CN110981958B - PD-L1 antibody - Google Patents
PD-L1 antibody Download PDFInfo
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- CN110981958B CN110981958B CN201910788886.3A CN201910788886A CN110981958B CN 110981958 B CN110981958 B CN 110981958B CN 201910788886 A CN201910788886 A CN 201910788886A CN 110981958 B CN110981958 B CN 110981958B
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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Abstract
The invention provides a novel anti-PD-L1 monoclonal recombinant single domain antibody, belonging to the field of tumor immunotherapy drugs. The PD-L1 antibody has the sequence shown in SEQ ID NO.1, and has short sequence and strong binding capacity to PD-L1. The PD-L1 can be theoretically used for preparing tumor immunotherapy drugs and has good application prospect.
Description
Technical Field
The invention belongs to the field of tumor immunotherapy drugs, and particularly relates to an anti-PD-L1 monoclonal recombinant single-domain antibody.
Background
Tumors are one of the major diseases endangering human health in the twenty-first century. Although there are currently treatment means for treating tumors, such as surgical treatment, radiotherapy, chemotherapy, targeted therapy, etc., the five-year survival rate of patients with advanced lung cancer is only 5%, in recent years, along with the development of tumor immunotherapy drugs, immunotherapy has become a new and more promising treatment means, and is recognized as the 4 th main pillar in the twenty-first century tumor treatment model. Currently, in immunotherapy, immune checkpoint inhibitors and cellular immunotherapy represented by Chimeric antigen receptor T cells (CAR-T) and T cell receptor T cells (TCR-T) have achieved good clinical efficacy.
Immune checkpoints (Immune checkpoints) are protein molecules and signaling pathways that function to inhibit the over-activation of Immune cells in Immune responses; the immune checkpoint inhibitor is a molecule capable of inhibiting the immune checkpoint, and can promote the anti-tumor immunity of an organism and kill tumor cells by enhancing the function of T lymphocytes.
Programmed death-1 molecule (PD-1) is a well-known immune checkpoint molecule expressed on the surface of activated T cells. Programmed cell death-Ligand1 (PD-L1) is a Ligand of PD-1, and the binding of the programmed cell death-Ligand1 and the PD-1 promotes the latter to play the role of immunosuppression, starts the apoptosis of antigen-specific T cells and reduces the apoptosis of regulatory T cells. PD-L1 is expressed on the surface of autologous cells, and its existence is to avoid the free clearance of autologous cells by immune cells. And PD-L1 is also expressed on the cell surface of various tumors (such as malignant tumors of lung cancer, melanoma, bladder cancer, kidney cancer and the like), so that tumor cells can be prevented from being eliminated by an immune system.
The PD-L1 antibody is one kind of medicine for tumor immunotherapy, and through combining with PD-L1, blocks the combination of PD-1 and PD-L1, it can reduce immunosuppression and make T cell play anticancer role. Based on similar principles, PD-L1 can also be used in the preparation of anti-infective drugs, particularly against pathogens for which no effective vaccine is currently available, such as HIV, hepatitis viruses (a, b, c), influenza virus, herpes virus, giardia, malaria, leishmania, staphylococcus aureus, pseudomonas aeruginosa, etc.
Disclosure of Invention
The invention aims to provide a novel anti-PD-L1 monoclonal recombinant single-domain antibody.
The technical scheme of the invention is as follows:
an anti-PD-L1 monoclonal recombinant single domain antibody, wherein the amino acid sequence of CDR1 is shown as SEQ ID NO.7, the amino acid sequence of CDR2 is shown as SEQ ID NO.8, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 9.
The anti-PD-L1 monoclonal recombinant single domain antibody has an amino acid sequence shown in SEQ ID NO. 10.
An anti-tumor biological immunity medicine, which is a medicinal preparation prepared by taking the PD-L1 antibody as an active ingredient and adding pharmaceutically acceptable auxiliary ingredients and/or carriers.
The use of the aforementioned recombinant single domain antibody in the preparation of a PD-L1 inhibitor.
The PD-L1 inhibitor is a drug that inhibits the binding of PD-L1 to PD-1 for the aforementioned uses.
The recombinant single domain antibody as described above is used for preparing anti-tumor drugs.
Further, the tumor is a malignant tumor.
Further, the malignant tumor is lung cancer, melanoma, bladder cancer, kidney cancer, liver cancer, nasopharyngeal cancer, esophageal cancer, cervical cancer, breast cancer or stomach cancer.
The invention has the following beneficial effects:
the invention provides a novel anti-PD-L1 monoclonal recombinant single-domain antibody, which only has 368 amino acids, has small molecular weight and is easy for industrial production.
The PD-L1 antibody of the invention can fully bind to PD-L1 on the surface of tumor cells, blocks the binding of PD-L1 and PD-1, and has the blocking effect equivalent to that of positive control Tecntriq (an anti-PD-L1 antibody) biological similar medicine.
The PD-L1 antibody can be used for preparing anti-tumor immunocyte medicaments and has good application prospect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The foregoing aspects of the present invention are explained in further detail below with reference to specific embodiments. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1SEC-HPLC chart
FIG. 2 is an electrophoresis image of RNA. M: DNAmarker; lane 1: NB 034; lane 2: NB 035.
FIG. 3 is a diagram of colony PCR identification. The upper part: NB 034; the following steps: NB 035; the first lane on the left is DNAmarker, which is the same size as FIG. 2.
FIG. 4 is a histogram of the fluorescence signal of PD-1/PD-L1 Bioassay.
Detailed Description
EXAMPLE 1 preparation of PD-L1 antibody B132205 of the present invention
The main steps for preparing the PD-L1 monoclonal recombinant single-domain antibody are as follows:
1. constructing library to obtain immune antibody protein
To ensure immunityResults were confirmed by immunizing two alpacas (Nos.: NB034 and NB035), collecting 50mL of peripheral blood after 4 immunizations, and isolating PBMC (peripheral blood mononuclear cells). PBMC total RNA was then extracted with TRIzol reagent. The RNA purity was determined by electrophoresis using 1. mu.g of RNA, and the results showed that the RNA purity was good (FIG. 1). See also
III First-StrandSynthesis System for RT-PCR instructions will extract RNA reverse transcription into cDNA. Amplification of nucleic acid fragments encoding the variable region of the heavy chain antibody by nested PCR:
first round PCR:
the upstream primer F1:
the downstream primer R1:
second round PCR:
the first round PCR product is used as a template,
the upstream primer F2:
the downstream primer R2:
the downstream primer R3:
first round of PCR amplification was performed using primers F1 and R1 to obtain a product of about 750bp, which was purified and second round of PCR amplification was performed using primers F2 and R2, R3(R2 and R3 were mixed together, 2 reverse primers were used because there were 2 subtypes of antibody) to obtain a VHH target fragment of about 450 bp.
The vector poc comb3Xss (NBbiolab, P001) and the target fragment were cleaved with SfiI, respectively, overnight at 50 ℃. After enzyme digestion, the vector and the target fragment are connected by using T4 ligase, electrophoresis is carried out, and then gel cutting is carried out to recover the target fragment. The ligation molar ratio was poc comb3 Xss: VHH 1: 3.
The aforementioned ligation products were then electroporated into e.coli TG1 competent cells, and a heavy chain single domain antibody phage library display library against PD-L1 was constructed and identified. The size of the reservoir was calculated to be 1.21X 10 by gradient dilution plating9. To examine the library insertion rate, 48 clones were randomly picked for colony PCR, and the results showed that the insertion rates were all 100% (FIG. 2).
2. PD-L1 heavy chain single domain antibody screening
The B132205 antibody was diluted to a final concentration of 5. mu.g/mL with carbonate buffer pH 9.6, added to the wells at 100. mu.L/well, and coated overnight at 4 ℃. Discarding the coating solution, adding 300. mu.L of 3% BSA-PBS blocking solution into each well, blocking at 37 ℃ for 1h, adding 100. mu.L of phage library, incubating at 37 ℃ for 1h, adding 100. mu.L of Gly-HCl eluent, transferring the eluent into a 1.5mL sterile centrifuge tube, and quickly neutralizing with 15. mu.L of Tris-HCl neutralization buffer. 10 μ L was diluted in gradient, titer was determined, and the elutriation recovery was calculated, and the remaining eluates were mixed and amplified and purified for the next round of affinity elutriation, with the conditions changed as in Table 1 for each round, for a total of three rounds.
TABLE 1 affinity panning conditions
The elutriation eluate was mixed with 20mL of e.colitg1 culture at the early stage of logarithmic growth, and allowed to stand at 37 ℃ for 30 min. Adding 1mL of 20% glucose, and culturing at 37 ℃ and 180r/min for 30min with shaking. M13K07 phage was added at a ratio of cell: phase of 1: 20, 4. mu.L of Amp + (100mg/mL) was added, the mixture was allowed to stand at 37 ℃ for 30min, and the mixture was cultured at 180r/min with shaking for 30 min. The culture was aliquoted into centrifuge tubes, the cell pellet resuspended overnight, the pellet suspended in 200 μ L PBS to give phage with positive vector and the titer was determined.
3. Identification and analysis of specific phage clones
The elutriated eluate was plated on a plate, 48 single colonies were randomly picked with sterilized toothpicks and inoculated into 1mL of 2 XYT-A medium, followed by shaking culture at 37 ℃ for 8 hours. Then 200. mu.L of the above culture was added with M13K07 phage at a ratio of cell to phase of 1: 20, and incubated at 37 ℃ for 15min and 220r/min for 45min with shaking. The following day supernatants were centrifuged and used for ELISA to identify positive phage clones. The recombinant single-domain antibody of B132205 was obtained by sequence analysis of the positive phage clone, etc. The amino acid sequence (SEQ ID NO.6) is as follows:
the key fragments of the SEQ ID NO.6 sequence are shown in Table 2,
TABLE 2 Key fragments of recombinant Single Domain antibodies of the invention
The advantageous effects of the present invention will be further described below by way of experimental examples.
Experimental example 1 verification of Effect of antibody B132205 on blocking the interaction between PD-1 and PD-L1
1 principle of experiment
PD-1/PD-L1Bioassay is a functional activity evaluation system of a cell-dependent bioluminescence detection means, and consists of two genetically engineered cell lines. PD-1 effector cells: jurkat T cells express the human luciferase reporter gene induced by the PD-1 and IL2 promoters. PD-L1aAPC/CHO-K1 cells: CHO-K1 cells expressed human PD-L1 and a cell surface protein capable of activating the TCR. When these two cells were co-cultured, PD-L1 interacted with ligand PD-1 and inhibited T cell activation signaling pathways, and inhibited luciferase expression regulated by the IL2 promoter. When the corresponding PD-1 antibody is added, it blocks the interaction between PD-1 and ligand PD-L1, removes the inhibitory signal, thereby activating T cells and activating luciferase expression induced by the IL2 promoter. The blocking functional activity of the anti-PD-1 or PD-L1 antibody was evaluated by evaluating the expression amount of luciferase by adding Bio-Glo reagent (including buffer and substrate).
This example uses Jurkat T cells expressing human PD-1 and NFAT-RE-induced luciferase reporter genes as effector cells and CHO-K1 cells expressing human PD-L1 and TCR activator proteins as antigen presenting cells. Biomimic of Tecntriq, an antibody against PD-L1, was used as a positive control to evaluate the in vitro blocking functional activity of PD1/PDL1 of antibody B132205.
Similar drug sequences to Tecentriq:
heavy Chain Sequence (Heavy Chain Sequence, SEQ ID NO. 11):
light Chain Sequence (Light Chain Sequence, SEQ ID NO.12)
2 Experimental procedures
1) The target cells were collected by centrifugation, resuspended and cell density adjusted using F12K Nutrient mix medium containing 10% FBS, and plated in 96-well plates.
2) The plates were transferred to a cell culture incubator (37 ℃/5% CO2) for 16-20 hours of incubation.
3) A positive control and a test article (B132205 prepared by the method of example 1) were prepared at 2X concentration using Assay Buffer.
4) Effector cells were harvested by centrifugation, resuspended using Assay Buffer and cell density adjusted.
5) And taking the 96-well experiment plate which is finished with incubation out of the cell culture box, removing the culture medium, and sequentially transferring the reference substance, the test substance working solution and the effector cell suspension to the 96-well experiment plate.
6) Transfer the assay plates to a cell incubator (37 ℃/5% CO)2) And incubated for about 6 hours.
7) Preparing luciferase substrate working solution.
8) The 96-well assay plate with completed incubation was removed from the cell incubator and the luciferase substrate working solution was transferred to the 96-well assay plate.
9) Chemiluminescence values were read on PHERA Star FSX and data were recorded.
10) And establishing a corresponding dose-effect curve chart according to the corresponding relation between the Relative chemiluminescence signal value (Relative Luminescence Unit) and the final detection concentration.
3 results
As shown in fig. 4, antibody B132205 was effective in blocking PD-1 and PD-L1 binding and activating downstream pathways to produce a signal. The EC50 of the dose-response curve is 0.1080 mu g/ml, which is equivalent to that of the positive drug.
In conclusion, the PD-L1 antibody can well recognize PD-L1 and effectively block the combination of PD-1 and PD-L1; the application of the derivative in the preparation of tumor immunotherapy medicaments can theoretically achieve good anti-tumor effects.
SEQUENCE LISTING
<110> Sichuan university Hospital in western China
<120> a PD-L1 antibody
<130>GYKH1352-2019P017256CC
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Claims (5)
1. An anti-PD-L1 monoclonal recombinant single domain antibody is characterized in that the amino acid sequence of CDR1 is shown as SEQ ID NO.7, the amino acid sequence of CDR2 is shown as SEQ ID NO.8, and the amino acid sequence of CDR3 is shown as SEQ ID NO. 9.
2. The anti-PD-L1 monoclonal, recombinant single domain antibody according to claim 1, having the amino acid sequence shown in SEQ id No. 10.
3. An anti-tumor biological immunity drug, which is characterized in that the anti-tumor biological immunity drug is a pharmaceutical preparation prepared by taking the PD-L1 antibody of claim 1 as an active ingredient and adding pharmaceutically acceptable auxiliary ingredients.
4. Use of the recombinant single domain antibody of claim 1 or 2 in the preparation of a PD-L1 inhibitor;
the PD-L1 inhibitor is a medicine for treating lung cancer, melanoma, bladder cancer, renal cancer, liver cancer, nasopharyngeal carcinoma, esophageal cancer, cervical cancer, breast cancer or gastric cancer.
5. The use of claim 4, wherein the PD-L1 inhibitor is a medicament that inhibits the binding of PD-L1 to PD-1.
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| CN111856032A (en) * | 2020-07-21 | 2020-10-30 | 成都华西海圻医药科技有限公司 | Method for rapidly detecting concentration of anti-PD-L1 monoclonal antibody |
| CN113527494B (en) * | 2020-11-26 | 2022-08-02 | 四川大学华西医院 | Novel anti-tumor transformation receptor T cell |
| CN113234162B (en) * | 2020-12-24 | 2022-05-13 | 四川大学华西医院 | Chimeric antigen receptor T cell targeting CD133 |
| CN115120744A (en) * | 2021-03-24 | 2022-09-30 | 四川大学 | Application of recombinant human endostatin adenovirus and anti-PD-1 antibody or anti-PD-L1 antibody in preparation of anti-tumor drugs |
| WO2022228431A1 (en) * | 2021-04-27 | 2022-11-03 | 上海君实生物医药科技股份有限公司 | Anti-pd-l1 single domain antibody and use thereof |
| CN114057880B (en) * | 2021-11-10 | 2022-09-09 | 四川大学华西医院 | DLL3 monoclonal antibody |
| CN115785269B (en) * | 2022-11-01 | 2023-09-22 | 四川大学 | anti-PD-L1 antibodies and uses thereof |
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