Mouse chimeric anti-canine PD-L1 antibody and application thereof

Abstract

The invention provides a mouse canine chimeric anti-canine PD-L1 antibody and application thereof. A heavy chain variable region and a light chain variable region of the mouse canine chimeric anti-canine PD-L1 antibody or a functional fragment of the mouse canine chimeric anti-canine PD-L1 antibody comprise amino acid sequences shown in SEQ ID NO.1-6. The obtained anti-canine PD-L1 antibody is formed by assembling a mouse antibody variable region and a canine antibody constant region sequence. The biological binding activity with the dog PD-L1 protein is maintained, meanwhile, the immunogenicity of dogs during use is reduced, and too fast exhaustion is avoided. The dog PD-L1 genetically engineered antibody provided by the invention, as a novel immunosuppressant, is expected to be applied to treatment of canine tumors and related immune diseases in the future.

Description

Technical Field

[0001] The present invention belongs to the field of tumor immunotherapy drugs, and relates to an anti-PD-L1 antibody. More specifically, it relates to a murine-canine chimeric anti-canine PD-L1 antibody and its applications. Background Art

[0002] Companion dogs naturally develop several cancers, which are clinically similar to human cancers in many aspects. Therefore, the study of immunooncology drugs in dogs can inform and prioritize new immunooncology treatments for humans. Immune checkpoint blockade therapy is one of the most promising tumor immunotherapies currently, and has shown significant clinical efficacy in various tumor types.

[0003] Currently, in the treatment of canine diseases, immunotherapy has gradually become an important means. As a new type of immunosuppressant, PD-L1 genetically engineered antibodies may be used to treat certain canine diseases in the future, such as canine-related tumors, autoimmune diseases, etc.

[0004] However, the challenge is that immunotherapy antibodies against canine immune checkpoint molecules, such as canine PD-L1 (cPD-L1), are not yet commercially available. Antibodies with too high affinity will cause rapid depletion of the antibody in the body when injected, preventing the antibody from exerting normal antibody effects and not being able to effectively induce immune effects or only inducing mild immune effects. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a murine-canine chimeric anti-canine PD-L1 antibody genetically engineered antibody, which can bind to canine PD-L1 protein. The variable region of the murine antibody is combined with the constant region of the canine antibody, reducing immunogenicity and reducing rejection reactions in dogs during application. It has a relatively high affinity for canine PD-L1 protein and may be used to treat certain canine diseases in the future, such as canine-related tumors, autoimmune diseases, etc.

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

[0007] In the first aspect, the present invention provides a murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment. The amino acid sequence of the heavy chain variable region (VH) of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment includes any one or a combination of at least two of SEQ ID NO.1-3;

[0008] The amino acid sequence of the light chain variable region (VL) of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment includes any one or a combination of at least two of SEQ ID NO.4-6.

[0009] This application has developed a new PD-L1 antibody as an immuno-oncology drug and characterized its functions and biological properties. This PD-L1 antibody has a high affinity for canine PD-L1, can bind to the canine PD-L1 protein, and can remain in the body to induce other immune responses, thereby enhancing the effectiveness of the antibody.

[0010] The specific sequences are shown in Table 1 below:

[0011] Table 1

[0012]

[0013] As a preferred technical solution of the present invention, the CDR1, CDR2, and CDR3 of the heavy chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment are the amino acid sequences shown in SEQ ID NO.1-3.

[0014] The CDR1, CDR2, and CDR3 of the light chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment are the amino acid sequences shown in SEQ ID NO.4-6.

[0015] As a preferred technical solution of the present invention, the amino acid sequence of the heavy chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment (SEQ ID NO.7)

[0016] EVQLQQSGAELVRPGSSVKMSCKTSGYTFT SYGIN WVKQRPGQGLEWIG YIYIGNGYSENNEKFKG KATLTSDTSSSTAYMQLSGLTSEDSANYFCAR LLFGGYSDY WGQGTTLTVSS (the underlined part is the CDR region)

[0017] As a preferred technical solution of the present invention, the amino acid sequence of the light chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment (SEQ ID NO.8)

[0018] DIQMTQSPSSLSASLGGNVTITC KASQDIHKYIA WYQHKPGKGPRLLIH YTSTLQP GIPSRFSGDGSGRDFSFSISNLEPEDIATYYC LQYDNLLRT FGGGTKLEIK (the underlined part is the CDR region)

[0019] The present invention relates to antibody modification technology. Compared with immunizing mice with canine PD-L1 protein as an antigen to obtain hybridoma cells, and then retrieving the variable region sequence of murine anti-canine PD-L1 antibody, and assembling the CDR region in the variable region with the canine antibody framework to obtain a canineized anti-canine PD-L1 antibody, the variable region of the murine anti-PD-L1 antibody in the present invention is obtained by immunizing mice with human PD-L1 as an antigen to obtain hybridoma cells, retrieving the variable region of murine anti-human PD-L1 antibody, and assembling and modifying the variable region of murine anti-human PD-L1 antibody with the canine antibody constant region into a chimeric antibody to reduce its immunogenicity and reduce heterologous reactions when used in dogs in the future. At the same time, it has a high affinity for canine PD-L1 protein, reduces the immunogenicity during the use of dogs and does not cause antibody depletion, and has biological binding activity, and can be used for the treatment of canine tumors and diseases involving the canine PD-1 / PD-L1 pathway in the future.

[0020] As a preferred technical solution of the present invention, the amino acid sequence of the heavy chain constant region of the murine chimeric anti-canine PD-L1 antibody or its functional fragment (SEQ ID NO.9)

[0021] ASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLHSLSSMVTVPSSRWPSETFTCNVVHPASNTKVDKPVFNECRCTDTPPCPVPEPLGGPSVLIFPPKPKDILRITRTPEVTCVVLDLGREDPEVQISWFVDGKEVHTAKTQSREQQFNGTYRVVSVLPIEHQDWLTGKEFKCRVNHIDLPSPIERTISKARGRAHKPSVYVLPPSPKELSSSDTVSITCLIKDFYPPDIDVEWQSNGQQEPERKHRMTPPQLDEDGSYFLYSKLSVDKSRWQQGDPFTCAVMHETLQNHYTDLSLSHSPGK

[0022] As a preferred technical solution of the present invention, the amino acid sequence of the light chain constant region of the murine chimeric anti-canine PD-L1 antibody or its functional fragment (SEQ ID NO.10)

[0023] RNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

[0024] In a second aspect, the present invention provides a gene sequence or its complementary sequence encoding the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment as described in the first aspect.

[0025] As a preferred technical solution of the present invention, the CDR1, CDR2, and CDR3 of the heavy chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment optimized according to CHO codons are the gene sequences shown in SEQ ID NOs. 11 to 13 or their complementary sequences.

[0026] 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 the 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. Multiple species exhibit specific biases towards certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is generally related to the translation efficiency of messenger RNA (mRNA), and the translation efficiency of mRNA is in turn thought to depend particularly on the characteristics of the translated codons and the availability of specific transfer RNA (tRNA) molecules. The predominance of the selected tRNA in a cell usually reflects the codons most frequently used in peptide synthesis. Therefore, genes can be customized based on codon optimization for 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 contribute to achieving faster translation speeds and higher accuracy.

[0027] In the present invention, the murine-canine chimeric anti-canine PD-L1 monoclonal antibody shown can be optimized according to the codon preference of the selected host cell, and is not limited to being optimized only using CHO codons.

[0028] The CDR1, CDR2, and CDR3 of the light chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment with the gene sequence optimized according to CHO codons are the gene sequences shown in SEQ ID NOs. 14 to 16 or their complementary sequences.

[0029] The specific sequences are shown in Table 2 below:

[0030] Table 2

[0031]

[0032] As a preferred technical solution of the present invention, the gene sequence is optimized according to CHO codons, the gene sequence shown in SEQ ID NO. 17 encodes the heavy chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment, and the gene sequence shown in SEQ ID NO. 18 encodes the light chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment.

[0033] Mouse-dog chimeric anti-dog PD-L1 antibody heavy chain variable region gene sequence (SEQ ID NO.17)

[0034] GAGGTGCAGCTGCAGCAGAGCGGCGCTGAGCTGGTGAGACCTGGCAGCAGCGTGAAGATGAGCTGTAAGACCAGCGGCTACACCTTCACA TCTTATGGCATCAAC TGGGTGAAGCAGAGACCAGGCCAGGGCCTGGAGTGGATTGGC TACATCTACATCGGCAACGGCTACTCTGAGAACAACGAGAAGTTCAAGGGC AAGGCTACACTGACATCTGATACCTCCAGCTCCACCGCCTACATGCAGCTGAGCGGCCTGACTTCCGAGGATTCCGCCAATTATTTCTGCGCCAGA CT GCTCTTTGGCGGATACTCTGACTAC TGGGGACAGGGAACCACCCTGACCGTGTCCTCT (The underlined part is the CDR region)

[0035] Mouse-dog chimeric anti-dog PD-L1 antibody light chain variable region gene sequence (SEQ ID NO.18)

[0036] GATATCCAGATGACACAGTCTCCTTCTTCTCTGTCTGCCTCTCTGGGCGGCAACGTGACCATCACCTGT AAGGCCAGCCAGGATATCCACAAGTACATCGCC TGGTATCAGCACAAGCCTGGCAAGGGCCCTAGACTGCTGATCCAC TACACCTCTACCCTGCAGCCT GGCATCCCTAGCAGATTCTCTGGCGACGGCTCTGGCAGAGATTTCTCTTTCTCTATCTCTAACCTGGAGCCTGAGGATATCGCCACCTACTACTGT CTGCAGTACGATAACCTGCTGAGAACC TTCGGCGGCGGCACAAAGCTGGAGATCAAG (The underlined part is the CDR region)

[0037] As a preferred technical solution of the present invention, the gene sequence is optimized according to CHO codons. The gene sequence shown in SEQ ID NO.19 encodes the heavy chain constant region of the mouse-dog chimeric anti-dog PD-L1 antibody or its functional fragment, and the gene sequence shown in SEQ ID NO.20 encodes the light chain constant region of the mouse-dog chimeric anti-dog PD-L1 antibody or its functional fragment.

[0038] Murine chimeric anti-canine PD-L1 antibody heavy chain constant region gene sequence (SEQ ID NO.19)

[0039] GCCTCTACCACCGCCCCATCTGTGTTCCCTCTGGCTCCCTCCTGTGGCAGCACATCCGGCTCTACAGTGGCCCTGGCCTGCCTGGTGAGCGGCTACTTCCCTGAGCCTGTGACAGTGAGCTGGAACAGTGGCTCTCTGACATCTGGAGTCCACACCTTCCCTTCTGTGCTGCAGAGCTCTGGCCTGCACTCCCTGTCCAGCATGGTGACAGTGCCTAGCAGCAGATGGCCTAGCGAGACCTTTACATGCAACGTGGTGCATCCTGCCTCCAACACCAAGGTGGACAAGCCTGTGTTTAACGAATGTAGATGTACCGATACACCACCTTGTCCTGTGCCTGAGCCTCTGGGCGGCCCTTCTGTGCTGATTTTTCCTCCTAAGCCTAAGGATATCCTGAGAATCACAAGAACTCCTGAGGTGACTTGTGTGGTGCTGGACCTGGGCAGAGAAGATCCTGAGGTGCAGATCTCTTGGTTTGTGGACGGCAAGGAGGTTCACACTGCCAAGACCCAGTCTAGAGAGCAGCAGTTTAACGGCACCTACAGAGTGGTGAGCGTGCTGCCTATCGAGCACCAGGACTGGCTGACTGGCAAGGAGTTCAAGTGTAGGGTGAACCACATCGATCTGCCATCCCCTATCGAGAGAACAATCAGCAAGGCCAGGGGCAGAGCCCATAAGCCTTCTGTGTACGTGCTGCCTCCTTCTCCTAAGGAGCTGAGCTCTTCTGATACAGTGTCTATCACCTGTCTGATCAAGGACTTCTATCCTCCTGATATCGATGTGGAGTGGCAGTCTAACGGCCAGCAGGAGCCTGAGAGAAAGCACAGAATGACCCCCCCTCAGCTGGACGAGGATGGCAGCTACTTTCTGTATTCTAAGCTGTCTGTGGATAAGAGCAGATGGCAGCAGGGCGATCCTTTCACCTGTGCCGTGATGCACGAGACCCTGCAGAACCACTACACAGACCTGAGCCTGAGCCACAGCCCTGGCAAGTGA

[0040] Mouse-derived chimeric anti-canine PD-L1 antibody light chain constant region gene sequence (SEQ ID NO.20)

[0041] AGAAACGATGCCCAGCCTGCCGTGTACCTGTTCCAGCCTTCTCCTGATCAGCTGCACACCGGCTCTGCCAGCGTGGTGTGTCTGCTGAACTCTTTCTACCCTAAGGATATCAACGTGAAGTGGAAGGTGGATGGCGTGATCCAGGATACAGGCATCCAGGAGTCTGTGACAGAGCAGGATAAGGATTCTACATACTCTCTGTCTAGCACACTGACCATGTCTAGCACCGAGTACCTGTCTCACGAGCTGTACTCTTGTGAGATCACCCACAAGTCTCTGCCTTCTACCCTGATCAAGTCTTTCCAGAGATCTGAGTGTCAGAGAGTGGATTGA

[0042] In a third aspect, the present invention also provides an expression vector containing the gene sequence or its complementary sequence as described in the second aspect.

[0043] In a fourth aspect, the present invention also provides a pharmaceutical composition, which includes the mouse-canine chimeric anti-canine PD-L1 antibody or its functional 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.

[0044] In a fifth aspect, the application of the mouse-canine chimeric anti-canine PD-L1 antibody or its functional 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 drugs or diagnostic kits for treating tumors or autoimmune diseases.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] In the present invention, the variable region of the mouse-derived antibody and the constant region sequence of the canine-derived antibody are retrieved from hybridoma cells for assembly, a eukaryotic expression vector is constructed and eukaryotic expression is carried out. After purification with an affinity chromatography Protein A column, SDS-PAGE, HPLC, and ELISA are used to identify the antibody assembly, purity, and biological activity. The results show that the chimeric antibody is correctly assembled and expressed and has biological binding activity with canine PD-L1 protein.

[0047] In the present invention, first, the variable region sequence of a murine antibody is assembled with the constant region sequence of a murine antibody, and the resulting expressed antibody can bind to canine PD-L1 protein, and the logarithm of the affinity for canine PD-L1 protein can reach 10 -8 M. To reduce immunogenicity, the present invention further assembles the variable region of a murine antibody with the constant region sequence of a canine antibody. The resulting expressed antibody has biological binding activity with canine PD-L1 protein, maintaining both the antigen-antibody binding effect and avoiding rapid depletion, filling the gap in immunosuppressive drugs for the treatment of canine tumors, and can be applied to the current treatment of canine diseases. Currently, immunotherapy has gradually become an important means, and the chimeric PD-L1 genetically engineered antibody (canine) as a new type of immunosuppressive agent is expected to be applied to the treatment of canine tumors and related immune diseases in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a detection graph of the affinity between a murine anti-PD-L1 antibody and PD-L1 proteins of different species.

[0049] Figure 2 It is a SDS-PAGE detection result graph of a murine-canine chimeric anti-canine PD-L1 chimeric antibody.

[0050] Figure 3 It is a HPLC detection result graph of a murine-canine chimeric anti-canine PD-L1 antibody.

[0051] Figure 4 It is an ELISA detection result graph of a murine-canine chimeric anti-canine PD-L1 antibody.

[0052] Figure 5 It is a dose-effect curve graph of a murine-canine chimeric anti-canine PD-L1 antibody in a reporter gene experiment. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0053] The technical solution of the present invention will be further described below with reference to the drawings and through specific embodiments. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0054] In the following examples, unless otherwise specified, the reagents and consumables used are purchased from conventional reagent manufacturers in the art; unless otherwise specified, the experimental methods and technical means used are conventional methods and means in the art.

[0055] Example 1 Reverse transcription to obtain the murine variable region sequence

[0056] For the hybridoma cells that were screened by immunizing mice with human PD-L1 protein as an antigen and can produce high-affinity antibodies, total RNA was extracted according to the technical manual of Trizol reagent, and the total RNA was reverse transcribed into cDNA according to the steps of the cDNA synthesis kit.

[0057] The heavy and light chain fragments of the antibody were obtained by 5'RACE PCR, and the gene sequences of the heavy and light chain variable regions were obtained after sequencing. The amino acid sequences corresponding to the gene sequences are shown in SEQ ID NO.7-8.

[0058] The sequences of the heavy and light chain variable regions were optimized according to the codons of CHO cells; the obtained gene sequences are shown in SEQ ID NO.17-18.

[0059] The variable region sequences of the antibody light and heavy chains were assembled with murine constant region sequences (the amino acid sequence of murine light chain constant region UniProtKB: P01837, the amino acid sequence of heavy chain constant region UniProtKB: P01868), and after codon optimization according to HEK-293 cells, they were constructed into an expression vector. The variable region sequences of the antibody light and heavy chains were assembled with canine constant region sequences (the amino acids at positions 138-468 of the heavy chain constant region amino acids: GenBank: AAL35301.1, the amino acids at positions 115-224 of the light chain amino acids UniProtKB: A0A8I3NNP1, that is, the amino acid sequences are shown in SEQ ID NO.9-10; the gene sequences after CHO codon optimization are shown in SEQ ID NO.19-20), and after assembly, they were constructed into an expression vector after codon optimization according to CHO cells.

[0060] Preparation of Murine Antibody in Example 2

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

[0062] Transient transfection and expression of the murine target antibody was carried out. When the density of HEK-293 cells was 2×10 6 / ml, transient transfection was carried out at a ratio of DNA:PEI = 1:3. When the cell viability was lower than 60%, the expressed cell suspension was harvested. After centrifugation at 4000 rpm for 30 min, purification was carried out using a Protein A affinity chromatography column.

[0063] The affinities of the murine antibody for human and canine PD-L1 proteins were detected. The detection results of two parallel experiments are as Figure 1 shown. Although the logarithm of the affinity of the murine PD-L1 antibody for human PD-L1 protein was slightly higher than that for canine PD-L1 protein in this result, the logarithm of the affinity of the murine PD-L1 antibody for human PD-L1 protein was between 1.0 -9 M and 1.0 -10 M, and it was a high-affinity antibody. However, the logarithm of its affinity for canine PD-L1 protein was between 1.0 -8 M and 1.0-9 Between M, it is still a high-affinity antibody, so it will not be depleted too quickly during its in vivo application, and it also provides data support for the modification of murine antibodies, indicating that the variable region of the murine anti-PD-L1 antibody can bind to the canine PD-L1 protein. Therefore, the murine-canine chimeric PD-L1 antibody retains the variable region of the murine antibody.

[0064] Example 3 Preparation of Chimeric Antibody

[0065] Assemble the variable region of the murine antibody and the canine constant region sequence, construct a vector, and perform transient transfection and expression after plasmid extraction and transformation. The specific steps are as follows:

[0066] 1. Inoculate CHO cells at a viable cell density of 2.5×10 6 cells / mL one day before transfection. On the day of transfection, after adjusting the cell density, prepare the Reagent-DNA mixture by mixing 1 μg of total plasmid and 7 μL of PEI (1 mg / mL) per milliliter of cells, and let it stand at room temperature for about 15 min. Then evenly add the incubated Reagent-DNA mixture to the cells.

[0067] 2. Add the feed supplement (MetaCell CHO TransFeed) in proportion on the day after transfection.

[0068] 3. Add the feed supplement again in the same proportion on the 5th day after transfection. Harvest the protein when the cell viability is lower than 60%.

[0069] 4. Purification: Purify the harvested cell supernatant using a Protein A affinity chromatography column, then replace the antibody with a concentration buffer of PBS. After measuring the antibody concentration, identify it by SDS-PAGE and HPLC.

[0070] 5. Identification by SDS and HPLC

[0071] (1) SDS-PAGE

[0072] Prepare the gel using a PAGE gel simple preparation kit (Beijing Yangguang Yingrui Biotechnology Co., Ltd.). Add the treated sample to the gel wells, set the initial voltage to 80 V, and adjust the voltage to 120 V until the sample enters the separation gel. After staining with Coomassie Brilliant Blue staining solution, decolorize with decolorizing solution until the bands are clear.

[0073] The results are as Figure 2 shown, indicating that the murine-canine chimeric anti-canine PD-L1 antibody was successfully expressed in eukaryotic cells. After the antibody was treated with reducing buffer, the heavy chain was around 55 KD and the light chain was around 30 KD, which was consistent with the expected size. After the antibody was treated with non-reducing buffer, the antibody molecule was around 180 kD, indicating that the light chain and heavy chain could be correctly assembled.

[0074] (2) HPLC detection

[0075] As Figure 3 shown, a thermo HPLC instrument was used for purity detection, and the purity was 91.66%.

[0076] Example 4 ELISA bioactivity identification

[0077] The antigen canine PD-L1 protein was dissolved and diluted with carbonate buffer (pH 9.6), coated onto a 96-well ELISA plate (100 μL / well) overnight at 4 °C, washed 5 times with PBST (200 μL / well), then blocked with blocking solution (100 μL / well) at 37 °C for 2 h, and washed 5 times with PBST (200 μL / well).

[0078] After diluting the antibody with PBS, 100 μL was added to each well and incubated at 37 °C for 1 h, then washed 5 times with PBST.

[0079] After diluting rabbit anti-canine IgG-HRP (Solarbio) with PBS, 100 μL was added to each well and incubated at 37 °C for 1 h, then washed 5 times with PBST (200 μL / well).

[0080] TMB chromogenic solution was added at 100 μL / well, incubated at 37 °C for 10 min, and then stop solution (0.5 mol / L sulfuric acid solution) was added to the ELISA plate wells at a ratio of 50 μL / well.

[0081] Finally, the absorbance at OD 450nm was measured with an ELISA reader.

[0082] The results were as Figure 4 shown, indicating that the murine-canine chimeric anti-canine PD-L1 antibody could bind to canine PD-L1 protein, and the murine-canine chimeric anti-canine PD-L1 antibody had biological binding activity with canine PD-L1 protein.

[0083] Example 5 Blocking functional activity identification of murine-canine chimeric anti-canine PD-L1 antibody

[0084] In this example, the GeneCopoeia PD-1 / PD-L1 reporter gene system was used to evaluate the blocking functional activity of the murine-canine chimeric anti-canine PD-L1 antibody against the PD-L1 target.

[0085] (1) After resuscitating the target cells GS-J2B / PD-1 (GeneCopoeia, product number: RD00871) and effector cells GS-C3 / PD-L1 (GeneCopoeia, product number: RD00703), they were cultured in a cell culture incubator at 37 °C with 5% CO2.

[0086] (2) Inoculate the target cells on the experimental plate and incubate them in a cell culture incubator (37 °C, 5% CO2) for 20 hours. Remove the supernatant, and successively add the positive control Tecentriq Biosimilar (Lot No.: GS0301 / DS03EC001), murine-canine chimeric anti-canine PD-L1 antibody, and effector cells. Incubate in the incubator for 6 hours, then add the working solution of the luciferase detection reagent to the corresponding wells of the experimental plate and incubate at room temperature for 5 minutes.

[0087] (3) Use a PHERAstar microplate reader to read the chemiluminescence value, record the data, and analyze the data. Establish a corresponding dose-response curve based on the corresponding relationship between the relative chemiluminescence signal value and the sample concentration as Figure 5 shown.

[0088] The experimental results show that the positive control antibody (Tecentriq Biosimilar) can effectively block the binding of PD-L1 to PD-1 and restore the inhibited downstream signaling pathway. The EC 50 value of its dose-response curve is 0.6595 nM; the murine-canine chimeric anti-canine PD-L1 antibody can also well block the binding of PD-L1 to PD-1 and restore the inhibited downstream signaling pathway. The EC 50 value of its dose-response curve is 1.042 nM. The above results indicate that the murine-canine chimeric anti-canine PD-L1 antibody maintains both high affinity and biological binding activity with the antigen and the ability to specifically block the PD-1 / PD-L1 signaling pathway between cells after modification.

[0089] The applicant declares that the above description is only the specific implementation manner of the present invention, but the protection scope 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 protection scope and the disclosure scope of the present invention.

Claims

1. A murine-canine chimeric anti-canine PD-L1 antibody or a functional fragment thereof, characterized in that, The amino acid sequence of the heavy chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment includes any one or a combination of at least two of SEQ ID NOs. 1 to 3; The amino acid sequence of the light chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment includes any one or a combination of at least two of SEQ ID NOs. 4 to 6.

2. The murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment according to claim 1, characterized in that, The CDR1, CDR2, and CDR3 of the heavy chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment are the amino acid sequences shown in SEQ ID NOs. 1 to 3; The CDR1, CDR2, and CDR3 of the light chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment are the amino acid sequences shown in SEQ ID NOs. 4 to 6.

3. The murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment according to claim 1 or 2, characterized in that, The heavy chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment includes the amino acid sequence shown in SEQ ID NO. 7; The light chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment includes the amino acid sequence shown in SEQ ID NO.

8.

4. The murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment according to any one of claims 1 to 3, characterized in that, The heavy chain constant region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment includes the amino acid sequence shown in SEQ ID NO. 9; The light chain constant region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment includes the amino acid sequence shown in SEQ ID NO.

10.

5. A gene sequence or its complementary sequence encoding the murine-canine chimeric anti-canine PD-L1 antibody or its functional 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 optimized according to CHO codons, and the CDR1, CDR2, and CDR3 of the heavy chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment are the gene sequences shown in SEQ ID NOs. 11 to 13 or their complementary sequences; The gene sequence is optimized according to CHO codons, and the CDR1, CDR2, and CDR3 of the light chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment are the gene sequences shown in SEQ ID NOs. 14 to 16 or their complementary sequences.

7. The gene sequence or its complementary sequence according to claim 5 or 6, characterized in that, The gene sequence is optimized according to CHO codons; Among them, the gene sequence shown in SEQ ID NO. 17 encodes the heavy chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment, and the gene sequence shown in SEQ ID NO. 18 encodes the light chain variable region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment; The gene sequence shown in SEQ ID NO. 19 encodes the heavy chain constant region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional fragment, and the gene sequence shown in SEQ ID NO. 20 encodes the light chain constant region of the murine-canine chimeric anti-canine PD-L1 antibody or its functional 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 comprises the murine-canine chimeric anti-canine PD-L1 antibody or its functional 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 murine-canine chimeric anti-canine PD-L1 antibody or its functional 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 medicament or a kit for treating tumors or autoimmune diseases.

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