Animal model and therapeutic molecule

Abstract

The present invention relates, inter alia, to rodents and cells engineered to contain feline exogenous DNA, their use in medical and disease research, methods for producing the rodents and cells, and antibodies and antibody chains produced by such rodents, and derivatives thereof.

Description

Technical Field

[0001] The present invention relates, inter alia, to rodents and cells engineered to contain feline exogenous DNA, their use in medical and disease research, methods for producing such rodents and cells, and antibodies and antibody chains produced by such rodents, and derivatives thereof.

Background Art

[0002] The insertion of human DNA into rodents is disclosed, for example, in Murphy et al, Vol 111 no 14,5153 - 5158, doi:10.1073 / pnas.1324022111; MacDonald et al vol.111 no.14,5147 - 5152, doi:10.1073 / pnas.1323896111; and Lee et al, Nature Biotechnology Volume:32, Pages:356 - 363 2014 DOI:, doi:10.1038 / nbt.2825. This approach is designed to prepare antibody products for use in human therapy. Methods for inserting canine and feline DNA into rodents are described in WO2018189520.

[0003] The present invention relates to rodents and cells, and antibody repertoires, antibodies and parts of antibodies (e.g., those produced from rodents containing feline immunoglobulin DNA including a complete feline antibody), and the use of such antibodies and parts thereof in cats for the prevention and treatment of diseases, and methods for producing such rodents, cells, antibodies, antibody chains and repertoires.

Summary of the Invention

[0004] i) a rodent or rodent cell having a genome comprising no more than 9 feline IGH V gene segments, one or more feline IGH D region gene segments (plural available), and one or more feline IGH J region gene segments (plural available), The rodent or rodent cell can express a feline IGH gene segment to form an antibody heavy chain, The rodent or rodent cell, wherein at least one of the feline IGH V gene segments is selected from the list including V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5.

[0005] i) A rodent or rodent cell having a genome containing 11 or fewer feline lambda V gene segments and one or more feline IGL J region gene segments (plural available), The rodent or rodent cell can express a feline lambda gene segment to form an antibody light chain, The rodent or rodent cell, wherein at least one of the feline lambda gene segments is selected from the list including V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126.

[0006] i) A rodent or rodent cell having a genome containing 6 or fewer feline kappa V gene segments and one or more feline IGK J region gene segments (plural available), The rodent or rodent cell can express a feline kappa gene segment to form an antibody light chain, The rodent or rodent cell, wherein at least one of the feline kappa gene segments is selected from V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4.

[0007] In one embodiment, the gene segment has the nucleic acid sequence shown in Table 1, and "V3-14", "V-124", etc. are indicated by the corresponding SEQ ID NOs. "Gene segment" also means "gene".

[0008] A method for producing a rodent or rodent cell disclosed herein, the method comprising, in the genome of a rodent cell, (i) nine or fewer feline IGH V gene segments, one or more feline IGH D region gene segments, and one or more feline IGH J region gene segments (at least one of the feline IGH V gene segments is selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3, and V3-5), and / or (ii) eleven or fewer feline lambda V gene segments and one or more feline lambda J region gene segments (at least one of the feline V gene segments is selected from the list comprising V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13, and V1-126), and / or (iii) six or fewer feline kappa V gene segments and one or more feline J region gene segments (at least one of the feline kappa V gene segments is selected from V4-1, V2-12, V2-5, V2-13, V2-9, and V2-4), wherein the rodent or rodent cell can express the feline variable region gene segment(s) in combination with a constant region to form an antibody chain.

[0009] References herein to feline variable region gene segments are, where appropriate, references to feline V region gene segments, feline D region gene segments, and / or feline J region gene segments. In one aspect, a feline variable region gene segment is at least one feline V region gene segment, at least one feline D region gene segment, and at least one feline J region gene segment.

[0010] A method for producing an antibody or antibody chain specific for a desired antigen, the method comprising immunizing a rodent disclosed herein with the desired antigen and recovering the antibody chain or antibody, or recovering the cell producing the antibody chain or antibody.

[0011] A method of making an antibody chain or antibody specific for a desired antigen, the method comprising immunizing a rodent disclosed herein with the desired antibody and then, optionally, manipulating the nucleic acid encoding the antibody to replace any rodent constant region of the antibody chain or antibody with a feline constant region.

[0012] Any reference to replacement of a rodent constant region herein means that, where the constant region is a rodent constant region, it is replaced, for example, with a feline constant region, and where the constant region is already, for example, a feline constant region, it means that such replacement is not necessary. This may apply, for example, in the case of the lambda locus.

[0013] A method of making an antibody, antibody chain or part thereof, wherein the antibody chain has a feline variable region, the method comprising expressing a nucleic acid, such as DNA, encoding the antibody, antibody chain or part thereof intracellularly,

[0014] The sequence of the nucleic acid encoding the variable region of the antibody chain is obtained from immunizing a rodent disclosed herein with an antigen, Optionally, i. purifying and / or isolating the antibody or antibody chain, and ii. then optionally comprising the subsequent step of formulating the antibody or antibody chain into a pharmaceutically acceptable formulation suitable for administration to a cat.

[0015] A method of preparing a pharmaceutical composition, the method comprising making an antibody, preferably a complete feline antibody, by the method disclosed herein and further comprising combining the antibody with a pharmaceutically acceptable carrier or other excipient to make the composition.

[0016] A nucleic acid encoding a chimeric antibody chain, wherein the nucleic acid comprises any one of the feline IGH V gene segments selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5, and a rodent constant region, and optionally, the feline V gene segment is a sequence that has undergone somatic hypermutation in a rodent.

[0017] A nucleic acid encoding a chimeric antibody chain, wherein the nucleic acid comprises any two, three, four, five, six, seven, eight or all nine of the feline IGH V gene segments selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5, and a rodent constant region, and optionally, the feline V gene segment is a sequence that has undergone somatic hypermutation in a rodent.

[0018] A nucleic acid encoding a chimeric antibody chain, wherein the nucleic acid comprises any one of the feline lambda V gene segments selected from the list comprising lambda V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126, and a rodent constant region, and optionally, the feline V gene segment sequence is a sequence that has undergone somatic hypermutation in a rodent.

[0019] A nucleic acid encoding a chimeric antibody chain, wherein the nucleic acid comprises any two, three, four, five, six, seven, eight, nine, ten or all eleven of the feline lambda V gene segments selected from the list comprising lambda V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126, and a rodent constant region, and optionally, the feline V gene segment is a sequence that has undergone somatic hypermutation in a rodent.

[0020] A nucleic acid encoding a chimeric antibody chain, wherein the nucleic acid comprises any one of the feline kappa V gene segments selected from the list comprising V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4, and a rodent constant region, and optionally, the feline V gene segment sequence is a sequence that has undergone somatic hypermutation in a rodent.

[0021] A nucleic acid encoding a chimeric antibody chain, wherein the nucleic acid comprises any two, three, four, five or all six of the feline kappa V gene segments selected from the list comprising V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4, and a rodent constant region, and optionally, the feline V gene segment is a sequence that has undergone somatic hypermutation in a rodent.

[0022] The antibody chain, in combination with a rodent constant region, has a variable region obtained by the expression of any one of feline IGH V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5 in a rodent.

[0023] The antibody chain, in combination with a rodent constant region, has a variable region obtained by expressing any two, three, four, five, six, seven, eight or all nine of feline IGH V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5 in a rodent.

[0024] The antibody chain, in combination with a rodent constant region, has a variable region obtained by the expression of any one of feline lambda V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126 in a rodent.

[0025] The antibody chain has a variable region obtained by expressing, in combination with a rodent constant region, any 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 of Nek lambda V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13, and V1-126 in rodents.

[0026] The antibody chain has a variable region obtained by expressing, in combination with a rodent constant region, any one of Nek kappa V4-1, V2-12, V2-5, V2-13, V2-9, and V2-4 in rodents.

[0027] The antibody chain has a variable region obtained by expressing, in combination with a rodent constant region, any 2, 3, 4, 5, or all 6 of Nek kappa V4-1, V2-12, V2-5, V2-13, V2-9, and V2-4 in rodents.

[0028] The pharmaceutical composition comprises an antibody chain having a variable region comprising any one, or any 2, 3, 4, 5, 6, 7, 8, or all 9 of Nek IGH V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3, and V3-5, in combination with a pharmaceutically acceptable excipient or carrier.

[0029] The pharmaceutical composition comprises an antibody chain having a variable region comprising any one, or any 2, 3, 4, 5, 6, 7, 8, 9, 10, or all 11 of Nek lambda V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13, and V1-126, in combination with a pharmaceutically acceptable excipient or carrier.

[0030] The pharmaceutical composition comprises an antibody chain having a variable region containing any one, or any two, three, four, five or all six of Nekokappa V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4, in combination with a pharmaceutically acceptable excipient or carrier.

[0031] A pharmaceutical composition comprising an antibody, wherein the antibody (i) has a heavy chain having a variable region obtained by the expression of any one of Nek oIGH V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5, (ii) has a lambda light chain obtained by the expression of any one of Nekolambda V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126, or (iii) has any one of kappa light chains obtained by the expression of any one of Nekokappa V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4, and the antibody is formulated in combination with a pharmaceutically acceptable excipient or carrier.

[0032] An antibody heavy chain repertoire, the repertoire comprising antibody heavy chains having Nek oIGH V gene segments from no more than 9 different Nek oIGH V gene segments, at least one of the Nek oIGH V gene segments being selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5, Optionally, the repertoire A, (i) V3-14 (optionally having up to 8 other Nek oIGH V gene segments), (ii) V3-14 and V3-11 (optionally having up to 7 other Nek oIGH V gene segments), (iii) V3-14, V3-11 and V3-12 (optionally having up to 6 other Nek oIGH V gene segments), (iv) V3-14, V3-11, V3-12, and V3-84 (optionally having up to 5 other feline IGH V gene segments), (v) V3-14, V3-11, V3-12, V3-84, and V3-20 (optionally having up to 4 other feline IGH V gene segments), (vi) V3-14, V3-11, V3-12, V3-84, V3-20, and V3-27 (optionally having up to 3 other feline IGH V gene segments), (vii) V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, and V4-3 (optionally having up to 2 other feline IGH V gene segments), (viii) V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, and V3-N3 (optionally having up to 1 other feline IGH V gene segment), or (ix) An antibody heavy chain having feline IGH V gene segments expressed from V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3, and V3-5, and / or B. Comprising an antibody heavy chain expressed from IGH V gene segments of feline IGH V families 3 and 4.

[0033] Gene families such as IGH V family 3 are terms well-known in the art. In one aspect, references to gene families such as IGH V family 3 refer to the IMGT (ImMunoGeneTics) notation, which is available (as of May 26, 2022) from the following: https: / / www.imgt.org / IMGTrepertoire / LocusGenes / #F.

[0034] An antibody heavy chain repertoire, the repertoire comprising antibody heavy chains having feline IGH V gene segments from 9 or fewer different feline IGH V gene segments, wherein one IGH V segment must be IGH V4-3 and at least one of the remaining IGH V segments is selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5.

[0035] An antibody lambda light chain repertoire, the repertoire comprising antibody light chains having feline lambda V gene segments from 11 or fewer different lambda V gene segments, wherein at least one of the V gene segments is selected from the list comprising V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126, Optionally, the repertoire A, (i) V1-124 (optionally having up to 10 other feline lambda V gene segments), (ii) V1-124 and V1-36 (optionally having up to 9 other feline lambda V gene segments), (iii) V1-124, V1-36 and V1-104 (optionally having up to 8 other feline lambda V gene segments), (iv) V1-124, V1-36, V1-104 and V1-33 (optionally having up to 7 other feline lambda V gene segments), (v) V1-124, V1-36, V1-104, V1-33 and V1-56 (optionally having up to 6 other feline lambda V gene segments), (vi) V1-124, V1-36, V1-104, V1-33, V1-56, V1-42 (optionally having up to 5 other feline lambda V gene segments), or (vii) An antibody lambda light chain having a lambda V gene segment comprising V1-124, V1-36, V1-104, V1-33, V1-56, V1-42 and V1-41 (optionally having up to 4 other Nekolambdav gene segments). and / or B. comprising an antibody lambda light chain expressed from lambda V gene segments of Nekolambdav families 1 and 3.

[0036] An antibody kappa light chain repertoire, the repertoire comprising an antibody kappa light chain having a Nekokappa V gene segment from up to 6 different Nekokappa V gene segments, wherein at least 1 of the V gene segments is selected from the list comprising V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4. Optionally, the repertoire A. (i) V4-1 (optionally having up to 5 other Nekokappa V gene segments), (ii) V4-1 and V2-12 (optionally having up to 4 other Nekokappa V gene segments), (iii) V4-1, V2-12 and V2-5 (optionally having up to 3 other Nekokappa V gene segments), (iv) V4-1, V2-12, V2-5 and V2-13 (optionally having up to 2 other Nekokappa V gene segments), (v) V4-1, V2-12, V2-5, V2-13 and V2-9 (optionally having up to 1 other Nekokappa V gene segment), or (vi) V4-1, V2-12, V2-5, V2-13, V2-4 and V2-4, or (vii) An antibody kappa light chain having a Nekokappa V gene segment comprising V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4. and / or B. comprising an antibody kappa light chain expressed from kappa V gene segments of Nekokappa V families 2 and 4.

[0037] An antibody kappa light chain repertoire, the repertoire comprising antibody kappa light chains having kappa V gene segments from 6 or fewer different feline kappa V gene segments, wherein one of the feline kappa V gene segments needs to be IGKV4-1, and at least 1 of the V gene segments is selected from the list comprising V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4.

[0038] An isolated antibody or antibody chain, or a part thereof, or a nucleic acid such as DNA encoding an antibody chain or a part thereof (obtained or obtainable from a rodent or rodent cell described herein, or from a repertoire described herein (e.g., a complete feline antibody chain or a complete feline antibody)), or a pharmaceutical composition comprising the same.

[0039] An antibody or antibody chain, or a part thereof, or a nucleic acid such as DNA encoding an antibody chain or a part thereof (obtained or obtainable from a rodent or rodent cell described herein, or from a repertoire described herein (e.g., a complete feline antibody chain or a complete feline antibody)), or a pharmaceutical composition comprising the same, for use in the treatment or prevention of a feline disease in a cat in need of treatment or prevention of the disease.

[0040] A method of treating a cat, the method comprising delivering an antibody or antibody chain, or a part thereof, to a cat in need thereof, wherein the antibody is obtained or obtainable from a rodent or rodent cell or repertoire disclosed herein.

[0041] The rodent or rodent cell has a genome comprising a feline IGH V gene segment selected from any one of the sequences listed in Table 1. The rodent or rodent cell has a genome comprising a feline IGH V gene segment selected from the list comprising IGH V3-14 (SEQ ID NO: 1), V1-36 (SEQ ID NO: 11), V1-104 (SEQ ID NO: 12), and V1-42 (SEQ ID NO: 15), one or more feline IGH D region gene segments (multiple possible) and one or more feline IGH J region gene segments (multiple possible), The rodent or rodent cell can express the feline gene segment to form an antibody heavy chain.

[0042] A rodent or rodent cell having a genome comprising a feline IGK V gene segment selected from any one of the sequences listed in Table 1. A rodent or rodent cell having a genome comprising the feline IGK V gene segment IGK V2-5 (SEQ ID NO: 23) and one or more feline IGH J region gene segments (multiple possible), The rodent or rodent cell can express the feline gene segment to form an antibody light chain.

[0043] Table Table 1 - List of Preferred V Gene Sequences Table 2a - Analysis of Healthy Feline PBMCs, List of Most Commonly Observed Heavy Chain and Light Chain Pairings Table 2b - Analysis of Healthy Feline PBMCs, List of Most Commonly Observed Heavy Chains Table 2c - Analysis of Healthy Feline PBMCs, List of Most Commonly Observed Light Chains Table 3 - Primer Sequences Table 4 - Analysis of Healthy Feline PBMCs, List of Light Chains Ranked by the Number of Different Heavy Chain Pairings Table 5a - Analysis of Healthy Feline PBMCs, List of Most Commonly Observed Heavy Chains Table 5b - Analysis of Healthy Feline PBMCs, List of Most Commonly Observed Lambda Chains Table 5c - Analysis of healthy cat PBMCs, list of the most commonly observed kappa chains

DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention relates to a rodent comprising a feline immunoglobulin gene segment. Conventional rodents contain both human IG DNA and feline IG DNA, and generally attempt to insert as many V gene segments as possible in order to optimize the range of antibodies produced. However, achieving complete insertion of large IG loci requires a significant amount of genomic engineering effort. In the present disclosure, the antibody profiles produced in European shorthair cats were analyzed, and it was shown that only a few V gene segments contribute disproportionately to the antibody pool. For example, the feline IGH V3-14 gene segment is found in more than 14% of the heavy chains of antibodies. Thus, it is not necessary to insert all V gene segments to create an antibody population that provides a repertoire suitable for the identification and selection of lead antibody candidates. This rationalizes the genetic engineering required to create a rodent model for antibody production and enables the creation of a rodent model with an IGHV and IG light chain V usage that reflects their use in cats that are not used in experiments.

[0045] When most (e.g., all) of the feline IG locus is inserted into a rodent, some V gene segments that are preferentially used in cats may be less frequently expressed in the rodent because they are far upstream from the (rodent) constant region. This can reduce the effectiveness of such rodents in producing therapeutically important antibodies. Thus, another advantage of the present invention is that preferred V gene segments can be inserted closer to the constant region than their natural position in the cat, so these preferred V gene segments are more likely to be used more frequently in the rodent than when a larger portion of the feline IG locus is inserted, and thus can more faithfully represent the natural repertoire of the cat.

[0046] The feline V gene segments selected for insertion into a rodent to create an antibody population can be based on the frequency of observation, the number of different pairings (between the heavy feline V gene segments and the light feline V gene segments), or a combination of these two. The feline V gene segment with the highest frequency of observation is preferred. Preferred heavy or light chain feline V gene segments are those contained within antibody chains that pair with the greatest number of different light or heavy chain feline V antibody chains (antibody chains containing different feline V gene segments), respectively.

[0047] Accordingly, the present invention relates to the following.

[0048] i) A rodent or rodent cell having a genome comprising no more than 9 feline IGH V gene segments, one or more feline D region gene segments, and one or more feline J region gene segments, ii) said rodent or rodent cell is capable of expressing the feline gene segments to form an antibody heavy chain, and iii) at least one of said feline IGH V gene segments is selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5.

[0049] Table 2b shows the frequency of observation of feline heavy chain V gene segments. One or more of the 9 preferred IGH V gene segments can be combined with other feline V gene segment(s) within the rodent genome. It is not necessary to limit to only the 9 preferred feline V gene segments, but at least one must be present. A preferred feline gene segment that is present is IGH V3-14. A preferred alternative feline gene segment is V3-11. These two sequences are found together in more than 23% of feline antibody heavy chains.

[0050] Further embodiments disclosed herein relate to the feline IGH disclosed herein (i) V3-14 (optionally having up to 8 other feline IGH V gene segments), (ii) V3-14 and V3-11 (optionally having up to 7 other feline IGH V gene segments), (iii) V3-14, V3-11 and V3-12 (optionally having up to 6 other feline IGH V gene segments), (iv) V3-14, V3-11, V3-12 and V3-84 (optionally having up to 5 other feline IGH V gene segments), (v) V3-14, V3-11, V3-12, V3-84 and V3-20 (optionally having up to 4 other feline IGH V gene segments), (vi) V3-14, V3-11, V3-12, V3-84, V3-20 and V3-27 (optionally having up to 3 other feline IGH V gene segments), (vii) V3-14, V3-11, V3-12, V3-84, V3-20, V3-27 and V4-3 (optionally having up to 2 other feline IGH V gene segments), (v) V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3 and V3-N3 (optionally having up to 1 other feline IGH V gene segment), or (ix) V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5, which are rodents or rodent cells and contain the same.

[0051] Further embodiments disclosed herein are rodents or rodent cells containing only 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 of the feline IGH V gene segment(s) V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5.

[0052] Further embodiments disclosed herein are rodents or rodent cells that have only one feline IGH V gene segment, only one feline IGH D gene segment, only one feline IGH J gene segment, and form a "common heavy chain".

[0053] In one aspect, a bispecific antibody can be generated using a common heavy chain approach.

[0054] Further embodiments disclosed herein are rodents or rodent cells that contain at least two IgH gene segments, e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, or all nine feline IGH V gene segments V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3, and V3-5. The rodent or rodent cell can contain one, two, three, four, five, six, seven, eight, or nine of the feline IGH V gene segments V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3, and V3-5.

[0055] In one embodiment of any aspect of the invention, the total number of feline V gene segments inserted into each locus (heavy chain, lambda chain, and / or kappa chain) is 9 or less.

[0056] In one embodiment of any aspect of the invention, the total number of feline V gene segments inserted into each locus (heavy chain, lambda chain, and / or kappa chain) is 11 or less.

[0057] In one embodiment of any aspect of the invention, the total number of feline V gene segments inserted into each locus (heavy chain, lambda chain, and / or kappa chain) is 6 or less.

[0058] In one embodiment of any aspect of the invention, the total number of feline V gene segments inserted into each locus (heavy chain, lambda chain, and / or kappa chain) is 20 or less.

[0059] In one embodiment of any aspect of the present invention, the total number of feline V gene segments inserted into each locus (heavy chain, lambda chain and / or kappa chain) is 35 or less.

[0060] In one embodiment of any aspect of the present invention, the total number of feline V gene segments inserted into each locus (heavy chain, lambda chain and / or kappa chain) is 50 or less.

[0061] Preferably, the rodent comprises at least one gene segment from each of feline IGH V gene families 3 and 4, and there are 9 or fewer feline IGH V gene segments in the genome of the rodent or rodent cells.

[0062] In one embodiment, all of the inserted V genes are functional V genes.

[0063] In one embodiment, the inserted V genes consist of functional V genes and non-functional V genes (including pseudogenes and open reading frames).

[0064] In one embodiment, a subset of the total number of preferred feline V gene segments is inserted. One, two, three, four, five, six, seven, eight or nine of the preferred feline IGH V gene segments may be inserted. One, two, three, four, five, six, seven, eight, nine, ten or eleven of the preferred feline IGL V gene segments may be inserted. One, two, three, four, five or six of the preferred feline IGK V gene segments may be inserted.

[0065] The total number of feline V gene segments inserted into each locus may be limited as disclosed herein, such that, in combination, for example, the present invention contemplates the insertion of 20 feline IGH V gene segments, and 6 of the inserted feline IGH V gene segments are preferred feline IGH V gene segments.

[0066] In a further embodiment, the feline V gene segments to be inserted are inserted together with other feline V gene segments and / or other regions of the feline IG locus. In particular, the chromosomal regions surrounding the preferred V genes may also be inserted together.

[0067] For example, feline V gene segments inserted as part of a single bacterial artificial chromosome (BAC). In one aspect, each BAC contains 3 to 5 feline V gene segments, including 1 of the preferred feline V gene segments.

[0068] In one aspect, reference to a BAC such as BAC FCAB-223I3 is a reference to the FCAB library of Amplicon Express, which is currently available from the College of Veterinary Medicine and Biomedical Sciences, Texas A&M University.

[0069] In one aspect, the insertion of the feline IGH V gene segment may involve the insertion of part or all of BAC FCAB-223I3.

[0070] In one aspect, the insertion of the feline IGH V gene segment may involve the insertion of part or all of BAC FCAB-253K11. In one aspect, the insertion of the feline IGH V gene segment may involve the insertion of part or all of BAC FCAB-253K11 and part or all of BAC FCAB-180D24. In one aspect, the insertion of the feline IGH V gene segment may involve the insertion of part or all of BAC FCAB-253K11, part or all of BAC FCAB-180D24, and part or all of BAC FCAB-74C2.

[0071] In one aspect, the insertion of the feline IGL V gene segment may involve the insertion of part or all of BAC FCAB-35I7.

[0072] In one aspect, the insertion of the feline IGL V gene segment may involve the insertion of some or all of BAC FCAB-166L22. In one aspect, the insertion of the feline IGL V gene segment may involve the insertion of some or all of BAC FCAB-166L22 and some or all of BAC FCAB-87D23. In one aspect, the insertion of the feline IGL V gene segment may involve the insertion of some or all of BAC FCAB-166L22, some or all of BAC FCAB-87D23, and some or all of BAC FCAB-526E12.

[0073] In one aspect, the insertion of the feline IGK V gene segment may involve the insertion of some or all of BAC FCAB-609P16.

[0074] In one embodiment, the feline gene segment to be inserted is inserted as part of an array or cluster of feline gene segments and, optionally, is embedded in a rodent or feline non-coding regulatory sequence or scaffold sequence.

[0075] In one embodiment, the feline gene segment to be inserted can be inserted as part of a minilocus.

[0076] Alternative methods of inserting gene segments into rodents are described for dogs in US20170306352, which is incorporated by reference and is equally applicable to cats.

[0077] In one embodiment, the feline V gene segment to be inserted is inserted as part of a concatemer. In one aspect, the concatemer is a series of preferred feline V gene segments and, optionally, comprises feline or rodent regulatory sequences associated with the feline V gene segment.

[0078] The present invention further relates to the following.

[0079] An isolated nucleotide molecule comprising the sequence of any one of SEQ ID NO: 1, 11, 12, 15 or 23, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, for example at least 95% identity.

[0080] A polypeptide comprising or consisting of an amino acid sequence encoded by the nucleotide molecule according to the invention. In one embodiment, the polypeptide can be an antibody.

[0081] A pharmaceutical composition comprising the antibody according to the invention.

[0082] A rodent or rodent cell having a genome comprising the sequence of any one of SEQ ID NO: 1, 11, 12, 15 or 23. Suitable such rodent cells include isolated rodent B cells or cell lines such as ES cells or iPS cells. Mouse ES cell lines are described, for example, in WO2018 / 1899520.

[0083] A vector comprising the sequence of any one of SEQ ID NO: 1, 11, 12, 15 or 23.

[0084] A rodent or rodent cell having a genome comprising a feline IGH V gene segment selected from the list comprising IGH V3-14 (SEQ ID NO: 1), V1-36 (SEQ ID NO: 11), V1-104 (SEQ ID NO: 12), and V1-42 (SEQ ID NO: 15), one or more feline IGH D region gene segments (plural available) and one or more feline IGH J region gene segments (plural available), wherein the rodent or rodent cell can express the feline gene segments to form an antibody heavy chain.

[0085] A rodent or rodent cell having a genome comprising a feline IGH V gene segment IGH V3-14 (SEQ ID NO: 1), a feline IGH V gene segment, one or more feline IGH D region gene segments (multiple possible), and one or more feline IGH J region gene segments (multiple possible), The rodent or rodent cell capable of expressing the feline gene segment to form an antibody heavy chain.

[0086] A rodent or rodent cell having a genome comprising a feline IGH V gene segment IGH V3-14 (SEQ ID NO: 1), and a feline IGH V gene segment IGH V1-36 (SEQ ID NO: 11), one or more feline IGH D region gene segments (multiple possible), and one or more feline IGH J region gene segments (multiple possible), The rodent or rodent cell capable of expressing the feline gene segment to form an antibody heavy chain.

[0087] A rodent or rodent cell having a genome comprising a feline IGH V gene segment IGH V3-14 (SEQ ID NO: 1), a feline IGH V gene segment IGH V1-36 (SEQ ID NO: 11), and a feline IGH V gene segment IGH V1-104 (SEQ ID NO: 12), one or more feline IGH D region gene segments (multiple possible), and one or more feline IGH J region gene segments (multiple possible), The rodent or rodent cell capable of expressing the feline gene segment to form an antibody heavy chain.

[0088] A rodent or rodent cell having a genome comprising a feline IGH V gene segment IGH V3-14 (SEQ ID NO: 1), a feline IGH V gene segment IGH V1-36 (SEQ ID NO: 11), a feline IGH V gene segment IGH V1-104 (SEQ ID NO: 12), and a feline IGH V gene segment IGH V1-42 (SEQ ID NO: 15), one or more feline IGH D region gene segments (multiple possible), and one or more feline IGH J region gene segments (multiple possible), The rodent or rodent cell capable of expressing a feline gene segment to form an antibody heavy chain.

[0089] A rodent or rodent cell having a genome containing the feline IGK V gene segment IGK V2-5 (SEQ ID NO: 23) and one or more feline IGH J region gene segments (plural possible), The rodent or rodent cell capable of expressing a feline gene segment to form an antibody light chain.

[0090] The present invention further relates to the following.

[0091] i) A rodent or rodent cell having a genome containing 11 or fewer feline lambda V gene segments and one or more feline J region gene segments, ii) The rodent or rodent cell can express a feline lambda gene segment to form an antibody light chain, iii) At least one of the feline lambda gene segments is selected from V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126.

[0092] Table 2c shows the observed frequencies of feline light chain V gene segments. One or more of the 11 preferred feline lambda gene segments can be combined with other feline V gene segment(s) within the rodent genome. It is not necessary to limit to only the 11 preferred feline V gene segments, but at least one must be present. A preferred feline gene segment present is IGH V-124. A preferred alternative feline gene segment is V1-36. A preferred alternative feline gene segment is V1-104. A preferred alternative feline gene segment is V1-33. These four sequences are found together in more than 17% of the lambda antibody chains.

[0093] Further embodiments disclosed herein relate to murine lambda V (i) V1-124 (optionally having up to 10 other murine lambda V gene segments), (ii) V1-124 and V1-36 (optionally having up to 9 other murine lambda V gene segments), (iii) V1-124, V1-36 and V1-104 (optionally having up to 8 other murine lambda V gene segments), (iv) V1-124, V1-36, V1-104 and V1-33 (optionally having up to 7 other murine lambda V gene segments), (v) V1-124, V1-36, V1-104, V1-33 and V1-56 (optionally having up to 6 other murine lambda V gene segments), (vi) V1-124, V1-36, V1-104, V1-33, V1-56, V1-42 (optionally having up to 5 other murine lambda V gene segments), or (vii) a rodent or rodent cell comprising V1-124, V1-36, V1-104, V1-33, V1-56, V1-42 and V1-41 (optionally having up to 4 other murine lambda V gene segments).

[0094] Further embodiments disclosed herein are rodent or rodent cells comprising 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or only 1 V gene segment selected from the list of V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126.

[0095] Further embodiments disclosed herein are rodent or rodent cells having only 1 murine lambda V gene segment and only 1 murine lambda J gene segment, forming a "common light chain", as shown above.

[0096] Further embodiments disclosed herein are rodents or rodent cells comprising at least two feline lambda V gene segments, such as at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 feline lambda V gene segments selected from V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13, and V1-126. The rodent or rodent cell may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the feline lambda gene segments V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13, and V1-126.

[0097] Preferably, the rodent comprises at least one lambda V gene segment from each of feline lambda V gene segment families 1 and 3 and has 11 or fewer feline lambda V gene segments in its genome.

[0098] The present invention further relates to the following.

[0099] i) A rodent or rodent cell having a genome comprising 6 or fewer feline kappa V gene segments and one or more feline J region gene segments (s), ii) The rodent or rodent cell is capable of expressing a feline kappa gene segment to form an antibody light chain, iii) At least one of the feline kappa gene segments is selected from V4-1, V2-12, V2-5, V2-13, V2-9, and V2-4.

[0100] Table 2c shows the observed frequencies of feline light chain V gene segments. One or more of the six preferred feline kappa gene segments can be combined with other feline V gene segments within the rodent genome. It is not necessary to limit to only the six preferred feline kappa V gene segments, but at least one must be present. The preferred feline gene segment that is present is V4-1, which is found in more than 27% of all kappa chain antibodies. A preferred alternative feline gene segment is V2-12. These two sequences are found together in more than 50% of lambda antibody chains.

[0101] Further embodiments disclosed herein are feline kappa V (i) V4-1 (optionally having up to five other feline kappa V gene segments), (ii) V4-1 and V2-12 (optionally having up to four other feline kappa V gene segments), (iii) V4-1, V2-12 and V2-5 (optionally having up to three other feline kappa V gene segments), (iv) V4-1, V2-12, V2-5 and V2-13 (optionally having up to two other feline kappa V gene segments), (v) V4-1, V2-12, V2-5, V2-13 and V2-9 (optionally having up to one other feline kappa V gene segment), or (vi) V4-1, V2-12, V2-5, V2-13, V2-4 and V2-4, or (vii) A rodent or rodent cell comprising V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4.

[0102] Further embodiments disclosed herein are rodent or rodent cells comprising five or fewer, four or fewer, three or fewer, two or fewer, or only one feline kappa V gene segment selected from V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4.

[0103] Further embodiments disclosed herein are rodents or rodent cells that have only one kappa V gene segment and only one kappa J gene segment and form a "common light chain".

[0104] Further embodiments disclosed herein are rodents or rodent cells that contain at least two kappa V gene segments, such as at least three, at least four, at least five or six kappa V gene segments selected from V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4.

[0105] Further embodiments disclosed herein are rodents or rodent cells that contain one, two, three, four, five or six kappa V gene segments selected from V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4.

[0106] The rodent contains kappa V gene segments from each of kappa V gene segment families 2 and 4, suitably including IGK V2-12 and IGK V4-1, and preferably has no more than six kappa V gene segments in the genome.

[0107] In another preferred embodiment, the rodent genome contains feline DNA encoding one or more of the following heavy and light chain pairs, which together form more than 7% of the observed antibody chain pairings.

[0108] IGH V3-14fc IGL V1-104fc IGH V3-14fc IGL V1-124fc IGH V3-14fc IGL V1-36fc IGH V3-14fc IGL V1-56fc IGH V3-14fc IGL V1-126fc IGH V3-14fc IGL V1-42fc IGH V3-14fc IGL V1-41fc IGH V3-14fc IGK V4-1fc IGH V3-14fc IGL V3-7fc IGH V3-14fc IGK V2-12fc IGH V3-12fc IGL V1-104fc IGH V3-11fc IGL V1-56fc IGH V3-14fc IGL V1-33fc IGH V3-11fc IGL V1-41fc

[0109] Inserted DNA with a limited number of V gene segments can be referred to herein as a restricted insertion or a restricted locus. In some embodiments of the invention, it will be understood that the number of inserted heavy chain V gene segments is not restricted (when the light chain is restricted), or vice versa, the number of inserted light chain V gene segments is not restricted when the heavy chain is restricted. All of the insertions of one, two or three cats can be restricted.

[0110] The number of cat V gene segments at a restricted locus suitably provides coverage of at least 80%, such as at least 85%, 90%, 95%, 97%, 98% or 99% of the observed antibody sequences, and optionally, the coverage of the observed antibody sequences is less than 100%.

[0111] The number of cat V gene segments at a restricted locus can be a single cat V gene segment and suitably provides coverage of at least 10%, such as at least 20%, 30%, 40%, 50%, 60% or 70% of the observed antibody sequences, and optionally, the coverage of the observed antibody sequences is less than 100%.

[0112] The number of feline V gene segments at the restricted locus preferably provides coverage of up to 80% of the observed antibody sequences, for example, up to 85%, 90%, 95%, 97%, 98% or 99% coverage.

[0113] The number of feline V gene segments at the restricted locus can be a single feline V gene segment and preferably provides coverage of about 11% of the observed antibody sequences, or about 20%, about 30%, about 40%, about 50%, about 60% or about 70% coverage.

[0114] For example, the genome of a rodent or rodent cell can comprise a combination of a restricted feline heavy chain insertion disclosed herein, a restricted feline lambda light chain insertion disclosed herein, and / or a restricted feline kappa light chain insertion disclosed herein.

[0115] The genome of a rodent or rodent cell of the invention can comprise one or more feline IGH V gene segments as restricted insertions as described herein, but without light chain feline DNA, or can comprise one or more feline IG light chain V gene segments as restricted insertions as described herein, but without heavy chain feline DNA.

[0116] If the immunoglobulin locus of the genome of a rodent or rodent cell is not restricted by the number of V gene segments, it can have any number including all of the feline V gene segments. Thus, the genome of a rodent or rodent cell can comprise a combination of a feline heavy chain inserted as disclosed herein and any light chain insertion (such as a complete feline light chain, or a feline light chain insertion having more than 11 feline V lambda gene segments and / or more than 6 feline kappa V gene segments). Alternatively, the genome of a rodent or rodent cell can comprise a feline light chain insertion disclosed herein in combination with any heavy chain (such as a complete feline heavy chain, or a feline heavy chain insertion having more than 9 feline V gene segments).

[0117] For example, if only heavy chain insertion is restricted by the number of V gene segments, the light chain locus can have one or more, or all, of the light chain V gene segments. This can include at least 50% of the feline kappa and / or lambda light chain variable (V) genes, such as at least 60%, at least 70%, at least 80%, at least 90%, and in one embodiment, all of the feline kappa and / or lambda V genes.

[0118] In one embodiment, the genome of a rodent or rodent cell can include at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or at least 100 feline IG lambda V region genes, optionally functional feline IG lambda V region genes. In a preferred embodiment, the rodent genome includes at least 78 feline IG light chain lambda V region genes, optionally functional feline IG light chain lambda V region genes.

[0119] In one embodiment, the genome of a rodent or rodent cell can include at least 5, 6, 7, 8, 9, or at least 10 feline IG kappa V region genes, optionally functional feline IG kappa V region genes. In a preferred embodiment, the rodent genome includes at least 10 feline IG light chain kappa V region genes, optionally functional feline IG light chain kappa V region genes.

[0120] For example, when only light chain insertion is restricted, the heavy chain locus may contain one or more or all of the heavy chain feline IGH V gene segments. This may include at least 50% of the feline heavy chain variable (V) genes, such as at least 60%, at least 70%, at least 80%, at least 90%, and in one embodiment, all of the feline heavy chain V genes. In this embodiment, the genome of the rodent or rodent cell may contain at least 4, 5, 10, 15 or 20 feline IGH V region genes, such as at least 20, 30, 40 or at least 50 feline IGH V region genes, optionally including functional feline IGH V region genes. In a preferred embodiment, the rodent genome contains at least 34 feline IG heavy chain V region genes, optionally including functional feline IG heavy chain V region genes.

[0121] References to genes herein may, where appropriate, be references to gene segments. The use of the term "gene" is not intended to exclude such features as are disclosed equivalently in relation to gene segments, unless the context or necessity otherwise indicates.

[0122] Similarly, references to gene segments herein may, where appropriate, be references to genes. The use of the term "gene segment" is not intended to exclude such features as are disclosed equivalently in relation to genes, unless the context or necessity otherwise indicates.

[0123] Preferred feline DNA is the DNA of the European shorthair cat, and thus preferred cells and rodents contain, as disclosed herein, DNA gene segments of the European shorthair cat. The use of regulatory sequences of the European shorthair cat is also preferred. The DNA of the European shorthair cat may be provided as genomic DNA.

[0124] In all embodiments and aspects of the present invention, the feline gene segment may be located upstream of the rodent constant region within the genome, suitably upstream of the heavy chain constant region for the inserted feline heavy chain variable region gene segment, and suitably upstream of the light chain constant region for the inserted feline light chain variable region gene segment, such that the rodent or rodent cell can produce a chimeric antibody heavy chain, or a chimeric light chain, or both, resulting from the expression of the inserted variable region gene segment and the host constant region.

[0125] Reference to the position of the variable region upstream of a constant region such as the rodent constant region means that there is an appropriate relative position of the two genomic portions encoding the variable and constant regions of the antibody, enabling the chimeric antibody chain to be expressed in the rodent in vivo. In this way, the inserted feline DNA and the constant region are arranged functionally with respect to each other for the production of an antibody or antibody chain.

[0126] Information regarding the variable region of the chimeric antibody chain, or a nucleic acid comprising the variable region of the chimeric antibody chain, can be obtained from cells expressing the chimeric antibody using standard techniques. These sequences can be used to generate a complete feline antibody by expressing a nucleic acid encoding the antibody variable region together with the feline constant region to generate a feline antibody, and can be used, for example, for the treatment of felines.

[0127] The feline DNA may be inserted between the wild-type rodent constant region located at the wild-type locus, suitably between the rodent constant region and the host VDJ or VJ region. The rodent constant region expressed together with the feline variable region is preferably the wild-type rodent constant region located at the wild-type locus, depending on the feline heavy or light chain VDJ or VJ. In one aspect, the IGH variable region gene is inserted downstream of the rodent heavy chain J region and upstream of the Emu enhancer.

[0128] In one aspect, the IGH variable region gene is inserted downstream of the rodent heavy chain J region and upstream of the Emu enhancer. In one aspect, the rodent is a mouse, and the insertion of the IGH V region gene is performed at position 114666435 on chromosome 12 of the mouse genome. In one aspect, the insertion of the IG lambda V region gene is performed at position 19047551 on chromosome 16 of the mouse genome. In one aspect, the insertion of the IG kappa V region gene or gene cluster is performed at position 70674755 on chromosome 6 of the mouse genome.

[0129] Preferably, the feline V(D)J gene segment to be inserted can undergo V(D)J rearrangement in the body of the rodent to form an antibody chain. However, if a single rearranged heavy or light chain variable region is inserted, or if a complete antibody chain VDJC or VJC is inserted, rearrangement of the gene segment may not be necessary.

[0130] In a preferred embodiment, the rodent (or rodent cell) of the present invention is a mouse (or mouse cell).

[0131] Alternatively, the feline gene segment to be inserted is located (inserted) in the genome in functional alignment with the feline constant region, such that the rodent can produce an antibody chain resulting from the expression of the inserted feline VDJ gene segment having the feline constant region, and / or an antibody chain resulting from the expression of the inserted feline VJ gene segment having the feline constant region.

[0132] One possibility is the expression of a complete feline antibody light chain having a chimeric heavy chain with feline VDJ and the rodent heavy chain constant region. One possibility is the expression of a complete feline antibody heavy chain having a chimeric light chain with feline VJ and the rodent heavy chain constant region.

[0133] References herein to "inserted feline gene segment" or "inserted feline DNA", etc. refer to feline sequences that are present in the genome of the rodent or rodent cell and are thus inserted into the genome of the rodent or rodent cell.

[0134] In one aspect, the inserted feline DNA includes at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, and in one aspect all, of the feline heavy chain diversity (D) genes.

[0135] In one aspect, the inserted feline DNA includes at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, and in one aspect all, of the feline heavy chain joining (J) genes.

[0136] In one aspect, the inserted feline DNA includes at least 50%, such as at least 60%, at least 70%, at least 80%, at least 90%, and in one aspect all, of the feline light chain joining (J) genes.

[0137] The genome of a rodent or rodent cell can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 IGH D region gene segments from a feline.

[0138] The genome of a rodent or rodent cell can include at least 1, 2, 3, 4, or 5 IGH J region genes from a feline.

[0139] The genome of a rodent or rodent cell can include at least 1, 2, 3, 4, or 5 IG kappa J region genes from a feline.

[0140] The genome of a rodent or rodent cell can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 IG lambda J region genes from a feline.

[0141] In any embodiment, the number of feline genes mentioned above can further increase, and in one embodiment, it doubles in the case of a homozygote having insertions in both alleles.

[0142] One possible insertion of the above feline V gene segment is upstream of the rodent constant region so as to generate a chimeric antibody chain expressed at the endogenous locus.

[0143] In other embodiments, the inserted feline DNA is located at a site different from the naturally occurring heavy or light chain constant regions, such as on a different chromosome within the rodent genome. The insertion can occur at a random position within the rodent genome. In this case, the insertion of the VDJ or VJ region gene is accompanied by a constant region, preferably also by a 3' enhancer from a rodent or a feline.

[0144] A preferred embodiment is to use a rodent constant region and a rodent 3' enhancer together with a feline VDJ or VJ region, or to use a feline constant region and a feline 3' enhancer together with a feline VDJ or VJ region. In one embodiment, since the feline gene segment(s) are functionally arranged with the constant region and are present within the genome, the rodent can produce antibody chains.

[0145] The present invention also particularly contemplates cells and rodents having an insertion of a feline gene (such as DNA encoding feline lambda V, J, and C genes) associated with a feline constant region (encoding a "complete" feline antibody chain) of the endogenous rodent IG locus.

[0146] In one embodiment, the above complete feline VJC lambda antibody chain is inserted between the rodent lambda locus, preferably between the last rodent C gene and the 3' enhancer.

[0147] In one aspect, a rodent or rodent cell comprises one or more feline IG lambda V-region genes, one or more feline IG lambda J-region genes, and one or more feline lambda constant-region genes located at the kappa locus of the rodent cell, for example within or upstream or downstream of the locus of the rodent kappa constant region. Preferably, the insertion is upstream of the constant region of the IG kappa locus such that the IG lambda V and J-region genes are expressed together with the IG kappa constant region.

[0148] Suitably, due to the insertion, the feline genes are placed at a position substantially the same as the native rodent kappa gene and those genes can be deleted or replaced. For example, the distance from the last inserted 3' lambda J gene to the rodent kappa constant-region gene is the same as or substantially the same as the distance from the last rodent kappa 3'J gene to the constant region.

[0149] In one aspect, the insertion is within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 kb from the boundary (upstream or downstream) of the rodent immunoglobulin kappa locus. The rodent kappa light chain is naturally expressed at a higher level than the rodent lambda light chain and insertion of feline DNA at this kappa locus can provide high-level expression of the feline lambda chain V-region genes.

[0150] In one aspect, the feline lambda V and / or J gene segments are associated with feline regulatory sequences or scaffold sequences. In another aspect, the feline lambda V and / or J gene segments are associated with regulatory sequences or scaffold sequences from a rodent, such as the native regulatory sequences or scaffold sequences of the rodent cell used for insertion of the feline DNA.

[0151] In one embodiment where the feline lambda V gene is inserted into the rodent kappa locus, the rodent kappa locus or a part thereof may be deleted, for example, by mutation, or may become non-functional. Inactivating or rendering non-functional the endogenous κ light chain locus in a mouse or rodent to increase the expression of lambda light chain is described, for example, in WO2021003149 with respect to dogs and can be similarly applied to cats.

[0152] As used herein, references to a gene segment being associated with a regulatory sequence or a scaffold sequence, and similar descriptions, refer to a gene segment that is operably linked to the regulatory sequence or the scaffold sequence. The term "associated with" in this context means "operably linked to" and may be interchangeable therewith.

[0153] In one embodiment, the rodent genome is homozygous for the inserted feline gene. Such homozygous rodents are preferably used for the production of feline antibodies by immunization.

[0154] The insertion can occur at the native rodent IG locus. In another embodiment, the rodent genome can be heterozygous for the insertion of the feline gene at one, two, or three loci such as the immunoglobulin locus.

[0155] The rodent genome can be heterozygous for the insertion of the feline gene at the kappa locus.

[0156] The inserted feline DNA at the restricted IgH locus contains at least four IGH variable region genes, preferably one of which is V3-14.

[0157] In one aspect, the rodent genome is modified to reduce or prevent the expression of a complete rodent antibody having both a rodent variable region and a constant region. This can occur by inversion of all or part of the rodent VDJ region and / or VJ region, deletion of all or part of the rodent VDJ region and / or VJ region, or insertion into the endogenous rodent VDJ region and / or VJ region of the genome. For example, insertion of feline DNA at the IG locus upstream of the rodent constant region causes the host rodent V(D) and J gene segments to move further away from the rodent constant region, reducing or inactivating the expression of host rodent antibodies from that locus.

[0158] In one aspect, the rodent VDJ or VJ region or a part thereof is deleted.

[0159] In one aspect, all or some of the rodent V region genes are deleted for kappa and / or lambda, for example,

[0160] at least 50%, preferably at least 75% or at least 90%, or all, of the rodent IGH V gene segment and / or D gene segment and / or J gene segment, and / or

[0161] at least 50%, preferably at least 75% or at least 90%, or all, of the rodent IG light chain V gene segment and / or J gene segment.

[0162] In one aspect, the rodent is a mouse and the genome includes deletion of one, some, or all of the mouse IGH V region genes, preferably deletion from V1-85 to V5-2.

[0163] In one aspect, the rodent is a mouse and the genome includes deletion of one, some, or all of the mouse IG kappa V region genes, preferably deletion from V3-1 to V2-137.

[0164] In one aspect, the rodent is a mouse, and the mouse heavy chain D and J region genes are retained in the genome upstream of the feline heavy chain variable region gene to be inserted.

[0165] In one aspect, the rodent IG lambda gene is not deleted from the rodent genome.

[0166] In one aspect, one or both alleles of the rodent kappa locus are wholly or partially deleted or inactivated by inserting feline DNA into the rodent kappa locus.

[0167] In one aspect, the rodent kappa locus is wholly or partially inactivated, for example, by insertion, deletion, or inversion.

[0168] In one aspect, the rodent lambda locus is wholly or partially inactivated, for example, by insertion, deletion, or inversion.

[0169] In one aspect, the rodent heavy chain locus is wholly or partially inactivated, for example, by insertion, deletion, or inversion.

[0170] The feline variable region gene segment(s) is / are suitably inserted upstream of the rodent constant region, which contains all the DNA necessary to encode the complete constant region or a part of the constant region sufficient to enable the formation of a functional chimeric antibody that can specifically recognize an antigen.

[0171] Accordingly, as used herein, reference to a chimeric antibody or antibody chain having a rodent constant region is not limited to an antibody chain having a complete constant region, but also includes chimeric antibodies or chains having a portion of a constant region sufficient to provide one or more effector functions found in antibodies that occur naturally in rodents. Effector functions include the ability to interact with Fc receptors and / or the ability to bind complement. Similarly, feline variable region DNA is located within the host genome and as a result can form chimeric antibody chains with all or part of a rodent constant region to form an antibody chain or a portion thereof, respectively.

[0172] Preferably, the rodent genome includes all of the feline heavy chain constant region DNA and intervening regions. Preferably, the rodent genome includes all of the feline heavy chain constant region gene and intervening regions.

[0173] Preferably, the rodent genome includes all of the feline lambda constant region DNA and intervening regions. Preferably, the rodent genome includes all of the feline lambda constant region gene and intervening regions.

[0174] Preferably, the rodent genome includes all of the feline kappa constant region DNA and intervening regions. Preferably, the rodent genome includes all of the feline kappa constant region gene and intervening regions.

[0175] In one aspect, the inserted feline DNA can be expressed in different rodent constant regions via isotype switching.

[0176] In one aspect, the inserted feline DNA can be expressed in different rodent constant regions via trans-switching.

[0177] In one embodiment, one or more, or all, of the inserted feline V, D, or J region gene segments are preferably associated with feline regulatory sequences from the same feline breed.

[0178] In one aspect, at least one rodent enhancer or other control sequences such as a switch region is arranged and maintained functionally with a rodent constant region. In this way, the effect of the enhancer or other control sequences can be exerted wholly or partially in cells or transgenic rodents.

[0179] In one aspect, one or more rodent control sequences such as an Emu enhancer sequence are maintained in their native position upstream of the rodent Mu constant region, suitably with respect to the distance from the constant region.

[0180] In one aspect, one or more rodent control sequences such as enhancer sequence(s) are maintained in their native position downstream of the rodent constant region, suitably with respect to the distance from the constant region.

[0181] In one aspect, the rodent Smu switch sequence is maintained in its native position upstream of the rodent Mu constant region, suitably with respect to the distance from the constant region. In such a position, the rodent enhancer or switch sequence operates in vivo suitably with the host constant region sequence(s).

[0182] In a further aspect, one or more promoter elements or other control elements of the feline V, D or J region genes are optimized in the genome to interact with the rodent transcriptional machinery.

[0183] In one aspect, the genome of a rodent or rodent cell contains one or more feline promoters or enhancers, and / or other control elements associated with feline V, D or J gene segments.

[0184] In one aspect, one or more feline control regions (elements) such as promoters, enhancers, switch regions, etc. replace one or more rodent promoters, enhancers, switch regions, respectively. The feline control array is appropriately maintained in a functional array with the constant region such that the effect of the control array(s) is exerted either wholly or in part in cells or transgenic rodents.

[0185] In one aspect, at least one or more of the feline V, D, or J gene segments to be inserted are associated with regulatory sequences such as recombination signal sequences (RSS) from the same feline, and optionally, the regulatory sequence indicates the success of recombination of the V, D, or J gene segment.

[0186] In one aspect, "the same" feline refers to a feline of the same breed. In one aspect, the same feline refers to exactly the same animal.

[0187] In one aspect, at least one or more of the feline V, D, or J gene segments to be inserted are directly associated with a regulatory sequence either in cis or in trans, or are located flanking the regulatory sequence on one or both sides, and optionally, the regulatory sequence is directly located flanking one or more gene segments.

[0188] In one aspect, the regulatory 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, flanking the D gene segment, or upstream of the J gene segment.

[0189] In one aspect, an RSS sequence from the same feline is placed flanking the V, D, or J sequence of the feline DNA to be inserted. For example, the feline RSS sequence can be used together with the feline V, D, and / or J sequences. It will be understood that this can be achieved by inserting a genomic fragment from a feline into the rodent genome.

[0190] In a further aspect, the invention provides a method of replacing all or part of an endogenous immunoglobulin variable region gene locus with a feline locus in a rodent cell, the method comprising obtaining a cloned genomic fragment or synthetic sequence that wholly or partially contains a companion locus comprising at least one V, or D (in the case of the heavy chain) or J gene segment, and at least one associated regulatory sequence, and inserting the feline DNA into the rodent genome, suitably into an endogenous rodent immunoglobulin gene locus, preferably a heavy or light chain rodent gene locus corresponding to the nature of the feline DNA to be inserted.

[0191] In one aspect, the feline DNA to be inserted is associated with rodent regulatory sequences that enable V(D)J recombination in the rodent. Such an approach is disclosed for dogs, for example, in US20170306352, which is incorporated by reference, and is similarly applicable to cats.

[0192] In one aspect, the regulatory sequence is a non-coding regulatory sequence comprising the following sequences of endogenous host origin (the promoter, intron, splice site, and recombination signal sequence for V(D)J recombination, preceding each V gene segment coding sequence). In another aspect, the regulatory sequence is one or more of the promoter, intron, splice site, and recombination signal sequence for V(D)J recombination, preceding the coding sequence of each V gene segment, all of which are of endogenous host origin.

[0193] In another aspect, the partially feline immunoglobulin gene locus generated by the invention comprises one or more of the following sequences of endogenous host origin, the ADAM6A or ADAM6B gene, the Pax-5 activating gene-intergenic repeat (PAIR) element, or the CTCF binding site from the intergenic control region 1 of the heavy chain gene.

[0194] In one aspect, the present invention relates to a transgenic rodent, such as a mouse, in which the entire endogenous immunoglobulin variable locus has been deleted in its genome and is replaced by an engineered, partially feline immunoglobulin locus that contains the feline immunoglobulin variable genes VH, D, and JH and / or feline VL and JL coding sequences, as described herein, as well as rodent immunoglobulin variable locus non-coding regulatory sequences. The engineered, partially feline immunoglobulin locus of the genetically engineered rodent functions and expresses immunoglobulin chains composed of feline variable domains and rodent constant domains.

[0195] The rodent can be the mouse described in US20170306352, the disclosure of which is incorporated by reference.

[0196] The rodent can be the mouse described in WO2021003149, the disclosure of which is incorporated by reference.

[0197] In one aspect, the feline DNA inserted into the restricted locus is not a genomic DNA fragment. To insert the gene segment(s) with the most natural expression, the DNA to be inserted can include DNA in which the gene segment is not in the natural germline configuration but is within genomic DNA. The feline DNA to be inserted can be, for example, a minigene having a plurality of different V gene segments.

[0198] The feline V gene segment inserted at the restricted locus can be arranged such that the gene segment(s) of most interest is closest to the constant region. For example, the IGH V 3-14 gene segment can be arranged closest to the constant region to maximize expression. Gene segments closer to the constant region are typically expressed at higher levels. Alternatively, the gene segments with the highest natural occurrence rate in the feline antibody population may be located away from the constant region to provide a balanced repertoire.

[0199] The present invention provides a rodent or rodent cell that expresses a limited number of preferred V gene segments, such as one gene segment, and as a preferred feature, contains only a single V, D, and J gene segment for the heavy chain variable region in the genome, and / or only a single V and J for the light chain variable region. In this way, the rodent produces only a single type of variable region for the heavy chain of the feline antibody chain produced and / or only a single type of variable region for the light chain.

[0200] In one aspect, the term "single type of variable region" refers to a variable region having only one V (one D) and one J gene segment sequence.

[0201] Such a sequence can provide a common light chain useful for generating a bispecific antibody where a single light chain is desired and is expressed with two different heavy chains in a bispecific antibody.

[0202] Accordingly, the present invention also relates to a method for generating a bispecific antibody, the method comprising immunizing a rodent disclosed herein with an antigen (the rodent contains only a single type of feline light chain variable region (a single feline V and J gene segment, feline VJ) in the rodent genome), and selecting a bispecific antibody that can bind to the antigen and, appropriately, also to a second preferred antigen target.

[0203] The present invention also relates to bispecific antibodies obtainable or obtained from the method of the present invention, wherein the antibody light chain is obtained from the expression of the preferred V gene segments disclosed herein and, appropriately, from the rodent of the present invention. Appropriately, the bispecific antibody has a preferred light and heavy chain pairing as disclosed herein.

[0204] Any bispecific antibody format can be used, such as those disclosed in Brinkmann U and Kontermann RE, MAbs. 2017 Feb - Mar;9(2):182 - 212.

[0205] The feline V gene segments selected for use with the common light chain are determined based on frequency (Tables 2b and 2c), the number of different heavy chain feline V gene segment pairings (Table 4), or a combination of both.

[0206] In another aspect, the present disclosure expressly contemplates the use of IGH V3-16 (SEQ ID NO: 45) instead of IGH V3-N3 (SEQ ID NO: 8). References herein to IGH V3-N3 or SEQ ID NO: 8 can be considered equivalent for the purposes of this aspect of the invention to references to IGH V3-16 or SEQ ID NO: 45.

[0207] In another aspect, the present disclosure expressly contemplates the use of IGL V1-N1 (SEQ ID NO: 46) instead of IGL V1-33 (SEQ ID NO: 13). References herein to IGL V1-33 or SEQ ID NO: 13 can be considered equivalent for the purposes of this aspect of the invention to references to IGH V1-N1 or SEQ ID NO: 46.

[0208] In another aspect, the present disclosure expressly contemplates the use of IGL V1-N14 (SEQ ID NO: 47) instead of IGL V1-41 (SEQ ID NO: 16). References herein to IGL V1-41 or SEQ ID NO: 16 can be considered equivalent for the purposes of this aspect of the invention to references to IGH V1-N14 or SEQ ID NO: 47.

[0209] The present disclosure also expressly contemplates the use of IGH V3-16 (SEQ ID NO: 45) instead of IGH V3-N3 (SEQ ID NO: 8), IGL V1-N1 (SEQ ID NO: 46) instead of IGL V1-33 (SEQ ID NO: 13), and IGL V1-N14 (SEQ ID NO: 47) instead of IGL V1-41 (SEQ ID NO: 16).

[0210] The term "bispecific antibody" means an antibody that includes specificity for two target molecules and includes formats such as DVD-Ig (see DiGiammarino et al., "Design and generation of DVD-Ig™ molecules for dual-specific targeting", Meth. Mo. Biol., 2012, 889, 145-156), mAb2 (see WO2008 / 003103), FIT-Ig (see WO2015 / 103072), mAb-dAb, dock-and-lock, Fab-arm exchange, SEEDbody, Triomab, LUZ-Y, Fcab, κλ-body, orthogonal Fab, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, Fab-scFv-Fc, Fab-scFv, intrabody, BiTE, diabody, DART, TandAb, sc diabody, sc diabody-CH3, diabody-CH3, triplebody, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, scFv-CH-CL-scFv, F(ab')2-scFv, scFv-KIH, Fab-scFv-Fc, tetravalent HCab, ImmTAC, knob-in-hole, knob-in-hole with a common light chain, knob-in-hole with a common light chain and a charge pair, charge pair, charge pair with a common light chain, DT-IgG, DutaMab, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody. See Spiess, C., et al., Mol. Immunol. (2015) for a review of bispecific formats.

[0211] In another embodiment, the bispecific antibody molecule is in another non-Ig format, such as a T cell receptor binding domain, an immunoglobulin superfamily domain, an agnathan variable lymphocyte receptor, a fibronectin domain (e.g., Adnectin™), an antibody constant domain (e.g., CH3 domain, e.g., CH2 and / or CH3 of Fcab™ (where the constant domain is not a functional CH1 domain), scFv, (scFv)2, sc-diabody, scFab, centyrin and epitope-binding domain (Evibody™) derived from scaffolds selected from CTLA-4, lipocalin domain, Protein A such as the Z domain of Protein A (e.g., Affibody™ or SpA), A domain (e.g., Avimer™ or Maxibody™), heat shock protein (such as an epitope-binding domain derived from GroEI and GroES), transferrin domain (e.g., transbody), ankyrin repeat protein (e.g., DARPin™), peptide aptamer, C-type lectin domain (e.g., Tetranectin™), human γ-crystallin or human ubiquitin (affilin), PDZ domain, scorpion toxin, and a kunitz-type domain fusion of a human protease inhibitor, including antibodies.

[0212] The structure of antibodies has been utilized to engineer various antibody formats targeting human diseases. An example of such engineered antibody formats is bispecific antibodies. Bispecific antibodies bind to two different targets and can thus simultaneously bind to two different epitopes. One area of interest is bispecific antibodies targeting T cells for efficient killing of tumors. Bispecific antibodies have a "two-target" function and can affect multiple disease pathways by binding to two different surface receptors or ligands.

[0213] Bispecific antibodies can also bring two targets into proximity to support the formation of protein complexes on a single cell or to cause cell-cell contact. Bispecific antibody formats differ in various respects, such as molecular weight, number of antigen-binding sites, spatial relationship between different binding sites, valence of each antigen, ability to support secondary immune functions, pharmacokinetic half-life, etc. These diverse formats provide excellent opportunities to customize the design of bispecific antibodies according to the proposed mechanism of action and intended clinical applications (Kontermann and Brinkmann Bispecific Antibodies Drug Discovery Today Volume 20, Number 7, 2015).

[0214] Producing IgG-type bispecific antibodies by co-expressing two light chains and two heavy chains within a single host cell can be very difficult because the yield of the desired bispecific IgG is low and it is difficult to remove closely related mispaired IgG contaminants. This reflects the fact that the heavy chains form not only the desired heterodimers but also homodimers, which is the so-called heavy chain pairing problem. Furthermore, the light chains may mispair with non-cognate heavy chains, which is the so-called light chain pairing problem. As a result, co-expression of the two antibodies can generate up to nine unwanted IgG species in addition to the desired bispecific antibody.

[0215] Various approaches have been described in the art to promote heterodimerization, i.e., the formation of specific bispecific antibodies of interest for human therapy, thereby reducing the content of unwanted homodimers in the resulting mixture.

[0216] In one embodiment, the antibody is a multispecific antibody or a fragment thereof. Multispecific proteins, such as multispecific antibodies, bind to at least two different targets, i.e., are at least bispecific. Thus, in one embodiment, the antibody is a bispecific antibody or a fragment thereof. In other embodiments, the multispecific antibody or a fragment thereof binds to three, four or more targets.

[0217] Bispecific antibodies have specificity for up to two epitopes. Bispecific antibodies are characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope.

[0218] In one embodiment, the first epitope and the second epitope are on the same antigen, for example, the same protein (or subunit of a multimeric protein). In one embodiment, the first epitope and the second epitope overlap. In one embodiment, the first epitope and the second epitope do not overlap. In one embodiment, the first epitope and the second epitope are on different antigens, for example, different proteins (or different subunits of a multimeric protein).

[0219] In another embodiment, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In a further embodiment, a bispecific antibody molecule comprises an antibody having binding specificity for a first epitope and an antibody having binding specificity for a second epitope. In one embodiment, a bispecific antibody molecule comprises an antibody or a fragment thereof having binding specificity for a first epitope and a half-antibody or a fragment thereof having binding specificity for a second epitope. In one embodiment, a bispecific antibody or a fragment thereof comprises a Fab having binding specificity for a first epitope and a Fab having binding specificity for a second epitope.

[0220] The bispecific antibodies of the present invention based on the IgG format (composed of two heavy chains and two light chains) can be produced by various methods known in the art. For example, bispecific antibodies can be produced by fusing two antibody-secreting cell lines to create a new cell line, or by expressing two antibodies in a single cell using recombinant DNA technology. In these approaches, each heavy chain from each antibody can form a monospecific dimer (also called a homodimer) containing two identical heavy chain pairs with the same specificity, and a bispecific dimer (also called a heterodimer) containing two different heavy chain pairs with different specificities, resulting in the generation of multiple antibody species. Furthermore, the light chains and heavy chains from each antibody can pair randomly, forming inappropriate non-functional combinations. This problem, known as heavy chain and light chain mispairing, can be solved by selecting antibodies that share a common light chain expressed as a bispecific antibody. Methods for addressing the problem of heavy chain-light chain mispairing include the generation of bispecific antibodies using a single light chain. This requires heavy chain-light chain engineering or a new antibody library that utilizes a single light chain that restricts diversity. Furthermore, antibodies with a common light chain have been identified from transgenic mice with a single light chain. Another approach is to exchange the CH1 domain of one heavy chain with the CL domain of its cognate light chain (Crossmab technology). The scFv format is also covered.

[0221] Methods for preparing bispecific antibodies are described in WO2019008123 and WO2014160179, which are incorporated by reference.

[0222] The present invention also relates to a method for generating bispecific antibodies, the method comprising immunizing a first rodent disclosed herein with a first antigen (the rodent comprising only a single feline lambda light chain variable region (a single feline V and J gene segment, feline VJ) within the rodent genome), and selecting an antibody capable of binding to the first antigen, and immunizing a second rodent disclosed herein with a second antigen (the rodent comprising the same single feline lambda light chain variable region (a single feline V and J gene segment, feline VJ) within the rodent genome), and selecting an antibody capable of binding to the second antigen, and optionally, the single feline lambda light chain V gene segment is selected from the list comprising feline lambda V1-124, V3-7, V1-42, V1-104, V1-56, V1-126, V3-13, V3-5, V3-36, V1-41 and V1-33.

[0223] In one embodiment, the single lambda feline light chain V gene segment is V1-124.

[0224] In one embodiment, the single lambda feline light chain V gene segment is V3-7.

[0225] The present invention also relates to bispecific antibodies obtainable or obtained from the method of the present invention, wherein the feline antibody light chain is obtained from the expression of a single preferred lambda V gene segment disclosed herein, suitably obtained from the rodent of the present invention. Suitably, the bispecific antibody has a preferred light and heavy chain pairing as disclosed herein. In one embodiment, the single lambda feline V gene segment is selected from the list comprising feline lambda V1-124, V3-7, V1-42, V1-104, V1-56, V1-126, V3-13, V3-5, V3-36, V1-41 and V1-33.

[0226] The present invention provides a rodent or rodent cell comprising a single feline lambda V and J in the light chain variable region within the genome. In this way, the rodent produces only a single type of variable region for the light chain of the feline antibody chain produced. In one aspect, the single feline lambda light chain V gene segment is V1-124. In one aspect, the single lambda feline light chain V gene segment is V3-7.

[0227] The present invention also relates to a method for generating a bispecific antibody, the method comprising immunizing a first rodent disclosed herein with a first antigen (the rodent comprising only a single feline kappa light chain variable region (a single feline V and J gene segment, feline VJ) within the rodent genome), and selecting an antibody capable of binding to the first antigen, and immunizing a second rodent disclosed herein with a second antigen (the rodent comprising the same single feline kappa light chain variable region (a single feline V and J gene segment, feline VJ) within the rodent genome), and selecting an antibody capable of binding to the second antigen, and optionally, the single feline kappa light chain V gene segment is selected from the list comprising feline kappa V2-5, V2-9, V2-12, V2-13, V4-1 and V2-4.

[0228] In one aspect, the single kappa feline light chain V gene segment is V4-1.

[0229] In one aspect, the single kappa feline light chain V gene segment is V2-5.

[0230] The present invention also relates to a bispecific antibody obtainable or obtained from the method of the present invention, wherein the feline antibody light chain is obtained from the expression of a single preferred kappa V gene segment disclosed herein, suitably obtained from a rodent of the present invention. Suitably, the bispecificity has a preferred light and heavy chain pairing as disclosed herein. In one aspect, the single kappa feline V gene segment is selected from the list comprising feline kappa V2-5, V2-9, V2-12, V2-13, V4-1 and V2-4.

[0231] The present invention provides a rodent or rodent cell comprising a single feline kappa V and J in the light chain variable region within the genome. In this way, the rodent generates only a single type of variable region for the light chain of the produced feline antibody chain. In one embodiment, the single feline kappa light chain V gene segment is V4-1. In one embodiment, the single lambda feline light chain V gene segment is V2-5.

[0232] The present invention also relates to bispecific antibodies, wherein both light chains are identical and are selected from the list comprising feline lambda V1-124, V3-7, V1-42, V1-104, V1-56, V1-126, V3-13, V3-5, V3-36, V1-41 and V1-33.

[0233] In one embodiment, both light chains are feline lambda V1-124.

[0234] In one embodiment, both light chains are feline lambda V3-7.

[0235] The present invention also relates to bispecific antibodies, wherein both light chains are identical and are selected from the list comprising feline kappa V2-5, V2-9, V2-12, V2-13, V4-1 and V2-4.

[0236] In one embodiment, both light chains are feline lambda V4-1.

[0237] In one embodiment, both light chains are feline lambda V2-5.

[0238] In one aspect, the rodent cells of the present invention are rodent ES cells, rodent hematopoietic stem cells or other cells capable of differentiating into a rodent that can produce a repertoire of antibody chains comprising variable regions encoded by feline DNA such as chimeric antibody heavy chains or chimeric antibody light chains, or complete feline antibody chains or antibodies encoded by feline variable regions having variable and constant regions.

[0239] In one aspect, the cells of the present invention are rodent ES cells or induced pluripotent stem cells (iPS cells). Such cells are suitable for inserting feline DNA to generate rodents that express the antibody chains described herein.

[0240] In one aspect, the cells are isolated rodent cells.

[0241] In one aspect, the cells are isolated rodent B cells.

[0242] The rodent or rodent cells of the present invention are preferably a mouse or a rat, or mouse or rat cells (such as mouse or rat ES cells), preferably a mouse or mouse ES cells. The ES cells can be of the mouse cell line 129 or C57BL (e.g., C57BL / 6N strain, C57BL / 6J strain, 129S5 strain, or 129Sv strain), or cells having a hybrid genome containing 129 and / or C57BL genomic DNA.

[0243] The present invention also relates to cell lines grown from or otherwise derived from the cells described herein, including immortalized cell lines.

[0244] The cells or cell lines of the present invention may contain feline V, (D), or J genes in an unrearranged configuration or after rearrangement after in vivo maturation.

[0245] The present invention also relates to antibody chains having variable regions that can be obtained by immunizing the rodents of the present invention with an antigen, or to cells or cell lines such as CHO cell lines from which the nucleic acid sequences of the variable regions can be identified or have been identified from the rodents or rodent cells described herein, or from the antibody repertoires described herein. The antibody chain can be a chimeric antibody heavy chain or, preferably, a complete feline antibody chain. The cell or cell line expressing the antibody chain or antibody can be a CHO cell or another mammalian cell line suitable for the production of veterinary drugs for animals. The cells can be immortalized by fusion with tumor cells to provide antibody-producing cells and cell lines, or can be produced by direct cell immortalization.

[0246] The present invention also relates to vectors for use in the present invention. In one aspect, such a vector is a bacterial artificial chromosome (BAC) containing all or part of the feline IG locus suitable for insertion into ES cells. It will be understood that other cloning vectors can also be used in the present invention. Thus, references to BACs herein can generally be construed to refer to any suitable vector. The vector can contain one or more selectable markers and / or one or more site-specific recombination sites. In one aspect, the vector contains two or more, for example three, heterospecific and non-compatible site-specific recombination sites. In one aspect, the site-specific recombination site can be a loxP site or a variant thereof, or an FRT site or a variant thereof. In one aspect, the vector contains one or more transposon ITR (inverted terminal repeat) sequences.

[0247] A suitable BAC containing feline DNA is provided as the FCAB library of Amplicon Express and is currently available from the College of Veterinary Medicine and Biomedical Sciences, Texas A&M University.

[0248] In one aspect, part or all of the DNA to be inserted is from the European shorthair cat.

[0249] Preferably, the cell or rodent comprises feline V, D, and J gene segments from the FCAB BAC library of Amplicon Express and is currently available from the College of Veterinary Medicine and Biomedical Sciences, Texas A&M University.

[0250] In one aspect, one or more feline gene segment alleles used in the present invention are the reference alleles of each feline gene. In one aspect, these are those of Felis Catus 8.0. Refer to the assembly accession number GCA_000181335.3 (assembled in November 2014). In one aspect, these are those of Felis Catus 9.0. Refer to the assembly accession number GCF_000181335.3 (assembled in November 2017).

[0251] In one aspect, at least 90%, at least 95%, preferably all, of the feline gene segments inserted are the feline reference alleles of CanFam3.1. In one aspect, at least 50, 60, 70, 80, 90, or 100% of the inserted V gene segments are feline V gene reference alleles, and / or at least 50, 60, 70, 80, 90, or 100% of the inserted D gene segments are feline D gene reference alleles, and / or at least 50, 60, 70, 80, 90, or 100% of the inserted J gene segments are feline J gene reference alleles, and combinations thereof.

[0252] In one aspect, references to feline gene segments (such as IGH V3-14) are references to equivalent gene segments of Felis Catus 8.0 or Felis Catus 9.0. However, it will be understood that such sequences may differ from the exact sequences of Felis Catus 8.0 or Felis Catus 9.0, for example, being 99%, 98%, 97%, 96% or 95% identical.

[0253] In one aspect, references to feline gene segments (such as IGH V3-14) are references to gene segments having the related sequences of Table 1.

[0254] In one aspect, references to feline gene segments (such as IGH V3-14) are references to gene segments having the related sequences of Table 1 or having 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0255] In one aspect, a rodent or rodent cell contains one or more reference alleles of feline gene segments.

[0256] Preferably, all of the gene segments of the V(D) and J regions inserted into the genome are from the same breed of cat or the same cat.

[0257] In one aspect, a rodent can generate a diversity of combinations of at least 1x10 6 different functional chimeric immunoglobulin sequences.

[0258] It should be noted that the terms "feline" or "felid" in this specification are interchangeable and both refer to the domestic cat.

[0259] The present invention also relates to methods for producing rodents containing inserted feline DNA, methods for producing antibodies and antibody chains from those rodents, and methods for producing pharmaceutical compositions containing feline antibody chains or antibodies.

[0260] A method for producing a rodent or rodent cell disclosed in this specification, the method comprising, in the genome of a rodent cell, (i) nine or fewer feline IGH V gene segments, one or more feline IGH D region gene segments, and one or more feline IGH J region gene segments (at least one of the feline IGH V gene segments is selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3, and V3-5), and / or (ii) eleven or fewer feline lambda V gene segments and one or more feline lambda J region gene segments (at least one of the feline IGH V gene segments is selected from the list comprising V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13, and V1-126), and / or (iii) six or fewer feline kappa V gene segments and one or more feline J region gene segments (at least one of the feline kappa gene segments is selected from V4-1, V2-12, V2-5, V2-13, V2-9, and V2-4), wherein the rodent or rodent cell can express the feline variable region gene segment(s) in combination with a constant region to form an antibody chain.

[0261] Methods for targeted insertion of foreign DNA at endogenous murine loci are well known in the art, and the inserted V, D, and J genes can be expressed in the host constant regions. The same principle can be applied to the insertion of feline DNA. See Murphy et al, Vol 111 no 14, 5153 - 5158, doi:10.1073 / pnas.1324022111; MacDonald et al vol.111 no.14, 5147 - 5152, doi:10.1073 / Pnas.1323896111; and Lee et al, Nature Biotechnology Volume:32, Pages:356 - 363 Year published:, 2014 DOI:, doi:10.1038 / nbt.2825. A method suitable for the construction of a chimeric IG locus encoding feline VDJ and / or VJ gene segments having constant regions is disclosed in WO2018189520, which method is incorporated herein by reference.

[0262] Preferably, this method inserts feline VDJ and VJ region genes for both the light and heavy chains, resulting in the production of antibodies having variable regions derived from the expression of feline DNA in both the light and heavy chains.

[0263] In one aspect, murine ES cells having one or more chimeric loci are used to create chimeras that generate host embryos from an RAG - 1 deficient background, or other suitable genetic background that prevents the production of mature host B and T lymphocytes. This allows all B and T cells to be derived from the injected ES cells.

[0264] The ES cells of the present invention can be used to generate animals using techniques well known in the art, which includes, for example, injecting the ES cells into blastocysts, subsequently transplanting the chimeric blastocysts into females to produce offspring, mating those offspring to produce heterozygous offspring, and then mating those offspring to generate homozygous recombinants having the necessary insertions. In one aspect, the host blastocysts are Rag deficient, such as RAG - 1 deficient.

[0265] The present invention relates to a chimeric rodent generated by injecting ES cells of the present invention into a blastocyst and subsequently transplanting the chimeric blastocyst into a female rodent to produce offspring.

[0266] In a preferred 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 deleted from and reinserted into the IGH locus previously.

[0267] In one embodiment, the rodent ADAM6a and ADAM6b genes are at the 5' position of one or more feline V, D, and J genes to be inserted.

[0268] In one embodiment, the rodent IGH D gene and J gene are present in the rodent genome. In one embodiment, the rodent IGH D gene and J gene have not been deleted from the rodent genome. In one embodiment, the rodent IGH D and J genes are at the 5' position of one or more feline V, D, and J gene segments to be inserted.

[0269] The present invention also relates to a method for producing an antibody or antibody chain specific for a desired antigen, the method comprising immunizing a rodent with the desired antigen and recovering the antibody or antibody chain alone or as part of a complete antibody, or recovering the cells that produce the antibody or antibody chain alone or as part of a complete antibody, as disclosed herein (see, for example, Harlow, E. & Lane, D. 1998, 5th edition, Antibodies: A Laboratory Manual, Cold Spring Harbor Lab. Press, Plainview, NY; and Pasqualini and Arap, Proceedings of the National Academy of Sciences (2004) 101:257-259).

[0270] Appropriately, an immunogenic amount of antigen is delivered. The invention also relates to a method of detecting a target antigen, which comprises detecting the antibody produced as described above with a secondary detection agent that recognizes a portion of the antibody.

[0271] The invention also relates to a method of producing an antibody chain or antibody specific for a desired antigen, the method comprising immunizing a rodent comprising a feline gene segment located upstream of a non-feline constant region as disclosed herein, and then, appropriately, manipulating the nucleic acid encoding the antibody to replace the constant region of the antibody chain or antibody with that of a feline constant region. Preferably, the constant region is from the same feline breed. Standard cloning techniques are known for replacing a non-human mammalian constant region with an appropriate feline constant region DNA sequence (see, for example, Sambrook, J and Russell, D. (2001, 3’d edition) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY)). Alternatively, direct nucleic acid synthesis can be used to generate a complete feline sequence using sequence information from DNA encoding a chimeric antibody.

[0272] The invention also relates to a method of identifying a feline variable region by single cell sequencing and generating a vector that expresses a complete antibody chain having the corresponding feline constant region. The invention also relates to obtaining these feline sequences by PCR and ligating them to an appropriate constant region such as a feline constant region by appropriate molecular biology techniques such as, but not limited to, bridge PCR and Gibson cloning. The DNA can also be synthesized for incorporation into an expression vector.

[0273] In yet another aspect, the chimeric antibodies or antibody chains produced by the invention are appropriately manipulated at the DNA level to generate molecules having properties or structures such as an antibody, for example, as follows: Feline variable regions from heavy or light chains lacking a constant region, domain antibodies, feline variable regions having a constant region from either a heavy or light chain from the same or a different species, feline variable regions and non-naturally occurring constant regions, or feline variable regions and other fusion partners. The present invention relates to all such chimeric antibody derivatives derived from chimeric antibodies identified according to the present invention.

[0274] The present invention also relates to a method of making an antibody or a portion thereof, the method comprising: (i) a nucleic acid encoding an antibody or a portion thereof obtained or obtainable according to the present invention, or (ii) providing sequence information capable of expressing a nucleic acid encoding an antibody obtained or obtainable according to the present invention or a portion thereof to produce an antibody, and expressing an antibody chain.

[0275] Also disclosed is a method of producing an antibody chain or a portion thereof, the antibody chain having a feline variable region, the method comprising expressing in a cell a nucleic acid such as DNA encoding the antibody chain or a portion thereof, the sequence of the nucleic acid encoding the variable region of the antibody chain is obtained or obtainable by immunizing a rodent disclosed herein with an antigen to generate an antibody chain, or is obtained from an antibody of the repertoire described herein, optionally, purifying and / or isolating the antibody chain, and optionally, further comprising the subsequent step of formulating the antibody into a pharmaceutically acceptable formulation suitable for administration to a cat.

[0276] Also disclosed is a method of preparing a pharmaceutical composition, the method comprising making an antibody by the method disclosed herein and further comprising combining the antibody with a pharmaceutically acceptable carrier or other excipient to make a composition.

[0277] The present invention further relates to chimeric antibodies or antibody chains expressed from gene segments identified as important for the production of feline antibodies, and nucleic acids encoding the same, for example, it is as follows.

[0278] A nucleic acid encoding a chimeric antibody chain, the nucleic acid having a variable domain comprising any one of the feline IGH V gene segments selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5, and a rodent constant region, and optionally, the feline V gene segment is a sequence that has undergone somatic hypermutation in the rodent.

[0279] The antibody chain, in combination with the rodent constant region, has a variable region obtained by the expression of any one of feline IGH V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5 in the rodent.

[0280] A nucleic acid encoding a chimeric antibody chain, the nucleic acid having a variable domain comprising any one of the feline lambda V gene segments selected from the list comprising lambda V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126, and a rodent constant region, and optionally, the feline V gene segment is a sequence that has undergone somatic hypermutation in the rodent.

[0281] The antibody chain, in combination with the rodent constant region, has a variable region obtained by the expression of any one of feline lambda V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126 in the rodent.

[0282] A nucleic acid encoding a chimeric antibody chain, the nucleic acid having a variable domain comprising any one of the feline kappa V gene segments selected from the list comprising V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4, and a rodent constant region, and optionally, the feline V gene segment is a sequence that has undergone somatic hypermutation in a rodent.

[0283] The antibody chain, in combination with the rodent constant region, has a variable region obtained by the expression of any one of feline kappa V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4 in a rodent.

[0284] The antibody, antibody chain or nucleic acid can be obtained from, or obtainable from, a rodent or rodent cell, or can be obtained from the antibody repertoire disclosed herein.

[0285] The invention also relates to a part or whole of an immunoglobulin molecule comprising a feline variable domain and a rodent constant domain derived from a restricted insertion of B cells from a rodent, and hybridoma cells obtainable from or obtained from such B cells, and a part or whole of an immunoglobulin molecule comprising a feline variable domain and a rodent constant domain obtainable from or obtained from such hybridoma cells.

[0286] In another aspect, the invention relates to fragments and functional derivatives (also referred to as parts of antibody chains) of the antibodies and chains disclosed herein, and to the use of antibodies, chains and fragments in medicine, including diagnosis and in vitro or ex vivo studies. Functional antibody fragments / parts include fragments that can specifically bind to an antigen.

[0287] Functional antibody fragments can be, for example, FAB or single-chain variable fragments (scFv). The fragment can comprise at least the variable region of the antibody. The fragment can comprise at least the CDR regions. Suitably, these portions are functional in that they can bind to the desired antigen (preferably the same antigen as that used to immunize the rodent to stimulate antibody production).

[0288] The invention also relates to nucleic acids such as DNA or RNA encoding the above antibodies, antibody chains, or portions thereof. In particular, the portion can be the variable portion of the antibody chain, which is the portion encoded by feline DNA within the rodent.

[0289] In one aspect, the antibody or fragment comprises any combination exemplified in the examples and tables herein, or any derivative thereof that is a complete feline antibody or fragment thereof capable of antigen binding.

[0290] The antibodies of the invention can be isolated, and in one aspect, are isolated from the cells or organisms in which the antibody is expressed.

[0291] The invention relates to both polyclonal and monoclonal antibodies of chimeric antibodies or complete feline antibodies, which are produced in response to antigen challenge in the rodents of the invention, or are derived therefrom as described herein, and / or contain V gene segments identified as being highly utilized herein, such as IGH V3-14. Methods for generating both monoclonal and polyclonal antibodies are well known in the art.

[0292] Also disclosed herein are pharmaceutical compositions comprising the above antibodies and antibody chains, or nucleic acids encoding the above antibodies or chains.

[0293] The present invention relates to a pharmaceutical composition comprising an antibody chain having a variable region containing any one of feline IGH V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5, in combination with a pharmaceutically acceptable excipient or carrier.

[0294] The pharmaceutical composition comprises an antibody chain having a variable region containing any one of feline lambda V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126, in combination with a pharmaceutically acceptable excipient or carrier.

[0295] The pharmaceutical composition comprises an antibody chain having a variable region containing any one of feline kappa V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4, in combination with a pharmaceutically acceptable excipient or carrier.

[0296] A pharmaceutical composition comprising an antibody, wherein the antibody (i) has a heavy chain having a variable region obtained by the expression of any one of feline IGH V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5, (ii) a lambda light chain obtained by the expression of any one of feline lambda V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126, or (iii) any one of a kappa light chain obtained by the expression of any one of feline kappa V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4, and the antibody is formulated in combination with a pharmaceutically acceptable excipient or carrier.

[0297] Suitable excipients and carriers are well known in the art and include water, surfactants, carbohydrates (e.g., cyclodextrin derivatives) and amino acids.

[0298] The present invention also relates to a pharmaceutical composition comprising a complete feline antibody packaged within a delivery vehicle such as an IV bag or infusion device.

[0299] The antibody chain can be a chimeric antibody chain or a complete feline antibody chain.

[0300] The present invention also relates to an antibody repertoire and methods of making the same. For example, the repertoire can be derived from a transgenic rodent such as a mouse or from a synthetic antibody repertoire such as a phage display system that utilizes the same limited set of immunoglobulin V gene segments disclosed herein for the heavy and / or light chain feline DNA to be inserted. Suitable methods for generating a synthetic antibody repertoire are described, for example, in WO2018234438, which is incorporated by reference and is applicable to felines as well.

[0301] Accordingly, the present invention relates to an antibody heavy chain repertoire, the repertoire comprising antibody heavy chains having feline IGH V gene segments from no more than 9 different feline IGH V gene segments, at least 1 of the feline IGH V gene segments being selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3, and V3-5, and optionally, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or all 9 feline IGH V gene segments being selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3, and V3-5.

[0302] Optionally, the repertoire (i) V3-14 (optionally having up to 8 other feline IGH V gene segments), (ii) V3-14 and V3-11 (optionally having up to 7 other feline IGH V gene segments), (iii) V3-14, V3-11, and V3-12 (optionally having up to 6 other feline IGH V gene segments), (iv) V3-14, V3-11, V3-12, and V3-84 (optionally having up to 5 other feline IGH V gene segments), (v) V3-14, V3-11, V3-12, V3-84, and V3-20 (optionally having up to 4 other feline IGH V gene segments), (vi) V3-14, V3-11, V3-12, V3-84, V3-20, and V3-27 (optionally having up to 3 other feline IGH V gene segments), (vii) V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, and V4-3 (optionally having up to 2 other feline IGH V gene segments), (v) V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, and V3-N3 (optionally having up to 1 other feline IGH V gene segment), or (ix) An antibody heavy chain comprising a feline IGH V gene segment expressed from V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3, and V3-5.

[0303] In one embodiment, the repertoire comprises antibody heavy chains expressed from IGH V gene segments of feline IGH V families 3 and 4.

[0304] Also disclosed is an antibody light chain repertoire, which repertoire comprises antibody light chains having lambda V gene segments from 11 or fewer different feline lambda V gene segments, wherein at least 1 of the feline lambda V gene segments is selected from the list comprising V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126, and optionally, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or all 11 feline lambda V gene segments are selected from the list comprising V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126.

[0305] Optionally, the repertoire comprises (i) V1-124 (optionally having up to 10 other feline lambda V gene segments), (ii) V1-124 and V1-36 (optionally having up to 9 other feline lambda V gene segments), (iii) V1-124, V1-36 and V1-104 (optionally having up to 8 other feline lambda V gene segments), (iv) V1-124, V1-36, V1-104 and V1-33 (optionally having up to 7 other feline lambda V gene segments), (v) V1-124, V1-36, V1-104, V1-33 and V1-56 (optionally having up to 6 other feline lambda V gene segments), (vi) V1-124, V1-36, V1-104, V1-33, V1-56, V1-42 (optionally having up to 5 other feline lambda V gene segments), or (vii) antibody lambda light chains having feline lambda V gene segments comprising V1-124, V1-36, V1-104, V1-33, V1-56, V1-42 and V1-41 (optionally having up to 4 other feline lambda V gene segments).

[0306] In another embodiment, the repertoire comprises antibody lambda light chains expressed from lambda V gene segments of the Nekolambdafamily 1 and 3.

[0307] An antibody light chain repertoire is also disclosed, the repertoire comprising antibody kappa light chains having kappa V gene segments from 6 or fewer different feline kappa V gene segments, at least 1 of the feline kappa V gene segments being selected from the list comprising V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4, and optionally, at least 2, at least 3, at least 4, at least 5 or all 6 of the feline kappa V gene segments being selected from the list comprising V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4.

[0308] Optionally, the repertoire comprises (i) V4-1 (optionally having up to 5 other feline kappa V gene segments), (ii) V4-1 and V2-12 (optionally having up to 4 other feline kappa V gene segments), (iii) V4-1, V2-12 and V2-5 (optionally having up to 3 other feline kappa V gene segments), (iv) V4-1, V2-12, V2-5 and V2-13 (optionally having up to 2 other feline kappa V gene segments), (v) V4-1, V2-12, V2-5, V2-13 and V2-9 (optionally having up to 1 other feline kappa V gene segment), or (vi) V4-1, V2-12, V2-5, V2-13, V2-4 and V2-4, or (vii) antibody kappa light chains having feline kappa V gene segments comprising V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4.

[0309] In another embodiment, the repertoire comprises antibody kappa light chains expressed from the kappa V gene segments of the Nekokappa V families 2 and 4.

[0310] The antibodies and antibody chains disclosed herein can be used for methods of preventing or treating feline diseases. In particular, intact feline antibodies having feline constant regions can be used.

[0311] Accordingly, the invention relates to an antibody or antibody chain, or a portion thereof, disclosed herein, for use in treating or preventing a feline disease in a cat in need of such treatment or prevention, e.g., an antibody obtainable or obtained from a rodent or rodent cell disclosed herein, or a repertoire disclosed herein (e.g., an intact feline antibody made using sequence information from a rodent or rodent cell).

[0312] The invention also relates to a method of treating a cat, the method comprising delivering to a cat in need of treatment an antibody or antibody chain, or a portion thereof, disclosed herein, wherein the antibody is, e.g., an antibody obtainable or obtained from a rodent or rodent cell disclosed herein, or a repertoire disclosed herein, or an intact feline antibody made using sequence information from a rodent or rodent cell.

[0313] In a further aspect, the invention relates to the use of a rodent described herein as a test model for drugs and vaccines. Accordingly, the invention relates to a method for identifying or validating a drug or vaccine, the method comprising delivering a vaccine or drug to a mammal of the invention (e.g., a cat) and monitoring one or more of an immune response, a safety profile, and an effect against a disease.

[0314] The present invention also relates to a kit comprising an antibody or antibody derivative disclosed herein, instructions for use of such an antibody, or a suitable laboratory reagent such as a buffer, antibody detection reagent, or excipient for formulation with the antibody.

[0315] Certain specific preferred gene segments are disclosed herein, and related sequences are provided.

[0316] The IGH V gene segment is selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5, or is selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-16 and V3-5.

[0317] The lambda gene segment is selected from the list comprising V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126, or is selected from the list comprising V1-124, V1-36, V1-104, V1-N1, V1-56, V1-42, V1-N14, V3-7, V3-5, V3-13 and V1-126.

[0318] The kappa gene segment is selected from the list comprising V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4.

[0319] The present invention also relates to the feline D gene segment (IGH D-N1). The feline D gene segment has the following DNA sequence (SEQ ID NO: 48).

Chemical formula

[0320] The present invention also relates to a vector or cell genome or cell line genome or rodent genome comprising the feline D gene segment IGH D-N1.

[0321] This disclosure expressly contemplates the use of IGH D-N1 (SEQ ID NO: 48) in combination with and apart from the V gene segments described herein in the rodents, rodent cells and methods, antibodies, pharmaceutical compositions, etc. described herein.

[0322] For example, in one embodiment, the invention relates to

[0323] a rodent or rodent cell having a genome comprising one or more feline IGH V gene segments, the feline D gene segment IGH D-N1 and one or more feline IGH J region gene segments (plural),

[0324] the rodent or rodent cell can express the feline gene segments to form an antibody heavy chain,

[0325] Optionally, the number of feline IGH V gene segments is 9 or less, and at least one of the feline IGH V gene segments is selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-16 and V3-5.

[0326] This disclosure also expressly contemplates the use of IGH D-N1 (SEQ ID NO: 48) in hosts other than rodents. Thus, as used herein, references to "rodents" can be construed as "non-human animals" (or "non-human mammals") in these contexts.

[0327] With respect to the feline V gene segments disclosed herein, the use of IGH D-N1 obviates the need to insert all feline D genes into the host to create an antibody population that provides a repertoire suitable for the identification and selection of antibody candidates for therapeutic and prophylactic treatment.

[0328] Table 1 TIFF2025520702000002.tif209169 TIFF2025520702000003.tif209169 TIFF2025520702000004.tif193169 TIFF2025520702000005.tif209169 TIFF2025520702000006.tif33169

[0329] It will be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine research, numerous procedures equivalent to the specific procedures described herein. Such equivalents are considered to be within the scope of the invention and are covered by the claims. All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which the invention pertains. All publications and patent applications are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0330] The use of the words "a" or "an" used in combination with the term "comprising" in the claims and / or the specification may mean "one" in some cases, but is also consistent with the meaning of "one or more", "at least one", "one or more". The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer only to alternative cases or the alternatives are mutually exclusive, but the disclosure supports a definition that refers only to the alternatives and "and / or". Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of the device's error, the method used to determine the value, or the variation that exists among the subjects being studied.

[0331] As used in this specification and the claims (if any), the terms "comprising" (and any form of comprising such as "comprise" or "comprises"), "having" (and any form of having such as "have" or "has"), "including" (and any form of including such as "includes" or "include"), or "containing" (and any form of containing such as "contains" or "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0332] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed prior to that 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 BA, CA, CB, CBA, BCA, ACB, BAC, or CAB if order is important in a particular context. Continuing with this example, combinations including repetitions of one or more items or terms such as BB, AAA, ABAB, BBC, AAABCCCC, CBBAAA, CABABB are explicitly included. One of ordinary skill in the art will understand that, unless otherwise apparent from the context, there is usually no limit to the number of items or terms being combined.

[0333] Unless the context dictates otherwise, any part of this disclosure can be read in combination with any other part of this disclosure.

[0334] All compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the invention are described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the composition and / or method and in the steps or the order of the steps of the methods described herein, without departing from the concept, spirit, and scope of the invention.

[0335] The invention is further described in detail in the following non-limiting examples.

Example

[0336] Example 1, Analysis of Healthy Feline PBMCs, Isolation of Intact Single B Cells, and Generation of VH:VL Pairing Data by 10x Immune Profiling Two frozen PBMC samples were obtained from Life Link Biologicals from two healthy European shorthair SPF cats.

[0337] After gating out dead cells stained with viability dye, live lymphocyte cells (including B cells) were enriched from the cell fraction using FACS sorting of the forward scatter-side scatter lymphocyte gate. Sample 1 yielded approximately 320,000 B cells (CD21-positive cells), and Sample 2 yielded approximately 120,000 B cells (CD21-positive cells). 40,000 cells from each sample were pelleted by centrifugation and processed using the microfluidic encapsulation protocol of the 10X Genomics Chromium Next Gem Single Cell 5’ v2 kit (10X Genomics) according to the manufacturer's protocol. The resulting cDNA was quality controlled by quantification using a Qubit fluorometer (ThermoFisher) and Bioanalyzer analysis (Agilent). Subsequently, as shown in Table 3, the cDNA was processed using feline constant region-specific PCR primers designed to cross-react with as many constant regions as possible to generate a 5’ VDJ NGS sequencing library. After pelleting, the number of cells loaded onto the 10X chip is expected to be approximately 28,000.

[0338] The 5’ VDJ library thus generated was sequenced on an Illumina NGS platform using 150bp paired-end sequencing. At this read length, a minimum of 2,000 read pairs per cell barcode is recommended. The resulting sequencing data was demultiplexed, and each library contained a pair of Read1 and Read2.fastq.gz files.

[0339] The resulting set of FASTQ files was processed via Cell Ranger software (10X Genomics), and in the case of the VDJ and 5’GEX library combination, this was processed via the “cellranger multi” command, which was provided with a list of V+D+J+C reference sequences for the heavy and light chains within the feline repertoire, and a set of references to the internal feline genome reference and annotations (incorporating information from FelisCatus8.0 and FelisCatus9.0 and internal sequence data), which included a list of validated IG genes for the three IG loci within the feline genome from internal curation, and a list of internal primers designed as parameters for this purpose. The results of the VDJ library that had gone through the “cellranger multi” step were QC’d and compared to the estimated cell count in the cell counting step. In sample 1, 14,562 10X V-J paired cells were recovered, and in sample 2, 1,503 10X V-J paired cells were recovered. The chains reconstructed from Cellranger (all_contig.fasta) were blasted against a set of heavy and light chain V+D+J reference sequences formatted to run the enclone software (10xGenomics). The results of the enclosing step were sorted by aa% and filtered against the v_name sequences corresponding to the highly reliable heavy + light V repertoire cell barcodes calls, and only the highly reliable cells were selected as the final result.

[0340] Results From the resulting heavy chain + light chain paired data, the usage of the heavy chain was plotted against the usage of the light chain. Some heavy chain / light chain pairing patterns emerged more dominantly than others.

[0341] Tables 2a - 2c show the paired data ranked by top pairing, heavy chain V gene segment, and light chain V gene segment. Table 4 shows the ranking of light chain V genes by the number of pairings of different heavy chain V genes.

[0342] Table 2a TIFF2025520702000007.tif89153

[0343] Table 2b TIFF2025520702000008.tif62153

[0344] Table 2c TIFF2025520702000009.tif106153

[0345] Table 3 TIFF2025520702000010.tif192153

[0346] Table 4 TIFF2025520702000011.tif106153

[0347] This experiment was repeated with subsequent data, and the following data were generated. Table 5a TIFF2025520702000012.tif62153

[0348] Table 5b TIFF2025520702000013.tif73153

[0349] Table 5c TIFF2025520702000014.tif45153

[0350] The present invention is further described in the following numbered clauses. 1. A rodent or rodent cell having a genome comprising no more than 9 feline IGH V gene segments, one or more feline IGH D region gene segments (plural available), and one or more feline IGH J region gene segments (plural available), wherein the rodent or rodent cell can express the feline gene segments to form an antibody heavy chain, At least one of the feline IGH V gene segments is selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5, said rodent or rodent cell.

[0351] 2. Feline IGH (i) V3-14 (optionally having up to 8 other feline IGH V gene segments), (ii) V3-14 and V3-11 (optionally having up to 7 other feline IGH V gene segments), (iii) V3-14, V3-11 and V3-12 (optionally having up to 6 other feline IGH V gene segments), (iv) V3-14, V3-11, V3-12 and V3-84 (optionally having up to 5 other feline IGH V gene segments), (v) V3-14, V3-11, V3-12, V3-84 and V3-20 (optionally having up to 4 other feline IGH V gene segments), (vi) V3-14, V3-11, V3-12, V3-84, V3-20 and V3-27 (optionally having up to 3 other feline IGH V gene segments), (vii) V3-14, V3-11, V3-12, V3-84, V3-20, V3-27 and V4-3 (optionally having up to 2 other feline IGH V gene segments), (viii) V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3 and V3-N3 (optionally having up to 1 other feline IGH V gene segment), or (ix) The rodent or rodent cell according to clause 1, comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5.

[0352] 3. The rodent or rodent cell according to clause 1, which contains 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or only 1 of the feline IGH V gene segment(s), and optionally, the rodent or rodent cell has a genome encoding only a single type of feline heavy chain VDJ variable region.

[0353] 4. The rodent or rodent cell according to clause 1, which contains at least 2 IGH gene segments, for example, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or 9 feline IGH V gene segments.

[0354] 5. The rodent or rodent cell according to any one of clauses 1 to 4, which contains at least 1 gene segment from the feline IGH V gene segments of families 3 and 4.

[0355] 6. A rodent or rodent cell having a genome containing 11 or less feline lambda V gene segments and one or more feline lambda J region gene segment(s), wherein the rodent or rodent cell can express the feline lambda gene segment to form an antibody light chain, and at least 1 of the feline lambda gene segments is selected from V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13, and V1-126.

[0356] 7. Feline lambda V (i) V1-124 (optionally having up to 10 other feline lambda V gene segments), (ii) V1-124 and V1-36 (optionally having up to 9 other feline lambda V gene segments), (iii) V1-124, V1-36, and V1-104 (optionally having up to 8 other feline lambda V gene segments), (iv) V1-124, V1-36, V1-104, and V1-33 (optionally having up to 7 other Nekolambdavirus V gene segments), (v) V1-124, V1-36, V1-104, V1-33, and V1-56 (optionally having up to 6 other Nekolambdavirus V gene segments), (vi) V1-124, V1-36, V1-104, V1-33, V1-56, V1-42 (optionally having up to 5 other Nekolambdavirus V gene segments), or (vii) a rodent or rodent cell according to clause 6, comprising V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, and V1-41 (optionally having up to 4 other Nekolambdavirus V gene segments).

[0357] 8. A rodent or rodent cell according to clause 6, comprising 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or only 1 V gene segment, and optionally, said rodent or rodent cell has a genome encoding only a single type of Nekolambdavirus light chain VJ variable region.

[0358] 9. A rodent or rodent cell according to clause 6, comprising at least 2 Nekolambdavirus V gene segments, for example, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or 11 Nekolambdavirus V gene segments.

[0359] 10. A rodent or rodent cell according to any one of clauses 6 to 9, comprising Nekolambdavirus V gene segments from each of lambda V families 1 and 3.

[0360] 11. A rodent or rodent cell having a genome comprising 6 or fewer Nekokappa V gene segments and one or more Nekokappa J region gene segments (plural available), The rodent or rodent cell can express the feline kappa gene segment to form an antibody light chain, The rodent or rodent cell, wherein at least one of the feline kappa gene segments is selected from V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4.

[0361] 12. Feline kappa V (i) V4-1 (optionally having up to 5 other feline kappa V gene segments), (ii) V4-1 and V2-12 (optionally having up to 4 other feline kappa V gene segments), (iii) V4-1, V2-12 and V2-5 (optionally having up to 3 other feline kappa V gene segments), (iv) V4-1, V2-12, V2-5 and V2-13 (optionally having up to 2 other feline kappa V gene segments), (v) V4-1, V2-12, V2-5, V2-13 and V2-9 (optionally having up to 1 other feline kappa V gene segment), or (vi) V4-1, V2-12, V2-5, V2-13, V2-4 and V2-4, or (vii) The rodent or rodent cell according to clause 11, comprising V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4. The rodent or rodent cell according to clause 11.

[0362] 13. Containing 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer or only 1 feline kappa V gene segment, and optionally, the rodent or rodent cell has a genome encoding only a single type of feline kappa light chain VJ variable region, The rodent or rodent cell according to clause 11.

[0363] 14. Containing at least 2 feline kappa V gene segments, for example, at least 3, at least 4, at least 5 or 6 feline kappa V gene segments, The rodent or rodent cell according to item 11.

[0364] 15. A feline kappa V gene segment from each of kappa V gene segment families 2 and 4, suitably including IGK V4-1 and IGK V2-12, The rodent or rodent cell according to any one of items 11 to 14.

[0365] 16. The rodent genome includes feline gene segments from both the heavy chain and at least one feline light chain, optionally, (i) the genome of the rodent or rodent cell is as described in any one of claims 6 to 10 for the feline lambda gene segment and / or as described in any one of claims 11 to 15 for the feline kappa V gene segment in combination with that described in any one of items 1 to 5, or (ii) the genome of the rodent or cell is as described in items 1 to 5 and further includes feline kappa V and J gene segments that can be expressed to form a kappa antibody light chain variable domain, and / or the genome of the rodent or cell is a rodent or rodent cell as described in any one of items 1 to 4 and further includes feline lambda V and J gene segments that can be expressed to form a lambda antibody light chain. The rodent or rodent cell according to any one of items 1 to 15.

[0366] 17. (i) The feline gene segment(s) is / are located upstream of the rodent constant region within the genome, suitably upstream of the heavy chain constant region for the inserted feline heavy chain variable region gene segment, and / or suitably upstream of the light chain constant region for the inserted feline light chain variable region gene segment, such that the rodent or rodent cell can produce a chimeric antibody heavy chain, or light chain, or both resulting from the expression of the inserted feline variable region gene segment and the host constant region, or (ii) The feline gene segment(s) is / are located in the genome in functional array with the feline constant region, such that the rodent can produce antibody chains resulting from the expression of the feline VDJ gene segment having the feline constant region, and / or antibody chains resulting from the expression of the feline VJ gene segment having the feline constant region. A rodent or rodent cell according to any one of clauses 1 to 16.

[0367] 18. A genome in which one or more feline V gene segments are regulated by one or more non-coding regulatory sequences selected from promoter, intron, splice site, and recombination signal sequences for V(D)J recombination that precede the coding sequence of each V gene segment, and the regulatory sequences are of endogenous host origin. A rodent or rodent cell according to any one of clauses 1 to 16.

[0368] 19. A nucleic acid encoding a chimeric antibody chain, the nucleic acid having a variable domain comprising any one of the feline IGH V gene segments selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3, and V3-5, and a rodent constant region, and optionally, the feline V gene segment has undergone somatic hypermutation in the rodent.

[0369] 20. An antibody chain having a variable region obtainable or obtained by the expression of any one of feline IGH V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3, and V3-5 in a rodent in combination with a rodent constant region.

[0370] 21. A nucleic acid encoding a chimeric antibody chain, wherein the nucleic acid has a variable domain comprising any one of the feline lambda V gene segments selected from the list consisting of lambda V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126, and a rodent constant region, and optionally, the feline V gene segment has undergone somatic hypermutation in a rodent, said nucleic acid.

[0371] 22. An antibody chain having a variable region obtained or obtainable by expression of any one of feline lambda V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126 in a rodent in combination with a rodent constant region.

[0372] 23. A nucleic acid encoding a chimeric antibody chain, wherein the nucleic acid has a variable domain comprising any one of the feline kappa V gene segments selected from the list consisting of V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4, and a rodent constant region, and optionally, the feline V gene segment has undergone somatic hypermutation in a rodent, said nucleic acid.

[0373] 24. An antibody chain having a variable region obtained or obtainable by expression of any one of feline kappa V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4 in a rodent in combination with a rodent constant region.

[0374] 25. A pharmaceutical composition comprising an antibody chain having a variable region comprising any one of feline IGH V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5 in combination with a pharmaceutically acceptable excipient or carrier.

[0375] A pharmaceutical composition comprising an antibody chain having a variable region containing any one of Nekolambda V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126, in combination with a pharmaceutically acceptable excipient or carrier.

[0376] A pharmaceutical composition comprising an antibody chain having a variable region containing any one of Nekokappa V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4, in combination with a pharmaceutically acceptable excipient or carrier.

[0377] 28. A pharmaceutical composition comprising an antibody, wherein the antibody (i) has a heavy chain having a variable region obtained by the expression of any one of Nek oIGH V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5, (ii) a lambda light chain having a variable region obtained by the expression of any one of Nekolambda V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126, or (iii) any one of kappa light chains having a variable region obtained by the expression of any one of Nekokappa V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4, and the antibody is formulated in combination with a pharmaceutically acceptable excipient or carrier, the pharmaceutical composition.

[0378] 29. An antibody heavy chain repertoire, wherein the repertoire comprises an antibody heavy chain having a Nek oIGH V gene segment from 9 or fewer different Nek oIGH V gene segments, and at least one of the Nek oIGH V gene segments is selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5, Optionally, the repertoire A, (i) V3-14 (optionally having up to 8 other Nek oIGH V gene segments), (ii) V3-14 and V3-11 (optionally having up to 7 other feline IGH V gene segments), (iii) V3-14, V3-11 and V3-12 (optionally having up to 6 other feline IGH V gene segments), (iv) V3-14, V3-11, V3-12 and V3-84 (optionally having up to 5 other feline IGH V gene segments), (v) V3-14, V3-11, V3-12, V3-84 and V3-20 (optionally having up to 4 other feline IGH V gene segments), (vi) V3-14, V3-11, V3-12, V3-84, V3-20 and V3-27 (optionally having up to 3 other feline IGH V gene segments), (vii) V3-14, V3-11, V3-12, V3-84, V3-20, V3-27 and V4-3 (optionally having up to 2 other feline IGH V gene segments), (viii) V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3 and V3-N3 (optionally having up to 1 other feline IGH V gene segment), or (ix) an antibody heavy chain having feline IGH V gene segments expressed from V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3 and V3-5, and / or B. said antibody heavy chain repertoire comprising antibody heavy chains expressed from IGH V gene segments of feline IGH V families 3 and 4.

[0379] 30. An antibody light chain repertoire, said repertoire comprising antibody light chains having feline lambda V gene segments from no more than 11 different feline lambda V gene segments, at least one of said feline lambda V gene segments being selected from the list comprising V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13 and V1-126, Optionally, the repertoire is A. (i) V1-124 (optionally having up to 10 other feline lambda V gene segments), (ii) V1-124 and V1-36 (optionally having up to 9 other feline lambda V gene segments), (iii) V1-124, V1-36 and V1-104 (optionally having up to 8 other feline lambda V gene segments), (iv) V1-124, V1-36, V1-104 and V1-33 (optionally having up to 7 other feline lambda V gene segments), (v) V1-124, V1-36, V1-104, V1-33 and V1-56 (optionally having up to 6 other feline lambda V gene segments), (vi) V1-124, V1-36, V1-104, V1-33, V1-56, V1-42 (optionally having up to 5 other feline lambda V gene segments), or (vii) an antibody lambda light chain having a feline lambda V gene segment comprising V1-124, V1-36, V1-104, V1-33, V1-56, V1-42 and V1-41 (optionally having up to 4 other feline lambda V gene segments), and / or B. the antibody light chain repertoire comprising an antibody lambda light chain expressed from feline lambda V gene segments of feline lambda families 1 and 3.

[0380] 31. An antibody light chain repertoire, wherein the repertoire comprises an antibody kappa light chain having a feline kappa V gene segment from 5 or fewer different feline kappa V gene segments, and at least one of the feline kappa V gene segments is selected from the list comprising V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4, Optionally, the repertoire is A. (i) V4-1 (optionally having up to 5 other feline kappa V gene segments), (ii) V4-1 and V2-12 (optionally having up to four other feline kappa V gene segments), (iii) V4-1, V2-12 and V2-5 (optionally having up to three other feline kappa V gene segments), (iv) V4-1, V2-12, V2-5 and V2-13 (optionally having up to two other feline kappa V gene segments), (v) V4-1, V2-12, V2-5, V2-13 and V2-9 (optionally having up to one other feline kappa V gene segment), or (vi) V4-1, V2-12, V2-5, V2-13, V2-4 and V2-4, or (vii) An antibody kappa light chain having a feline kappa V gene segment comprising V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4, and / or B. The antibody light chain repertoire comprising an antibody kappa light chain expressed from feline kappa V gene segments of feline kappa V families 2 and 4.

[0381] 32. An antibody or antibody chain, or a part thereof, or DNA encoding an antibody chain or a part thereof, obtained from or obtainable from a rodent or rodent cell according to any one of clauses 1 to 18, or from a repertoire according to any one of clauses 29 to 31.

[0382] 33. An antibody or antibody chain, or a part thereof, obtained from or obtainable from a rodent or cell according to any one of clauses 1 to 18, or from a repertoire according to any one of clauses 29 to 31, for use in the treatment or prevention of a feline disease that requires treatment or prevention of a disease.

[0383] 34. A method for producing a rodent or rodent cell according to any one of clauses 1 to 33, wherein into the genome of a rodent cell, (i) Nine or fewer feline IGH V gene segments, one or more feline IGH D region gene segments, and one or more feline IGH J region gene segments (at least one of said feline IGH V gene segments is selected from the list comprising V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-N3, and V3-5), and / or (ii) Eleven or fewer feline lambda V gene segments and one or more feline lambda J region gene segments (at least one of said feline V gene segments is selected from the list comprising V1-124, V1-36, V1-104, V1-33, V1-56, V1-42, V1-41, V3-7, V3-5, V3-13, and V1-126), and / or (iii) Six or fewer feline kappa V gene segments and one or more feline kappa J region gene segments (at least one of said feline kappa gene segments is selected from V4-1, V2-12, V2-5, V2-13, V2-9, and V2-4), comprising inserting, Said rodent or rodent cell can express the feline variable region gene segment(s) in combination with a constant region to form an antibody chain, said method.

[0384] 35. A method for producing an antibody or antibody chain specific for a desired antigen, comprising immunizing a rodent according to any one of clauses 1 to 18 with said desired antigen and recovering said antibody chain or said antibody, or recovering the cells producing said antibody chain or said antibody, said method.

[0385] 36. A method for producing an antibody chain or antibody specific for a desired antigen, comprising immunizing a rodent according to any one of clauses 1 to 18 with said desired antibody and then, appropriately, replacing the rodent constant region of said antibody chain or antibody with a feline constant region by manipulating the nucleic acid encoding said antibody, said method.

[0386] 37. A method for producing an antibody chain or a part thereof, wherein the antibody chain has a feline variable region, and the method includes expressing in a cell a nucleic acid encoding the antibody chain or a part thereof, The sequence of the nucleic acid encoding the variable region of the antibody chain is obtained from, or can be obtained from, a rodent according to any one of items 1 to 18 immunized with an antigen, or from an antibody of the antibody repertoire according to any one of items 29 to 31, and each contains one of the feline heavy chain, kappa or lambda gene segments according to any one of items 1, 5 or 10, Optionally, purifying and / or isolating the antibody chain, and Next, optionally, the method includes a subsequent step of formulating the antibody into a pharmaceutically acceptable formulation suitable for administration to a cat.

[0387] 38. A method for preparing a pharmaceutical composition, which includes producing an antibody by the method according to any one of items 35 to 37, and further includes preparing the pharmaceutical composition by combining the antibody with a pharmaceutically acceptable carrier or other excipient.

[0388] 39. A pharmaceutical composition containing the antibody according to any one of items 25 to 28, which is a monoclonal antibody.

[0389] 40. A method for treating a cat, which includes delivering an antibody or an antibody chain or a functional part thereof to a cat in need of treatment, and the antibody is obtained from, or can be obtained from, a rodent or rodent cell according to any one of items 1 to 18. Optionally, the antibody chain is as described in any one of items 20, 22, 24 or 32, and / or the antibody or antibody chain is in the form of a pharmaceutical composition according to any one of items 25 to 28, and / or the antibody or antibody chain is a complete feline antibody or antibody chain having a feline variable region and a constant region, The method, preferably comprising delivering the antibody in the form of a pharmaceutical composition having a complete feline heavy chain and a complete feline light chain.

[0390] 41. A rodent or rodent cell having a genome comprising a feline IGH V gene segment selected from the list comprising IGH V3-14 (SEQ ID NO: 1), V1-36 (SEQ ID NO: 11), V1-104 (SEQ ID NO: 12), and V1-42 (SEQ ID NO: 15), one or more feline IGH D region gene segments (plural available) and one or more feline IGH J region gene segments (plural available), wherein the feline gene segment can be expressed to form an antibody heavy chain, the rodent or rodent cell having the genome.

[0391] 42. A rodent or rodent cell having a genome comprising the feline IGK V gene segment IGK V2-5 (SEQ ID NO: 23), and one or more feline IGH J region gene segments (plural available), wherein the feline gene segment can be expressed to form an antibody light chain, the rodent or rodent cell having the genome.

Claims

1. A rodent or rodent cell having a genome comprising nine or fewer feline IGH V gene segments, one or more feline IGH D region gene segments, and one or more feline IGH J region gene segments, The aforementioned feline gene segment can be expressed to form an antibody heavy chain. At least one of the feline IGH V gene segments is selected from the list including V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-16, and V3-5. The aforementioned rodent or rodent cell.

2. Cat IGH (i) V3-14, (ii) V3-14 and V3-11, (iii) V3-14, V3-11 and V3-12, (iv) V3-14, V3-11, V3-12 and V3-84, (v) V3-14, V3-11, V3-12, V3-84 and V3-20, (vi) V3-14, V3-11, V3-12, V3-84, V3-20 and V3-27, (vii) V3-14, V3-11, V3-12, V3-84, V3-20, V3-27 and V4-3, (viiii) V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3 and V3-16, or (ix) V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-16 and V3-5, The rodent or rodent cell according to claim 1.

3. A rodent or rodent cell having a genome comprising 11 or fewer Nekoramuda V gene segments and one or more Nekoramuda J region gene segments, The aforementioned rodent or rodent cell can express the Nekoramuda gene segment to form an antibody light chain. The rodent or rodent cell wherein at least one of the Nekoramuda gene segments is selected from V1-124, V1-36, V1-104, V1-N1, V1-56, V1-42, V1-N14, V3-7, V3-5, V3-13 and V1-126.

4. Nekoramuda V (i) V1-124, (ii) V1-124 and V1-36, (iii) V1-124, V1-36 ​​and V1-104, (iv) V1-124, V1-36, V1-104 and V1-N1, (v) V1-124, V1-36, V1-104, V1-N1 and V1-56, (vi) V1-124, V1-36, V1-104, V1-N1, V1-56, V1-42, or (vii) Including V1-124, V1-36, V1-104, V1-N1, V1-56, V1-42 and V1-N14, The rodent or rodent cell according to claim 3.

5. A rodent or rodent cell having a genome comprising six or fewer Necrokappa V gene segments and one or more Necrokappa J region gene segments, The aforementioned cat kappa gene segment can be expressed to form an antibody light chain. At least one of the aforementioned cat kappa gene segments is selected from V4-1, V2-12, V2-5, V2-13, V2-9, and V2-4. The aforementioned rodent or rodent cell.

6. Nekokappa V (i) V4-1, (ii) V4-1 and V2-12, (iii) V4-1, V2-12 and V2-5, (iv) V4-1, V2-12, V2-5 and V2-13, (v) V4-1, V2-12, V2-5, V2-13 and V2-9, or (vi) Including V4-1, V2-12, V2-5, V2-13, V2-9 and V2-4, The rodent or rodent cell according to claim 5.

7. The rodent genome comprises a feline gene segment from both the heavy chain and at least one feline light chain, according to any one of claims 1 to 6, of the rodent or rodent cell.

8. (i) The genome of the rodent or rodent cell is the genome according to claim 1 or 2, which is a combination of the Nekoramuda gene segment according to claim 3 or 4 and / or the Nekokappa V gene segment according to claim 5 or 6, or (ii) The genome of the rodent or cell is the genome defined in claim 1 or 2, further comprising feline kappa V and J gene segments that can be expressed to form a kappa antibody light chain variable domain, and / or the genome of the rodent or cell is the genome defined in claim 1 or 2, further comprising feline lambda V and J gene segments that can be expressed to form a lambda antibody light chain. The rodent or rodent cell according to claim 7.

9. (i) The feline gene segment(s) is located upstream of the rodent constant region within the genome, preferably upstream of the heavy chain constant region for the inserted feline heavy chain variable region gene segment, and / or preferably upstream of the light chain constant region for the inserted feline light chain variable region gene segment, and as a result, the rodent or rodent cell can produce a chimeric antibody heavy chain or light chain, or both, resulting from the expression of the inserted feline variable region gene segment and the host constant region, (ii) The rodent or rodent cell according to any one of claims 1 to 6, wherein the feline gene segment(s) are functionally aligned with the feline constant region and located within the genome, and as a result, the rodent can produce antibody chains resulting from the expression of the feline VDJ gene segment having the feline constant region, and / or antibody chains resulting from the expression of the feline VJ gene segment having the feline constant region.

10. (i) The feline gene segment(s) are located upstream of the rodent constant region within the genome, preferably upstream of the heavy chain constant region with respect to the inserted feline heavy chain variable region gene segment, and / or preferably upstream of the light chain constant region with respect to the inserted feline light chain variable region gene segment, and as a result, the rodent or rodent cell can produce a chimeric antibody heavy chain or light chain, or both, resulting from the expression of the inserted feline variable region gene segment and the host constant region, (ii) The rodent or rodent cell according to claim 7, wherein the feline gene segment(s) are functionally aligned with the feline constant region and located within the genome, and as a result, the rodent can produce antibody chains resulting from the expression of the feline VDJ gene segment having the feline constant region, and / or antibody chains resulting from the expression of the feline VJ gene segment having the feline constant region.

11. (i) The feline gene segment(s) are located upstream of the rodent constant region in the genome, preferably upstream of the heavy chain constant region with respect to the inserted feline heavy chain variable region gene segment, and / or preferably upstream of the light chain constant region with respect to the inserted feline light chain variable region gene segment, and as a result, the rodent or rodent cell can produce a chimeric antibody heavy chain or light chain, or both, resulting from the expression of the inserted feline variable region gene segment and the host constant region, (ii) The rodent or rodent cell according to claim 8, wherein the feline gene segment(s) are functionally aligned with the feline constant region and located within the genome, and as a result, the rodent can produce antibody chains resulting from the expression of the feline VDJ gene segment having the feline constant region, and / or antibody chains resulting from the expression of the feline VJ gene segment having the feline constant region.

12. A method for producing an antibody or antibody chain specific to a desired antigen, comprising immunizing a rodent according to any one of claims 1 to 6 with the desired antigen, recovering the antibody chain or the antibody, or recovering cells that produce the antibody chain or the antibody.

13. A method for producing an antibody chain or antibody specific to a desired antigen, comprising immunizing a rodent according to any one of claims 1 to 6 with the desired antigen, and then replacing the rodent constant region of the antibody chain or antibody with the feline constant region.

14. A method for preparing a pharmaceutical composition, comprising producing an antibody by the method of claim 12, and further comprising combining the antibody with a pharmaceutically acceptable carrier or other excipient to produce the pharmaceutical composition.

15. A vector, cell genome, cell line genome, or rodent genome containing the feline D gene segment IGH D-N1 (SEQ ID NO: 48).

16. Having a genome containing one or more feline IGH V gene segments, feline D gene segment IGH D-N1, and one or more feline IGH J region gene segments, A rodent or rodent cell capable of expressing the aforementioned feline gene segment to form an antibody heavy chain.

17. The rodent or rodent cell according to claim 16, wherein the feline IGH V gene segments number nine or less, and at least one of the feline IGH V gene segments is selected from the list including V3-14, V3-11, V3-12, V3-84, V3-20, V3-27, V4-3, V3-16N3 and V3-5.