AR loci. A large number of genomic sequences will be useful for future research and enabled statistically powerful conclusions to be drawn 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, 5,0o0 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. [[ID=z]] <000o0904><o000905>2. Materials and Methods 2.1.1 Annotation by Bioinformatics Initially, seated positions were annotated using a method similar to that of Das et al., but with principles equivalent to those of Olivieri et al.'s algorithm. 1、2 In short, we investigated the reference genome (CanFam3.1) of canids using human and mouse sequences. Once a region was identified, we searched locally for AR genes. Initially, mouse and human AR genes and RSS consensus were used, but as more canid genes were identified, these were used instead. Next, annotations were validated and added based on the alignment of RNA sequencing data.

[0239] 2.1.2 Dogs Peripheral blood samples were obtained from 26 dogs. The samples were unused clinical surplus from mandatory veterinary blood collections from patient animals at the University of Cambridge Veterinary Hospital. This study had been pre-approved by the Ethics Committee of the University of Cambridge Veterinary School.

[0240] 2.1.3 Sequencing Mononuclear cells were isolated from peripheral blood using Ficoll-Paque (GE Healthcare) according to the manufacturer's instructions. The cells were processed into mRNA using polyA pulldown by the core sequencing team at Wellcome Trust Sanger Institute, cleaved, and sequenced on a HiSeq 2500 machine (Illumina) using 250 bp paired-end reads.

[0241] 2.1.4 Gene Nomenclature The AR gene was divided into families, including Bao et al. 3 They were assigned as functional, pseudogene, or ORF using the same criteria. Family numbers were assigned based on homology with human families, and new numbers were given where no obvious match was found. All gene names are IMGT 4 It was assigned according to the naming system.

[0242] 2.1.5 Non-reference alleles 10 7 The mapping of variant call files from the whole genome sequences of individual canid animals to AR loci was kindly provided by Steven Friedenberg of the University of Minnesota.

[0243] 2.1.6 Interspecies and intraspecies seating alignment I used RepeatMasker to mask the array and PipMaker to generate an alignment plot. 5、6 .

[0244] 2.1.7 Phylogenetic tree analysis I used Clustal Omega to align the array and Interactive Tree of Life to visualize the output tree. 7、8 .

[0245] 3.Results 3.1.1 Number of genes Within previously annotated loci, three novel IGHJ genes were identified. 162 IGLV genes were identified across seven gene families, 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, along with five IGKJ genes and one IGKC gene.

[0246] 3.1.2 Non-reference alleles Whole genome sequences from 107 dogs of 19 breeds, all aligned with the current reference build (CanFam3.1), were investigated for novel AR alleles, and 4,074 were identified across three loci. Regarding allele distribution, the reference allele was found 53,311 times out of 68,908 alleles called (77%). No significant breed specificity was identified in the allele distribution.

[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%), that is, the novel alleles were pseudogenes rather than the novel alleles being functional "gain" (3.4%). Since there are far more ways to lose a function than to gain one, this is not surprising, and thus mutations are likely to bring about that direction of change. Changes from a function to an ORF were classified as loss, and changes from a pseudogene to an ORF were classified as gain.

[0248] Non-reference V alleles were called 13,129 times in all samples and loci. Interestingly, IGKV2-S13, and eight IGLV genes were only detected as non-reference alleles including in boxer samples. This is probably an error in the reference genome, but it is also possible that the reference dog has nine rare alleles that just happened not to 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 at 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 In canids, the IGH locus is located at the subtelomere of chromosome 8 on the antisense strand. This telomere location is observed in all mammals except monotremes and marsupials. 1 However, light chain loci do not show strong conservation of chromosome locations among humans, mice, and dogs.

[0251] Similar to the IGH locus in humans and mice, the IGH locus in canids contains all functional gene segments that are transcribed identically to the constant region, with transcription reversed in one pseudogene (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 while there are slight differences in the location and expected function of the V gene, 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 most recent publicly available build (CanFam 3.1).

[0252] The IGK locus in canids is small (approximately 400 kbp) and has an unusual structure (Figure 2). There are 11 V genes upstream of the J and C genes, and all eight distal C genes of the IGKV2 family are functional, and unlike the three proximal C genes, they are in the same transcriptional direction as the J and C genes. However, there are also eight V genes downstream of other genes, and most are inverted relative to those genes. This is reminiscent of the IGL locus in equids, and it was found that the orientation does not affect the use of the V genes.

[0253] Inversion and block duplication appear to be characteristic of light chain loci, particularly the IGK locus. The IGK locus in humans, pigs, mice, horses, and dogs all contain the V gene, which has a transcription direction opposite to that of the C gene. Furthermore, the loci in dogs, humans, and pigs undergo inversion duplication of the entire block. 10、11 In pig and human loci, the genes in two blocks do not diverge sufficiently, so some or all of them are known by the same name as the pair 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 with respect to the J-C cluster (Figure 3). The inversion appears to be under some locus-specific selection pressure, as only 3 out of 116 of the most C-proximal IGLV genes in the opposite transcriptional orientation to the J-C genes are inverted, and there are 26 inverted V genes out of the last 46. This increase is partly due to the duplication of blocks in which segments of IGLV1 and IGLV8, which show a very high level of sequence identity, occur in a pattern where the members are positionally conserved within the repetitive blocks.

[0255] From a broader perspective of trends, the canid IGL locus is consistent with other published loci. For previously annotated IGHV, the gene-to-pseudogene ratio was approximately 1:1, which was found to be consistent with 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 so far. 14、15 . For example, the mouse IGL locus, which contains only 9 functional genes, reflects its use in only 5% of the expressed antibodies. 14 . The canid repertoire shows a similar bias, but conversely, it has been reported that the IGK chain is used in 91% depending on the relative sizes of the two light chain repertoires of 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、18Alignment was performed using the IGK, IGL, and TRA / D sitting positions of canid animals, both relative to themselves and to the mouse and human sitting positions, respectively.

[0257] In IGK self-alignment, three comparisons are noteworthy: self-alignment of upstream and downstream blocks (green and red boxes in Figure 8, respectively), and their relative alignment (blue box in Figure 8). Within the identity percentage plot (PIP), lines indicate areas of identity, and therefore sequences aligned to themselves always have a solid line running along the diagonal. Solid lines deviating from the main diagonal indicate a high probability of duplication events, and gaps represent indels and other mutations accumulated since the duplication.

[0258] The multiple dashed diagonals in the upstream self-alignment are characteristic of a block, similar to the block duplication of three gene cassettes within the TCRB locus in canids, in which case a single V and its adjacent sequences are locally duplicated multiple times. 16 In the self-alignment of the downstream block, the lines are shorter and sometimes perpendicular, indicating local inverted homology. Finally, homology comparison of the upstream and downstream blocks revealed a degree of good homology between the C gene and the upstream IgKV2 gene, particularly IGKV2-S18 and IGKV-S19. This pattern should be seen when a single gene is replicated multiple times upstream of the C gene, followed by block inversion replication in a manner reminiscent of human IGK and equid IGL. Thus, this downstream block likely has reduced selective pressure and accumulates mutations and local inversions at a higher rate, except for the IGKV2-S18 and IGKV-S19 genes, which have less divergence. While this is not the only possible explanation, it is consistent with proposed explanations for similar features across other species and AR loci.

[0259] Two things are striking in the comparison of canine and human IGL loci: a nearly unbroken diagonal near the midpoint of the human sequence and a very high degree of homology characterized by the coding sequence or numerous lines near it (Figure 9a). Further analysis of the diagonal in the human / canine PIP revealed that it extends to the region of the human locus containing the non-AR genes ZNF280A, ZNF280B, and PRAME (Figure 9b). The sequences of these genes, and most of the regions surrounding them, are highly conserved between dogs and humans, suggesting that they likely have similar functional importance in dogs as in humans. Specific comparisons indicate that the ZNF280B and PRAME genes are likely to function in dogs, although ZNF280A cannot be definitively identified at this locus (data not shown). Further investigation identifies another non-IGL gene, PCBP2, located near ZNF280B.

[0260] While the locations and genes in question are not always conserved, non-AR genes scattered across AR loci are a common feature across species. For example, the ADAM6 gene is found at the IGH locus between the IGHV and IGHD genes in humans and mice, but the human orthologue is non-functional. In dogs, no orthologue has been characterized, but there are two candidates. One is located between IGHV3-4 and IGHV3-5, and the other is located upstream of the entire IGH locus. Due to the limited study of the canine ADAM gene family, this potential orthologue has not been added to the IGH annotation, but it will be maintained as a candidate for future research. No other non-AR genes have been identified at the IGK or IGL loci in canids at present.

[0261] 3.1.5 Distribution of alleles One of the striking characteristics of using dogs as a model organism is the high degree of heterogeneity between breeds, while the high degree of uniformity within breeds is estimated to be due to a 35% loss in nucleotide diversity caused by breed formation. 19Because selection is extremely rigorous, recent studies have identified 22 homozygous blocks longer than 1 megabase in certain varieties, which the authors attribute to the selective pressure imposed by breeders. 20 This is in contrast to the diversity seen in humans. Even in geographically isolated populations, the variation attributable to this segregation accounts for 5-10%, whereas more than a quarter of canine genomic variation is attributable to breed variation rather than individual variation. 21 .

[0262] Given the level of breed specificity in canid genomics, it is perhaps surprising that the non-reference AR allele did not appear to follow a strong breed-specific haplotype. The two most prevalent breeds in this sample were the Boxer and the Standard Poodle (22 and 20 dogs, respectively), which followed a similar pattern: a small number of non-reference alleles were found relatively frequently in both breeds, while the rarer alleles were found on a single chromosome in one dog of one breed. Less prevalent breeds followed a similar distribution, and given the number of alleles that appeared only once, the non-reference alleles appeared to be typically found in heterozygotes.

[0263] Larger breed-specific cohort sizes may reveal trends not evident in this dataset, but currently, the selective pressures forming AR gene loci appear to be breed-independent and dominant over breed itself. A possible exception is an evolutionary bottleneck increasing the abundance of a less common allele. For example, IGLC1*01 (the reference allele) is found on 19 of the 44 Boxer chromosomes sequenced and on one of the 6 Toy Poodle chromosomes, but not on any of the other breeds. Of the 217 alleles called across all breeds, 194 are represented by the other allele IGLC1*02. It is possible that Toy Poodles were previously crossed with Boxers, or there is a sequencing error, and this is indeed a Boxer-specific allele, but it remains to be seen if a larger dataset may be needed to finally answer these questions in any way.

[0264] Considering the distribution of non-reference alleles, these are all consistent with what is expected. The fact that changes are not observed as frequently as expected suggests that the genes in question are under selective pressure to maintain their current state, and therefore further deviations are undesirable. This could be either the loss of a useful functional gene or the reactivation of an autoreactive gene, both of which are selected to be eliminated because they reduce the organism's fitness. Similarly, AR genes are crucial to the organism's fitness, and their loss comes with a selection cost.

[0265] The fact that the acquisition of function is more common than expected lends weight to the proposed theory 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 acquire functionality in recombination and act as substrates for gene conversion. 22Assuming that the reference allele is the original, aligned with its majority use, the acquisition of a functional non-reference allele is an example of a pseudogene, which is a mutable initiation pool of novel, beneficial alleles, and is under such selective 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, MP 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, HG Genomic analyzes 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 loci in mouse cells The IG loci of mouse ES cells were modified by BAC insertion to introduce heavy chain DNA from canids into the mouse IGH locus and light chain immunoglobulin DNA from canids into the mouse IGL kappa and lambda loci, as follows:

[0268] IGH insertion in canids Insertion of canid DNA from chromosome 8 was performed at the mouse IGH locus using BAC insertion. The inserted DNA contained nucleotides 72,988,807–73,128,041, including IGHV4-1 to IGHV3-4, as well as IGHV1-6 and IGHJ1-6. See Figures 1 and 10.

[0269] IGL lambda DNA insertion in canids Insertion of canid DNA from chromosome 26 was performed at the IGL-lambda locus on mouse chromosome 16. The inserted DNA contained nucleotides 27,509,860–27,646,373 and included IGLV3-1 to IGLV4-6, as well as IGLJ1-9 and IGLC1-9. See Figures 2 and 13.

[0270] The coordinates are from the GRCm38 / mm10 assembly in December 2011 for mice, and from Canfam3.1 for dogs.

[0271] The DNA insertion from canids was performed at the landing pad located at the following position in the mouse genome. Heavy chain DNA of a canid: inserted just upstream of position 114,666435 on mouse chromosome 12. Kappa DNA from a canid: Inserted just upstream of position 70,674,7.55 on mouse chromosome 6. Lambda DNA of a canid: inserted just upstream of position 19,047,551 on mouse chromosome 16.

[0272] The expression of chimeric transcripts has been confirmed by PCR analysis from chimeric sublocations - see Figure 15.

[0273] The method was as follows:

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

[0275] All bacteria, including BACs, were cultured at 32°C on Luria Bertani (LB) medium or LB agar supplemented with 12.5 μg / ml chloramphenicol. BAC-containing cells exhibited recombination ability upon addition of the pSIM18 plasmid to the cells using a standard CaCl2 heat shock protocol, and maintenance of pSIM18 was selected by supplementing the medium with 75 μg / ml hygromycin.

[0276] Recombinant plasmids such as pRMCE38 were linearized, and a 1 kbp BAC-specific homology arm was introduced using Gibson assembly. The plasmid contained sequences necessary for downstream recombination, such as the cre-lox and PiggbyBac sequences, as well as selection markers for the BAC or ESC engineering process. The completed plasmids were restriction digested and gel-purified to obtain fragments spanning the BAC-specific homology arm, the recombinant sequence to be introduced, and the homology arm to the vector backbone of the BAC. These fragments were inserted into BAC by electroporation, and those that successfully incorporated into BAC were selected.

[0277] Successful modification of resistant clones was verified by PCR across the junction between the inserted DNA and endogenous DNA. Once both ends of the BAC were modified, the BAC DNA was purified and electroporated into Electromax® DH10B cells (Life Technologies). Clones that lost hygromycin resistance and were therefore pSIM18 negative were selected for further analysis. PCR was performed on the DNA of these clones to ensure correct modification at both ends, as well as the presence of exons inserted using the appropriate BAC. Clones that passed this quality control check were used for genetic engineering of embryonic stem cells (ESCs).

[0278] ESC gene manipulation The ESC culture, electroporation, and drug selection processes were carried out as described by Lee et al., 2014. Male mouse AB2.1 cells, already containing SRMCE landing pads at each immunoglobulin locus, were 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] Each transfection has 1 x 10 7Individual cells were used, and all transfections were performed using a Bio-Rad electroporator (GenePulser Xcell) at 500 μF and 230 V. For BAC introduction, 10 μg of BAC DNA and 25 μg of pCAGGS-iCRE were used per transfection. After 24 hours, cells were selected with 3 μg / ml puromycin for 1 week, and colonies were selected for proliferation and testing. Successful integration of BAC into the landing pad was confirmed by PCR using primers spanning the junction between the mouse and BAC DNA.

[0280] Next, the positive clones were subjected to excision of the 3' landing pad using PBase. The clones were grown and electroporated with 10 μg of PiggyBac transposase plasmid at a rate of 1 × 10⁶ per unit. 5 Individual cells were used. After a 3-day recovery period in M15, the cells were divided, seeded at low density, selected by FIAU the following day, and maintained for 10 days. Normal excision of the 3' end of the landing pad was confirmed by junction PCR. Positive clones were then subjected to the same exon assay as the original BAC to confirm that no deletions had occurred.

[0281] Mouse generation and analysis Positive ES cell clones were injected into blastocysts of C57BL / 6 Tyrc-Brd mouse strains using standard procedures. The injected blastocysts were transferred to the uterus of pseudopregnant female B6 / CBA F1 recipients. Approximately 40 blastocysts were injected from each clone. To enable identification of puppies derived from injected ESCs based on coat pigment, a high percentage of chimeric males were mated with albino C57BL / 6 Tyrc-Brd females. Mice derived from 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 employed. Blood, spleen, and femur were collected from mice confirmed to have BAC and associated wild-type controls. Bone marrow samples were obtained by aspirating the femur with PBS. 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 targeting the 5' end of the constant region of the mouse associated with the inserted BAC (e.g., IGLC from mice containing IGL BAC) or the polyA tail of the mRNA. These transcripts were then amplified using barcode primers nested in the mouse C region and priming from the inserted leader region derived from the V gene of a canid. This confirmed that only chimeric transcripts, and not all mouse immunoglobulin transcripts, were amplified. These PCR products were visualized on a gel to confirm the presence of chimeric transcripts in BAC-containing mice, and not in wild-type mice. See Figure 15 - Confirmation of IGH Chimeric Transcripts. The first eight lanes correspond to RNA from blood samples, the second eight lanes to bone marrow samples, and the last eight lanes to spleen samples. Of the given eight, the first four are primed with the C region, and the second four are primed with the polyA tail. Of the four, the first two are controls, and the latter two are mice expected to produce chimeric transcripts.

[0283] All animal testing and breeding were conducted under the accreditation of the Wellcome Trust Sanger Institute (AWERB), an animal welfare and ethics review body. Approval from the UK Home Office is provided under project license 80 / 2432.

[0284] (Example 3) Figures 4, 5, and 6 show the annotation of Ig loci in cats. The annotation method is the same as that outlined in 2.1.1 of Example 1, except that the cat genome was investigated instead of the canine genome, and RNA sequencing data was not used.

[0285] Annotation provides tools and information that allow the use of cat DNA in rodent genomes using the methods described above.

[0286] (Example 4) Rapid amplification of the 5' end of cDNA (5'RACE) Substances and methods blood sample Baseline samples: Peripheral blood was collected from 13 dogs at the University of Minnesota School of Veterinary Medicine. RNA was extracted in-situ and transported on dry ice. This study was pre-approved by the Ethics Committee of the University of Minnesota School of Veterinary Medicine.

[0287] Library preparation First-strand cDNA was synthesized using a mixed pool of C-specific primers and template switch oligos (TSO). The reaction contained: 666.7 μM dNTPs (Sigma Aldrich), 666.7 nM TSO, 333.3 nM heavy and light chain RT primer mixtures, 1–5 μg RNA, 2 mM DTT (Invitrogen), 3 mM MgCl2, 40 units RNaseOUT (Invitrogen), and 100 units SuperScript II reverse transcriptase (Invitrogen) in 30 μl volume. Extension was performed at 42°C for 60 minutes, followed by the addition of 1 μl RNase A / T1 mixture (Thermo Scientific), and incubation 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: AMPure XP solution ratio 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 25 μl volumes 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 for either the heavy-chain or light-chain. The cycling conditions were as follows: 98°C for 30 seconds, followed by 20 cycles (98°C for 10 seconds, 63°C for 30 seconds, 72°C for 20 seconds), followed by a final extension at 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 performed in 25 μL of PCR reaction using Q5 polymerase and 400 nM each of forward primers containing 5'RRVA and 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 in 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) by the Wellcome Trust Sanger Institute's core sequencing team using 300 bp paired-end reads (including 10% PhiX spike-in). The demultiplexed data was then filtered for quality before being sent to IMGT V-Quest software (using "HighV-Quest" software, selecting "Species" = "Canis lupus familiaris (dog)" and "Receptor Type or Locus = "IG""). 14 .

Claims

1. i) IGH V region genes of one or more companion animals, D region genes of one or more companion animals, and J region genes of one or more companion animals A rodent or rodent cell having a genome containing, The aforementioned rodent or rodent cell can express the variable region gene of the companion animal and form part of the antibody chain. One or more V, D, or J region genes of the inserted companion animal are accompanied by a recombinant signal sequence and promoter derived from the same companion animal. The companion animal is a dog. A rodent or a rodent cell.

2. The rodent or rodent cell according to claim 1, wherein the genome of the rodent comprises a companion animal gene derived from both the heavy chain and at least one light chain.

3. A rodent or rodent cell according to claim 1 or 2, comprising at least 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, or at least 80 companion animal IGH V region genes.

4. A rodent or rodent cell according to any one of claims 1 to 3, comprising at least 4, 5, 10, 15, 16, 17, 18, or 19 companion animal IGL kappa V region genes.

5. A rodent or rodent cell according to any one of claims 1 to 4, comprising at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or at least 160 companion animal IGL lambda V region genes.

6. The rodent or rodent cell according to any one of claims 1 to 5, wherein the gene of the companion animal is located upstream of the genome of the constant region of the rodent, thereby enabling the rodent or rodent cell to produce a portion of the chimeric antibody chain resulting from the expression of the inserted variable region gene and the constant region of the host.

7. The rodent or rodent cell according to claim 6, wherein the genes of the companion animal are located upstream of the heavy chain constant region in the genome of the heavy chain variable region gene of the companion animal to be inserted, and upstream of the light chain constant region in the genome of the light chain variable region gene of the companion animal to be inserted.

8. A rodent or rodent cell according to any one of claims 1 to 5, having a genome in which the genes of the companion animal are functionally arranged in the genome together with a constant region derived from the same companion animal, thereby enabling the rodent to produce a portion of the antibody chain resulting from the expression of the inserted VDJ or VJ region gene of the companion animal and the constant region of the companion animal.

9. A rodent or rodent cell according to any one of claims 1 to 8, comprising one or more IGL-lambda V region genes of a companion animal, one or more IGL-lambda J region genes of a companion animal, and one or more constant lambda region genes of a companion animal, located at the kappa locus of the rodent cell.

10. The rodent or rodent cell according to claim 9, wherein 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 gene of one or more companion animals are located downstream of the kappa constant region of the rodent.

11. A rodent or rodent cell according to any one of claims 1 to 10, comprising kappa variable region genes of a canid, wherein all kappa variable region genes of a canid in the genome of the rodent are located upstream of a constant region in which the variable region genes are expressed together.

12. A rodent or rodent cell according to any one of claims 1 to 11, wherein all of the gene segments of the companion animal correspond to the reference alleles of canids in CanFam 3.1, or the gene segments of the companion animal include non-reference alleles derived from one or more of the following gene segments: IGKV2-S13, IGLV 1-57, IGLV 1-68, IGLV 1-72, IGLV 1-88, IGLV 1-96, IGLV 8-60, IGLV 8-90, and IGLV 8-120.

13. The rodent or rodent cell according to any one of claims 1 to 12, wherein the rodent is a mouse.

14. A method for producing a rodent or rodent cell according to any one of claims 1 to 13, wherein the method includes the step of inserting one or more companion animal IGH V region genes, one or more companion animal IGH D region genes, and one or more companion animal IGH J region genes into the genome of a rodent cell, wherein the rodent or rodent cell can express the variable region genes of the companion animal in combination with a constant region and form part of an antibody chain.

15. A method for producing a rodent or rodent cell according to claim 14, wherein the method comprises the step of inserting IGL V region genes of one or more companion animals and IGL J region genes of one or more companion animals into the genome of a rodent cell, and the rodent or rodent cell can express the variable region genes of the companion animal in combination with a constant region and form part of an antibody chain.

16. A method for producing a portion of an antibody chain specific to a desired antigen, the method comprising the steps of: immunizing a rodent according to any one of claims 1 to 13 with the desired antigen; and recovering a portion of the antibody chain or recovering cells that produce a portion of the antibody chain.

17. A method for producing a portion of an antibody chain specific to a desired antigen and derived from a single companion animal, the method comprising the steps of: immunizing a rodent containing the genes of the companion animal described in any one of claims 1 to 13; and then replacing a constant region of a portion of the antibody chain of the rodent with a constant region of a companion animal derived from the same companion animal.

18. The method according to claim 17, wherein the step of replacing a constant region of a portion of the antibody chain of the rodent with a constant region of a companion animal derived from the same companion animal is performed by genetically modifying the nucleic acid encoding a portion of the antibody chain.

19. A method for producing a portion of an antibody chain, wherein the portion of the antibody chain has a variable region of a companion animal, and the method includes the step of expressing DNA encoding the portion of the antibody chain in a cell. The DNA sequence encoding the variable region of a portion of the antibody chain is obtained by immunizing a rodent according to any one of claims 1 to 13 with an antigen to produce a portion of the antibody chain, or can be obtained therefrom. method.

20. A method for treating a companion animal, the method comprising the step of delivering a portion of an antibody chain to a companion animal in need thereof, wherein the portion of the antibody chain is obtained or can be obtained from a rodent or cell as described in any one of claims 1 to 13, or from the method described in any one of claims 16 to 19.

21. A method for partially replacing the locus of an endogenous immunoglobulin variable region gene in a rodent or rodent cell with the locus, recombinant signal sequence, and promoter of a homologous or orthologous companion animal gene, including the IGH V region gene, the D region gene, and the J region gene of one or more companion animals, of one or more companion animals, i) A step of obtaining a cloned genome fragment or synthetic sequence that partially includes the locus, recombinant signal sequence, and promoter of the homologous or orthologous companion animal gene, which includes 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) A step of genetically modifying the cloned genome fragment of (i) using homologous recombination to produce a large targeting vector for use in the rodent or rodent cells, and iii) The step of introducing the vector of (ii) into the rodent or rodent cell to partially replace the locus of the endogenous immunoglobulin variable gene. Includes, The companion animal is a dog, in this manner.

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