Anti-PD-L1 antibodies, their preparation methods and applications

Abstract

This invention belongs to the field of biotechnology, and particularly relates to anti-PD-L1 antibodies, their preparation methods, and applications. The antibody consists of a heavy chain and a light chain; the amino acid sequence of the heavy chain has 85-100% identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; the amino acid sequence of the light chain has 85-100% identity with SEQ ID NO: 4 or SEQ ID NO: 5. This invention provides an anti-PD-L1 antibody with high specificity and strong competitive activity, while ensuring good thermostability. It can effectively detect the presence of PD-L1 and block the PD-L1 / PD-1 pathway, achieving the blocking / treatment of related diseases. Its high thermostability allows it to be combined with most existing combination therapies, giving it broader application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to anti-PD-L1 antibodies, their preparation methods and applications. Background Technology

[0002] The immune system must maintain a balance between an effective response to eliminate pathogens and the maintenance of tolerance to prevent autoimmune diseases. T cells are crucial for maintaining this balance, and their proper regulation is primarily achieved through molecular coordination within the B7-CD28 family. Interactions between B7 family members (which function as ligands) and CD28 family members (which function as receptors) provide not only critical positive signals that initiate, enhance, and maintain T cell responses, but also, when appropriate, critical negative signals that promote the restriction, termination, and / or attenuation of T cell responses. One member of the CD28 family, called PD-1 (also known as programmed cell death-1), is incrementally regulated on activated T cells, B cells, and monocytes; the B7 family ligand, PD-L1 (also known as B7H1 or programmed cell death-1 ligand 1), interacts with the receptor PD-1 on T cells, playing a significant role in the negative regulation of the immune response.

[0003] Existing results show that PD-L1, highly expressed in tumor cells, plays a crucial role in tumor immune escape by increasing T cell apoptosis. For example, in numerous samples from ovarian cancer, kidney cancer, colorectal cancer, pancreatic cancer, liver cancer, and melanoma, PD-L1 expression is associated with poor prognosis and short overall survival, regardless of subsequent treatment. Given the important role of PD-L1 in cancer development and immune system regulation, there is a continuous need to monitor the presence of PD-L1 and to develop effective antibodies that block the PD-L1 / PD-1 pathway. Therefore, anti-PD-L1 antibodies have emerged.

[0004] Anti-PD-L1 antibodies have significant applications in cancer diagnosis and treatment. For example, they have shown remarkable efficacy in the treatment of advanced non-small cell lung cancer, and based on this, they have also been successfully applied to various other lung cancer diseases, including small cell lung cancer and locally advanced non-small cell lung cancer.

[0005] Anti-PD-L1 antibodies can be used in various treatments, such as in combination with chemotherapy, targeted drugs, and radiotherapy. However, many shortcomings of anti-PD-L1 antibodies still need to be addressed. One particularly prominent issue is their poor thermal stability. Therefore, improving the thermal stability and competitiveness of anti-PD-L1 antibodies is crucial for further expanding their application areas and enhancing their efficacy. Summary of the Invention

[0006] To address the issues of relatively poor thermal stability of existing anti-PD-L1 antibodies, weak competitiveness of most anti-PD-L1 antibodies, and limited efficacy in treatment, this invention provides an anti-PD-L1 antibody, its preparation method, and its application.

[0007] The purpose of this invention is:

[0008] I. Preparation to obtain a highly competitive anti-PD-L1 antibody;

[0009] Second, ensure that the anti-PD-L1 antibody has good thermal stability.

[0010] To achieve the above objectives, the present invention provides the following technical solutions.

[0011] Anti-PD-L1 antibody,

[0012] The antibody is composed of heavy and light chains;

[0013] The heavy chain includes at least one of the following:

[0014] The first heavy chain (4D11_huVH4) has an amino acid sequence that is 85-100% identical to SEQ ID NO: 1;

[0015] The second chain (4D11_huVH5) has an amino acid sequence that is 85-100% identical to SEQ ID NO: 2;

[0016] The third chain (4D11_huVH6) has an amino acid sequence that is 85-100% identical to SEQ ID NO: 3;

[0017] The light chain includes at least one of the following:

[0018] The first light chain (4D11_huVL3) has an amino acid sequence that is 85-100% identical to SEQ ID NO: 4;

[0019] The second light chain (4D11_huVL4) has an amino acid sequence that is 85-100% identical to SEQ ID NO: 5.

[0020] As a preferred option

[0021] The amino acid sequence of the 4D11_huVH4 is shown in SEQ ID NO: 1;

[0022] The amino acid sequence of the 4D11_huVH5 is shown in SEQ ID NO: 2;

[0023] The amino acid sequence of the 4D11_huVH6 is shown in SEQ ID NO: 3;

[0024] The amino acid sequence of the 4D11_huVL3 is shown in SEQ ID NO: 4;

[0025] The amino acid sequence of the 4D11_huVL4 is shown in SEQ ID NO: 5.

[0026] As a preferred option

[0027] The antibody is a humanized antibody.

[0028] Preparation method of anti-PD-L1 antibody

[0029] The method includes:

[0030] 1) Construct non-human hybridoma cells, purify RNA from them, reverse transcribe the purified RNA into cDNA as a template, and then amplify the target fragment in vitro using specific PCR primers. Ligate the positive PCR product into a T vector for plasmid recombination and transformation. Select positive PCR products for cloning and sequencing through blue-white screening and PCR verification to obtain the original sequence of the non-human antibody.

[0031] 2) Humanized sequence construction: Based on the original non-human antibody sequence obtained in step 1), a model was built and compared with the database to mutate the original non-human antibody sequence into a humanized sequence;

[0032] 3) Antibody construction: Combine the humanized sequences obtained in step 2) into humanized antibodies;

[0033] 4) Antibody expression: The humanized antibody obtained in step 3) was expressed in the Expi293 mammalian expression system;

[0034] 5) Antibody purification: Collect the cell supernatant after expression in Expi293 cells in step 4) and purify it to antibody purity ≥90%.

[0035] As a preferred option

[0036] Step 1) The non-human hybridoma cells were obtained by culturing them using the following method:

[0037] 1-1) Mice were immunized with the PD-L1-his antigen to induce the production of B cells in mice that could secrete antibodies against the specific target protein.

[0038] 1-2) Cell fusion was performed on spleen cells from mice that had generated an immune response to obtain fused cells;

[0039] 1-3) The obtained fusion cells were screened by ELISA and selected to subclone the fusion cells that could compete with PD1-hFc for binding to PD-L1-his. After subcloning, the cells were screened again by ELISA and selected to establish lineages, thus obtaining non-human hybridoma cells.

[0040] As a preferred option

[0041] Step 1) The original sequence of the non-human antibody includes:

[0042] Heavy chain nucleic acid sequence, light chain nucleic acid sequence, heavy chain amino acid sequence, and light chain amino acid sequence.

[0043] As a preferred option

[0044] The heavy chain nucleic acid sequence is shown in SEQ ID NO: 6;

[0045] The light chain nucleic acid sequence is shown in SEQ ID NO: 7;

[0046] The heavy chain amino acid sequence is shown in SEQ ID NO: 8;

[0047] The light chain amino acid sequence is shown in SEQ ID NO: 9.

[0048] As a preferred option

[0049] Step 2) The humanized sequence includes human heavy chain sequences and human light chain sequences.

[0050] Application of anti-PD-L1 antibodies

[0051] The anti-PD-L1 antibody is used to promote the secretion of IFN-γ in cell supernatant.

[0052] As a preferred option

[0053] The anti-PD-L1 antibody is used to prepare drugs and / or drug compositions;

[0054] The drug and / or drug composition are used for targeted therapy of tumors and / or cancers and / or immune diseases and / or infectious diseases.

[0055] The beneficial effects of this invention are:

[0056] This invention provides an anti-PD-L1 antibody with high specificity and strong competitive activity, while ensuring good thermal stability. It can effectively detect the presence of PD-L1 and block the PD-L1 / PD-1 pathway, achieving the blocking / treatment of related diseases. Its high thermal stability allows it to be combined with most existing combination therapies, giving it broader application value. In terms of competitiveness, it is generally superior to existing commercially available anti-PD-L1 antibodies such as atezolizumab. Attached Figure Description

[0057] Figure 1 This is a schematic diagram showing the purification results of the murine hybridoma antibody described in Example 2;

[0058] Figure 2 The results of ELISA assay for determining the binding activity of mouse PD-L1 antibody to the target antigen;

[0059] Figure 3 The results of the ELISA assay for the competitive activity of mouse PD-L1 antibody against 9.0 µg / ml PD-1-hFc ligand were obtained.

[0060] Figure 4 The results of FACS assay were used to detect the binding activity of mouse PD-L1 antibody to CHO-PD-L1 cells.

[0061] Figure 5 The results of FACS assay for detecting the competitive activity of murine PD-L1 antibody against PD1-hFc;

[0062] Figure 6 The results of the detection of CD80-hFc activity blocked by mouse PD-L1 antibody;

[0063] Figure 7 This is a schematic diagram showing the purification results of the 4D11 humanized anti-PD-L1 antibody;

[0064] Figure 8 The results of ELISA assay were used to detect the binding activity of 4D11 humanized anti-PD-L1 antibody to PD-L1-his.

[0065] Figure 9 The results of FACS assay were used to detect the binding activity of 4D11 humanized anti-PD-L1 antibody to CHO-PD-L1 cells.

[0066] Figure 10 The results of the ELISA assay were used to detect the competitive activity of 4D11 humanized anti-PD-L1 antibody against PD-1-mFc.

[0067] Figure 11The results of the FACS assay for the competitive activity of 4D11 humanized anti-PD-L1 antibody against PD-1-mFc were obtained.

[0068] Figure 12 The results of characterization of the activity of the 4D11 humanized anti-PD-L1 antibody were obtained for the Luciferase Assay.

[0069] Figure 13 The results of the thermostability test of the 4D11 humanized anti-PD-L1 antibody;

[0070] Figure 14 The results of the MLR experiment for the 4D11 humanized anti-PD-L1 antibody are shown. Detailed Implementation

[0071] The present invention will be further described clearly and in detail below with reference to specific embodiments and the accompanying drawings. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0072] Example 1

[0073] Primary immunization of mice:

[0074] Three Bal B / C mice (Shanghai Xipu-Bikai Experimental Animal Co., Ltd., females, 8 weeks old) were immunized with the PD-L1-his antigen to induce the production of B cells in the mice that can secrete antibodies against the specific target protein.

[0075] Boosted immunization in mice:

[0076] Based on the initial immunization results, mouse number 61, which performed best in the initial immunization, was selected and given a booster immunization via subcutaneous injection for subsequent cell fusion.

[0077] Example 2

[0078] Hybridoma fusion assay:

[0079] Cell fusion:

[0080] The 61 immunized mice described in Example 1 were sacrificed, and spleen cells were collected. Cell fusion of the immunized mice was performed according to the standard operating procedure for cell fusion based on the principle of SP2 / 0:spleen cells = 1:5. The fusion number was marked as H009 fusion cell plate, batch number: 20210806.

[0081] Cell screening:

[0082] Fusion cells from the H009 fusion cell plate were taken and analyzed by ELISA. 34 wells with strong binding to PD-L1-his and strong competition with PD1-hFc ligands were selected for subcloning.

[0083] Subclone:

[0084] To obtain stable clones, subcloned cells were detected, and positive cells were cloned and further purified. After two cycles of subcloning, ELISA was used to identify 4D11 clones that strongly bind to PD-L1-his (OD450=2.200) and strongly compete with PD1-hFc (OD450=0.690) for cell line establishment and testing, resulting in murine hybridoma cells.

[0085] purification:

[0086] The aforementioned murine hybridoma cells were expanded and cultured. After expansion, the cell supernatant was collected, and antibody purification was performed according to standard protein purification procedures. The purification results are as follows: Figure 1 As shown, from Figure 1 As can be seen from this, the purity of the anti-PD-L1 antibody obtained from murine hybridoma cells in this purification is >90%.

[0087] Example 3

[0088] Detection of PD-L1 antibody activity in murine hybridoma cells:

[0089] (1) ELISA method for detecting the binding activity of mouse PD-L1 antibody to PD-L1-his:

[0090] The ELISA method was used to coat the test plate with 1.0 µg / ml PD-L1-his, and a concentration gradient of mouse PD-L1 antibody samples was set up. The binding activity OD value was measured to detect the affinity between the mouse PD-L1 antibody and the target antigen. The results are as follows: Figure 2 As shown, from Figure 2 The data shows that the murine PD-L1 antibody binds strongly to the antigen (EC50=0.00301), with clear upper and lower plateaus on the curve, a large window, and good activity.

[0091] (2) ELISA method for detecting the competitive activity of mouse PD-L1 antibody against PD1-hFc:

[0092] The test plate was coated with 4.0 µg / ml PD-L1-his, and a concentration gradient of murine PD-L1 antibody was set up. The OD value was measured to detect the competitive activity between the murine PD-L1 antibody and 9.0 µg / ml PD-1-hFc ligand. Results are as follows: Figure 3 As shown, from Figure 3 The data shown indicates that the OD value of this mouse-derived PD-L1 antibody shows a clear trend, with distinct upper and lower plateaus on the curve, indicating strong competitive activity (IC50=0.02759).

[0093] (3) FACS method for detecting the binding activity of mouse PD-L1 antibody to CHO-PD-L1 cells:

[0094] A series of concentrations of murine PD-L1 antibody were prepared and incubated with CHO-PD-L1 cells. The binding signal intensity was measured using FACS to detect the binding activity of the murine PD-L1 antibody to the target protein. Results are as follows: Figure 4 As shown, from Figure 4 The data shows that the murine PD-L1 antibody binds strongly to CHO-PD-L1 and has good activity (IC50=0.1281).

[0095] (4) FACS method for detecting the competitive activity of mouse PD-L1 antibody against PD1-hFc:

[0096] CHO-PD-L1 cells were uniformly seeded into 96-well cell culture plates using the FACS method. A constant concentration of PD1-hFc and a gradient of mouse PD-L1 antibody concentrations were set to bind to the cells. Then, 3 µg / ml of secondary antibody was added to each well. The competitive activity OD value was measured after binding to determine the competitiveness between the mouse PD-L1 antibody and PD1-hFc. Results are as follows: Figure 5 As shown, from Figure 5 The data shown indicates that this murine PD-L1 antibody is highly competitive (IC50=0.2174).

[0097] (5) Detection of CD80-hFc activity blocked by murine PD-L1 antibody:

[0098] The test plate was coated with 2.0 µg / ml PD-L1-mFc, a sample concentration gradient was set, and the OD value was measured to detect the blocking activity of mouse PD-L1 antibody and 2.0 µg / ml CD80-hFc. The results are as follows: Figure 6 As shown, from Figure 6 The data shows that the OD value of this murine PD-L1 antibody exhibits a clear trend, with distinct upper and lower plateaus on the curve, indicating strong competitive activity (IC50=0.1942). Its blocking activity against CD80 is superior to that of the positive control.

[0099] As can be seen from the detection results of (1) to (5) above, the murine PD-L1 antibody obtained by the present invention has strong competitiveness and has great application potential in the treatment of related diseases.

[0100] Example 4

[0101] Sequencing of PD-L1 antibody in murine hybridoma cells:

[0102] Hybridoma cells were cultured to obtain a small amount of purified high-purity RNA, which was reverse transcribed into cDNA. This cDNA was then used as a template for in vitro amplification of the target fragment using specific PCR primers. The positive PCR product was ligated into a T-vector for plasmid recombination and transformation. Positive clones were selected through blue-white screening and PCR verification, cultured, and sequenced. The sequencing results were analyzed, and the correct sequences were determined, completing the antibody sequencing.

[0103] The sequencing results are shown in the table below:

[0104] Biological information sequencing results Serial Number Nucleic acid sequence of mouse antibody heavy chain. CAGGTGCAGCTGAAGGAGTCCGGCCCCAGCCTGGTGAAGCCCTCCCAGACACTGAGCCTGACATGTTCCGTGACAGGCGATTCCATCACCAGGCGCTACTGGAGCTGGATCAGAAAGTTCCCTGGCAGCAAGTTCGAGTACATGGGCTACATCTCCTACACCGGCAGCACATACCAGA ACCCCTCCCTGAAGAGCAGAATCTCCATCACAAGAGACACCAGCAAGAATCAGTACTACCTGCAGCTGAACAGCGTGACCAGCGAGGATACAGCCACCTACTTCTGTGCCAGGTCCAGCGATTGGCTGTACCCCTTCGCCGATTGGGGCCAGGGCACCCTGGTGACAGTGTCCGCCGCC SEQ ID NO: 6 Nucleic acid sequence of mouse antibody light chain. CAGATCGTGCTGACACAGAGCCCCGCCATCATGAGCGCCAGCCCCGGAGAGAAGGTGACCATGACATGCTCCGCCTCCAGCAGCGTGAGCTACATGCACTGGTACCAGCAGAAGAGCTCCACATCCCCTAAGCTGTGGATCTACGACACATCCAAGCTG GCCTCCGGCGTGCCCGGCAGATTCTCCGGAAGCGGCTCCGGCAATTCCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGTGGCCACATACTACTGTTTCCAGGGCAGCGGCTACCCCCTGACATTTGGCGCCGGCACAAAGCTGGAGCTGAAG SEQ ID NO: 7 Protein sequence of mouse antibody heavy chain. QVQLKESGPSLVKPSQTLSLTCSVTGDSITSGYWSWIRKFPGSKFEYMGYISYTGSTYQNPSLKSRISITRDTSKNQYYLQLNSVTSEDTATYFCARSSDWLYPFAD SEQ ID NO: 8 Protein sequence of mouse antibody light chain. QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSSTSPKLWIYDTSKLASGVPGRFSGSGSGNSYSLTISSMEAEDVATYYCFQGSGYPLTFGAGTKLELK SEQ ID NO: 9

[0105] Example 5

[0106] Human sequence mutations:

[0107] Using 3D modeling and database comparison, the original murine sequence was mutated into a human sequence. The original murine hybridoma cell PD-L1 antibody sequence was designed into multiple humanized amino acids (huVH4, huVH5, huVH6, huVL3, huVL4, specific sequences are shown below), and the designed sequences were combined into a humanized antibody, which was then expressed in the Expi 293 mammalian expression system.

[0108] The sequencing results of the above humanized amino acid sequences are shown in the table below.

[0109] Biological information sequencing results Serial Number 4D11_huVH4 QVQLKESGPGLVKPSQTLSLTCSVTGDSITSGYWSWIRQHPGKKFEYMGYISYTGSTYQNPSLKSRISITRDTSKNQYSLKLSSVTAADTAVYYCARSSDWLYPFADWGQGTTVTVSS SEQ ID NO: 1 4D11_huVH5 QVQLKESGPGLVKPSQTLSLTCTVTGDSITSGYWSWIRQHPGKGFEYMGYISYTGSTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSSDWLYPFADWGQGTTVTVSS SEQ ID NO: 2 4D11_huVH6 QVQLQESGPGLVKPSQTLSLTCTVSGGSITSGYWSWIRQHPGKGLEYMGYISYTGSTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSSDWLYPFADWGQGTTVTVSS SEQ ID NO: 3 4D11_huVL3 EIVLTQSPATLSLSPGERATMTCSASSSVSYMHWYQQKPGQAPRLWIYDTSKLASGVPARFSGSGSGNDYTLTISSLEPEDFAVYYCFQGSGYPLTFGQGTKLEIK SEQ ID NO: 4 4D11_huVL4 EIVLTQSPATLSLSPGERATMSCRASSSVSYMHWYQQKPGQAPRLWIYDTSKLATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSGYPLTFGQGTKLEIK SEQ ID NO: 5

[0110] The 4D11 humanized anti-PD-L1 antibody was purified as follows: The 4D11 humanized anti-PD-L1 antibody expressed in Expi 293 cells was purified using cell supernatant following standard protein purification procedures. The experimental results were characterized as follows: Figure 7 As shown in the figure. The experimental results indicate that the purity of the purified 4D11 humanized anti-PD-L1 antibody is >90%.

[0111] Example 6

[0112] 4D11 Humanized Anti-PD-L1 Antibody Activity Assay:

[0113] (1) ELISA method for detecting the binding activity of 4D11 humanized anti-PD-L1 antibody to PD-L1-his:

[0114] The ELISA method was used to coat the test plate with 1.0 µg / ml PD-L1-his. A concentration gradient of 4D11 humanized anti-PD-L1 antibody samples was set up, and the binding activity OD value was measured to detect the affinity between the 4D11 humanized anti-PD-L1 antibody and the target antigen. The experimental results were characterized, and the characterization results are as follows: Figure 8 As shown, the 4D11 humanized anti-PD-L1 antibody binds strongly to the antigen, with clear upper and lower plateaus on the curve, a large window, and good activity. Among this series of antibodies, 4D11-huVH4 / 3, 4D11-huV5 / L3, 4D11-huV6 / L3, and 4D11-huV4 / L4 all exhibit superior activity compared to the positive antibody Atezolizumab.

[0115] (2) FACS method was used to detect the binding activity of 4D11 humanized anti-PD-L1 antibody to CHO-PD-L1 cells:

[0116] A series of concentrations of 4D11 humanized anti-PD-L1 antibody were prepared and incubated with CHO-PD-L1 cells. The binding signal intensity was measured using FACS to detect the binding activity of the 4D11 humanized anti-PD-L1 antibody to the target protein. The experimental results were characterized as follows: Figure 9 As shown, this series of 4D11 humanized anti-PD-L1 antibodies exhibits strong binding to CHO-PD-L1 and good activity. Among these 4D11 humanized anti-PD-L1 antibodies, 4D11-huVH4 / 3, 4D11-huV5 / L3, 4D11-huV6 / L3, 4D11-huV4 / L4, and 4D11-huV5 / L4 all demonstrate superior binding activity compared to the positive antibody Atezolizumab.

[0117] (3) ELISA method for detecting the competitive activity of 4D11 humanized anti-PD-L1 antibody against PD-1-mFc:

[0118] The test plate was coated with 4.0 µg / ml PD-L1-his, and a sample concentration gradient was set up. The OD value was measured to detect the competitive activity between the 4D11 series humanized anti-PD-L1 antibody and 9.0 µg / ml PD-1-mFc ligand. Data showed that the antibody's OD value exhibited a clear trend, with distinct upper and lower plateaus on the curve, indicating strong competitive activity. The experimental results were characterized, as shown below. Figure 10 As shown, 4D11-huVH4 / 3, 4D11-huV6 / L3, and 4D11-huV4 / L4 exhibited superior competitive activity compared to the positive antibody Atezolizumab.

[0119] (4) FACS method for detecting the competitive activity of 4D11 humanized anti-PD-L1 antibody against PD-1-mFc:

[0120] CHO-PD-L1 cells were uniformly seeded into 96-well cell culture plates using the FACS method. A constant concentration of PD-1-mFc (3.0 µg / ml) and a gradient of 4D11 humanized anti-PD-L1 antibody concentrations were set to bind to the cells. Alex488-labeled goat anti-mouse secondary antibody was then added. The competitive activity OD value was measured after binding to determine the competitiveness of 4D11 humanized anti-PD-L1 antibody against PD-1-mFc. The experimental results were characterized as follows: Figure 11 As shown, the 4D11 humanized anti-PD-L1 antibodies in this series are all highly competitive, with 4D11-huVH4 / 3, 4D11-huV5 / L3, 4D11-huV6 / L3, 4D11-huV4 / L4, and 4D11-huV5 / L4 exhibiting superior competitive activity compared to the positive antibody Atezolizumab.

[0121] (5) Luciferase Assay for detecting the activity of 4D11 humanized anti-PD-L1 antibody:

[0122] 50 μl / well of a mixture of huPD-1-NF-AT-Juakat and huPD-L1 artificial APC cells were added to each well of a 96-well plate. The final density of huPD-1-NF-AT-Juakat cells was 1E5 (cells / well), and the final density of huPD-L1 artificial APC cells was 2E4 (cells / well). The 4D11 humanized anti-PD-L1 antibody was diluted to a specific concentration and added to the 96-well plate for incubation with the cells. After incubation, a fluorescence reaction solution was added. The 96-well cell culture plate was then placed in a chemiluminescence analyzer to read the chemiluminescence values. The data were analyzed using Graphpad Prism software for four-parameter curve fitting to obtain the EC50 value. The experimental results were characterized as follows: Figure 12 As shown, the curve of the 4D11 humanized anti-PD-L1 antibody has clear upper and lower plateaus, a large window, and its activity is superior to that of the positive control Atezolizumab.

[0123] Example 7

[0124] Thermostability assay of humanized antibodies:

[0125] The thermal stability of proteins was monitored by detecting fluorescence signal intensity using the Protein Thermal Shift™ dye kit and a real-time quantitative PCR method. The experimental results were characterized, and the characterization results are as follows: Figure 13 As shown in the table below, except for 4D11-huVH5 / L4 and 4D11-huVH6 / L4, the Tm values ​​of the other four humanized anti-PD-L1 antibodies of 4D11 are higher than those of the parent antibody. Among them, 4D11-huVH4 / L4 has the highest thermal stability, followed by 4D11-huVH4 / L3.

[0126] protein name Tm value 1 Tm value 2 average 4D11-H / L 70.55 70.41 70.48 4D11-huVH4 / L3 71.59 71.55 71.57 4D11-huVH5 / L3 70.74 70.65 70.70 4D11-huVH6 / L3 70.70 70.68 70.69 4D11-huVH4 / L4 71.69 71.63 71.66 4D11-huVH5 / L4 70.51 70.45 70.48 4D11-huVH6 / L4 70.50 70.44 70.47

[0127] From the data in the table above and Figure 13 The data clearly show that, compared to the parent antibody and conventional anti-PD-L1 antibodies, such as atezolizumab, the thermal stability can be improved by more than 30%, which greatly expands the application field and scope of anti-PD-L1 antibodies, making them more effective in combination therapy.

[0128] Example 8

[0129] 4D11 humanized anti-PD-L1 antibody MLR experiment:

[0130] All 4D11 humanized anti-PD-L1 antibody samples were set up with concentration gradients and incubated with fully differentiated cells. OD values ​​were measured using an ELISA kit to detect IFN-γ secretion in the cell supernatant. The experimental results were characterized, and the characteristics are as follows: Figure 14 As shown, both groups of 4D11 humanized anti-PD-L1 antibodies can promote the secretion of IFN-γ in cell supernatant, and the secretion is dose-dependent.

Claims

1. An anti-PD-L1 antibody, characterized in that, The antibody is composed of heavy and light chains; The heavy chain includes at least one of the following: The first heavy chain contains the heavy chain variable region as shown in SEQ ID NO: 1; The second heavy chain contains the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 2; The third heavy chain contains the heavy chain variable region as shown in SEQ ID NO: 3; The light chain includes at least one of the following: The first light chain contains a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 4; The second light chain contains the light chain variable region as shown in SEQ ID NO: 5, which contains an amino acid sequence.

2. The application of the anti-PD-L1 antibody according to claim 1, characterized in that... ， The anti-PD-L1 antibody of claim 1 is used to prepare pharmaceuticals and / or pharmaceutical compositions; The drug and / or drug composition are used for targeted therapy of cancer.

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