ANTI-CANINE PD-1 ANTIBODY BINDING WITH CANINE PD-1

Abstract

Provided is an anti-canine PD-1 antibody, capable of binding with canine PD-1 and comprising three light-chain complementary determining regions (CDR1-3) or a conservatively modified variant maintaining its function and/or three heavy-chain complementary determining regions (CDR1-3) or a conservatively modified variant maintaining its function. Further provided is an application of the anti-canine PD-1 antibody in the preparation of medicines for treating canine cancers.

Claims

1-23. (canceled)

24. An anti-canine PD-1 antibody capable of binding with canine PD-1, comprising: three light-chain complementary determining regions (CDR1-3) or a conservatively modified variant maintaining the function thereof; and/or three heavy-chain complementary determining regions (CDR1-3) or a conservatively modified variant maintaining functions thereof.

25. The antibody according to claim 24, wherein the light-chain complementary determining region CDR1 is selected from a group consisting of: TABLE-US-00020 (SEQ ID NO: 1)   TCAAGTGTAACTTAC, (SEQ ID NO: 2) TCAAGTGTAAGTTAC and (SEQ ID NO: 3) TCAAGTATAACTTAC; the light-chain complementary determining region CDR2 is selected from a group consisting of: TABLE-US-00021 (SEQ ID NO: 4)   GACACATCC; the light-chain complementary determining region CDR3 is selected from a group consisting of: TABLE-US-00022 (SEQ ID NO: 5) CAGCAGTACAGTGGTCACCCATCCTCG, (SEQ ID NO: 6) CAACAGTACAGTGGTTACCCGTACACG and (SEQ ID NO: 7) CAGCAGTACAGTGGTTACCCATCCACG; the heavy-chain complementary determining region CDR1 is selected from a group consisting of: TABLE-US-00023 (SEQ ID NO: 8) GGCTTCAACATCAAAGACACCTAT and  (SEQ ID NO: 9) GGCTTCAAC ATTAAAGACACCTAT; the heavy-chain complementary determining region CDR2 is selected from a group consisting of: TABLE-US-00024 (SEQ ID NO: 10) ATTGATCCTGCGATTGATAATACT. (SEQ ID NO: 11) ATTGATCCTGCGA TTGGTAATACT and (SEQ ID NO: 12) ATTGATCCTGCGATTGGTAATCCT; the heavy-chain complementary determining region CDR3 is selected from a group consisting of: TABLE-US-00025 (SEQ ID NO: 13) GCTTCTGGGTTCTATACTATGGACTAC, (SEQ ID NO: 14)  GCTAGAGGGTTCTATGGTATGGACTAC and (SEQ ID NO: 15) GCTTCTGGGTTCTATGCTATGGACTGC.

26. The antibody according to claim 24, wherein the anti-canine PD-1 antibody comprises the following combination of the three light-chain complementary determining regions (CDR1-3): TABLE-US-00026 (1) CDR1: (SEQ ID NO: 1) TCAAGTGTAACTTAC, CDR2: (SEQ ID NO: 4) GACACATCC, CDR3: (SEQ ID NO: 5) CAGCAGTACAGTGGTCACCCATCCTCG; (2) CDR1: (SEQ ID NO: 2) TCAAGTGTAAGTTAC, CDR2: (SEQ ID NO: 4) GACACATCC, CDR3: (SEQ ID NO: 6) CAACAGTACAGTGGTTACCCGTACACG; or (3) CDR1: (SEQ ID NO: 3) TCAAGTATAACTTAC, CDR2: (SEQ ID NO: 4) GACACATCC, CDR3: (SEQ ID NO: 7) CAGCAGTACAGTGGTTACCCATCCACG.

27. The antibody according to claim 24, wherein the anti-canine PD-1 antibody comprises the following combination of the three heavy-chain complementary determining regions (CDR1-3): TABLE-US-00027 (1) CDR1: (SEQ ID NO: 8) GGCTTCAACATCAAAGACACCTAT, CDR2: (SEQ ID NO: 10) ATTGATCCTGCGATTGATAATACT, CDR3: (SEQ ID NO: 13) GCTTCTGGGTTCTATACT ATGGACTAC; (2) CDR1: (SEQ ID NO: 9)  GGCTTCAACATTAAAGACACCTAT, CDR2: (SEQ ID NO: 11) ATTGATCCTGCGATTGGTAATACT, CDR3: (SEQ ID NO: 13)  GCTTCTGGGTTCTATACTATGGACTAC; or (3) CDR1: (SEQ ID NO: 9) GGCTTCAACATTAAAGACACCTAT, CDR2: (SEQ ID NO: 12) ATTGATCCTGCGATTGGTAATCCT, CDR3: (SEQ ID NO: 15) GCTTCTGGGTTCTATGCTATGGACTGC.

28. The antibody according to claim 24, wherein the anti-canine PD-1 antibody comprises the following combination of the following three light-chain complementary determining regions (CDR1-3) and three heavy-chain complementary determining regions (CDR1-3): (1) light chain CDR1: TCAAGTGTAACTTAC (SEQ ID NO: 1), light chain CDR2: GACACATCC(SEQ ID NO: 4), light chain CDR3: CAGCAGTACAGTGGTCACCCATCCTCG (SEQ ID NO: 5), heavy chain CDR1: GGCTTCAACATCAAAG ACACCTAT (SEQ ID NO: 8), heavy chain CDR2: ATTGATCCTGCGATTGATA ATACT (SEQ ID NO: 10) and heavy chain CDR3: GCTTCTGGGTTCTATACTATGG ACTAC (SEQ ID NO: 13); (2) light chain CDR1: TCAAGTGTAAGTTAC (SEQ ID NO: 2), light chain CDR2: GACACATCC(SEQ ID NO: 4), light chain CDR3: CAACAGTACAGTGGTTACCCGTACACG (SEQ ID NO: 6), heavy chain CDR1: GGCTTCAACATTAAAGACACCTAT (SEQ ID NO: 9), heavy chain CDR2: ATTGA TCCTGCGATTGGTAATACT (SEQ ID NO: 11), heavy chain CDR3: GCTTCTGGGTT CTATACTATGGACTAC (SEQ ID NO: 13); or (3) light chain CDR1: TCAAGTATAA CTTAC(SEQ ID NO: 3), light chain CDR2: GACACATCC (SEQ ID NO: 4), light chain CDR3: CAGCAGTACAGTGGTTACCCATCCACG (SEQ ID NO: 7), heavy chain CDR1: GGCTTCAACATTAAAGACACCTAT (SEQ ID NO: 9), heavy chain CDR2: ATTG ATCCTGCGATTGGTAATCCT (SEQ ID NO: 12) and heavy chain CDR3: GCTTCTGGG TTCTATGCTATGGACTGC (SEQ ID NO: 15).

29. The antibody according to claim 24, wherein the anti-canine PD-1 antibody comprises a light chain variable region selected from a group consisting of: TABLE-US-00028 (1) (SEQ ID NO: 16) GCAAATTGTTCTCACCCAGTCTCCAGCAATCATGT CTGCATCTCCAGGGGAGAAGGTCACCATGACCTGC AGTGCCAGCTCAAGTGTAACTTACATGTATTGGTT CCAGCAGAAGCCAGGCTCCTCCCCCAGACTCTGGA TTTATGACACATCCAACCTGGTTTCTGGAGTCCCT GCTCGCTTCAGTGGCAGTAGGTCTGGGACCTCTTA TTCTCTCACAATCAGCATATGGAGGCTGAAGATGC TGCCACTTATTACTGCCAGCAGTACAGTGGTCACC CATCCTCGTTCGGCTCGGGGACAAAGTTGGAAATT AAA; (2) (SEQ ID NO: 17) CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTC TGCATCTCCAGGGGAGAAGGTCACCATGACCTGCA GTGCCAGCTCAAGTGTAAGTTACATGTTCTGGTAC CAGCAGAAGCCAGGCTCCTCCCCCAGACTCTGGAT TTATGACACATCCAACCTGGTTTCTGGAGTCCCTG CTCGCTTCAGTGGCAGTAGGTCTGGGACCTCTTAT TCTCTCACAATCAGCAGCATGGAGGCTGAAGATGC TGCCACTTATTACTGCCAACAGTACAGTGGTTACC CGTACACGTTCGGAGGGGGGACCAAGCTGGAAATA AAG; and (3) (SEQ ID NO: 18) CAAATTGTTCTCACCCAGTCTCCAGCCATCATGTC TGCATCTCCAGGGGAAAAGGTCACCATGACCTGCA GTGCCAGCTCAAGTATAACTTACATGTTCTGGTAC CAGCAGAAGCCAGGCTCCTCCCCCAGACTCTGGAT TTATGACACATCCAACCTGGTTTCTGGAGTCCCTG CTCGCTTCAGTGGCAGTAAGTCTGGGACCTCTTAT TCTCTCACAATCACCAGCATGGAGGCTGAAGATGC TGCCACTTATTACTGCCAGCAGTACAGTGGTTACC CATCCACGTTCGGCTCGGGGACAAAGTTGGAAATA AAA.

30. The antibody according to claim 24, wherein the anti-canine PD-1 antibody comprises a heavy chain variable region nucleotide sequence selected from a group consisting of: TABLE-US-00029 (1) (SEQ ID NO: 19) GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGT GAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAG CTTCTGGCTTCAACATCAAAGACACCTATATGCAT TGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTG GATTGGAAGGATTGATCCTGCGATTGATAATACTA AATATGACCCGAAGTTCCAGGGCAAGGCCACTATA ACAGCTGACACATCCTCCAACACAGCCTACCTGCA GCTCAGCAGCCTGACATCTGAGGACACTGCCGTCT ATTACTGTGCTTCTGGGTTCTATACTATGGACTAC TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA; (2) (SEQ ID NO: 20) GAGGTTCGGCT GCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGG CCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTC AACATTAAAGACACCTATTTACACTGGTTGAAGGA GAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGA TTGATCCTGCGATTGGTAATACTAGATATGACCCG AAGTTCCAGGTCAAGGCCACTATAACAGCAGACAC ATCCTCCAACACAGCCTACCTGCAACTCAGCAGCC TGACATCTGAGGACTCTGCCGTCTATTACTGTGCT AGAGGGTTCTATGGTATGGACTACTGGGGTCAAGG AACCTCAGTCACCGTCTCCTCA; and (3) (SEQ ID NO: 21) GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGGTTGT GAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAG CTTCTGGCTTCAACATTAAAGACACCTATATGCAC TGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTG GATTGGAAGGATTGATCCTGCGATTGGTAATCCTA AATATGACCCGAAGTTCCAGGGCAGGGCCACTATA ACTGCTGACACATCCTCCAACACAGCCTACCTGCA GCTCAGCAGCCTGACATCTGAGGACACTGCCGTCT ATTACTGTGCTTCTGGGTTCTATGCTATGGACTGC TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

31. The antibody according to claim 24, wherein the anti-canine PD-1 antibody comprises the following combination of the light chain variable regions and heavy chain variable regions: (1) a light chain variable region as shown by SEQ ID NO: 16 and a heavy chain variable region as shown by SEQ ID NO: 19; (2) a light chain variable region as shown by SEQ ID NO: 17 and a heavy chain variable region as shown by SEQ ID NO: 20; or (3) a light chain variable region as shown by SEQ ID NO: 18 and a heavy chain variable region as shown by SEQ ID NO: 21.

32. The antibody according to claim 24, wherein the anti-canine PD-1 antibody is a mouse-anti-canine PD-1 antibody, preferably, the anti-canine PD-1 antibody is a mouse-anti-canine PD-1 monoclonal antibody, and the anti-canine PD-1 antibody blocks the binding of canine PD-1 to canine PD-L1.

33. The antibody according to claim 24, wherein the anti-canine PD-1 antibody has an affinity of 1.000 E.sup.07-1.000 E.sup.12.

34. The antibody according to claim 24, wherein the anti-canine PD-1 antibody is a caninized anti-canine PD-1 monoclonal antibody, and the caninized anti-canine PD-1 monoclonal antibody block the binding of the canine PD-1 to canine PD-L1.

35. The antibody according to claim 34, wherein the caninized anti-canine PD-1 monoclonal antibody comprises a heavy chain constant region sequence and/or a light chain constant region sequence of the canine PD-1 monoclonal antibody; preferably, the heavy chain constant region has a sequence as shown by SEQ ID NO: 28, and the light chain constant region has a sequence as shown by SEQ ID NO: 29.

36. A functional fragment of an anti-canine PD-1 antibody capable of binding with canine PD-1, comprising three light-chain complementary determining regions (CDR1-3) or a combination of conservatively modified variants maintaining the function thereof, or comprising three heavy-chain complementary determining regions (CDR1-3) or a combination of conservatively modified variants maintaining functions thereof; the light-chain complementary determining region CDR1 is selected from a group consisting of: TABLE-US-00030 (SEQ ID NO: 1) TCAAGTGTAACTTAC (SEQ ID NO: 2) TCAAGTGTAAGTTAC and (SEQ ID NO: 3) TCAAGTATAACTTAC; the light-chain complementary determining region CDR2 is: GACACATCC (SEQ ID NO: 4); the light-chain complementary determining region CDR3 is selected from a group consisting of: TABLE-US-00031 (SEQ ID NO: 5) CAGCAGTACAGTGGTCACCCATCCTCG, (SEQ ID NO: 6) CAACAGTACAGTGGTTACCCGTACACG and (SEQ ID NO: 7) CAGCAGTACAGTGGTTACCCATCCACG; the heavy-chain complementary determining region CDR1 is selected from a group consisting of: TABLE-US-00032 (SEQ ID NO: 8) GGCTTCAACATCAAAGACACCTAT and (SEQ ID NO: 9) GGCTTCAACATTAAAGACACCTAT; the heavy-chain complementary determining region CDR2 is selected from a group consisting of: TABLE-US-00033 (SEQ ID NO: 10) ATTGATCCTGCGATTGATAATACT, (SEQ ID NO: 11) ATTGATCCTGCGATTGGTAATACT and (SEQ ID NO: 12) ATTGATCCTGCGATTGGTAATCCT; the heavy-chain complementary determining region CDR3 is selected from a group consisting of: TABLE-US-00034 (SEQ ID NO: 13) GCTTCTGGGTTCTATACTATGGACTAC, (SEQ ID NO: 14) GCTAGAGGGTTCTATGGTATGGACTAC and (SEQ ID NO: 15) GCTTCTGGGTTCTATGCTATGGACTGC.

37. The fragment according to claim 36, wherein the functional fragment of the anti-canine PD-1 antibody comprises the following combination of the three light-chain complementary determining regions (CDR1-3): TABLE-US-00035 (1) CDR1: (SEQ ID NO: 1)  TCAAGTGTAACTTAC, CDR2: (SEQ ID NO: 4) GACACATCC, CDR3: (SEQ ID NO: 5) CAGCAGTACAGTGGTC ACCCATCCTCG; (2) CDR1: (SEQ ID NO: 2) TCAAGTGTAAGTTAC, CDR2: (SEQ ID NO: 4) GACACATCC, CDR3: (SEQ ID NO: 6) CAACAGTACAGT GGTTACCCGTACACG; or (3) CDR1: (SEQ ID NO: 3) TCAAGTATAAC TTAC, CDR2: (SEQ ID NO: 4) GACACATCC, CDR3: (SEQ ID NO: 7) CAGCAGTACAGTGGTTACCCATCCACG  or a combination of the conservatively modified variants maintaining functions thereof; and/or, wherein the functional fragment of the anti-canine PD-1 antibody comprises the following combination of the three heavy-chain complementary determining regions (CDR1-3): TABLE-US-00036 (1) CDR1: (SEQ ID NO: 8) GGCTTCAACATCAAAGACACCTAT, CDR2: (SEQ ID NO: 10) ATTGATCCTGCGATTGATAATACT, CDR3: (SEQ ID NO: 13) GCTTCTGGGTTCTATACTATGGACTAC; (2) CDR1: (SEQ ID NO: 9)  GGCTTCAACATTAAAGACACCTAT, CDR2: (SEQ ID NO: 11) ATTGATCCTGCGATTGGTAATACT, CDR3: (SEQ ID NO: 13) GCTTCTGGGTTCTATACTATGGACTAC; or (3) CDR1: (SEQ ID NO: 9) GGCTT CAACATTAAAGACACCTAT; CDR2: (SEQ ID NO: 12)  ATTGATCCTGCGATTGGTAATCCT; CDR3: (SEQ ID NO: 15) GCTTCTGGGTTCTATGCTATGGACTGC, or a combination of the conservatively modified variants maintaining functions thereof.

38. The fragment according to claim 36, wherein the functional fragment of the anti-canine PD-1 antibody comprises the following combination of the three light-chain complementary determining regions (CDR1-3) and three heavy-chain complementary determining regions (CDR1-3): (1) light chain CDR1: TCAAGTGTAACTTAC (SEQ ID NO: 1), light chain CDR2: GACACATCC (SEQ ID NO: 4), light chain CDR3: CAGCAGTACAGTGGTCACCCATCCTCG (SEQ ID NO: 5), heavy chain CDR1: GGCTTCAACATCAAAGACACCTAT (SEQ ID NO: 8), heavy chain CDR2: ATTGATCCTGCGATTGATAATACT (SEQ ID NO: 10), heavy chain CDR3: GCTTCTGGGTTCTATACTATGGACTAC (SEQ ID NO: 13); (2) light chain CDR1: TCAAGTGTAAGTTAC (SEQ ID NO: 2), light chain CDR2: GACACATCC (SEQ ID NO: 4), light chain CDR3: CAACAGTACAGTGGTTACCC GTACACG (SEQ ID NO: 6), heavy chain CDR1: GGCTTCAACATTAAAGACACC TAT (SEQ ID NO: 9), heavy chain CDR2: ATTGATCCTGCGATTGGTAATACT (SEQ ID NO: 11), heavy chain CDR3: GCTTCTGGGTTCTATACTATGGACTAC (SEQ ID NO: 13); or (3) light chain CDR1: TCAAGTATAACTTAC (SEQ ID NO: 3), light chain CDR2: GACACATCC (SEQ ID NO: 4), light chain CDR3: CAGCAGTACAGTGGTTACCCATCCACG (SEQ ID NO: 7), heavy chain CDR1: GGCTTCAACATTAA AGACACCTAT (SEQ ID NO: 9), heavy chain CDR2: ATTGATCCTGCGATTGGTA ATCCT (SEQ ID NO: 12), heavy chain CDR3: GCTTCTGGGTTCTATGCTATGGAC TGC (SEQ ID NO: 15), or a combination of the conservatively modified variants maintaining functions thereof.

39. The fragment according to claim 36, wherein the functional fragment of the anti-canine PD-1 antibody comprises the following combination of the three light-chain complementary determining regions (CDR1-3) and three heavy-chain complementary determining regions (CDR1-3): (1) light chain CDR1: TCAAGTGTAACTTAC (SEQ ID NO: 1), light chain CDR2: GACACATCC (SEQ ID NO: 4), light chain CDR3: CAGCAGTACAGTGGTCACCCATCCTCG (SEQ ID NO: 5), heavy chain CDR1: GGCTTCAACATCAAAGACACCTAT (SEQ ID NO: 8), heavy chain CDR2: ATTGATCCTGCGATT GATAATACT (SEQ ID NO: 10), heavy chain CDR3: GCTTCTGGGTTCTATACTAT GGACTAC (SEQ ID NO: 13); (2) light chain CDR1: TCAAGTGTAAGTTAC (SEQ ID NO: 2), light chain CDR2: GACACATCC (SEQ ID NO: 4), light chain CDR3: CAACAGTACAGTGGTTACCCGTACACG (SEQ ID NO: 6), heavy chain CDR1: GGCTTCAACATTAAAGACACCTAT (SEQ ID NO: 9), heavy chain CDR2: ATTGA TCCTGCGATTGGTAATACT (SEQ ID NO: 11), heavy chain CDR3: GCTTCTGGGT TCTATACTATGGACTAC (SEQ ID NO: 13); or (3) light chain CDR1: TCAAGTATA ACTTAC (SEQ ID NO: 3), light chain CDR2: GACACATCC (SEQ ID NO: 4), light chain CDR3: CAGCAGTACAGTGGTTACCCATCCACG (SEQ ID NO: 7), heavy chain CDR1: GGCTTCAACATTAAAGACACCTAT (SEQ ID NO: 9), heavy chain CDR2: ATTGATCCTGCGATTGGTAATCCT (SEQ ID NO: 12), heavy chain CDR3: GCTTCTGGGTTCTATGCTATGGACTGC (SEQ ID NO: 15).

40. The fragment according to claim 36, wherein the functional fragment of the anti-canine PD-1 antibody comprises a light chain variable region nucleotide sequence selected from a group consisting of: (1) a nucleotide sequence as shown by SEQ ID NO: 16; (2) a nucleotide sequence as shown by SEQ ID NO: 17; and (3) a nucleotide sequence as shown by SEQ ID NO: 18; and/or, wherein the functional fragment of the anti-canine PD-1 antibody comprises a heavy chain variable region nucleotide sequence selected from a group consisting of: (1) a nucleotide sequence as shown by SEQ ID NO: 19; (2) a nucleotide sequence as shown by SEQ ID NO: 20; and (3) a nucleotide sequence as shown by SEQ ID NO: 21.

41. The fragment according to claim 36, wherein the functional fragment of the anti-canine PD-1 antibody comprises the following combination of the light chain variable regions and heavy chain variable regions: (1) a light chain variable region as shown by SEQ ID NO: 16 and a heavy chain variable region as shown by SEQ ID NO: 19; (2) a light chain variable region as shown by SEQ ID NO: 17 and a heavy chain variable region as shown by SEQ ID NO: 20; or (3) a light chain variable region as shown by SEQ ID NO: 18 and a heavy chain variable region as shown by SEQ ID NO: 21, or a combination of the conservatively modified variants maintaining functions thereof.

42. The fragment according to claim 36, wherein the functional fragment is an antigen-binding fragment.

43. A method for treating canine cancers by using the antibody according to claim 24.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG. 1 shows the SDS-PAGE electrophoresis pattern and western blotting pattern obtained by using an antigen fragment which was expressed by expressing a vector in Expi293F cells and purified.

[0056] FIG. 2 shows the ELISA experimental results of six mice from two groups 21 days after immunization.

[0057] FIG. 3 shows the ELISA detection results of the immunoreaction between the supernatant from two hybridoma libraries and canine PD-1.

[0058] FIG. 4 shows the binding determined over time.

[0059] FIGS. 5-9 show the responses of 30 antibodies from Nos. M1 to M30 to PD-1 His over time.

[0060] FIG. 10 shows the binding determined over time during the measurement.

[0061] FIGS. 11-12 show the test results of M1-M for the analyte PD1 Fc.

[0062] FIG. 13 shows that the binding of the antibodies C1, C3 and C11 of the present disclosure to PBMC do not influence the proliferation of lymphocyte.

[0063] FIG. 14 shows that the antibodies C1, C3 and C11 of the present disclosure can be effectively bound to PD1 on the surface of T cells.

[0064] FIG. 15 shows that the addition of the antibodies C1, C3 and C11 of the present disclosure does not influence the release of interferon.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0065] The technical solution of the present disclosure will be further described in combination with examples and accompanying drawings of the description. These examples are merely used to describe the present disclosure, but not intent to limit the protection scope of the present disclosure.

EXAMPLES

Experimental Materials and Methods:

[0066] 1. Plasmid Preparation [0067] 1) design a target sequence, optimize and synthesize; [0068] 2) subclone the complete sequence into pcDNA3.1 plasmid; and [0069] 3) prepare a large number of plasmids required by transfecting Expi293F cells for expression.

[0070] 2. Cell Culture and Transient Transfection [0071] 1) culture Expi293F cells in a serum-free Expi293 expression medium; [0072] 2) put cell culture flasks into an incubator at 37° C. and 8% CO.sub.2 for shaking culture, and the day before transfection, inoculate the cells into Corning cell culture flasks with proper density; [0073] 3) mix DNA and transfection reagents in an optimal proportion on the day of transfection, and then add the mixed solution into the culture flask for cell transfection; [0074] 4) transiently transfect the plasmids encoding the target recombinant protein to suspension Expi293F cell culture; and [0075] 5) collect the cell culture supernatant on the 6th day for purification.

[0076] 3. Purification and Analysis [0077] 1) centrifuge the cell culture; [0078] 2) slowly add the supernatant to an affinity purification column; [0079] 3) use proper buffer solution for cleaning and elution, combine multiple eluates and change the buffer solution to the final formulation to prepare a buffer solution; [0080] 4) subject the purified protein to SDS-PAGE and Western blotting analysis to measure the molecular weight and purity; and [0081] 5) measure the protein concentration by means of the BCA method using BSA as a standard.

[0082] 4. Preparation and Screening of Mouse-Anti-Canine PD-1 Monoclonal Antibody [0083] 1) rapidly immunize mice, 3 Balb/C mice in each group, and two groups in total; [0084] 2) detect serum antigen titers from all the 6 mice by means of ELISA on day 21; [0085] 3) rapidly immunize mice, fusion, select hybridomas; [0086] 4) detect antigen titers in the supernatant of hybridoma cells by using ELISA; [0087] 5) screen the supernatant of hybridoma cell by using flow cytometry; [0088] 6) culture mice monoclonal antibodies, 1 cell/well, thirty 96-well plates in total; [0089] 7) detect antigen titers of the mice monoclonal antibodies by using ELISA, ten plates in total for screening; [0090] 8) select monoclonal antibodies for amplification and freeze preservation; [0091] 9) perform dynamics affinity analysis on mice monoclonal antibody supernatant; and [0092] 10) perform dynamics cross-block analysis on the mice monoclonal antibody supernatant.

[0093] 5. PBMC Separation [0094] Reagents: Lymphoprep™, PBS and RPMI-1640 [0095] Experimental process: [0096] 1) collect 5 mL canine peripheral blood with a heparin sodium-treated anticoagulant tube; [0097] 2) add an equal volume of PBS buffer solution and mix with canine anticoagulation; [0098] 3) add 5 mL lymphocyte separation solution in a density of 1.077 g/mL into a 15 mL centrifugal tube, and add 5 mL blood diluent into the centrifugal tube, centrifuge at 800 g for 20 min at room temperature; [0099] 4) gently insert a capillary pipet into an albuginea layer on the interface, carefully withdraw the middle leucocyte layer, add an equal amount of RPMI-1640, mix well, centrifuge at 800 g for 20 min at room temperature, and discard supernatant; and [0100] 5) resuspend with RPMI-1640, centrifuge at 800 g for 20 min at room temperature, and discard supernatant; then repeat the above steps once.

[0101] 6. PBMC Culture [0102] 1) count PBMC obtained from the lymphocyte separation solution and culture in a 12-well plate at the conditions of 10% FBS, 37° C. and 5% CO.sub.2; [0103] 2) divide PBMC into two groups, in which one group was added with ConA at a final concentration of 0.5 μg/mL and the other group was without ConA; and [0104] 3) optionally, divide the group added with ConA into several treatment groups, for example, for the addition of different antibodies, according to the requirements of the experiment.

[0105] 7. PBMC PD-1 Flow Cytometry Detection [0106] Antibodies: CD3-FITC; M1, M3 and M11 monoclonal antibodies; and PE-labeled anti-canine IgG antibody [0107] 1) count ConA-activated and nonactivated PBMC, respectively, take 5×10 E3 cells respectively and resuspend in 100 μL PBS; [0108] 2) stain PBMC with M1, M3, M11 and CD3-FITC respectively, including non-staining tubes, single staining tubes, and double staining tubes; incubate the antibodies in dark place for 30 min at room temperature; [0109] 3) centrifuge cells at 200 g for 5 min, and discard supernatant, resuspend the cells with 100 μL PBS, add the PE-labeled anti-canine IgG antibody and incubate in dark place for 20 min at room temperature; and [0110] 4) centrifuge cells at 200 g for 5 min, discard supernatant, and resuspend the cells with 500 μL PBS, and detect on a flow cytometer.

Example 1. Preparation of Antigens and Standards

[0111] The amino acids 25-168 of the canine PD-1 protein were picked, and the former signal peptide was removed. This amino acid sequence was add with a His tag at the terminal end for purification and detection. A gene fragment encoding this amino acid sequence was synthesized and cloned into a pUC57 plasmid for preservation, and then subcloned into pcDNA 3.1 plasmid. A large number of transfection plasmids were prepared for the expression in Expi293F cells. Expi293F cells were maintained in suspension culture, and transiently transfected with the plasmids. Then the expressed protein was purified and analyzed for the concentration. Results are shown in FIGS. 1A and 1B. FIG. 1A shows SDS-PAGE electrophoresis pattern, in which lane M is protein markers corresponding to 200 kD, 116 kD, 971d), 66 kD, 44 kD, 29 kD, 20 kD, 14 kD and 6 kD respectively; lane 1 shows purified proteins under reduction conditions; lane 2 shows purified proteins under non-reduction conditions. FIG. 1A shows Western Blotting pattern, in which lane M is protein markers corresponding to 120 kD, 80 kD, 60 kD, 50 kD, 421d), 321(1) and 18 kD respectively; lane P shows a His-tag protein positive control; lane 1 shows purified proteins under reduction conditions; lane 2 shows purified proteins under non-reduction conditions. The primary antibody: mouse-anti-His mAb.

Optimization of codon or not: Codon optimization was performed [0112] Expression system and vector: [0113] Mammal, and expression vector pcDNA3.1 [0114] Host cell line: Expi293F [0115] Optimization of the expression or not: [0116] Expression optimization was performed [0117] Expression system: 40 mL serum-free Expi293F™ expression medium [0118] Protein were obtained from the supernatant of cell culture, and further Purification: purified by HisTrap™ FF Crude [0119] Concentration: 2.4 mg/mL, determined by BCA™ (BSA used as standard) [0120] Purity: About 95%, Coomassie blue-stained SDS-PAGE gel was subjected to densitometric analysis for estimation under non-reduction conditions [0121] Sterilization: Filtered by a 0.22 μm filter and aseptic packaging was performed [0122] Storage: Preserved at −80° C., subpackaged and freeze preserved to avoid repeated freeze thawing [0123] Storage of the buffer solution: 1×PBS, pH 7.2

Example 2. Preparation of Monoclonal Antibodies

[0124] The protein prepared in Example 1 was used as an antigen to immunize mice rapidly, 3 mice in each group, and two groups in total. On day 21, the serum antigen titers were determined for the immunized mice, fused and selected to build a hybridoma library. Supernatant antigen titers of the hybridoma library were determined and screened with flow cytometry. Then monoclonal antibodies were selected for culture, amplified and froze for preservation. Finally, the dynamics affinity and dynamics cross-block analysis were performed for the mice monoclonal antibody supernatant.

[0125] 1. Description of the Parameters for Analyzing the Serum from the Mice on Day 21: [0126] Coat EIA/RIA polystyrene plates with antigen--->block--->primary antibody--->detection antibody--->substrate--->stop and read. [0127] Analysis type: a solid phase antigen ELISA [0128] Ag ID: PD-1 His [0129] Ab ID: PD-1A mice serum, D21 m1-m3 Ab [0130] Ab dilution: 1/1000 [0131] Ab ID: PD-1S mice serum, D21 m1-m3 Ab [0132] Ab dilution: 1/1000 [0133] Ab ID: normal mice serum Ab [0134] Ab dilution: 1/1000 [0135] Detection reagent ID: goat-anti-mouse HRP [0136] Dilution: 1/5000

TABLE-US-00008 TABLE 1 Test results of the serum of mice on day 21 Normal mice Ag PD-1A PD-1A PD-1A PD-1s PD-1s PD-1s serum (ng/mL) m1 m2 m3 m1 m2 m3 Control 1000 3.45 3.43 3.38 3.24 3.45 3.36 0.18 333 3.37 3.31 3.4 2.59 3.29 2.98 0.14 111 3.22 2.44 2.53 1.09 2.22 1.32 0.12 37 2.26 1 1.21 0.48 0.83 0.53 0.11 12 0.8 0.28 0.34 0.18 0.22 0.2 0.08 4 0.35 0.16 0.19 0.13 0.14 0.14 0.07 1 0.18 0.1 0.11 0.11 0.09 0.09 0.09 No 0.1 0.12 0.07 0.09 0.07 0.11 0.08

[0137] Furthermore, referring to FIG. 2, it can be seen from the ELISA experimental results that all 6 mice of the two groups produce good antibody responses 21 days after immunization, and can be subjected to the next experiment.

[0138] 2. Description of the Parameters of the Immunoassay for the Supernatant of the Hybridoma Library [0139] Coat EIA/RIA polystyrene plates with antigen--->block--->primary antibody--->detection antibody--->substrate--->stop and read. [0140] Analysis type: a solid phase antigen ELISA [0141] Ag ID: PD-1 His [0142] Ab ID: PD-1A mice serum, D21 m1-m3 Ab [0143] Ab dilution: 1/1000 [0144] Ab ID: PD-1S mice serum, D21 m1-m3 Ab [0145] Ab dilution: 1/1000 [0146] Ab ID: PD-1A hybridoma library [0147] Ab dilution: undiluted [0148] Ab ID: PD-1S hybridoma library [0149] Ab dilution: undiluted [0150] Detection reagent ID: goat-anti-mouse HRP [0151] Dilution: 1/5000

TABLE-US-00009 TABLE 2 Immunoassay results of the supernatant of the hybridoma library Ag PD-1 A PD-1 A PD-1 S Concentration fused PD-1 S hybridoma hybridoma (ng/mL) serum fused serum library library Medium 1000 3.51 3.50 3.43 3.40 0.05  333 3.39 3.39 3.34 3.23 0.04  111 3.19 2.72 2.75 2.62 0.04  37 2.02 1.12 1.54 1.43 0.05  12 0.88 0.41 0.65 0.59 0.06   4 0.31 0.17 0.30 0.28 0.05   1 0.14 0.15 0.17 0.16 0.05 No 0.08 0.08 0.13 0.12 0.09

[0152] Moreover, referring to FIG. 3, it can be seen from the ELISA results that the supernatants from two hybridoma libraries have good immunoreactions to canine PD-1. Next, monoclonal antibodies can be generated by cloning. The antibodies were cloned in 30 plates of 96-well in total, and each well had a single cell. The single cell was cultured, amplified and preserved. ELISA was performed on the supernatant of each cell culture, so as to screen out monoclonal antibodies having strong activities against canine PD-1.

[0153] 3. Screening of the Monoclonal Antibodies: [0154] Test type: a solid phase antigen ELISA [0155] Ag ID: PD-1 His [0156] Ag concentration/dilution: 100 ng/mL [0157] Ag ID: PD-1A mice serum, D21 m1-m3 [0158] Ab concentration/dilution: 1/1000 [0159] Ab ID: PD-1A hybridoma library [0160] Ab concentration/dilution: undiluted [0161] Ab ID: PD-1A McAb supernatant [0162] Detection reagent ID: goat-anti-mouse HRP-Fc [0163] Dilution: 1/5000

[0164] After ELISA, 30 cell lines were selected, which had good immunoreactions and were likely to produce different antibodies, and renumbered from M1 to M30 as shown by Table 3 below. The antibodies produced by these cell lines were used for the following testing and screening.

TABLE-US-00010 TABLE 3 Screening results of the monoclonal antibodies O.D. determined by ELISA Clone names ELISA/T.C. vessel compared with PD-1 His PD-1 A-M1 2C08 2.65 PD-1 A-M2 3G04 3.11 PD-1 A-M3 4G01 2.87 PD-1 A-M4 4G08 3.23 PD-1 A-M5 5B05 3.11 PD-1 A-M6 5F09 2.71 PD-1 A-M7 6A10 2.57 PD-1 A-M8 7H06 2.80 PD-1 A-M9 8F02 3.09 PD-1 A-M10 8H01 2.83 PD-1 A-M11 9B04 2.66 PD-1 A-M12 9E06 3.06 PD-1 A-M13 10A04 2.74 PD-1 A-M14 10G01 3.30 PD-1 A-M15 11E10 2.34 PD-1 A-M16 11F06 2.78 PD-1 A-M17 13B03 3.00 PD-1 A-M18 13C10 2.50 PD-1 A-M19 13E11 2.87 PD-1 A-M20 14A06 2.93 PD-1 A-M21 15A03 2.89 PD-1 A-M22 15G11 3.02 PD-1 A-M23 18A08 2.58 PD-1 A-M24 19B12 3.16 PD-1 A-M25 21D02 2.54 PD-1 A-M26 21G04 2.81 PD-1 A-M27 23F10 2.64 PD-1 A-M28 26F03 2.56 PD-1 A-M29 29D02 3.03 PD-1 A-M30 29E08 2.73 Negative control HT Medium 0.07 Positive control Positive serum 3.16 Positive control Hybridoma library 3.25

[0165] 4. Affinity Screening of the Monoclonal Antibodies:

[0166] Test Procedures: [0167] Sensor detect (30 s)--->load Ab (700 s)--->negative quench (480 s)--->base line (300 s)--->Ab bind (600 s)-->dissociate (600 s)-->repeat

[0168] The binding or dissociation with the surface can cause migration, and thus the binding was determined by measuring the migration changes over time (see FIG. 4).

[0169] Octet BMIA was used to determine the affinity of the supernatant of the PD-1A clone culture to PD-1 His. Anti-mouse IgG Fc was added to each clone supernatant to capture competence factors.

[0170] The following clones show no PD-1 His binding:

TABLE-US-00011   PD-1A-M2 PD-1A-M4 PD-1A-M5 PD-1A-M6 PD-1A-M7 PD-1A-M9 PD-1A-M12 PD-1A-M13 PD-1A-M14 PD-1A-M16 PD-1 A-M18 PD-1 A-M19 PD-1 A-M20 PD-1 A-M21 PD-1 A-M24 PD-1 A-M25 PD-1 A-M26 PD-1 A-M28 PD-1 A-M29

[0171] FIGS. 5-9 show the changes in the responses of 30 antibodies from Nos. M1 to M30 (M cAb supernatant amplified by undiluted PD-1A) to PD-1 His (a concentration of 150 μg/mL) over time.

TABLE-US-00012 TABLE 4 Sorting of the clone supernatants according to KDs and responses to PD-1 His Analyte concentration Response Antibody ID (nM) (nm) K.sub.D k.sub.on k.sub.dis R.sup.2 PD-1 A-M3 20 0.041  <1.0E−12 1.740E+05  <1.0E−07 0.921 PD-1 A-M10 20 0.044 3.063E−11 2.272E+05 6.959E−06 0.914 PD-1 A-M23 20 0.061 5.371E−10 2.957E+05 1.588E−04 0.900 PD-1 A-M11 20 0.049 9.035E−10 3.456E+05 3.123E−04 0.827 PD-1 A-M30 20 0.059 2.904E−09 8.543E+04 2.481E−04 0.964 PD-1 A-M15 20 0.068 1.039E−08 5.851E+04 6.079E−04 0.964 PD-1 A-M1 20 0.049 1.438E−07 3.059E+03 4.399E−04 0.925 PD-1 A-M8 20 0.069 2.211E−07 3.231E+03 7.143E−04 0.918 PD-1 A-M27 20 0.037 2.333E−07 2.042E+03 4.764E−04 0.922 PD-1 A-M22 20 0.060 2.472E−07 2.700E+03 6.674E−04 0.949 PD-1 A-M17 20 0.012 1.428E−05 2.458E+04 3.509E−01 0.800 PD-1 A-M2 20 −0.011 — — — — PD-1 A-M4 20 0.002 — — — — PD-1 A-MS 20 −0.005 — — — — PD-1 A-M6 20 −0.003 — — — — PD-1 A-M7 20 0.005 — — — — PD-1 A-M9 20 −0.006 — — — — PD-1 A-M12 20 −0.005 — — — — PD-1 A-M13 20 −0.002 — — — — PD-1 A-M14 20 0.006 — — — — PD-1 A-M16 20 0.008 — — — — PD-1 A-M18 20 0.011 — — — — PD-1 A-M19 20 0.000 — — — — PD-1 A-M20 20 0.012 — — — — PD-1 A-M21 20 0.013 — — — — PD-1 A-M24 20 0.010 — — — — PD-1 A-M25 20 0.006 — — — — PD-1 A-M26 20 −0.004 — — — — PD-1 A-M28 20 0.004 — — — — PD-1 A-M29 20 −0.012 — — — — Note: response refers to the response (nm) calculated during the binding step; K.sub.D refers to an affinity constant (M = k.sub.dis/k.sub.on) k.sub.on refers to a binding rate (1/Ms) k.sub.dis refers to a dissociation rate (1/s) Full-R.sup.2 refers to a curve-fitting correlation coefficient

[0172] Thus, antibodies having high affinities for canine PD-1 were obtained, as shown by Table 5 below.

TABLE-US-00013 TABLE 5 Antibodies having high affinities for canine PD-1 PD-1 A-M3 PD-1 A-M10 PD-1 A-M23 PD-1 A-M11 PD-1 A-M30 PD-1 A-M15 PD-1 A-M1 PD-1 A-M8 PD-1 A-M27 PD-1 A-M22

[0173] 5. Screening of the Block Function of the Monoclonal Antibodies [0174] Preincubate of antibodies and antigens (1 h)--->balance (30 s)--->Load Lead Ab(600 s)--->quench (300 s)--->base line (300 s)-->Ab+Ag bind (300 s)-->repeat

[0175] The binding or dissociation with the surface can cause migration, and thus the binding was determined by measuring the migration changes over time (see FIG. 10).

[0176] D-L1 Fc (5p g/mL) was loaded on an AHC sensor for cross-linking assay. Epitope analysis was used to determine the binding of antibodies loaded on the sensor to recombinant antibodies preincubated with PD1-Fc. The binding (response) degrees observed were compared to that of antigen only under the same conditions. If the total response of an antibody was higher than that of antigen only, the antibody would be matched. If the response of an antibody was lower than 80% of that of antigen, the antibody would be blocked with each other.

[0177] Sorted by the lowest to highest % of the response, the following clones blocking the binding of PD1-Fc to PD-L1 were obtained: [0178] PD1 A-M27 [0179] PD1 A-M1 [0180] PD1 A-M22 [0181] PD1 A-M8 [0182] PD1 A-M11 [0183] PD1 A-M15 [0184] PD1 A-M3 [0185] PD1 A-M23 [0186] PD1 A-M10 [0187] PD1 A-M13 [0188] PD1 A-M21 [0189] PD1 A-M19 [0190] PD1 A-M18 [0191] PD1 A-M30 [0192] PD1 A-M24 [0193] PD1 A-M6 [0194] PD1 A-M16 [0195] PD1 A-M20 [0196] PD1 A-M7 [0197] PD1 A-M17

[0198] FIGS. 11-12 show the test results for which the loaded Ab is PD-L1 Fc, the concentration (μg/mL) is 5; Ab is PD1A McAb supernatant, the dilution ratio is ½, quenching Ab is human γ-immunoglobulin in a concentration (μg/mL) of 150, the analyte is PD1 Fc in a concentration (nM) of 50.

TABLE-US-00014 TABLE 6 Sorting of response by % Response (lowest to highest) Lead Ab ID Second Ab ID (nm) % response Block/pair Lead Ab ID Secondary Ab ID Response (nm) % response Block/pair PD-Ll Fc PD1 A - M1 −0.009 -21.90% Block PD-Ll Fc PD1 A - M27 −0.012 −22.04% Block PD-Ll Fc PD1 A - M2 0.050 118.57% Pair PD-Ll Fc PD1 A - M1 −0.009 −21.90% Block PD-Ll Fc PD1 A - M3 −0.002 -5.00% Block PD-Ll Fc PD1 A - M22 −0.013 −20.53% Block PD-Ll Fc PD1 A - M4 0.034 81.19% N/A PD-Ll Fc PD1 A - M8 −0.009 −18.56% Block PD-Ll Fc PD1 A - M5 0.047 111.19% Pair PD-Ll Fc PD1 A - M11 −0.008 −15.97% Block PD-Ll Fc PD1 A - M6 0.025 58.81% Block PD-Ll Fc PD1 A - M15 −0.005 -8.21% Block PD-Ll Fc Only Ag (PD1 Fc) 0.042 100.00% N/A PD-Ll Fc PD1 A - M3 −0.002 -5.00% Block PD-Ll Fc PD1 A - M7 0.036 71.26% Block PD-Ll Fc PD1 A - M23 0.000 −0.47% Block PD-Ll Fc PD1 A - M8 −0.009 −18.56% Block PD-Ll Fc PD1 A - M10 0.006 11.98% Block PD-Ll Fc PD1 A - M9 0.048 95.41% N/A PD-Ll Fc PD1 A - M13 0.013 22.11% Block PD-Ll Fc PD1 A - M10 0.006 11.98% Block PD-Ll Fc PD1 A - M21 0.015 23.48% Block PD-Ll Fc PD1 A - M11 −0.008 −15.97% Block PD-Ll Fc PD1 A - M19 0.017 27.06% Block PD-Ll Fc PD1 A - M12 0.052 102.99% Pair PD-Ll Fc PD1 A - M18 0.025 42.38% Block PD-Ll Fc Only Ag (PD1 Fc) 0.050 100.00% N/A PD-Ll Fc PD1 A - M30 0.023 43.15% Block PD-Ll Fc PD1 A - M13 0.013 22.11% Block PD-Ll Fc PD1 A - M24 0.037 57.23% Block PD-Ll Fc PD1 A - M14 0.052 87.60% N/A PD-Ll Fc PD1 A - M6 0.025 58.81% Block PD-Ll Fc PD1 A - M15 −0.005 -8.21% Block PD-Ll Fc PD1 A - M16 0.037 61.81% Block PD-Ll Fc PD1 A - M16 0.037 61.81% Block PD-Ll Fc PD1 A - M20 0.042 64.85% Block PD-Ll Fc PD1 A - M17 0.044 74.20% Block PD-Ll Fc PD1 A - M7 0.036 71.26% Block PD-Ll Fc PD1 A - M18 0.025 42.38% Block PD-Ll Fc PD1 A - M17 0.044 74.20% Block PD-Ll Fc Only Ag (PD1 Fc) 0.060 100.00% N/A PD-Ll Fc PD1 A - M4 0.034 81.19% N/A PD-Ll Fc PD1 A - M19 0.017 27.06% Block PD-Ll Fc PD1 A - M14 0.052 87.60% N/A PD-Ll Fc PD1 A - M20 0.042 64.85% Block PD-Ll Fc PD1 A - M9 0.048 95.41% N/A PD-Ll Fc PD1 A - M21 0.015 23.48% Block PD-Ll Fc PD1 A - M26 0.053 98.70% N/A PD-Ll Fc PD1 A - M22 −0.013 −20.53% Block PD-Ll Fc PD1 A - M12 0.052 102.99% Pair PD-Ll Fc PD1 A - M23 0.000 −0.47% Block PD-Ll Fc PD1 A - M25 0.056 104.44% Pair PD-Ll Fc PD1 A - M24 0.037 57.23% Block PD-Ll Fc PD1 A - M28 0.057 105.56% Pair PD-Ll Fc Only Ag (PD1 Fc) 0.064 100.00% N/A PD-Ll Fc PD1 A - M5 0.047 111.19% Pair PD-Ll Fc PD1 A - M25 0.056 104.44% Pair PD-Ll Fc PD1 A - M2 0.050 118.57% Pair PD-Ll Fc PD1 A - M26 0.053 98.70% N/A PD-Ll Fc PD1 A - M29 0.067 124.26% Pair PD-Ll Fc PD1 A - M27 −0.012 −22.04% Block PD-Ll Fc Only Ag (PD1 Fc) 0.042 100.00% N/A PD-Ll Fc PD1 A - M28 0.057 105.56% Pair PD-Ll Fc Only Ag (PD1 Fc) 0.050 100.00% N/A PD-Ll Fc PD1 A - M29 0.067 124.26% Pair PD-Ll Fc Only Ag (PD1 Fc) 0.060 100.00% N/A PD-Ll Fc PD1 A - M30 0.023 43.15% Block PD-Ll Fc Only Ag (PD1 Fc) 0.064 100.00% N/A PD-Ll Fc Only Ag (PD1 Fc) 0.054 100.00% N/A PD-Ll Fc Only Ag (PD1 Fc) 0.054 100.00% N/A

Abbreviation

[0199] Abbreviation: [0200] Keywords: Response Response (nm) calculated during the binding step [0201] KD Affinity constant (M)=k.sub.dis/k.sub.on [0202] k.sub.on Binding rate (1/Ms) [0203] k.sub.dis Dissociation rate (1/s) [0204] Full-R.sup.2 Curve-fitting correlation coefficient

[0205] The antibodies having strong ability to block the binding of canine PD-1 to PD L-1 were finally confirmed below: [0206] PD1 A-M27 [0207] PD1 A-M1 [0208] PD1 A-M22 [0209] PD1 A-M8 [0210] PD1 A-M11 [0211] PD1 A-M15 [0212] PD1 A-M3 [0213] PD1 A-M23 [0214] PD1 A-M10 [0215] PD1 A-M13

[0216] 6. Final Screening of the Monoclonal Antibodies

[0217] The following 10 monoclonal antibodies were selected for antibody subtype tests, according to the affinity test results and block function screen results.

TABLE-US-00015 TABLE 7 Antibody light Monoclonal antibody Affinity Block Function chain subtype M3 <1.000E.sup.−12 −5.00% IgG1 kappa M10 3.063E.sup.−11 11.98% IgG1 kappa M23 5.371E.sup.−10 −0.47% IgG1 κ M11 9.035E.sup.−10 −15.97% IgG1 κ M13 / 22.11% IgG2b κ M15 1.039E.sup.−08 −8.21% IgG1 κ M1 1.438E.sup.−07 −21.90% IgG1 κ M8 2.211E.sup.−07 −18.56% IgG1 κ M27 2.333E.sup.−07 −22.04% IgG1 κ M22 2.472E−07 −20.53% IgG1 κ

[0218] Then, six cell lines were selected from the 10 antibodies for DNA sequence analysis. The selected 6 antibodies were M1, M3, M10, M11, M15 and M23.

Example 3. Determination, Caninization and Expression of the Monoclonal Antibodies

[0219] (1) Sequence Determination of the Monoclonal Antibodies [0220] 1) extract RNA after the centrifugation and precipitation of hybridoma cell lines; [0221] 2) perform reverse transcription RT-PCR (5′RACE) and amplification of the light- and heavy chain variable regions of the hybridoma antibodies; [0222] 3) clone DNA fragments produced by PCR into a sequencing plasmids and transform into Escherichia coli; [0223] 4) pick 6-12 bacterial colonies and extract plasmid DNA, respectively; [0224] 5) design primer sequences for the plasmid to sequence the cloned DNA fragments; [0225] 6) determine the common sequences shared by several bacterial colones; and [0226] 7) perform analysis to obtain the cDNA sequences of the antibody variable regions.

[0227] (2) cDNA Sequences of the Variable Regions of the Mouse-Anti-Canine PD-1 Monoclonal Antibodies

[0228] 1) Protein Sequences in the Variable Regions of the Selected 6 Antibodies

TABLE-US-00016 M1K QIVLTQSPAIMSASPEKVTMTCSASSSVTYMYWFQQKPGSSPRLWIYDTSNLVSGVPARFSGSRSGTSYSLTISS MEAEDAATYYCQQYSGHPSSFGSGTKLEIK M3K QIVLTQSPAIMSASPEKVTMTCSASSSVSYMFWYQQKPGSSPRLWIYDTSNLVSGVPARFSGSRSGTSYSLTISS MEAEDAATYYCQQYSGHPSSFGSGTKLEIK M10K QIVLTQSPAIMSASPEKVTMTCSASSSVSYMFWYQQKPGSSPRLWIYDTSNLVSGVPARFSGSRSGTSYSLTISS MEAEDAATYYCQQYSGHPSSFGSGTKLEIK M11K QIVLTQSPAIMSASPEKVTMTCSASSSVTYMFWYQQKPGSSPRLWIYDTSNLVSGVPARFSGSRSGTSYSLTISS MEAEDAATYYCQQYSGHPSSFGSGTKLEIK M15K QIVLTQSPAIMSASPEKVTMTCSASSSVSYMFWYQQKPGSSPRLWIYDTSNLVSGVPARFSGSRSGTSYSLTISS MEAEDAATYYCQQYSGHPSSFGSGTKLEIK M23K QIVLTQSPAIMSASPEKVTMTCSASSSVSYMFWYQQKPGSSPRLWIYDTSNLVSGVPARFSGSRSGTSYSLTISS MEAEDAATYYCQQYSGHPSSFGSGTKLEIK M1H EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQLEWIGRIDPAIDNTKYDPKFQGKATITADTSS NTAYQLSSTSEDTAVYYCASGFYTMDYWGQGTSVTVSS M3H EVRLQQSGAELVKPGASVKLSCTASGFNIKDTYLHWLKERPEQLEWIGRIDPAIGNTRYDPKFQVKATITADTSS NTAYQLSSTSEDSAVYYCARGFYGMDYWGQGTSVTVSS M10H EVRLQQSGAELVKPGASVKLSCTASGFNIKDTYLHWLKERPEQLEWIGRIDPAIGNTRYDPKFQVKATITADTSS NTAYQLSSTSEDSAVYYCARGFYGMDYWGQGTSVTVSS M11H EVQLQQSGAEVVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQLEWIGRIDPAIDNPKYDPKFQGKATITADTSS NTAYQLSSTSEDTAVYYCASGFYAMDCWGQGTSVTVSS M15H EVRLQQSGAELVKPGASVKLSCTASGFNIKDTYLHWLKERPEQLEWIGRIDPAIDNTRYDPKFQVKATITADTSS NTAYQLSSTSEDSAVYYCARGFYGMDYWGQGTSVTVSS M23H EVRLQQSGAELVKPGASVKLSCTASGFNIKDTYLHWLKERPEQLEWIGRIDPAIDNTRYDPKFQVKATITADTSS NTAYQLSSTSEDSAVYYCARGFYGMDYWGQGTSVTVSS

[0229] The selected 6 antibodies were M1, M3, M10, M11, M15 and M23. K: light chain, H: heavy chain.

[0230] (2) cDNA Sequences of the Variable Regions of the Selected 6 Antibodies

TABLE-US-00017 M1K: GCAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCA TGACCTGCAGTGCCAGCTCAAGTGTAACTTACATGTATTGGTTCCAGCAGAAGCCAGGCTCC TCCCCCAGACTCTGGATTTATGACACATCCAACCTGGTTTCTGGAGTCCCTGCTCGCTTCAG TGGCAGTAGGTCTGGGACCTCTTATTCTCTCACAATCAGCATATGGAGGCTGAAGATGCTGC CACTTATTACTGCCAGCAGTACAGTGGTCACCCATCCTCGTTCGGCTCGGGGACAAAGTTGG AAATTAAA M3K: CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCAT GACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTTCTGGTACCAGCAGAAGCCAGGCTCCT CCCCCAGACTCTGGATTTATGACACATCCAACCTGGTTTCTGGAGTCCCTGCTCGCTTCAGT GGCAGTAGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGC CACTTATTACTGCCAACAGTACAGTGGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTGG AAATAAAG M10K: CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCAT GACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTTCTGGTACCAGCAGAAGCCAGGCTCCT CCCCCAGACTCTGGATTTATGACACATCCAACCTGGTTTCTGGAGTCCCTGCTCGCTTCAGT GGCAGTAGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGC CACTTATTACTGCCAACAGTACAGTGGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTGG AAATAAAG M11K: CAAATTGTTCTCACCCAGTCTCCAGCCATCATGTCTGCATCTCCAGGGGAAAAGGTCACCAT GACCTGCAGTGCCAGCTCAAGTATAACTTACATGTTCTGGTACCAGCAGAAGCCAGGCTCCT CCCCCAGACTCTGGATTTATGACACATCCAACCTGGTTTCTGGAGTCCCTGCTCGCTTCAGT GGCAGTAAGTCTGGGACCTCTTATTCTCTCACAATCACCAGCATGGAGGCTGAAGATGCTGC CACTTATTACTGCCAGCAGTACAGTGGTTACCCATCCACGTTCGGCTCGGGGACAAAGTTGG AAATAAAA M15K: CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCAT GACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTTCTGGTACCAGCAGAAGCCAGGCTCCT CCCCCAGACTCTGGATTTATGACACATCCAACCTGGTTTCTGGAGTCCCTGCTCGCTTCAGT GGCAGTAGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGC CACTTATTACTGCCAACAGTACAGTGGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTGG AAATAAAG M23K: CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCAT GACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTTCTGGTACCAGCAGAAGCCAGGCTCCT CCCCCAGACTCTGGATTTATGACACATCCAACCTGGTTTCTGGAGTCCCTGCTCGCTTCAGT GGCAGTAGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGC CACTTATTACTGCCAACAGTACAGTGGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTGG AAATAAAG M1H: GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTC CTGCACAGCTTCTGGCTTCAACATCAAAGACACCTATATGCATTGGGTGAAGCAGAGGCCTG AACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGATTGATAATACTAAATATGACCCG AAGTTCCAGGGCAAGGCCACTATAACAGCTGACACATCCTCCAACACAGCCTACCTGCAGCT CAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTTCTGGGTTCTATACTATGG ACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA M3H: GAGGTTCGGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTC CTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATTTACACTGGTTGAAGGAGAGGCCTG AACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGATTGGTAATACTAGATATGACCCG AAGTTCCAGGTCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAACT CAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCTAGAGGGTTCTATGGTATGG ACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA M10H: GAGGTTCGGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTC CTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATTTACACTGGTTGAAGGAGAGGCCTG AACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGATTGGTAATACTAGATATGACCCG AAGTTCCAGGTCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAACT CAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCTAGAGGGTTCTATGGTATGG ACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA M11H: GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGGTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTC CTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATATGCACTGGGTGAAGCAGAGGCCTG AACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGATTGGTAATCCTAAATATGACCCG AAGTTCCAGGGCAGGGCCACTATAACTGCTGACACATCCTCCAACACAGCCTACCTGCAGCT CAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTTCTGGGTTCTATGCTATGG ACTGCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA M15H: GAGGTTCGGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTC CTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATTTACACTGGTTGAAGGAGAGGCCTG AACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGATTGGTAATACTAGATATGACCCG AAGTTCCAGGTCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAACT CAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCTAGAGGGTTCTATGGTATGG ACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA M23H: GAGGTTCGGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTC CTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATTTACACTGGTTGAAGGAGAGGCCTG AACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGATTGGTAATACTAGATATGACCCG AAGTTCCAGGTCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAACT CAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCTAGAGGGTTCTATGGTATGG ACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0231] (3) Sequencing Result Analysis on the Selected Antibodies

[0232] Based on the antibody sequencing results, it was found that, in the 6 antibodies, M3, M10, M15 and M23 were the same antibody. Therefore, the finally confirmed antibodies were M1, M3 and M11.

[0233] (4) Caninization and Affinity Experimental Results of the Monoclonal Antibodies:

[0234] Heavy chain constant regions and light chain constant regions of the three monoclonal antibodies M1, M3 and M11 were replaced with a heavy chain constant region (as shown by SEQ ID NO: 28) and a light chain constant region (as shown by SEQ ID NO: 29) of the canine PD-1 monoclonal antibody. The resulted caninized monoclonal antibodies were respectively named C1, C3 and C11. C1 corresponds to the monoclonal antibody (NO. 35.38) in the table below; C3 corresponds to the monoclonal antibody (NO. 36.39) in the table below; and C11 corresponds to the monoclonal antibody (NO. 37.40) in Table 8 below.

TABLE-US-00018 TABLE 8 Antibody ID K.sub.D k.sub.on k.sub.dis R.sup.2 C1(35.38) 1.595E.sup.−09 2.512E.sup.+08 4.007E.sup.−01 0.778 C3(36.39) <1.0E.sup.−12 6.265E.sup.+02 <1.0E.sup.−07 0.714 C11(37.40) <1.0E.sup.−12 1.190.sup.+03 <1.0E.sup.−07 0.667

TABLE-US-00019 TABLE 9 PD-1 His Response Antibody ID Conc. (nM) (nm) K.sub.D k.sub.on k.sub.dis R.sup.2 35.38 1000.0 0.001 — — — — 35.38 333.3 −0.004 — — — — 35.38 111.3 0.016 2.060E−09  4.300E+05 8.859E−04  0.377 35.38 37.3 0.008 2.647E−09  3.599E+06 9.528E−03  0.819 35.38 12.4 0.001 2.456E−09  8.046E+06 1.976E−02  0.380 35.38 4.0 0.005 1.835E−08  6.932E+07 1.272E+00  0.843 35.38 1.1 −0.031 — — — — Global Fit: 1.595E−09  2.512E+08 4.007E−01  0.778 36.39 1000.0 0.007 <1.0E−12 3.296E+02 <1.0E−07 0.334 36.39 333.3 0.010 <1.0E−12 1.765E+03 <1.0E−07 0.661 36.39 111.3 0.026 <1.0E−12 1.470E+04 <1.0E−07 0.804 36.39 37.3 0.017 <1.0E−12 4.051E+04 <1.0E−07 0.887 36.39 12.4 0.005 — — — — 36.39 4.0 0.009 — — — — 36.39 1.1 0.002 — — — — Global Fit: <1.0E−12 6.265E+02 <1.0E−07 0.714 37.40 1000.0 0.009 <1.0E−12 1.474E+03 <1.0E−07 0.368 37.40 333.3 0.012 <1.0E−12 9.474E+02 <1.0E−07 0.838 37.40 111.3 0.025 <1.0E−12 2.375E+04 <1.0E−07 0.715 37.40 37.3 0.015 <1.0E−12 6.755E+04 <1.0E−07 0.703 37.40 12.4 0.008 <1.0E−12 5.010E+04 <1.0E−07 0.588 37.40 4.0 0.004 — — — — 37.40 1.1 −0.003 — — — — Global Fit: <1.0E−12 1.190E+03 <1.0E−07 0.667 K.sub.D Affinity constant (M) = k.sub.dis/k.sub.on k.sub.on Binding rate (1/Ms) k.sub.dis Dissociation rate (1/s) Full-R.sup.2 Curve-fitting correlation coefficient

Example 4. The Monoclonal Antibodies Did not Influence the Normal Growth of Lymphocyte

[0235] After being stimulated with 0.5 μg/mL ConA (concanavalin A), lymphocyte were amplified. The binding of caninized monoclonal antibodies C1, C3 and C11 to PBMC did not influence the amplification of lymphocyte. The upper and lower pictures in FIG. 13 correspond to the growth of PBMC observed under 4× and 10× lens, respectively. It can be seen that the cells are in a rapid growth period, and cell cloning is obvious. Therefore, the caninized monoclonal antibodies C1, C3 and C11 obtained by the present application do not influence the normal growth of lymphocyte.

Example 5. The Monoclonal Antibodies can Effectively Bind to PD1 on the Surface of T Cells

[0236] The cells were stimulated by 0.5 μg/mL ConA for 3 days, and collected. PBMC was incubated with canine-anti-CD3-FITC and caninized monoclonal antibodies C1, C3 and C11 respectively. Then the cells were incubated again with PE-labeled canine-IgG antibody. The detection results show that T cells account for 62.1%, 56.0% and 65.4%, respectively, in total PBMC. PD1 positive cells account for 94.6%, 96.2% and 96.7%, respectively, in T cells, indicating that the caninized monoclonal antibodies C1, C3 and C11 can effectively bind to PD1 on the surface of T cells. Moreover, the results also indicate that the caninized monoclonal antibodies C1, C3 and C11 have potential blocking effect on the PD1/PD-L1 signaling pathway, and thus have protective effect on the function of T cells (see FIG. 14).

Example 6. The Addition of the Monoclonal Antibodies Did not Influence the Release of Interferon

[0237] After being stimulated by 0.5 μg/mL ConA, lymphocyte were amplified. The amplified PBMC were taken, and incubated with the caninized monoclonal antibodies C1, C3 and C11 respectively to detect the influence of the addition of the antibodies on the release capacity of PBMC interferon. After the antibodies were bound to PBMC for 72 h, the supernatant was collected to detect the level of IFNγ. The results show that the interferon release levels of the activated PBMC are higher than that of the unactivated PBMC. Thus, the addition of the antibodies do not influence the release of interferon. In addition, there is no distinct difference among the three antibodies (see FIG. 15).