WO2020259566A1 - 抗PD-L1纳米抗体及其Fc融合蛋白和应用 - Google Patents

抗PD-L1纳米抗体及其Fc融合蛋白和应用 Download PDF

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WO2020259566A1
WO2020259566A1 PCT/CN2020/098051 CN2020098051W WO2020259566A1 WO 2020259566 A1 WO2020259566 A1 WO 2020259566A1 CN 2020098051 W CN2020098051 W CN 2020098051W WO 2020259566 A1 WO2020259566 A1 WO 2020259566A1
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nanobody
cells
fusion protein
sequence
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屈向东
潘琴
金后聪
都业杰
郑翰
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启愈生物技术(上海)有限公司
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Priority to US17/597,130 priority Critical patent/US20220242974A1/en
Priority to EP20833039.9A priority patent/EP4023671A4/en
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Definitions

  • the present invention relates to an anti-PD-L1 Nanobody.
  • the present invention also relates to an anti-PD-L1 Nanobody Fc fusion protein.
  • the present invention also relates to the application of an anti-PD-L1 Nanobody.
  • the present invention also relates to an anti-PD-L1 Nanobody.
  • the application of Nanobody Fc fusion protein belongs to the field of biomedicine.
  • the immune system can recognize tumor antigens and eliminate them. If the immune system can completely eliminate tumor cells, then immune clearance can proceed stably. If tumor cells escape the immune system's clearance through mutations, the immune system will rebalance. In this process, the immunogenicity of tumor cells gradually decreases. The ability of tumor cells to proliferate becomes weaker under the pressure of the immune system, making the detection of tumor cells more difficult.
  • oncogenes causes tumor cells to change themselves and the tumor microenvironment, which breaks the balance between the immune system and tumor cells.
  • the tumor microenvironment can also suppress the immune system by releasing immunosuppressive factors, such as IL-10 and TGF- ⁇ .
  • immunosuppressive factors such as IL-10 and TGF- ⁇ .
  • the surface of tumor cells also highly express immunosuppressive proteins (such as programmed death ligand-1, PD-L1).
  • PD-L1 interacts with PD-1 and induces T cells to undergo apoptosis.
  • tumors grow rapidly and metastasize. If the host's immune system is artificially activated and redirected to tumor cells, the tumor tissue can theoretically be eliminated, and the theory of immunotherapy has been widely proven in clinical treatment.
  • Immunotherapy can be divided into two categories: specific treatment and non-specific treatment.
  • Specific therapies in the category include the following treatment strategies: tumor vaccines activate immune cells against the patient’s antigen by injecting tumors.
  • Tumor vaccines include: inactivated tumor cell vaccines, tumor antigen vaccines, tumor DNA vaccines, dendritic cell (DC) vaccines and bacterial vaccines.
  • Specific ACT immunotherapy mainly includes three treatment methods,
  • TIL Tumor infiltrating lymphocytes
  • T cell receptor (TCR) therapy T cells recognize tumor antigens through their single-chain antibody fragments (scFv), and clone the single-chain antibody fragment TCR into normal T cells through viral vectors. Therefore, normal T cells become specific tumor killer T cells.
  • scFv single-chain antibody fragments
  • CAR-T T cells are genetically modified to obtain tumor-specific receptor T cells. Different from the conventional T cell recognition mechanism, CAR-T cell recognition of tumor antigens is not restricted by MHC molecules. Therefore, CAR-T cells can overcome the immune escape mechanism of tumors by increasing costimulatory signal molecules, and enhance the killing ability of T cells against tumor cells.
  • LAK lymphokine activated killer
  • CIK cytokine induced killer
  • LAK cell therapy On the one hand, LAK cells use IL-2 to stimulate immune cells in peripheral blood lymphocytes, including NK cells and T cells, and on the other hand, by overexpressing FAS ligands, they can enhance their ability to recognize target cells. Release perforin and granzyme cells to kill tumor cells.
  • CIK cell therapy CIK cells are derived from peripheral blood lymphocytes (PBL) of patients or healthy people, and are expanded under the stimulation of anti-CD3 antibodies, IFN- ⁇ and IL-2 under ex vivo conditions. CIK cells mainly exert anti-tumor effects through FasL and perforin.
  • PBL peripheral blood lymphocytes
  • Immune checkpoints are protective molecules in the human immune system that prevent inflammatory damage caused by excessive activation of T cells in the normal body. Tumor cells can take advantage of this feature to overexpress immune checkpoint molecules, inhibit the body's immune response, escape the human immune system's surveillance and killing, and promote the growth of tumor cells. Immune checkpoint inhibitor therapy can inhibit the activity of immune checkpoints in the tumor microenvironment and reactivate the immune response of T cells to tumors to achieve anti-tumor effects.
  • the complete activation of T cells is regulated by the "dual signal" system: the first signal comes from the specific binding of its own TCR (T cell receptor) to the MHC of the antigen, that is, the T cell recognizes the antigen; the second signal comes from a costimulatory molecule, the The signal is involved in the interaction of costimulatory molecules expressed by antigen-presenting cells (APC) with corresponding receptors or ligands (eg CD28) on the surface of T cells.
  • APC antigen-presenting cells
  • CD28-B7 is a positive costimulatory signal
  • negative costimulatory molecules are mainly CTLA4-B7 pathway and PD-1/PD-L1 pathway. After tumor cells invade, this inhibitory pathway is favored by tumor cells to inhibit T cell activation, thereby evading the clearance of the immune system.
  • PD-1 (CD279) was first reported in 1992.
  • the human PD-1 encoding gene PDCD1 is located at 2q37.3, with a total length of 2097bp, composed of 6 exons, and the translation product is a PD-1 precursor consisting of 288 amino acids.
  • Protein, the mature protein is obtained by cutting the signal peptide composed of the first 20 amino acids.
  • PD-1 includes the extracellular immunoglobulin variable region IgV domain, hydrophobic transmembrane domain and intracellular domain.
  • the N-terminal ITIM motif of the intracellular tail domain contains 2 phosphorylation sites, and the C-terminal is one ITSM motif.
  • PD-1 is a membrane protein that belongs to the CD28 immunoglobulin superfamily.
  • PD-1 has two ligands, namely PD-L1 (CD274, B7-H1) and PD-L2 (CD273, B7-DC) of the B7 protein family.
  • PD-L1 and PD-L2 are 40% identical . The difference between the two is mainly due to the different expression patterns.
  • PD-L1 is constitutively under-expressed in APCs, non-hematopoietic cells (such as vascular endothelial cells, pancreatic islet cells) and immune-privileged sites (such as placenta, testis, and eyes).
  • non-hematopoietic cells such as vascular endothelial cells, pancreatic islet cells
  • immune-privileged sites such as placenta, testis, and eyes.
  • Type I and type II interferons, TNF- ⁇ and VEGF can all induce the expression of PD-L1.
  • PD-L2 is only expressed in activated macrophages and dendritic cells.
  • the ITSM motif of PD-1 undergoes tyrosine phosphorylation, which in turn leads to the dephosphorylation of downstream protein kinases Syk and PI3K, and inhibits downstream AKT, ERK and other pathways. Activation, and ultimately inhibit the transcription and translation of genes and cytokines required for T cell activation, and play a negative role in regulating T cell activity.
  • tumor cells and tumor microenvironment up-regulate the expression of PD-L1 and bind to PD-1 on the surface of tumor-specific CD8+ T cells to negatively regulate T cell activity and inhibit immune response.
  • Tumor cells can up-regulate the expression of PD-L1 through the following four ways: 1. Gene amplification (9p24.1) encoding PD-L1; 2. EGFR, MAPK, PI3K-Akt signaling pathway activation, HIF-1 transcription factor, etc. Up-regulate the expression of PD-L1 from the transcriptional level; 3. Induction of Epstein-Barr virus (EB virus-positive gastric cancer and nasopharyngeal carcinoma show high PD-L1 expression); 4. Regulation of epigenetics.
  • stimulation of inflammatory factors such as interferon- ⁇ can also induce the expression of PD-L1 and PD-L2.
  • Inflammatory factors can induce other cells in the tumor microenvironment, including macrophages, dendritic cells and stromal cells to express PD-L1 and PD-L2, and tumor-infiltrating T cells that can recognize tumor antigens can secrete interferon- ⁇ , and then Inducing the up-regulation of PD-L1 expression, this process is called "adaptive immune resistance", tumor cells can achieve self-protection through this mechanism.
  • PD-L1 and PD-L2 have been found in various solid tumors and hematological malignancies.
  • the expression of PD-Ls has a strong correlation with the poor prognosis of tumor cells, which proves to include esophageal cancer, stomach cancer, kidney cancer, ovarian cancer, bladder cancer, pancreatic cancer and melanoma.
  • the PD-1 therapeutic monoclonal antibodies that have been approved by the FDA for marketing include Nivolumab (Opdivo, September 2014), Pembrolizumab (Keytruda, December 2014) and Cemiplimab (Libtayo, September 2018).
  • PD-L1 has been marketed.
  • Therapeutic monoclonal antibodies include Atezolizumab (Tecentriq, September 2014), avelumab (Bavencio, May 2016) and Duravulumab (Imfinzi, May 2017). The approved indications are shown in the table below
  • PD-1 monoclonal antibodies such as Pidilizumab, AMP-224, AMP-514, PDR001 and PD-L1 monoclonal antibodies such as BMS-936559 and CK-301, which are under development and clinical trials.
  • the purpose of the present invention is to provide an anti-PD-L1 Nanobody and its Fc fusion protein and application to solve the above problems.
  • the present invention provides an anti-PD-L1 Nanobody, which is characterized in that it contains at least one VHH fragment, and the VHH fragment contains three amino acid fragments of CDR1, CDR2 and CDR3, and CDR1, CDR2 and CDR3 are respectively selected from the following sequences :
  • the anti-PD-L1 Nanobody of the present invention is characterized in that its sequence is as shown in SEQ ID No. 1 to SEQ No. 41.
  • the present invention also provides an anti-PD-L1 Nanobody Fc fusion protein, characterized in that it comprises the anti-PD-L1 Nanobody according to claim 1 or 2 and an Fc segment, the sequence of the Fc segment is as SEQ ID No. 42 shown.
  • the anti-PD-L1 Nanobody of the present invention is characterized in that in its sequence, except for CDR1, CDR2 and CDR3, 80% of the amino acid sequence is identical to the sequence shown in SEQ ID No. 1 to SEQ No. 41.
  • the invention also provides an application of the anti-PD-L1 nanobody in the preparation of a reagent for blocking the binding of PD-L1 and PD-1.
  • the invention also provides an application of the Fc fusion protein of the anti-PD-L1 nanobody in the preparation of a reagent for blocking the binding of PD-L1 and PD-1.
  • the anti-PD-L1 Nanobody of the present invention is characterized in that its dosage is 20ug/ml to 0.000128ug/ml.
  • the anti-PD-L1 Nanobody of the present invention is characterized in that: its sequence is as shown in SEQ ID No. 100 to SEQ ID No. 105.
  • the present invention also provides a humanized anti-PD-L1 Nanobody Fc fusion protein, characterized in that it comprises the anti-PD-L1 Nanobody according to claim 8 and an Fc segment, the sequence of the Fc segment is as SEQ ID No. 42 is shown.
  • Nanobody or its Fc fusion protein as described in any one of the above in the preparation of medicines for the treatment of cancer, infection or immunomodulatory diseases.
  • cancer or tumor is selected from the following tissues or parts: colorectal, breast, ovarian, pancreas, stomach, esophagus, prostate, kidney, cervix, bone marrow cancer, Lymphoma, leukemia, thyroid, endometrial, uterus, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous Cellular skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome.
  • the nanobody and its Fc fusion protein of the invention have strong specificity, high affinity, weak immunogenicity to humans, and significant anti-tumor effects.
  • Figure 1A is the binding curve of human Fc fusion proteins with PD-L1 Nanobody numbers QP1120, QP1122, QP1123, QP1124, QP1125, QP1126, QP1127, QP1128, QP1129, QP1130 and QP1139 to PD-L1 protein;
  • Figure 1B is the binding curve of human Fc fusion protein with PD-L1 Nanobody numbers QP1140, QP1141, QP1142, QP1143, QP1144, QP1145, QP1146, QP1147, QP1148, QP1149, and QP1150 to PD-L1 protein;
  • Figure 1C is the binding curve of human Fc fusion proteins of PD-L1 Nanobody numbers QP1151, QP1152, QP1153, QP1154, QP1155, QP1156, QP1157, QP1158, QP1159, QP1160, and QP1161 to PD-L1 protein;
  • Figure 1D is the binding curve of human Fc fusion protein of PD-L1 Nanobody numbered QP1162, QP1163, QP1164, QP1165, QP1166, QP1168 and QP1169 to PD-L1 protein;
  • Figure 2A is the binding curve of human Fc fusion proteins of PD-L1 Nanobody numbers QP1120, QP1122, QP1123, QP1124, QP1125, QP1126, QP1127, QP1128, QP1129 and QP1130 to biotinylated mouse PD-L1 protein;
  • Figure 2B is the binding curve of human Fc fusion proteins with PD-L1 Nanobody numbers QP1122, QP1142, QP1143, QP1144, QP1145, QP1146, QP1147, QP1148, QP1149, QP1150 and QP1151 to biotinylated mouse PD-L1 protein;
  • Figure 2C is the binding curve of human Fc fusion protein of PD-L1 Nanobody numbers QP1126, QP1152, QP1153, QP1154, QP1155, QP1156, QP1157, QP1158, QP1159, QP1160, QP1161 to biotinylated mouse PD-L1 protein;
  • Figure 3A is the PD-1/PD-L1 interaction blocking curve of PD-L1 Nanobody numbers QP1120, QP1162, QP1163, QP1164, QP1165, QP1166, QP1168, QP1169, QP1139, QP1140 and QP1141;
  • FIG. 3B PD-L1 Nanobody: the blocking curve of human Fc fusion proteins numbered QP1122, QP1142, QP1143, QP1144, QP1145, QP1146, QP1147, QP1148, QP1149, QP1150 and QP1151 against PD-1/PD-L1 interaction;
  • FIG. 3C PD-L1 Nanobody No. QP1126, QP1152, QP1153, QP1154, QP1155, QP1156, QP1157, QP1158, QP1159, QP1160, QP1161 human Fc fusion protein blocking PD-1/PD-L1 interaction curve;
  • Figure 4A is the PD-1/PD-L1 interaction blocking curve of human Fc fusion proteins with PD-L1 Nanobody numbers QP1120, QP1162, QP1163, QP1164 and QP1165;
  • Figure 4B is the PD-1/PD-L1 interaction blocking curve of human Fc fusion proteins with PD-L1 Nanobody numbers QP1166, QP1168, QP1169, QP1122, QP1126;
  • Figure 4C is the PD-1/PD-L1 interaction blocking curve of human Fc fusion proteins with PD-L1 Nanobody numbers QP1139, QP1140, QP1141, QP1142, QP1143;
  • Figure 4D is the human Fc fusion protein of PD-L1 Nanobody numbered QP1144, QP1145, QP1146, QP1147 and QP1148: blocking curve of PD-1/PD-L1 interaction;
  • Figure 4E is the PD-1/PD-L1 interaction blocking curve of human Fc fusion proteins with PD-L1 Nanobody numbers QP1149, QP1150, QP1151, QP1152 and QP1153;
  • Figure 4F is the PD-1/PD-L1 interaction blocking curve of human Fc fusion proteins with PD-L1 Nanobody numbers QP1149, QP1155, QP1156, QP1157 and QP1158;
  • Figure 4G is the PD-1/PD-L1 interaction blocking curve of human Fc fusion proteins with PD-L1 Nanobody numbers QP1159, QP1160 and QP1161;
  • Figure 5A is the human Fc fusion protein of PD-L1 Nanobody numbers QP1120, QP1162, QP1163, QP1164, QP1165 and QP1166: binding curve to human non-small cell lung cancer cell HCC827;
  • Figure 5B is the human Fc fusion protein of PD-L1 Nanobody numbers QP1168, QP1169, QP1122, QP1126, QP1139 and QP1141: binding curve to human non-small cell lung cancer cell HCC827;
  • Figure 5C is the binding curve of human Fc fusion protein of PD-L1 Nanobody numbered QP1142, QP1149, QP1151, QP1156, QP1157, QP1158 to human non-small cell lung cancer cell HCC827;
  • Figure 6 is the humanized PD-L1 Nanobody numbered QP1162 The binding curves of human Fc fusion proteins of QP320, QP321, QP322, QP1166, QP323, QP324, QP325 to biotinylated human PDL1 protein;
  • Figure 7 is the blocking curve of human Fc fusion protein of humanized PD-L1 Nanobody numbered QP1162, QP320, QP321, QP322, QP1166, QP323, QP324, QP325 against PD-1/PD-L1 interaction;
  • Figure 8 is a schematic diagram of the structure of an anti-CLDN18.2/anti-PD-L1 bispecific antibody molecule
  • Figure 9 is the result of the identification of the functional activity of the anti-CLDN18.2/anti-PD-L1 bispecific antibody PD-L1 (QP3711461 in the mixed lymphocyte reaction MLR is dependent on the concentration of the cytokine IFN ⁇ produced after T cell activation.
  • Figure 10 is the result of the functional activity identification of anti-CLDN18.2/anti-PD-L1 bispecific antibody PD-L1 (QP3711461 in the mixed lymphocyte reaction MLR is dependent on the concentration of the cytokine IL-2 produced after T cell activation.
  • Figure 11 shows the results of the tumor growth curve of the anti-CLDN18.2/anti-PD-L1 bispecific antibody in the humanized transgenic mouse C57BL/6-hPDL1 model MC38-hPDL1.
  • the PD-L1-his fusion protein of immune camel is purified by nickel column, and the sequence is shown in SEQ ID NO.43.
  • One Xinjiang Bactrian camel was selected for subcutaneous multi-point injection immunization, and 50 mL of peripheral blood was collected after four immunizations to separate PBMC;
  • RNA extract total RNA with TRIzol reagent. After electrophoresis to verify that the RNA is sufficiently pure, use III First-Strand Synthesis System for RT-PCR transcribe 8ug RNA, after 2 rounds of nested PCR, the target nucleic acid fragment is recovered and purified with DNA product purification kit.
  • the obtained phage clones that bind to PD-L1 are spread on a 96-well plate, and 150ul 2 ⁇ TY-Amp-0.1% sucrose medium is added, and cultured at 37°C for 3 hours;
  • the ELISA plate is coated with PD-L1-his protein 2ng/ul, 50ul/well, 4°C overnight;
  • the sample well has a reading value that is more than 2 times higher than the control well. It is considered a positive clone.
  • the positive hole bacteria solution is expanded and the plasmid is extracted for sequencing; the same sequence of CDR1, 2, 3 is regarded as the same clone, so Obtain 41 unique Nanobody sequences, as shown in SEQ ID NO: 1-41.
  • Table 1 EC50 value of each antibody corresponding to the experimental result in Figure 1A
  • Mouse PDL1 was expressed by HEK293. Use Thermo's Biotinlytion kit to obtain the biotinylated protein mPDL1-Biotin;
  • Table 5 is the EC50 value of each antibody corresponding to the experimental result in Figure 2A
  • Table 6 is the EC50 value of each antibody corresponding to the experimental result in Figure 2B
  • Table 7 is the EC50 value of each antibody corresponding to the experimental result in Figure 2C
  • Table 8 is the EC50 value of each antibody corresponding to the experimental result in Figure 2D
  • Table 9 is the IC50 value of each antibody corresponding to the experimental result in Figure 3A
  • Table 10 is the IC50 value of each antibody corresponding to the experimental result in Figure 3B
  • Table 11 is the IC50 value of each antibody corresponding to the experimental result in Figure 3C
  • Table 12 is the IC50 value of each antibody corresponding to the experimental result in Figure 4A
  • Table 13 is the IC50 value of each antibody corresponding to the experimental result in Figure 4B
  • Table 14 is the IC50 value of each antibody corresponding to the experimental result in Figure 4C
  • Table 15 is the IC50 value of each antibody corresponding to the experimental result in Figure 4D
  • Table 16 is the IC50 value of each antibody corresponding to the experimental result in Figure 4E
  • Table 17 is the IC50 value of each antibody corresponding to the experimental result in Figure 4F
  • Table 18 is the IC50 value of each antibody corresponding to the experimental result in Figure 4G
  • HCC827 naturally highly expresses PD-L1.
  • Table 19 is the EC50 value of each antibody corresponding to the experimental result in Figure 5A
  • Table 20 is the EC50 value of each antibody corresponding to the experimental result in Figure 5B
  • Table 20 is the EC50 value of each antibody corresponding to the experimental result in Figure 5C
  • the heavy and light chain variable with high homology to QP1162 SEQ ID No. 35
  • QP1166 SEQ ID No. 39
  • the region germline gene is used as a template, and the CDRs of the murine antibody are transplanted into the corresponding human template to form the variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Then select some important amino acid residues to make back mutation combinations. The amino acid residues are identified and annotated by the Kabat numbering system.
  • VH containing gene fragments required for recombination and digestion with BsmBI to recover the expression vector pQD were added to DH5H competent cells at a ratio of 3:1, ice bath at 0°C for 30 minutes, and heat at 42°C Press for 90s, add 5 times the volume of LB medium, incubate at 37°C for 45min, spread on LB-Amp plate, culture overnight at 37°C, pick a single clone and send it to sequencing to obtain each target clone.
  • variable region sequence and protein expression number of the humanized design of each clone are shown below.
  • the antibodies in this table are fused with the human IgG1-FC constant region at the C-terminus:
  • the culture density of 293E cells is maintained between 0.2-3 ⁇ 10 6 /ml, and the maintenance phase medium (GIBCO Freestyle 293 expression medium) is cultured.
  • the maintenance phase medium GIBCO Freestyle 293 expression medium
  • the day before transfection the cells to be transfected are centrifuged to change the medium, and the cell density is adjusted to 0.5- 0.8 ⁇ 10 6 /ml.
  • the density of 293E cells was 1-1.5 ⁇ 10 6 /ml.
  • Prepare plasmid and transfection reagent PEI The amount of plasmid to be transfected is 100ug/100ml cells, and the mass ratio of PEI to plasmid is 2:1. Mix the plasmid and PEI, and let stand for 15 minutes, not more than 20 minutes.
  • the plasmid and PEI mixture was slowly added to the 293E cells, cultured in a shaker at 8% CO2, 120 rpm, 37° C., on the fifth day of transfection, the cell supernatant was collected by centrifugation at 4700 rpm in a horizontal centrifuge for 20 minutes.
  • Coating protein QP1138 (PD1-FC) 2ug/ml 50ul/well, 4°C overnight. Wash 3 times with PBS. Blocking: 3% BSA 250ul/well, incubate at room temperature for 1h. Prepare 2ug/ml biotin-PDL1-FC and different concentrations of QP1120 15ug/ml, QP11801181 30ug/ml, 1:3 dilution, equal volume mixing, and incubate at room temperature for 1h. Wash with PBST 3 times and PBS 3 times. Incubate the secondary antibody: HRP-strepavidin (1:5000) 50ul/well, wash 6 times with PBST and 3 times with PBS. Color rendering: TMB 100ul/hole, color rendering 10min. 2M H2SO4 50ul/hole termination. The results are shown in Figure 7 and Table 24.
  • SPR Surface plasmon resonance
  • Biacore T200 (GE) is used to determine the affinity of the molecule to be tested with the protein human PD-L1 and cynoPD-L1
  • the antigen information is as follows:
  • DC (donor1) cells Resuscitate PBMC, separate monocytes with EasySep TM Human Monocyte Isolation Kit (Stemcell 19359), add rhGM-CSF (1000U/ml) and rhIL4 (500U/ml), culture cells at 37°C for 6 days Induce into iDC; change the medium every 2-3 days and add rhGM-CSF (1000U/ml) and rhIL4 (500U/ml) at the same time; collect the cells by centrifugation at 300x g for 5min, add rhGM-CSF (1000U/ml) and rhIL4 (500U/ml) medium was resuspended, while adding LPS (1 ⁇ g/ml), the cells were cultured at 37°C for 1 day to induce mature DC; the cells were collected and counted for later use.
  • donor1 cells Resuscitate PBMC, separate monocytes with EasySep TM Human Monocyte Isolation Kit (Stemcell 19359)
  • T (donor2) cells resuscitate PBMC, and isolate CD4+Tcell with EasySep TM Human CD4+T Cell Isolation Kit (Stemcell 17952).
  • QP3711461 is obviously dependent on the concentration of the cytokines IFN ⁇ and IL-2 produced by T cells after activation. Prove the biological function of the PD-L1 antibody in QP3711461. As shown in Figure 9 and Figure 10.
  • mouse colon cancer cell MC38-hPDL1 in the transgenic mouse C57BL/6-hPDL1 subcutaneous model to evaluate the in vivo efficacy of the anti-PD-L1 nanobody.
  • mice Take logarithmic growth phase mouse colon cancer cell MC38-hPDL1 cells (the cells knock out PDL1 in mice and express human PDL1), remove the culture medium and wash twice with PBS, and then inoculate them in C57BL/6-hPDL1 cells.
  • the right flank of the mouse is subcutaneously, and the inoculation volume is 5 ⁇ 10 5 /100 ⁇ L/mouse.
  • the mice after inoculation were observed and the tumor growth was monitored.
  • On the 8th day after inoculation when the average tumor volume reached 92.9 mm 3 , they were randomly divided into 4 groups according to the tumor volume, with 9 mice in each group. The grouping day was defined as D0 day, and the administration was started on D0 day.
  • the molecule to be tested is anti-CLDN18.2/anti-PD-L1 bispecific antibody QP3711461, and the dosages are 4mpk, 10mpk, 25mpk, BIW ⁇ 3, ip administration.
  • the average tumor volume of the PBS group (negative control group) reached 1445.20mm 3
  • the average tumor volume of the QP3711461 (4mpk) group was 477.00mm 3
  • TGI 72.00%
  • the average tumor volume of the QP3711461 (10mpk) group was 279.97mm 3
  • TGI 86.14%
  • the average tumor volume of the QP3711461 (25mpk) group was 293.96mm 3
  • TGI 85.14%
  • the tumor volume of each dose group and the PBS group were statistically significantly different (t test, p ⁇ 0.01).

Abstract

提供一种抗PD-L1纳米抗体及其Fc融合蛋白和应用，该抗PD-L1纳米抗体及其Fc融合蛋白特异性强，亲和力高，对人的免疫原性弱。并且稳定性高，具有显著的抗肿瘤效果。

Description

抗PD-L1纳米抗体及其Fc融合蛋白和应用 技术领域
本发明涉及一种抗PD-L1纳米抗体，本发明还涉及一种抗PD-L1纳米抗体的Fc融合蛋白，本发明还涉及抗PD-L1纳米抗体的应用，本发明还涉及抗PD-L1纳米抗体的Fc融合蛋白的应用，属于生物医药领域。
背景技术
在经典免疫监视理论中，免疫系统可以识别肿瘤抗原并将其消除。如果免疫系统能够完全消除肿瘤细胞，那么免疫清除可以稳定进行。如果肿瘤细胞通过突变逃避免疫系统的清除，免疫系统将会进行重新平衡(rebalance)。在这一过程中，肿瘤细胞的免疫原性逐渐降低。肿瘤细胞的增殖能力在免疫系统的压力下变弱，使得肿瘤细胞的侦测变得更为困难。
致癌基因的激活导致肿瘤细胞改变自身及肿瘤微环境，使得免疫系统和肿瘤细胞间的平衡被打破。当免疫系统和肿瘤细胞进入逃逸阶段，肿瘤细胞的恶性程度会提高，而肿瘤细胞丢失MHC分子使其避免被免疫细胞识别和消除。肿瘤微环境也可以通过释放免疫抑制因子抑制免疫系统，如IL-10，TGF-β等。肿瘤细胞表面也会高表达免疫抑制蛋白(如程序化死亡配体-1，PD-L1)，当效应T细胞与肿瘤细胞结合时，PD-L1与PD-1相互作用并诱导T细胞发生凋亡，这是肿瘤对免疫系统产生耐受的主要原因之一，肿瘤迅速生长，发生转移。如果人为激活宿主的免疫系统并将其重定向到肿瘤细胞，从理论上来讲肿瘤组织就能够被清除， 而免疫治疗的理论已经在临床治疗中得到广泛证明。
免疫疗法可分为两类：特异性治疗和非特异性治疗。在类别中特定疗法，包括以下治疗策略：肿瘤疫苗通过注射肿瘤激活免疫细胞对患者的抗原。肿瘤疫苗包括：灭活的肿瘤细胞疫苗，肿瘤抗原疫苗，肿瘤DNA疫苗，树突状细胞(DC)疫苗和细菌疫苗。特异性ACT免疫疗法主要包括三种治疗方法，
肿瘤浸润淋巴细胞(TIL)：淋巴细胞从肿瘤组织中分离并在体外培养。TIL可以分泌具有特异性抗肿瘤的IL-2能力。
T细胞受体(TCR)治疗：T细胞识别肿瘤抗原通过其单链抗体片段(scFv)，并将单链抗体片段TCR通过病毒载体克隆到正常T细胞中。因此，正常T细胞变为特异性肿瘤杀伤T细胞。
CAR-T：T细胞通过基因修饰获得具有肿瘤特异性受体T细胞。与常规T细胞识别机制不同，CAR-T细胞识别肿瘤抗原不受到MHC分子的限制。因此，CAR-T细胞能够通过增加共刺激信号分子克服肿瘤的免疫逃逸机制，增强T细胞对肿瘤细胞的杀伤能能力。
在非特异性ACT免疫疗法中，有两种主要应用治疗方法：淋巴因子激活的杀手(lymphokine activated killer,LAK)细胞治疗和细胞因子诱导的杀手(cytokine induced killer,CIK)细胞治疗。
LAK细胞治疗：LAK细胞一方面利用IL-2刺激外周血淋巴细胞中的免疫细胞，包括NK细胞和T细胞等，另一方面通过过表达FAS配体，增强对靶细胞的识别能力，并通过释放穿孔素和颗粒酶细胞杀死肿瘤细胞。
CIK细胞治疗：CIK细胞来源于病人或健康人的外周血淋巴细胞 (PBL)，在ex vivo条件下受到anti-CD3抗体、IFN-γ和IL-2刺激下扩增。CIK细胞主要通过FasL和穿孔素发挥抗肿瘤的作用。
免疫检查点是人体免疫系统中的保护性分子，在正常机体中防止由于T细胞过度活化引起的炎性损伤。肿瘤细胞能够利用这一特性，过度表达免疫检查点分子，抑制机体的免疫反应，逃避人体免疫系统的监视和杀伤，从而促进肿瘤细胞的生长。免疫检查点抑制剂治疗可以通过抑制肿瘤微环境中的免疫检查点活性，重新激活T细胞对肿瘤的免疫应答反应，实现抗肿瘤的效果。T细胞的完全活化受“双信号”系统调节：第一信号来自其自身TCR(T细胞受体)与抗原的MHC的特异性结合，即T细胞识别抗原；第二信号来自共刺激分子，该信号参与由抗原呈递细胞(APC)表达的共刺激分子与T细胞表面上的相应受体或配体(例如CD28)的相互作用。例如CD28-B7是正向共刺激信号，而负向共刺激分子主要是CTLA4-B7途径和PD-1/PD-L1途径。在肿瘤细胞侵入后，这种抑制途径被肿瘤细胞利于抑制T细胞活化，从而逃避免疫系统的清除作用。
PD-1(CD279)最早于1992年被报道，人PD-1编码基因PDCD1位于2q37.3，全长2097bp，由6个外显子组成，翻译产物为288个氨基酸组成的PD-1前体蛋白，剪切前20个氨基酸组成的信号肽后得到成熟蛋白质。PD-1包括胞外免疫球蛋白可变区IgV结构域，疏水跨膜结构域和胞内结构域，胞内尾部结构域N端ITIM基序包含2个磷酸化位点，C端则是一个ITSM基序。PD-1是膜蛋白，属于CD28免疫球蛋白超家族，主要表达在激活后的T细胞表面，此外还在胸腺的CD4-CD8-T细胞、活化 的NK细胞和单核细胞有低丰度表达。PD-1有2个配体，分别是B7蛋白家族的PD-L1(CD274,B7-H1)和PD-L2(CD273,B7-DC)，PD-L1和PD-L2氨基酸序列有40％相同。两者区别主要在于表达模式不同，PD-L1组成性的低表达于APCs、非造血细胞(如血管内皮细胞、胰岛细胞)和免疫豁免部位(如胎盘、睾丸和眼睛)，炎性细胞因子如I型和II型干扰素、TNF-α和VEGF等均可以诱导PD-L1的表达。PD-L2则只在被激活的巨噬细胞和树突细胞中有表达。PD-1与PD-L1在激活的T细胞结合后，PD-1的ITSM基序发生酪氨酸磷酸化，进而导致下游蛋白激酶Syk和PI3K的去磷酸化，抑制下游AKT、ERK等通路的活化，最终抑制T细胞活化所需基因及细胞因子的转录和翻译，发挥负向调控T细胞活性的作用。
在肿瘤细胞中，肿瘤细胞及肿瘤微环境通过上调PD-L1表达并与肿瘤特异的CD8+T细胞表面的PD-1结合，负调控T细胞活性，抑制免疫反应。肿瘤细胞可以通过以下4种途径上调PD-L1表达：1.编码PD-L1的基因扩增(9p24.1)；2.EGFR、MAPK、PI3K-Akt信号通路激活，HIF-1转录因子等可以从转录水平上调PD-L1的表达；3.EB病毒的诱导(EB病毒阳性的胃癌和鼻咽癌表现为PD-L1高表达)；4.表观遗传学的调控。在肿瘤微环境中，interferon-γ等炎症因子的刺激同样可以诱导PD-L1和PD-L2的表达。炎症因子可以诱导肿瘤微环境中其他细胞，包括巨噬细胞、树突状细胞和基质细胞表达PD-L1和PD-L2，而能够识别肿瘤抗原的肿瘤浸润性T细胞能够分泌interferon-γ，进而诱导PD-L1表达上调，这一过程被称为“适应性免疫抵抗”，肿瘤细胞通过这一机制可以实现自我保护。有越来越多的证据表明肿瘤利用PD-1依赖的免疫抑制 免疫逃避。在各种实体瘤和血液系统恶性肿瘤种均已经发现PD-L1和PD-L2的高表达。此外，PD-Ls的表达与肿瘤细胞的不良预后之间具有很强相关性，证明了包括食道癌、胃癌、肾癌、卵巢癌、膀胱癌、胰腺癌和黑色素瘤等。
目前FDA已经批准上市的PD-1治疗性单抗有Nivolumab(Opdivo，2014年9月)，Pembrolizumab(Keytruda，2014年12月)和Cemiplimab(Libtayo，2018年9月)，已上市的PD-L1治疗性单抗有Atezolizumab(Tecentriq，2014年9月)，avelumab(Bavencio，2016年5月)以及Duravulumab(Imfinzi，2017年5月)，已批准的适应症见下表所示
Figure PCTCN2020098051-appb-000001
此外还有Pidilizumab，AMP-224，AMP-514，PDR001等PD-1单抗和BMS-936559，CK-301等PD-L1单抗处于研发和临床试验中。
然而现有的单抗，亲和力并未达到理想状态，并且由于体积较大，因而免疫原性较强。
发明内容
本发明的目的在于提供一种抗PD-L1纳米抗体及其Fc融合蛋白和应 用，以解决上述问题。
本发明提供一种抗PD-L1纳米抗体，其特征在于：至少包含一个VHH片段，在所述VHH片段中，包含CDR1、CDR2和CDR3三个氨基酸片段,CDR1、CDR2、CDR3分别选自以下序列：
1)SEQ ID No.44至SEQ ID No.60所示的CDR1；
2)SEQ ID No.61至SEQ ID No.82所示的CDR2；
3)SEQ ID No.83至SEQ ID No.99所示的CDR3。
进一步，本发明的抗PD-L1纳米抗体，其特征在于：其序列如：SEQIDNo.1至SEQNo.41所示。
本发明还提供一种抗PD-L1纳米抗体的Fc融合蛋白，其特征在于：包含如权利要求1或2所述的抗PD-L1纳米抗体以及Fc段，所述Fc段的序列如SEQ ID No.42所示。
进一步，本发明的抗PD-L1纳米抗体，其特征在于：其序列中，除CDR1、CDR2和CDR3以外，有80％的氨基酸序列与SEQIDNo.1至SEQNo.41所示的序列相同。
本发明还提供一种抗PD-L1纳米抗体在制备阻断PD-L1和PD-1结合试剂中的应用。
本发明还提供一种抗PD-L1纳米抗体的Fc融合蛋白在制备阻断PD-L1和PD-1结合试剂中的应用。
进一步，本发明的抗PD-L1纳米抗体其特征在于:其用量为20ug/ml至0.000128ug/ml。
进一步，本发明的抗PD-L1纳米抗体人源化改造后，其特征在于： 其序列如SEQ ID No.100至SEQ ID No.105所示。
本发明还提供一种人源化抗PD-L1纳米抗体的Fc融合蛋白，其特征在于：包含如权利要求8所述的抗PD-L1纳米抗体以及Fc段，所述Fc段的序列如SEQ ID No.42所示。
上述任一项所述的纳米抗体或者其Fc融合蛋白，在制备治疗癌症、感染或免疫调节疾病的药物中的应用。
上述任一项所述的纳米抗体，在制备抑制肿瘤生长的药物中的应用。
上述任一项所述的纳米抗体或者其Fc融合蛋白的应用中：癌症或肿瘤选自下组织或部位：结直肠、乳腺、卵巢、胰腺、胃、食管、前列腺、肾、宫颈、骨髓癌、淋巴癌、白血病、甲状腺、子宫内膜、子宫、膀胱、神经内分泌、头部颈部、肝、鼻咽、睾丸、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤，或者骨髓增生异常综合症。
发明的有益效果
本发明的纳米抗体及其Fc融合蛋白特异性强，亲和力高，对人的免疫原性弱，具有显著的抗肿瘤效果。
附图说明
图1A是PD-L1纳米抗体编号QP1120、QP1122、QP1123、QP1124、QP1125、QP1126、QP1127、QP1128、QP1129、QP1130和QP1139的human Fc融合蛋白对PD-L1蛋白的结合曲线；
图1B是PD-L1纳米抗体编号QP1140、QP1141、QP1142、QP1143、QP1144、QP1145、QP1146、QP1147、QP1148、QP1149、和QP1150的human Fc融 合蛋白对PD-L1蛋白的结合曲线；
图1C是PD-L1纳米抗体编号QP1151、QP1152、QP1153、QP1154、QP1155、QP1156、QP1157、QP1158、QP1159、QP1160、和QP1161的human Fc融合蛋白对PD-L1蛋白的结合曲线；
图1D是PD-L1纳米抗体编号QP1162、QP1163、QP1164、QP1165、QP1166、QP1168和QP1169的human Fc融合蛋白对PD-L1蛋白的结合曲线；
图2A是PD-L1纳米抗体编号QP1120、QP1122、QP1123、QP1124、QP1125、QP1126、QP1127、QP1128、QP1129和QP1130的human Fc融合蛋白对生物素化小鼠PD-L1蛋白结合曲线；
图2B是PD-L1纳米抗体编号QP1122、QP1142、QP1143、QP1144、QP1145、QP1146、QP1147、QP1148、QP1149、QP1150和QP1151的human Fc融合蛋白对生物素化小鼠PD-L1蛋白结合曲线；
图2C是PD-L1纳米抗体编号QP1126、QP1152、QP1153、QP1154、QP1155、QP1156、QP1157、QP1158、QP1159、QP1160、QP1161的human Fc融合蛋白对生物素化小鼠PD-L1蛋白结合曲线；
图3A是PD-L1纳米抗体编号QP1120、QP1162、QP1163、QP1164、QP1165、QP1166、QP1168、QP1169、QP1139、QP1140和QP1141对PD-1/PD-L1相互作用阻断曲线；
图3B PD-L1纳米抗体：编号QP1122、QP1142、QP1143、QP1144、QP1145、QP1146、QP1147、QP1148、QP1149、QP1150和QP1151的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图3C PD-L1纳米抗体编号QP1126、QP1152、QP1153、QP1154、QP1155、 QP1156、QP1157、QP1158、QP1159、QP1160、QP1161的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图4A是PD-L1纳米抗体编号QP1120、QP1162、QP1163、QP1164和QP1165的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图4B是PD-L1纳米抗体编号QP1166、QP1168、QP1169、QP1122、QP1126的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图4C是PD-L1纳米抗体编号QP1139、QP1140、QP1141、QP1142、QP1143的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图4D是PD-L1纳米抗体编号QP1144、QP1145、QP1146、QP1147和QP1148的human Fc融合蛋白:对PD-1/PD-L1相互作用阻断曲线；
图4E是PD-L1纳米抗体编号QP1149、QP1150、QP1151、QP1152和QP1153的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图4F是PD-L1纳米抗体编号QP1149、QP1155、QP1156、QP1157和QP1158的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图4G是PD-L1纳米抗体编号QP1159、QP1160和QP1161的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图5A是PD-L1纳米抗体编号QP1120、QP1162、QP1163、QP1164、QP1165和QP1166的human Fc融合蛋白:对人非小细胞肺癌细胞HCC827结合曲线；
图5B是PD-L1纳米抗体编号QP1168、QP1169、QP1122、QP1126、QP1139和QP1141的human Fc融合蛋白:对人非小细胞肺癌细胞HCC827结合曲线；
图5C是PD-L1纳米抗体编号QP1142、QP1149、QP1151、QP1156、QP1157、QP1158的human Fc融合蛋白对人非小细胞肺癌细胞HCC827结合曲线；图6是人源化PD-L1纳米抗体编号QP1162、QP320、QP321、QP322、QP1166、QP323、QP324、QP325的human Fc融合蛋白对生物素化人PDL1蛋白结合曲线；
图7是人源化PD-L1纳米抗体编号QP1162、QP320、QP321、QP322、QP1166、QP323、QP324、QP325的human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线；
图8是抗CLDN18.2/抗PD-L1双特异抗体分子的结构示意图；
图9是抗CLDN18.2/抗PD-L1双特异抗体PD-L1功能活性鉴定(混合淋巴细胞反应MLR中QP3711461对T细胞激活后产生的细胞因子IFNγ的浓度依赖结果。
图10是抗CLDN18.2/抗PD-L1双特异抗体PD-L1功能活性鉴定(混合淋巴细胞反应MLR中QP3711461对T细胞激活后产生的细胞因子IL-2的浓度依赖结果。
图11是抗CLDN18.2/抗PD-L1双特异抗体在免疫靶点人源化转基因小鼠C57BL/6-hPDL1模型MC38-hPDL1中的肿瘤生长曲线结果。
具体实施方式
以下进一步详细描述本发明的技术方案。
实施例1 PD-L1纳米抗体Fc融合蛋白的制备方法:
1.针对PD-L1的纳米抗体的筛选
1.1文库构建
a)经镍柱纯化得到免疫骆驼的PD-L1-his融合蛋白，序列如SEQ ID NO.43所示。选1只新疆双峰驼进行皮下多点注射免疫，四次免疫后采集50mL外周血分离PBMC；
b)用TRIzol试剂提取总RNA。电泳鉴定RNA纯度足够后，使用
Figure PCTCN2020098051-appb-000002
III First-Strand Synthesis System for RT-PCR转录8ug RNA，2轮巢式PCR后用DNA产物纯化试剂盒回收、纯化得到目的核酸片段。
c)将噬菌体载体pComb3XSS与目的片段分别用sfiI进行酶切，50℃过夜酶切，然后割胶回收目的片段。连接摩尔比例为Vector：VHH＝1:3。
d)电转化至TG1大肠杆菌后立即加入1mL SOC培养基复苏，37℃，180rpm复苏45min，然后离心，加入5mL SOC重悬，取10μL测定库容量，其余涂布于200mm的平板上共8块。第二天10-5共有104个克隆，因此库容量为5.04×10 9(104*5*100*10 5)。随机从滴度平板上挑取48个克隆进行鉴定，结果表明插入率100％，且大小正确。
1.2针对PD-L1的纳米抗体淘选
a)经过3轮淘选后，获得的与PD-L1结合的噬菌体克隆铺在一块96孔板中，并加入150ul 2×TY-Amp-0.1％蔗糖培养基，37℃培养3h；
b)每孔加入30ul 2×TY-Amp-5mM IPTG(IPTG最终浓度为1mM)，诱导可溶性抗体片段表达，30℃培养过夜；
c)ELISA板包被PD-L1-his蛋白2ng/ul，50ul/孔，4℃过夜；
d)1×PBS洗板1次，加入200ul 2％Milk-PBS封闭ELISA板，37℃孵育1h。
e)过夜培养的菌液，4000g离心10mins，转移上清至新的96孔板中。
f)1×PBST洗板2次，加入25ul 2％Milk-PBS封闭液，再加入25ul培养上清，混匀。25℃孵育1h。
g)1×PBST洗板3次，加入100ul anti-Fab-HRP antibody(1:5000in 2％Milk-PBS)，25℃孵育1h。
h)1×PBST洗板4次，加入TMB 100ul/孔，室温避光显色10min，加入2M H 2SO 450ul/孔，终止反应，上机于450nm读值；
i)样品孔读值高于对照孔读值2倍以上认为是阳性克隆孔，将阳性孔菌液扩大培养并提取质粒测序；将CDR1、2、3均相同的序列视为同一个克隆，从而获得41个独特的纳米抗体序列，序列如SEQ ID NO：1-41所示。
为了方便筛选进行，将41个克隆转化为C端为人IgG1的PD-L1-FC融合蛋白。重新构建的质粒在HEK293细胞中进行表达，通过protein A亲和层析纯化，除QP1121-FC外，一共获得40个候选PD-L1-FC融合蛋白，序列为SEQ ID NO.1-41所示的纳米抗体后端连接SEQ ID NO.42的Fc段所组成。相应的纳米抗体的Fc融合蛋白的编号在相应的纳米抗体后加Fc后缀形成。
2.PD-L1纳米抗体Fc融合蛋白对PD-L1蛋白结合曲线
a)包被PDL1-mFc融合蛋白，1ug/ml，100ul/孔，4℃过夜；
b)1×PBS洗板3次，加入3％BSA封闭，250ul/孔，室温孵育1h；
c)1×PBST洗板3次，1×PBS洗板3次，加入5倍梯度稀释(10ug/ml至0.000128ug/ml)的待测抗体，室温孵育1h；
d)1×PBST洗板3次，1×PBS洗板3次，加入HRP-anti-hFc(1：2500)，50ul/孔，室温孵育1h；
e)1×PBST洗板3次，1×PBS洗板3次，加入TMB 100ul/孔，室温避光显色10min，加入2M H 2SO 450ul/孔，终止反应，上机于450nm读值。
结果如图1A至图1D，和表1至表4所示。
表1：与图1A中实验结果对应的各抗体的EC50值
  EC50
QP1120-FC 0.0008491
QP1122-FC 0.001204
QP1123-FC 0.001286
QP1124-FC 0.001256
QP1125-FC 0.001066
QP1126-FC 0.001212
QP1127-FC 0.4651
QP1128-FC 0.00333
QP1129-FC 0.003955
QP1130-FC 0.005762
QP1139-FC 0.001327
3280A 0.002289
表2与图1B中实验结果对应的各抗体的EC50值
  EC50
QP1140-FC 0.001131
QP1141-FC 0.001456
QP1142-FC 0.001027
QP1143-FC 0.001669
QP1144-FC 0.001197
QP1145-FC 0.001776
QP1146-FC 0.001938
QP1147-FC 0.001695
QP1148-FC 0.00116
QP1149-FC 0.001819
QP1150-FC 0.001391
3280A 0.00246
表3与图1C中实验结果对应的各抗体的EC50值
  EC50
QP1151-FC 0.001037
QP1152-FC 0.001559
QP1153-FC 0.001419
QP1154-FC 0.00112
QP1155-FC 0.001707
QP1156-FC 0.001241
QP1157-FC 0.001216
QP1158-FC 0.001674
QP1159-FC 0.0009905
QP1160-FC 0.0007973
QP1161-FC 0.001055
3280A 0.002356
表4与图1D中实验结果对应的各抗体的EC50值
  EC50
QP1162-FC 0.0007951
QP1163-FC 0.0007316
QP1164-FC 0.0007242
QP1165-FC 0.0008409
QP1166-FC 0.0008137
QP1168-FC 0.0008302
QP1169-FC 0.0007612
3280A 0.002705
3.PD-L1纳米抗体human Fc融合蛋白对生物素化小鼠PD-L1蛋白结合曲线
a)小鼠PDL1由HEK293表达获得。使用Thermo公司Biotinlytion试剂盒得到生物素化蛋白mPDL1-Biotin；
b)包被Strepavidin，4ug/ml，50ul/孔，4℃过夜；
c)1×PBS洗板3次，加入3％BSA封闭，250ul/孔，室温孵育1h；
d)1×PBST洗板3次，1×PBS洗板3次，加入mPDL1-Biotin，1ug/ml，50ul/孔，室温孵育1h；
e)1×PBST洗板3次，1×PBS洗板3次，加入5倍梯度稀释10ug/ml至0.000128ug/ml的待测抗体，室温孵育1h；
f)1×PBST洗板3次，1×PBS洗板3次，加入HRP-anti-hFc(1：2500)，50ul/孔，室温孵育1h；
g)1×PBST洗板6次，1×PBS洗板3次，加入TMB 100ul/孔，室温避光显色10min，加入2M H 2SO 450ul/孔，终止反应，上机于450nm读值。
结果如图2A至图2D所示。
表5是与图2A中实验结果对应的各抗体的EC50值
  EC50
QP1120-FC N/A
QP1122-FC ～37681
QP1123-FC 0.01144
QP1124-FC ～185556
QP1125-FC 16.29
QP1126-FC N/A
QP1127-FC 26.98
QP1128-FC 94261
QP1129-FC 0.07072
QP1130-FC ～126139
3280A 0.01098
表6是图2B中实验结果对应的各抗体的EC50值
  EC50
QP1122-FC N/A
QP1142-FC N/A
QP1143-FC ～26132
QP1144-FC ～95487
QP1145-FC ～257223
QP1146-FC ～61912
QP1147-FC ～63480
QP1148-FC ～69726
QP1149-FC ～36588
QP1150-FC N/A
QP1151-FC ～44027
3280A 0.008206
表7是图2C中实验结果对应的各抗体的EC50值
  EC50
QP1126-FC ～6.932e-008
QP1152-FC ～118144
QP1153-FC 7.139
QP1154-FC 72.92
QP1155-FC ～55064
QP1156-FC 0.09957
QP1157-FC 0.1189
QP1158-FC N/A
QP1159-FC 3.502
QP1160-FC ～47090
QP1161-FC 0.06164
3280A 0.01322
表8是图2D中实验结果对应的各抗体的EC50值
  EC50
QP1162-FC 0.04891
QP1163-FC 0.007373
QP1164-FC 0.01443
QP1165-FC 0.01341
QP1166-FC 0.01765
QP1168-FC 0.04416
QP1169-FC 0.1067
QP1139-FC 0.02131
QP1140-FC 0.08768
QP1141-FC ～44849
3280A 0.00701
4.PD-L1纳米抗体human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线采用竞争性ELISA进行检测
a)包被PD1-hFc融合蛋白，1ug/ml，100ul/孔，4℃过夜；
b)1×PBS洗板3次，加入3％BSA封闭，250ul/孔，室温孵育1h；
c)1×PBST洗板3次，1×PBS洗板3次，加入PDL1-mFc，2ug/ml，50ul/孔，同时加入等体积5倍梯度稀释(20ug/ml至0.000256ug/ml)待测抗体，室温孵育1h；
d)1×PBST洗板3次，1×PBS洗板3次，加入HRP-anti-mFc(1：2500)，50ul/孔，室温孵育1h；
e)1×PBST洗板3次，1×PBS洗板3次，加入TMB 100ul/孔，室温避光显色10min，加入2M H 2SO 450ul/孔，终止反应，上机于450nm读值。
结果如图3A至图3C所示。
表9是与图3A中实验结果对应的各抗体的IC50值
IC50
QP1120-FC 0.3191
QP1162-FC ～1.037
QP1163-FC 0.9799
QP1164-FC 0.3334
QP1165-FC 0.3223
QP1166-FC 0.6531
QP1168-FC 0.3524
QP1169-FC ～0.5939
QP1139-FC 0.3279
QP1140-FC 0.6766
QP1141-FC 0.3986
3280A ～1.01
表10是与图3B中实验结果对应的各抗体的IC50值
IC50
QP1122-FC 0.6305
QP1142-FC 0.2909
QP1143-FC ～0.572
QP1144-FC 0.3105
QP1145-FC 0.967
QP1146-FC 0.3395
QP1147-FC ～0.711
QP1148-FC 0.3541
QP1149-FC 0.442
QP1150-FC 0.4183
QP1151-FC 0.3254
3280A ～1.006
表11是与图3C中实验结果对应的各抗体的IC50值
  IC50
QP1126-FC 0.3245
QP1152-FC 0.3295
QP1153-FC 0.4048
QF1154-FC 0.2776
QP1155-FC 1.074
QP1156-FC 0.3237
QP1157-FC 0.3289
QP1158-FC 0.247
QP1159-FC 0.2817
QP1160-FC 0.3077
QF1161-FC 0.3263
328QA ～1.07
5.PD-L1纳米抗体human Fc融合蛋白对PD-1/PD-L1相互作用阻断曲线，采用竞争性ELISA，biotin检测
a)使用thermo公司Biotinlytion试剂盒得到生物素化蛋白PDL1-Biotin；
b)包被PD1-hFc融合蛋白，1ug/ml，100ul/孔，4℃过夜；
c)1×PBS洗板3次，加入3％BSA封闭，250ul/孔，室温孵育1h；
d)1×PBST洗板3次，1×PBS洗板3次，加入PDL1-Biotin，2ug/ml，50ul/孔，同时加入等体积3倍梯度稀释(100nM至0.0457nM，对应质量浓度为15ug/ml至0.00686ug/ml)待测抗体，室温孵育 1h；
e)1×PBST洗板3次，1×PBS洗板3次，加入HRP-Strepavidin(1∶5000)，50ul/孔，室温孵育1h；
f)1×PBST洗板6次，1×PBS洗板3次，加入TMB 100ul/孔，室温避光显色10min，加入2M H 2SO 450ul/孔，终止反应，上机于450nm读值。
结果如图4A至图4G所示。
表12是与图4A中实验结果对应的各抗体的IC50值
  IC50
QP1120-FC 4.903
QP1162-FC 4.754
QP1163-FC 14.18
QP1164-FC 11.46
QP1165-FC 13.66
3280A 4.914
表13是与图4B中实验结果对应的各抗体的IC50值
  IC50
QP1166-FC 12.75
QP1168-FC 13.45
QP1169-FC 4.551
QP1122-FC 4.085
QP1126-FC 4.291
3280A 4.273
表14是与图4C中实验结果对应的各抗体的IC50值
  IC50
QP1139-FC ～11.74
QP1140-FC 14.58
QP1141-FC 13.91
QP1142-FC 13.49
QP1143-FC 14.01
3280A 5.44
表15是与图4D中实验结果对应的各抗体的IC50值
  IC50
QP1144-FC 13.3
QP1145-FC 21.99
QP1146-FC 15.24
QP1147-FC 14.39
QP1148-FC 14.15
3280A 4.557
表16是与图4E中实验结果对应的各抗体的IC50值
  IC50
QP1149-FC 13.33
QP1150-FC 13.99
QP1151-FC 14.03
QP1152-FC 13.55
QP1153-FC 13.19
3280A 4.721
表17是与图4F中实验结果对应的各抗体的IC50值
  IC50
QP1149-FC 14.14
QP1155-FC ～11.71
QP1156-FC ～11.88
QP1157-FC 13.46
QP1158-FC 4.652
3280A 4.881
表18是与图4G中实验结果对应的各抗体的IC50值
  IC50
QP1159-FC ～11.66
QP1160-FC 11.94
QP1161-FC 11.94
3280A 4.189
6.PD-L1纳米抗体human Fc融合蛋白对人非小细胞肺癌细胞HCC827结合曲线
a)人非小细胞肺癌细胞HCC827天然高表达PD-L1。准备对数期生长HCC827细胞(汇合度80％)，调整浓度并铺板种于costar 96孔板中，1E5细胞/孔，1×PBS洗板一次，加入3％BSA，250ul/孔， 37℃孵育1h；
b)加入4倍梯度稀释(33.33nM至0.008nM，对应质量浓度为5ug/mL至0.001ug/mL)待测抗体，50ul/孔，冰上孵育1h；
c)1×PBS洗板2次，加入PE-anti hFc(1∶200)，50ul/孔，冰上孵育1h；
d)1×PBS洗板3次，上机读数。
结果如图5A至图5C所示。
表19是与图5A中实验结果对应的各抗体的EC50值
  EC50
QP1120-FC 0.2912
QP1162-FC 0.2302
QP1163-FC 0.1946
QP1164-FC 0.1926
QP1165-FC 0.2325
QP1166-FC 0.161
3280A 0.3181
表20是与图5B中实验结果对应的各抗体的EC50值
  EC50
QP1168-FC 0.1927
QP1169-FC 0.1898
QP1122-FC 0.1719
QP1126-FC 0.1714
QP1139-FC 0.2182
QP1141-FC 0.2044
3280A 0.3181
表20是与图5C中实验结果对应的各抗体的EC50值
  EC50
QP1142-FC 0.203
QP1149-FC 0.248
QP1151-FC 0.2359
QP1156-FC 0.1605
QP1157-FC 0.161
QP1158-FC 0.1788
3280A 0.3181
实施例2抗PD-L1纳米抗体的人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选与QP1162(SEQ ID No.35)、QP1166(SEQ ID No.39)同源性高的重轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。再选择一些重要的氨基酸残基做回复突变组合。其中氨基酸残基由Kabat编号系统确定并注释。
设计引物PCR搭建各人源化抗体VH基因片段，再与带信号肽及恒定区基因(FC)片段的表达载体pQD进行同源重组，构建抗体全长表达载体VH-FC-pQD。
利用在线软件DNAWorks(v3.2.2)(http：//helixweb.nih.gov/dnaworks/)设计多条引物合成VH/VK含重组所需基因片段：5’-30bp信号肽+VH+30bp FC-3’。按照TaKaRa公司Primer STAR GXL DNA聚合酶操作说明书，用上面设计的多条引物，分两步PCR扩增得到VH/VK含重组所需基因片段。带信号肽及恒定区基因(FC)片段的表达载体pQD的构建及酶切，利用限制性内切酶，如BsmBI，识别序列与酶切位点不同的特性设计构建带信号肽及恒定区基因(FC)片段的表达载体pQD。BsmBI酶切载体，切胶回收备用。重组构建表达载体VH-FC-pQD。VH含重组所需基因片段与BsmBI酶切回收表达载体pQD(带信号肽及恒定区基因(FC)片段)按3：1比例分别加入DH5H感受态细胞中，0℃冰浴30min，42℃热击90s，加入5倍体积LB介质，37℃孵育45min，涂布LB-Amp平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。
各克隆人源化设计轻重链可变区序列及蛋白表达编号如下所示，此表中抗体在其C端融合人IgG1-FC恒定区：
表21：QP1162和QP1166人源化设计
Figure PCTCN2020098051-appb-000003
同时设计克隆表达人源化前嵌合抗体及对照抗体如下表所示：
表22：人源化前嵌合抗体及对照抗体
Figure PCTCN2020098051-appb-000004
2.抗PD-L1纳米抗体人源化蛋白表达
293E细胞培养密度维持在0.2-3×10 6/ml之间，维护阶段培养基(GIBCO Freestyle 293 expression medium)进行培养，转染前一天将待转染细胞离心换液，调整细胞密度为0.5-0.8×10 6/ml。转染当天，293E细胞密度为1-1.5×10 6/ml。准备质粒和转染试剂PEI，需转染质粒量为 100ug/100ml细胞，使用PEI和质粒的质量比为2：1。将质粒和PEI进行混匀，静置15min，不宜超过20min。将质粒和PEI混合物缓慢加入293E的细胞中，放入8％CO2，120rpm，37℃的摇床中培养，转染第五天，水平离心机4700rpm离心20min收集细胞上清。
3.抗PD-L1纳米抗体人源化蛋白纯化
Protein A亲和层析纯化
用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；将离心后培养液上清过柱，上样40ml，流速0.33ml/min；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速0.33ml/min；用洗脱液过柱，UV280上升至15mAU时开始收集洗脱峰(PAC-EP)，UV280下降至15mAU时停止收集，流速1ml/min。样品收集完成后，用pH调节液将PAC-EP调至中性。
4.人源化抗PD-L1纳米抗体活性鉴定(Binding-ELISA)
包被抗体QP1162/QP320/QP321/QP322/QP1166/QP323/QP324/QP325 0.75ug/ml，QP11801181 1.5ug/ml 50ul/孔，4℃过夜。PBS 3 times。封闭：3％BSA 250ul/孔，RT 1h。分别孵育2ug/ml biotin-PDL1-FC 1：4稀释至不同浓度，室温孵育1h。PBST洗3次，PBS洗3次。孵育二抗：HRP-strepavidin(1：5000)50ul/孔，PBST洗6次，PBS洗3次。显色：TMB 100ul/孔，显色10min。2M H2SO4 50ul/孔终止。结果如图6和表23所示。
表23：人源化抗PD-L1纳米抗体活性鉴定(Binding-ELISA)结果
QP1162 1162-V1 1162-V2 1162-V3 QP1166 1166-V1 1166-V2 1166-V3  
conc.(ug/ml) QP1162 QP320 QP321 QP322 QP1166 QP323 QP324 QP325 QP11801181
2.0000 3.4366 0.2297 0.2251 3.0042 3.4143 2.8564 0.4130 3.4216 3.4911
0.5000 3.4482 0.1419 0.1378 2.7861 3.2805 1.8773 0.2012 3.2090 3.4778
0.1667 3.2597 0.0957 0.0836 2.7811 3.1343 0.7893 0.1285 3.1059 3.7196
0.0556 2.8355 0.0843 0.0756 2.7008 3.2327 0.3141 0.0882 2.7828 3.2350
0.0185 2.5016 0.0792 0.0960 2.0715 2.5660 0.1810 0.1070 2.1246 2.5747
0.0062 1.5396 0.1076 0.1201 1.2735 1.5464 0.1663 0.1502 1.3189 1.9496
0.0021 0.5232 0.1140 0.1561 0.4107 0.5605 0.2577 0.1414 0.5978 0.8702
0.0007 0.1214 0.1248 0.1562 0.1756 0.2169 0.1810 0.1917 0.1817 0.1986
5.人源化抗PD-L1纳米抗体活性鉴定(Blocking-ELISA)
包被蛋白QP1138(PD1-FC)2ug/ml 50ul/孔，4℃过夜。PBS洗3次。封闭：3％BSA 250ul/孔，室温孵育1h。分别配制2ug/ml biotin-PDL1-FC和不同浓度QP1120 15ug/ml，QP11801181 30 ug/ml，1∶3稀释，等体积混匀，室温孵育1h。PBST洗3次，PBS洗3次。孵育二抗：HRP-strepavidin(1∶5000)50ul/孔，PBST洗6次，PBS洗3次。显色：TMB 100ul/孔，显色10min。2M H2SO4 50ul/孔终止。结果如图7和表24所示。
表24：人源化抗PD-L1纳米抗体活性鉴定(Blocking-ELISA)
Figure PCTCN2020098051-appb-000005
7.人源化抗PD-L1纳米抗体SPR鉴定亲和力
表面等离子体共振(SPR)检测亲和力
通过Biacore T200(GE)测定待检分子与蛋白人PD-L1及cynoPD-L1的亲和力
抗原信息如下：
表25：蛋白编号
蛋白编号 蛋白描述 货号
QPP09.1 PD-L1 Protein，Human，Recombinant(His Tag) SinoBiologic，10084-H08H
QPP10.1 PD-L1 Protein，Cynomolgus，Recombinant(His Tag) SinoBiologic，90251-C08H
表26：SPR亲和力结果
Figure PCTCN2020098051-appb-000006
实施例3人源化抗PD-L1纳米抗体活性鉴定
构建设计抗CLDN18.2/抗PD-L1双特异抗体分子QP3711461，形式如图8所示。
Figure PCTCN2020098051-appb-000007
1.PD-L1功能体外活性鉴定(混合淋巴细胞反应MLR)
准备DC(donor1)细胞：复苏PBMC，用EasySep TMHuman Monocyte  Isolation Kit(Stemcell 19359)分离单核细胞monocytes，加入rhGM-CSF(1000U/ml)和rhIL4(500U/ml)，37℃培养细胞6天诱导为iDC；每2-3天半换液，同时补充rhGM-CSF(1000U/ml)和rhIL4(500U/ml)；收集细胞300x g离心5min，用加入rhGM-CSF(1000U/ml)和rhIL4(500U/ml)的培养基重悬，同时加入LPS(1μg/ml)，37℃继续培养细胞1天诱导为成熟DC；收集细胞，计数备用。
准备T(donor2)细胞：复苏PBMC，用EasySep TMHuman CD4+T Cell Isolation Kit(Stemcell 17952)分离CD4+Tcell。
准备抗体：用培养基1：5梯度稀释抗体(初始浓度10ug/ml)6个浓度。将DC细胞：T细胞为1：10的比例混合，加入不同浓度的抗体，混合培养，第2天检测培养上清中IL2的表达，第5天检测培养上清中IFNg的表达。
在混合淋巴细胞反应实验中，QP3711461对T细胞激活后产生的细胞因子IFNγ及IL-2的浓度有明显的抗体浓度依赖。证明QP3711461中PD-L1抗体的生物学功能。如图9和图10所示。
2.PD-L1功能体内活性鉴定
利用小鼠结肠癌细胞MC38-hPDL1在转基因小鼠C57BL/6-hPDL1皮下模型，评估抗PD-L1纳米抗体的体内药效。
实验方法：取对数生长期小鼠结肠癌细胞MC38-hPDL1细胞(该细胞敲除小鼠的PDL1，表达人的PDL1)，去除培养液并用PBS洗两次后接种于C57BL/6-hPDL1小鼠右侧胁腹部皮下，接种量：5×10 5/100μL/只。观察接种后小鼠并监测肿瘤的生长，接种后第8天，平均肿瘤体积达到 92.9mm 3时，根据肿瘤体积随机分成4组，每组9只。分组当天定义为D0天，并于D0天开始给药。
实验结果：如图11和表25所示。
表25：肿瘤体积
分组 第27天肿瘤体积 第27天TGI％ P值(ttest)
溶媒对照组(PBS)   -- --
QP1461371-4mpk 477.00 72.00％ 0.0095**
QP1461371-10mpk 279.97 86.14％ 0.0018**
QP1461371-25mpk 293.96 85.14％ 0.0037**
待测分子为抗CLDN18.2/抗PD-L1双特异抗体QP3711461，给药剂量分别为4mpk、10mpk、25mpk，BIW×3，i.p.给药。给药后第27天PBS组(阴性对照组)平均肿瘤体积达到1445.20mm 3，QP3711461(4mpk)组平均肿瘤体积477.00mm 3，TGI＝72.00％，QP3711461(10mpk)组平均肿瘤体积279.97mm 3，TGI＝86.14％，QP3711461(25mpk)组平均肿瘤体积293.96mm 3，TGI＝85.14％；各剂量给药组与PBS组的肿瘤体积均有统计学意义极显著差异(t检验，p<0.01)。
该实验说明在免疫靶点人源化转基因小鼠的MC38-hPDL1模型中，本发明的抗CLDN18.2/抗PD-L1双特异抗体中的抗PD-L1分子表现出优越的抗肿瘤能力。
本发明所提供的抗PD-L1纳米抗体编号及对应序列如下所示。
>SEQ ID No.1 QP1120
Figure PCTCN2020098051-appb-000008
>SEQ ID No.2 QP1121
Figure PCTCN2020098051-appb-000009
>SEQ ID No.3 QP1122
Figure PCTCN2020098051-appb-000010
>SEQ ID No.4 QP1123
Figure PCTCN2020098051-appb-000011
>SEQ ID No.5 QP1124
Figure PCTCN2020098051-appb-000012
>SEQ ID No.6 QP1125
Figure PCTCN2020098051-appb-000013
>SEQ ID No.7 QP1126
Figure PCTCN2020098051-appb-000014
>SEQ ID No.8 QP1127
Figure PCTCN2020098051-appb-000015
>SEQ ID No.9 QP1128
Figure PCTCN2020098051-appb-000016
>SEQ ID No.10 QP1129
Figure PCTCN2020098051-appb-000017
>SEQ ID No.11 QP1130
Figure PCTCN2020098051-appb-000018
>SEQ ID No.12 QP1139
Figure PCTCN2020098051-appb-000019
>SEQ ID No.13 QP1140
Figure PCTCN2020098051-appb-000020
>SEQ ID No.14 QP1141
Figure PCTCN2020098051-appb-000021
>SEQ ID No.15 QP1142
Figure PCTCN2020098051-appb-000022
>SEQ ID No.16 QP1143
Figure PCTCN2020098051-appb-000023
>SEQ ID No.17 QP1144
Figure PCTCN2020098051-appb-000024
>SEQ ID No.18 QP1145
Figure PCTCN2020098051-appb-000025
>SEQ ID No.19 QP1146
Figure PCTCN2020098051-appb-000026
>SEQ ID No.20 QP1147
Figure PCTCN2020098051-appb-000027
>SEQ ID No.21 QP1148
Figure PCTCN2020098051-appb-000028
>SEQ ID No.22 QP1149
Figure PCTCN2020098051-appb-000029
>SEQ ID No.23 QP1150
Figure PCTCN2020098051-appb-000030
>SEQ ID No.24 QP1151
Figure PCTCN2020098051-appb-000031
>SEQ ID No.25 QP1152
Figure PCTCN2020098051-appb-000032
>SEQ ID No.26 QP1153
Figure PCTCN2020098051-appb-000033
>SEQ ID No.27 QP1154
Figure PCTCN2020098051-appb-000034
>SEQ ID No.28 QP1155
Figure PCTCN2020098051-appb-000035
>SEQ ID No.29 QP1156
Figure PCTCN2020098051-appb-000036
>SEQ ID No.30 QP1157
Figure PCTCN2020098051-appb-000037
>SEQ ID No.31 QP1158
Figure PCTCN2020098051-appb-000038
>SEQ ID No.32 QP1159
Figure PCTCN2020098051-appb-000039
>SEQ ID No.33 QP1160
Figure PCTCN2020098051-appb-000040
>SEQ ID No.34 QP1161
Figure PCTCN2020098051-appb-000041
>SEQ ID No.35 QP1162
Figure PCTCN2020098051-appb-000042
>SEQ ID No.36 QP1163
Figure PCTCN2020098051-appb-000043
>SEQ ID No.37 QP1164
Figure PCTCN2020098051-appb-000044
>SEQ ID No.38 QP1165
Figure PCTCN2020098051-appb-000045
>SEQ ID No.39 QP1166
Figure PCTCN2020098051-appb-000046
>SEQ ID No.40 QP1168
Figure PCTCN2020098051-appb-000047
>SEQ ID No.41 QP1169
Figure PCTCN2020098051-appb-000048
>SEQ ID No.42 Human IgG1：
Figure PCTCN2020098051-appb-000049
>SEQ ID No.43 PD-L1-his
Figure PCTCN2020098051-appb-000050
>SEQ ID No.44 QP1120-CDR1
TYAMS
>SEQ ID No.45 QP1121-CDR1
GTCMA
>SEQ ID No.46 QP1122-CDR1
TNVMG
>SEQ ID No.47 QP1123-CDR1
HYCMG
>SEQ ID No.48 QP1124-CDR1
TCAMG
>SEQ ID No.49 QP1125-CDR1
TCAMA
>SEQ ID No.50 QP1126-CDR1
TKYMS
>SEQ ID No.51 QP1127-CDR1
TYSMA
>SEQ ID No.52 QP1128-CDR1
AYYMA
>SEQ ID No.53 QP1129-CDR1
RYSVG
>SEQ ID No.54 QP1130-CDR1
TSTMM
>SEQ ID No.55 QP1139-CDR1
TNAMG
>SEQ ID No.56 QP1140-CDR1
TKYMG
>SEQ ID No.57 QP1142-CDR1
TNIMG
>SEQ ID No.58 QP1145-CDR1
TKYMA
>SEQ ID No.59 QP1154-CDR1
TNYMA
>SEQ ID No.60 QP1165-CDR1
AYAMS
>SEQ ID No.61 QP1120-CDR2
CIDIYGRTSYTDPVKG
>SEQ ID No.62 QP1121-CDR2
GLWTGDGVTYYADSVKG
>SEQ ID No.63 QP1122-CDR2
AILAGGRNTYYADSVKG
>SEQ ID No.64 QP1123-CDR2
SIDTFGIPKYADSVKG
>SEQ ID No.65 QP1124-CDR2
SISKYGITTYANSVKG
>SEQ ID No.66 QP1125-CDR2
SISTLGTTNYASSVKG
>SEQ ID No.67 QP1127-CDR2
AINSDGHTTYVDSVKG
>SEQ ID No.68 QP1128-CDR2
AINRDGDTKYADSVKG
>SEQ ID No.69 QP1129-CDR2
GQTPRGTTTYADSVKD
>SEQ ID No.70 QP1130-CDR2
GIHNDGGPIAYADSVKG
>SEQ ID No.71 QP1139-CDR2
AILGGGRNTYYADSVKG
>SEQ ID No.72 QP1143-CDR2
AILAGGRDTYYADSVKG
>SEQ ID No.73 QP1144-CDR2
AILAGGRNTNYADSVKG
>SEQ ID No.74 QP1145-CDR2
AILAGGRNTSYADSVKG
>SEQ ID No.75 QP1146-CDR2
AILVGGRNTYYADSVKG
>SEQ ID No.76 QP1148-CDR2
AILAGGRNTAYADSVKG
>SEQ ID No.77 QP1150-CDR2
AILAGGRNTHYADSVKG
>SEQ ID No.78 QP1151-CDR2
AILTGGRNTYYADSVKG
>SEQ ID No.79 QP1153-CDR2
AILAGGRNTDYADSVKG
>SEQ ID No.80 QP1157-CDR2
AILVGGRNTYYADPVKG
>SEQ ID No.81 QP1161-CDR2
AIRVGGRNTDYADSVKG
>SEQ ID No.82 QP1162-CDR2
CIDIYGRASYTDPVKG
>SEQ ID No.83 QP1120-CDR3
ARDFGYCTASWVHEGFSRY
>SEQ ID No.84 QP1121-CDR3
SNGMCGQYWALEDEYKY
>SEQ ID No.85 QP1122-CDR3
ADTRAAFWNIGPLNSDQYNI
>SEQ ID No.86 QP1123-CDR3
GRSYTNCRDGPPSASHYSH
>SEQ ID No.87 QP1124-CDR3
KTFSCRNRGGAYLADA
>SEQ ID No.88 QP1126-CDR3
ADTRAALWYIGPLNSDQYNT
>SEQ ID No.89 QP1127-CDR3
ATSQLGFWAQKLWEAIRDGTWSPSTTDFGF
>SEQ ID No.90 QP1128-CDR3
ASDWSRLYKIYWLDDNYYVR
>SEQ ID No.91 QP1129-CDR3
AGQALLWASLRQTSYQF
>SEQ ID No.92 QP1130-CDR3
GWYFSGDYVP
>SEQ ID No.93 QP1143-CDR3
ADTRAAFWYIGPLNSDQYNI
>SEQ ID No.94 QP1145-CDR3
ADTRAAFWYIGPLNSHQYNI
>SEQ ID No.95 QP1152-CDR3
ADTRAAFWYIGPLNSDQYNL
>SEQ ID No.96 QP1153-CDR3
ADTRAAFWSIGPLNSDQYNI
>SEQ ID No.97 QP1156-CDR3
ADTRAAFWYIGPLNSDQYNS
>SEQ ID No.98 QP1160-CDR3
ADARAAFWYIGPLNSDQYNI
>SEQ ID No.99 QP1163-CDR3
ARDFGYCTASWVHAGFSRY
>SEQ ID No.100 QP320
Figure PCTCN2020098051-appb-000051
>SEQ ID No.101 QP321
Figure PCTCN2020098051-appb-000052
>SEQ ID No.102 QP322
Figure PCTCN2020098051-appb-000053
>SEQ ID No.103 QP323
Figure PCTCN2020098051-appb-000054
>SEQ ID No.104 QP324
Figure PCTCN2020098051-appb-000055
>SEQ ID No.105 QP325
Figure PCTCN2020098051-appb-000056
>SEQ ID No.106 QP1180
Figure PCTCN2020098051-appb-000057
>SEQ ID No.107 QP1181
Figure PCTCN2020098051-appb-000058
>SEQ ID No.108 QP1461
Figure PCTCN2020098051-appb-000059
>SEQ ID No.109 QP371
Figure PCTCN2020098051-appb-000060
Figure PCTCN2020098051-appb-000061
Figure PCTCN2020098051-appb-000062
Figure PCTCN2020098051-appb-000063
Figure PCTCN2020098051-appb-000064
Figure PCTCN2020098051-appb-000065
Figure PCTCN2020098051-appb-000066
Figure PCTCN2020098051-appb-000067
Figure PCTCN2020098051-appb-000068
Figure PCTCN2020098051-appb-000069
Figure PCTCN2020098051-appb-000070
Figure PCTCN2020098051-appb-000071
Figure PCTCN2020098051-appb-000072
Figure PCTCN2020098051-appb-000073
Figure PCTCN2020098051-appb-000074
Figure PCTCN2020098051-appb-000075
Figure PCTCN2020098051-appb-000076
Figure PCTCN2020098051-appb-000077
Figure PCTCN2020098051-appb-000078
Figure PCTCN2020098051-appb-000079
Figure PCTCN2020098051-appb-000080
Figure PCTCN2020098051-appb-000081
Figure PCTCN2020098051-appb-000082
Figure PCTCN2020098051-appb-000083
Figure PCTCN2020098051-appb-000084
Figure PCTCN2020098051-appb-000085
Figure PCTCN2020098051-appb-000086
Figure PCTCN2020098051-appb-000087
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Claims (12)

  1. 一种抗PD-L1纳米抗体，其特征在于：
    至少包含一个VHH片段，在所述VHH片段中，包含CDR1、CDR2和CDR3三个氨基酸片段,CDR1、CDR2、CDR3分别选自以下序列：
    1)SEQ ID No.44至SEQ ID No.60所示的CDR1；
    2)SEQ ID No.61至SEQ ID No.82所示的CDR2；
    3)SEQ ID No.83至SEQ ID No.99所示的CDR3。
  2. 如权利要求1所述的抗PD-L1纳米抗体，其特征在于：
    其序列如：SEQIDNo.1至SEQNo.41所示。
  3. 一种抗PD-L1纳米抗体的Fc融合蛋白，其特征在于：包含如权利要求1或2所述的抗PD-L1纳米抗体以及Fc段，所述Fc段的序列如SEQ ID No.42所示。
  4. 如权利要求1所述的抗PD-L1纳米抗体，其特征在于：其序列中，除CDR1、CDR2和CDR3以外，有80％的氨基酸序列与SEQIDNo.1至SEQNo.41所示的序列相同。
  5. 抗PD-L1纳米抗体在制备阻断PD-L1和PD-1结合试剂中的应用。
  6. 抗PD-L1纳米抗体的Fc融合蛋白在制备阻断PD-L1和PD-1结合试剂中的应用。
  7. 如权利要求5所述的抗PD-L1纳米抗体其特征在于:其用量为20ug/ml至0.000128ug/ml。
  8. 如权利要求1所述的抗PD-L1纳米抗体人源化改造后，其特征在于：其序列如SEQ ID No.100至SEQ ID No.105所示。
  9. 一种人源化抗PD-L1纳米抗体的Fc融合蛋白，其特征在于：包含 如权利要求8所述的抗PD-L1纳米抗体以及Fc段，所述Fc段的序列如SEQ ID No.42所示。
  10. 如权利要求1-4或权利要求9中任一项所述的纳米抗体或者其Fc融合蛋白，在制备治疗癌症、感染或免疫调节疾病的药物中的应用。
  11. 如权利要求1-4或权利要求9中任一项所述的纳米抗体，在制备抑制肿瘤生长的药物中的应用。
  12. 如权利要求10所述的应用，其特征在于：
    所述癌症或肿瘤选自下组织或部位：结直肠、乳腺、卵巢、胰腺、胃、食管、前列腺、肾、宫颈、骨髓癌、淋巴癌、白血病、甲状腺、子宫内膜、子宫、膀胱、神经内分泌、头部颈部、肝、鼻咽、睾丸、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤，或者骨髓增生异常综合症。
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