Anti-LAG-3 antibody

Abstract

The present invention provides an anti-LAG-3 antibody capable of repeated administration even to animals other than rat. An anti-LAG-3 antibody comprising (a) a light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLLDSDGNTY (SEQ ID NO: 16), CDR2 having the amino acid sequence of SVS and CDR3 having the amino acid sequence of MQATHVPFT (SEQ ID NO: 17) and the light chain constant region of an antibody of an animal other than rat; and (b) a heavy chain comprising a heavy chain variable region containing CDR1 having the amino acid sequence of GFDFDTYP (SEQ ID NO: 18), CDR2 having the amino acid sequence of ITIKTHNYAT (SEQ ID NO: 19) and CDR3 having the amino acid sequence of NREDFDY (SEQ ID NO: 20) and the heavy chain constant region of an antibody of an animal other than rat. A pharmaceutical composition comprising the above anti-LAG-3 antibody as an active ingredient. A method for preparing the above anti-LAG-3 antibody is also provided.

Claims

1. An anti-LAG-3 antibody comprising (a) a light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLLDSDGNTY (SEQ ID NO: 16), CDR2 having the amino acid sequence of SVS and CDR3 having the amino acid sequence of MQATHVPFT (SEQ ID NO: 17) and the light chain constant region of an antibody of bovine; and (b) a heavy chain comprising a heavy chain variable region containing CDR1 having the amino acid sequence of GFDFDTYP (SEQ ID NO: 18), CDR2 having the amino acid sequence of ITIKTHNYAT (SEQ ID NO: 19) and CDR3 having the amino acid sequence of NREDFDY (SEQ ID NO: 20) and the heavy chain constant region of an antibody of bovine, wherein the light chain constant region of the bovine antibody has the amino acid sequence as shown in SEO ID NO: 3 and the heavy chain constant region of the bovine antibody has the amino acid sequence as shown in SEO ID NO: 4.

2. The antibody of claim 1, wherein the light chain variable region and the heavy chain variable region are derived from rat.

3. The antibody of claim 2, wherein the light chain variable region is the light chain variable region of a rat anti-bovine LAG-3 antibody and the heavy chain variable region is the heavy chain variable region of a rat anti-bovine LAG-3 antibody.

4. The antibody of claim 3, wherein the light chain variable region has the amino acid sequence as shown in SEQ ID NO. 1 and the heavy chain variable region has the amino acid sequence as shown in SEQ ID NO: 2.

5. The antibody of claim 1 which has a four-chain structure comprising two light chains and two heavy chains.

6. A pharmaceutical composition comprising the antibody of claim 1 as an active ingredient.

7. A method to treat cancer and/or infection in cattle, buffalo or sheep comprising administering an effective amount of the pharmaceutical composition of claim 6 to cattle, buffalo or sheep in need thereof so as to treat said cancer and/or infection.

8. The method of claim 7, wherein the cancer and/or infection is selected from the group consisting of neoplastic diseases, leukemia, Johne's disease, anaplasmosis, bacterial mastitis, mycotic mastitis, mycoplasma infections, tuberculosis, Theileria orientalis infection, cryptosporidiosis, coccidiosis, trypanosomiasis and leishmaniasis.

9. An artificial genetic DNA encoding the antibody of claim 1.

10. A vector comprising the artificial genetic DNA of claim 9.

11. A host cell transformed with the vector of claim 10.

12. A method of preparing an antibody, comprising culturing the host cell of claim 11 and collecting an anti-LAG-3 antibody from the resultant culture.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 The amino acid sequence of rat-bovine chimeric anti-bovine LAG-3 antibody ch2D8. CDR1, CDR2 and CDR3 regions in the light chain variable region and the heavy chain variable region of rat anti-bovine LAG-3 antibody 2D8 are shown. Further, amino acids introduced as mutations to bovine IgG1 (CH2 domain) are also shown (amino acid numbers and mutations: 247 E.fwdarw.P, 248 L.fwdarw.V, 249 P.fwdarw.A, 250 G.fwdarw.deletion, 344 A.fwdarw.S, 345 P.fwdarw.S).

(2) FIG. 2 Schematic drawings of pDC6 vector and rat-bovine chimeric anti-bovine LAG-3 antibody ch2D8.

(3) FIG. 3 The purity of purified rat-bovine chimeric anti-bovine LAG-3 antibody ch2D8.

(4) FIG. 4 Binding specificity of rat-bovine chimeric anti-bovine LAG-3 antibody ch2D8.

(5) FIG. 5 Inhibitory activity of rat-bovine chimeric anti-bovine LAG-3 antibody ch2D8 against bovine LAG-3/MHC II binding

(6) FIG. 6 Changes in IFN-γ response due to rat-bovine chimeric anti-bovine LAG-3 antibody ch2D8.

(7) FIG. 7 Cross-reactivity of rat anti-bovine LAG-3 antibody 2D8 with water buffalo LAG-3

(8) FIG. 8 Cross-reactivity of rat anti-bovine LAG-3 antibody 2D8 with ovine T cells

BEST MODES FOR CARRYING OUT THE INVENTION

(9) Hereinbelow, the present invention will be described in detail.

(10) The present invention provides an anti-LAG-3 antibody comprising (a) a light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLLDSDGNTY (SEQ ID NO: 16). CDR2 having the amino acid sequence of SVS and CDR3 having the amino acid sequence of MQATHVPFT (SEQ ID NO: 17) and the light chain constant region of an antibody of an animal other than rat; and (b) a heavy chain comprising a heavy chain variable region containing CDR1 having the amino acid sequence of GFDFDTYP (SEQ ID NO: 18), CDR2 having the amino acid sequence of ITIKTHNYAT (SEQ ID NO: 19) and CDR3 having the amino acid sequence of NREDFDY (SEQ ID NO: 20) and the heavy chain constant region of an antibody of an animal other than rat.

(11) CDR1, CDR2 and CDR3 in the light chain variable region (VL) of rat anti-bovine LAG-3 antibody 2D8 are a region consisting of the amino acid sequence of QSLLDSDGNTY (SEQ ID NO: 16), a region consisting of the amino acid sequence of SVS and a region consisting of the amino acid sequence of MQATHVPFT (SEQ ID NO: 17), respectively (see FIG. 1).

(12) Further, CDR1, CDR2 and CDR3 in the heavy chain variable region (VH) of rat anti-bovine LAG-3 antibody 2D8 are a region consisting of the amino acid sequence of GFDFDTYP (SEQ ID NO: 18), a region consisting of the amino acid sequence of ITIKTHNYAT (SEQ ID NO: 19) and a region consisting of the amino acid sequence of NREDFDY (SEQ ID NO: 20), respectively (see FIG. 1).

(13) In the amino acid sequences of QSLLDSDGNTY (SEQ ID NO: 16), SVS and MQATHVPFT (SEQ ID NO: 17), as well as the amino acid sequences of GFDFDTYP (SEQ ID NO: 18). ITIKTHNYAT (SEQ ID NO: 19) and NREDFDY (SEQ ID NO: 20), one, two, three, four or five amino acids may be deleted, substituted or added. Even when such mutations have been introduced, the resulting amino acid sequences are capable of having the function as a CDR of VL or a CDR of VH of the LAG-3 antibody.

(14) As used herein, the term “antibody” is a concept encompassing not only full-length antibodies but also antibodies of smaller molecular sizes such as Fab, F(ab)′.sub.2, ScFv, Diabody, V.sub.H, V.sub.L, Sc(Fv).sub.2. Bispecific sc(Fv).sub.2, Minibody, scFv-Fc monomer and scFv-Fc dimer.

(15) In the anti-LAG-3 antibody of the present invention, VL and VH thereof may be derived from rat. For example, VL thereof may be the VL of a rat anti-bovine LAG-3 antibody, and VH thereof may be the VH of the rat anti-bovine LAG-3 antibody.

(16) The amino acid sequence of the VL and the amino acid sequence of the VH of the rat anti-bovine LAG-3 antibody are shown in SEQ ID NOS: 1 and 2, respectively. The amino acid sequences as shown in SEQ ID NOS: 1 and 2 may have deletion(s), substitution(s) or addition(s) of one or several (e.g., up to five, about 10 at the most) amino acids. Even when such mutations have been introduced, the resulting amino acid sequences are capable of having the function as VL or VH of the LAG-3 antibody.

(17) There are two types of immunoglobulin light chain, which are called Kappa chain (K) and Lambda chain (λ). In the anti-LAG-3 antibody of the present invention, the light chain constant region (CL) of an antibody of an animal other than rat may have the amino acid sequence of the constant region of either Kappa chain or Lambda chain. However, the relative abundance of Lambda chain is higher in bovine, ovine, feline, canine and equine, and that of Kappa chain is higher in mouse, rat, human and porcine. Since a chain with a higher relative abundance is considered to be preferable, a bovine, ovine, feline, canine or equine antibody preferably has the amino acid sequence of the constant region of Lambda chain whereas a mouse, rat, human or porcine antibody preferably has the amino acid sequence of the constant region of Kappa chain (κ).

(18) The heavy chain constant region (CH) of an antibody of an animal other than rat may have the amino acid sequence of the constant region of an immunoglobulin equivalent to human IgG4. Immunoglobulin heavy chain is classified into γ chain, μ chain, α chain, δ chain and ε chain depending on the difference in constant region. According to the type of heavy chain present, five classes (isotypes) of immunoglobulin are formed; they are IgG, IgM, IgA, IgD and IgE.

(19) Immunoglobulin G (IgG) accounts for 70-75% of human immunoglobulins and is the most abundantly found monomeric antibody in plasma. IgG has a four-chain structure consisting of two light chains and two heavy chains. Human IgG1, IgG2 and IgG4 have molecular weights of about 146,000, whereas human IgG3 has a long hinge region that connects Fab region and Fc region and has a larger molecular weight of 170,000. Human IgG1 accounts for about 65%, human IgG2 about 25%, human IgG3 about 7%, and human IgG4 about 3% of human IgG. They are uniformly distributed inside and outside of blood vessels. Having a strong affinity for Fc receptors and complement factors on effector cell surfaces, human IgG1 induces antibody-dependent cell cytotoxicity (ADCC) and also activates complements to induce complement-dependent cell cytotoxicity (CDC). Human IgG2 and IgG4 are low at ADCC and CDC activities because their affinity for Fc receptors and complement factors is low.

(20) Immunoglobulin M (IgM), which accounts for about 10% of human immunoglobulins, is a pentameric antibody consisting of five basic four-chain structures joined together. It has a molecular weight of 970.000. Usually occurring only in blood, IgM is produced against infectious microorganisms and takes charge of early stage immunity.

(21) Immunoglobulin A (IgA) accounts for 10-15% of human immunoglobulins. It has a molecular weight of 160,000. Secreted IgA is a dimeric antibody consisting of two IgA molecules joined together. IgA1 is found in serum, nasal discharge, saliva and breast milk. In intestinal juice, IgA2 is found abundantly.

(22) Immunoglobulin D (IgD) is a monomeric antibody accounting for no more than 1% of human immunoglobulins. IgD is found on B cell surfaces and involved in induction of antibody production.

(23) Immunoglobulin E (IgE) is a monomeric antibody that occurs in an extremely small amount, accounting for only 0.001% or less of human immunoglobulins. Immunoglobulin E is considered to be involved in immune response to parasites but in advanced countries where parasites are rare, IgE is largely involved in bronchial asthma and allergy among other things.

(24) With respect to canine, sequences of IgG-A (equivalent to human IgG2), IgG-B (equivalent to human IgG1), IgG-C (equivalent to human IgG3) and IgG-D (equivalent to human IgG4) have been identified as the heavy chain of IgG. In the antibody of the present invention, an IgG's heavy chain constant region with neither ADCC activity nor CDC activity is preferable (IgG4 in human). In the case where the constant region of an immunoglobulin equivalent to human IgG4 has not been identified, one may use a constant region that has lost both ADCC activity and CDC activity as a result of introducing mutations into the relevant region of an immunoglobulin equivalent to human IgG4.

(25) With respect to bovine, sequences of IgG1, IgG2 and IgG3 have been identified as the heavy chain of IgG. In the antibody of the present invention, an IgG's heavy chain constant region with neither ADCC activity nor CDC activity is preferable (IgG4 in human). Although the constant region of wild-type human IgG1 has ADCC activity and CDC activity, it is known that these activities can be reduced by introducing amino acid substitutions or deletions into specific sites. In bovine, the constant region of an immunoglobulin equivalent to human IgG4 has not been identified, so mutations may be added at the relevant region of an immunoglobulin equivalent to human IgG1 and the resultant constant region then used. As one example, the amino acid sequence of the CH of a bovine antibody (IgG1 chain. GenBank: X62916) having mutations introduced into CH2 domain and a nucleotide sequence for such amino acid sequence (after codon optimization) are shown in SEQ ID NOS: 4 and 8, respectively.

(26) When an animal other than rat is canine, an anti-LAG-3 antibody is more preferable in which (i) the CL of a canine antibody has the amino acid sequence of the constant region of Lambda chain and (ii) the CH of the canine antibody has the amino acid sequence of the constant region of an immunoglobulin equivalent to human IgG4.

(27) When an animal other than rat is bovine, an anti-LAG-3 antibody is more preferable in which (i) the CL of a bovine antibody has the amino acid sequence of the constant region of Lambda chain and (ii) the CH of the bovine antibody has mutations introduced thereinto that reduce ADCC activity and/or CDC activity.

(28) The anti-LAG-3 antibody of the present invention encompasses rat-bovine chimeric antibodies, bovinized antibodies and complete bovine-type antibodies. However, animals are not limited to bovine and may be exemplified by human, canine, porcine, simian, mouse, feline, equine, goat, sheep, water buffalo, rabbit, hamster, guinea pig and the like.

(29) For example, the anti-LAG-3 antibody of the present invention may be an anti-LAG-3 antibody in which the CL of a bovine antibody has the amino acid sequence as shown in SEQ ID NO: 3 and the CH of the bovine antibody has the amino acid sequence as shown in SEQ ID NO: 4.

(30) The amino acid sequences as shown in SEQ ID NOS: 3 and 4 may have deletion(s), substitution(s) or addition(s) of one or several (e.g., up to five, about 10 at the most) amino acids. Even when such mutations have been introduced, the resulting amino acid sequences are capable of having the function as CL or CH of the LAG-3 antibody.

(31) The anti-LAG-3 antibody of the present invention may have a four-chain structure comprising two light chains and two heavy chains.

(32) The anti-LAG-3 antibody of the present invention may be prepared as described below. Briefly, an artificial gene is synthesized which comprises (i) the identified variable region sequences of a rat anti-bovine LAG-3 antibody and (ii) the constant region sequences of an antibody of an animal other than rat (e.g., bovine) (preferably, an immunoglobulin equivalent to human IgG1, in which mutations have been introduced into the relevant region to reduce ADCC activity and/or CDC activity). The resultant gene is inserted into a vector (e.g., plasmid), which is then introduced into a host cell (e.g., mammal cell such as CHO cell). The host cell is cultured, and the antibody of interest is collected from the resultant culture.

(33) The amino acid sequence and the nucleotide sequence of the VL of the rat anti-bovine LAG-3 antibody identified by the present inventors are shown in SEQ ID NOS: 1 and 5, respectively. Further, the nucleotide sequence after codon optimization is shown in SEQ ID NO: 11.

(34) The amino acid sequence and the nucleotide sequence of the VH of the rat anti-bovine LAG-3 antibody identified by the present inventors are shown in SEQ ID NOS: 2 and 6, respectively. Further, the nucleotide sequence after codon optimization is shown in SEQ ID NO: 12.

(35) The amino acid sequence and the nucleotide sequence of the CL (Lambda chain, GenBank: X62917) of a bovine antibody are shown in SEQ ID NOS: 3 and 7, respectively. Further, the nucleotide sequence after codon optimization is shown in SEQ ID NO: 13.

(36) The amino acid sequence and the nucleotide sequence (after codon optimization) of the CH (IgG1 chain, modified from GenBank: X62916) of the bovine antibody are shown in SEQ ID NOS: 4 and 8, respectively.

(37) Further. SEQ ID NO: 9 shows the amino acid sequence of a chimeric light chain consisting of the VL of the rat anti-bovine LAG-3 antibody and the CL (Lambda chain, GenBank: X62917) of the bovine antibody. The nucleotide sequence (after codon optimization) of the chimeric light chain consisting of the VL of the rat anti-bovine LAG-3 antibody and the CL (Lambda chain, GenBank: X62917) of the bovine antibody is shown in SEQ ID NO: 14.

(38) SEQ ID NO: 10 shows the amino acid sequence of a chimeric heavy chain consisting of the VH of the rat anti-bovine LAG-3 antibody and the CH (IgG1 chain, modified from GenBank: X62916) of the bovine antibody. The nucleotide sequence (after codon optimization) of the chimeric heavy chain consisting of the VH of the rat anti-bovine LAG-3 antibody and the CH (IgG1 chain, modified from GenBank: X62916) of the bovine antibody is shown in SEQ ID NO: 15.

(39) Amino acid sequences and nucleotide sequences of CLs and CHs for various animals other than rat may be obtained from known databases for use in the present invention.

(40) Amino acid sequences and nucleotide sequences of bovine CLs and CHs are summarized in the table below.

(41) Table.

(42) TABLE-US-00001 TABLE GenBank Accession Species Ig Domain  Nucleotide Sequence Amino Acid Sequence No.  IMGT Database Reference Bovine Bovine Ig IgG1 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGAGTTC ASTTAPKVYPLSGGGDKSSSTVTLGC X62916 http://www. Symons DB (Scientific heavy chain variant 1 TTGCTGCGGGGACAAGTCCAGCTCCACCGTGACCCTGG LVSSYMPEPVTVTWNSGALKSGVHTFP imgt.org/ et al., J Name Bos constant GCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTGACC AVLQSSGLYSLSSMVTVPGSTSGQTFT IMGT Immogenet, taurus) region GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA CNVAHPASSTKVDKAVDPTCKPSPCD repertoire/ 14, 273- CH1~CH3 CACCTTCCCGGCTGTCCTTCAGTCCTCCGGGCTGTACT CCPPPELPGGPSVFIFPPKPKDTLTISG index.php? 283 (1987) CTCTCAGCAGCATGGTGACCGTGCCCGGCAGCACCTCA TPEVTCVVVDVGHDDPEVKFSWFVDD section= PMID: GGACAGACCTTCACCTGCAACGTAGCCCACCCGGCCAG VEVNTATTKPREEQFNSTYRVVSALRI LocusGenes& 3141517 CAGCACCAAGGTGGACAAGGCTGTTGATCCCACATGCA QHQDWTGGKEFKCKVHNEGLPAPTVRT repertoire= Symons DB AACCATCACCCTGTGACTGTTGCCCACCCCCTGAGTC ISRTKGPAREPQVYVLAPPQEELSKST genetable& et al., CCCGGAGGACCCTCTGTCTTCATCTTCCCACCGAAACC VSLTCMVTSFYPDYIAVEWQRNGQPES species= Mol. CAAGGACACCCTCACAATCTGGGGAACGCCCGAGGTCA EDKYGTTPPQLDADSSYFLYSKLRVDR bovine&group= Immunol., CGTGTGTGGTGGTGGACGTGGGCCACGATGACCCCGAG NSWQEGDTYTCVVMHEALHNHYTQKS IGHC 841-850 GTGAAGTTCTCCTGGTTCGTGGACGACGTGGAGGTAAA TSKSAGK* (1989). CACAGCCACGAGGAAGCCGAGAGAGGAGCAGTTCAACA (SEQ ID NO: 21) PMID: GCACCTACCGCGTGGTCAGCGCCCTGCGCATCCAGCAC 2513487 CAGGACTGGACTGGAGGAAAGGAGTTCAAGTGCAAGGT Kacskovica CCACAACGAAGGCCTCCCGGCCCCCATCGTGAGGACCA L. and TCTCCAGGACCAAAGGGCCGGCCCGGGAGCCGCAGGT Butler JE., GTATGTCCTGGCCCCACCCCAGGAAGAGCTCAGCAAAA Mol. GCAGGGTCAGCCTCACCTGCATGGTCACCAGCTTCTAC Immunol., CCAGACTACATCGCCGTGGAGTGGCAGAGAAAGGGGCA 33, 189- GCCTGAGTCGGAGGACAAGTACGGCACCACCCCGCCC 195 (1996). CAGCTGGACGCCGACAGCTCCTACTTCCTGTACAGCAA PMID: GCTCAGGGTGGACAGGAACAGCTGGCAGGAAGGAGAC 3649440 ACCTACACGTGTGTGGTGATGCACGAGGCCCTGCACAA Rabbani H. TCACTACACGCAGAAGTCCACCTCTAAGTCTGCGGGTA et al., AATGA Immuno- (SEQ ID NO: 29) genetics, IgG1 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGAGTTC ASTTAPKVYPLSSCCGDKSSSTVTLGC X16701 46. 326- variant 2 TTGCTGCGGGGACAAGTCCAGCTCCACCGTGACCCTGG LVSSYMPEPVTVTWNSGALKSGVHTFP (M25278) 331 (1997). GCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTGACC AVLQSSGLYSLSSMVTVPGSTSGQTFT PMID: GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA CNVAHPASSTKVDKAVDPTCKPSPCD 9218535 CACCTTCCCGGCCGTCCTTCAGTCCTCCGGGCTGTACT CCPPPELPGGPSVFIFPPKPKDTLTISG Saini S.S. CTCTCAGCAGCATGGTGACCGTGCCCGGCAGCACCTCA TPEVTCVVVDVGHDDPEVKFSWFVDD et al., GGACAGACCTTCACCTGCAACGTAGCCCACCCGGCCAG VEVNTATTKPREEQFNSTYRVVSALRI Scand J. CAGCACCAAGGTGGACAAGGCTGTTGATCCCACATGCA QHQDWTGGKEFKCKVHNEGLPAPIVRT Immunol. AACCATCACCCTGTGACTGTTGCCCACCCCCTGAGCTC ISRTKGPAREPQVYVLAPPQEELSKST 65, 32-8 CCCGGAGGACCCTCTGTCTTCATCTTCCCACCGAAACC VSLTCMVTSFYPDYIAVEWQRNGQPES (2007). CAAGGACACCCTCACAATCTCGGGAACGCCCGAGGTCA EDKYGTTPPQLDADSSYFLYSKLRVDR PMID: CGTGTGTGGTGGTGGACGTGGGCCACGATGACCCCGAG NSWQEGDTYTCVVMHEALHNHYTQKS 17212764 GTGAAGTTCTCCTGGTTCGTGGACGACGTGGAGGTAAA TSKSAGK* CACAGCCACGACGAAGCCGAGAGAGGAGCAGTTCAACA (SEQ ID NO: 22) GCACCTACCGCGTGGTCAGCGCCCTGCGCATCCAGCAC CAGGACTGGACTGGAGGAAAGGAGTTCAAGTGCAAGGT CCACAACGAAGGCCTCCCGGCCCCCATCGTGAGGACCA TCTCCAGGACCAAAGGGCCGGCCCGGGAGCCGCAGGT GTATGTCCTGGCCCCACCCCAGGAAGAGCTCAGCAAAA GCACGGTCAGCCTCACCTGCATGGTCACCAGCTTCTAC CCAGACTACATCGCCGTGGAGTGGCAGAGAAACGGGCA GCCTGAGTCGGAGGACAAGTACGGCACGACCCCGCCC CAGCTGGACGCCGACAGCTCCTACTTCCTGTACAGCAA GCTCAGGGTGGACAGGAACAGCTGGCAGGAAGGAGAC ACCTACACGTGTGTGGTGATGCACGAGGCCCTGCACAA TCACTACACGCAGAAGTCCACCTCTAAGTCTGCGGGTA AATGA (SEQ ID NO: 30) IgG1 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGAGTTC ASTTAPKVYPLSSCCGDKSSSTVTLGC 582409 variant 3 TTGCTGCGGGGACAAGTCCAGCTCCACCGTGACCCTGG LVSSYMPEPVTVTWNSGALKSGVHTFP GCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTGACC AVLQSSGLYSLSSMVTVPGSTSGTQTF GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA TCNVAHPASSTKVDKAVDPRCKTTCD CACCTTCCCGGCCGTCCTTCAGTCCTCCGGGCTCTACT CCPPPELPGGPSVFIFPPKPKDTLTISG CTCTCAGCAGCATGGTGACCGTGCCCGGCAGCACCTCA TPEVTCVVVDVGHDDPEVKFSWFVDD GGAACCCAGACCTTCACCTGCAACGTAGCCCACCCGGC VEVNTATTKPREEQFNSTYRVVSALRI CAGCAGCACCAAGGTGGACAAGGCTGTTGATCCCAGAT QHQDWTGGKEFKCKVHNEGLPAPIVRT GCAAAACAACCTGTGACTGTTGCCCACCGCCTGAGCTC ISRTKGPAREPQVYVLAPPQEELSKST CCTGGAGGACCCTCTGTCTTCATCTTCCCACCGAAACC VSLTCMVTSFYPDYIAVEWQRNGQPES CAAGGACACCCTCACAATCTCGGGAACGCCCGAGGTCA EDKYGTTPPQLDADGSYFLYSRLRVDR CGTGTGTGGTGGTGGACGTGGGCCACGATGACCCCGAG NSWQEGDTYTCVVMHEALHNHYTQKS GTGAAGTTCTCCTGGTTCGTGGACGACGTGGAGGTAAA TSKSAGK* CACAGCCACGACGAAGCCGAGAGAGGAGCAGTTCAACA (SEQ ID NO: 23) GCACCTACCGCGTGGTCAGCGCCCTGCGCATCCAGCAC CAGGACTGGACTGGAGGAAAGGAGTTCAAGTGCAAGGT CCACAACGAAGGCCTCCCAGCCCCCATCGTGAGGACCA TCTCCAGGACCAAAGGGCCGGCCCGGGAGCCGCAGGT GTATGTCCTGGCCCCACCCCAGGAAGAGCTCAGCAAAA GCACGGTCAGCCTCACCTGCATGGTCACCAGCTTCTAC CCAGACTACATCGCCGTGGAGTGGCAGAGAAATGGGCA GCCTGAGTCAGAGGACAAGTACGGCACGACCCCTCCCC AGCTGGACGCCGACGGCTCCTACTTCCTGTACAGCAGG CTCAGGGTGGACAGGAACAGCTGGCAGGAAGGAGACA CCTACACGTGTGTGGTGATGCACGAGGCCCTGCACAAT CACTACACGCAGAAGTCCACCTCTAAGTCTGCGGGTAA ATGA (SEQ ID NO: 31) IgG2 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGGCATC ASTTAPKVYPLASSCGDTSSSTVTLGC 582407 variant 1 CAGCTGCGGAGACACATCCAGCTCCACCGTGACCCTGG LVSSYMPEPVTVTWNSGALKSGVHTFP GCTGCCTGGTGTCCAGCTACATGCCCGAGCCGGTGACC AVLQSSGLYSLSSMVTVPASSSGQTFT GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA CNVAHPASSTKVDKAVGVSIDCSKCHN CACCTTCCCGGCTGTCCTTCAGTCCTCCGGGCTCTACT QPCVREPSVFIFPPKPKDTLMITGTPEV CTCTCAGCAGCATGGTGACCGTGCCCGCCAGCAGCTCA TCVVVNVGHDNPEVQFSWFVDDVEVH GGACAGACCTTCACCTGCAACGTAGCCCACCCGGCCAG TARSKPREEQFNSTYRVVSALPIQHQD CAGCACCAAGGTGGACAAGGCTGTTGGGGTCTCCATTG WTGGKEFKCKVNNKGLSAPIVRISRSK ACTGCTCCAAGTGTCATAACCAGCCTTGCGTGAGGGAA GPAREPQVYVLDPPKEELSKSTLSVTC CCATCTGTCTTCATCTTCCCACCGAAACCCAAAGACAC MVTGFYPEDVAVEWQRNRQTESEDKY CCTGATGATCACAGGAACGCCCGAGGTCACGTGTGTGG RTTPPQLDTDRSYFLYSKLRVDRNSWQ TGGTGAACGTGGGCCACGATAACCCCGAGGTGCAGTTC EGDAYTCVVMHEALHNHYMQKSTSKS TCCTGGTTCGTGGATGACGTGGAGGTGCACACGGCCAG AGK* GTCGAAGCCAAGAGAGGAGCAGTTCAACAGCACGTACC (SEQ ID NO: 24) GCGTGGTCAGCGCCCTGCCCATCCAGCACCAGGACTGG ACTGGAGGAAAGGAGTTCAAGTGCAAGGTCAACAACAA AGGCCTCTGGGCCCCCATCGTGAGGATCATCTCCAGGA GCAAAGGGCCGGCCCGGGAGCCGCAGGTGTATGTCCT GGACCCACCCAAGGAAGAGCTCAGCAAAAGCACGCTCA GCGTCACCTGCATGGTCACCGGCTTCTACCCAGAAGAT GTAGCCGTGGAGTGGCAGAGAAACCGGCAGACTGAGTC GGAGGACAAGTACCGCACGACCCCGCCCCAGCTGGAC ACCGACCGCTCCTACTTCCGTACAGCAAGCTCAGGGT GGACAGGAACAGCTGGCAGGAAGGAGACGCCTACACG TGTGTGGTGATGCACGAGGCCCTGCACAATCACTACAT GCAGAAGTCCACCTCTAAGTCTGCGGGTAAATGA (SEQ ID NO: 32) IgG2 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGAGTTC ASTTAPVKYPLSSCCGDKSSSGVTLGC X16702 variant 3 TTGCTGCGGGGACAAGTCCAGCTCGGGGGTGACCCTGG LVSSYPEPVTVTWNSGALKSGVHTFP (M25279) GCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTGACC AVLQSSGLYSLSSMVTVPASSSGTQTF GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA TCNVHAPASSTKVDKAVGVSSDCSKP CACCTTCCCGGCCGTCCTTCAGTCCTCCGGGCTCTACT NNQHCVREPSVFIFPPKPKDTLMITGTP CTCTCAGCAGCATGGTGACCGTGCCCGCCAGCAGCTCA EVTCVVVNVGHDNPEVQFSWFVDDVE GGAACCCAGACCTTCACCTGCAACGTAGCCCACCCGGC VHTARTKPREEQFNSTYRVVSALPIQH CAGCAGCACCAAGGTGGACAAGGCTGTTGGGGTCTCCA QDWTGGKEFKCKVNIKGLSASIVRIISRS GTGACTGCTCCAAGCCTAATAACCAGCATTGCGTGAGG KGPAREPQVYVLDPPKEELSKSTVSLT GAACCATCTGTCTTCATCTTCCCACCGAAACCCAAAGA CMVIGFYPEDVDVEWQRDRQTESEDKY CACCCTGATGATCACAGGAACGCCCGAGGTCACGTGTG RTTPPQLDADRSYFLYSKLRVDRNSWQ TGGTGGTGAACGTGGGCCACGATAACCCCGAGGTGCAG RGDTYTCVVMHEALHNHYMQKSTSKS TTCTCCTGGTTCGTGGACGACGTGGAGGTGCACACGGC AGK* CAGGACGAAGCCGAGAGAGGAGCAGTTCAACAGCACGT (SEQ ID NO: 26) ACCGCGTGGTCAGCGCCCTGCCCATCCAGCACCAGGAC TGGACTGGAGGAAAGGAGTTCAAGTGCAAGGTCAACAT CAAAGGCCTCTCGGCCTCCATCGTGAGGATCATCTCCA GGAGCAAAGGGCCGGCCCGGGAGCCGCAGGTGTATGT CCTGGACCCACCCAGGAAGAGCTCAGCAAAAGCACGG TCAGCCTCACCTGCATGGTCATCGGCTTCTACCCAGAA GATGTAGACGTGGAGTGGCAGAGAGACCGGCAGACTGA GTCGGAGGACAAGTACCGCACGACCCCGCCCCAGCTG GACGCCGACCGCTCCTACTTCCTGTACAGCAAGCTCAG GGTGGACAGGAACAGCTGGCAGAGAGGAGACACCTAC ACGTGTGTGGTGATGCACGAGGCCCTGCACAATCACTA CATGCAGAAGTCCACCTCTAAGTCTGCGGGTAAATGA (SEQ ID NO: 34) IgG3 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGGCATC ASTTAPKVYPLASSCGDTSSSTVTLGC U63638 variant 1 CAGCTGCGGAGACACATCCAGCTCCACCGTGACCCTGG LVSSYMPEPVTVTWNSGALKSGVHTFP GCTGCCTGGTCTCCAGCTACATGCCCGAGGGGGTGACC AVRQSSGLYSLSSMVTVPASSSETQTF GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA TCNVAHPASSTKVDKAVTARRPVPTTP CACCTTCCCGGCCGTCCGGCAGTCCTCTGGGCTGTACT KTTIPPGKPTTPKSEVEKTPCQCSKCP CTCTCAGCAGCATGGTGACTGTGCCCGCCAGCAGCTCA EPLGGLSVFIFPPKPKDTLTISGTPEVT GAAACCCAGACCTTCACCTGCAACGTAGCCCACCCGGC CVVVDVGQDDPEVQFSWFVDDVEVHT CAGCAGCACCAAGGTGGACAAGGCTGTCACTGCAAGGC ARTKPREEQFNSTYRVVSALRIQHQDW GTCCAGTCCCGACGACGCCAAAGACAACTATCCCTCCT LQGKEFKCKVNNKGLPAPIVRTISRTKG GGAAAACCCACAACCCCAAAGTCTGAAGTTGAAAAGAC QAREPQVYVLAPPREELSKSTLSLTCLI ACCCTGCCAGTGTTCCAAATGCCCAGAACCTCTGGGAG TGFYPEEIDVEWQRNGQPESEDKYHITT GACTGTCTGTCTTCATCTTCCCACCGAAACCCAAGGAC APQLDADGSYFLYSKLRVNKSSWQEG ACCCTCACAATCTCGGGAACGCCCGAGGTCAGGTGTGT DHYTCAVMHEALRNHYKEKSISRSPGK* GGTGGTGGACGTGGGCCAGGATGACCCCGAGGTGCAG (SEQ ID NO: 27) TTCTCCTGGTTCGTGGACGACGTGGAGGTGCACACGGC CAGGACGAAGCCGAGAGAGGAGCAGTTCAACAGCACCT ACCGCGTGGTCAGCGCCCTGCGCATCCAGCACCAGGA CTGGCTGCAGGGAAAGGAGTTCAAGTGCAAGGTCAACA ACAAAGGCCTCCCGGCCCCCATTGTGAGGACCATCTCC AGGACCAAAGGGCAGGCCCGGGAGCCGCAGGTGTATG TCCTGGCCCCACCCCGGGAAGAGCTCAGCAAAGCACG CTCAGCCTCACCTGCCTGATCACCGGTTTCTACCCAGA AGAGATAGACGTGGAGTGGCAGAGAAATGGGCAGCCTG AGTCGGAGGACAAGTACCACACGACCGCACCCCAGCTG GATGCTGACGGCTCCTACTTCCTGTACAGCAAGCTCAG GGTGAACAAGAGCAGCTGGCAGGAAGGAGACCACTACA CGTGTGCAGTGATGCACGAAGCTTTACGGAATCACTAC AAAGAGAAGTCCATCTCGAGGTCTCCGGGTAAATGA (SEQ ID NO: 35) IgG3 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGGCATC ASTTAPKVYPLASRCGDTSSSTVTLGC U63639 variant 2 CCGCTGCGGAGACACATCCAGCTCCACCGTGACCCTGG LVSSYMPEPVTVTWNSGALKSGVHTFP GCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTGACC AVLQSSGLYSLSSMVTVPASTSETQTF GTGACCTGGAACTCGGGTGCCCTGAAGAGTGGCGTGCA TCNVAHPASSTKVDKAVTARRPVPTTP CACCTTCCCGGCCGTCCTTCAGTCCTCCGGGCTGTACT KTTIPPGKPTTQESEVEKTPCQCSKCP CTCTCAGCAGCATGGTGACCGTGCCCGCCAGCACCTCA EPLGGLSVFIFPPKPKDTLTISGTPEVT GAAACCCAGACCTTCACCTGCAACGTAGCCCACCCGGC CVVVDVGQDDPEVQFSWFVDDVEVHT CAGCAGCACCAAGGTGGACAAGGCTGTCACTGCAAGGC ARTKPREEQFNSTYRVVSALRIQHQDW GTCCAGTCCCGACGACGCCAAAGACAACCATCCCTCCT LQGKEFKCKVNNKGLPAPIVRTISRTKG GGAAAACCCACAACCCAGGAGTCTGAAGTTGAAAAGAC QAREPQVYVLAPPPREELSKSTLSLTCLI ACCCTGCCAGTGTTCCAAATGCCCAGAACCTCTGGGAG TGFYPEEIDVEWQRNGQPESEDKYHTT GACTGTCTGTCTTGATCTTCCCACCGAAACCCAAGGAC APQLDADGSYFLYSRLRVNKSSWQEG ACCCTCACAATCTCGGGAACGCCCGAGGTCACGTGTGT DYYTCAVMHEALRNHYKEKSISRSPGK* GGTGGTGGACGTGGGCCAGGATGACCCCGAGGTGCAG (SEQ ID NO: 28) TTCTCCTGGTTGGTGGACGACGTGGAGGTGCACACGGC CAGGACGAAGCCGAGAGAGGAGCAGTTCAACAGCACCT ACCGCGTGGTCAGCGCCCTGCGCATCCAGCACCAGGA CTGGCTGCAGGGAAAGGAGTTCAAGTGCAAGGTCAACA ACAAAGGCCTCCCGGCCCCCATTGTGAGGACCATCTCC AGGACCAAAGGGCAGGCCCGGGAGCCGCAGGTGTATG TCCTGGCCCCACCCCGGGAAGAGCTCAGCAAAAGCACG CTCAGCCTCACCTGCCTGATCACCGGTTTCTACCCAGA AGAGATAGACGTGGAGTGGCAGAGAAATGGGCAGCCTG AGTCGGAGGACAAGTACCACACGACCGCACCCCAGCTG GATGCTGACGGCTCCTACTTCCTGTACAGCAGGCTCAG GGTGAACAAGAGCAGCTGGCAGGAAGGAGACCACTACA CGTGTGCAGTGATGCATGAAGCTTTACGGAATCACTAC AAAGAGAAGTCCATCTCGAGGTCTCCGGGTAAATGA (SEQ ID NO: 36) Bovine Ig Ig lambda CAGCCCAAGTCCCCACCCTCGGTCACCCTGTTCCCGCC QPKSPPSVTLFPPSTEELNGNKATLVC X62917 Not Chen L. light chain CTCCACGGAGGAGCTCAACGGCAACAAGGCCACCCTG LISDFYPGSVTVVWKADGSTITRNVETT registered et al., constant GTGTGTCTCATCAGCGACTTCTACCCGGGTAGCGTGAC RASKQSNSKYAASSYLSLTSSDWKSKG Vet, region (CL) CGTGGTCTGGAAGGCAGACGGCAGCACCATCACCCGCA SYSCEVTHEGSTVTKTVKPSECS* Immunol. ACGTGGAGACCACCCGGGCCTCCAAACAGAGCAACAG (SEQ ID NO: 3) Immuno- CAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGAGCA pathol. GCGACTGGAAATCGAAAGGCAGTTACAGCTGCGAGGTC 124, 284- ACGCACGAGGGGAGCACCGTGACGAAGACAGTGAAGC 294 (2008). CCTCAGAGTGTTCTTAG PMID: (SEQ ID NO: 7) 18538861

(43) TABLE-US-00002 TABLE GenBank Nucleotide Amino Acid Accession IMGT Species Ig Domain Sequence Sequence No. Database Reference Ovine Ovine Ig IgG1 GCCTCAACAACACCCC ASTTPPKVYPLTSCC X69797 http:// Dufour V. (Scien- heavy  CGAAAGTCTACCCTCT GDTSSSIVTLGCLVSS www.imgt.org/ et al., J.  tific chain GACTTCTTGCTGCGGG YMPEPVTVTWNSGA IMGTreper- Immunol., Name: constant GACACGTCCAGCTCCA LTSGVHTFPAILQSSG toire/ 156, 2163- Ovis  region TCGTGACCCTGGGCTG LYSLSSVVTVPASTSG index.php? 2170  aries) (CH1.sup.~CH3) CCTGGTCTCCAGCTATA AQTFICNVAHPASST section= (1996). TGCCCGAGCCGGTGAC KVDKRVEPGCPDPC LocusGenes& PMID: CGTGACCTGGAACTCT KHCRCPPPELPGGPS repertoire= 8690905 GGTGCCCTGACCAGCG VFIFPPKPKDTLTISGT genetable& GCGTGCACACCTTCCC PEVTCVVVDVGQDD species= GGCCATCCTGCAGTCC PEVQFSWFVDNVEV sheep&group= TCCGGGCTCTACTCTC RTARTKPREEQFNSTF IGHC TCAGCAGCGTGGTGAC RVVSALPIQHQDWT CGTGCCGGCCAGCACC GGKEFKCKVHNEAL TCAGGAGCCCAGACCT PAPIVRTISRTKGQAR TCATCTGCAACGTAGC EPQVYVLAPPQEELS CCACCCGGCCAGCAGC KSTLSVTCLVTGFYP ACCAAGGTGGACAAG DYIAVEWQKNGQPE CGTGTTGAGCCCGGAT SEDKYGTTTSQLDAD GCCCGGACCCATGCAA GSYFLYSRLRVDKNS ACATTGCCGATGCCCA WQEGDTYACVVMH CCCCCTGAGCTCCCCG EALHNHYTQKSISKP GAGGACCGTCTGTCTT PGK* (SEQ ID  CATCTTCCCACCGAAA NO: 37) CCCAAGGACACCCTTA CAATCTCTGGAACGCC CGAGGTCACGTGTGTG GTGGTGGACGTGGGCC AGGATGACCCCGAGGT GCAGTTCTCCTGGTTC GTGGACAACGTGGAG GTGCGCACGGCCAGG ACAAAGCCGAGAGAG GAGCAGTTCAACAGC ACCTTCCGCGTGGTCA GCGCCCTGCCCATCCA GCACCAAGACTGGACT GGAGGAAAGGAGTTC AAGTGCAAGGTCCAC AACGAAGCCCTCCCGG CCCCCATCGTGAGGAC CATCTCCAGGACCAAA GGGCAGGCCCGGGAG CCGCAGGTGTACGTCC TGGCCCCACCCCAGGA AGAGCTCAGCAAAAG CACGCTCAGCGTCACC TGCCTGGTCACCGGCT TCTACCCAGACTACAT CGCCGTGGAGTGGCA GAAAAATGGGCAGCCT GAGTCGGAGGACAAG TACGGCACGACCACAT CCCAGCTGGACGCCGA CGGCTCCTACTTCCTGT ACAGCAGGCTCAGGG TGGACAAGAACAGCT GGCAAGAAGGAGACA CCTACGCGTGTGTGGT GATGCACGAGGCTCTG CACAACCACTACACAC AGAAGTCGATCTCTAA GCCTCCGGGTAAATGA (SEQ ID NO: 38) IgG2 GCCTCCACCACAGCCC ASTTAPKVYPLTSCC X70983 Clarkson  CGAAAGTCTACCCTCT GDTSSSSSIVTLGCLV C. A. et GACTTCTTGCTGCGGG SSYMPEPVTVTWNS al., Mol. GACACGTCCAGCTCCA GALTSGVHTFPAILQS Immunol.,  GCTCCATCGTGACCCT SGLYSLSSVVTVPAST 30, 1195- GGGCTGCCTGGTCTCC SGAQTFICNVAHPASS 1204  AGCTATATGCCCGAGC AKVDKRVGISSDYSK (1993). CGGTGACCGTGACCTG CSKPPCVSRPSVFIFP PMID: GAACTCTGGTGCCCTG PKPKDSLMITGTPEV 8413324 ACCAGCGGCGTGCAC TCVVVDVGQGDPEV ACCTTCCCGGCCATCC QFSWFVDNVEVRTA TGCAGTCCTCCGGGCT RTKPREEQFNSTFRV CTACTCTCTCAGCAGC VSALPIQHDHWTGG GTGGTGACCGTGCCGG KEFKCKVHSKGLPAP CCAGCACCTCAGGAGC IVRTISRAKGQAREP CCAGACCTTCATCTGC QVYVLAPPQEELSKS AACGTAGCCCACCCGG TLSVTCLVTGFYPDYI CCAGCAGCGCCAAGG AVEWQRARQPESED TGGACAAGCGTGTTGG KYGTTTSQLDADGS GATCTCCAGTGACTAC YFLYSRLRVDKSSWQ TCCAAGTGTTCTAAAC RGDTYACVVMHEAL CGCCTTGCGTGAGCCG HNHYTQKSISKPPGK* ACCGTCTGTCTTCATCT (SEQ ID NO: 39) TCCCCCCGAAACCCAA GGACAGCCTCATGATC ACAGGAACGCCCGAG GTCACGTGTGTGGTGG TGGACGTGGGCCAGG GTGACCCCGAGGTGCA GTTCTCCTGGTTCGTG GACAACGTGGAGGTG CGCACGGCCAGGACA AAGCCGAGAGAGGAG CAGTTCAACAGCACCT TCCGCGTGGTCAGCGC CCTGCCCATCCAGCAC GACCACTGGACTGGA GGAAAGGAGTTCAAG TGCAAGGTCCACAGCA AAGGCCTCCCGGCCCC CATCGTGAGGACCATC TCCAGGGCCAAAGGG CAGGCCCGGGAGCCG CAGGTGTACGTCCTGG CCCCACCCCAGGAAG AGCTCAGCAAAAGCA CGCTCAGCGTCACCTG CCTGGTCACCGGCTTC TACCCAGACTACATCG CCGTGGAGTGGCAGA GAGCGCGGCAGCCTG AGTCGGAGGACAAGT ACGGCACGACCACATC CCAGCTGGACGCCGAC GGCTCCTACTTCCTGT ACAGCAGGCTCAGGG TGGACAAGAGCAGCT GGCAAAGAGGAGACA CCTACGCGTGTGTGGT GATGCACGAGGCTCTG CACAACCACTACACAC AGAAGTCGATCTCTAA GCCTCCGGGTAAATGA (SEQ ID NO: 40) Ovine Ig Ig CCATCCGTCTTCCTCTT PSVFLFKPSEEQLRTG X54110 Not Jenne C. N.  light  kappa CAAACCATCTGAGGAA TVSVVCLVNDFYPKD registered et al., chain (CK) CAGCTGAGGACCGGA INVKVKVDGVTQNS Dev. Comp. constant ACTGTCTCTGTCGTGT NFQNSFTDQDSKKST Immunol.  region GCTTGGTGAATGATTT YSLSSTLTLSSSEYQS 30(1-2),  CTACCCCAAAGATATC HNAYACEVSHKSLPT 165-174 AATGTCAAGGTGAAAG ALVKSFNKNEC* (2006).  TGGATGGGGTTACCCA (SEQ ID NO: 41) PMID: GAACAGCAACTTCCAG 16083958 AACAGCTTCACAGACC AGGACAGCAAGAAAA GCACCTACAGCCTCAG CAGCACCCTGACACTG TCCAGCTCAGAGTACC AGAGCCATAACGCCTA TGCGTGTGAGGTCAGC CACAAGAGCCTGCCCA CCGCCCTCGTCAAGAG CTTCAATAAGAATGAA TGTTAG (SEQ ID  NO: 42) Ig GGTCAGCCCAAGTCCG GQPKSAPSVTLFPPST AY734681 lambda  CACCCTCGGTCACCCT EELSTNKATVVCLIN (CL) GTTCCCGCCTTCCACG DFYPGSVNVVWKAD GAGGAGCTCAGTACCA GSTINQNVKTTQASK ACAAGGCCACCGTGGT QSNSKYAASSYLTLT GTGTCTCATCAACGAC GSEWKSKSSYTCEVT TTCTACCCGGGTAGCG HEGSTVTKTVKPSEC TGAACGTGGTCTGGAA S* (SEQ ID NO: 43) GGCAGATGGCAGCACC ATCAATCAGAACGTGA AGACCACCCAGGCCTC CAAACAGAGCAACAG CAAGTACGCGGCCAGC AGCTACCTGACCCTGA CGGGCAGCGAGTGGA AGTCTAAGAGCAGTTA CACCTGCGAGGTCACG CACGAGGGGAGCACC GTGACGAAGACAGTG AAGCCCTCAGAGTGTT CTTAG (SEQ ID  NO: 44)

(44) TABLE-US-00003 TABLE GenBank Nucleotide Amino Acid  Accession IMGT Species Ig Domain Sequence Sequence No. Database Reference Ovine Ovine Ig IgG1 GCCTCAACAACACCCC ASTTPPKVYPLTSCC X69797 http:// Dufour V.  (Scien- heavy  CGAAAGTCTACCCTCT GDTSSSIVTLGCLVSS www.imgt.org/ et al., J. tific chain GACTTCTTGCTGCGGG YMPEPVTVTWNSGA IMGTreper- Immunol., Name: constant GACACGTCCAGCTCCA LTSGVHTFPAILQSSG toire/ 156,  Ovis  region TCGTGACCCTGGGCTG LYSLSSVVTVPASTSG index.php? 2163-2170 aries) (CH1.sup.~CH3) CCTGGTCTCCAGCTATA AQTFICNVAHPASST section= (1996).  TGCCCGAGCCGGTGAC KVDKRVEPGCPDPC LocusGenes& PMID: CGTGACCTGGAACTCT KHCRCPPPELPGGPS repertoire= 8690905 GGTGCCCTGACCAGCG VFIFPPKPKDTLTISGT genetable& GCGTGCACACCTTCCC PEVTCVVVDVGQDD species= GGCCATCCTGCAGTCC PEVQFSWFVDNVEV sheep&group= TCCGGGCTCTACTCTC RTARTKPREEQFNSTF IGHC TCAGCAGCGTGGTGAC RVVSALPIQHQDWT CGTGCCGGCCAGCACC GGKEFKCKVHNEAL TCAGGAGCCCAGACCT PAPIVRTISRTKGQAR TCATCTGCAACGTAGC EPQVYVLAPPQEELS CCACCCGGCCAGCAGC KSTLSVTCLVTGFYP ACCAAGGTGGACAAG DYIAVEWQKNGQPE CGTGTTGAGCCCGGAT SEDKYGTTTSQLDAD GCCCGGACCCATGCAA GSYFLYSRLRVDKNS ACATTGCCGATGCCCA WQEGDTYACVVMH CCCCCTGAGCTCCCCG EALHNHYTQKSISKP GAGGACCGTCTGTCTT PGK* (SEQ ID  CATCTTCCCACCGAAA NO: 37) CCCAAGGACACCCTTA CAATCTCTGGAACGCC CGAGGTCACGTGTGTG GTGGTGGACGTGGGCC AGGATGACCCCGAGGT GCAGTTCTCCTGGTTC GTGGACAACGTGGAG GTGCGCACGGCCAGG ACAAAGCCGAGAGAG GAGCAGTTCAACAGC ACCTTCCGCGTGGTCA GCGCCCTGCCCATCCA GCACCAAGACTGGACT GGAGGAAAGGAGTTC AAGTGCAAGGTCCAC AACGAAGCCCTCCCGG CCCCCATCGTGAGGAC CATCTCCAGGACCAAA GGGCAGGCCCGGGAG CCGCAGGTGTACGTCC TGGCCCCACCCCAGGA AGAGCTCAGCAAAAG CACGCTCAGCGTCACC TGCCTGGTCACCGGCT TCTACCCAGACTACAT CGCCGTGGAGTGGCA GAAAAATGGGCAGCCT GAGTCGGAGGACAAG TACGGCACGACCACAT CCCAGCTGGACGCCGA CGGCTCCTACTTCCTGT ACAGCAGGCTCAGGG TGGACAAGAACAGCT GGCAAGAAGGAGACA CCTACGCGTGTGTGGT GATGCACGAGGCTCTG CACAACCACTACACAC AGAAGTCGATCTCTAA GCCTCCGGGTAAATGA (SEQ ID NO: 38) IgG2 GCCTCCACCACAGCCC ASTTAPKVYPLTSCC X70983 Clarkson   CGAAAGTCTACCCTCT GDTSSSSSIVTLGCLV C. A. et  GACTTCTTGCTGCGGG SSYMPEPVTVTWNS al., Mol. GACACGTCCAGCTCCA GALTSGVHTFPAILQS Immunol., GCTCCATCGTGACCCT SGLYSLSSVVTVPAST 30, GGGCTGCCTGGTCTCC SGAQTFICNVAHPASS 1195-1204 AGCTATATGCCCGAGC AKVDKRVGISSDYSK (1993).  CGGTGACCGTGACCTG CSKPPCVSRPSVFIFP PMID: GAACTCTGGTGCCCTG PKPKDSLMITGTPEV 8413324 ACCAGCGGCGTGCAC TCVVVDVGQGDPEV ACCTTCCCGGCCATCC QFSWFVDNVEVRTA TGCAGTCCTCCGGGCT RTKPREEQFNSTFRV CTACTCTCTCAGCAGC VSALPIQHDHWTGG GTGGTGACCGTGCCGG KEFKCKVHSKGLPAP CCAGCACCTCAGGAGC IVRTISRAKGQAREP CCAGACCTTCATCTGC QVYVLAPPQEELSKS AACGTAGCCCACCCGG TLSVTCLVTGFYPDYI CCAGCAGCGCCAAGG AVEWQRARQPESED TGGACAAGCGTGTTGG KYGTTTSQLDADGS GATCTCCAGTGACTAC YFLYSRLRVDKSSWQ TCCAAGTGTTCTAAAC RGDTYACVVMHEAL CGCCTTGCGTGAGCCG HNHYTQKSISKPPGK* ACCGTCTGTCTTCATCT (SEQ ID NO: 39) TCCCCCCGAAACCCAA GGACAGCCTCATGATC ACAGGAACGCCCGAG GTCACGTGTGTGGTGG TGGACGTGGGCCAGG GTGACCCCGAGGTGCA GTTCTCCTGGTTCGTG GACAACGTGGAGGTG CGCACGGCCAGGACA AAGCCGAGAGAGGAG CAGTTCAACAGCACCT TCCGCGTGGTCAGCGC CCTGCCCATCCAGCAC GACCACTGGACTGGA GGAAAGGAGTTCAAG TGCAAGGTCCACAGCA AAGGCCTCCCGGCCCC CATCGTGAGGACCATC TCCAGGGCCAAAGGG CAGGCCCGGGAGCCG CAGGTGTACGTCCTGG CCCCACCCCAGGAAG AGCTCAGCAAAAGCA CGCTCAGCGTCACCTG CCTGGTCACCGGCTTC TACCCAGACTACATCG CCGTGGAGTGGCAGA GAGCGCGGCAGCCTG AGTCGGAGGACAAGT ACGGCACGACCACATC CCAGCTGGACGCCGAC GGCTCCTACTTCCTGT ACAGCAGGCTCAGGG TGGACAAGAGCAGCT GGCAAAGAGGAGACA CCTACGCGTGTGTGGT GATGCACGAGGCTCTG CACAACCACTACACAC AGAAGTCGATCTCTAA GCCTCCGGGTAAATGA (SEQ ID NO: 40) Ovine Ig Ig CCATCCGTCTTCCTCTT PSVFLFKPSEEQLRTG X54110 Not Jenne C. N.  light  kappa CAAACCATCTGAGGAA TVSVVCLVNDFYPKD registered et al., chain (CK) CAGCTGAGGACCGGA INVKVKVDGVTQNS Dev. Comp. constant ACTGTCTCTGTCGTGT NFQNSFTDQDSKKST Immunol.  region GCTTGGTGAATGATTT YSLSSTLTLSSSEYQS 30 (1-2), CTACCCCAAAGATATC HNAYACEVSHKSLPT 165-174 AATGTCAAGGTGAAAG ALVKSFNKNEC* (2006).  TGGATGGGGTTACCCA (SEQ ID NO: 41) PMID: GAACAGCAACTTCCAG 16083958 AACAGCTTCACAGACC AGGACAGCAAGAAAA GCACCTACAGCCTCAG CAGCACCCTGACACTG TCCAGCTCAGAGTACC AGAGCCATAACGCCTA TGCGTGTGAGGTCAGC CACAAGAGCCTGCCCA CCGCCCTCGTCAAGAG CTTCAATAAGAATGAA TGTTAG (SEQ ID  NO: 42) Ig GGTCAGCCCAAGTCCG GQPKSAPSVTLFPPST AY734681 lambda CACCCTCGGTCACCCT EELSTNKATVVCLIN (CL) GTTCCCGCCTTCCACG DFYPGSVNVVWKAD GAGGAGCTCAGTACCA GSTINQNVKTTQASK ACAAGGCCACCGTGGT QSNSKYAASSYLTLT GTGTCTCATCAACGAC GSEWKSKSSYTCEVT TTCTACCCGGGTAGCG HEGSTVTKTVKPSEC TGAACGTGGTCTGGAA S* (SEQ ID NO: 43) GGCAGATGGCAGCACC ATCAATCAGAACGTGA AGACCACCCAGGCCTC CAAACAGAGCAACAG CAAGTACGCGGCCAGC AGCTACCTGACCCTGA CGGGCAGCGAGTGGA AGTCTAAGAGCAGTTA CACCTGCGAGGTCACG CACGAGGGGAGCACC GTGACGAAGACAGTG AAGCCCTCAGAGTGTT CTTAG (SEQ ID  NO: 44)

(45) TABLE-US-00004 TABLE GenBank Nucleotide  Amino Acid Accession IMGT Species Ig Domain Sequence Sequence No. Database Reference Ovine Ovine Ig IgG1 GCCTCAACAACACCCC ASTTPPKVYPLTSCC X69797 http:// Dufour V. (Scien- heavy CGAAAGTCTACCCTCT GDTSSSIVTLGCLVSS www.imgt.org/ et al., tific chain GACTTCTTGCTGCGGG YMPEPVTVTWNSGA IMGTreper- J. Immunol., Name: constant GACACGTCCAGCTCCA LTSGVHTFPAILQSSG toire/ 156,  Ovis region TCGTGACCCTGGGCTG LYSLSSVVTVPASTSG index.php? 2163-2170 aries) (CH1.sup.~CH3) CCTGGTCTCCAGCTATA AQTFICNVAHPASST section= (1996).  TGCCCGAGCCGGTGAC KVDKRVEPGCPDPC LocusGenes& PMID: CGTGACCTGGAACTCT KHCRCPPPELPGGPS repertoire= 8690905 GGTGCCCTGACCAGCG VFIFPPKPKDTLTISGT genetable& GCGTGCACACCTTCCC PEVTCVVVDVGQDD species= GGCCATCCTGCAGTCC PEVQFSWFVDNVEV sheep&group= TCCGGGCTCTACTCTC RTARTKPREEQFNSTF IGHC TCAGCAGCGTGGTGAC RVVSALPIQHQDWT CGTGCCGGCCAGCACC GGKEFKCKVHNEAL TCAGGAGCCCAGACCT PAPIVRTISRTKGQAR TCATCTGCAACGTAGC EPQVYVLAPPQEELS CCACCCGGCCAGCAGC KSTLSVTCLVTGFYP ACCAAGGTGGACAAG DYIAVEWQKNGQPE CGTGTTGAGCCCGGAT SEDKYGTTTSQLDAD GCCCGGACCCATGCAA GSYFLYSRLRVDKNS ACATTGCCGATGCCCA WQEGDTYACVVMH CCCCCTGAGCTCCCCG EALHNHYTQKSISKP GAGGACCGTCTGTCTT PGK* (SEQ ID  CATCTTCCCACCGAAA NO: 37) CCCAGGACACCCTTA CAATCTCTGGAACGCC CGAGGTCACGTGTGTG GTGGTGGACGTGGGCC AGGATGACCCCGAGGT GCAGTTCTCCTGGTTC GTGGACAACGTGGAG GTGCGCACGGCCAGG ACAAAGCCGAGAGAG GAGCAGTTCAACAGC GAGCAGTTCAACAGC ACCTTCCGCGTGGTCA GCGCCCTGCCCATCCA GCACCAAGACTGGACT GGAGGAAAGGAGTTC AAGTGCAAGGTCCAC AACGAAGCCCTCCCGG CCCCCATCGTGAGGAC CATCTCCAGGACCAAA GGGCAGGCCCGGGAG CCGCAGGTGTACGTCC TGGCCCCACCCCAGGA AGAGCTCAGCAAAAG CACGCTCAGCGTCACC TGCCTGGTCACCGGCT TCTACCCAGACTACAT CGCCGTGGAGTGGCA GAAAAATGGGCAGCCT GAGTCGGAGGACAAG TACGGCACGACCACAT CCCAGCTGGACGCCGA CGGCTCCTACTTCCTGT ACAGCAGGCTCAGGG TGGACAAGAACAGCT GGCAAGAAGGAGACA CCTACGCGTGTGTGGT GATGCACGAGGCTCTG CACAACCACTACACAC AGAAGTCGATCTCTAA GCCTCCGGGTAATGA (SEQ ID NO: 38) IgG2 GCCTCCACCACAGCCC ASTTAPKVYPLTSCC X70983 Clarkson   CGAAAGTCTACCCTCT GDTSSSSSIVTLGCLV C. A. GACTTCTTGCTGCGGG SSYMPEPVTVTWNS et al., Mol. GACACGTCCAGCTCCA GALTSGVHTFPAILQS Immunol.,  GCTCCATCGTGACCCT SGLYSLSSVVTVPAST 30, GGGCTGCCTGGTCTCC SGAQTFICNVAHPASS 1195-1204 AGCTATATGCCCGAGC AKVDKRVGISSDYSK (1993).  CGGTGACCGTGACCTG CSKPPCVSRPSVFIFP PMID: GAACTCTGGTGCCCTG PKPKDSLMITGTPEV 8413324 ACCAGCGGCGTGCAC TCVVVDVGQGDPEV ACCTTCCCGGCCATCC QFSWFVDNVEVRTA TGCAGTCCTCCGGGCT RTPREEQFNSTFRV CTACTCTCTCAGCAGC VSALPIQHDHWTGG GTGGTGACCGTGCCGG KEFKCKVHSKGLPAP CCAGCACCTCAGGAGC IVRTISRAKGQAREP CCAGACCTTCATCTGC QVYVLAPPQEELSKS AACGTAGCCCACCCGG TLSVTCLVTGFYPDYI CCAGCAGCGCCAAGG AVEWQRARQPESED TGGACAAGCGTGTTGG KYGTTTSQLDADGS GATCTCCAGTGACTAC YFLYSRLRVDKSSWQ TCCAAGTGTTCTAAAC RGDTYACVVMHEAL CGCCTTGCGTGAGCCG HNHYTQKSISKPPGK* ACCGTCTGTCTTCATCT (SEQ ID NO: 39) TCCCCCCGAAACCCAA GGACAGCCTCATGATC ACAGGAACGCCCGAG GTCACGTGTGTGGTGG TGGACGTGGGCCAGG GTGACCCCGAGGTGCA GTTCTCCTGTTCGTG GACAACGTGGAGGTG CGCACGGCCAGGACA AAGCCGAGAGAGGAG CAGTTCAACAGCACCT TCCGCGTGGTCAGCGC CCTGCCCATCCAGCAC GACCACTGGACTGGA GGAAAGGAGTTCAAG TGCAAGGTCCACAGCA AAGGCCTCCCGGCCCC CATCGTGAGGACCATC TCCAGGGCCAAAGGG CAGGCCCGGGAGCCG CAGGTGTACGTCCTGG CCCCACCCCAGGAAG AGCTCAGCAAAAGCA CGCTCAGCGTCACCTG CCTGGTCACCGGCTTC TACCCAGACTACATCG CCGTGGAGTGGCAGA GAGCGCGGCAGCCTG AGTCGGAGGACAAGT ACGGCACGACCACATC CCAGCTGGACGCCGAC GGCTCCTACTTCCTGT ACAGCAGGCTCAGGG TGGACAAGAGCAGCT GGCAAAGAGGAGACA CCTACGCGTGTGTGGT GATGCACGAGGCTCTG CACAACCACTACACAC AGAAGTCGATCTCTAA GCCTCCGGGTAAATGA (SEQ ID NO: 40) Ovine Ig Ig CCATCCGTCTTCCTCTT PSVFLFKPSEEQLRTG X54110 Not Jenne C. N. light kappa CAAACCATCTGAGGAA TVSVVCLVNDFYPKD registered et al., chain (CK) CAGCTGAGGACCGGA INVKVKVDGVTQNS Immunol. constant ACTGTCTCTGTCGTGT NFQNSFTDQDSKKST 30 (1-2), region GCTTGGTGAATGATTT YSLSSTLTLSSSEYQS 165-174 CTACCCCAAAGATATC HNAYACEVSHKSLPT (2006).  AATGTCAAGGTGAAAG ALVKSFNKNEC* PMID: TGGATGGGGTTACCCA (SEQ ID NO: 41) 16083958 GAACAGCAACTTCCAG AACAGCTTCACAGACC AGGACAGCAAGAAAA GCACCTACAGCCTCAG CAGCACCCTGACACTG TCCAGCTCAGAGTACC AGAGCCATAACGCCTA TGCGTGTGAGGTCAGC CACAAGAGCCTGCCCA CCGCCCTCGTCAAGAG CTTCAATAAGAATGAA TGTTAG (SEQ ID NO: 42) Ig GGTCAGCCCAAGTCCG GQPKSAPSVTLFPPST AY734681 lambda CACCCTCGGTCACCCT EELSTNKATVVCLIN (CL) GTTCCCGCCTTCCACG DFYPGSVNVVWKAD GAGGAGCTCAGTACCA GSTINQNVKTTQASK ACAAGGCCACCGTGGT QSNSKYAASSYLTLT GTGTCTCATCAACGAC GSEWKSKSSYTCEVT TTCTACCCGGGTAGCG HEGSTVTKTVKPSEC TGAACGTGGTCTGGAA S* (SEQ ID NO: 43) GGCAGATGGCAGCACC ATCAATCAGAACGTGA AGACCACCCAGGCCTC CAAACAGAGCAACAG CAAGTACGCGGCCAGC AGCTACCTGACCCTGA CGGGCAGCGAGTGGA AGTCTAAGAGCAGTTA CACCTGCGAGGTCACG CACGAGGGGAGCACC GTGACGAAGACAGTG AAGCCCTCAGAGTGTT CTTAG (SEQ ID NO: 44)

(46) TABLE-US-00005 TABLE GenBank Nucleotide  Amino Acid  Accession IMGT Species Ig Domain Sequence Sequence No. Database Reference Human Human Ig IgG4 GAGTCCAAATATGGTC ESKYGPPCPSCPAPEF K01316 http:// Ellison J.  (Scien- heavy  variant CCCCATGCCCATCATG LGGPSVFLFPPKPKD www.imgt.org/ et al., DNA,  tific chain 1 CCCAGCACCTGAGTTC TLMISRTPEVTCVVV IMGTreper- 1, 11-18 Name: constant CTGGGGGGACCATCAG DVSQEDPEVQFNWY toire/ (1981). PMID: Homo region TCTTCCTGTTCCCCCC VDGVEVHNAKTKPR index.php? 6299662 sapiens) (CH1.sup.~CH3) AAAACCCAAGGACAC EEQFNSTYRVVSVLT section= TCTCATGATCTCCCGG VLHQDWLNGKEYKC LocusGenes& ACCCCTGAGGTCACGT KVSNKGLPSSIEKTIS repertoire= GCGTGGTGGTGGACGT KAKGQPREPQVYTLP genetable& GAGCCAGGAAGACCC PSQEEMTKNQVSLTC species= CGAGGTCCAGTTCAAC LVKGFYPSDIAVEWE human&group= TGGTACGTGGATGGCG SNGQPENNYKTTPPV IGHC TGGAGGTGCATAATGC LDSDGSFFLYSRLTV CAAGACAAAGCCGCG DKSRWQEGNVFSCS GGAGGAGCAGTTCAA VMHEALHNHYTQKS CAGCACGTACCGTGTG LSLSLGK* (SEQ  GTCAGCGTCCTCACCG ID NO: 53) TCCTGCACCAGGACTG GCTGAACGGCAAGGA GTACAAGTGCAAGGTC TCCAACAAAGGCCTCC CGTCCTCCATCGAGAA AACCATCTCCAAAGCC AAAGGGCAGCCCCGA GAGCCACAGGTGTACA CCCTGCCCCCATCCCA GGAGGAGATGACCAA GAACCAGGTCAGCCTG ACCTGCCTGGTCAAAG GCTTCTACCCCAGCGA CATCGCCGTGGAGTGG GAGAGCAATGGGCAG CCGGAGAACAACTACA AGACCACGCCTCCCGT GCTGGACTCCGACGGC TCCTTCTTCCTCTACAG CAGGCTAACCGTGGAC AAGAGCAGGTGGCAG GAGGGGAATGTCTTCT CATGCTCCGTGATGCA TGAGGCTCTGCACAAC CACTACACACAGAAGA GCCTCTCCCTGTCTCT GGGTAAATGA (SEQ  ID NO: 54) IgG4 GAGTCCAAATATGGTC ESKYGPPCPSCPAPEF AJ001563 Brusco A.  variant CCCCGTGCCCATCATG LGGPSVFLFPPKPKD et al., 2 CCCAGCACCTGAGTTC TLMISRTPEVTCVVV Eur. J. CTGGGGGGACCATCAG DVSQEDPEVQFNWY Immunogenet., TCTTCCTGTTCCCCCC VDGVEVHNAKTKPR 25, 349-355 AAAACCCAAGGACAC EEQFNSTYRVVSVLT (1998). PMID: TCTCATGATCTCCCGG VVHQDWLNGKEYK 9805657 ACCCCTGAGGTCACGT CKVSNKGLPSSIEKTI GCGTGGTGGTGGACGT SKAKGQPREPQVYTL GAGCCAGGAAGACCC PPSQEEMTKNQVSLT CGAGGTCCAGTTCAAC CLVKGFYPSDIAVEW TGGTACGTGGATGGCG ESNGQPENNYKTTPP TGGAGGTGCATAATGC VLDSDGSFFLYSRLT CAAGACAAAGCCGCG VDKSRWQEGNVFSC GGAGGAGCAGTTCAA SVMHEALHNHYTQK CAGCACGTACCGTGTG SLSLSLGK* (SEQ  GTCAGCGTCCTCACCG ID NO: 55) TCGTGCACCAGGACTG GCTGAACGGCAAGGA GTACAAGTGCAAGGTC TCCAACAAAGGCCTCC CGTCCTCCATCGAGAA AACCATCTCCAAAGCC AAAGGGCAGCCCCGA GAGCCACAGGTGTACA CCCTGCCCCCATCCCA GGAGGAGATGACCAA GAACCAGGTCAGCCTG ACCTGCCTGGTCAAAG GCTTCTACCCCAGCGA CATCGCCGTGGAGTGG GAGAGCAATGGGCAG CCGGAGAACAACTACA AGACCACGCCTCCCGT GCTGGACTCCGACGGC TCCTTCTTCCTCTACAG CAGGCTAACCGTGGAC AAGAGCAGGTGGCAG GAGGGGAATGTCTTCT CATGCTCCGTGATGCA TGAGGCTCTGCACAAC CACTACACGCAGAAGA GCCTCTCCCTGTCTCT GGGTAAATGA (SEQ  ID NO: 56) IgG4 GCACCTGAGTTCCTGG APEFLGGPSVFLFPPK AJ001564 variant GGGGACCATCAGTCTT PKDTLMISRTPEVTC 3 CCTGTTCCCCCCAAAA VVVDVSQEDPEVQF CCCAAGGACACTCTCA NWYVDGVEVHNAK TGATCTCCCGGACCCC TKPREEQFNSTYRVV TGAGGTCACGTGCGTG SVLTVLHQDWLNGK GTGGTGGACGTGAGCC EYKCKVSNKGLPSSI AGGAAGACCCCGAGG EKTISKAKGQPREPQ TCCAGTTCAACTGGTA VYTLPPSQEEMTKNQ CGTGGATGGCGTGGAG VSLTCLVKGFYPSDIA GTGCATAATGCCAAGA VEWESNGQPENNYK CAAAGCCGCGGGAGG TTPPVLDSDGSFFLYS AGCAGTTCAACAGCAC KLTVDKSRWQEGNV GTACCGTGTGGTCAGC FSCSVMHEALHNHY GTCCTCACCGTCCTGC TQKSLSLSLGK* ACCAGGACTGGCTGA (SEQ ID NO: 57) ACGGCAAGGAGTACA AGTGCAAGGTCTCCAA CAAAGGCCTCCCGTCC TCCATCGAGAAAACCA TCTCCAAAGCCAAAGG GCAGCCCCGAGAGCC ACAGGTGTACACCCTG CCCCCATCCCAGGAGG AGATGACCAAGAACC AGGTCAGCCTGACCTG CCTGGTCAAAGGCTTC TACCCCAGCGACATCG CCGTGGAGTGGGAGA GCAATGGGCAGCCGG AGAACAACTACAAGA CCACGCCTCCCGTGCT GGACTCCGACGGCTCC TTCTTCCTCTACAGCA AGCTCACCGTGGACAA GAGCAGGTGGCAGGA GGGGAACGTCTTCTCA TGCTCCGTGATGCATG AGGCTCTGCACAACCA CTACACGCAGAAGAGC CTCTCCCTGTCTCTGG GTAAATGA (SEQ ID NO: 58) Human Ig Ig ACTGTGGCTGCACCAT TVAAPSVFIFPPSDEQ X96754 http:// None light  kappa CTGTCTTCATCTTCCCG LKSGTASVVCLLNNF www.imgt.org/ chain (CK) CCATCTGATGAGCAGT YPREAKVQWKVDN IMGTreper- constant TGAAATCTGGAACTGC ALQSGNSQESVTEQD toire/ region CTCTGTTGTGTGCCTG  SKDSTYSLSSTLTLSK index.php? CTGAATAACTTCTATCC ADYEKHKVYACEVT section= CAGAGAGGCCAAAGT HQGLSSPVTKSFNRG LocusGenes& ACAGTGGAAGGTGGAT EC* (SEQ ID  repertoire= AACGCCCTCCAATCGG NO: 59) genetable& GTAACTCCCAGGAGAG species= TGTCACAGAGCAGGA human&group= CAGCAAGGACAGCAC IGKC CTACAGCCTCAGCAGC ACCCTGACGCTGAGCA AAGCAGACTACGAGA AACACAAAGTCTACGC CTGCGAAGTCACCCAT CAGGGCCTGAGCTCGC CCGTCACAAAGAGCTT CAACAGGGGAGAGTG TTAG (SEQ ID  NO: 60)

(47) The amino acid sequences as shown in SEQ ID NOS: 3, 21-28, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 and 59 may have deletion(s), substitution(s) or addition(s) of one or several (e.g., up to five, about 10 at the most) amino acids. Even when such mutations have been introduced, the resulting amino acid sequences are capable of having the function as the constant region of Ig heavy chain or light chain.

(48) Although the constant region of wild-type human IgG1 has ADCC activity and CDC activity, it is known that these activities can be reduced by introducing amino acid substitutions and deletions into specific sites. In the case of animals other than human where the constant region of an immunoglobulin equivalent to human IgG4 has not been identified, mutations may be introduced into the relevant region of an immunoglobulin equivalent to human IgG1 so that the resultant constant region with reduced ADCC activity and CDC activity can be used.

(49) The present invention provides an artificial genetic DNA comprising (a′) a DNA encoding a light chain comprising a light chain variable region (VL) containing CDR1 having the amino acid sequence of QSLLDSDGNTY (SEQ ID NO: 16), CDR2 having the amino acid sequence of SVS and CDR3 having the amino acid sequence of MQATHVPFT (SEQ ID NO: 17) and the light chain constant region (CL) of an antibody of an animal other than rat: and (b′) a DNA encoding a heavy chain comprising a heavy chain variable region (VH) containing CDR1 having the amino acid sequence of GFDFDTYP (SEQ ID NO: 18). CDR2 having the amino acid sequence of ITIKTHNYAT (SEQ ID NO: 19) and CDR3 having the amino acid sequence of NREDFDY (SEQ ID NO: 20) and the heavy chain constant region (CH) of an antibody of an animal other than rat. The present invention also provides a DNA encoding a light chain comprising a VL containing CDR1 having the amino acid sequence of QSLLDSDGNTY (SEQ ID NO: 16), CDR2 having the amino acid sequence of SVS and CDR3 having the amino acid sequence of MQATHVPFT (SEQ ID NO: 17) and the CL of an antibody of an animal other than rat. Further, the present invention also provides a DNA encoding a heavy chain comprising a VH containing CDR1 having the amino acid sequence of GFDFDTYP (SEQ ID NO: 18), CDR2 having the amino acid sequence of ITIKTHNYAT (SEQ ID NO: 19) and CDR3 having the amino acid sequence of NREDFDY (SEQ ID NO: 20) and the CH of an antibody of an animal other than rat.

(50) For (a) a light chain comprising a light chain variable region containing CDR1 having the amino acid sequence of QSLLDSDGNTY (SEQ ID NO: 16), CDR2 having the amino acid sequence of SVS and CDR3 having the amino acid sequence of MQATHVPFT (SEQ ID NO: 17) and the light chain constant region of an antibody of an animal other than rat; and (b) a heavy chain comprising a heavy chain variable region containing CDR1 having the amino acid sequence of GFDFDTYP (SEQ ID NO: 18), CDR2 having the amino acid sequence of ITIKTHNYAT (SEQ ID NO: 19) and CDR3 having the amino acid sequence of NREDFDY (SEQ ID NO: 20) and the heavy chain constant region of an antibody of an animal other than rat, reference should be had to the foregoing description. The DNA of (a′) is a DNA (gene) encoding the light chain of (a); and the DNA of (b′) is a DNA (gene) encoding the heavy chain of (b). An artificial genetic DNA comprising the DNA of (a′) and the DNA of (′b) may be synthesized on commercial synthesizer. Restriction enzyme recognition sites. KOZAK sequences, poly-A addition signal sequences, promoter sequences, intron sequences or the like may be added to the artificial genetic DNA.

(51) The present invention also provides a vector comprising the above-mentioned artificial genetic DNA.

(52) As the vector, Escherichia coli-derived plasmids (e.g., pBR322, pBR325, pUC12 or pUC13); Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5 or pC194), yeast-derived plasmids (e.g., pSH19 or pSH15); bacteriophages such as λ phage; animal viruses such as retrovirus or vaccinia virus, or insect pathogen viruses such as baculovirus may be used. In the Examples described later. pDC6 (Japanese Patent No. 5704753, U.S. Pat. No. 9,096,878, EU Patent 2385115, Hong Kong (China) patent HK1163739 and Australia Patent 2009331326) was used.

(53) The vector may also comprise promoters, enhancers, splicing signals, poly-A addition signals, intron sequences, selection markers, SV40 replication origins, and so forth.

(54) The present invention also provides a host cell transformed by the above vector. It is possible to prepare the anti-LAG-3 antibody of the invention by culturing the host cell and collecting the antibody of interest from the resultant culture. Therefore, the present invention also provides a method of preparing an antibody, comprising culturing the above-described host cell and collecting the anti-LAG-3 antibody of the invention from the culture. In the method of the present invention for preparing an antibody, a vector incorporating an artificial genetic DNA comprising a DNA encoding the light chain and a DNA encoding the heavy chain may be transfected into a host cell. Alternatively, a vector incorporating a DNA encoding the light chain and a vector incorporating a DNA encoding the heavy chain may be co-transfected into a host cell.

(55) Examples of the host cell include, but are not limited to, bacterial cells (such as Escherichia bacteria. Bacillus bacteria or Bacillus subtilis), fungal cells (such as yeast or Aspergillus), insect cells (such as S2 cells or Sf cells), animal cells (such as CHO cells, COS cells, HeLa cells. C127 cells, 3T3 cells, BHK cells or HEK 293 cells) and plant cells. Among these. CHO-DG44 cell (CHO-DG44(dfhr.sup.−/−)) which is a dihydrofolate reductase deficient cell is preferable.

(56) Introduction of a recombinant vector into a host cell may be performed by the methods disclosed in Molecular Cloning 2nd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989 (e.g., the calcium phosphate method, the DEAE-dextran method, transfection, microinjection, lipofection, electroporation, transduction, scrape loading, the shotgun method, etc.) or by infection.

(57) The resultant transformant may be cultured in a medium, followed by collection of the anti-LAG-3 antibody of the present invention from the culture. When the antibody is secreted into the medium, the medium may be recovered, followed by isolation and purification of the antibody from the medium. When the antibody is produced within the transformed cells, the cells may be lysed, followed by isolation and purification of the antibody from the cell lysate.

(58) Examples of the medium include, but are not limited to. OptiCHO medium. Dynamis medium, CD CHO medium, ActiCHO medium, FortiCHO medium, Ex-Cell CD CHO medium, BalanCD CHO medium, ProCHO 5 medium and Cellvento CHO-100 medium.

(59) The pH of the medium varies depending on the cell to be cultured. Generally, a pH range from 6.8 to 7.6 is used; mostly, a pH range from 7.0 to 7.4 is appropriate.

(60) When the cell to be cultured is CHO cells, culture may be performed by methods known to those skilled in the art. For example, it is usually possible to perform culturing in a gas-phase atmosphere having a CO.sub.2 concentration of 0-40%, preferably 2-10%, at 30-39° C., preferably around 37° C.

(61) The appropriate period of culture is usually from one day to three months, preferably from one day to three weeks.

(62) Isolation and purification of the antibody may be performed by known methods. Known isolation/purification methods which may be used in the present invention include, but are not limited to, methods using difference in solubility (such as salting-out or solvent precipitation); methods using difference in molecular weight (such as dialysis, ultrafiltration, gel filtration or SDS-polyacrylamide gel electrophoresis); methods using difference in electric charge (such as ion exchange chromatography); methods using specific affinity (such as affinity chromatography); methods using difference in hydrophobicity (such as reversed phase high performance liquid chromatography); and methods using difference in isoelectric point (such as isoelectric focusing).

(63) The anti-LAG-3 antibody of the present invention may be used as an antibody drug for animals or human. Therefore, the present invention provides a pharmaceutical composition comprising the above-described anti-LAG-3 antibody as an active ingredient.

(64) The pharmaceutical composition of the present invention may be used for prevention and/or treatment of cancers and/or infections. Examples of cancers and/or infections include, but are not limited to, neoplastic diseases (e.g., malignant melanoma, lung cancer, gastric cancer, renal cancer, breast cancer, bladder cancer, esophageal cancer, ovarian cancer and the like), leukemia, Johne's disease, anaplasmosis, bacterial mastitis, mycotic mastitis, mycoplasma infections (such as mycoplasma mastitis, mycoplasma pneumonia or the like), tuberculosis, Theileria orientalis infection, cryptosporidiosis, coccidiosis, trypanosomiasis and leishmaniasis.

(65) The anti-LAG-3 antibody of the present invention may be dissolved in buffers such as PBS, physiological saline or sterile water, optionally filter-sterilized with a filter or the like, and then administered to animal subjects (including human) by injection. To the solution of this antibody, additives (such as coloring agents, emulsifiers, suspending agents, surfactants, solubilizers, stabilizers, preservatives, antioxidants, buffers, isotonizing agents, pH adjusters and the like) may be added. As routes of administration, intravenous, intramuscular, intraperitoneal, subcutaneous or intradermal administration and the like may be selected. Transnasal or oral administration may also be used.

(66) The dose and the number of times and frequency of administration of the anti-LAG-3 antibody of the present invention may vary depending on the symptoms, age and body weight of the animal subject, the method of administration, the dosage form and so on. For example, 0.1-100 mg/kg body weight, preferably 1-10 mg/kg body weight, per adult animal may usually be administered at least once at such a frequency that enables confirmation of the desired effect.

(67) While the pharmaceutical composition of the present invention may be used alone, it may be used in combination with surgical operations, radiation therapies, other immunotherapies such as cancer vaccine, or molecular target drugs. Synergistic effect can be expected from such combinations.

EXAMPLES

(68) Hereinbelow, the present invention will be described in more detail with reference to the following Examples. However, the present invention is not limited to these Examples.

[Example 1] Establishment of Rat-Bovine Chimeric Anti-Bovine LAG-3 Antibody

(69) 1. Introduction

(70) Lymphocyte activation gene 3 (LAG-3), an immunoinhibitory receptor, was identified as a molecule closely related to CD4. Recently, it has been elucidated that LAG-3 is involved in immunosuppression in chronic infections and tumors. In the subject Example, for the purpose of establishing a novel therapy for bovine infections, the present inventors have prepared a chimeric antibody gene by linking the variable region genes of rat anti-bovine LAG-3 monoclonal antibody 2D8 capable of inhibiting the binding of bovine LAG-3 and MHC class II to the constant region genes of bovine immunoglobulins (bovine IgG1 and Igλ; however, mutations have been introduced into the putative binding sites for Fcγ receptors in CH2 domain of bovine IgG1 to inhibit ADCC activity; see FIG. 1 for amino acid numbers and mutations: 247 E.fwdarw.P, 248 L.fwdarw.V, 249 P.fwdarw.A, 250 G.fwdarw.deletion, 344 A.fwdarw.S, 345 P.fwdarw.S; Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y. Murata S, Ohashi K. Immunology, 142(4):551-561; August 2014). This chimeric antibody gene was introduced into Chinese hamster ovary cells (CHO cells). By culturing/proliferating the resultant cells, the present inventors have obtained a rat-bovine chimeric anti-bovine LAG-3 antibody ch2D8 and confirmed its binding inhibitory activity and biological activities.

(71) 2. Materials, Methods and Experimental Results

(72) 2.1. Construction of Bovine LAG-3 Expressing Cells

(73) The nucleotide sequence of the full length cDNA molecule of bovine LAG-3 gene (GenBank accession number AB608099; Shirai T, Konnai S, Ikebuchi R, Okagawa T, Sunden Y, Onuma M, Murata S. Ohashi K. Vet. Immunol. Immunopathol, 144(3-4):462-467, Dec. 15, 2011) was determined. Based on the resultant genetic information, bovine LAG-3 expressing cells were prepared. First, for preparing a bovine LAG-3 expressing plasmid, PCR was performed using a synthesized bovine peripheral blood mononuclear cell (PBMC)-derived cDNA as a template and designed primers having BglII and EcoRI recognition sites on the 5′ side (boLAG-3-EGFP F and R). The PCR products were digested with BglII (Takara) and EcoRI (Takara), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into pEGFP-N2 vector (Clontech) treated with restriction enzymes in the same manner. The resultant expression plasmid of interest was extracted with QIAGEN Plasmid Midi kit (Qiagen) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pEGFP-N2-boLAG-3.

(74) TABLE-US-00006 Primer (boLAG-3-EGFP F): (SEQ ID NO: 61) GAAAGATCTATGCTGTGGGAGGCTTGGTT  Primer (boLAG-3-EGFP R): (SEQ ID NO: 62) CCGGAATTCGGGTTGCTCTGGCTGCAGCT

(75) Bovine LAG-3 expressing cells were prepared by the procedures described below. First, COS-7 cells at a density of 5×10.sup.4 cells/cm.sup.2 were subcultured in 6-well plates, and then cultured overnight in RPMI 1640 medium (Sigma-Aldrich) containing 10% inactivated fetal bovine serum (Cell Culture Technologies), penicillin 200 U/ml, streptomycin 200 μg/ml and 0.01% L-glutamine (Life Technologies) at 37° C. in the presence of 5% CO.sub.2. Subsequently, the pEGFP-N2-boLAG-3 or pEGFP-N2 (negative control) was introduced into COS-7 cells at 0.4 μg/cm.sup.2 using Lipofectamine 2000 (Invitrogen). The cells were cultured for 48 hours (bovine LAG-3-EGFP expressing cells). In order to confirm the expression of bovine LAG-3 in the thus prepared expressing cells, intracellular localization of EGFP was visualized with an inverted confocal laser microscope LSM700 (ZEISS).

(76) 2.2. Construction of Soluble Bovine LAG-3

(77) Bovine LAG-3-Ig expressing plasmid was constructed by the procedures described below. In order to amplify the signal peptide and the extracellular region of bovine LAG-3 (GenBank accession number AB608099), primers were designed that had NheI and NsiI recognition sites added on the 5′ side (boLAG-3-Ig F and R). PCR was performed using a synthesized bovine PBMC-derived cDNA as a template. The PCR products were digested with NheI (Takara) and NsiI (Takara), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into pCXN2.1-Rabbit IgG1 Fc vector (Niwa H, Yamamura K, Miyazaki J. Gene, 108(2):193-199; Dec. 15, 1991; kindly provided by Dr. T Yokomizo, Juntendo University Graduate School of Medicine, and modified in the inventors' laboratory) treated with restriction enzymes in the same manner. The expression plasmid was purified with FastGene Xpress Plasmid PLUS Kit (NIPPON Genetics) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pCXN2.1-boLAG-3-Ig.

(78) TABLE-US-00007 Primer (boLAG-3-EGFP F): (SEQ ID NO: 63) CTAGCTAGCCGCCCACCATGCTGTGGGAGGCTTGGTT Primer (boLAG-3-EGFP R): (SEQ ID NO: 64) TGCATGCATCAGAACAGCTAGGTTGTACG

(79) Soluble bovine LAG-3-Ig expressing cells were prepared by the procedures described below. Briefly, 30 μg of pCXN2.1-boLAG-3-Ig was introduced into 7.5×10.sup.7 Expi293F cells (Life Technologies) using Expifectamine (Life Technologies). After 7-day culture under shaking, the culture supernatant was collected. The recombinant protein was purified from the supernatant using Ab-Capcher Extra (ProteNova). After purification, the buffer was exchanged with PBS (pH 7.4) using PD MiniTrap G-25 (GE Healthcare). The resultant protein was stored at −30° C. until use in experiments (bovine LAG-3-Ig). The concentration of the purified bovine LAG-3-Ig was measured using Rabbit IgG ELISA Quantitation Set (Bethyl). For each washing operation in ELISA, Auto Plate Washer BIO WASHER 50 (DS Pharma Biomedical) was used. Absorbance was measured with Microplate Reader MTP-650FA (Corona Electric).

(80) 2.3. Preparation of Rat Anti-Bovine LAG-3 Monoclonal Antibody Producing Cells

(81) A peptide chain was synthesized from a part of the extracellular region of bovine LAG-3 (amino acid numbers 71 to 99; amino acid sequence: GSAAPTPRGPGPRRYTVLRLAPGGLRIGK (SEQ ID NO: 72)) by adding a cysteine residue to the NH.sub.2 terminus, and was linked to keyhole limpet hemocyanin (a carrier protein). An emulsion of this peptide chain and TiterMax Gold Adjuvant (Sigma-Aldrich) was prepared. Rat was immunized in the footpad with the resulting emulsion. Subsequently, hybridomas were established by the iliac lymph node method to thereby obtain rat anti-bovine LAG-3 monoclonal antibody producing hybridoma 2D8. With respect to the method of establishment of rat anti-bovine LAG-3 monoclonal antibody, details are disclosed in the following non-patent document (Okagawa T, Konnai S, Nishimori A. Ikebuchi R. Mizorogi S, Nagata R, Kawaji S. Tanaka S, Kagawa Y, Murata S, Mori Y. Ohashi K. Infect. Immun., 84(1):77-89; Oct. 19, 2015).

(82) 2.4. Preparation of Rat-Bovine Chimeric Anti-Bovine LAG-3 Antibody Expressing Vector

(83) Rat-bovine chimeric anti-bovine LAG-3 antibody ch2D8 was established by fusing the antibody constant regions of bovine IgG1 and Igλ with rat anti-bovine LAG-3 antibody 2D8 being used as an antibody variable region.

(84) First, the genes of heavy chain and light chain variable regions were identified by RACE from a hybridoma that would produce rat anti-bovine LAG-3 antibody 2D8. Subsequently, a gene sequence was prepared in which the heavy chain and the light chain variable regions of rat anti-bovine LAG-3 antibody 2D8 were linked to known constant regions of bovine IgG1 (heavy chain; modified from GenBank Accession number X62916) and bovine Ig. (light chain; GenBank Accession number X62917), respectively, and codon optimization was carried out [SEQ ID NOS: 9 and 10 (amino acid sequences). SEQ ID NOS: 14 and 15 (nucleotide sequences after codon optimization)]. It should be noted that in order to suppress the ADCC activity of bovine IgG1, mutations were added to the putative binding sites of Fcγ receptors in CH2 domain (See FIG. 1 for amino acid numbers and mutations: 247 E.fwdarw.P, 248 L.fwdarw.V, 249 P.fwdarw.A, 250 G.fwdarw.deletion, 344 A.fwdarw.S, 345 P.fwdarw.S; Ikebuchi R, Konnai S. Okagawa T. Yokoyama K, Nakajima C. Suzuki Y, Murata S, Ohashi K. Immunology, 142(4):551-561; August 2014). Then, the gene was artificially synthesized in such a manner that NotI recognition sequence, KOZAK sequence, chimeric antibody light chain sequence, poly-A addition signal sequence (PABGH), promoter sequence (PCMV), SacI recognition sequence, intron sequence (INRBG), KOZAK sequence, chimeric antibody heavy chain sequence and XbaI recognition sequence would be located in this order. The synthesized gene strand was digested with NotI (Takara) and XbaI (Takara), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into the cloning site (Noll and XbaI restriction enzyme recognition sequences downstream of PCMV and between INRBG and PABGH) of expression plasmid pDC6 (kindly provided by Prof. S. Suzuki, Hokkaido University Research Center for Zoonosis Control) treated with restriction enzymes in the same manner (FIG. 2). The resultant plasmid was extracted with QIAGEN Plasmid Midi kit (Qiagen) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pDC6-boLAG-3ch2D8.

(85) 2.5. Expression of Rat-Bovine Chimeric Anti-Bovine LAG-3 Antibody

(86) The pDC6-boLAG-3ch2D8 prepared above was transfected into CHO-DG44 cells (CHO-DG44 (dfhr.sup.−/−)) which are a dihydrofolate reductase deficient cell. Forty-eight hours later, the medium was exchanged with CD OptiCHO medium (Life Technologies) containing 20 mM GlutaMAX supplement (Life Technologies). After cultured for 3 weeks, the cells were subjected to selection and cloning by limiting dilution. Subsequently, the concentrations of the chimeric antibody in the culture supernatants were measured by dot blotting and ELISA using anti-bovine IgG F(c) rabbit polyclonal antibody (Rockland) to thereby select high expression clones. The thus established cell clone stably expressing rat-bovine chimeric anti-bovine LAG-3 antibody was transferred into CD OptiCHO medium and cultured under shaking for 14 days (125 rpm, 37° C., 5% CO.sub.2). Chimeric antibody production in the culture supernatant was measured by ELISA using anti-bovine IgG F(c) rabbit polyclonal antibody (Rockland). For each washing operation in ELISA, Auto Plate Washer BIO WASHER 50 (DS Pharma Biomedical) was used. Absorbance was measured with Microplate Reader MTP-650FA (Corona Electric). The culture supernatant at day 14 was centrifuged at 10,000 g for 10 min to remove cells, and the centrifugal supernatant was passed through a Steritop-GP 0.22 μm filter (Millipore) for sterilization and then stored at 4° C. until it was subjected to purification.

(87) 2.6. Purification of Rat-Bovine Chimeric Anti-Bovine LAG-3 Antibody

(88) From the culture supernatant prepared as described above, each chimeric antibody was purified using Ab Capcher Extra (ProteNova). An open column method was used for binding to resin; PBS pH 7.4 was used as an equilibration buffer and a wash buffer. As an elution buffer, 0.1 M Glycine-HCl (pH 2.8) was used. As a neutralization buffer, 1M Tris (pH 9.0) was used. The purified antibody was subjected to buffer exchange with PBS (pH 7.4) using PD-10 Desalting Column (GE Healthcare) and concentrated using Amicon Ultra-15 (50 kDa, Millipore). The thus purified chimeric antibody was passed through a 0.22 μm syringe filter (Millipore) for sterilization and stored at 4° C. until use in experiments.

(89) 2.7. Confirmation of the Purity of Purified Rat-Bovine Chimeric Anti-Bovine LAG-3 Antibody (FIG. 3)

(90) In order to confirm the purity of purified rat-bovine chimeric anti-bovine LAG-3 antibody, antibody proteins were detected by SDS-PAGE and CBB staining. The purified rat-bovine chimeric anti-bovine LAG-3 antibody ch2D8 was suspended in Laemmli Sample Buffer (Bio-Rad) and denatured at 95° C. for 5 min under reducing conditions (reduced with 2-mercaptoethaanol; Sigma-Aldrich) or under non-reducing conditions. Using 10% acrylamide gel, the prepared samples were electrophoresed. As a molecular weight marker, Precision Plus Protein All Blue Standards (Bio-Rad) was used. After electrophoresis, the gels were stained with Quick-CBB (Wako) and decolored in distilled water.

(91) The results are shown in FIG. 3. Bands of rat-bovine chimeric anti-bovine LAG-3 antibody were observed at predicted positions, that is, at 25 kDa (light chain) and 50 kDa (heavy chain) under reducing conditions and at 150-250 kDa under non-reducing conditions.

(92) 2.8. Binding Specificity of Rat-Bovine Chimeric Anti-Bovine LAG-3 Antibody (FIG. 4)

(93) It was confirmed by flow cytometry that the rat-bovine chimeric anti-bovine LAG-3 antibody specifically binds to the bovine LAG-3 expressing cells (described above). First, rat anti-bovine LAG-3 antibody 2D8 or rat-bovine chimeric anti-bovine LAG-3 antibody ch2D8 was reacted with bovine LAG-3 expressing cells at room temperature for 30 min. After washing, Allophycocyanine (APC)-labeled anti-rat Ig goat antibody (Southern Biotech) or Alexa Fluor 647-labeled anti-bovine IgG (H+L) goat F(ab′)2 (Jackson ImmunoResearch) was reacted at room temperature for 30 min. As negative control antibody, rat IgG2a (K) isotype control (BD Biosciences) or bovine IgG1 antibody (Bethyl) was used. After washing, each rat antibody or rat-bovine chimeric antibody bound to cell surfaces was detected by FACS Verse (BD Biosciences). For every washing operation and dilution of antibodies, PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used.

(94) The experimental results are shown in FIG. 4. It was revealed that rat-bovine chimeric anti-bovine LAG-3 antibody ch2D8 binds to bovine LAG-3 expressing cells in the same manner as rat anti-bovine LAG-3 antibody 2D8.

(95) 2.9. Inhibitory Activity of Rat-Bovine Chimeric Anti-Bovine LAG-3 Antibody against Bovine LAG-3/MHC class II Binding (FIG. 5)

(96) Using BL3.1 cell clone (bovine B-cell lymphoma-derived cell clone which highly expresses MHC class II) and bovine LAG-3-Ig (described above), bovine LAG-3/MHC class II binding inhibition by anti-LAG-3 antibodies was tested. First, rat anti-bovine LAG-3 antibody 2D8 or rat-bovine chimeric anti-bovine LAG-3 antibody ch2D8 at a final concentration of 0, 1.56, 3.12, 6.25, 12.5 or 25 μg/ml and bovine LAG-3-Ig at a final concentration of 3.3 μg/ml were mixed in 96-well plates, where they were reacted at 37° C. for 30 min. Subsequently, BL3.1 cell clone (1×10.sup.5 cells) was blocked with 10% inactivated goat serum (Life Technologies)-supplemented PBS at room temperature for 15 min, and reacted with the above reaction mixture at room temperature for 30 min. As negative control antibody, rat IgG1 (κ) isotype control (BD Biosciences) or bovine IgG1 antibody (Bethyl) was used. After washing, Alexa Fluor 647-labeled anti-rabbit IgG (H+L) goat F(ab′).sub.2 (Life Technologies) which had been subjected to absorption treatment (37° C. 30 min) in advance with rat serum-derived IgG (Sigma-Aldrich) and bovine serum-derived IgG (Sigma-Aldrich) was reacted at room temperature for 30 min to thereby detect bovine LAG-3-Ig bound to cell surfaces. For analysis, FACS Verse (BD Biosciences) was used. For every washing operation and dilution of antibodies, PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used. Taking the proportion of LAG-3-Ig bound cells without antibody addition as 100%, the proportion of LAG-3-Ig bound cells at each antibody concentration was shown as relative value.

(97) The experimental results are shown in FIG. 5. It was revealed that rat-bovine chimeric anti-bovine LAG-3 antibody ch2D8 inhibited the binding of LAG-3-Ig to LAG-3 expressing cells by a comparable level to rat anti-bovine LAG-3 antibody 2D8,

(98) 2.10. Biological Activity Test Using Rat-Bovine Chimeric Anti-Bovine LAG-3 Antibody (FIG. 6)

(99) In order to confirm that bovine LAG-3/MHC class 11 binding inhibition by rat-bovine chimeric anti-bovine LAG-3 antibody activates lymphocytes, a biological activity test was performed using IFN-γ production as an indicator. Briefly, PBMCs isolated from bovine peripheral blood were suspended in RPMI 1640 medium (Sigma-Aldrich) containing 10% inactivated fetal bovine serum (Cell Culture Technologies), penicillin 200 U/ml, streptomycin 200 μg/ml and 0.01% L-glutamine (Life Technologies) to give a concentration of 2×10.sup.6 cells/ml. To the PBMCs, 10 μg/ml of rat anti-bovine LAG-3 antibody 2D8 or rat-bovine chimeric anti-bovine LAG-3 antibody ch2D8 was added. Culturing was then performed at 37° C. under 5% CO.sub.2 for 2 days. As control antibodies, rat serum-derived IgG (Sigma-Aldrich) and bovine serum-derived IgG (Sigma-Aldrich) were used. Two days later, a culture supernatant was collected, and IFN-γ production was measured with Bovine IFN-γ ELISA Kit (BETYL). For each washing operation in ELISA, Auto Plate Washer BIO WASHER 50 (DS Pharma Biomedical) was used. Absorbance was measured with Microplate Reader MTP-650FA (Corona Electric).

(100) The experimental results are shown in FIG. 6. It was revealed that rat-bovine chimeric anti-bovine LAG-3 antibody ch2D8 increased bovine PBMCs' IFN-γ response in the same manner as rat anti-bovine LAG-3 antibody 2D8.

(101) 2.11. CDR Analysis of Rat Anti-Bovine LAG-3 Antibody

(102) The complementarity-determining regions (CDRs) of rat anti-bovine LAG-3 antibody 2D8 were determined using NCBI IGBLAST (http://www.ncbi.nlm.nih.gov/igblast/). The results are shown in FIG. 1.

[Example 2] Application of Anti-LAG-3 Antibody to Other Animal Species

(103) 1. Materials, Methods and Experimental Results

(104) 1.1. Identification of Ovine and Water Buffalo LAG-3 Genes

(105) In order to determine the full-lengths of the coding sequences (CDSs) of water buffalo (Bubalus bubalis: Asian water buffalo) and ovine LAG-3 cDNAs, primers for amplifying the full lengths of CDSs from the nucleotide sequences of water buffalo and ovine LAG-3 genes (GenBank accession number AB608099 and XM_012129455) were first designed (buLAG-3 CDS F and R, ovLAG-3 CDS F and R), and then PCR was performed using a synthesized water buffalo or ovine PBMC-derived cDNA as a template. For the resultant amplified products, nucleotide sequences were determined with a capillary sequencer according to conventional methods.

(106) TABLE-US-00008 Primer (buLAG-3 CDS F): (SEQ ID NO: 65) ATGCTGTGGGAGGCTTGGTTC Primer (buLAG-3 CDS R): (SEQ ID NO: 66) TCAGGGATGCTCTGGCTGCA Primer (ovLAG-3 CDS F): (SEQ ID NO: 67) ATGCTGTGGGAGGCTCAGTTCCAGG Primer (ovLAG-3 CDS R): (SEQ ID NO: 68) TCAGGGTTGCTCCGGCTGCA
1.2. Construction of Water Buffalo LAG-3 Expressing COS-7 Cells

(107) In order to prepare a water buffalo LAG-3 expressing plasmid, PCR was performed using a synthesized water buffalo PBMC-derived cDNA as a template and primers designed by adding Sac and EcoRI recognition sites on the 5′ side (buLAG-3-EGFP F and R). The resultant PCR products were digested with SacI (Takara) and EcoRI (Takara), then purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into pEGFP-N2 vector (Clontech) treated with restriction enzymes in the same manner. The expression plasmid of interest was extracted using FastGene Xpress Plasmid PLUS Kit (NIPPON Genetics) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared plasmid is designated as pEGFP-N2-buLAG-3.

(108) TABLE-US-00009 Primer (buLAG-3-EGFP F): (SEQ ID NO: 69) ATTGAGCTCATGCTGTGGGAGGCTTGGTT Primer (buLAG-3-EGFP R): (SEQ ID NO: 70) AATGAATTCGGGATGCTCTGGCTGCAGC

(109) COS-7 cells at a density of 5×10.sup.4 cells/cm.sup.2 were subcultured in 6-well plates, and then cultured overnight in RPMI 1640 medium containing 10% inactivated fetal bovine serum (Invitrogen) and 0.01% L-glutamine (Life Technologies) at 37° C. in the presence of 5% CO.sub.2. The pEGFP-N2-buLAG-3 or pEGFP-N2 (negative control) was introduced into COS-7 cells at 0.4 μg/cm.sup.2 using Lipofectamine 2000 (Invitrogen). The cells were cultured for 48 hours (buLAG-3-EGFP expressing cells). In order to confirm the expression of water buffalo LAG-3 in the thus prepared expressing cells, intracellular localization of EGFP was visualized with an all-in-one fluorescence microscope BZ-9000 (KEYENCE).

(110) 1.3. Reactivity of Rat Anti-Bovine LAG-3 Antibody 2D8 with Water Buffalo LAG-3 (FIG. 7)

(111) It was confirmed by flow cytometry that rat anti-bovine LAG-3 monoclonal antibody cross-reacts with water buffalo LAG-3. Water buffalo LAG-3-EGFP expressing COS cells were blocked with 10% inactivated goat serum (Invitrogen) supplemented PBS at room temperature for 15 min and reacted with 10 μg/ml of rat anti-bovine LAG-3 antibody 2D8 at room temperature for 30 min. After washing, the cells were reacted with APC-labeled anti-rat Ig goat antibody (Beckman Coulter) at room temperature for 30 min. As a negative control antibody, rat IgG1 (κ) isotype control (BD Bioscience) was used. For analysis. FACS Verse (BD Bioscience) was used. For every washing operation and dilution of antibodies, 1% bovine serum albumin (Sigma-Aldrich) supplemented PBS was used.

(112) Experimental results are shown in FIG. 7. It was confirmed that rat anti-bovine LAG-3 antibody 2D8 binds to water buffalo LAG-3 expressing cells.

(113) 1.4. Reactivity of Rat Anti-Bovine LAG-3 Antibody 2D8 with Ovine Lymphocytes (FIG. 8)

(114) Peripheral blood mononuclear cells (PBMCs) were isolated from ovine peripheral blood by density gradient centrifugation using Percoll (GE Healthcare). The isolated ovine PBMCs were suspended in RPMI 1640 medium (Sigma-Aldrich) containing 10% inactivated fetal calf serum (Invitrogen), penicillin 200 U/ml, streptomycin 200 μg/ml and 0.01% L-glutamine (Life Technologies). Cell density was adjusted to 2×10.sup.6 cells/ml. To these PBMCs, phorbol 12-myristate acetate (PMA) 20 ng/ml and ionomycin 1 μg/ml (Sigma-Aldrich) were added, followed by culturing overnight at 37° C. under 5% CO.sub.2. Cultured PBMCs were harvested and blocked with 10% inactivated goat serum (Invitrogen) supplemented PBS at room temperature for 15 min. Then, rat anti-bovine LAG-3 antibody 2D8 was reacted at room temperature for 30 min. As a negative control, rat serum-derived IgG (Sigma-Aldrich) was used. After washing, labeling was performed using APC-labeled goat anti-rat Ig antibody (Beckman Coulter) at room temperature for 30 min. Subsequently, a reaction was performed with mouse anti-ovine CD8 antibody (38.65, AbD Serotec) at room temperature for 30 min. After washing, labeling was performed using PerCP/Cy5.5-labeled goat anti-mouse IgG2a antibody (Santa Cruz) at room temperature for 30 min. After further washing, a reaction was performed with Alexa Flour 488-labled anti-ovine CD21 mouse antibody (GB25A, VMRD) at room temperature for 30 min. For the labeling of GB25A, Zenon Labeling Kit (Life Technologies) was used. For analysis, FACS Verse (BD Biosciences) was used. Every washing operation and dilution of antibodies, 1% bovine serum albumin (Sigma-Aldrich) supplemented PBS was used.

(115) The experimental results are shown in FIG. 8. Rat anti-bovine LAG-3 antibody 2D8 strongly bound to ovine CD8.sup.+ T cells (CD21.sup.− CD8.sup.+ cells) and CD8.sup.− T cells (CD21.sup.− CD8.sup.− cells; i.e., a cell population containing CD4.sup.+ T cells and γ δ T cells), both activated by PMA/ionomycin stimulation.

(116) All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

(117) The anti-LAG-3 antibody of the present invention is applicable to prevention and/or treatment of cancers and infections in animals.

SEQUENCE LISTING FREE TEXT

(118) <SEQ ID NO: 1>

(119) SEQ ID NO: 1 shows the amino acid sequence of the light chain variable region (VL) of rat anti-bovine LAG-3 antibody. Underlined parts: CDR1, CDR2 and CDR3 in this order from the NH2 terminus.

(120) TABLE-US-00010 MMSPVQSLFLLLLWILGTNGDVVLTQTPPTLSATIGQSVSISCRSSQSLL DSDGNTYLNWLLQRPGQSPQLLIYSVSNLESGVPNRFSGSGSETDFTLKI SGVEAEDLGVYYCMQATHVPFTFGSGTKLEIK
<SEQ ID NO: 2>
SEQ ID NO: 2 shows the amino acid sequence of the heavy chain variable region (VH) of rat anti-bovine LAG-3 antibody. Underlined parts: CDR1, CDR2 and CDR3 in this order from the NH2 terminus.

(121) TABLE-US-00011 MVLLELVSVIALFQGVHCEVQLVESGGGLVQPKGSLRLSCAASGFDFDTY PMSWVRQAPGKGLDWVASITIKTHNYATLYAASVKERFTISRDDSQSMVY LQMNNLKTEDTALYYCNREDFDYWGQGVMVTVSS
<SEQ ID NO: 3>
SEQ ID NO: 3 shows the amino acid sequence of the light chain constant region (CL) of a bovine antibody (bovine Ig lambda, GenBank: X62917).

(122) TABLE-US-00012 QPKSPPSVTLFPPSTEELNGNKATLVCLISDFYPGSVTVVWKADGSTITR NVETTRASKQSNSKYAASSYLSLTSSDWKSKGSYSCEVTHEGSTVTKTVK PSECS
<SEQ ID NO: 4>
SEQ ID NO: 4 shows the amino acid sequence of the heavy chain constant region (CH) of a bovine antibody (bovine IgG1, modified from GenBank: X62916). Mutated sites are underlined. Amino acid sequences and mutations: 119 E.fwdarw.P, 120 L.fwdarw.V, 121 P.fwdarw.A, 122 G.fwdarw.deletion, 216 A.fwdarw.S, 217 P.fwdarw.S

(123) TABLE-US-00013 ASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGV HTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVDPT CKPSPCDCCPPPPVAGPSVFIFPPKPKDTLTISGTPEVTCVVVDVGHDDP EVKFSWFVDDVEVNTATTKPREEQFNSTYRVVSALRIQHQDWTGGKEFKC KVHNEGLPSSIVRTISRTKGPAREPQVYVLAPPQEELSKSTVSLTCMVTS FYPDYIAVEWQRNGQPESEDKYGTTPPQLDADSSYFLYSKLRVDRNSWQE GDTYTCVVMHEALHNHYTQKSTSKSAGK
<SEQ ID NO: 5>
SEQ ID NO: 5 shows the nucleotide sequence of the VL of rat anti-bovine LAG-3 antibody-.

(124) TABLE-US-00014 ATGATGAGTCCTGTCCAATCCCTGTTTTTGTTATTGCTTTGGATTCTGGG AACCAATGGTGATGTTGTGCTGACCCAGACTCCACCCACTTTATCGGCTA CCATTGGACAATCGGTCTCCATCTCTTGCAGGTCAAGTCAGAGTCTCTTA GATAGTGATGGAAATACCTATTTAAATTGGTTGCTACAGAGGCCAGGCCA ATCTCCACAGCTTCTAATTTATTCGGTATCCAACCTGGAATCTGGGGTCC CCAACAGGTTCAGTGGCAGTGGGTCAGAAACAGATTTCACACTCAAAATC AGTGGAGTGGAGGCTGAAGATTTGGGAGTTTATTACTGCATGCAAGCTAC CCATGTTCCATTCACGTTCGGCTCAGGGACGAAGTTGGAAATAAAA
The nucleotide sequence of SEQ ID NO: 5 after codon optimization is shown in <SEQ ID NO: 11>.

(125) TABLE-US-00015 ATGATGTCTCCCGTCCAAAGCTTGTTCCTGCTTCTCCTCTGGATTCTGGG CACAAACGGAGATGTGGTTCTCACCCAGACCCCCCCTACTCTGTCTGCCA CCATCGGCCAGAGCGTGTCCATATCCTGTCGCAGCTCCCAAAGCCTGCTG GACTCCGATGGGAATACTTACCTGAATTGGCTGTTGCAGCGGCCTGGCCA GTCCCCCCAGCTGTTGATCTACAGCGTTAGCAATCTGGAAAGCGGGGTCC CCAACCGATTCTCCGGAAGCGGCTCCGAGACCGATTTTACCCTCAAGATC TCCGGCGTGGAAGCCGAGGACCTGGGAGTGTATTATTGCATGCAGGCCAC CCATGTGCCCTTCACCTTCGGTAGCGGTACCAAGTTGGAGATCAAG
<SEQ ID NO: 6>
SEQ ID NO: 6 shows the nucleotide sequence of the VH of rat anti-bovine LAG-3 antibody.

(126) TABLE-US-00016 ATGGTTCTCCTGGAGTTGGTTTCCGTGATTGCTCTTTTTCAAGGCGTGCA TTGTGAGGTGCAGCTTGTTGAGTCTGGTGGAGGGCTGGTGCAGCCTAAGG GGTCATTGAGACTCTCATGTGCAGCCTCTGGATTTGACTTCGATACTTAT CCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGTCTGGATTGGGTTGC TAGTATAACCATTAAGACTCATAATTATGCAACACTTTATGCTGCTTCAG TGAAAGAGAGATTCACCATCTCCAGAGATGACTCACAAAGCATGGTTTAC TTGCAAATGAACAACTTGAAAACTGAGGACACAGCCTTGTATTACTGTAA CAGGGAGGACTTTGATTACTGGGGCCAAGGAGTCATGGTCACAGTCTCCT CA
The nucleotide sequence of SEQ ID NO: 6 after codon optimization is shown in <SEQ ID NO: 12>.

(127) TABLE-US-00017 ATGGTGCTTCTCGAGCTGGTCAGCGTGATTGCTCTGTTTCAGGGCGTGCA CTGCGAAGTGCAGCTGGTGGAGAGTGGTGGTGGGCTCGTGCAACCAAAAG GCAGTCTCAGGCTGAGTTGTGCCGCCTCCGGATTCGATTTCGACACCTAC CCAATGAGCTGGGTCAGGCAAGCCCCAGGGAAAGGACTCGATTGGGTGGC AAGCATTACCATCAAGACACACAATTATGCTACCCTGTATGCCGCAAGCG TAAAGGAACGCTTTACCATCTCCCGCGATGATAGCCAGTCCATGGTATAT TTGCAAATGAATAATTTGAAGACAGAAGATACCGCTTTGTATTATTGCAA CAGAGAAGATTTTGATTATTGGGGGCAGGGGGTGATGGTAACCGTGTCCA GC
<SEQ ID NO: 7>
SEQ ID NO: 7 shows the nucleotide sequence of the CL of a bovine antibody (bovine Ig lambda, GenBank: X62917).

(128) TABLE-US-00018 CAGCCCAAGTCCCCACCCTCGGTCACCCTGTTCCCGCCCTCCACGGAGGA GCTCAACGGCAACAAGGCCACCCTGGTGTGTCTCATCAGCGACTTCTACC CGGGTAGCGTGACCGTGGTCTGGAAGGCAGACGGCAGCACCATCACCCGC AACGTGGAGACCACCCGGGCCTCCAAACAGAGCAACAGCAAGTACGCGGC CAGCAGCTACCTGAGCCTGACGAGCAGCGACTGGAAATCGAAAGGCAGTT ACAGCTGCGAGGTCACGCACGAGGGGAGCACCGTGACGAAGACAGTGAAG CCCTCAGAGTGTTCTTAG
The nucleotide sequence of SEQ ID NO: 7 after codon optimization is shown in <SEQ ID NO: 13>.

(129) TABLE-US-00019 CAGCCTAAGTCCCCTCCTTCAGTCACCCTGITTCCACCATCTACCGAAGA ACTCAACGGGAATAAAGCAACACTGGTGTGCCTTATTTCTGATTTTTACC CAGGGTCTGTGACAGTGGTTTGGAAAGCTGACGGTTCAACAATTACAAGA AACGTGGAGACAACAAGGGCTTCTAAGCAGTCAAACTCTAAGTATGCTGC AAGTTCTTACCTTTCTCTTACAAGTAGTGACTGGAAAAGTAAGGGCAGTT ATTCATGCGAGGTCACTCACGAGGGAAGTACTGTAACTAAAACTGTAAAA CCATCAGAGTGTTCATAG
<SEQ ID NO: 8>
SEQ ID NO: 8 shows the nucleotide sequence (after codon optimization) of the CH of a bovine antibody (bovine IgG1, modified from GenBank: X62916).

(130) TABLE-US-00020 GCTAGCACCACAGCACCTAAAGTTTACCCTCTGTCTTCCTGCTGCGGCGA CAAGTCTTCATCAACTGTTACTCTTGGATGCCTGGTCTCAAGTTACATGC CCGAGCCCGTGACAGTGACCTGGAACTCAGGCGCTCTGAAGTCTGGAGTG CACACATTTCCAGCTGTGCTTCAGTCTAGCGGCCTGTATTCCCTCAGCTC TATGGTTACTGTACCTGGTAGCACCAGCGGACAGACTTTCACCTGTAATG TTGCCCATCCCGCATCTTCTACCAAGGTCGATAAAGCCGTTGACCCCACT TGCAAACCATCCCCTTGTGATTGTTGTCCACCCCCTCCAGTGGCTGGCCC TTCCGTCTTCATTTTCCCTCCTAAACCTAAGGATACTCTGACCATCTCAG GGACACCCGAGGTCACCTGTGTCGTCGTGGACGTGGGACATGACGACCCA GAAGTCAAGTTCTCATGGTTCGTGGACGATGTGGAGGTGAACACAGCAAC AACAAAGCCCAGAGAAGAACAGTTTAACAGCACATATCGGGTGGTCAGCG CCTTGCGTATTCAGCACCAGGACTGGACTGGTGGCAAGGAGTTTAAGTGC AAGGTGCATAACGAAGGTCTGCCCTCTTCTATAGTGAGAACTATCTCCCG AACTAAGGGCCCCGCTCGGGAGCCCCAGGTTTACGTCCTTGCTCCCCCTC AGGAGGAACTGAGTAAATCAACCGTGAGTCTCACCTGTATGGTTACCTCA TTTTACCCAGACTACATCGCCGTAGAGTGGCAGAGGAATGGACAGCCAGA GTCTGAGGACAAATACGGCACTACTCCTCCCCAACTGGATGCCGACTCTT CCTACTTCCTCTACTCCAAATTGCGAGTTGACCGGAACTCATGGCAGGAG GGGGACACATACACATGCGTCGTTATGCACGAGGCCCTGCACAACCATTA CACCCAGAAGTCCACATCTAAAAGTGCAGGTAAGTAA
<SEQ ID NO: 9>
SEQ ID NO: 9 shows the amino acid sequence of a chimeric light chain consisting of the VL of rat anti-bovine LAG-3 antibody and the CL of a bovine antibody.

(131) TABLE-US-00021 MMSPVQSLFLLLLWILGTNGDVVLIQTPPTLSATIGQSVSISCRSSQSLL DSDGNTYLNWLLQRPGQSPQLLIYSVSNLESGVPNRFSGSGSETDFTLKI SGVEAEDLGVYYCMQATHVPFTFGSGTKLEIKQPKSPPSVTLFPPSTEEL NGNKATLVCLISDFYPGSVTVVWKADGSTITRNVETTRASKQSNSKYAAS SYLSLTSSDWKSKGSYSCEVTHEGSTVTKTVKPSECS
<SEQ ID NO: 10>
SEQ ID NO: 10 shows the amino acid sequence of a chimeric heavy chain consisting of the VH of rat anti-bovine LAG-3 antibody and the CH of a bovine antibody (bovine IgG1, modified from GenBank: X62916).

(132) TABLE-US-00022 MVLLELVSVIALFQGVHCEVQLVESGGGLVQPKGSLRLSCAASGFDFDTY PMSWVRQAPGKGLDWVASITIKTHNYATLYAASVKERFTISRDDSQSMVY LQMNNLKTEDTALYYCNREDFDYWGQGVMVTVSSASTTAPKVYPLSSCCG DKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLS SMVTVPGSTSGQTFTCNVAHPASSTKVDKAVDPTCKPSPCDCCPPPPVAG PSVFIFPPKPKDTLTISGTPEVTCVVVDVGHDDPEVKFSWFVDDVEVNTA TTKPREEQFNSTYRVVSALRIQHQDWTGGKEFKCKVHNEGLPSSIVRTIS RTKGPAREPQVYVLAPPQEELSKSTVSLTCMVTSFYPDYIAVEWQRNGQP ESEDKYGTTPPQLDADSSYFLYSKLRVDRNSWQEGDTYTCVVMHEALHNH YTQKSTSKSAGK
<SEQ ID NO: 14>
SEQ ID NO: 14 shows the nucleotide sequence (after codon optimization) of a chimeric light chain consisting of the VL of rat anti-bovine LAG-3 antibody and the CL of a bovine antibody.

(133) TABLE-US-00023 ATGATGTCTCCCGTCCAAAGCTTGTTCCTGCTTCTCCTCTGGATTCTGGG CACAAACGGAGATGTGGTTCTCACCCAGACCCCCCCTACTCTGTCTGCCA CCATCGGCCAGAGCGTGTCCATATCCTGTCGCAGCTCCCAAAGCCTGCTG GACTCCGATGGGAATACTTACCTGAATTGGCTGTTGCAGCGGCCTGGCCA GTCCCCCCAGCTGTTGATCTACAGCGTTAGCAATCTGGAAAGCGGGGTCC CCAACCGATTCTCCGGAAGCGGCTCCGAGACCGATTTTACCCTCAAGATC TCCGGCGTGGAAGCCGAGGACCTGGGAGTGTATTATTGCATGCAGGCCAC CCATGTGCCCTTCACCTTCGGTAGCGGTACCAAGTTGGAGATCAAGCAGC CTAAGTCCCCTCCTTCAGTCACCCTGTTTCCACCATCTACCGAAGAACTC AACGGGAATAAAGCAACACTGGTGTGCCTTATTTCTGATTTTTACCCAGG GTCTGTGACAGTGGTTTGGAAAGCTGACGGTTCAACAATTACAAGAAACG TGGAGACAACAAGGGCTTCTAAGCAGTCAAACTCTAAGTATGCTGCAAGT TCTTACCTTTCTCTTACAAGTAGTGACTGGAAAAGTAAGGGCAGTTATTC ATGCGAGGTCACTCACGAGGGAAGTACTGTAACTAAAACTGTAAAACCAT CAGAGTGTTCATAG
<SEQ ID NO: 15>
SEQ ID NO: 15 shows the nucleotide sequence (after codon optimization) of a chimeric heavy chain consisting of the VH of rat anti-bovine LAG-3 antibody and the CH of a bovine antibody (bovine IgG1, modified from GenBank: X62916).

(134) TABLE-US-00024 ATGGTGCTTCTCGAGCTGGTCAGCGTGATTGCTCTGTTTCAGGGCGTGCA CTGCGAAGTGCAGCTGGTGGAGAGTGGTGGTGGGCTCGTGCAACCAAAAG GCAGTCTCAGGCTGAGTTGTGCCGCCTCCGGATTCGATTTCGACACCTAC CCAATGAGCTGGGTCAGGCAAGCCCCAGGGAAAGGACTCGATTGGGTGGC AAGCATTACCATCAAGACACACAATTATGCTACCCTGTATGCCGCAAGCG TAAAGGAACGCTTTACCATCTCCCGCGATGATAGCCAGTCCATGGTATAT TTGCAAATGAATAATTTGAAGACAGAAGATACCGCTTTGTATTATTGCAA CAGAGAAGATTTTGATTATTGGGGGCAGGGGGTGATGGTAACCGTGTCCA GCGCTAGCACCACAGCACCTAAAGTTTACCCTCTGTCTTCCTGCTGCGGC GACAAGTCTTCATCAACTGTTACTCTTGGATGCCTGGTCTCAAGTTACAT GCCCGAGCCCGTGACAGTGACCTGGAACTCAGGCGCTCTGAAGTCTGGAG TGCACACATTTCCAGCTGTGCTTCAGTCTAGCGGCCTGTATTCCCTCAGC TCTATGGTTACTGTACCTGGTAGCACCAGCGGACAGACTTTCACCTGTAA TGTTGCCCATCCCGCATCTTCTACCAAGGTCGATAAAGCCGTTGACCCCA CTTGCAAACCATCCCCTTGTGATTGTTGTCCACCCCCTCCAGTGGCTGGC CCTTCCGTCTTCATTTTCCCTCCTAAACCTAAGGATACTCTGACCATCTC AGGGACACCCGAGGTCACCTGTGTCGTCGTGGACGTGGGACATGACGACC CAGAAGTCAAGTTCTCATGGTTCGTGGACGATGTGGAGGTGAACACAGCA ACAACAAAGCCCAGAGAAGAACAGTTTAACAGCACATATCGGGTGGTCAG CGCCTTGCGTATTCAGCACCAGGACTGGACTGGTGGCAAGGAGTTTAAGT GCAAGGTGCATAACGAAGGTCTGCCCTCTTCTATAGTGAGAACTATCTCC CGAACTAAGGGCCCCGCTCGGGAGCCCCAGGTTTACGTCCTTGCTCCCCC TCAGGAGGAACTGAGTAAATCAACCGTGAGTCTCACCTGTATGGTTACCT CATTTTACCCAGACTACATCGCCGTAGAGTGGCAGAGGAATGGACAGCCA GAGTCTGAGGACAAATACGGCACTACTCCTCCCCAACTGGATGCCGACTC TTCCTACTTCCTCTACTCCAAATTGCGAGTTGACCGGAACTCATGGCAGG AGGGGGACACATACACATGCGTCGTTATGCACGAGGCCCTGCACAACCAT TACACCCAGAAGTCCACATCTAAAAGTGCAGGTAAGTAA
<SEQ ID NO: 16>
SEQ ID NO: 16 shows the amino acid sequence (QSLLDSDGNTY) of CDR11 of the VL of rat anti-bovine LAG-3 antibody 2D8.
<SEQ ID NO: 17>
SEQ ID NO: 17 shows the amino acid sequence

(135) TABLE-US-00025 (MQATHVPFT)
of CDR3 of the VL of rat anti-bovine LAG-3 antibody 2D8.
<SEQ ID NO: 18>
SEQ ID NO: 18 shows the amino acid sequence

(136) TABLE-US-00026 (GFDFDTYP)
of CDR1 of the VH of rat anti-bovine LAG-3 antibody 2D8.
<SEQ ID NO: 19>
SEQ ID NO: 19 shows the amino acid sequence

(137) TABLE-US-00027 (ITIKTHNYAT)
of CDR2 of the VH of rat anti-bovine LAG-3 antibody 2D8.
<SEQ ID NO: 20>
SEQ ID NO: 20 shows the amino acid sequence

(138) TABLE-US-00028 (NREDFDY)
of CDR3 of the VH of rat anti-bovine LAG-3 antibody 2D8.
<SEQ ID NO: 21>
SEQ ID NO: 21 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG1 variant 1).
<SEQ ID NO: 22>
SEQ ID NO: 22 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG1 variant 2).
<SEQ ID NO: 23>
SEQ ID NO: 23 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG1 variant 3).
<SEQ ID NO: 24>
SEQ ID NO: 24 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG2 variant 1).
<SEQ ID NO: 25>
SEQ ID NO: 25 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG2 variant 2).
<SEQ ID NO: 26>
SEQ ID NO: 26 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG2 variant 3).
<SEQ ID NO: 27>
SEQ ID NO: 27 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG3 variant 1).
<SEQ ID NO: 28>
SEQ ID NO: 28 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG3 variant 2).
<SEQ ID NO: 29>
SEQ ID NO: 29 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG1 variant 1).
<SEQ ID NO: 30>
SEQ ID NO: 30 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG1 variant 2).
<SEQ ID NO: 31>
SEQ ID NO: 31 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG1 variant 3).
<SEQ ID NO: 32>
SEQ ID NO: 32 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG2 variant 1).
<SEQ ID NO: 33>
SEQ ID NO: 33 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG2 variant 2).
<SEQ ID NO: 34>
SEQ ID NO: 34 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG2 variant 3).
<SEQ ID NO: 35>
SEQ ID NO: 35 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG3 variant 1).
<SEQ ID NO: 36>
SEQ ID NO: 36 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG3 variant 2).
<SEQ ID NO: 37>
SEQ ID NO: 37 shows the amino acid sequence of the CH (CH1-CH3) of an ovine antibody (IgG1).
<SEQ ID NO: 38>
SEQ ID NO: 38 shows the nucleotide sequence of the CH (CH1-CH3) of an ovine antibody (IgG1).
<SEQ ID NO: 39>
SEQ ID NO: 39 shows the amino acid sequence of the CH (CH1-CH3) of an ovine antibody (IgG2).
<SEQ ID NO: 40>
SEQ ID NO: 40 shows the nucleotide sequence of the CH (CH1-CH3) of an ovine antibody (IgG2).
<SEQ ID NO: 41>
SEQ ID NO: 41 shows the amino acid sequence of the light chain (Ig kappa(CK)) constant region of an ovine antibody.
<SEQ ID NO: 42>
SEQ ID NO: 42 shows the nucleotide sequence of the light chain (Ig kappa(CK)) constant region of an ovine antibody.
<SEQ ID NO: 43>
SEQ ID NO: 43 shows the amino acid sequence of the light chain (Ig lambda(CL)) constant region of an ovine antibody.
<SEQ ID NO: 44>
SEQ ID NO: 44 shows the nucleotide sequence of the light chain (Ig lambda(CL)) constant region of an ovine antibody.
<SEQ ID NO: 45>
SEQ ID NO: 45 shows the amino acid sequence of the CH (CH1-CH3) of a water buffalo antibody (presumed to be IgG1).
<SEQ ID NO: 46>
SEQ ID NO: 46 shows the nucleotide sequence of the CH (CH1-CH3) of a water buffalo antibody (presumed to be IgG1).
<SEQ ID NO: 47>
SEQ ID NO: 47 shows the amino acid sequence of the CH (CH1-CH3) of a water buffalo antibody (presumed to be IgG2).
<SEQ ID NO: 48>
SEQ ID NO: 48 shows the nucleotide sequence of the CH (CH1-CH3) of a water buffalo antibody (presumed to be IgG2).
<SEQ ID NO: 49>
SEQ ID NO: 49 shows the amino acid sequence of the CH (CH1-CH3) of a water buffalo antibody (presumed to be IgG3).
<SEQ ID NO: 50>
SEQ ID NO: 50 shows the nucleotide sequence of the CH (CH1-CH3) of a water buffalo antibody (presumed to be IgG3).
<SEQ ID NO: 51>
SEQ ID NO: 51 shows the amino acid sequence of the light chain (presumed to be Ig lambda) constant region (CL) of a water buffalo antibody.
<SEQ ID NO: 52>
SEQ ID NO: 52 shows the nucleotide sequence of the light chain (presumed to be Ig lambda) constant region (CL) of a water buffalo antibody.
<SEQ ID NO: 53>
SEQ ID NO: 53 shows the amino acid sequence of the CH (CH1-CH3) of a human antibody (IgG4 variant 1).
<SEQ ID NO: 54>
SEQ ID NO: 54 shows the nucleotide sequence of the CH (CH1-CH3) of a human antibody (IgG4 variant 1).
<SEQ ID NO: 55>
SEQ ID NO: 55 shows the amino acid sequence of the CH (CH1-CH3) of a human antibody (IgG4 variant 2).
<SEQ ID NO: 56>
SEQ ID NO: 56 shows the nucleotide sequence of the CH (CH1-CH3) of a human antibody (IgG4 variant 2).
<SEQ ID NO: 57>
SEQ ID NO: 57 shows the amino acid sequence of the CH (CH1-CH3) of a human antibody (IgG4 variant 3).
<SEQ ID NO: 58>
SEQ ID NO: 58 shows the nucleotide sequence of the CH (CH1-CH3) of a human antibody (IgG4 variant 3).
<SEQ ID NO: 59>
SEQ ID NO: 59 shows the amino acid sequence of the CL of a human antibody.
<SEQ ID NO: 60>
SEQ ID NO: 60 shows the nucleotide sequence of the CL of a human antibody.
<SEQ ID NOS: 61-70>
SEQ ID NOS: 61-70 show the nucleotide sequences of primers boLAG-3-EGFP F, boLAG-3-EGFP R, boLAG-3-Ig F, boLAG-3-Ig R, buLAG-3 CDS F, buLAG-3 CDS R, ovLAG-3 CDS F, ovLAG-3 CDS R, buLAG-3-EGFP F, and buLAG-3-EGFP R in this order.
<SEQ ID NO: 71>
SEQ ID NO: 71 shows the amino acid sequence of the full length of bovine LAG-3.

(139) TABLE-US-00029 MLWEAWFQVWLFLQLLWAAAVEAPEPGAEVPVVWAQEGAPAQLPCSPTIP LQDLSLPRTRQVTWQHVPESGSAAPTPRGPGPRRYTVLRLAPGGLRIGKL PLQPRVQLEEMGLQRGDFSLWLRPARRADAGEYHAAVRFGNRALACRLRL RVGQAAVTASPPGPLWTSSWVVLNCSFSRPDLPASVHWFRGPGRVPVQES PHHHLVGNFLFLPQVSSLDSGTWGCSLTYRDGFNVSITYNLAVLGLEPRA TLTVYAGAGSKVELPCRLPPGVGIQSSLTAMWTPPGEGPDLLVAGDRNNF TLRLEAVGQAQAGTYTCRVHLQGRQLSATVTLAVITVTPKPYGSSGSLRK PFCEVTPASGQERFVWSPLDKRSQRRSPGPWLLTPDARPLSQPWQCHLYQ GERLLGTAVYLTELSHPGAQRSGRALGAGRTAHLPLLILGLLFLLLLVTG ASSFHLWRRQWRPRRFSALEHGTHPSQASSKTGELEPELEPEPDPEVEPE PEPEPESQPQLQPEQP*
<SEQ ID NO: 72>
SEQ ID NO: 72 shows the amino acid sequence of a part of the extracellular region of bovine LAG-3 which corresponds to amino acid numbers 71 to 99).

(140) TABLE-US-00030 GSAAPTPRGPGPRRYTVLRLAPGGLRIGK.