Anti-pd-l1 nanobodies, fc fusion proteins thereof, and uses thereof

Abstract

The application provides an anti-PD-L1 nanobody and an Fc fusion protein thereof and application, the anti-PD-L1 nanobody and the Fc fusion protein thereof of the application are high in specificity and affinity, weak in immunogenicity to human, high in stability and have remarkable anti-tumor effect.

Description

[0001] This application is a divisional application of the invention patent application filed on June 22, 2020, with application number 202010574992.4 and invention title "Anti-PD-L1 Nanobody and its Fc Fusion Protein and Application". Technical Field

[0002] This invention relates to an anti-PD-L1 nanobody, an Fc fusion protein of an anti-PD-L1 nanobody, and applications of the anti-PD-L1 nanobody and its Fc fusion protein, belonging to the field of biomedicine. Background Technology

[0003] In the classical immune surveillance theory, the immune system recognizes and eliminates tumor antigens. If the immune system can completely eliminate tumor cells, immune clearance can proceed stably. If tumor cells evade clearance through mutation, the immune system will rebalance. During this process, the immunogenicity of tumor cells gradually decreases. The proliferative capacity of tumor cells weakens under the pressure of the immune system, making tumor cell detection more difficult.

[0004] Activation of oncogenes leads to alterations in tumor cells and the tumor microenvironment, disrupting the balance between the immune system and tumor cells. When the immune system and tumor cells enter an escape phase, the malignancy of tumor cells increases, and the loss of MHC molecules prevents them from being recognized and eliminated by immune cells. The tumor microenvironment can also suppress the immune system by releasing immunosuppressive factors such as IL-10 and TGF-β. Tumor cells also highly express immunosuppressive proteins (such as programmed death-ligand-1 and PD-L1). When effector T cells bind to tumor cells, PD-L1 interacts with PD-1 and induces T cell apoptosis, which is one of the main reasons why tumors develop resistance to the immune system, leading to rapid tumor growth and metastasis. Theoretically, if the host's immune system were artificially activated and redirected to tumor cells, tumor tissue could be eliminated, and the theory of immunotherapy has been widely proven in clinical practice.

[0005] Immunotherapy can be divided into two categories: specific and non-specific treatments. Specific therapies within this category include the following treatment strategies: tumor vaccines, which activate immune cells against a patient's antigens through injection of tumor-specific immunotherapies. Tumor vaccines include: inactivated tumor cell vaccines, tumor antigen vaccines, tumor DNA vaccines, dendritic cell (DC) vaccines, and bacterial vaccines. Specific ACT immunotherapy primarily includes three treatment methods.

[0006] Tumor-infiltrating lymphocytes (TILs): Lymphocytes isolated from tumor tissue and cultured in vitro. TILs can secrete IL-2, which has specific anti-tumor properties.

[0007] T-cell receptor (TCR) therapy: T cells recognize tumor antigens via their single-chain antibody fragments (scFv), and clone the single-chain antibody fragment TCR into normal T cells using a viral vector. Thus, normal T cells are transformed into specific tumor-killing T cells.

[0008] CAR-T: T cells are genetically modified to acquire tumor-specific receptor T cells. Unlike conventional T cell recognition mechanisms, CAR-T cells are not restricted by MHC molecules to recognize tumor antigens. Therefore, CAR-T cells can overcome tumor immune escape mechanisms by increasing co-stimulatory signaling molecules, thereby enhancing the T cell's ability to kill tumor cells.

[0009] In nonspecific ACT immunotherapy, there are two main treatment methods: lymphokine-activated killer (LAK) cell therapy and cytokine-induced killer (CIK) cell therapy.

[0010] LAK cell therapy: LAK cells use IL-2 to stimulate immune cells in peripheral blood lymphocytes, including NK cells and T cells, and overexpress FAS ligands to enhance their ability to recognize target cells. They also kill tumor cells by releasing perforin and granzymes.

[0011] CIK cell therapy: CIK cells are derived from peripheral blood lymphocytes (PBLs) of patients or healthy individuals and expand under ex vivo conditions upon stimulation with anti-CD3 antibodies, IFN-γ, and IL-2. CIK cells primarily exert their anti-tumor effects through FasL and perforin.

[0012] Immune checkpoints are protective molecules in the human immune system, preventing inflammatory damage caused by excessive T cell activation in a healthy body. Tumor cells can exploit this property by overexpressing immune checkpoint molecules, suppressing the body's immune response, evading the surveillance and killing effects of the immune system, and thus promoting tumor cell growth. Immune checkpoint inhibitor therapy can inhibit the activity of immune checkpoints in the tumor microenvironment, reactivating the T cell immune response against the tumor and achieving an anti-tumor effect. Complete T cell activation is regulated by a "dual signaling" system: the first signal comes from the specific binding of the T cell's own TCR (T cell receptor) to the MHC of the antigen, i.e., T cell recognition of the antigen; the second signal comes from co-stimulatory molecules, which participate in the interaction between co-stimulatory molecules expressed by antigen-presenting cells (APCs) and corresponding receptors or ligands (such as CD28) on the surface of T cells. For example, CD28-B7 is a positive co-stimulatory signal, while negative co-stimulatory molecules are mainly the CTLA4-B7 pathway and the PD-1 / PD-L1 pathway. After tumor cell invasion, this inhibitory pathway is used by tumor cells to suppress T cell activation, thereby evading the clearance action of the immune system.

[0013] PD-1 (CD279) was first reported in 1992. The human PD-1 encoding gene, PDCD1, is located at 2q37.3, with a full length of 2097 bp, consisting of 6 exons. The translation product is a 288-amino acid precursor protein of PD-1, which is cleaved into a signal peptide consisting of the first 20 amino acids to yield the mature protein. PD-1 includes an extracellular immunoglobulin variable region (IgV domain), a hydrophobic transmembrane domain, and an intracellular domain. The N-terminal ITIM motif of the intracellular tail domain contains two phosphorylation sites, while the C-terminus contains an ITSM motif. PD-1 is a membrane protein belonging to the CD28 immunoglobulin superfamily. It is mainly expressed on the surface of activated T cells, and also shows low-abundance expression on CD4-CD8-T cells in the thymus, activated NK cells, and monocytes. PD-1 has two ligands: PD-L1 (CD274, B7-H1) and PD-L2 (CD273, B7-DC) from the B7 protein family. PD-L1 and PD-L2 share 40% of their amino acid sequences. The main difference lies in their expression patterns. PD-L1 is constitutively lowly expressed in APCs, non-hematopoietic cells (such as vascular endothelial cells and pancreatic islet cells), and immune-exempt sites (such as the placenta, testis, and eye). Inflammatory cytokines such as type I and type II interferons, TNF-α, and VEGF can all induce PD-L1 expression. PD-L2, on the other hand, is only expressed in activated macrophages and dendritic cells. After PD-1 binds to activated T cells, the ITSM motif of PD-1 undergoes tyrosine phosphorylation, which in turn leads to the dephosphorylation of downstream protein kinases Syk and PI3K, inhibiting the activation of downstream pathways such as AKT and ERK. Ultimately, this inhibits the transcription and translation of genes and cytokines required for T cell activation, thus exerting a negative regulatory effect on T cell activity.

[0014] In tumor cells, tumor cells and the tumor microenvironment negatively regulate T cell activity and suppress the immune response by upregulating PD-L1 expression and binding it to PD-1 on the surface of tumor-specific CD8+ T cells. Tumor cells can upregulate PD-L1 expression through the following four pathways: 1. Amplification of the gene encoding PD-L1 (9p24.1); 2. Activation of EGFR, MAPK, and PI3K-Akt signaling pathways, and HIF-1 transcription factor can upregulate PD-L1 expression at the transcriptional level; 3. Induction by EBV (EBV-positive gastric and nasopharyngeal carcinomas show high PD-L1 expression); 4. Epigenetic regulation. In the tumor microenvironment, stimulation by inflammatory factors such as interferon-γ 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. Tumor-infiltrating T cells, capable of recognizing tumor antigens, can secrete interferon-γ, thereby inducing upregulation of PD-L1 expression. This process is known as "adaptive immune resistance," a mechanism through which tumor cells can protect themselves. Increasing evidence suggests that tumors utilize PD-1-dependent immunosuppression to evade the immune system. High expression of PD-L1 and PD-L2 has been found in various solid tumors and hematologic malignancies. Furthermore, PD-L expression is strongly correlated with poor prognosis in tumor cells, including esophageal cancer, gastric cancer, renal cancer, ovarian cancer, bladder cancer, pancreatic cancer, and melanoma.

[0015] Currently, the FDA has approved the following PD-1 therapeutic monoclonal antibodies: Nivolumab (Opdivo, September 2014), Pembrolizumab (Keytruda, December 2014), and Cemiplimab (Libtayo, September 2018). The FDA has also approved the following PD-L1 therapeutic monoclonal antibodies: Atezolizumab (Tecentriq, September 2014), avelumab (Bavencio, May 2016), and Duravirulumab (Imfinzi, May 2017). The approved indications are shown in the table below.

[0016]

[0017] In addition, PD-1 monoclonal antibodies such as Pidilizumab, AMP-224, AMP-514, and PDR001, and PD-L1 monoclonal antibodies such as BMS-936559 and CK-301 are under development and in clinical trials.

[0018] However, existing monoclonal antibodies do not have ideal affinity and are more immunogenic due to their large size. Summary of the Invention

[0019] The purpose of this invention is to provide an anti-PD-L1 nanobody and its Fc fusion protein and applications to solve the above-mentioned problems.

[0020] This invention provides an anti-PD-L1 nanobody, characterized in that it comprises at least one VHH fragment, wherein the VHH fragment contains three amino acid fragments, CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 are selected from the following sequences:

[0021] 1) CDR1 shown in SEQ ID No. 44 to SEQ ID No. 60;

[0022] 2) CDR2 shown in SEQ ID No. 61 to SEQ ID No. 82;

[0023] 3) CDR3 shown in SEQ ID No. 83 to SEQ ID No. 99.

[0024] Furthermore, 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.

[0025] The present invention also provides an Fc fusion protein of an anti-PD-L1 nanobody, characterized in that it comprises the anti-PD-L1 nanobody as described in claim 1 or 2 and an Fc segment, wherein the sequence of the Fc segment is shown in SEQ ID No. 42.

[0026] Furthermore, the anti-PD-L1 nanobody of the present invention is characterized in that, except for CDR1, CDR2 and CDR3, 80% of its amino acid sequence is identical to the sequences shown in SEQ ID No. 1 to SEQ No. 41.

[0027] The present invention also provides the application of anti-PD-L1 nanobody in the preparation of reagents that block PD-L1 and PD-1 binding.

[0028] The present invention also provides the application of an Fc fusion protein of an anti-PD-L1 nanobody in the preparation of a reagent that blocks PD-L1 and PD-1 binding.

[0029] Furthermore, the anti-PD-L1 nanobody of the present invention is characterized in that its dosage is from 20ug / ml to 0.000128ug / ml.

[0030] Furthermore, the humanized anti-PD-L1 nanobody of the present invention is characterized in that its sequence is shown in SEQ ID No. 100 to SEQ ID No. 105.

[0031] The present invention also provides an Fc fusion protein of a humanized anti-PD-L1 nanobody, characterized in that it comprises the anti-PD-L1 nanobody as described in claim 8 and an Fc segment, wherein the sequence of the Fc segment is shown in SEQ ID No. 42.

[0032] The use of any of the nanobodies or their Fc fusion proteins described above in the preparation of drugs for treating cancer, infections, or immunomodulatory diseases.

[0033] The application of any of the above-mentioned nanobodies in the preparation of drugs that inhibit tumor growth.

[0034] In the application of any of the nanobodies or their Fc fusion proteins described above: the cancer or tumor is selected from the following tissues or sites: colorectal, breast, ovary, pancreas, stomach, esophagus, prostate, kidney, cervix, bone marrow cancer, lymphoma, leukemia, thyroid, endometrium, uterus, bladder, neuroendocrine, head and neck, liver, nasopharynx, testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell carcinoma, squamous cell carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome.

[0035] Beneficial effects of the invention

[0036] The nanobody and its Fc fusion protein of the present invention have high specificity, high affinity, weak immunogenicity in humans, and significant anti-tumor effects. Attached Figure Description

[0037] Figure 1A These are the binding curves of human Fc fusion proteins of PD-L1 nanobodies numbered QP1120, QP1122, QP1123, QP1124, QP1125, QP1126, QP1127, QP1128, QP1129, QP1130 and QP1139 to PD-L1 protein;

[0038] Figure 1B These are the binding curves of the human Fc fusion proteins of PD-L1 nanobodies numbered QP1140, QP1141, QP1142, QP1143, QP1144, QP1145, QP1146, QP1147, QP1148, QP1149, and QP1150 to the PD-L1 protein.

[0039] Figure 1CThese are the binding curves of the human Fc fusion proteins of PD-L1 nanobodies numbered QP1151, QP1152, QP1153, QP1154, QP1155, QP1156, QP1157, QP1158, QP1159, QP1160, and QP1161 to the PD-L1 protein.

[0040] Figure 1D The binding curves of human Fc fusion proteins of PD-L1 nanobodies numbered QP1162, QP1163, QP1164, QP1165, QP1166, QP1168 and QP1169 to PD-L1 protein are shown.

[0041] Figure 2A The binding curves of human Fc fusion proteins of PD-L1 nanobodies numbered QP1120, QP1122, QP1123, QP1124, QP1125, QP1126, QP1127, QP1128, QP1129 and QP1130 to biotinylated mouse PD-L1 protein are shown.

[0042] Figure 2B The binding curves of human Fc fusion proteins of PD-L1 nanobodies numbered QP1122, QP1142, QP1143, QP1144, QP1145, QP1146, QP1147, QP1148, QP1149, QP1150 and QP1151 to biotinylated mouse PD-L1 protein are shown.

[0043] Figure 2C The binding curves of human Fc fusion proteins of PD-L1 nanobodies numbered QP1126, QP1152, QP1153, QP1154, QP1155, QP1156, QP1157, QP1158, QP1159, QP1160, and QP1161 to biotinylated mouse PD-L1 protein are shown.

[0044] Figure 2D The binding curves of human Fc fusion proteins of QP1162, QP1163, QP1164, QP1165, QP1166, QP1168, QP1169, QP1139, QP1140, and QP1141 to biotinylated mouse PD-L1 protein are shown.

[0045] Figure 3A These are the blocking curves of PD-L1 nanobodies numbered QP1120, QP1162, QP1163, QP1164, QP1165, QP1166, QP1168, QP1169, QP1139, QP1140 and QP1141 against PD-1 / PD-L1 interaction.

[0046] Figure 3B PD-L1 nanobodies: human Fc fusion proteins numbered QP1122, QP1142, QP1143, QP1144, QP1145, QP1146, QP1147, QP1148, QP1149, QP1150 and QP1151, blocking the interaction between human Fc proteins and PD-1 / PD-L1;

[0047] Figure 3C The human Fc fusion proteins of PD-L1 nanobodies numbered QP1126, QP1152, QP1153, QP1154, QP1155, QP1156, QP1157, QP1158, QP1159, QP1160, and QP1161 block the interaction between PD-1 and PD-L1;

[0048] Figure 4A These are the human Fc fusion proteins of PD-L1 nanobodies numbered QP1120, QP1162, QP1163, QP1164, and QP1165, which block the interaction between PD-1 and PD-L1.

[0049] Figure 4B These are the PD-L1 / PD-L1 interaction blocking curves of the human Fc fusion proteins of PD-L1 nanobodies numbered QP1166, QP1168, QP1169, QP1122, and QP1126;

[0050] Figure 4C These are the PD-L1 / PD-L1 interaction blocking curves of the human Fc fusion proteins of PD-L1 nanobodies numbered QP1139, QP1140, QP1141, QP1142, and QP1143;

[0051] Figure 4D These are the human Fc fusion proteins of PD-L1 nanobodies numbered QP1144, QP1145, QP1146, QP1147, and QP1148: blocking curves for PD-1 / PD-L1 interaction.

[0052] Figure 4E These are the human Fc fusion proteins of PD-L1 nanobodies numbered QP1149, QP1150, QP1151, QP1152, and QP1153, which block the interaction between PD-1 and PD-L1.

[0053] Figure 4FThese are the human Fc fusion proteins of PD-L1 nanobodies numbered QP1149, QP1155, QP1156, QP1157, and QP1158, which block the interaction between PD-1 and PD-L1.

[0054] Figure 4G These are the curves showing the blocking effect of human Fc fusion proteins of PD-L1 nanobodies numbered QP1159, QP1160, and QP1161 on PD-1 / PD-L1 interaction.

[0055] Figure 5A The human Fc fusion proteins of PD-L1 nanobodies numbered QP1120, QP1162, QP1163, QP1164, QP1165, and QP1166: binding curves against human non-small cell lung cancer cells HCC827.

[0056] Figure 5B The human Fc fusion proteins of PD-L1 nanobodies numbered QP1168, QP1169, QP1122, QP1126, QP1139, and QP1141: binding curves to human non-small cell lung cancer cells HCC827.

[0057] Figure 5C The binding curves of human Fc fusion proteins of PD-L1 nanobodies numbered QP1142, QP1149, QP1151, QP1156, QP1157, and QP1158 to human non-small cell lung cancer cells HCC827 are shown. Figure 6 The binding curves of human Fc fusion proteins of humanized PD-L1 nanobodies numbered QP1162, QP320, QP321, QP322, QP1166, QP323, QP324, and QP325 to biotinylated human PDL1 protein are shown.

[0058] Figure 7 These are the human Fc fusion proteins of humanized PD-L1 nanobodies numbered QP1162, QP320, QP321, QP322, QP1166, QP323, QP324, and QP325, which block the interaction between human Fc proteins and PD-1 / PD-L1.

[0059] Figure 8 This is a schematic diagram of the structure of the anti-CLDN18.2 / anti-PD-L1 bispecific antibody molecule;

[0060] Figure 9 This is the concentration-dependent result of the anti-CLDN18.2 / anti-PD-L1 bispecific antibody PD-L1 functional activity assay (mixed lymphocyte reaction MLR) on the cytokine IFNγ produced after T cell activation by QP3711461.

[0061] Figure 10 This is a concentration-dependent result of the anti-CLDN18.2 / anti-PD-L1 bispecific antibody PD-L1 functional activity assay (mixed lymphocyte reaction MLR) on the cytokine IL-2 produced after T cell activation by QP3711461.

[0062] Figure 11 The results show the tumor growth curves of the anti-CLDN18.2 / anti-PD-L1 bispecific antibody in the MC38-hPDL1 model of humanized transgenic mice with immune targets, C57BL / 6-hPDL1. Detailed Implementation

[0063] The technical solution of the present invention will be further described in detail below.

[0064] Example 1: Preparation method of PD-L1 nanobody Fc fusion protein:

[0065] 1. Screening of nanobodies targeting PD-L1

[0066] 1.1 Library Construction

[0067] a) The PD-L1-his fusion protein of immunized camels was obtained by nickel column purification, and its sequence is shown in SEQ ID NO.43. One Bactrian camel from Xinjiang was selected for subcutaneous multi-site immunization. After four immunizations, 50 mL of peripheral blood was collected to separate PBMCs.

[0068] b) Extract total RNA using TRIzol reagent. After electrophoresis confirms sufficient RNA purity, use... The III First-Strand Synthesis System for RT-PCR was used to transcribe 8ug RNA, followed by two rounds of nested PCR. The target nucleic acid fragment was then recovered and purified using a DNA product purification kit.

[0069] c) The phage vector pComb3XSS and the target fragment were digested separately with sfiI enzyme and incubated overnight at 50°C. The target fragment was then recovered by gel extraction. The ligation molar ratio was Vector:VHH = 1:3.

[0070] d) Immediately after electroporation into TG1 *E. coli*, 1 mL of SOC medium was added for resuscitation. The culture was incubated at 37°C and 180 rpm for 45 min, followed by centrifugation. The culture was then resuspended in 5 mL of SOC, and 10 μL was used to determine the library capacity. The remainder was plated onto 8 200 mm plates. On the second day, 10⁻⁵ clones were found, resulting in a library capacity of 5.04 × 10⁻⁵. 9 (104*5*100*10 5Forty-eight clones were randomly selected from the titration plate for identification, and the results showed that the insertion rate was 100% and the size was correct.

[0071] 1.2 Screening of Nanobodies Targeting PD-L1

[0072] a) After three rounds of panning, the phage clones that bind to PD-L1 were plated in a 96-well plate, and 150 μL of 2×TY-Amp-0.1% sucrose medium was added. The plate was then incubated at 37°C.

[0073] Incubate for 3 hours;

[0074] b) Add 30 μL of 2×TY-Amp-5 mM IPTG (final IPTG concentration is 1 mM) to each well to induce expression of soluble antibody fragments and incubate overnight at 30°C;

[0075] c) Coat ELISA plates with PD-L1-his protein at 2 ng / ul, 50ul / well, and incubate overnight at 4°C;

[0076] d) Wash the plate once with 1×PBS, add 200ul of 2% Milk-PBS to block the ELISA plate, and incubate at 37°C for 1h.

[0077] e) Centrifuge the overnight culture at 4000g for 10 mins and transfer the supernatant to a new 96-well plate.

[0078] f) Wash the plate twice with 1×PBST, add 25 μL of 2% Milk-PBS blocking buffer, then add 25 μL of culture supernatant and mix well. Incubate at 25°C for 1 hour.

[0079] g) Wash the plate 3 times with 1×PBST, add 100ul of anti-Fab-HRP antibody (1:5000in 2% Milk-PBS), and incubate at 25°C for 1 hour.

[0080] h) Wash the plate 4 times with 1×PBST, add 100ul of TMB / well, develop color at room temperature in the dark for 10min, add 50ul of 2M H2SO4 / well to stop the reaction, and read the value at 450nm; i) The sample well reading is considered to be a positive clone well if it is more than twice the reading of the control well. The bacterial culture of the positive well is expanded and plasmids are extracted and sequenced; the sequences that are all the same in CDR1, 2 and 3 are regarded as the same clone, thus obtaining 41 unique nanobody sequences, as shown in SEQ ID NO: 1-41.

[0081] To facilitate screening, 41 clones were converted into PD-L1-FC fusion proteins with a C-terminus of human IgG1. The reconstructed plasmids were expressed in HFK293 cells and purified by protein A affinity chromatography. Except for QP1121-FC, a total of 40 candidate PD-L1-FC fusion proteins were obtained, with sequences consisting of the nanobody shown in SEQ ID NO. 1-41 linked to the Fc segment of SEQ ID NO. 42. The Fc fusion protein numbers of the corresponding nanobody were formed by adding the suffix "Fc" to the corresponding nanobody.

[0082] 2. Binding curve of PD-L1 nanobody Fc fusion protein to PD-L1 protein

[0083] a) Coat PDL1-mFc fusion protein at 1ug / ml, 100ul / well, and incubate overnight at 4°C;

[0084] b) Wash the plate 3 times with 1×PBS, add 3% BSA to block, 250ul / well, and incubate at room temperature for 1h;

[0085] c) Wash the plate 3 times with 1×PBST, wash the plate 3 times with 1×PBS, add the test antibody in a 5-fold serial dilution (10ug / ml to 0.000128ug / ml), and incubate at room temperature for 1 h;

[0086] d) Wash the plate 3 times with 1×PBST, wash the plate 3 times with 1×PBS, add HRP-anti-hFc (1:2500), 50ul / well, and incubate at room temperature for 1h;

[0087] e) Wash the plate 3 times with 1×PBST, then 3 times with 1×PBS. Add 100 μL of TMB per well and incubate at room temperature in the dark for 10 min. Add 50 μL of 2M H2SO4 per well to terminate the reaction. Read the value at 450 nm.

[0088] The results are as follows Figures 1A to 1D As shown in Tables 1 to 4.

[0089] Table 1: and Figure 1A EC50 values ​​of each antibody corresponding to the experimental results

[0090] QP1120-FC 0.0008491 QP1122-FC 0.001204 QP1123FC 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

[0091] Table 2 and Figure 1B EC50 values ​​of each antibody corresponding to the experimental results

[0092] 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 328QA 0.00246

[0093] Table 3 and Figure 1C EC50 values ​​of each antibody corresponding to the experimental results

[0094] 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 0.000g905 QP1160-FC 0.0007973 QP1161-FC 0.001055 3280A 0.002356

[0095] Table 4 and Figure 1D EC50 values ​​of each antibody corresponding to the experimental results

[0096] 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

[0097] 3. Binding curve of PD-L1 nanobody human Fc fusion protein to biotinylated mouse PD-L1 protein

[0098] a) Mouse PDL1 was obtained through HEK293 expression. Biotinylated protein mPDL1-Biotin was obtained using the Thermo Biotinylation Kit.

[0099] b) Coating with Strepavidin, 4ug / ml, 50ul / well, overnight at 4°C;

[0100] c) Wash the plate 3 times with 1×PBS, add 3% BSA to block, 250ul / well, and incubate at room temperature for 1h;

[0101] d) Wash the plate 3 times with 1×PBST, then wash it 3 times with 1×PBS. Add mPDL1-Biotin, 1ug / ml, 50ul / well, and incubate at room temperature for 1h.

[0102] e) Wash the plate 3 times with 1×PBST, wash the plate 3 times with 1×PBS, add the test antibody diluted 5-fold from 10ug / ml to 0.000128ug / ml, and incubate at room temperature for 1h;

[0103] f) Wash the plate 3 times with 1×PBST, wash the plate 3 times with 1×PBS, add HRP-anti-hFc (1:2500), 50ul / well, and incubate at room temperature for 1h;

[0104] g) Wash the plate 6 times with 1×PBST, then 3 times with 1×PBS. Add 100 μL of TMB per well and incubate at room temperature in the dark for 10 min. Add 50 μL of 2M H2SO4 per well to terminate the reaction. Read the value at 450 nm.

[0105] The results are as follows Figures 2A to 2D As shown.

[0106] Table 5 is related to Figure 2A EC50 values ​​of each antibody corresponding to the experimental results

[0107] 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

[0108] Table 6 is... Figure 2B EC50 values ​​of each antibody corresponding to the experimental results

[0109] 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

[0110] Table 7 is... Figure 2C EC50 values ​​of each antibody corresponding to the experimental results

[0111] 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

[0112] Table 8 is... Figure 2D EC50 values ​​of each antibody corresponding to the experimental results

[0113] 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

[0114] 4. The blocking curve of PD-1 / PD-L1 interaction between the PD-L1 nanobody human Fc fusion protein and the PD-1 / PD-L1 was detected by competitive ELISA.

[0115] a) Coat PD1-hFc fusion protein at 1ug / ml, 100ul / well, and incubate overnight at 4°C;

[0116] b) Wash the plate 3 times with 1×PBS, add 3% BSA to block, 250ul / well, and incubate at room temperature for 1h;

[0117] c) Wash the plate 3 times with 1×PBST, wash the plate 3 times with 1×PBS, add PDL1-mFc, 2ug / ml, 50ul / well, and add an equal volume of 5-fold serially diluted (20ug / ml to 0.000256ug / ml) antibody to be tested, and incubate at room temperature for 1h;

[0118] d) Wash the plate 3 times with 1×PBST, wash the plate 3 times with 1×PBS, add HRP-anti-mFc (1:2500), 50ul / well, and incubate at room temperature for 1h;

[0119] e) Wash the plate 3 times with 1×PBST, then 3 times with 1×PBS. Add 100 μL of TMB per well and incubate at room temperature in the dark for 10 min. Add 50 μL of 2MH2SO4 per well to terminate the reaction. Read the value at 450 nm.

[0120] The results are as follows Figures 3A to 3C As shown.

[0121] Table 9 is related to Figure 3A IC50 values ​​of each antibody corresponding to the experimental results

[0122] 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

[0123] Table 10 is related to Figure 3B IC50 values ​​of each antibody corresponding to the experimental results

[0124] 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

[0125] Table 11 is related to Figure 3C IC50 values ​​of each antibody corresponding to the experimental results

[0126] QP1126-FC 0.3245 QP1152-FC 0.3295 QP1153-FC 0.4048 QP1154-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 QP1161-FC 0.3263 328QA ～1.07

[0127] 5. The blocking curve of PD-1 / PD.L1 interaction between the PD-L1 nanobody human Fc fusion protein and biotin was obtained by competitive ELISA detection.

[0128] a) Biotinylated protein PDL1-Biotin was obtained using Thermo Biotinylation Kit;

[0129] b) Coat PD1-hFc fusion protein at 1ug / ml, 100ul / well, and incubate overnight at 4°C;

[0130] c) Wash the plate 3 times with 1×PBS, add 3% BSA to block, 250ul / well, and incubate at room temperature for 1h;

[0131] d) Wash the plate 3 times with 1×PBST, wash the plate 3 times with 1×PBS, add PDL1-Biotin, 2ug / ml, 50ul / well, and add an equal volume of 3-fold serially diluted (100nM to 0.0457nM, corresponding to a mass concentration of 15ug / ml to 0.00686ug / ml) antibody to be tested, and incubate at room temperature for 1h;

[0132] e) Wash the plate 3 times with 1×PBST, wash the plate 3 times with 1×PBS, add HRP-Strepavidin (1:5000), 50ul / well, and incubate at room temperature for 1h;

[0133] f) Wash the plate 6 times with 1×PBST, then 3 times with 1×PBS. Add 100 μL of TMB per well and incubate at room temperature in the dark for 10 min. Add 50 μL of 2M H2SO4 per well to terminate the reaction. Read the value at 450 nm.

[0134] The results are as follows Figures 4A to 4G As shown.

[0135] Table 12 is related to Figure 4A IC50 values ​​of each antibody corresponding to the experimental results

[0136] QP1120-FC 4.903 QP1162-FC 4.754 QP1163-FC 14.18 QP1164-FC 11.46 QP1165-FC 13.66 3280A 4.914

[0137] Table 13 is related to Figure 4B IC50 values ​​of each antibody corresponding to the experimental results

[0138] QP1166-FC 12.75 QP1168-FC 13.45 QP1169-FC 4.551 QP1122-FC 4.085 QP1126-FC 4.291 3280A 4.273

[0139] Table 14 is related to Figure 4CIC50 values ​​of each antibody corresponding to the experimental results

[0140] QP1139-FC ～11.74 QP1140-FC 14.58 QP1141-FC 13.91 QP1142-FC 13.49 QP1143-FC 14.01 3280A 5.44

[0141] Table 15 is related to Figure 4D IC50 values ​​of each antibody corresponding to the experimental results

[0142] QP1144-FC 13.3 QP1145-FC 21.99 QP1146-FC 15.24 QP1147-FC 14.39 QP1148-FC 14.15 3280A 4.557

[0143] Table 16 is related to Figure 4E IC50 values ​​of each antibody corresponding to the experimental results

[0144] QP1149-FC 13.33 QP1150-FC 13.99 QP1151-FC 14.03 QP1152-FC 13.55 QP1153-FC 13.19 3280A 4.721

[0145] Table 17 is related to Figure 4F IC50 values ​​of each antibody corresponding to the experimental results

[0146] QP1149-FC 14.14 QP1155-FC ～11.71 QP1156-FC ～11.88 QP1157-FC 13.46 QP1158-FC 4.652 3280A 4.881

[0147] Table 18 is related to Figure 4G IC50 values ​​of each antibody corresponding to the experimental results

[0148] QP1159-FC ～11.66 QP1160-FC 11.94 QP1161-FC 11.94 3280A 4.189

[0149] 6. Binding curve of PD-L1 nanobody human Fc fusion protein to human non-small cell lung cancer cells HCC827

[0150] a) Human non-small cell lung cancer cells HCC827 naturally overexpress PD-L1. HCC827 cells in logarithmic growth phase (80% confluence) were prepared, their concentration adjusted, and seeded in Costar 96-well plates at 1E5 cells / well. The plates were washed once with 1×PBS, and 250 μL of 3% BSA was added to each well. The plates were incubated at 37°C for 1 h.

[0151] b) Add 4-fold serial dilutions (33.33 nM to 0.008 nM, corresponding to a mass concentration of 5 μg / mL to 0.001 μg / mL) of the antibody to be tested, 50 μL / well, and incubate on ice for 1 h;

[0152] c) Wash the plate twice with 1×PBS, add PE-antihFc (1:200), 50ul / well, and incubate on ice for 1h;

[0153] d) Wash the plate 3 times with 1×PBS and read the values ​​on the instrument.

[0154] The results are as follows Figures 5A to 5C As shown.

[0155] Table 19 is related to Figure 5A EC50 values ​​of each antibody corresponding to the experimental results

[0156] 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

[0157] Table 20 is related to Figure 5B EC50 values ​​of each antibody corresponding to the experimental results

[0158] 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

[0159] Table 20 is related to Figure 5C EC50 values ​​of each antibody corresponding to the experimental results

[0160] 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

[0161] Example 2: Humanization of anti-PD-L1 nanobody

[0162] By comparing the germline gene database of heavy and light chain variable regions of human antibodies with IMGT human antibody germline genes and MOE software, germline genes of heavy and light chain variable regions with high homology to QP1162 (SEQ ID No. 35) and QP1166 (SEQ ID No. 39) were selected as templates. The CDRs of murine antibodies were transplanted into the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Then, some important amino acid residues were selected for reverse mutation combinations. The amino acid residues were identified and annotated using the Kabat numbering system.

[0163] Primers were designed and PCR was used to construct the VH gene fragments of each humanized antibody. Then, homologous recombination was performed with the expression vector pQD containing the signal peptide and constant region gene (FC) fragments to construct the full-length antibody expression vector VH-FC-pQD.

[0164] Multiple primers were designed using the online software DNAWorks (v3.2.2) (http: / / helixweb.nih.gov / dnaworks / ) to synthesize the VH / VK gene fragment containing the required recombination gene: 5'-30bp signal peptide + VH + 30bp FC-3'. Following the instructions for TaKaRa Primer STAR GXL DNA polymerase, the designed primers were used for two-step PCR amplification to obtain the VH / VK gene fragment containing the required recombination gene. The expression vector pQD containing the signal peptide and constant region gene (FC) fragment was constructed and digested using restriction endonucleases, such as BsmBI, which recognize sequences different from their digestion sites. The vector was digested with BsmBI, and the gel was recovered for later use. The recombinant expression vector VH-FC-pQD was then constructed. VH containing the gene fragment required for recombination and the expression vector pQD (with signal peptide and constant region gene (FC) fragment) recovered by BsmBI restriction enzyme digestion were added to DH5H competent cells at a ratio of 3:1. The cells were incubated at 0°C on ice for 30 min, followed by heat shock at 42°C for 90 s. Five volumes of LB medium were added, and the cells were incubated at 37°C for 45 min. The cells were then plated on LB-Amp plates and cultured overnight at 37°C. Single clones were picked and sent for sequencing to obtain the target clones.

[0165] The sequences and protein expression numbers of the light and heavy chain variable regions of each clone's humanized design are shown below. In this table, the antibody is fused with the human IgG1-FC constant region at its C-terminus:

[0166] Table 21: Human-centered design of QP1162 and QP1166

[0167]

[0168] Simultaneously, the designed clones and expression of pre-humanized chimeric antibodies and control antibodies are shown in the table below:

[0169] Table 22: Pre-humanized chimeric antibodies and control antibodies

[0170]

[0171] 2. Expression of humanized protein from anti-PD-L1 nanobody

[0172] The 293E cell culture density was maintained at 0.2-3 × 10⁻⁶. 6 The cells were cultured in maintenance phase medium (GIBCO Freestyle 293 expression medium) between 0.5 and 0.8 × 10⁶ cells / ml. One day before transfection, the cells were centrifuged and the medium changed to adjust the cell density to 0.5-0.8 × 10⁶ cells / ml. 6 / ml. On the day of transfection, the density of 293E cells was 1-1.5×10⁻⁶. 6 / ml. Prepare plasmid and transfection reagent PEI. The required plasmid quantity for transfection is 100ug / 100ml of cells, using a PEI to plasmid mass ratio of 2:1. Mix the plasmid and PEI thoroughly and let stand for 15 minutes, not exceeding 20 minutes. Slowly add the plasmid and PEI mixture to 293E cells and incubate in a shaker at 37°C, 120 rpm, with 8% CO2. On the fifth day after transfection, collect the cell supernatant by centrifuging at 4700 rpm for 20 minutes.

[0173] 3. Purification of humanized protein from anti-PD-L1 nanobody

[0174] Protein A affinity chromatography purification

[0175] Pass the equilibration solution through the column at a flow rate of at least 3 CV (20 ml), ensuring the pH and conductivity of the final effluent from the instrument are consistent with the equilibration solution. The flow rate is 1 ml / min. Pass the supernatant of the centrifuged culture solution through the column, loading 40 ml at a flow rate of 0.33 ml / min. Pass the equilibration solution through the column again at a flow rate of at least 3 CV (20 ml), ensuring the pH and conductivity of the final effluent from the instrument are consistent with the equilibration solution. The flow rate is 0.33 ml / min. Pass the elution solution through the column. Begin collecting the elution peak (PAC-EP) when UV280 rises to 15 mAU and stop collecting when UV280 falls to 15 mAU. The flow rate is 1 ml / min. After sample collection, adjust the PAC-EP to neutral using pH adjustment solution.

[0176] 4. Identification of the activity of humanized anti-PD-L1 nanobodies (Binding-ELISA)

[0177] Coating antibodies QP1162 / QP320 / QP321 / QP322 / QP1166 / QP323 / QP324 / QP325 0.75ug / ml, QP11801181 1.5ug / ml 50ul / well, incubated overnight at 4℃. PBS 3 times. Blocking: 3% BSA 250ul / well, RT 1h. Incubate separately with 2ug / ml biotin-PDL1-FC diluted 1:4 to different concentrations, incubated at room temperature for 1h. Wash 3 times with PBST, then 3 times with PBS. Secondary antibody incubation: HRP-strepavidin (1:5000) 50ul / well, washed 6 times with PBST, then 3 times with PBS. Color development: TMB 100ul / well, development for 10min. Terminate with 2M H2SO4 50ul / well. Results as shown. Figure 6 As shown in Table 23.

[0178] Table 23: Results of activity identification of humanized anti-PD-L1 nanobodies (Binding-ELISA)

[0179]

[0180] 5. Identification of the activity of humanized anti-PD-L1 nanobodies (Blocking-ELISA)

[0181] Coating protein QP1138 (PD1-FC) 2ug / ml 50ul / well, incubated overnight at 4℃. Wash 3 times with PBS. Blocking: 3% BSA 250ul / well, incubated at room temperature for 1h. Prepare 2ug / ml biotin-PDL1-FC and different concentrations of QP1120 15ug / ml, QP11801181 30ug / ml, diluted 1:3, mixed in equal volumes, and incubated at room temperature for 1h. Wash 3 times with PBST, then 3 times with PBS. Secondary antibody incubation: HRP-strepavidin (1:5000) 50ul / well, washed 6 times with PBST, then 3 times with PBS. Color development: TMB 100ul / well, developed for 10min. Stopped with 2M H2SO4 50ul / well. Results as shown. Figure 7 As shown in Table 24.

[0182] Table 24: Activity identification of humanized anti-PD-L1 nanobodies (Blocking-ELISA)

[0183]

[0184] 7. Affinity assessment of humanized anti-PD-L1 nanobody using SPR

[0185] Surface plasmon resonance (SPR) detection of affinity

[0186] The affinity of the test molecules for human PD-L1 and cynoPD-L1 proteins was determined using a Biacore T200 (GE) instrument.

[0187] The antigen information is as follows:

[0188] Table 25: Protein Numbering

[0189] QPP09.1 PD-L1 Protein, Human, Recombinant (His Tag) SinoBiologic, 10084-H08H QPP10.1 PD-L1 Protein, Cynomolgus, Recombinant (His Tag) SinoBiologic, 90251-C08H

[0190] Table 26: SPR Affinity Results

[0191]

[0192] Example 3: Identification of the activity of humanized anti-PD-L1 nanobody

[0193] The anti-CLDN18.2 / anti-PD-L1 bispecific antibody molecule QP3711461 was constructed and designed, in the form of... Figure 8 As shown.

[0194]

[0195] 1. In vitro activity assessment of PD-L1 function (mixed lymphocyte response, MLR)

[0196] Preparation of DC (donor1) cells: Resuscitate PBMCs using EasySep TM Monocytes were isolated using the Human Monocyte Isolation Kit (Stemcell 19359). rhGM-CSF (1000 U / ml) and rhIL4 (500 U / ml) were added, and the cells were cultured at 37°C for 6 days to induce iDCs. The medium was changed halfway every 2-3 days, with additional rhGM-CSF (1000 U / ml) and rhIL4 (500 U / ml). Cells were collected, centrifuged at 300 x g for 5 min, resuspended in medium containing rhGM-CSF (1000 U / ml) and rhIL4 (500 U / ml), and LPS (1 μg / ml) was added. The cells were cultured at 37°C for another day to induce mature DCs. Cells were collected, counted, and stored for later use.

[0197] Preparation of T(donor2) cells: Resuscitate PBMCs using EasySep TM Human CD4+T Cell Isolation Kit (Stemcell 17952) isolates CD4+T cells.

[0198] Antibody preparation: Dilute the antibody 1:5 in culture medium to obtain 6 concentrations (initial concentration 10ug / ml). Mix DC cells and T cells at a ratio of 1:10, add different concentrations of antibody, and culture. Detect IL2 expression in the culture supernatant on day 2 and IFNg expression in the culture supernatant on day 5.

[0199] In mixed lymphocyte reaction assays, QP3711461 showed a significant antibody concentration-dependent effect on the concentrations of cytokines IFNγ and IL-2 produced after T cell activation. This demonstrates the biological function of the PD-L1 antibody in QP3711461. Figure 9 and Figure 10 As shown.

[0200] 2. Identification of PD-L1 functional activity in vivo

[0201] The in vivo efficacy of anti-PD-L1 nanobody was evaluated using mouse colon cancer cells MC38-hPDL1 in a transgenic mouse C57BL / 6-hPDL1 subcutaneous model.

[0202] Experimental methods: Log-phase mouse colon cancer cells MC38-hPDL1 (these cells knock out mouse PDL1 and express human PDL1) were harvested, the culture medium was removed, and the cells were washed twice with PBS before being subcutaneously inoculated into the right flank of C57BL / 6-hPDL1 mice at a dose of 5 × 10⁻⁶ cells / cm². 5 / 100μL / mouse. Mice were observed after inoculation and tumor growth was monitored. On day 8 post-inoculation, the average tumor volume reached 92.9 mm. 3 At that time, the tumors were randomly divided into 4 groups of 9 each, based on their tumor volume. The day of grouping was defined as day D0, and drug administration began on day D0.

[0203] Experimental results: such as Figure 11 As shown in Table 25.

[0204] Table 25: Tumor Volume

[0205] Solvent control group (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**

[0206] The test molecule was the anti-CLDN18.2 / anti-PD-L1 bispecific antibody QP3711461, administered at doses of 4 mpk, 10 mpk, and 25 mpk, with BIW×3 administration per intraperitoneal (ip). On day 27 post-administration, the mean tumor volume in the PBS group (negative control group) reached 1445.20 mmHg. 3 The average tumor volume in the QP3711461 (4mpk) group was 477.00 mm. 3 The TGI was 72.00%, and the mean tumor volume in the QP3711461 (10 mpk) group was 279.97 mm. 3 The TGI was 86.14%, and the mean tumor volume in the QP3711461 (25 mpk) group was 293.96 mm. 3 TGI = 85.14%; there were statistically significant differences in tumor volume between each dose group and the PBS group (t-test, p < 0.01).

[0207] This experiment demonstrates that in the MC38-hPDL1 model of humanized transgenic mice with immune targets, the anti-PD-L1 molecule in the anti-CLDN18.2 / anti-PD-L1 bispecific antibody of this invention exhibits superior anti-tumor activity.

[0208] The anti-PD-L1 nanobody provided by this invention is numbered and its corresponding sequence is shown below.

[0209] >SEQ ID No.1 QP1120

[0210] QVQLVESGGGSVQSGGSLRLSCAASGFTYG TYAMS WFRQAPGKEREGVA CIDIYGRTSYTDPVKGRFTISQDNAKNTLYLQMNSLKPEDTAMYYCA ARDFGYCTASWVHEGFSRY WGQGTQVTVSS

[0211] >SEQ ID No.2 QP1121

[0212] QVQLVESGGGSVHAGGSLRLSCVRVSMYTYT GTCMA WFRQAPGKEREGVA GLWTGDGVTYYADSVKG RFTISQDDAKNTLYLQMDSLKPEDTAMYYCA SNGMCGQYWALEDEYKY WGQGTQVTVSS

[0213] >SEQ ID No.3 QP1122

[0214] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TNVMG WFRQAPGKEREGVA AILAGGRNTYYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCA ADTRAAFWNIGPLNSDQYNI WGQGTQVTVSS

[0215] >SEQ ID No.4 QP1123

[0216] QVQLVESGGGSVQSGGSLRLSCAASGYTVR HYCMG WFRQTPGKEREGVA SIDTFGIPKYADSVKG RFTISQDNAKNTLNLQMDSLKPEDTAMYFCA GRSYTNCRDGPPSASHYSH WGQGTQVTVSS

[0217] >SEQ ID No.5 QP1124

[0218] QVQLVESGGGLVQPGGSLRLSCTAPGFTSN TCAMG WYRQAAGMQREWVS SISKYGITTYANSVKG RFTISKDKAEDTVYLQMNNLKPEDTAMYVC KTFSCRNRGGAYLADA WGQGTQVTVSS

[0219] >SEQ ID No.6 QP1125

[0220] QVQLVESGGGLVQPGGSLRLSCTAPGFTDK TCAMA WYRQVAGIEREWVS SISTLGTTNYASSVKG RFTISKDNAKDTVYLQMNNLKPEDTAMYVC KTFSCRNRGGSYLPDT WGQGTQVTVSS

[0221] >SEQ ID No.7 QP1126

[0222] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TKYMS WFRQAPGKEREGVA AILAGGRNTYYADSVKG RFTISQDNAKNTVYLRMNSLKPEDTAMYYCAADTRAALWYIGPLNS DQYNT WGQGTQVTVSS

[0223] >SEQ ID No.8 QP1127

[0224] QVQLVESGGGSVQSGGSLTLSCAISGYAYA TYSMA WFRQAHGKEREGVA AINSDGHTTYVDSVKG RFTISRDNTNKNSYTLTLTMNNLNPEDTAMYYCAATSQLGFWAQKLW EAIRDGTWSPSTTDFGF WGRGTQVTVSS

[0225] >SEQ ID No.9 QP1128

[0226] QVQLVESGGGSVQSGGSLRLSCAASGYTAS AYYMA WFRQNSRKQREGVA AINRDGDTKYADSVKG RFTISRDDAKNTLYLQMNSLKPEDTAVYYCA ASDWSRLYKIYWLDDNYYVR WGQGTQVTVSS

[0227] >SEQ ID No.10 QP1129

[0228] QVQLVESGGGSVQSGGSLRLSCAASGYSSS RYSVG WFRQAPGKEREGVA GQTPRGTTTYADSVKD RFTISRDNAKNTVYLQMNSLKPEDTAMYYCA AGQALLWASLRQTSYQF WGQGTQVTVSS

[0229] >SEQ ID No.11 QP1130

[0230] QVQLVESGGGLVQPGGSLRLSCAASGFTFS TSTMM WVRRAPGKGLEWVS GIHNDGGPIAYADSVKG RFTISRDNAKNTLYLQMTSLKSEDTALYYCAR GWYFSGDYVP MTQGTQVTVSS

[0231] >SEQ ID No.12 QP1139

[0232] QVQLVESGGGSVQAGGSLTLSCAVSGNTYG TNAMG WFRQAPGKEREGVA AILGGGRNTYYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCA ADTRAAFWNIGPLNSDQYNI WGQGTQVTVSS

[0233] >SEQ ID No.13 QP1140

[0234] QVQLVESGGGSVQAGGSLTLSCVVSGNTYS TKYMG WFRQAPGKEREGVA AILAGGRNTYYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCA ADTRAAFWNIGPLNSDQYNI WGQGTQVTVSS

[0235] >SEQ ID No.14 QP1141

[0236] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TNAMG WFRQAPGKEREGVA AILGGGRNTYYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCA ADTRAAFWNIGPLNSDQYNI WGQGTQVTVSS

[0237] >SEQ ID No.15 QP1142

[0238] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TNIMG WFRQAPGKEREGVA AILAGGRNTYYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCA ADTRAAFWNIGPLNSDQYNI WGQGTQVTVSS

[0239] >SEQ ID No.16 QP1143

[0240] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TKYMG WFRQAPGKEREGVA AILAGGRDTYYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCA ADTRAAFWYIGPLNSDQYNI WGQGTQVTVSS

[0241] >SEQ ID No.17 QP1144

[0242] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TKYMG YFRQAPGKEREGVA AILAGGRNTNYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLNS DQYNI WGQGTQVTVSS

[0243] >SEQ ID No.18 QP1145

[0244] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TKYMA WFRQAPGKEREGVA AILAGGRNTSYADSVKG RFTISQDNAKNTVYLQTNSLKPEDTAMYYCA ADTRAAFWYIGPLNSHQYNI WGQGTQVTVSS

[0245] >SEQ ID No.19 QP1146

[0246] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TNYMG WFRQAPGKEREGVA AILVGGRNTYYADSVKG RFTISQDNAKNLVYLQMNSLKPEDTAMYYCA ADTRAAFWNIGPLNSDQYNI WGQGTQVTVSS

[0247] >SEQ ID No.20 QP1147

[0248] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TKYMG WFRQAPGKEREGVA AILAGGRNTYYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCA ADTRAAFWYIGPLNSDQYNI WGQGTQVTVSS

[0249] >SEQ ID No.21 QP1148

[0250] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TNYMG WFRQAPGKEREGVA AILAGGRNTAYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCA ADTRAAFWNIGPLNSDQYNI WGQGTQVTVSS

[0251] >SEQ ID No.22 QP1149

[0252] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TNYMG WFRQAPGKEREGVA AILAGGRNTYYADSVKG RFTISQDNAKDTVYLQMNSLKPEDTAMYYCA ADTRAAFWNIGPLNSDQYNI WGQGTQVTVSS

[0253] >SEQ ID No.23 QP1150

[0254] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TKYMG WFRQAPGKEREGVA AILAGGRNTHYADSVKG RFAISQDNAKNTVYLQMNSLKPEDTAMYYCA ADTRAAFWNIGPLNSDQYNI WGQGTQVTVSS

[0255] >SEQ ID No.24 QP1151

[0256] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TNYMG WFRQAPGKEREGVA AILTGGRNTYYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCA ADTRAAFWNIGPLNSDQYNI WGQGTQVTVSS

[0257] >SEQ ID No.25 QP1152

[0258] QVQLVESGGGSVQAGGSLTLSCAISGNTYS TKYMG WFRQAPGKEREGVA AILAGGRNTYYADSVKG RFTISQDNAKNTVYLQMHSLKPEDTAMYYCAADTRAAFWYIGPLNS DQYNL WGQGTQVTVSS

[0259] >SEQ ID No.26 QP1153

[0260] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TKYMG WFRQAPGKEREGVA AILAGGRNTDYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCA ADTRAAFWSIGPLNSDQYNI WGQGTQVTVSS

[0261] >SEQ ID No.27 QP1154

[0262] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TNYMA WFRQAPGKEREGVA AIRAGGRNTDYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCA ADTRAAFWYIGPLNSDQYNI WGQGTQVTVSS

[0263] >SEQ ID No.28 QP1155

[0264] QVQLVESGGGSVQAGGSLTLSCEVSGSTYS TNYMG WFRQAPGKEREGVA AILAGGRNTDYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCA ADTRAAFWYIGPLNSDQYNI WGQGTQVTVSS

[0265] >SEQ ID No.29 QP1156

[0266] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TKYMG WFRQAPGKEREGVA AILAGGRNTYYADSVKG RFTISQDSAKNTVYLQMNSLKPEDTAMYYCA ADTRAAFWYIGPLNSDQYNS WGQGTQVTVSS

[0267] >SEQ ID No.30 QP1157

[0268] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TNYMG WFRQAPGKEREGVA AILVGGRNTYYADPVKG RFTISQDNAKNLVYLQMNSLKPEDTAMYYCA ADTRAAFWNIGPLNSDQYNI WGQGTQVTVSS

[0269] >SEQ ID No.31 QP1158

[0270] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TKYMG WFRQAPGKEREGVA AILAGGRNTYYADSVKG RFTISQDNAKNTVYLRMNSLKPEDTAMYYCAADTRAALWYIGPLNS DQYNT WGQGTQVTVSS

[0271] >SEQ ID No.32 QP1159

[0272] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TKYMG WFRQAPGKEREGVA AILAGGRNTSYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCAADTRAAFWYIGPLN SDQYNI WGQGTQVTVSS

[0273] >SEQ ID No.33 QP1160

[0274] QVQLVESGGGSVQAGGSLTLSCAISGNTYS TKYMG WFRQAPGKEREGVA AILAGGRNTYYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCA ADARAAFWYIGPLNSDQYNI WGQGTQVTVSS

[0275] >SEQ ID No.34 QP1161

[0276] QVQLVESGGGSVQAGGSLTLSCAVSGNTYS TNYMA WFRQAPGKEREGVA AIRVGGRNTDYADSVKG RFTISQDNAKNTVYLQMNSLKPEDTAMYYCA ADTRAAFWYIGPLNSDQYNI WGQGTQVTVSS

[0277] >SEQ ID No.35 QP1162

[0278] QVQLVESGGGSVQSGGSLRLSCAASGFTYG TYAMS WFRQAPGKEREGVA CIDIYGRASYTDPVKG RFTISQDNAKNTLYLQMNSLKPEDTAMYYCA ARDFGYCTASWVHEGFSRY WGQGTQVTVSS

[0279] >SEQ ID No.36 QP1163

[0280] QVQLVESGGGSVQSGGSLRLSCAASGFTYG TYAMS WFRQAPGKEREGVA CIDIYGRTSYTDPVKG RFTISQDNAKNTLYLQMNSLKPEDTAMYYCA ARDFGYCTASWVHAGFSRY WGQGTQVTVSS

[0281] >SEQ ID No.37 QP1164

[0282] QVQLVESGGGSVQSGGSLRLSCAASGFTYG TYAMS WFRQTPGKEREGVA CIDIYGRTSYTDPVKG RFTISQDNAKNTLYLQMNSLKPEDTAMYYCA ARDFGYCTASWVHEGFSRY WGQGTQVTVSS

[0283] >SEQ ID No.38 QP1165

[0284] QVQLVESGGGSVQSGGSLRLSCAASGFTYG AYAMS WFRQAPGKEREGVA CIDIYGRTSYTDPVKG RFTISQDNAKNTLYLQMNSLKPEDTAMYYCA ARDFGYCTASWVHEGFSRY WGQGTQVTVSS

[0285] >SEQ ID No.39 QP1166

[0286] QVQLVESGGDSVQPGGSLRLSCAASGFTYG TYAMS WFRQAPGKEREGVA CIDIYGRTSYTDPVKG RFTISQDNAKNTLYLQMNSLKPEDTAMYYCA ARDFGYCTASWVHEGFSRY WGQGTQVTVSS

[0287] >SEQ ID No.40 QP1168

[0288] QVQLVESGGGSVQSGGSLRLSCAASGFTYE TYAMS WFRQAPGKEREGVA CIDIYGRTSYTDPVKG RFTISQDNAKNTLYLQMNSLKPEDTAMYYCA ARDFGYCTASWVHEGFSRY WGQGTQVTVSS

[0289] >SEQ ID No.41 QP1169

[0290] QVQLVESGGGSVQSGGSLRLSCAASGFTYG TYAMS WFRQAPGKEREGVA CIDIYGRTSYTDPVKG RFTISQDNAKNTLYLQMNSLKPGDTAMYYCA ARDFGYCTASWVHEGFSRY WGQGTQVTVSS

[0291] >SEQ ID No.42 Human IgG1:

[0292] EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0293] >SEQ ID No.43 PD-L1-his

[0294] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNEREQKLISEEDLHHHHHH

[0295] >SEQ ID No.44 QP1120-CDR1

[0296] TYAMS

[0297] >SEQ ID No.45 QP1121-CDR1

[0298] GTCMA

[0299] >SEQ ID No.46 QP1122-CDR1

[0300] TNVMG

[0301] >SEQ ID No.47 QP1123-CDR1

[0302] HYCMG

[0303] >SEQ ID No.48 QP1124-CDR1

[0304] TCAMG

[0305] >SEQ ID No.49 QP1125-CDR1

[0306] TCAMA

[0307] >SEQ ID No.50 QP1126-CDR1

[0308] TKYMS

[0309] >SEQ ID No.51 QP1127-CDR1

[0310] TYSMA

[0311] >SEQ ID No.52 QP1128-CDR1

[0312] AYYMA

[0313] >SEQ ID No.53 QP1129-CDR1

[0314] RYSVG

[0315] >SEQ ID No.54 QP1130-CDR1

[0316] TSTMM

[0317] >SEQ ID No.55 QP1139-CDR1

[0318] TNAMG

[0319] >SEQ ID No.56 QP1140-CDR1

[0320] TKYMG

[0321] >SEQ ID No.57 QP1142-CDR1

[0322] TNIMG

[0323] >SEQ ID No.58 QP1145-CDR1

[0324] TKYMA

[0325] >SEQ ID No.59 QP1154-CDR1

[0326] TNYMA

[0327] >SEQ ID No.60 QP1165-CDR1

[0328] AYAMS

[0329] >SEQ ID No.61 QP1120-CDR2

[0330] CIDIYGRTSYTDPVKG

[0331] >SEQ ID No.62 QP1121-CDR2

[0332] GLWTGDGVTYYADSVKG

[0333] >SEQ ID No.63 QP1122-CDR2

[0334] AILAGGRNTYYADSVKG

[0335] >SEQ ID No.64 QP1123-CDR2

[0336] SIDTFGIPKYADSVKG

[0337] >SEQ ID No.65 QP1124-CDR2

[0338] SISKYGITTYANSVKG

[0339] >SEQ ID No.66 QP1125-CDR2

[0340] SISTLGTTNYASSVKG

[0341] >SEQ ID No.67 QP1127-CDR2

[0342] AINSDGHTTYVDSVKG

[0343] >SEQ ID No.68 QP1128-CDR2

[0344] AINRDGDTKYADSVKG

[0345] >SEQ ID No.69 QP1129-CDR2

[0346] GQTPRGTTTYADSVKD

[0347] >SEQ ID No.70 QP1130-CDR2

[0348] GIHNDGGPIAYADSVKG

[0349] >SEQ ID No.71 QP1139-CDR2

[0350] AILGGGRNTYYADSVKG

[0351] >SEQ ID No.72 QP1143-CDR2

[0352] AILAGGRDTYYADSVKG

[0353] >SEQ ID No.73 QP1144-CDR2

[0354] AILAGGRNTNYADSVKG

[0355] >SEQ ID No.74 QP1145-CDR2

[0356] AILAGGRNTSYADSVKG

[0357] >SEQ ID No.75 QP1146-CDR2

[0358] AILVGGRNTYYADSVKG

[0359] >SEQ ID No.76 QP1148-CDR2

[0360] AILAGGRNTAYADSVKG

[0361] >SEQ ID No.77 QP1150-CDR2

[0362] AILAGGRNTHYADSVKG

[0363] >SEQ ID No.78 QP1151-CDR2

[0364] AILTGGRNTYYADSVKG

[0365] >SEQ ID No.79 QP1153-CDR2

[0366] AILAGGRNTDYADSVKG

[0367] >SEQ ID No.80 QP1157-CDR2

[0368] AILVGGRNTYYADPVKG

[0369] >SEQ ID No.81 QP1161-CDR2

[0370] AIRVGGRNTDYADSVKG

[0371] >SEQ ID No.82 QP1162-CDR2

[0372] CIDIYGRASYTDPVKG

[0373] >SEQ ID No.83 QP1120-CDR3

[0374] ARDFGYCTASWVHEGFSRY

[0375] >SEQ ID No.84 QP1121-CDR3

[0376] SNGMCGQYWALEDEYKY

[0377] >SEQ ID No.85 QP1122-CDR3

[0378] ADTRAAFWNIGPLNSDQYNI

[0379] >SEQ ID No.86 QP1123-CDR3

[0380] GRSYTNCRDGPPSASHYSH

[0381] >SEQ ID No.87 QP1124-CDR3

[0382] KTFSCRNRGGAYLADA

[0383] >SEQ ID No.88 QP1126-CDR3

[0384] ADTRAALWYIGPLNSDQYNT

[0385] >SEQ ID No.89 QP1127-CDR3

[0386] ATSQLGFWAQKLWEAIRDGTWSPSTTDFGF

[0387] >SEQ ID No.90 QP1128-CDR3

[0388] ASDWSRLYKIYWLDDNYYVR

[0389] >SEQ ID No.91 QP1129-CDR3

[0390] AGQALLWASLRQTSYQF

[0391] >SEQ ID NO.92 QP1130-CDR3

[0392] GWYFSGDYVP

[0393] >SEQ ID NO.93 QP1143-CDR3

[0394] ADTRAAFWYIGPLNSDQYNI

[0395] >SEQ ID No.94 QP1145-CDR3

[0396] ADTRAAFWYIGPLNSHQYNI

[0397] >SEQ ID NO.95 QP1152-CDR3

[0398] ADTRAAFWYIGPLNSDQYNL

[0399] >SEQ ID No.96 QP1153-CDR3

[0400] ADTRAAFWSIGPLNSDQYNI

[0401] >SEQ ID NO.97 QP1156-CDR3

[0402] ADTRAAFWYIGPLNSDQYNS

[0403] >SEQ ID NO.98 QP1160-CDR3

[0404] PLEASEWYIGPLNSDQYNI

[0405] >SEQ ID No.99 QP1163-CDR3

[0406] ARDFGYCTASWVHAGFSRY

[0407] >SEQ ID NO.100 QP320

[0408] EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWVRQAPGKGLEWVSCIDIYGRASYTDPVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSS

[0409] >SEQ ID No.101 QP321

[0410] EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWVRQAPGKGLEWVACIDIYGRASYTDPVKGRFTISRDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSS

[0411] >SEQ ID No.102 QP322

[0412] EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGREGVACIDIYGRASYTDPVKGRFTISQDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSS

[0413] >SEQ ID No.103 QP323

[0414] EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWVRQAPGKGLEWVSCIDIYGRTSYTDPVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSS

[0415] >SEQ ID No.104 QP324

[0416] EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWVRQAPGKGLEWVACIDIYGRTSYTDPVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSS

[0417] >SEQ ID No.105 QP325

[0418] EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGLEGVACIDIYGRTSYTDPVKGRFTISQDNSKNTLYLQMNSLRAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSS

[0419] >SEQ ID No.106 QP1180

[0420] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0421] >SEQ ID No.107 QP1181

[0422] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0423] >SEQ ID No.108 QP1461

[0424] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDHSYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0425] >SEQ ID No.109 QP371

[0426] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYIMHWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLGFTTRNAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGREGVACIDIYGRASYTDPVKGRFTISQDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSS

Claims

1. An anti-PD-L1 nanobody, characterized in that: The anti-PD-L1 nanobody comprises CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 are: 1) CDR1 shown in SEQ ID No. 44; CDR2 as shown in SEQ ID No. 61; CDR3 as shown in SEQ ID No. 83; or 2) CDR1 shown in SEQ ID No. 60; CDR2 as shown in SEQ ID No. 61; CDR3 as shown in SEQ ID No.

83.

2. The anti-PD-L1 nanobody as described in claim 1, characterized in that: Its sequence is shown as SEQ ID No. 39 or 38.

3. The anti-PD-L1 nanobody as described in claim 1, characterized in that: Except for CDR1, CDR2 and CDR3, 80% of its amino acid sequence is identical to the sequence shown in SEQ ID No. 39 or 38.

4. An Fc fusion protein of an anti-PD-L1 nanobody, characterized in that: The nanobody comprises the anti-PD-L1 nanobody as described in any one of claims 1-3 and the Fc segment, the sequence of which is shown in SEQ ID No.

42.

5. The use of the anti-PD-L1 nanobody as described in any one of claims 1-3, or the Fc fusion protein of the anti-PD-L1 nanobody as described in claim 4, in the preparation of a medicament for treating cancer or tumors; The cancer or tumor is selected from the following group: small cell lung cancer, non-small cell lung cancer, melanoma, basal cell carcinoma, squamous cell carcinoma, Merkel cell carcinoma, glioma, sarcoma, and mesothelioma.

6. A nucleic acid encoding an anti-PD-L1 nanobody as described in any one of claims 1-3, or a nucleic acid encoding an Fc fusion protein of an anti-PD-L1 nanobody as described in claim 4.

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