Targeting Claudin18.2 nano antibody and application thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a Claudin18.2 targeting nano antibody and application thereof. According to the present invention, the number of the provided nanometer antibodies capable of specifically binding Claudin18.2 is nine, the molecular weights of the nanometer antibodies are 12.9-14.9 kDa, the nanometer antibodies are respectively 2D11, 3A2, 3A5, 3C6, 3E6, 3H3, 4D4, 4F4 and 4G1, the amino acid sequences of the nanometer antibodies are respectively represented by SEQ ID NO.50-58, and the nucleotide sequences for coding the amino acid sequences are respectively represented by SEQ ID NO.59-67. The nano antibody provided by the invention is stable in structure and good in heat resistance, and can be used for preparing medicines for diagnosing and treating gastric cancer, pancreatic cancer, esophageal cancer, biliary tract cancer, colorectal cancer and lung cancer, and related products of the nano antibody can be used for preparing diagnostic reagents for immunodetection, flow detection and cellular immunofluorescence detection and medicines for treating diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a nanobody targeting Claudin18.2 and application thereof. BACKGROUND

[0002] The treatment field of advanced-stage gastric cancer (AGC) has been facing severe challenges, and the prognosis of patients is still not optimistic. The combination of HER2 targeted therapy, VEGF targeted drugs and PD-1 monoclonal antibody has been approved as a first-line treatment regimen, but it is mainly suitable for HER2 positive, PD-L1 positive or microsatellite instability cancer patients, and these patient groups are relatively limited. The detection rate of HER2 in gastric cancer or gastroesophageal junction cancer is only 15-25%, and the heterogeneity expression of receptor tyrosine kinase also limits the application range of molecular targeted therapy. In addition, the PD-1 antibody nivolumab combined with chemotherapy has been approved as a first-line treatment regimen for AGC patients in some countries, but in the guidelines of some countries and regions, the actual beneficiaries are mainly tumor patients with microsatellite instability (usually less than 5% of patients) and / or PD-L1 positive (combined positive score CPS>5). In addition, pancreatic cancer, a digestive system tumor, is known as the king of cancer, with a high degree of malignancy, and is the 12th most common malignant tumor and the 7th leading cause of cancer death worldwide. The 5-year survival rate under the current treatment regimen of surgery, chemotherapy and immunotherapy is only 10%.

[0003] Claudin18.2 is a highly selective marker protein, which is mainly expressed in differentiated gastric mucosa epithelial cells in normal tissues, but the expression of Claudin18.2 also increases accordingly in the case of tissue carcinogenesis. Especially in digestive tract tumors such as gastric cancer, pancreatic cancer, esophageal cancer, cholangiocarcinoma and gallbladder cancer, the expression level of Claudin18.2 is often high. Normal pancreatic tissue has no Claudin18.2 expression, but Claudin18.2 expression is abnormally activated when pancreatic malignant transformation occurs. The expression of Claudin18.2 in normal tissues is strictly limited to the tight junction of gastric mucosa, so it is masked in supramolecular complexes and most of the antibodies injected intravenously cannot be contacted. However, after cell malignant transformation, the change of cell polarity exposes the epitope of Claudin18.2 to the cell surface so that it can be targeted by antibodies. Claudin18.2 is expressed in a variety of human tumors and is a major subtype of gastric and pancreatic cancer. 50% to 70% of gastric cancer and pancreatic cancer show significant expression of Claudin18.2. Claudin18.2 expression has also been observed in lymph node metastases and liver metastases of gastric cancer and pancreatic adenocarcinoma. Therefore, Claudin18.2 is a potential therapeutic target for gastric cancer and pancreatic cancer.

[0004] In addition to the Claudin18.2 targeting monoclonal antibody, other new treatment methods include bispecific antibodies, antibody-drug conjugates (ADCs) and chimeric antigen receptor T cells (CAR-T), etc. These treatment methods have shown potential efficacy in Claudin18.2 expressing gastric cancer patients in early clinical trials. For example, in CAR-T cell therapy, CT041 showed an objective response rate of 33.3% and a median progression-free survival of 130 days in a phase I clinical trial.

[0005] Therefore, screening of nanobodies that can specifically bind to Claudin18.2 and preparing CAR-T cells can specifically recognize and kill Claudin18.2 expressing gastric cancer / pancreatic cancer cells, achieving strong anti-tumor effect, and having great clinical transformation value for Claudin18.2 targeted immunotherapy methods. SUMMARY

[0006] To solve the above problems, the present application provides a nanobody that specifically targets Claudin18.2. The molecular weight of these antibodies is 12.9-14.9 kDa, has the advantages of structural stability, good heat resistance, tolerance to strong denaturants or high temperature conditions, and high affinity, and can be used in the preparation process of various tumor-related drugs.

[0007] To achieve the above effects, the technical scheme adopted by the present application is as follows:

[0008] A nanobody targeting Claudin18.2, the nanobody comprising a CDR1 region, a CDR2 region and a CDR3 region, which are separated by FR regions, the sequence of the CDR region and the FR region comprising the following sequence, or a sequence with a homology of ≥80% to the following sequence:

[0009] The nanobody is 2D11, the amino acid sequence of the CDR1 region is as shown in SEQ ID NO. 1, the amino acid sequence of the CDR2 region is as shown in SEQ ID NO. 9, the amino acid sequence of the CDR3 region is as shown in SEQ ID NO. 16, the amino acid sequence of the FR1 region is as shown in SEQ ID NO. 23, the amino acid sequence of the FR2 region is as shown in SEQ ID NO. 30, the amino acid sequence of the FR3 region is as shown in SEQ ID NO. 38, and the amino acid sequence of the FR4 region is as shown in SEQ ID NO. 45; or

[0010] the amino acid sequence of the CDR1 region is shown as SEQ ID NO. 2, the amino acid sequence of the CDR2 region is shown as SEQ ID NO. 10; the amino acid sequence of the CDR3 region is shown as SEQ ID NO. 17, the amino acid sequence of the FR1 region is shown as SEQ ID NO. 24, the amino acid sequence of the FR2 region is shown as SEQ ID NO. 31, the amino acid sequence of the FR3 region is shown as SEQ ID NO. 39, and the amino acid sequence of the FR4 region is shown as SEQ ID NO. 46; or;

[0011] the amino acid sequence of the CDR1 region is shown as SEQ ID NO. 3, the amino acid sequence of the CDR2 region is shown as SEQ ID NO. 11; the amino acid sequence of the CDR3 region is shown as SEQ ID NO. 18, the amino acid sequence of the FR1 region is shown as SEQ ID NO. 25, the amino acid sequence of the FR2 region is shown as SEQ ID NO. 32, the amino acid sequence of the FR3 region is shown as SEQ ID NO. 40, and the amino acid sequence of the FR4 region is shown as SEQ ID NO. 45; or;

[0012] the amino acid sequence of the CDR1 region is shown as SEQ ID NO. 2, the amino acid sequence of the CDR2 region is shown as SEQ ID NO. 10; the amino acid sequence of the CDR3 region is shown as SEQ ID NO. 17, the amino acid sequence of the FR1 region is shown as SEQ ID NO. 26, the amino acid sequence of the FR2 region is shown as SEQ ID NO. 31, the amino acid sequence of the FR3 region is shown as SEQ ID NO. 39, and the amino acid sequence of the FR4 region is shown as SEQ ID NO. 45; or;

[0013] the amino acid sequence of the CDR1 region is shown as SEQ ID NO. 4, the amino acid sequence of the CDR2 region is shown as SEQ ID NO. 12; the amino acid sequence of the CDR3 region is shown as SEQ ID NO. 19, the amino acid sequence of the FR1 region is shown as SEQ ID NO. 27, the amino acid sequence of the FR2 region is shown as SEQ ID NO. 33, the amino acid sequence of the FR3 region is shown as SEQ ID NO. 41, and the amino acid sequence of the FR4 region is shown as SEQ ID NO. 47; or;

[0014] the amino acid sequence of the CDR1 region is shown as SEQ ID NO. 5, the amino acid sequence of the CDR2 region is shown as SEQ ID NO. 13; the amino acid sequence of the CDR3 region is shown as SEQ ID NO. 20, the amino acid sequence of the FR1 region is shown as SEQ ID NO. 24, the amino acid sequence of the FR2 region is shown as SEQ ID NO. 34, the amino acid sequence of the FR3 region is shown as SEQ ID NO. 42, and the amino acid sequence of the FR4 region is shown as SEQ ID NO. 45; or;

[0015] the amino acid sequence of the CDR1 region is shown as SEQ ID NO. 6, the amino acid sequence of the CDR2 region is shown as SEQ ID NO. 14; the amino acid sequence of the CDR3 region is shown as SEQ ID NO. 21, the amino acid sequence of the FR1 region is shown as SEQ ID NO. 28, the amino acid sequence of the FR2 region is shown as SEQ ID NO. 35, the amino acid sequence of the FR3 region is shown as SEQ ID NO. 43, and the amino acid sequence of the FR4 region is shown as SEQ ID NO. 48; or;

[0016] the amino acid sequence of the CDR1 region is shown as SEQ ID NO. 7, the amino acid sequence of the CDR2 region is shown as SEQ ID NO. 9; the amino acid sequence of the CDR3 region is shown as SEQ ID NO. 16, the amino acid sequence of the FR1 region is shown as SEQ ID NO. 29, the amino acid sequence of the FR2 region is shown as SEQ ID NO. 36, the amino acid sequence of the FR3 region is shown as SEQ ID NO. 38, and the amino acid sequence of the FR4 region is shown as SEQ ID NO. 45; or;

[0017] the amino acid sequence of the CDR1 region is shown as SEQ ID NO. 8, the amino acid sequence of the CDR2 region is shown as SEQ ID NO. 15; the amino acid sequence of the CDR3 region is shown as SEQ ID NO. 22, the amino acid sequence of the FR1 region is shown as SEQ ID NO. 24, the amino acid sequence of the FR2 region is shown as SEQ ID NO. 37, the amino acid sequence of the FR3 region is shown as SEQ ID NO. 44, and the amino acid sequence of the FR4 region is shown as SEQ ID NO. 49.

[0018] The code of these antibodies are 2D11 (SEQ ID NO. 50), 3A2 (SEQ ID NO. 51), 3A5 (SEQ ID NO. 52), 3C6 (SEQ ID NO. 53), 3E6 (SEQ ID NO. 54), 3H3 (SEQ ID NO. 55), 4D4 (SEQ ID NO. 56), 4F4 (SEQ ID NO. 57), 4G1 (SEQ ID NO. 58).

[0019] Preferably, the amino acid sequence of the nanobody is selected from one of SEQ ID NO. 50-SEQ ID NO. 58.

[0020] The nanobody obtained by the present application is a heavy chain variable region, comprising an antigenic determinant complementarity region (Complementarity-determining region, CDR) and a framework region (Framework frgion, FR), wherein the framework region is selected from the group consisting of FR1, FR2, FR3 and FR4, and an amino acid sequence having not less than 80%, preferably not less than 90%, more preferably not less than 95%, further preferably not less than 99% homology thereto, and the antigenic determinant complementarity region is selected from the group consisting of CDR1, CDR2 and CDR3, and an amino acid sequence having not less than 80%, preferably not less than 90%, more preferably not less than 95%, further preferably not less than 99% homology thereto.

[0021] The present application also provides a nucleic acid molecule comprising a nucleotide sequence encoding the nanobody, wherein the nucleotide sequence is selected from one of SEQ ID NO. 59-SEQ ID NO. 67.

[0022] The present application also provides a vector comprising the nucleic acid molecule.

[0023] The present application also provides a host cell comprising the nucleic acid molecule, or comprising the vector.

[0024] Preferably, the host cell is a chimeric antigen receptor immune cell; the chimeric antigen receptor immune cell is selected from one of a chimeric antigen receptor T cell, a chimeric antigen receptor NK cell, a chimeric antigen receptor macrophage, a chimeric antigen receptor NKT cell, a chimeric antigen receptor γδT cell, a chimeric antigen receptor B cell, a chimeric antigen receptor dendritic cell, and a chimeric antigen receptor red blood cell.

[0025] Preferably, the chimeric antigen receptor comprises a transmembrane domain, an intracellular domain and an extracellular domain, the extracellular domain comprises a signal peptide, the antibody targeting Claudin18.2 and a CD8a hinge region; the transmembrane domain is a CD8a transmembrane domain; the intracellular domain is one of 4-1BB and CD3 zeta signaling domain, CD28 costimulatory domain + CD3 zeta signaling domain.

[0026] The application also provides application of the nanobody, the nucleic acid molecule, the vector or the host cell in preparation of a drug for treating and / or preventing a tumor.

[0027] Preferably, the tumor is one of gastric cancer, pancreatic cancer, esophageal cancer, biliary tract cancer, colorectal cancer, lung cancer.

[0028] The application also provides a kit comprising the nanobody, the nucleic acid molecule, the vector or the host cell.

[0029] The application provides application of the Claudin18.2 antibody in preparation or screening of a therapeutic drug, or in preparation of a diagnostic drug. The therapeutic drug can be a drug that takes Claudin18.2 antigen as a target, binds to or acts on Claudin18.2 antigen, thereby treating and / or preventing indications. The therapeutic drug can be a tumor treatment drug. The tumor is a tumor expressing Claudin18.2. The tumor treatment drug can be a drug that takes Claudin18.2 antigen on the functional surface of the tumor cell surface as a target, binds to or acts on Claudin18.2 antigen, thereby treating and / or preventing tumors. The tumor can be a tumor positive for Claudin18.2, such as gastric cancer, pancreatic cancer, esophageal cancer, biliary tract cancer, colorectal cancer, lung cancer, etc.

[0030] Compared with the prior art, the application has the following advantages:

[0031] (1) The nanobody provided by the application has a small molecular weight of 12.9-14.9 kDa, stable structure, good heat resistance, tolerance to strong denaturants or high temperature conditions, and high affinity, which is convenient for storage and transportation;

[0032] (2) The nanobody provided by the application has weak immunogenicity in the human body and strong tumor tissue penetration, has an anti-gastric cancer and pancreatic cancer cell proliferation effect, is easy to express and genetically engineered, and is suitable for use as a diagnostic reagent or therapeutic antibody;

[0033] (3) The Claudin 18.2 protein expressed by eukaryotic cells (293F cells) is used for immunization, the protein expressed by eukaryotic cells is closer to the natural conformation of the protein than the protein expressed by prokaryotic cells, more epitopes are reserved, the alpaca is stimulated to produce high-titer antibodies, and the diversity of the gene library is ensured;

[0034] (4) The nanobody and the related products thereof can be used for preparing drugs for diagnosing and treating gastric cancer, pancreatic cancer, esophageal cancer, biliary tract cancer, colorectal cancer and lung cancer, the gene encoding the nanobody or the plasmid containing the gene or the recombinant plasmid containing the gene or the recombinant cell containing the gene can be used for preparing diagnostic reagents for immunodetection, flow detection and cell immunofluorescence detection and drugs for treating diseases. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is an amplification result graph of a VHH fragment;

[0036] Figure 2 is a sequencing quality control detection result of a phage display library;

[0037] Figure 3 is a phage FACS monoclonal detection result graph of cell panning of a phage display library;

[0038] Figure 4 is a binding result graph of a recombinant antibody and a target protein by flow detection;

[0039] Figure 5 is a virus transduction efficiency result graph;

[0040] Figure 6 is a flow cytometry detection CAR-T cell transduction efficiency result graph;

[0041] Figure 7 is a CAR-T cell in vitro killing AGS gastric cancer cell effect detection result graph;

[0042] Figure 8 is a CAR-T cell in vitro killing MKN45 gastric cancer cell effect detection result graph. DETAILED DESCRIPTION

[0043] The application will be further explained below in conjunction with specific examples, but it should be noted that the following examples are only used to explain the application and cannot be used to limit the application, and all technical solutions identical or similar to the application are within the protection scope of the application. If the specific technology or condition is not specified in the examples, the operation is performed according to the conventional technical method and the content of the instrument instruction manual; if the reagent or instrument is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.

[0044] Example 1: Preparation of Nanobodies

[0045] The nanobody targeting Claudin18.2 of this invention is prepared as follows:

[0046] (1) Alpaca immunization and detection of immune titer:

[0047] a. Alpaca were immunized five times with human Claudin 18.2 Protein-VLP (KACTUS, Cat. No. CLD-HM0P37) at 14-day intervals.

[0048] b. Seven days after the last immunization, collect 5 mL of peripheral blood and place the centrifuge tube containing the blood sample in a 37°C incubator for 1 hour; then transfer the blood sample to 4°C overnight.

[0049] c. Place the centrifuge tube containing the blood sample in a centrifuge and centrifuge at 5000 rpm for 20 min; separate the supernatant serum and transfer it to a new sterile centrifuge tube to collect the immune serum. Dilute human Claudin 18.2 Protein-VLP (KACTUS, Cat. No. CLD-HM0P37) with sterile PBS to a final concentration of 5 μg / mL. Take a new 96-well microplate and add 100 μL of Claudin 18.2 antigen protein (i.e., human Claudin 18.2 Protein-VLP) to each 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 PBS containing 2% BSA and block at 37°C for 2 hours. After removing the blocking buffer, wash the plate 5 times with PBST. Add 100 μL of serially diluted serum (100 μL / well) and incubate at room temperature for 1 hour. Use PBS for the control wells. Remove the liquid from the wells and wash 5 times with PBST. Add 100 μL of HRP anti-Llama IgG (H+L) antibody (1:50000 dilution) and incubate at room temperature for 1 hour. Remove the liquid from the wells and wash the plate 5 times with PBST. Add 100 μL / well of TMB chromogenic buffer. Incubate at room temperature in the dark for 10–15 minutes, then add 50 μL / well of stop solution. Read the OD450 values ​​in the wells using a microplate reader. The results are shown in Table 1.

[0050] Table 1. Results of Alpaca Immune Titer Detection

[0051]

[0052] After isolating serum from immunized alpacas, it was subjected to limiting dilutions according to the dilution gradient in Table 1 above, and ELISA was performed on 96-well plates pre-coated with human Claudin 18.2 Protein-VLP (KACTUS, Cat. No. CLD-HM0P37). According to the ELISA results, the immune serum can bind to the recombinant Claudin 18.2 protein, and the OD value changes gradient with the gradient dilution of the immune serum, reaching an immune titer of over 1:32K. The titer stopped increasing after four immunizations.

[0053] (2) PBMC isolation and VHH antibody fragment cloning

[0054] a. Collect 100 mL of anticoagulated peripheral blood sample from alpacas after the fourth immunization, and separate PBMC cells using lymphocyte separation medium;

[0055] b. Extract RNA using PrimeScript TM II. Reverse transcription was performed using the 1st Strand cDNA Synthesis Kit (TaKaRa, Cat.#6210B). After mixing the samples by pipetting, 80 μL / tube was dispensed and placed in a PCR instrument at 42°C for 1 h, followed by heat inactivation at 70°C for 15 min. Finally, the cDNA samples were stored on ice or at -20°C for long-term preservation.

[0056] c. Amplification of the VHH fragment

[0057] 1) Prepare the first round of PCR reaction system (50 μL / tube) as shown in Table 2 below.

[0058] Table 2 First-round PCR reaction system

[0059]

[0060] After configuring the PCR reaction system, set up the PCR instrument according to the following procedure, as shown in Table 3 below.

[0061] Table 3. First-round PCR amplification procedure

[0062]

[0063] 2) Agarose gel electrophoresis of PCR products

[0064] PCR products were analyzed by electrophoresis using 1% agarose gel to separate fragments with a molecular weight of approximately 750 bp. PCR products were recovered using a gel extraction kit, and their concentrations were determined using NanoDrop.

[0065] 3) Prepare the two-round PCR reaction system (50 μL / tube) as shown in Table 4 below.

[0066] Table 4. Second round PCR reaction system

[0067]

[0068]

[0069] After configuring the PCR reaction system, set up the PCR instrument according to the following procedure, as shown in Table 5 below.

[0070] Table 5. Second-round PCR amplification procedure

[0071]

[0072] 4) Agarose gel electrophoresis analysis of the second round of PCR products

[0073] PCR products were analyzed by electrophoresis using 1% agarose gel to isolate the VHH fragment with a molecular weight of approximately 400 bp. The VHH PCR product was recovered using a gel extraction kit, and its concentration was determined using NanoDrop.

[0074] The results of the two rounds of PCR are as follows Figure 1 As shown, peripheral blood was collected from immunized alpacas, total RNA was extracted, reverse transcribed into cDNA, and then subjected to two rounds of PCR using single-domain antibody amplification primers. The PCR products were then analyzed by agarose gel electrophoresis. Figure 1 It can be seen that the first round of PCR yielded PCR bands of approximately 1000bp and 750bp, respectively, and the 750bp fragment was recovered as a template for the second round of PCR. The second round of PCR yielded a band of approximately 500bp, which was due to the addition of the homologous arm VHH fragment, and subsequent homologous recombination was performed into the phage display vector pDisplay.

[0075] (3) Construction of a single-domain antibody phage display library:

[0076] a. Linearization of the phage display vector pDisplay, and the enzyme digestion system are shown in Table 6 below:

[0077] Table 6 Enzyme digestion reaction system

[0078]

[0079] The pDisplay vector was digested with SfiI enzyme, 100 μL / tube, and incubated overnight at 50°C. The pDisplay vector fragment was separated using a 1% agarose gel, and a 5000 bp fragment was recovered and its concentration determined using NanoDrop. The recovered pDisplay digestion product was aliquoted into 200 μL units in each 1.5 mL centrifuge tube, and 1 / 10 volume (20 μL) of 3M sodium acetate and 1 μg / μL glycogen were added. The mixture was then pipetted and aspirated to obtain the linearized vector fragment, which was stored at -80°C.

[0080] b. Electroporation to construct a phage display library:

[0081] 1) Take out the SS320 competent cells frozen at -80℃, place them on ice for 5 minutes to thaw, add 10 μL of the linearized vector fragment prepared in step a and 10 μL of the PCR product in step 3), and gently mix by tapping the bottom of the EP tube to avoid generating air bubbles, to obtain the competent cell-DNA mixture, and immediately insert it into ice.

[0082] 2) Quickly transfer the competent-DNA mixture from step 1) into the electroporation vessel (avoid generating air bubbles), gently shake to keep the liquid level, cover the vessel, electroporate, and after electroporation, remove the electroporation vessel and let it sit at room temperature. Open the vessel and add 100 μL of preheated 2YT medium within 15 seconds. Mix well and transfer to a 50 mL centrifuge tube. Add 2YT medium to the centrifuge tube to a final volume of 10 mL. Incubate at 37°C and 220 rpm for 60 min.

[0083] 3) Centrifuge at 5000 rpm for 1 min to collect bacteria. Take 100-200 μL of bacterial suspension, resuspend it, and spread it on a plate containing ampicillin. Invert the plate and incubate it overnight at 37℃ for 16 h to obtain plate single colonies. Calculate the library capacity.

[0084] 4) Preservation of bacteria: Collect the remaining bacterial culture into a 50mL centrifuge tube, centrifuge at 5000rpm for 5min, discard the supernatant, add 10mL of 2YT culture medium to resuspend, mix with 50% glycerol: resuspension = 1:1, and freeze at -80℃.

[0085] (4) Cell panning of phage display library

[0086] a. Phage display library QC: Randomly select plate clones obtained above for sequencing. The obtained sequences are all antibody sequences. The sequencing results are as follows: Figure 2 As shown, these antibody sequences are all differential sequences, indicating that the library has good diversity.

[0087] b. Four rounds of panning for positive antigen cells: In the first round, 100 μL of antibody library culture stored at -80℃ was added to 30 mL of 2YT medium and incubated at 37℃ and 220 rpm for 15 h. Then, 1×10⁻⁶ cells were added to the culture medium. 7 CHO-K1 negative cells were mixed with the antibody library bacterial culture and incubated for 1 hour. The mixture was then centrifuged at 1000 rpm for 3 minutes to collect the antibody library supernatant. The antibody library supernatant was then mixed with 5 × 10⁻⁶ cells. 6 CHO-K1 and CHO-K1-Claudin18.2 cells (constructed CHO-K1 cells stably transfected with human Claudin18.2 antigen) were mixed and incubated at 4°C for 1 h. The supernatant was removed by centrifugation at 1000 rpm for 3 min. The cells were resuspended and washed repeatedly with PBS 6 times. Finally, the cell pellet was retained.

[0088] The cell pellet was resuspended in SS320 bacterial culture (antibiotic-free) cultured in 2YT medium and incubated at 37°C, 220 rpm for 1 h. Then, M13K07 helper phage was added to the bacterial culture and incubated at 37°C, 220 rpm for 1 h. 20 μL of the bacterial culture from each EP tube was serially diluted and used for titer assays on LB agar plates containing ampicillin. The plates were incubated overnight at 37°C. The remaining bacterial culture in the CHO-K1-Claudin18.2 cell EP tubes was added to 2YT medium containing ampicillin resistance and incubated by shaking. The titers of the overnight cultured plates were measured, and the enrichment level was statistically analyzed. The second round of panning uses the antibody library culture from the first round of incubation. The concentration of the added phage antibody library is adjusted based on the colony plate titer detection data from the previous round (e.g., by dilution at multiples of 10×, 100×, 1000×, etc.) to remove phages that do not specifically bind to negative cells as much as possible. The remaining operations are the same as before. A total of 4 rounds of panning are performed, and the results are shown in Table 7 below.

[0089] Table 7. Selection results of the phage display library of this invention

[0090] Round Input (PFU) Output (PFU) Enrichment factor Round 1 2.76E+12 4.16E+05 6.63E+07 Round 2 2.87E+12 6.35E+07 4.25E+04 Round 3 3.11E+12 1.26E+08 2.47E+04 Round 4 3.21E+12 1.94E+08 1.65E+04

[0091] c. Experimental group: CHO-K1-18.2 cells; primary antibody: monoclonal bacterial supernatant; secondary antibody: PE-Goat anti-Human IgG Fc (invitrogen, Cat#:12-4998-82) (1:1000 dilution). Control group: CHO-K1 cells; primary antibody: monoclonal bacterial supernatant; secondary antibody: PE-Goat anti-Human IgG Fc (invitrogen, Cat#:12-4998-82) (1:1000 dilution). After four rounds of cell panning, single clones were selected from the bacterial culture and incubated with shaking. The primary antibody was used as the supernatant. The supernatant and cells were co-incubated for 1 hour, and the secondary antibody was incubated at 4℃ for half an hour. Flow cytometry was used to detect positive clones of E7, A2, A9, B6, B8, D11, F5, G11, G12, and H9, which were then sent for sequencing. The detection results are as follows: Figure 3 .

[0092] (5) Protein panning of phage display library:

[0093] a. Antigen protein panning: Human Claudin 18.2 Protein-VLP (KACTUS, Cat. No. CLD-HM0P37) was diluted to 5 μg / mL with sterile PBS, and 100 μl / well was added to a 96-well plate. Eight wells were used as positive controls. The plate was coated overnight at 4°C. 100 μL / well of PBS blocking buffer containing 2% BSA was added as negative controls. After overnight incubation, the coating buffer of human Claudin 18.2 Protein-VLP was removed, and 200 μL / well of PBS blocking buffer containing 2% BSA was added. The plate was incubated at room temperature for 2 h. The blocking buffer was removed by inverting the plate. Each well was washed 3 times with PBS. 100 μL / well of the phage antibody library obtained in step (3) was added to the antigen wells and control wells. The plate was incubated at room temperature for 1 h. The antibody library liquid was removed, and the plate was washed 8 times with PBS. 100 μL of 100 mM HCl was added to each well. Incubate at room temperature for 5 min, then transfer the liquid to an EP tube containing 35 μL of 1M Tris-HCl, ensuring the pH is neutral after neutralization. Add SS320 bacterial suspension to the EP tube and incubate at 37°C and 220 rpm for 1 h. Then add M13K07 helper phage to the bacterial suspension and incubate at 37°C and 220 rpm for 1 h. Take 20 μL of bacterial suspension from each EP tube for serial dilution and perform titer tests on LB agar plates containing ampicillin, incubating overnight at 37°C. Add the remaining bacterial suspension from the positive antigen EP tube to 2YT medium containing ampicillin for further shaking incubation. Perform titer analysis on the overnight incubated plates and calculate the enrichment level. The second round of panning selects the antibody library bacterial suspension from the first round of incubation. The concentration of the added phage antibody library is adjusted according to the colony plate titer detection data from the previous round (e.g., by performing dilutions of 10×, 100×, 1000×, etc.). The remaining operations are the same as before. A total of 3 rounds of panning are performed. The panning results are shown in Table 8 below.

[0094] Table 8. Protein panning results of the phage display library of this invention.

[0095]

[0096] b. Phage ELISA monoclonal assay. Human Claudin 18.2 Protein-VLP (KACTUS, Cat. No. CLD-HM0P37) and BSA negative control protein were coated, blocked, and washed in 96-well plates at 4°C. Add 500 μL of 2YT medium containing ampicillin to each well of a 96-well deep-well plate. Pick single clones of the output product titer after three rounds of protein panning and place them in separate 96-well deep-well plates. Incubate overnight at 37°C and 220 rpm. Centrifuge the overnight 96-well plates at 4000 rpm for 10 min. Add 50 μL of phage supernatant to the coated 96-well plates and incubate at room temperature for 1 h. Discard the liquid and wash the 96-well plates three times. Add 100 μL of HPR-M13 antibody to each well and incubate at room temperature for 1 h. Wash six times with PBS. Add 100 μL of TMB to each well and incubate at 37°C for 10 min. Add 50 μL of 1M phosphate to each well to stop the reaction. Detect the OD450 absorbance using a microplate reader and select positive single clones for sequencing. From the phage display library enriched through three rounds of protein panning, single-clone cells were randomly selected, amplified, induced, and then tested using antigens. The results are shown in Tables 9 and 10 below. This allows us to determine the binding of single-domain antibodies on the surface of single-clone phage cells to the target antigen. S / N = OD450 of positive antigen coating / OD450 of BSA coating wells. Clones with S / N-highlighted red markings were selected for testing.

[0097] Table 9. Monoclonal antibody test results for plate #3

[0098]

[0099] Table 10. Monoclonal antibody test results for plate #4

[0100]

[0101] (6) Construction of antibody eukaryotic expression vector:

[0102] Positive phage clones were amplified by PCR to obtain antibody sequences, which were then digested with SfiI and ligated into the eukaryotic expression vector pcDNA3.4-human IgG1Fc to construct the antibody expression vector. After the vector was verified by sequencing, endotoxin-free plasmids were prepared using the Qiagen plasmid desorption kit for later use.

[0103] (7) Preparation of antibody expression supernatant

[0104] Remove the LVTransm transfection reagent and antibody expression plasmid (i.e., the endotoxin-free plasmid obtained in step (6)) from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting. Remove the PBS buffer and warm it to room temperature. Take 20 μL of PBS into a 1.5 mL sterile EP tube, add 3 μg of antibody expression plasmid, mix thoroughly by pipetting, add 12 μL of LVTransm, and immediately mix by pipetting. Let stand at room temperature for 10 min. Add the above DNA / LVTransm complex to 3 mL of 293F cells and gently shake to mix thoroughly. Place the cells in a 37℃, 5% CO2 incubator and culture at 130 rpm. After continuous culture for 48 h, centrifuge to collect the culture supernatant, filter through a 0.45 μm filter membrane, and transfer the filtrate to a sterile centrifuge tube for later use, which is the antibody expression supernatant. Thus, the eukaryotic expression vector of nanobodies and the nanobodies expression supernatant have been prepared.

[0105] Example 2: Flow cytometry detection of the binding of recombinant antibody to target protein

[0106] The monoclonal sequences sent from the previous cell and protein screening were sequenced, and 12 differential sequences were obtained. Vectors were then constructed and validated.

[0107] 1. Experimental samples: Cells: CHO-K1, CHO-K1-CLDN18.1, CHO-K1-CLDN18.2, 2×10⁻⁶ 5 100 μL / well. Primary antibody: CLDN18.2 transfection supernatant, 100 μL / well; Secondary antibody: PE-Goat anti-Human IgG Fc (invitrogen, Cat#:12-4998-82) (1:1000 dilution).

[0108] 2. Experimental procedure: CHO-K1 cell lines overexpressing full-length Claudin18.2 (CHO-K1-Claudin18.2) and CHO-K1 cell lines overexpressing full-length Claudin18.1 (CHO-K1-Claudin18.1) were constructed. CHO-K1 cells were revived, cultured, and adjusted to the logarithmic growth phase.

[0109] The three types of cells were divided into several groups, with each group containing 2 × 10⁻⁶ cells. 5Cells were incubated with 100 μL of the supernatant containing the antibody obtained in Example 1. After thorough mixing, the cells were incubated at room temperature for 1 hour. After centrifugation at 800g for 5 min at room temperature, the supernatant containing the antibody was removed, and the cells were washed three times with PBS. 100 μL of PE-labeled PE-Goat anti-HumanIgG Fc (invitrogen, Cat#:12-4998-82) (1:1000 dilution) was added, thoroughly mixed, and incubated at room temperature in the dark for 30 min. After centrifugation at 800g for 5 min at room temperature, the supernatant containing the secondary antibody was removed, and the cells were washed three times with PBS. The cells were resuspended in 500 μL of PBS and analyzed by flow cytometry.

[0110] 3. Test Results: The test results are as follows Figure 4 As shown, by Figure 4 It is known that clones 2-D11, 3-A5, 3-E6, 3-H3, 4-D4, 4-F4, 3-A2, 3-C6, and 4-G1 specifically bind to CHO-K1-CLDN18.2 cells.

[0111] Example 3: Preparation of a lentiviral vector expressing a chimeric antigen receptor targeting Claudin18.2.

[0112] The DNA sequence of 1 μg nanobody and the chimeric antigen receptor plasmid were digested with SfiⅠ enzyme, and the reaction system is shown in Table 11.

[0113] Table 11 Enzyme digestion systems of nanobodies and chimeric antigen receptor plasmids

[0114]

[0115]

[0116] The above process was carried out on ice, then mixed, briefly separated, then treated at 50°C for 1 hour, and stored at 4°C.

[0117] After the reaction, the enzyme digestion products were detected by 1% agarose gel electrophoresis. The nanobody fragments of about 400 bp were recovered, and the chimeric antigen receptor backbone fragments of about 8000 bp were recovered. The results were quantified by ultraviolet absorption.

[0118] The nanobody small fragments (insert DNA) and the chimeric antigen receptor backbone large fragments (vector DNA) were ligated using T4 DNA ligase, and the reaction system is shown in Table 3.

[0119] Table 3. Linkage system of small fragments of antibody and large fragments of chimeric antigen receptor backbone.

[0120]

[0121] The reaction was carried out at room temperature for 2 hours, and then the ligation product was transformed into E. coli Tstbl3 competent cells. Single clones were selected from LB solid medium containing ampicillin, cultured overnight, and positive clones were selected for sequencing identification. The sequencing results were as expected. Thus, a chimeric antigen receptor lentiviral expression vector against Claudin18.2 was constructed.

[0122] Example 4 Lentiviral Packaging and Titer Detection

[0123] 293T cells were cultured in 15cm culture dishes until they were almost fully confluent, at which point lentivirus was packaged. 22 μg of the lentiviral expression vector constructed in Example 4, 15 μg of the envelope plasmid VGV-G, and 15 μg of the helper plasmid Δ8.9 were added to 2500 μL of Opti-MEM serum-depleted medium (Gibco). 135 μL of 1 μg / μL PEI was added to another 2500 μL of Opti-MEM serum-depleted medium, and both were mixed thoroughly. The PEI mixture was added to the DNA mixture at a mass ratio of PEI:total plasmid = 3:1, and the mixture was gently mixed and incubated at room temperature for 25 minutes. 5 mL of the transfection complex was added to a 15cm culture dish of 293T cells and cultured at 37°C and 5% CO2 for 24 h. After 24 h, the medium was replaced with fresh DMEM + 10% FBS medium and cultured further. Viral supernatants were collected at 48 h and 72 h, and the lentivirus was concentrated by ultracentrifugation to obtain a concentrated lentivirus solution.

[0124] 293T cells (5×10) 5 The cells were inoculated into 24-well plates, 1 μL of lentiviral concentrate was added, and polybrene was added to a final concentration of 10 ng / μL. The cells were incubated at 37°C in 5% CO2, 10% FBS·1% P / S·DMEM medium for 24 h. After 24 h, the medium was replaced with fresh DMEM + 10% FBS medium. After 48 h, the GFP positivity rate of 293T cells was detected by flow cytometry, which represents the viral transduction efficiency. The GFP positivity rate results are shown below. Figure 5 Calculate the titer using the formula:

[0125] Titer = Number of transfected cells (5 × 10⁻⁶) 5 ) × Positive rate / Viral volume (mL)

[0126] Depend on Figure 5 It can be seen that GFP+ indicates that the 293T cells were successfully transduced with the lentiviral vector.

[0127] Example 5: Lentiviral transduction of human primary T lymphocytes

[0128] CD3 was isolated from peripheral blood of healthy individuals. + T lymphocytes were diluted to 1×10⁻⁶ with T cell culture medium (TexMacs + 50 IU / mL human IL-2). 6Primary T cells were stimulated with 100×TranAct per mL of T cells. After 48 h, T cells were centrifuged and counted. Concentrated lentivirus solution prepared in Example 4 was added at an MOI of 10, and vectofusin was added to a final concentration of 10 ng / μL to promote transfection. The cells were centrifuged at 800×g for 60 min at 32°C and cultured at 37°C with 5% CO2 for 24 h. After 24 h, the culture medium was replaced with TexMacs + 50 IU / mL humanIL-2 fresh medium and cultured for 48 h. After 72 h of T cell transduction, 1×10⁶ cells were collected. 5 Cells were resuspended in 100 μL PBS (2% FBS), 100 μL dapi was added, and flow cytometry analysis was performed.

[0129] Test results as follows Figure 6 ,Depend on Figure 6 It can be seen that GFP+ indicates CAR-T cells that have been successfully transduced with lentiviral vectors.

[0130] Example 6: Detection of the in vitro antitumor effect of CAR-T cells

[0131] CAR-T cells prepared using Claudin18.2 nanobody were used to verify the efficacy and specificity of in vitro tumor cell killing. All tumor cells used overexpressed luciferase; AGS-WT: wild-type AGS gastric cancer cells; AGS-hCLDN18.1: AGS gastric cancer cells overexpressing human Claudin18.1; AGS-hCLDN18.2: AGS gastric cancer cells overexpressing human claudin18.2; MKN45-WT: wild-type MKN45 gastric cancer cells; MKN45-hCLDN18.1: MKN45 gastric cancer cells overexpressing human Claudin18.1; MKN45-hCLDN18.2: MKN45 gastric cancer cells overexpressing human claudin18.2.

[0132] Different CAR-T cells or untransduced T cells (UTDs) were co-cultured with constructed luciferase-expressing AGS-WT / AGS-hCLDN18.1 / AGS-hCLDN18.2 gastric cancer cells and MKN45-WT / MKN45-hCLDN18.1 / MKN45-hCLDN18.2 gastric cancer cells at an effector-to-target ratio of 1:1 or 10:1 for 18 hours. Cells were then lysed, luciferase substrate was added, and the luminescence signal was detected using a microplate reader. The specific procedure is as follows:

[0133] a. Target cell seeding: Six cell lines (AGS-WT / AGS-hCLDN18.1 / AGS-hCLDN18.2 and MKN45-WT / MKN45-hCLDN18.1 / MKN45-hCLDN18.2) were each adjusted to a concentration of 1×10⁻⁶. 5 / mL, take 100μL and inoculate it into a 96-well plate;

[0134] b. Effector cell seeding: CAR-T cells and untransduced T cells were added to 96-well plates at effector-to-target ratios (E:T) of 1:1 and 10:1, with a volume of 100 μL per well.

[0135] c. After co-culturing effector cells and target cells for 18 hours, the cells were lysed, luciferase substrate was added, and the luminescence signal was detected using a microplate reader. The CAR-T cell killing effect was characterized by the number of remaining target cells. Each group had three replicates, and the average value of the three replicates was taken.

[0136] The filtering results are as follows Figure 7-8 As shown, Figure 7 The image shows the results of CAR-T cell killing AGS gastric cancer cells in each group. Figure 8 The image shows the results of CAR-T cell killing MKN45 gastric cancer cells in each group. Ultimately, 3H3, 4D4, and 4F4 were selected as nanobodies possessing both efficacy and specificity.

[0137] Finally, it should be noted that the above embodiments are merely illustrative of the principles, performance, and effects of the present invention, and are not intended to limit the present invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A nanobody targeting Claudin18.2, characterized in that, The nanobody comprises CDR1, CDR2, and CDR3 regions, separated by FR regions. The CDR and FR regions contain sequences that are ≥80% homologous to the following sequences: The amino acid sequence of the CDR1 region is shown in SEQ ID NO.1, the amino acid sequence of the CDR2 region is shown in SEQ ID NO.9; the amino acid sequence of the CDR3 region is shown in SEQ ID NO.16, the amino acid sequence of the FR1 region is shown in SEQ ID NO.23, the amino acid sequence of the FR2 region is shown in SEQ ID NO.30, the amino acid sequence of the FR3 region is shown in SEQ ID NO.38, and the amino acid sequence of the FR4 region is shown in SEQ ID NO.45; or; The amino acid sequence of the CDR1 region is shown in SEQ ID NO.2, the amino acid sequence of the CDR2 region is shown in SEQ ID NO.10; the amino acid sequence of the CDR3 region is shown in SEQ ID NO.17, the amino acid sequence of the FR1 region is shown in SEQ ID NO.24, the amino acid sequence of the FR2 region is shown in SEQ ID NO.31, the amino acid sequence of the FR3 region is shown in SEQ ID NO.39, and the amino acid sequence of the FR4 region is shown in SEQ ID NO.46; or; The amino acid sequence of the CDR1 region is shown in SEQ ID NO.3, the amino acid sequence of the CDR2 region is shown in SEQ ID NO.11; the amino acid sequence of the CDR3 region is shown in SEQ ID NO.18, the amino acid sequence of the FR1 region is shown in SEQ ID NO.25, the amino acid sequence of the FR2 region is shown in SEQ ID NO.32, the amino acid sequence of the FR3 region is shown in SEQ ID NO.40, and the amino acid sequence of the FR4 region is shown in SEQ ID NO.45; or; The amino acid sequence of the CDR1 region is shown in SEQ ID NO.2, the amino acid sequence of the CDR2 region is shown in SEQ ID NO.10; the amino acid sequence of the CDR3 region is shown in SEQ ID NO.17, the amino acid sequence of the FR1 region is shown in SEQ ID NO.26, the amino acid sequence of the FR2 region is shown in SEQ ID NO.31, the amino acid sequence of the FR3 region is shown in SEQ ID NO.39, and the amino acid sequence of the FR4 region is shown in SEQ ID NO.45; or; The amino acid sequence of the CDR1 region is shown in SEQ ID NO.4, the amino acid sequence of the CDR2 region is shown in SEQ ID NO.12; the amino acid sequence of the CDR3 region is shown in SEQ ID NO.19, the amino acid sequence of the FR1 region is shown in SEQ ID NO.27, the amino acid sequence of the FR2 region is shown in SEQ ID NO.33, the amino acid sequence of the FR3 region is shown in SEQ ID NO.41, and the amino acid sequence of the FR4 region is shown in SEQ ID NO.47; or; The amino acid sequence of the CDR1 region is shown in SEQ ID NO. 5, the amino acid sequence of the CDR2 region is shown in SEQ ID NO. 13; the amino acid sequence of the CDR3 region is shown in SEQ ID NO. 20, the amino acid sequence of the FR1 region is shown in SEQ ID NO. 24, the amino acid sequence of the FR2 region is shown in SEQ ID NO. 34, the amino acid sequence of the FR3 region is shown in SEQ ID NO. 42, and the amino acid sequence of the FR4 region is shown in SEQ ID NO. 45; or; The amino acid sequence of the CDR1 region is shown in SEQ ID NO. 6, the amino acid sequence of the CDR2 region is shown in SEQ ID NO. 14; the amino acid sequence of the CDR3 region is shown in SEQ ID NO. 21, the amino acid sequence of the FR1 region is shown in SEQ ID NO. 28, the amino acid sequence of the FR2 region is shown in SEQ ID NO. 35, the amino acid sequence of the FR3 region is shown in SEQ ID NO. 43, and the amino acid sequence of the FR4 region is shown in SEQ ID NO. 48; or; The amino acid sequence of the CDR1 region is shown in SEQ ID NO.7, the amino acid sequence of the CDR2 region is shown in SEQ ID NO.9; the amino acid sequence of the CDR3 region is shown in SEQ ID NO.16, the amino acid sequence of the FR1 region is shown in SEQ ID NO.29, the amino acid sequence of the FR2 region is shown in SEQ ID NO.36, the amino acid sequence of the FR3 region is shown in SEQ ID NO.38, and the amino acid sequence of the FR4 region is shown in SEQ ID NO.45; or The amino acid sequence of the CDR1 region is shown in SEQ ID NO. 8, the amino acid sequence of the CDR2 region is shown in SEQ ID NO. 15, the amino acid sequence of the CDR3 region is shown in SEQ ID NO. 22, the amino acid sequence of the FR1 region is shown in SEQ ID NO. 24, the amino acid sequence of the FR2 region is shown in SEQ ID NO. 37, the amino acid sequence of the FR3 region is shown in SEQ ID NO. 44, and the amino acid sequence of the FR4 region is shown in SEQ ID NO.

49.

2. The nanobody targeting Claudin18.2 as described in claim 1, characterized in that, The amino acid sequence of the nanobody is selected from one of SEQ ID NO.50 to SEQ ID NO.

58.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleotide sequence encoding the nanobody of claim 2, wherein the nucleotide sequence is selected from one of SEQ ID NO. 59 to SEQ ID NO.

67.

4. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 3.

5. A host cell, characterized in that, The host cell comprises the nucleic acid molecule of claim 3, or the vector of claim 4.

6. The host cell as described in claim 5, characterized in that, The host cell is a chimeric antigen receptor immune cell; the chimeric antigen receptor immune cell is selected from one of the following: chimeric antigen receptor T cells, chimeric antigen receptor NK cells, chimeric antigen receptor macrophages, chimeric antigen receptor NKT cells, chimeric antigen receptor γδT cells, chimeric antigen receptor B cells, chimeric antigen receptor dendritic cells, and chimeric antigen receptor erythrocytes.

7. The host cell as described in claim 6, characterized in that, The chimeric antigen receptor comprises a transmembrane domain, an intracellular domain, and an extracellular domain. The extracellular domain comprises a signal peptide, the antibody targeting Claudin18.2 as described in claim 1, and a CD8a hinge region. The transmembrane domain is the CD8a transmembrane domain. The intracellular domain is one of the following: 4-1BB and CD3ζ signaling domain, or CD28 co-stimulatory domain + CD3ζ signaling domain.

8. The use of a nanobody according to any one of claims 1 to 2, a nucleic acid molecule according to claim 3, a carrier according to claim 4, or a host cell according to any one of claims 5 to 7 in the preparation of a drug for treating and / or preventing tumors.

9. The application as described in claim 8, characterized in that, The tumor is one of the following: stomach cancer, pancreatic cancer, esophageal cancer, biliary tract cancer, colorectal cancer, or lung cancer.

10. A reagent kit, characterized in that, The kit comprises the nanobody according to any one of claims 1 to 2, the nucleic acid molecule according to claim 3, the vector according to claim 4, or the host cell according to any one of claims 5 to 7.

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