Nanoantibody targeting phosphatidylinositol proteoglycan 3 and use thereof

Abstract

The present invention relates to a nanoantibody targeting phosphatidylinositol proteoglycan 3 and the use thereof. The amino acid sequence of the nanoantibody comprises an antigenic complementarity determining region and a framework region of a special structure. First, immunizing a llama by means of constructing GPC3 antigens so as to obtain llama PBMC cell constructs; then, screening out GPC3-targeting antibodies by means of a yeast display technique; and finally, obtaining an antibody having high sensitivity and specificity by means of functional detection and sequencing result analysis. The provided nanoantibody has specific recognition and binding ability for GPC3, has a small molecular weight, low immunogenicity, high stability, and a significant tumor inhibition effect, and provides a potential strategy for treating GPC3-expressing tumor diseases. The present invention is used for developing or screening for a drug for the diagnosis or treatment of GPC3-expressing cells or tumors, and the obtained drug has good specificity and affinity and a significant tumor inhibition effect.

Description

Nanobody targeting glypican-3 and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a nanobody targeting glypican-3 and application thereof. BACKGROUND

[0002] Liver cancer is the eighth most common cancer in the world and the third leading cause of cancer death globally. Among them, hepatocellular carcinoma is the most common histopathological type, accounting for 80% of all liver cancers. At present, surgical radical treatment is still the most effective treatment for hepatocellular carcinoma. Early liver cancer patients are suitable for surgical resection, local ablation, liver transplantation and other radical treatments. However, liver cancer has insidious onset, resulting in more than 70% of liver cancer patients in China being in the middle and advanced stages at the time of initial diagnosis, and the tumor cannot be radically treated by surgery. In addition, the recurrence rate of liver cancer after surgery is high, and the total recurrence rate within 5 years is as high as about 70%. Although current tyrosine kinase inhibitors have become the first-line immunotherapy drugs for unresectable liver cancer and have brought survival benefits to patients with advanced liver cancer, their efficacy and side effects are significantly related. And due to long-term inflammation and antigen stimulation, a special immune tolerance microenvironment is formed in liver cancer, which promotes the immune escape of tumor cells and limits the efficacy of immune checkpoint inhibitor therapy.

[0003] Glypican-3 (GPC3) is a heparan sulfate (HS) glycoprotein anchored on the cell membrane surface, which is overexpressed in liver cancer cells, but not expressed or lowly expressed in normal liver tissue, making it an ideal diagnostic and therapeutic target for hepatocellular carcinoma. GPC3 is highly expressed in the tissues and serum of liver cancer patients, but not highly expressed in normal liver tissue or serum. Compared with alpha-fetoprotein, GPC3 may have better practical value in detecting early liver cancer. Therefore, GPC3 has become an auxiliary diagnostic marker and therapeutic target for liver cancer.

[0004] Currently, the methods for treating GPC3 in the clinic include: monoclonal antibodies targeting GPC3, GPC3-derived polypeptide / DNA vaccines, immunotoxins, etc., but in clinical trials and preclinical trials, they all show limited efficacy. From the perspective of monoclonal antibodies, although some studies have shown that phase I clinical trials of anti-GPC3 monoclonal antibodies combined with other drugs show good tolerance and can effectively reduce tumor growth in patients with advanced hepatocellular carcinoma, monoclonal antibodies alone may not completely eliminate tumors, and the durability of the therapeutic effect has not been fully verified. Secondly, regarding GPC3-derived polypeptide / DNA vaccines, although their safety and certain immunogenicity response have been proven in clinical trials, the number of complete and partial remission cases is limited, and the median survival is relatively short. This indicates that although the vaccine can stimulate the immune system to attack tumors, the therapeutic effect is not significant, and it may not be suitable for all liver cancer patients. Furthermore, although immunotoxins have the ability to specifically kill tumor cells, their side effects and toxicity cannot be ignored. In clinical trials, the use of immunotoxins can lead to overactivation of the immune system, causing a series of adverse reactions such as fever, chills, nausea, etc. In addition, the preparation and purification of immunotoxins also face technical challenges, limiting their widespread use in the clinic. Although some progress has been made in the current clinical treatment of GPC3, there are still limitations in terms of efficacy, safety, and technical feasibility.

[0005] Therefore, although there are various treatment methods for liver cancer at present, there are still certain limitations, especially in terms of efficient and low side effect treatment targeting specific tumor markers or targets. In this context, nanobodies, as a new type of biological drug, are gradually showing their great potential in the treatment of liver cancer. Nanobodies have the advantages of small size, high affinity, easy modification and production, and can more accurately recognize and attack liver cancer cells while reducing damage to normal cells.

[0006] Therefore, exploring and screening nanobodies that can specifically bind to GPC3 and applying them in the treatment of liver cancer are expected to provide more efficient and safe treatment options for liver cancer patients, overcoming the limitations of existing treatment methods and opening up new avenues for the treatment of liver cancer. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application aims to provide a kind of nanobody targeting phosphatidylinositol glycoprotein 3 and its application, the present application is developed for Glypican-3 (GPC3) Nanobody drug, by constructing Glypican-3 antigen to immunize alpaca, obtain alpaca PBMC cell construct;Secondly, specific binding Glypican-3 high affinity nanobody is screened out by yeast display technology.The nanobody can safely and effectively target Glypican-3, and has good specificity and affinity when applied to the preparation or screening of tumor diagnosis or treatment drugs, and has obvious tumor inhibition effect.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] The first object of the present application is to provide a kind of nanobody targeting Glypican-3, including at least one of the heavy chain variable region CDR sequence selected from the following or amino acid sequence with at least 80% identity: SEQ ID NO.1-23.

[0010] Preferably, the nanobody includes:

[0011] Heavy chain variable region CDR1 sequence as shown in SEQ ID NO.1-7 amino acid sequence or amino acid sequence with at least 80% identity with SEQ ID NO.1-7;

[0012] Heavy chain variable region CDR2 sequence as shown in SEQ ID NO.8-15 amino acid sequence or amino acid sequence with at least 80% identity with SEQ ID NO.8-15;

[0013] Heavy chain variable region CDR3 sequence as shown in SEQ ID NO.16-23 amino acid sequence or amino acid sequence with at least 80% identity with SEQ ID NO.16-23.

[0014] Preferably, the complementarity determining region CDR of the nanobody targeting Glypican-3 includes CDR1-CDR3 as shown in the following amino acid sequence:

[0015] (1) CDR1 with amino acid sequence as shown in SEQ ID NO.1, CDR2 with amino acid sequence as shown in SEQ ID NO.8, and CDR3 with amino acid as shown in SEQ ID NO.16;Or

[0016] (2) CDR1 of an amino acid sequence shown as SEQ ID NO. 2, CDR2 of an amino acid sequence shown as SEQ ID NO. 9, and CDR3 of an amino acid sequence shown as SEQ ID NO. 17; or

[0017] (3) CDR1 of an amino acid sequence shown as SEQ ID NO. 3, CDR2 of an amino acid sequence shown as SEQ ID NO. 10, and CDR3 of an amino acid sequence shown as SEQ ID NO. 18; or

[0018] (4) CDR1 of an amino acid sequence shown as SEQ ID NO. 4, CDR2 of an amino acid sequence shown as SEQ ID NO. 11, and CDR3 of an amino acid sequence shown as SEQ ID NO. 19; or

[0019] (5) CDR1 of an amino acid sequence shown as SEQ ID NO. 2, CDR2 of an amino acid sequence shown as SEQ ID NO. 12, and CDR3 of an amino acid sequence shown as SEQ ID NO. 20; or

[0020] (6) CDR1 of an amino acid sequence shown as SEQ ID NO. 5, CDR2 of an amino acid sequence shown as SEQ ID NO. 13, and CDR3 of an amino acid sequence shown as SEQ ID NO. 21; or

[0021] (7) CDR1 of an amino acid sequence shown as SEQ ID NO. 6, CDR2 of an amino acid sequence shown as SEQ ID NO. 14, and CDR3 of an amino acid sequence shown as SEQ ID NO. 22; or

[0022] (8) CDR1 of an amino acid sequence shown as SEQ ID NO. 7, CDR2 of an amino acid sequence shown as SEQ ID NO. 15, and CDR3 of an amino acid sequence shown as SEQ ID NO. 23.

[0023] Preferably, the nanobody comprises a framework region FR sequence selected from at least one of the following or an amino acid sequence having at least 80% identity thereto: SEQ ID NO. 24-55.

[0024] Preferably, the nanobody comprises:

[0025] a framework region FR1 sequence shown as an amino acid sequence of SEQ ID NO. 24-32 or an amino acid sequence having at least 80% identity to SEQ ID NO. 24-32;

[0026] a framework region FR2 sequence as set forth in any one of SEQ ID NO. 33-40 or an amino acid sequence having at least 80% identity to SEQ ID NO. 33-40;

[0027] a framework region FR3 sequence as set forth in any one of SEQ ID NO. 41-48 or an amino acid sequence having at least 80% identity to SEQ ID NO. 41-48;

[0028] a framework region FR4 sequence as set forth in any one of SEQ ID NO. 49-55 or an amino acid sequence having at least 80% identity to SEQ ID NO. 49-55.

[0029] Preferably, the Glypican-3 targeting Nanobody comprises a framework region FR comprising FR1-FR4 with amino acid sequences as set forth in:

[0030] (1) FR1 as set forth in SEQ ID NO. 24, FR2 as set forth in SEQ ID NO. 33, FR3 as set forth in SEQ ID NO. 41, and FR4 as set forth in SEQ ID NO. 49; or

[0031] (2) FR1 as set forth in SEQ ID NO. 25, FR2 as set forth in SEQ ID NO. 33, FR3 as set forth in SEQ ID NO. 41, and FR4 as set forth in SEQ ID NO. 50; or

[0032] (3) FR1 as set forth in SEQ ID NO. 26, FR2 as set forth in SEQ ID NO. 34, FR3 as set forth in SEQ ID NO. 42, and FR4 as set forth in SEQ ID NO. 51; or

[0033] (4) FR1 as set forth in SEQ ID NO. 27, FR2 as set forth in SEQ ID NO. 34, FR3 as set forth in SEQ ID NO. 42, and FR4 as set forth in SEQ ID NO. 51; or

[0034] (5) FR1 as set forth in SEQ ID NO. 28, FR2 as set forth in SEQ ID NO. 35, FR3 as set forth in SEQ ID NO. 43, and FR4 as set forth in SEQ ID NO. 50; or

[0035] (6) FR1 of an amino acid sequence shown in SEQ ID NO. 29, FR2 of an amino acid shown in SEQ ID NO. 36, FR3 of an amino acid shown in SEQ ID NO. 44, and FR4 of an amino acid shown in SEQ ID NO. 50; or

[0036] (7) FR1 of an amino acid sequence shown in SEQ ID NO. 30, FR2 of an amino acid shown in SEQ ID NO. 36, FR3 of an amino acid shown in SEQ ID NO. 44, and FR4 of an amino acid shown in SEQ ID NO. 50; or

[0037] (8) FR1 of an amino acid sequence shown in SEQ ID NO. 31, FR2 of an amino acid shown in SEQ ID NO. 37, FR3 of an amino acid shown in SEQ ID NO. 45, and FR4 of an amino acid shown in SEQ ID NO. 52; or

[0038] (9) FR1 of an amino acid sequence shown in SEQ ID NO. 28, FR2 of an amino acid shown in SEQ ID NO. 38, FR3 of an amino acid shown in SEQ ID NO. 46, and FR4 of an amino acid shown in SEQ ID NO. 53; or

[0039] (10) FR1 of an amino acid sequence shown in SEQ ID NO. 32, FR2 of an amino acid shown in SEQ ID NO. 39, FR3 of an amino acid shown in SEQ ID NO. 47, and FR4 of an amino acid shown in SEQ ID NO. 54; or

[0040] (11) FR1 of an amino acid sequence shown in SEQ ID NO. 31, FR2 of an amino acid shown in SEQ ID NO. 40, FR3 of an amino acid shown in SEQ ID NO. 48, and FR4 of an amino acid shown in SEQ ID NO. 55.

[0041] Another object of the present application is to provide a polypeptide comprising the above-described nanobody.

[0042] Another object of the present application is to provide a polypeptide comprising the above-described nanobody.

[0043] Another object of the present application is to provide a polypeptide comprising the above-described nanobody. Another object of the present application is to provide a nucleic acid molecule encoding the above-described nanobody.

[0044] Another object of the present application is to provide an expression vector comprising the above-mentioned nucleic acid molecule.

[0045] Another object of the present application is to provide a host cell transformed or transfected with the above-mentioned expression vector.

[0046] Another object of the present application is to provide a pharmaceutical composition comprising the above-mentioned Nanobody and a pharmaceutically acceptable carrier and / or excipient.

[0047] Another object of the present application is to provide the use of the above-mentioned Nanobody, polypeptide, nucleic acid molecule, expression vector, host cell, and pharmaceutical composition in the preparation or screening of a diagnostic or therapeutic drug or kit for a tumor expressing Glypican-3.

[0048] Compared with the prior art, the present application has the following beneficial effects:

[0049] (1) The present application firstly immunizes a lama with a Glypican-3 antigen to obtain a lama PBMC cell construct; secondly, a Glypican-3-targeting antibody is screened through yeast display technology; and finally, an antibody with high sensitivity and specificity is obtained through functional detection and sequencing result analysis. The Nanobody provided by the present application has specific recognition and binding capacity for Glypican-3, has a molecular weight of 13.3-14.7 kDa, has small immunogenicity, has better stability, has obvious tumor inhibition effect, and the like, and provides a potential treatment strategy for a tumor disease expressing Glypican-3.

[0050] (2) The Nanobody provided by the present application can be used for preparing or screening a drug for diagnosing or treating a Glypican-3-expressing tumor, such as liver cancer, has good specificity and affinity, and has an obvious tumor inhibition effect. BRIEF DESCRIPTION OF DRAWINGS

[0051] Fig. 1 is a GPC3-His recombinant protein Reduced SDS-PAGE detection result graph;

[0052] Fig. 2 is a GPC3-His recombinant protein ELISA detection result graph;

[0053] Fig. 3 is a lama immunization titer detection result graph;

[0054] Fig. 4 is a VHH fragment amplification graph;

[0055] Fig. 5 is a first-time yeast display library screening graph;

[0056] Fig. 6 is a first-time positive epitope competition screening graph of the yeast display library;

[0057] Figure 7 is a diagram of the second time selection of the yeast display library;

[0058] Figure 8 is a diagram of the second time positive epitope competition screening of the yeast display library;

[0059] Figure 9 is a diagram of the first time single clone detection result of the yeast library;

[0060] Figure 10 is a diagram of the second time single clone detection result of the yeast library;

[0061] Figure 11 is a diagram of the flow detection of the binding of the recombinant antibody to the target protein;

[0062] Figure 12 is a diagram of the flow detection of the binding of the recombinant antibody to the target protein;

[0063] Figure 13 is a diagram of the flow detection of the binding of the recombinant antibody to the target protein;

[0064] Figure 14 is a diagram of the flow detection of the binding of the recombinant antibody to the target protein;

[0065] Figure 15 is a diagram of the flow detection of the binding of the recombinant antibody to the target protein;

[0066] Figure 16 is a diagram of the ELISA detection of the binding of the recombinant antibody to the target protein;

[0067] Figure 17 is a diagram of the flow cytometry detection of the binding efficiency of the nanobody to the target cells Huh7, HepG2 and non-target cells SK-HEP-1;

[0068] Figure 18 is a diagram of the flow detection of the binding of the nanobody to 293T-mGPC3;

[0069] Figure 19 is a diagram of the vector map of the chimeric antigen receptor expressing targeting GPC3;

[0070] Figure 20 is a diagram of the flow detection of the transduction efficiency of the CAR-T cells;

[0071] Figure 21 is a diagram of the in vitro anti-tumor effect detection of the CAR-T cells. DETAILED DESCRIPTION

[0072] The above content of the present application will be further explained in detail by the specific embodiments in the form of examples. However, it should not be understood that the above-mentioned subject matter of the present application is limited to the following examples only.

[0073] 1. Cell line

[0074] The 293T (human embryonic kidney cells) cells, HepG2 (human hepatoma cells) cells were purchased from the Chinese Academy of Sciences Stem Cell Bank; the Huh7 (human hepatoma cells) cells were gifted from the team of Professor Zhaoliang of Southern Medical University; the Jurkat cells were gifted from the team of Professor Jiang Qianli of Southern Medical University; the CHO-S cells and 293F cells were purchased from Aikangde Biotechnology Co., Ltd.

[0075] 2. Reagents, instruments and experimental consumables

[0076] Table 1. Reagents and their suppliers and item numbers

[0077] Table 2. Instruments, consumables and their suppliers and item numbers

[0078] Design principles or design ideas of the present application:

[0079] Camels were immunized with antigens, peripheral blood mononuclear cells (PBMCs) were isolated and total RNA was extracted for reverse transcription, the reverse transcription product was used as a template to amplify the variable domain of the heavy-chain of heavy chain antibody (VHH) and connected into a yeast display vector, and then electroporated into yeast competent cells to construct a yeast display library.

[0080] The constructed yeast display library was screened by magnetic sorting and flow sorting method to obtain specific anti-GPC3 nanobodies. Through primary library presentation and screening and identification, 12 strains of yeast display nanobodies capable of binding to GPC3 recombinant protein were obtained.

[0081] Through ELISA detection of the binding of nanobodies to target antigens, it can be known that the nanobodies can effectively bind to GPC3 antigens; through flow cytometry detection of the binding efficiency of nanobodies to target cells and non-target cells, it can be known that the nanobodies can specifically bind to target cells, proving that the GPC3-targeted nanobodies provided by the present application can specifically bind to human and mouse GPC3; through the construction of CAR-T cells, co-culture with target cells and non-target cells, it is concluded that the CAR-T cells constructed by nanobodies can effectively kill target cells, proving that the GPC3-targeted nanobodies provided by the present application can be used to construct CAR-T cells and have the expected function. As can be seen, the nanobodies provided by the present application have the effect of specific recognition and binding ability to GPC3.

[0082] Example 1. Expression and purification of target protein

[0083] GPC3 recombinant protein was prepared by entrusting Aikangde Biotechnology Co., Ltd., including the following steps:

[0084] a. Retrieve the extracellular segment sequence information (Gln 25-His 559) of Human GPC3 (P51654-1) from the UniProt database, add a His tag at the C-terminal, optimize according to the human codon bias, then perform gene synthesis, and cut at the Xba I and EcoR V sites and clone into the pcDNA3.4 vector, wherein the sequence information of the GPC3 gene is shown as SEQ ID NO. 67-68, and the sequence information of the GPC3-pcDNA3.4 vector is shown as SEQ ID NO. 69.

[0085] The amino acid sequence of the optimized GPC3 gene GPC3 25-559AA (S359F)-His is as follows:

[0086] The nucleotide sequence of the optimized GPC3 gene GPC3 25-559AA (S359F)-His is as follows:

[0087] The sequence of pcDNA3.4-GPC3-His (S359-F) is as follows:

[0088] After Sanger sequencing verification, plasmid extraction is prepared for standby.

[0089] b. Take the LVTransm transfection reagent and GPC3 expression vector from the refrigerator, thaw at room temperature, and mix thoroughly with a pipette gun. Take PBS buffer and warm it to room temperature. Take 2 mL of PBS into one well of a 6-well plate, add 100 μg of expression vector, mix thoroughly with a pipette gun, then add 300 μL of LVTransm, immediately mix with a pipette, and stand at room temperature for 10 minutes. Add the above DNA / LVTransm complex to 100 mL of 293F cells, mix thoroughly, and continue to culture at 37°C, 5% CO2 incubator, 130 RPM. After continuous culture for 5-7 days, centrifuge to collect the culture supernatant, filter with a 0.45 μm filter membrane, transfer the filtrate to a sterile centrifuge tube, and use Ni-NTA to purify the protein.

[0090] c. SDS-PAGE to detect the purity of the target antibody protein, as shown in Figure 1, the purity of the Human GPC3 recombinant protein obtained by the present application is greater than 95%, which can be used for alpaca immunization and antibody screening. The constructed protein eukaryotic expression vector is transiently transfected into 293F cells, and the protein expression supernatant is collected and the target protein is purified by nickel column.

[0091] d. 100ul GPC3-His antigen is coated at 4°C overnight using a 96-well plate, and the coating concentration is 2ug / mL; ELISA experiment is performed to detect the binding of GPC3-His and positive antibody. The experimental results are shown in Figure 2, the GPC3 purity is greater than 90% and has good binding activity with the positive control antibody, and the next step of immunization experiment can be arranged.

[0092] Example 2 Construction of yeast nanobody library

[0093] The GPC3 recombinant protein prepared in Example 1 is used to immunize alpaca twice, then peripheral blood lymphocytes are separated, total RNA is extracted, the RNA is reverse transcribed into cDNA, and then the nanobody VHH sequence is amplified using primers. The VHH sequence is used to construct a yeast display nanobody library.

[0094] The specific steps are as follows:

[0095] (1) Alpaca immunization and detection of immune titer

[0096] a. The recombinant protein prepared above is used for subcutaneous multi-point injection immunization of alpaca (Alpaca), with an interval of 21 days for immunization once, a total of 4 times, with an injection of 500ug for the first immunization, and an injection of 250ug for the second to fourth immunization.

[0097] b. Ten days after the last immunization, 5mL of peripheral blood is collected, and the centrifuge tube containing the blood sample is placed in a 37°C incubator for 1 hour; then the blood sample is transferred to 4°C overnight.

[0098] c. Place the centrifuge tube containing the blood sample in the centrifuge and centrifuge at 5000 rpm for 20 min; separate the upper serum and transfer the serum to a new sterile centrifuge tube to collect the immune serum. Dilute the target recombinant protein to a final concentration of 1 pg / mL using sterile CBS (carbonate buffer). Take a new 96-well enzyme plate and add 100 pL / well of 4°C overnight coating. Remove the antigen coating solution and wash 5 times using PBST (containing 0.05% Tween 20). Add 200 pL / well of 3% MPBS at 37°C for 2 hours. After removing the blocking buffer, wash the plate 5 times using PBST. Add 100 pL of gradient-diluted serum (100 pL / well) and incubate at room temperature for 1 hour. The control wells are PBS. Remove the liquid in the wells and wash 5 times using PBST. Add 100 pL of HRP anti-Llama IgG (H+L) antibody (1:50000 dilution) and incubate at room temperature for 1 hour. After removing the liquid in the wells, wash the plate 5 times using PBST. Add 100 pL / well of TMB developing solution. Incubate at room temperature for 10-15 minutes in the dark, add 50 pL / well of stop solution, and use an enzyme label to read the OD450 value in the wells.

[0099] The serum was isolated from the immunized llama and subjected to limited dilution according to the dilution gradient shown in FIG. 3, and ELISA detection was performed with the antigen pre-coated 96-well plate. According to the ELISA detection results, the immune serum can bind to the GPC3 recombinant protein, and the OD value changes in a gradient with the gradient dilution of the immune serum. The immune titer reaches more than 1:32K, and the titer stops growing after four immunizations, as shown in FIG. 3. The llama PBMC was subsequently isolated to extract RNA, reverse transcribed to prepare a cDNA library, and used for the construction of an antibody display library.

[0100] (2) PBMC isolation and VHH antibody fragment cloning

[0101] a. Collect 100 mL of peripheral blood anticoagulant sample and separate PBMC cells using lymphocyte separation medium.

[0102] b. Extract RNA and perform reverse transcription using PrimeScript TM II 1st Strand cDNA Synthesis Kit to prepare cDNA.

[0103] 1) Prepare the following reaction mixture Mix1 in 200 pL PCR:

[0104] Table 3

[0105] 2) After 5 min of incubation at 65°C, quickly cool on ice.

[0106] 3) Prepare the following reaction solution in the above PCR tube

[0107] Table 4

[0108] 4) After mixing, 80 μL / tube was aliquoted and placed in a PCR machine at 42°C for 1 hour and 70°C for 15 minutes. Finally, the cDNA sample was placed on ice or stored at -20°C for long-term preservation.

[0109] c. Amplification of VHH fragments

[0110] Table 5 Primers for two-round PCR reaction

[0111] Using the cDNA as a template, the first round of PCR was performed using specific primers, according to the following method:

[0112] 1) Preparation of the first round of PCR reaction system (50 μL / tube)

[0113] Table 6

[0114] After the PCR reaction system was prepared, the PCR machine was set according to the following program:

[0115] Table 7

[0116] 2) Agarose electrophoresis of PCR products

[0117] The PCR products were analyzed by electrophoresis using 1% agarose, and fragments with a molecular weight of about 750 bp were separated. The PCR products were recovered using a gel recovery kit, and the concentration was determined using a NanoDrop.

[0118] 3) Preparation of the second round of PCR reaction system (50 μL / tube)

[0119] Table 8

[0120] After the PCR reaction system was prepared, the PCR machine was set according to the following program:

[0121] Table 9

[0122] 4) Agarose electrophoresis analysis of the second round of PCR products

[0123] The PCR products were analyzed by electrophoresis using 1% agarose, and VHH fragments with a molecular weight of about 400 bp were separated. The VHH PCR products were recovered using a gel recovery kit, and the concentration was determined using a NanoDrop.

[0124] As shown in Figure 4, the peripheral blood of immunized alpaca was collected, total RNA was extracted, and after reverse transcription into cDNA, three rounds of PCR were performed using single-domain antibody amplification primers, and the results of agarose gel electrophoresis of the PCR products were obtained. The first round of PCR obtained PCR bands of about 1000 bp and 750 bp, respectively, and the 750 bp fragment was recovered as a template for the second round of PCR. The second round of PCR obtained a band of about 500 bp, which was a VHH fragment with added homologous arms for subsequent homologous recombination into the yeast display vector pDisplay.

[0125] (3) Construction of single-domain antibody yeast display library

[0126] a. Linearization of yeast display vector pDisplay, enzyme digestion system as follows:

[0127] Table 10

[0128] The pDisplay vector was digested with Sfil, 100 μL / tube, 50°C overnight. The pDisplay vector fragments were separated using a 1% agarose gel, and the 5000 bp vector fragments were cut and gel recovered, and the concentration was determined using NanoDrop. The recovered pDisplay digestion products were divided into 200 μL per 1.5 mL centrifuge tube, 1 / 10 volume (20 μL) of 3M sodium acetate was added, 1 μg / μL glycogen was added, mixed well by blowing and sucking, 880 μL of anhydrous ethanol was added, mixed well by inverting, and stored at -80°C.

[0129] b. Electroporation to construct yeast display library

[0130] 1) The -80°C frozen yeast competent strain was streaked onto YPD solid medium plates and incubated at 30°C for 3-5 days.

[0131] 2) Inoculate single colony yeast competent into 50 mL YPD medium, 250 rpm, 30°C, shake culture for 1-2 days.

[0132] 3) Prepare the yeast competent strain. After mixing the linearized vector fragments and PCR products, add to the electroporation cup, electroporate; the yeast competent strain after electroporation was transfected into a culture bottle, 220 rpm, 30°C, shake culture for 1 h. Take 20 μL of resuspension, dilute 5000 times with SDCAA, take 100 μL, spread on SDCAA plates, and incubate for 2-3 days. Calculate the library capacity, and continue to culture the remaining bacterial solution for 24 h.

[0133] 4) Preserve the bacteria: collect the remaining bacterial solution into a 50 mL centrifuge tube, centrifuge at 3000g for 5 min, discard the supernatant, resuspend with 10 mL of SDCAA, mix with 50% glycerol: resuspension = 1:1, and store at -80°C.

[0134] Finally, a library of 1.902*10 9 camel anti-GPC3 antibody immunized library was constructed for screening of specific anti-GPC3 nanobody.

[0135] Example 3 Panning of yeast antibody library

[0136] Using yeast display technology, the single domain antibody yeast display library obtained in Example 2 was subjected to one round of magnetic bead sorting and one round of flow sorting to obtain positive clones that bind to GPC3 recombinant protein.

[0137] (I) Yeast display library screening

[0138] a. Magnetic sorting. The yeast cultured in the SDCAA of Example 2 was taken and added to a 250 mL shake flask containing 50 mL of SGCAA medium, and cultured at 30°C, 240 rpm for 16 h. After centrifugation, the supernatant was discarded, and 1 mL of 0.5% PBSA was used for resuspension, which was added to a 1.5 mL centrifuge tube, centrifuged at 3000 g for 5 min, and the supernatant was discarded. It was washed again with 0.5% PBSA. The streptavidin magnetic beads incubated with the antigen were washed twice with 0.5% PBSA (4°C rotation incubation for 5 min each time), placed on a magnetic stand for 5 min, and the supernatant was discarded. The yeast bacterial solution was added to the magnetic beads combined with the antigen, and incubated at 4°C for 60 min, and placed on a magnetic stand for 15 min. The yeast bacterial solution was discarded and the magnetic beads were washed three times with 0.5% PBSA (4°C rotation incubation for 5 min each time). The magnetic beads were resuspended with 1 mL of SDCAA medium, and 0.5-5 μL of the resuspension was taken to 100 μL of SDCAA medium for plating. The resuspension was divided into two parts, one was added with 500 μL of 50% glycerol (-80°C storage); the other was added to a shake flask, supplemented with 2 mL of SDCAA medium, and cultured at 30°C, 240 rpm for 16 h. The bacterial solution in the shake flask was transferred to 50 mL of SDCAA medium (250 mL shake flask), and cultured at 30°C, 240 rpm overnight. The OD600 value of the bacterial solution was measured, and a portion of the bacterial solution was centrifuged according to the OD600 value, resuspended with SGCAA, and transferred to 50 mL of SGCAA medium to make the final OD600 value 1, and cultured at 30°C, 240 rpm overnight. The remaining bacterial solution was resuspended with SDCAA: 50% glycerol = 1:1, and stored at -80°C.

[0139] b. Flow sorting. The yeast cells after magnetic sorting were plated and cultured at the same time, and after induction of expression, incubated with Human GPC3-His-Biotin for 1 h, and the secondary antibody was APC Streptavidin. The first round of flow analysis was performed for detection, and positive clones were sorted out.

[0140] As shown in Figure 5, the yeast display library was first screened, wherein A: NC group: primary antibody: none, secondary antibody: APC-streptavidin; B: original library experimental group: primary antibody: GPC3-His-Biotin, anti V5 mouse IgG, secondary antibody: APC-streptavidin, PE-anti mouse IgG; C: 1MACS experimental group: primary antibody: GPC3-His-Biotin, secondary antibody: APC-streptavidin, FITC-anti V5; D: 1MACS+1FACS experimental group: primary antibody: GPC3-His-Biotin, secondary antibody: APC-streptavidin, FITC-anti V5.

[0141] According to the flow detection results, after one round of magnetic sorting and one round of flow sorting, the positive rate of GPC3-His-Biotin (Human) increased to 37.65%, and the positive clones were significantly enriched.

[0142] c. Positive epitope competition screening. After pre-incubation of the proteins in a ratio of Biotin-GPC3-His and hYP7-VH-hIgG1 + hYP7-VL-hIgG1 = 1:4, the sorted yeast cells were plated and cultured, and after induction of expression, incubated with Human GPC3-His-Biotin for 1 h, and the secondary antibody was APC Streptavidin, and flow analysis detection was performed.

[0143] The results of the first positive epitope competition screening of the yeast display library are shown in Figure 6. After pre-incubation of the proteins in a ratio of Biotin-GPC3-His and hYP7-VH-hIgG1 + hYP7-VL-hIgG1 = 1:4, the yeast cells 108522 were obtained after flow sorting. The sorted yeast cells were plated and cultured, and after induction of expression, incubated with Human GPC3-His-Biotin for 1 h, and the secondary antibody was APC Streptavidin, and flow analysis detection was performed.

[0144] d. Second screening. After one round of magnetic sorting and one round of flow sorting using Bio-GPC3-His protein, the positive rate of GPC3 was 27.340%, and the positive clones were sorted out, and the positive epitope competition screening was also performed.

[0145] The second screening result of the yeast display library is shown in Figure 7, wherein A: NC group: primary antibody: no addition, secondary antibody: APC-streptavidin; B: original library experimental group: primary antibody: GPC3-His-Biotin, anti-V5 mouse IgG, secondary antibody: APC-streptavidin, PE-anti-mouse IgG; C: 1MACS experimental group: primary antibody: GPC3-His-Biotin, secondary antibody: APC-streptavidin, FITC-anti-V5; D: 1MACS+1FACS experimental group: primary antibody: GPC3-His-Biotin, secondary antibody: APC-streptavidin, FITC-anti-V5. After one round of magnetic sorting and one round of flow sorting using Bio-GPC3-His protein, the positive rate of GPC3 was 27.340%.

[0146] The second positive epitope competition screening result of the yeast display library is shown in Figure 8, wherein after pre-incubation of the proteins in a ratio of Biotin-GPC3-His and hYP7-VH-hIgG1+hYP7-VL-hIgG1=1:4 and flow sorting, the positive rate of GPC3 was 13.58%.

[0147] (II) Screening of positive clones by yeast monoclonal flow detection

[0148] After sorting, the yeast bacterial solution was spread on SDCAA plates, and single clones were picked and cultured. After 48 hours of induction and expression, the single clones were incubated with Biotin-GPC3 antigen, and PE-Streptavidin was used as the secondary antibody. After incubation, flow detection was performed to determine the binding of the single-domain antibody displayed on the surface of the single clone yeast cells to the target antigen.

[0149] From the yeast display library enriched after the first sorting, single clone cells were randomly selected, amplified and induced, and then detected by antigens to determine the binding of the single-domain antibody displayed on the surface of the single clone yeast cells to the target antigen. The single clone detection result of the first screening of the yeast library is shown in Figure 9, wherein A is the result of plate 1, and B is the result of plate 2. The yeast single clone can be named as plate number+row+column. As shown in the figure, flow detection can distinguish positive clones from negative clones, and positive yeast single clones are screened for subsequent experiments.

[0150] From the second enrichment of the yeast display library, single clone cells were randomly selected, amplified and induced, and then detected by antigens to determine the binding of single-domain antibody displayed on the surface of the single clone yeast cells to the target antigen. The second screening of the yeast library was detected by single clone, and the results are shown in Figure 10. The figure is the result of plate 8, and the yeast single clone can be named as plate number + row + column. As can be seen from the figure, the flow detection can distinguish positive clones from negative clones, and the positive yeast single clone is screened for subsequent experiments.

[0151] The 11 potential positive clones screened by the application are respectively named GPC3-VHH-2B7 nanobody (abbreviated as 2B7), GPC3-VHH-2A10 nanobody (abbreviated as 2A10), GPC3-VHH-2G2 nanobody (abbreviated as 2G2), GPC3-VHH-1F7 nanobody (abbreviated as 1F7), GPC3-VHH-1D8 nanobody (abbreviated as 1D8), GPC3-VHH-2E10 nanobody (abbreviated as 2E10), GPC3-VHH-2B10 nanobody (abbreviated as 2B10), GPC3-VHH-2A5 nanobody (abbreviated as 2A5), GPC3-VHH-2C1 nanobody (abbreviated as 2C1), GPC3-VHH-2C5 nanobody (abbreviated as 2C5), GPC3-VHH-2E11 nanobody (abbreviated as 2E11) and GPC3-VHH-8D10 nanobody (abbreviated as 8D10).

[0152] The sequence of the screened clone verified by sequencing is as follows:

[0153] (1) The CDR sequence of the heavy chain variable region of the amino acid sequence of the 2B7 nanobody is: CDR1 with the amino acid sequence as shown in SEQ ID NO. 1, CDR2 with the amino acid sequence as shown in SEQ ID NO. 8, and CDR3 with the amino acid as shown in SEQ ID NO. 16; the framework region is FR1 with the amino acid sequence as shown in SEQ ID NO. 24, FR2 with the amino acid as shown in SEQ ID NO. 33, FR3 with the amino acid as shown in SEQ ID NO. 41, and FR4 with the amino acid as shown in SEQ ID NO. 49. The amino acid sequence of the 2B7 nanobody is as shown in SEQ ID NO. 56.

[0154] (2) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 2A10 nanobody are: CDR1 with an amino acid sequence as shown in SEQ ID NO. 1, CDR2 with an amino acid sequence as shown in SEQ ID NO. 8, and CDR3 with an amino acid as shown in SEQ ID NO. 16; the framework region is FR1 with an amino acid sequence as shown in SEQ ID NO. 25, FR2 with an amino acid as shown in SEQ ID NO. 33, FR3 with an amino acid as shown in SEQ ID NO. 41, and FR4 with an amino acid as shown in SEQ ID NO. 50. The amino acid sequence of the 2A10 nanobody is shown in SEQ ID NO. 57.

[0155] (3) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 2G2 nanobody are: CDR1 with an amino acid sequence as shown in SEQ ID NO. 2, CDR2 with an amino acid sequence as shown in SEQ ID NO. 9, and CDR3 with an amino acid as shown in SEQ ID NO. 17; the framework region is FR1 with an amino acid sequence as shown in SEQ ID NO. 26, FR2 with an amino acid as shown in SEQ ID NO. 34, FR3 with an amino acid as shown in SEQ ID NO. 42, and FR4 with an amino acid as shown in SEQ ID NO. 51. The amino acid sequence of the 2G2 nanobody is shown in SEQ ID NO. 58.

[0156] (4) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 1F7 nanobody are: CDR1 with an amino acid sequence as shown in SEQ ID NO. 2, CDR2 with an amino acid sequence as shown in SEQ ID NO. 9, and CDR3 with an amino acid as shown in SEQ ID NO. 17; the framework region is FR1 with an amino acid sequence as shown in SEQ ID NO. 27, FR2 with an amino acid as shown in SEQ ID NO. 34, FR3 with an amino acid as shown in SEQ ID NO. 42, and FR4 with an amino acid as shown in SEQ ID NO. 51. The amino acid sequence of the 1F7 nanobody is shown in SEQ ID NO. 59.

[0157] (5) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 1D8 nanobody are: CDR1 with an amino acid sequence as shown in SEQ ID NO. 3, CDR2 with an amino acid sequence as shown in SEQ ID NO. 10, and CDR3 with an amino acid as shown in SEQ ID NO. 18; and the framework regions are FR1 with an amino acid sequence as shown in SEQ ID NO. 28, FR2 with an amino acid as shown in SEQ ID NO. 35, FR3 with an amino acid as shown in SEQ ID NO. 43, and FR4 with an amino acid as shown in SEQ ID NO. 50. The amino acid sequence of the 1D8 nanobody is shown in SEQ ID NO. 60.

[0158] (6) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 2E10 nanobody are: CDR1 with an amino acid sequence as shown in SEQ ID NO. 4, CDR2 with an amino acid sequence as shown in SEQ ID NO. 11, and CDR3 with an amino acid as shown in SEQ ID NO. 19; and the framework regions are FR1 with an amino acid sequence as shown in SEQ ID NO. 29, FR2 with an amino acid as shown in SEQ ID NO. 36, FR3 with an amino acid as shown in SEQ ID NO. 44, and FR4 with an amino acid as shown in SEQ ID NO. 50. The amino acid sequence of the 2E10 nanobody is shown in SEQ ID NO. 61.

[0159] (7) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 2B10 nanobody are: CDR1 with an amino acid sequence as shown in SEQ ID NO. 4, CDR2 with an amino acid sequence as shown in SEQ ID NO. 11, and CDR3 with an amino acid as shown in SEQ ID NO. 19; and the framework regions are FR1 with an amino acid sequence as shown in SEQ ID NO. 30, FR2 with an amino acid as shown in SEQ ID NO. 36, FR3 with an amino acid as shown in SEQ ID NO. 44, and FR4 with an amino acid as shown in SEQ ID NO. 50. The amino acid sequence of the 2B10 nanobody is shown in SEQ ID NO. 62.

[0160] (8) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 2A5 nanobody are: CDR1 with the amino acid sequence as shown in SEQ ID NO. 2, CDR2 with the amino acid sequence as shown in SEQ ID NO. 12, and CDR3 with the amino acid sequence as shown in SEQ ID NO. 20; and the framework regions are FR1 with the amino acid sequence as shown in SEQ ID NO. 31, FR2 with the amino acid sequence as shown in SEQ ID NO. 37, FR3 with the amino acid sequence as shown in SEQ ID NO. 45, and FR4 with the amino acid sequence as shown in SEQ ID NO. 52. The amino acid sequence of the 2A5 nanobody is shown in SEQ ID NO. 63.

[0161] (9) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 2C1 nanobody are: CDR1 with the amino acid sequence as shown in SEQ ID NO. 5, CDR2 with the amino acid sequence as shown in SEQ ID NO. 13, and CDR3 with the amino acid sequence as shown in SEQ ID NO. 21; and the framework regions are FR1 with the amino acid sequence as shown in SEQ ID NO. 28, FR2 with the amino acid sequence as shown in SEQ ID NO. 38, FR3 with the amino acid sequence as shown in SEQ ID NO. 46, and FR4 with the amino acid sequence as shown in SEQ ID NO. 53. The amino acid sequence of the 2C1 nanobody is shown in SEQ ID NO. 64.

[0162] (10) The CDR sequences of the heavy chain variable region of the amino acid sequence of the 2C5 or 2E11 nanobody are: CDR1 with the amino acid sequence as shown in SEQ ID NO. 6, CDR2 with the amino acid sequence as shown in SEQ ID NO. 14, and CDR3 with the amino acid sequence as shown in SEQ ID NO. 22; and the framework regions are FR1 with the amino acid sequence as shown in SEQ ID NO. 32, FR2 with the amino acid sequence as shown in SEQ ID NO. 39, FR3 with the amino acid sequence as shown in SEQ ID NO. 47, and FR4 with the amino acid sequence as shown in SEQ ID NO. 54. The amino acid sequence of the 2C5 or 2E11 nanobody is shown in SEQ ID NO. 65.

[0163] The CDR sequences of the heavy chain variable region of the amino acid sequence of the 8D10 nanobody are as follows: CDR1 with an amino acid sequence as shown in SEQ ID NO. 7, CDR2 with an amino acid sequence as shown in SEQ ID NO. 15, and CDR3 with an amino acid sequence as shown in SEQ ID NO. 23; the framework region is FR1 with an amino acid sequence as shown in SEQ ID NO. 31, FR2 with an amino acid sequence as shown in SEQ ID NO. 40, FR3 with an amino acid sequence as shown in SEQ ID NO. 48, and FR4 with an amino acid sequence as shown in SEQ ID NO. 55. The amino acid sequence of the 8D10 nanobody is shown in SEQ ID NO. 66.

[0164] (III) Construction of antibody eukaryotic expression vector

[0165] The vector construction was entrusted to Aikangde Biotechnology Co., Ltd. to provide the construction of the vector. The positive yeast clone was subjected to PCR to obtain the antibody sequence, the PCR primers are shown in SEQ ID NO. 72 and SEQ ID NO. 73, the PCR reaction system and procedure are shown in Tables 11 and 12, and the antibody expression vector was constructed by connecting the Sfil enzyme-digested product with the eukaryotic expression vector pcDNA3.4-human IgG1 Fc. After the vector was verified by sequencing to be correct, a Qiagen plasmid maxi kit was used to prepare a endotoxin-free plasmid for standby use.

[0166] Table 11 PCR reaction system

[0167] After the PCR reaction system was prepared, the PCR instrument was set according to the following procedure:

[0168] Table 12

[0169] (IV) Preparation of antibody expression supernatant

[0170] The LVTransm transfection reagent and single-chain antibody expression vector were taken out from the refrigerator, thawed at room temperature, and then completely mixed by blowing up and down with a pipette gun. The PBS buffer was taken out and warmed to room temperature. 20 uL of PBS was taken into a 1.5 mL sterile EP tube, 3 ug of the antibody expression plasmid was added, and then mixed by blowing up and down with a pipette gun. Then, 12 uL of LVTransm was added, immediately mixed by blowing up and down with a pipette, and then placed at room temperature for 10 minutes. The above DNA / LVTransm complex was added to 3 mL of 293F cells, and then mixed by gently shaking. The cells were cultured at 37°C in a 5% CO2 incubator at 130 rpm. After continuous culture for 48 hours, the culture medium supernatant was collected by centrifugation, filtered with a 0.45 um filter membrane, and then transferred to a sterile centrifuge tube for standby use, which was the antibody expression supernatant. Thus, the nanobody eukaryotic expression vector and the nanobody expression supernatant were prepared.

[0171] Example 4 Flow cytometric detection of binding of recombinant antibodies to target proteins

[0172] (1) Construction of CHO-S cell lines overexpressing full-length GPC3 or truncated GPC3

[0173] a. Construction of overexpression vectors

[0174] According to the full-length sequence information of Human GPC3 (P51654-1) retrieved in the UniProt database, the gene was synthesized and subcloned into the lentiviral expression vector Lenti-CMV-puro after codon optimization, to construct the overexpression lentiviral vectors Lenti-CMV-GPC3 (full)-puro and Lenti-CMV-GPC3 (359-559)-puro. After sequence verification, the endotoxin-free plasmid was prepared for storage. The full-length GPC3 and truncated GPC3 gene sequences are shown below as SEQ ID NO. 74-77.

[0175] Full-length GPC3 amino acid sequence:

[0176] Full-length GPC3 nucleotide sequence:

[0177] Truncated GPC3 359-559 amino acid sequence:

[0178] Truncated GPC3 359-559 nucleotide sequence:

[0179] b. Packaging of overexpression lentivirus

[0180] Prepare a 15 cm dish and inoculate 1 x 10 7293T cells, add complete culture medium (DMEM high sugar, 10% FBS, double antibody), place in 37℃, 5% CO2 incubator, overnight culture. Take LVTransm transfection reagent and lentivirus packaging plasmid (Lenti-GOI, lentivirus packaging plasmid mix) from the refrigerator, thaw at room temperature, and mix thoroughly with a pipette gun. Take PBS buffer and warm it to room temperature. Take 2 mL of PBS into one well of a 6-well plate, add 20 μg of Lenti-GOI, 30 μL of lentivirus packaging plasmid mix, mix thoroughly with a pipette gun, then add 150 μL of LVTransm, immediately mix with a pipette, and stand at room temperature for 10 minutes. Add the above DNA / PEI complex dropwise to a 15 cm culture dish, gently shake the culture dish, and mix thoroughly. Place the culture dish in a 37℃, 5% CO2 incubator, and after 6-8 hours of culture, remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium. After continuous culture for 48 hours, collect the virus-containing culture supernatant, filter it with a 0.45 μm filter membrane, transfer it to a centrifuge tube, and after equilibration, centrifuge at 20000xg for 2 hours at 4℃. After centrifugation, carefully aspirate the liquid in the centrifuge tube in a biological safety cabinet, add 500 μL of PBS buffer to resuspend the precipitate, and store the virus at -80℃.

[0181] c. Stable cell line screening

[0182] Recover CHO-S cells from liquid nitrogen, and subculture for 3 times in succession to make the cells in the logarithmic growth phase. Take a new 6-well plate, and inoculate the target cells into the 6-well plate at a density of 5x10 5 Cells / mL. Inoculate 3 mL of cells per well of the 6-well plate. Add 200 μL of lentivirus to the inoculated 6-well plate, mix gently with a pipette, and then place the plate in a centrifuge and centrifuge at 800xg for 1 hour at room temperature. After centrifugation, take out the plate and continue to culture in a shaker for 24 hours. After overnight culture, replace the culture medium in the plate with fresh culture medium and continue to culture for 24 hours. Replace the culture medium with culture medium containing 10 μg / mL of Puromycin, and continuously culture for 5 days until all uninfected target cells are killed. Continue to expand the remaining live cells, and when the cells grow to 2x10 7 cells, collect the cells for cryopreservation.

[0183] (2) Flow cytometry detection of recombinant antibody binding to target protein

[0184] Recovery, culture of CHO-S cell strain, CHO-S cell strain overexpressing full-length GPC3 (CHO-S-GPC3), CHO-S cell strain overexpressing truncated GPC3 (CHO-S-GPC3(359-559)), 293F cell strain, Huh7-luc cell strain, and adjustment of cell state to logarithmic growth phase.

[0185] The five kinds of cells were respectively divided into several parts, and the number of cells in each part was 3 x 10 5 The expressed antibodies were respectively incubated with the target cells, and after sufficient mixing, the cells were incubated at room temperature for 1 hour. After centrifugation at 800 x g at room temperature for 5 minutes, the supernatant containing the antibodies was removed, and the cells were washed with PBS for 3 times. 100 μL of PE-labeled Anti-human IgG (1:500 dilution) was added, and after sufficient mixing, the cells were incubated at room temperature in the dark for 30 minutes; after centrifugation at 800 x g at room temperature for 5 minutes, the supernatant containing the secondary antibody was removed, and the cells were washed with PBS for 3 times; the cells were resuspended with 500 μL of PBS, and flow cytometry analysis was performed.

[0186] The results of flow cytometry detection of the binding of the recombinant antibodies to the target proteins are shown in Figures 11-15. According to the experimental data, the 168-2-G2, 168-2-A8, 168-2-A5, 168-2-C1, 168-2-B7, 168-2-C5, 168-1-D8, 168-2-E11-1, 168-2-B10, 168-2-G2, 168-2-A8, 168-2-A5, 168-2-C1, 168-2-B7, 168-2-C5, 168-1-D8, 168-2-E11-1, 168-2-B10 candidate clones were detected to bind to Huh7-Luc cells. Except for the 168-2-A8, 168-2-A5, 168-2-C1, and 168-1-H5 candidate clones, the other clones all had strong binding to Huh7-Luc cells. The 168-8-D10 and 168-8-F02 candidate clones cross-bound to CHO-S-GPC3, CHO-S-GPC3(359-559), and Huh7-luc cells, and sequencing found that the sequences of the two clones were completely identical.

[0187] Example 5 ELISA detection of the binding of recombinant antibodies to target proteins

[0188] Dilute the recombinant protein to a final concentration of 2 μg / mL using sterile PBS, take a new 96-well enzyme plate, add 100 μL per well, and coat overnight at 4°C. Remove the antigen coating solution and wash 5 times with PBST (containing 0.05% Tween 20). Add 200 μL / well of 3% MPBS and incubate at 37°C for 2 hours; after removing the blocking buffer, wash the plate 5 times with PBST; add the expressed recombinant antibody, 100 μL / well of transfection supernatant, and incubate at room temperature for 1 hour; the control wells are PBS; remove the liquid in the wells and wash 5 times with PBST; add 100 μL / well of HRP-Protein A antibody (1:50000 dilution), and incubate at room temperature for 1 hour; after removing the liquid in the wells, wash the plate 5 times with PBST; add 100 μL / well of TMB color developing solution; incubate at room temperature in the dark for 10-15 minutes; add 50 μL / well of stop solution; and read the OD450 value in the wells using an enzyme-labeled instrument.

[0189] The results of ELISA detection of the binding of the recombinant antibody to the target protein are shown in Figure 16. According to the ELISA results, the 12 candidate clones 168-2-G2, 168-2-A5, 168-2-C1, 168-2-B7, 168-2-C5, 168-1-D8, 168-2-E11-1, 168-2-E10, 168-1-F7, 168-2-A10-3, 168-8-D10, and 168-8-F02 bind to the GPC3 (511-560) antigen.

[0190] Example 6 Flow cytometry detection of the binding efficiency of the nanobody to target cells Huh7, HepG2, and non-target cells SK-HEP-1

[0191] Huh7 and HepG2 cells, which highly express GPC3, were selected as target cells, and SK-HEP-1 cells, which do not express GPC3, were selected as non-target cells, to detect the binding efficiency and binding specificity of the nanobody to the GPC3 antigen.

[0192] 2 x 105Huh7 cells, HepG2 cells, or SK-HEP-1 cells were collected and resuspended in 100 μL of PBS (2% FBS) containing 10 μg / mL of the nanobody supernatant, and incubated for 60 min. After washing the cells with PBS, Goat Anti-Human (H+L) (Alexa Flour 647 Conjugate) was added to the cell culture, incubated for 30 min, and the cells were washed and subjected to flow cytometry analysis. 5 Huh7 cells, HepG2 cells, or SK-HEP-1 cells were resuspended in 100 μL of PBS (2% FBS), and the nanobody supernatant was added, and incubated for binding for 60 min. After washing the cells with PBS, Goat Anti-Human (H+L) (Alexa Flour 647 Conjugate) was added to the cell culture, incubated for 30 min, and the cells were washed and subjected to flow cytometry analysis.

[0193] The flow cytometry detection of the binding efficiency of the nanobody to target cells Huh7, HepG2, and non-target cells SK-HEP-1 is shown in Figure 17. 2 x 105Huh7 cells, HepG2 cells, or SK-HEP-1 cells were collected and resuspended in 100 μL of PBS (2% FBS) containing 10 μg / mL of the nanobody supernatant, and incubated for 60 min. After washing the cells with PBS, Goat Anti-Human (H+L) (Alexa Flour 647 Conjugate) was added to the cell culture, incubated for 30 min, and the cells were washed and subjected to flow cytometry analysis. 5Huh7 cells, HepG2 cells or SK-HEP-1 cells were resuspended in 100 μL PBS (2% FBS) and added with the supernatant of the nanobodies, and incubated for 60 min. After washing the cells with PBS, Goat Anti-Human (H+L) (Alexa Flour 647 Conjugate) was added to the cell culture, and incubated for 30 min. The cells were washed and subjected to flow cytometry analysis. The nanobodies could effectively bind to GPC3-positive cells Huh7 and HepG2, but not to GPC3-negative cells SK-HEP-1, indicating that the nanobodies provided by the application have good effectiveness and specificity.

[0194] Example 7 Construction of mGPC3 overexpression cell line 293T-mGPC3 and detection of binding with nanobodies

[0195] A pHIV-mGPC3-P2A-GFP lentiviral vector was constructed, and the amino acid sequence of the mGPC3 is shown as SEQ ID NO. 78.

[0196] The lentivirus overexpressing mGPC3-GFP was packaged, and 293T cells were transfected at an MOI of 10. The GFP expression level, i.e., the transduction efficiency, was detected by flow cytometry.

[0197] As shown in FIG. 18(A), the transduction efficiency was detected by flow cytometry after lentivirus transduction of 293T cells. The transduction efficiency of 293T-mGPC3 was 66%.

[0198] 2 x 10 5 Hepa1-6-mGPC3 cells were resuspended in 100 μL PBS (2% FBS) and added with the supernatant of the nanobodies, and incubated for 60 min. After washing the cells with PBS, Goat Anti-Human (H+L) (Alexa Flour 647 Conjugate) was added to the cell culture, and incubated for 30 min. The cells were washed and subjected to flow cytometry analysis. As shown in FIG. 18(B), it can be seen that the nanobody 2E10 can effectively bind to mGPC3-positive cells.

[0199] Example 8 Preparation of a lentiviral vector expressing a chimeric antigen receptor targeting GPC3

[0200] The vector map expressing a chimeric antigen receptor targeting GPC3 is shown in FIG. 19. The CAR comprises a GPC3 nanobody, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory molecule and a CD3ζ intracellular activation signal domain.

[0201] 1 μg of the nanobodies and the chimeric antigen receptor plasmid were digested with Sfi I enzyme, and the reaction system was as shown in Table 13.

[0202] Table 13

[0203] Add sample on ice, mix, and immediately separate. Perform reaction according to Table 14.

[0204] Table 14

[0205] After the reaction, the enzyme-digested product was subjected to 1% agarose gel electrophoresis. Nanobodies were recovered from a fragment of about 400 bp, and the chimeric antigen receptor skeleton was recovered from a fragment of about 8000 bp. The amount of each was quantified by UV absorption.

[0206] The nanobody small fragment (insert DNA) and the chimeric antigen receptor skeleton large fragment (vector DNA) were connected using T4 DNA ligase. The reaction system is shown in Table 15.

[0207] Table 15

[0208] The reaction was carried out at room temperature for 2 hours. The transformed E. coli Tstbl3 competent cells were selected from solid medium, and overnight culture was performed. Positive clones were selected for sequencing identification. The sequencing results were as expected. Thus, the anti-GPC3 chimeric antigen receptor lentiviral expression vector was constructed.

[0209] Example 9 Lentivirus packaging and titer detection

[0210] 293T cells were cultured in a 15 cm dish. When the cells were almost fully grown, lentivirus packaging was performed.

[0211] Twenty-two μg of the lentiviral expression vector constructed in Example 6, 15 μg of the envelope plasmid VGV-G, and 15 μg of the helper plasmid Δ8.9 were mixed in 2500 μl of Opti-MEM reduced serum medium. One hundred thirty-five μl of PEI was mixed in 2500 μl of Opti-MEM reduced serum medium. The PEI mixture was added to the DNA mixture, mixed gently, and incubated at room temperature for 25 minutes. The transfection complex was dropped into 293T 15 cm dishes, and the dishes were incubated at 37°C / 5% CO2 for 24 hours. After 24 hours, the medium was replaced with fresh DMEM+10% FBS, and the incubation was continued. The virus supernatant was collected at 48 hours and 72 hours, and the lentivirus was concentrated by ultracentrifugation.

[0212] Five hundred μl of Jurkat cells (1×10 6) seeded in 24-well plates, added 1 μl of concentrated lentivirus, and added polybrene to a final concentration of 10 ng / μl, centrifuged at 800 x g for 60 min at 32°C, and incubated at 37°C / 5% CO2for 24 h. The medium was replaced with fresh RPMI 1640 + 10% FBS 24 h later. Flow cytometry was performed 48 h later to determine the percentage of GFP-positive Jurkat cells, which was the virus transduction efficiency. The titer was calculated according to the formula: titer = number of transfected cells (1 x 10 6 ) x positive rate / virus volume (mL).

[0213] Example 10 Lentivirus transduction of T lymphocytes

[0214] CD3+ T lymphocytes were isolated from the peripheral blood of healthy people, diluted to 1 x 10 6 cells / mL with T cell medium (TexMacs + 50 IU / mL human IL-2), and 100 x TranAct was added to stimulate the primary T cells. After 48 h, the T cells were centrifuged, counted, and the concentrated lentivirus solution prepared in Example 7 was added at an MOI of 20, with vectofusin added to a final concentration of 10 ng / μl for transfection promotion. The cells were centrifuged at 800 x g for 60 min at 32°C, and incubated at 37°C / 5% CO2for 24 h. The medium was replaced with fresh TexMacs + 50 IU / mL human IL-2 24 h later.

[0215] Example 11 Flow cytometry detection of CAR-T cell transduction efficiency

[0216] T cells were transduced, and 72 h later, 1 x 10 5 The cells were resuspended in 100 μl of PBS (2% FBS), biotin-GPC3 antigen was added, and the cells were incubated for 30 min. After the cells were washed with PBS, APC-Streptavidin was added to the cell culture, incubated for 30 min, the cells were washed, DAPI staining solution was added, and flow cytometry analysis was performed.

[0217] As shown in Figure 20, the CAR-T cell transduction efficiency was detected by flow cytometry. The untransduced T cells were used as a control, and the voltage and gating strategy were adjusted. GFP positivity confirmed that the vector was introduced into the T cells, and APC positivity confirmed that the cells expressed CAR, so the cell population that was positive for both GFP and APC was the CAR-T cells that were successfully transduced with the lentivirus vector.

[0218] Example 12 Detection of the anti-tumor effect of CAR-T cells in vitro

[0219] a. Target cell seeding: Huh7-luc (GPC3+) and SK-HEP-1-luc (GPC3-) were seeded at a concentration of 5 x 10 4 / mL, 100 μL was inoculated into a 96-well plate.

[0220] b. Effector cell inoculation: GPC3-CAR-T and untransduced T cells were used as effector cells, and CAR-T cells and untransduced T cells were added to a 96-well plate at an effector-target ratio of 1:1 and 10:1.

[0221] c. After co-culturing the effector cells and target cells for 24 hours, the cells were lysed, luciferase substrate was added, and the luminescence signal was detected by a microplate reader. The number of remaining target cells was used to characterize the killing effect of CAR-T cells. Each group had three replicates, and the average value of the three replicates was taken. 2G2, 2E10, 2C5 and 2A5 were screened as nanobodies with both effectiveness and specificity.

[0222] The results of the in vitro anti-tumor effect of CAR-T cells are shown in Figure 21. The GPC3 nanobody prepared CAR-T cells were verified for their effectiveness and specificity in killing tumor cells in vitro in three batches. Different CAR-T cells or untransduced T cells (UTD) were co-cultured with GPC3 + After co-culturing Huh7-luc cells or GPC3-SK-HEP-1-luc cells at an effector-target ratio of 1:1 or 10:1 for 24 hours, the cells were lysed, luciferase substrate was added, and the luminescence signal was detected by a microplate reader. 2G2, 2E11-1 and 2E10 were screened as three GPC3 nanobodies with both effectiveness and specificity.

[0223] In summary, the present application screened 11 specific anti-GPC3 nanobodies from immunized alpacas combined with a yeast surface display system, of which three had both effectiveness and specificity. CAR-T cells constructed based on these nanobodies can specifically kill target cells expressing GPC3.

[0224] The above examples are only illustrative of the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical idea disclosed by the present application should be covered by the claims of the present application.

Claims

1. A Nanobody targeting Glypican-3, characterized in that, the heavy chain variable region CDR sequences selected from at least one of the following or amino acid sequences at least 80% identical thereto: SEQ ID NO. 1-23.

2. The Nanobody according to claim 1, characterized in that, the nanobody comprises: a heavy chain variable region CDR1 sequence as set forth in or an amino acid sequence at least 80% identical to SEQ ID NO. 1-7; a heavy chain variable region CDR2 sequence as set forth in or an amino acid sequence at least 80% identical to SEQ ID NO. 8-15; a heavy chain variable region CDR3 sequence as set forth in or an amino acid sequence at least 80% identical to SEQ ID NO. 16-23.

3. The Nanobody according to claim 1, characterized in that, the nanobody comprises a framework region FR sequence selected from at least one of the following or an amino acid sequence at least 80% identical thereto: SEQ ID NO. 24-55.

4. The Nanobody of claim 1, wherein the nanobody comprises: a framework region FR1 sequence as set forth in or an amino acid sequence at least 80% identical to SEQ ID NO. 24-32; a framework region FR2 sequence as set forth in or an amino acid sequence at least 80% identical to SEQ ID NO. 33-40; a framework region FR3 sequence as set forth in or an amino acid sequence at least 80% identical to SEQ ID NO. 41-48; a framework region FR4 sequence as set forth in or an amino acid sequence at least 80% identical to SEQ ID NO. 49-55.

5. A Nanobody targeting Glypican-3, characterized in that, the amino acid sequence of the nanobody is selected from at least one of the following sequences or an amino acid sequence at least 80% identical thereto: SEQ ID NO. 56-66.

6. A polypeptide, characterized in that, the nanobody of any one of claims 1-5.

7. A nucleic acid molecule encoding the nanobody of any one of claims 1-5.

8. An expression vector, characterized by, the nucleic acid molecule of claim 7.

9. A host cell transformed or transfected with the expression vector of claim 8.

10. A pharmaceutical composition, characterized by, which contains the nanobody as set forth in any one of claims 1-5 and a pharmaceutically acceptable carrier and / or excipient.

11. Use of the Nanobody according to any one of claims 1 to 5, the polypeptide according to claim 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the host cell according to claim 9, the pharmaceutical composition according to claim 10 for the manufacture of a diagnostic or therapeutic medicament or kit for the diagnosis or treatment of a tumor, characterized in that, the tumor expresses Glypican-3. the tumor expresses Glypican-3.

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