Anti-canine PD-L1 monoclonal antibody or antigen binding fragment and application thereof

Abstract

The invention provides an anti-canine PD-L1 monoclonal antibody or an antigen binding fragment thereof and an application of the anti-canine PD-L1 monoclonal antibody or the antigen binding fragment. A heavy chain variable region and a light chain variable region of the anti-canine PD-L1 monoclonal antibody or the antigen binding fragment of the anti-canine PD-L1 monoclonal antibody comprise amino acid sequences as shown in SEQ ID NO.1-6. The anti-canine PD-L1 monoclonal antibody is formed by assembling a murine antibody variable region and a murine antibody constant region sequence, and has biological binding activity with canine PD-L1 protein, so that the anti-canine PD-L1 gene engineering monoclonal antibody provided by the invention can provide a basis for modification and development of canine antibodies, and can be used for preparing the anti-canine PD-L1 gene engineering monoclonal antibody. The compound can play a role in treating canine tumors or canine autoimmune diseases.

Description

Technical Field

[0001] The present invention belongs to the field of canine tumor immunotherapy drugs and relates to anti-canine PD-L1 antibodies, and more specifically, to anti-canine PD-L1 monoclonal antibodies or antigen-binding fragments thereof and applications. Background Art

[0002] PD-1 and PD-L1 in the PD-1 / PD-L1 signaling pathway have been widely developed as immunosuppressant drugs for the treatment of human cancer and immune diseases. For example, the FDA has approved three PD-1 antibodies, namely nivolumab, pembrolizumab, and cemiplimab; and three PD-L1 antibodies, namely atezolizumab, avelumab, and durvalumab.

[0003] When it comes to human and veterinary drugs, veterinary drugs are not simply cheap versions of human drugs. Veterinary drugs are specially made to prevent and treat animal diseases. They are suitable for the physiological functions, metabolic characteristics and some unique biological diseases of animals. Moreover, humans and animals of different species have very different responses to drugs. Based on the development of human PD-1 and PD-L1 immunosuppressant drugs, researchers explored the homology between pet dog and human cancers and found that pet dog and human cancers have common disease biological characteristics. Similar signaling pathways were also found to be altered in pet dog cancers. The most important signaling pathways found in canine cancers are PI3K, RTK / RAS / MAPK, WNT / b-catenin or cell cycle signals.

[0004] Currently, research on canine immunosuppressants is in its infancy, and the mechanism of action of canine PD-L1 needs to be fully explored. Therefore, the anti-canine PD-L1 monoclonal antibody provided by the present invention may provide a new approach to the research and development of canine immunosuppressants. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a genetically engineered anti-canine PD-L1 monoclonal antibody.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an anti-canine PD-L1 monoclonal antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region (VH) of the anti-canine PD-L1 monoclonal antibody or the antigen-binding fragment thereof comprises VH CDR1, VH CDR2 and VHCDR3, wherein:

[0008] VH CDR1 is the amino acid sequence shown in SEQ ID NO.1;

[0009] VH CDR2 is the amino acid sequence shown in SEQ ID NO. 2;

[0010] VH CDR3 has the amino acid sequence shown in SEQ ID NO.3.

[0011] As a preferred technical solution of the present invention, the light chain variable region (VL) of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment includes VL CDR1, VL CDR2 and VL CDR3, wherein:

[0012] VL CDR1 is the amino acid sequence shown in SEQ ID NO. 4;

[0013] VL CDR2 is the amino acid sequence shown in SEQ ID NO.5;

[0014] VL CDR3 has the amino acid sequence shown in SEQ ID NO.6.

[0015] The anti-canine PD-L1 monoclonal antibody provided in the present invention is composed of a combination of a murine antibody variable region and a murine antibody constant region. Experimental studies have shown that it can bind to the canine PD-L1 protein and has a high affinity for the canine PD-L1 protein. Therefore, it can provide researchers with a new idea and research target in the study of canine immunosuppressants.

[0016] The specific sequence is shown in Table 1 below:

[0017] Table 1

[0018]

[0019]

[0020] As a preferred technical solution of the present invention, the amino acid sequence of the heavy chain variable region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment (SEQ ID NO.7) is:

[0021] EVQLQQSGAELVRPGSSVKMSCKTSGYTFT SYGIN WVKQRPGQGLEWI G YIYIGNGYSENNEKFKG KATLTSDTSSSTAYMQLSGLTSEDSANYFCAR LLF GGYSDY WGQGTTLTVSS (the underlined part is the CDR region)

[0022] As a preferred technical solution of the present invention, the amino acid sequence of the heavy chain constant region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment (SEQ ID NO.8) is:

[0023] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVH TAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0024] As a preferred technical solution of the present invention, the amino acid sequence of the light chain variable region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment (SEQ ID NO.9) is:

[0025] DIQMTQSPSSLSASLGGNVTITC KASQDIHKYIA WYQHKPGKGPRLLIH Y TSTLQP GIPSRFSGDGGSGRDFSFSISNLEPEDIATYYC LQYDNLLRT FGGGTKLE IK (the underlined part is the CDR region)

[0026] As a preferred technical solution of the present invention, the amino acid sequence of the light chain constant region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment (SEQ ID NO.10)

[0027] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0028] The present invention relates to genetically engineered antibody technology. The mouse anti-PD-L1 antibody variable region of the present invention is obtained by immunizing mice with human PD-L1 as an antigen to obtain hybridoma cells, retrieving the mouse anti-human PD-L1 antibody variable region, and assembling the mouse anti-human PD-L1 antibody variable region with the mouse antibody constant region to obtain a mouse anti-canine PD-L1 monoclonal antibody. After sequence optimization, the anti-canine PD-L1 genetically engineered antibody is obtained by eukaryotic expression, which can provide a basis for the modification and development of canine antibodies.

[0029] In a second aspect, the present invention provides a gene sequence encoding the anti-canine PD-L1 monoclonal antibody or antigen-binding fragment thereof as described in the first aspect, or its complementary sequence.

[0030] As a preferred technical solution of the present invention, the gene sequence is codon-optimized according to the preference of the host cell, and the host cell includes HEK-293 cells.

[0031] It should be noted that codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell by replacing at least one codon of a native sequence with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the amino acid sequence. Various species exhibit specific biases for certain codons of specific amino acids. Codon bias (differences in codon selection between organisms) is generally associated with the translation efficiency of messenger RNA (mRNA), which in turn is believed to depend, in particular, on the properties of the translated codons and the availability of specific transfer RNA (tRNA) molecules. The predominance of the selected tRNA in the cell generally reflects the codons that are most frequently used in peptide synthesis. Therefore, genes can be customized based on codon optimization to perform optimal gene expression in a given organism. Codon optimization can be achieved by converting the nucleotide sequence of one species into the genetic sequence of a different species. Optimized codons help achieve faster translation speeds and higher accuracy.

[0032] In the present invention, the anti-canine PD-L1 monoclonal antibody can be optimized according to the codon preference of the selected host cell and is not limited to being optimized using only HEK-293 codons.

[0033] As a preferred technical solution of the present invention, the gene sequence is optimized according to the HEK-293 codon, the gene sequence shown in SEQ ID NO.11 encodes the heavy chain variable region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment, and the gene sequence shown in SEQ ID NO.12 encodes the light chain variable region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment.

[0034] Anti-canine PD-L1 monoclonal antibody heavy chain variable region gene sequence (SEQ ID NO.11)

[0035] GAAGTCCAGTTGCAGCAAAGCGGAGCAGAACTTGTGCGCCCTGGAAGCAGCGTTAAAATGTCCTGCAAAACCTCTGGGTATACCTTCACC TCATACGGCATCAAT TGGGTTAAGCAACGCCCTGGACAAGGACTTGAGTGGATCGGA TACATTTACATTGGAAACGGGTACTCCGAGAATAACGAAAAGTTCAAAGGG AAAGCCAACTCACCAGCGACACTTCTTCTTCCACCGCTTATATGCAGCTGTCAGGACTGACATCCGAGGACTCTGCAAACTACTTCTGTGCCAGA CT CCTGTTCGGGGGATATAGTGACTAT TGGGGCCAGGGGACCACTCTGACCGTAAGCTCC (the underlined part is the CDR region)

[0036] Anti-canine PD-L1 monoclonal antibody light chain variable region gene sequence (SEQ ID NO.12)

[0037] GATATCCAAATGACGCAATCCCCTTTCTAGCCTCTCTGCCTCCCTCGGCGGAAATGTCACCATTACCTGT AAGGCCTCACAGGACATTCATAAGTACATCGCT TGGTATCAGCACAAGCCCGGGAAAGGCCCAAGGCTGCTGATTCAC TATACCTCTACACTGCAACCT GGCATACCTAGCAGATTCAGTGGAGATGGAAGTGGTCGCGACTTCAGCTTAGTATTTCCAACTTGGAGCCTGAGGACATTGCTA CATACTACTGT CTTCAGTACGATAACTTGCTCCGAACA TTTGGTGGAGGCACTAAGCTGGAGATTAAG (the underlined part is the CDR region)

[0038] As a preferred technical solution of the present invention, the gene sequence is optimized according to the HEK-293 codon, the gene sequence shown in SEQ ID NO.13 encodes the heavy chain constant region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment, and the gene sequence shown in SEQ ID NO.14 encodes the light chain constant region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment.

[0039] Anti-canine PD-L1 monoclonal antibody heavy chain constant region gene sequence (SEQ ID NO.13)

[0040] GCCAAGACTACCCCCCCATCTGTCTACCCACTTGCACCCGGTTCTGCAGCGCAGACAAATAGTATGGTGACTCTCGGGTGTTTGGTTAAGGGGTACTTCCCTGAACCCGTCACTGTGACTTGGAATAGCGGATCCCTCTCCAGTGGAGTTCACACCTTCCCAGCCGTGCTCCAGAGTGATCTGTATACTCTCTCCAGCTCCGTTACGGTCCCTTCTAGCCCTCGGCCTTCCGAGACTGTGACTTGTAATGTGGCACATCCCGCATCATCAACCAAAGTCGACAAGAAAATCGTCCCTCGGGACTGTGGATGCAAACCTTGCATCTGCACTGTCCCCGAAGTCTCTTCAGTGTTTATCTTCCCACCCAAACCAAAAGACGTCCTGACAATCACACTGACCCCCAAGGTCACATGCGTGGTAGTGGATATCAGCAAAGACGATCCCGAGGTACAGTTTTCTTGGTTTGTCGACGATGTGGAGGTCCATACAGCGCAGACCCAGCCTAGGGAGGAACAGTTTAACTCCACTTTCCGCTCTGTTAGCGAACTCCCAATTATGCATCAGGATTGGCTGAATGGCAAAGAGTTCAAATGCCGCGTCAACAGTGCCGCTTTCCCCGCGCCAATCGAGAAGACAATCAGTAAGACGAAGGGCCGACCAAAAGCTCCACAGGTGTACACGATTCCCCCTCCAAAGGAGCAGATGGCTAAGGATAAGGTGAGCCTCACTTGCATGATTACGGATTTTTTCCCCGAAGATATTACTGTCGAATGGCAGTGGAACGGTCAGCCCGCAGAGAACTACAA GAATACCCAACCCATCATGAATACCAACGGCTCCTATTTCGTGTACTCTAAGCTGAATGTCCAGAAAAGCAATTGGGAAGCCGGGAATACCTTTACATGTTCTGTGCTGCATGAGGGCCTCCACAACCATCACACTGAAAAGTCTCTGTCACATTCTCCCGGCAAATGA

[0041] Anti-canine PD-L1 monoclonal antibody light chain constant region gene sequence (SEQ ID NO.14)

[0042] AGGGCCGACGCAGCACCTACCGTGAGTATTTTCCCCCCCAAGTAGTGAGCAGTTGACCTCTGGAGGCGCCTCCGGTGGTGCTTCCTGAACAATTTTTACCCAAAAGACATCAACGTCAAGTGGAAAATCGATGGGTCCGAAAGGCAGAACGGTGTGCTGAAT AGTTGGACCGACCAGGACTCAAAAGACAGTACATACTCTATGTCCTCCACGCTGACACTTACCAAAGATGAATACGAGCGACACAACTCCTACACGTGCGAGGCTACCCATAAGACCTCAACCTCTCCCATTGTCAAGAGCTTTAACCGGAATGAGTGTTGA

[0043] In a third aspect, the present invention further provides an expression vector comprising the gene sequence as described in the second aspect or its complementary sequence.

[0044] In a fourth aspect, the present invention further provides a pharmaceutical composition, comprising the anti-canine PD-L1 monoclonal antibody or antigen-binding fragment thereof according to the first aspect, the gene sequence or its complementary sequence according to the second aspect, or the expression vector according to the third aspect.

[0045] In a fifth aspect, the use of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment as described in the first aspect, the gene sequence or its complementary sequence as described in the second aspect, or the expression vector as described in the third aspect in the preparation of anti-canine PD-L1 chimeric antibodies and drugs for treating canine tumors or canine autoimmune diseases.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] As immunotherapy gradually becomes an important treatment method, the present invention immunizes mice with human PD-L1 protein as an antigen, retrieves the variable region of the mouse antibody and the constant region sequence of the mouse antibody from the obtained hybridoma cells, assembles them, constructs a eukaryotic expression vector and performs eukaryotic expression, and uses ELISA to identify the biological activity of the antibody. The results show that the expressed genetically engineered monoclonal antibody has biological binding activity with canine PD-L1 protein. This PD-L1 genetically engineered antibody may be developed into a new type of immunosuppressant in the future, playing a role in the treatment of canine tumors or canine autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the SDS-PAGE detection result of canine PD-L1 protein.

[0049] Figure 2 This is the HPLC detection result of canine PD-L1 protein.

[0050] Figure 3 This is the result of ELISA test of anti-canine PD-L1 monoclonal antibody.

[0051] Figure 4 This is a graph showing the affinity test between mouse anti-PD-L1 antibodies and PD-L1 proteins from different species. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further illustrated below with reference to the accompanying drawings and through specific implementation methods. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0053] In the following examples, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and technical means used were conventional methods and means in the field.

[0054] Example 1 Reverse transcription to obtain the mouse variable region sequence

[0055] Hybridoma cells that can produce high-affinity antibodies were screened by immunizing mice with human PD-L1 protein as an antigen. Total RNA was extracted according to the Trizol reagent technical manual and reverse transcribed into cDNA according to the cDNA synthesis kit steps.

[0056] The antibody heavy and light chain fragments were obtained by 5' RACE PCR, and the heavy and light chain variable region gene sequences were obtained after sequencing. The amino acid sequences corresponding to the gene sequences are shown in SEQ ID NOs. 7 and 9.

[0057] The heavy and light chain variable region sequences were codon-optimized according to the HEK-293 cell line; the resulting gene sequences are shown in SEQ ID NOs. 11-12.

[0058] The antibody heavy and light chain variable region sequences are assembled with murine constant region sequences (the murine heavy chain constant region amino acid sequence is selected from UniProtKB: P01868, as shown in SEQ ID NO. 8, and the light chain constant region amino acid sequence is selected from UniProtKB: P01837, as shown in SEQ ID NO. 10). Since the IgG1 constant region Fc has the highest affinity for Fc receptors, it is beneficial for inducing antibody-dependent cellular cytotoxicity (ADCC). Studies have shown that this effect is the basis of the anti-tumor effectiveness of antibodies in vivo. Therefore, the IgG1 constant region selected in the present invention can play a role in treating canine tumors or canine autoimmune diseases, providing a basis for the development of anti-tumor drugs.

[0059] The codons were then optimized according to HEK-293 cells and constructed into an expression vector.

[0060] Example 2 Preparation of mouse antibodies

[0061] The target plasmid was transformed into HST08 Escherichia coli competent cells, and positive colonies were picked and amplified into LB ampicillin resistant liquid medium for large-scale plasmid extraction (Full Gold Endotoxin-Free Plasmid Extraction Kit).

[0062] Mouse target antibodies were transiently transfected and expressed, and the density of HEK-293 cells was 2×10 6 When the cell viability was lower than 60%, the expressing cell suspension was harvested, centrifuged at 4000 rpm for 30 min, and then purified by Protein A affinity chromatography column.

[0063] Example 3 Preparation of Canine PD-L1 Protein

[0064] The extracellular domain sequence of the canine PD-L1 protein (amino acid sequence UniProtKB: E2RKZ5) was selected and constructed into an expression vector after CHO codon optimization. The protein was expressed in CHO cells by transient transfection and purified using a nickel column, followed by SDS-PAGE and HPLC analysis.

[0065] The results of SDS-PAGE identification are as follows Figure 1 As shown in the figure, the target band is about 35kDd~40kD, which is consistent with the expected band. The results show that the canine PD-L1 protein is correctly expressed; the HPLC identification results are as follows Figure 2 As shown, the purity is 83.81%.

[0066] Example 4 ELISA biological activity identification

[0067] The antigen canine PD-L1 protein was dissolved and diluted with carbonate buffer (pH 9.6), coated on a 96-well ELISA plate (100 μL / well) at 4°C overnight, washed five times with 200 μL / well of PBST, then blocked with 100 μL / well of blocking solution at 37°C for 2 h, and washed five times with 200 μL / well of PBST.

[0068] The antibody was diluted with PBS, 100 μL was added to each well, incubated at 37°C for 1 h, and washed 5 times with PBST.

[0069] Goat anti-mouse IgG-HRP (Solyb) was diluted with PBS and added to each well at 100 μL. The plates were incubated at 37°C for 1.5 h, and then washed five times with 200 μL / well of PBST.

[0070] TMB color development solution was added at 100 μL / well and incubated for several minutes. Stop solution (0.5 mol / L sulfuric acid solution) was added to the ELISA plate at a ratio of 50 μL / well.

[0071] Finally, the OD was measured by microplate reader 450nm The absorbance value at .

[0072] The results are as follows Figure 3 As shown, it shows that the anti-canine PD-L1 monoclonal antibody can bind to the canine PD-L1 protein, and the anti-canine PD-L1 monoclonal antibody has biological binding activity with the canine PD-L1 protein.

[0073] Furthermore, the inventors tested the affinity of the mouse antibody to human and canine PD-L1 proteins. The results of two parallel tests were as follows: Figure 4 As shown in the results, although the logarithmic value of the affinity of mouse PD-L1 antibody to human PD-L1 protein is slightly higher than that of canine PD-L1 protein, the logarithmic value of the affinity of mouse PD-L1 antibody to human PD-L1 protein is 1.0 -9 M and 1.0 -10 M, which is a high-affinity antibody, but the logarithm of its affinity to canine PD-L1 protein is 1.0 -8 M and 1.0 -9 M, it is still a higher affinity antibody.

[0074] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. An anti-canine PD-L1 monoclonal antibody or an antigen-binding fragment thereof, characterized in that, The heavy chain variable region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment includes VH CDR1, VH CDR2, and VH CDR3, where: VH CDR1 is the amino acid sequence shown in SEQ ID NO.1; VH CDR2 is the amino acid sequence shown in SEQ ID NO.2; VH CDR3 is the amino acid sequence shown in SEQ ID NO.

3.

2. The anti-canine PD-L1 monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein The light chain variable region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment includes VL CDR1, VL CDR2, and VL CDR3, where: VL CDR1 is the amino acid sequence shown in SEQ ID NO.4; VL CDR2 is the amino acid sequence shown in SEQ ID NO.5; VL CDR3 is the amino acid sequence shown in SEQ ID NO.

6.

3. The anti-canine PD-L1 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that, The heavy chain variable region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID NO.7, and the heavy chain constant region includes the amino acid sequence shown in SEQ ID NO.

8.

4. The anti-canine PD-L1 monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that The light chain variable region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment includes the amino acid sequence shown in SEQ ID NO.9, and the light chain constant region includes the amino acid sequence shown in SEQ ID NO.

10.

5. A gene sequence or its complementary sequence encoding the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 4.

6. The gene sequence or its complementary sequence according to claim 5, characterized in that, The gene sequence is codon-optimized according to the preference of the host cell, and the host cell includes HEK-293 cells.

7. The gene sequence or its complementary sequence according to claim 5 or 6, characterized in that, The gene sequence is optimized according to HEK-293 codons; Among them, the gene sequence shown in SEQ ID NO.11 encodes the heavy chain variable region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment, and the gene sequence shown in SEQ ID NO.12 encodes the light chain variable region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment; The gene sequence shown in SEQ ID NO.13 encodes the heavy chain constant region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment, and the gene sequence shown in SEQ ID NO.14 encodes the light chain constant region of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment.

8. An expression vector, characterized in that, Containing the gene sequence or its complementary sequence according to any one of claims 5 to 7.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 4, the gene sequence or its complementary sequence according to any one of claims 5 to 7, or the expression vector according to claim 8.

10. Use of the anti-canine PD-L1 monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 4, the gene sequence or its complementary sequence according to any one of claims 5 to 7, or the expression vector according to claim 8 in the preparation of a drug for anti-canine PD-L1 chimeric antibody, treating canine tumors or canine autoimmune diseases.

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