Nanobodies targeting claudin 18.2 and uses thereof

Abstract

The application relates to a nanobody targeting claudin 18.2 and application thereof, and relates to the technical field of immunology and molecular biology. The amino acid sequence of the complementarity determining region of the nanobody targeting claudin 18.2 comprises CDR1 as shown in any one of SEQ ID NO:1-4, CDR2 as shown in any one of SEQ ID NO:5-9, and CDR3 as shown in any one of SEQ ID NO:10-15. The application constructs an alpaca immune phage display library, screens a series of specific nanobody molecules targeting claudin 18.2 from the library, and verifies the function and application of the nanobody at the CLDN18.2 antigen protein and CLDN18.2 target cell levels.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology and molecular biology, in particular to a nanobody targeting claudin 18.2 and its use. BACKGROUND

[0002] Tight junctions (TJs) are an important cell adhesion structure, mainly present in the junction complex between epithelial cells and endothelial cells, which makes the adjacent cell membranes close together to form a physical barrier structure around the cells. The tight junction membrane domain contains at least three different proteins, which are called occludins, claudins and junctional adhesion molecules (JAMs), respectively. Among them, claudins (CLDNs) are a class of transmembrane proteins first discovered in 1998, with at least 27 family members. CLDNs have four transmembrane domains, and the N- and C-termini are both located in the cytoplasm. CLDN18 is a claudin in the CLDNs protein family that has been widely studied, and was first identified in 2001. There are two splice variants of human CLDN18 gene, which encode two protein subtypes of CLDN18.1 and CLDN18.2 (claudin 18.2), both of which consist of 261 amino acids and have two extracellular loops, with only 8 amino acid differences in extracellular loop 1. In the human body, the expression of CLDN18.1 and CLDN18.2 has tissue specificity, CLDN18.1 is mainly expressed in lung tissue, and CLDN18.2 is only expressed in the differentiated epithelial cells of the normal gastric mucosa, not in the gastric stem cells. However, in various tumor tissues, CLDN18.2 is highly expressed, such as non-small cell lung cancer (25%), gastric cancer (70-80%), pancreatic cancer (60%), and esophageal cancer (30%). Although CLDN18.2 is also expressed in normal gastric mucosa, the CLDN18.2 expressed in the gastric mucosa exists in the cell tight junction supermolecular complex, and the antigen epitope is difficult to contact the targeted drugs, while the malignant tumor cells lose polarity, leading to the exposure of CLDN18.2 epitopes, which can contact the targeted drugs. This characteristic makes CLDN18.2 an ideal target for targeted therapy of digestive tract tumors.

[0003] Up to now, the global research and development pipeline of CLDN18.2 target covers all mainstream technical routes, including monoclonal antibodies, antibody drug conjugates (ADC), bispecific antibodies and chimeric antigen receptor T cells (CAR-T), among which the monoclonal antibody and ADC route is the most popular. The fastest progressing targeted drug is the CLDN18.2 monoclonal antibody drug IMAB362 (Zolbetuximab, Satumomab) developed by the Japanese pharmaceutical company Anstel, which has been approved by the Japanese Ministry of Health, Labour and Welfare (MHLW) and the US FDA for the treatment of CLDN18.2-positive gastric cancer patients this year, becoming the world's first CLDN18.2-targeted drug. The fastest progressing CAR-T direction for CLDN18.2 is a self-CAR-T cell candidate product CT041 developed by Kozhi Pharmaceutical for the treatment of gastric cancer, pancreatic cancer and other solid tumors, and the related clinical trial has entered phase II. These studies show the safety and effectiveness of CLDN18.2 targeting therapy.

[0004] Heavy chain antibodies are naturally light chain-deleted single heavy chain domain antibodies found in animals of the order Carnivora, nurse sharks, bigeye sand sharks and chondrichthyes such as stingrays. Cloning of the heavy chain antibody variable region can obtain a single domain antibody (Single domain antibody, sdAb) composed of only the heavy chain variable region, which is also called nanobody (Nanobodies, Nbs) due to its small molecular weight. Although the molecular weight of nanobody is only 1 / 10 (about 15 kDa) of that of the complete traditional antibody, it retains the complete antigen recognition and binding capacity of the heavy chain antibody, and compared with traditional antibodies, it has the advantages of strong specificity, strong affinity, high stability, strong targeting and tissue penetration, low immunogenicity, easy humanization modification, etc. The disadvantage of nanobody is short half-life, but it can be fused with anti-serum albumin or the Fc segment of the antibody for expression to prolong its half-life in blood. Nanobodies have been widely used in biochemical mechanism and structural biology research, as well as in the development of diagnostic reagents and therapeutic drugs for diseases such as tumors.

[0005] Gastrointestinal tumors are high-incidence solid tumors in the world, and China is a high-incidence country of gastrointestinal cancers such as gastric cancer and pancreatic cancer. Although there are currently antibody and CAR-T drugs targeting the CLDN18.2 target in clinical research, there is only one marketed drug, and there is an urgent need to continue to develop antibodies targeting the CLDN18.2 target. The present application aims to develop new antibodies targeting CLDN18.2, especially candidate nanobody molecules that specifically recognize CLDN18.2 but not CLDN18.1, for the development of diagnostic reagents, targeted antibody drugs and CAR-T / NK immune cell drugs for diseases such as digestive system tumors. In view of this, the present application discloses a nanobody specifically targeting claudin 18.2 and its application. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a nanobody specifically targeting claudin 18.2 without recognizing CLDN18.1 and application thereof.

[0007] The technical solution of the present application to solve the above technical problem is as follows:

[0008] In a first aspect, a nanobody targeting claudin 18.2, the amino acid sequence of the complementarity determining region of the nanobody targeting claudin 18.2 comprises CDR1 as shown in any one of SEQ ID NO: 1-4, CDR2 as shown in any one of SEQ ID NO: 5-9, and CDR3 with an amino acid sequence as shown in any one of SEQ ID NO: 10-15.

[0009] Further, the amino acid sequence of the nanobody targeting claudin 18.2 is as shown in any one of SEQ ID NO: 16-21.

[0010] In a second aspect, a nucleic acid comprising a nucleic acid sequence encoding the nanobody targeting claudin 18.2 or a complementary sequence thereof.

[0011] Further, the nucleotide sequence encoding the nanobody targeting claudin 18.2 is as shown in any one of SEQ ID NO: 28-33.

[0012] In a third aspect, a fusion protein, the fusion protein being a recombinant protein produced by fusing the nanobody targeting claudin 18.2 with an Fc fragment of an immunoglobulin (IgG, IgA, etc.).

[0013] Further, the amino acid sequence of the Fc fragment of the immunoglobulin is as shown in SEQ ID NO: 40.

[0014] Further, the encoding nucleotide sequence of the Fc fragment of the immunoglobulin is as shown in SEQ ID NO: 41.

[0015] In a fourth aspect, a pharmaceutical composition comprising the nanobody targeting claudin 18.2, or the nucleic acid, or the fusion protein.

[0016] Further, the dosage form of the pharmaceutical composition comprises at least one of a microsphere preparation, a microcapsule preparation, and a nanocapsule preparation. It also comprises a pharmaceutically acceptable excipient and / or carrier.

[0017] In a fifth aspect, the application provides use of the nanobody targeting CLDN18.2, the nucleic acid, the fusion protein or the pharmaceutical composition in preparation of a detection reagent, an in vivo imaging probe or a therapeutic product targeting CLDN18.2.

[0018] The core innovation of the application is to provide a group of six nanobodies that specifically target CLDN18.2 (CLDN18.2) and do not recognize CLDN18.1. There is no antibody in the prior art that has the same CDR sequence as the antibody of the application. The problem of lack of candidate nanobody molecules that specifically target CLDN18.2 in the prior art is solved.

[0019] The application has the following advantages:

[0020] (1) The CLDN18.2 target selected by the application is the most safe and effective potential treatment target for digestive system tumors: CLDN18.2 is only expressed in the intercellular tight junction of the gastric mucosa epithelial tissue in normal tissues, and cannot contact the surrounding targeted drugs, while it is highly expressed in gastric cancer, pancreatic cancer, esophageal cancer and non-small cell lung cancer, and is exposed to targeted drugs; studies have shown that CAR-T or antibody drugs targeting CLDN18.2 have no damage to normal expression tissues, but have specific killing effect on CLDN18.2 expressing tumor tissues.

[0021] (2) The application provides candidate nanobody molecules for targeted therapy of digestive system tumors: compared with hematological tumors, the development of specific immunotherapy drugs for solid tumors is slow, and one of the constraints is the lack of effective and safe targets; current studies have shown that CLDN18.2 is a safe and effective treatment target for digestive system tumors, but the research on targeted drugs for CLDN18.2 target started late, and only one monoclonal antibody drug has just been marketed in a few countries, the bottleneck is in the antibody discovery stage, it is difficult to develop antibodies that specifically target CLDN18.2 and do not recognize CLDN18.1, and the nanobodies involved are even rare.

[0022] (3) The nanobodies of the present application have wide application fields: (a) The present application screened 6 candidate nanobodies against CLDN18.2 through llama immune library, and fused the C-terminus of the nanobodies with the Fc fragment of human IgG1. The binding activity (ELISA detection) and affinity (SPR detection) of the nanobodies to CLDN18.2 antigen were detected at the level of antigen protein CLDN18.2-VLP, and the results were better or comparable to those of the traditional control antibody IMAB362. At the level of various cells overexpressing CLDN18.2, the binding activity of the Fc fusion antibodies to CLDN18.2 antigen was detected by FACS, and 4 nanobodies were better than the traditional control antibody IMAB362. (b) Nanobodies have significant advantages over other antibodies, such as the half-life can be regulated by chemical modification or protein fusion modification, the penetration is strong, the hidden epitopes that cannot be recognized by ordinary antibodies can be recognized, the resistance to pepsin, acid and heat is high, the production is easy, and because it is a single chain, the molecular weight is small, and it is easy to assemble with other types of antibodies into bivalent antibodies / chimeric antigen receptors (CAR) and multivalent antibodies / CAR. Therefore, the nanobodies of the present application can be developed into immunodetection reagents, CAR-T / NK immune cell drugs and antibody drugs. In combination with the characteristics of CLDN18.2 target, the candidate antibodies of the present application will have better effects if used for immunodiagnostic reagents for cancer; if developed into cell or antibody immunotherapy drugs, the toxic side effects will be lower and the clinical efficacy will be better, thereby providing more drug options for patients.

[0023] (4) Can be used to develop double-target drugs: research has found that claudin 18.2 (Claudin18.2) is a safe and effective target for the treatment of digestive system tumors, and it is co-expressed with CDH17 in esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer and other digestive system tumors, and clinical or preclinical tests have proved that targeting these two targets can specifically kill tumor cells without harming normal tissues, so the anti-CLDN18.2 candidate nanobodies of the present application can be used in combination with CDH17 antibodies to develop anti-CLDN18.2 / CDH17 double-antibody drugs and double-target CAR-T / NK drugs, which are expected to enhance the efficacy of monoclonal antibodies or single-target CAR-T / NK immune cell drugs. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Flow chart for constructing a single-domain antibody library for CLDN18.2 llama immunization;

[0025] Figure 2 ELISA detection results of the candidate antibody protein expressed by Fc fusion and the antigen Hu-18.2-VLP binding;

[0026] Figure 3ELISA results of candidate antibody proteins expressed as Fc fusion binding to negative control antigen GAG-VLP;

[0027] Figure 4 FACS detection results of candidate antibody proteins expressed as Fc fusion binding to cells Hu-18.2-HEK293 overexpressing human antigen CLDN18.2;

[0028] Figure 5 FACS detection results of candidate antibody proteins expressed as Fc fusion binding to cells Hu-CLDN18.1-HEK293 overexpressing human antigen CLDN18.1;

[0029] Figure 6 FACS detection results of candidate antibody proteins expressed as Fc fusion binding to cells Hu-18.2-BxPC-3 overexpressing human antigen CLDN18.2;

[0030] Figure 7 FACS detection results of candidate antibody proteins expressed as Fc fusion binding to cells Hu-18.2-NUGC4 overexpressing human antigen CLDN18.2. DETAILED DESCRIPTION

[0031] The principles and features of the present application are described below, and the examples are only used to explain the present application, and are not used to limit the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art, or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be purchased through regular channels.

[0032] EXAMPLE

[0033] 1. Alpaca immunization

[0034] 1.1 Alpaca immunization method

[0035] Two alpacas (numbered: #1, #2) were cross-immunized with virus-like particles (VLP) Hu-18.2-VLP (purchased from Sino Biological) of full-length human CLDN18.2 antigen, cell strains Hu-18.2-HEK293 and Hu-18.2-KATO III (purchased from Sino Biological) overexpressing full-length human CLDN18.2, 500 μg of antigen or 1.00 × 10 7 Cells. The first antigen immunization was Freund's complete adjuvant (Sigma), the subsequent immunization was Freund's incomplete adjuvant (Sigma), and the cell immunization did not add adjuvant. The injection method was subcutaneous multi-point injection. The alpaca immunization scheme is shown in Table 1.

[0036] Table 1 Alpaca immunization scheme

[0037]

[0038] 1.2 ELISA serum titer detection

[0039] Plate coating with Hu-18.2-VLP antigen (2 pg / mL, 30 pL), 4°C overnight incubation; blocking solution 5% PBSM (PBS, 5% skim milk powder) room temperature blocking for 2 h; add 3-fold gradient dilution serum, incubate at room temperature for 1 h; add secondary antibody Goat anti-Llama IgG (H+L) Secondary Antibody, HRP (ThermoFisher, A16060) for 1 h; add 30 pL TMB (purchased from SurModics) for color development, room temperature for 30 min; add 50 pL TMB color development termination solution (purchased from Biyun, P0215) to terminate the reaction, read the OD450 value of the enzyme label (Molecular Devices). According to the 1.65-fold NC calibration serum titer, the titer greater than 32K is qualified. ELISA detection results of two immunized alpacas are shown in Table 2. ELISA serum titer detection results show that the titer of two alpacas is qualified.

[0040] Table 2 Hu-18.2-VLP immune serum titer value

[0041] Serum to be tested Number of immunizations Titer (k) #1 4 64 #2 4 128

[0042] 2, Alpaca immune library construction

[0043] 2.1 Immune library construction

[0044] After 4 immunizations, 2 alpacas were bled respectively, and immune libraries were constructed. Peripheral blood 80 ml was collected from each alpaca, and peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Paque density gradient separation liquid (GE, 17144003S). The separated PBMC was extracted RNA, and reverse transcribed into cDNA by reverse transcription kit (TaKaRa, 6210A), and all antibody nucleotide sequences were obtained. Primers were designed on the front end of germline gene VH and CH2, and two different size DNA fragments were obtained by PCR, and the smaller target fragment was recovered by gel cutting; by comparing all sequences of VHH antibody V gene and J gene, a degenerate primer containing NcoI and NotI restriction sites was designed, and then all VHH gene fragments were amplified with the recovered DNA fragment product as the template. The phage display vector and VHH fragment were digested and ligated, and the ligation product was recovered by recovery kit (Omega, D6492-02), and the competent E. coli SS320 (Lucigen, MC1061F) was transformed by using an electroporator (Bio-Rad, MicroPulser), so as to obtain camel immune phage display library (RA044 and RA045). The above process is shown in Figure 1 .

[0045] 2.2 Single-domain antibody library capacity determination

[0046] The capacity of the single-domain antibody library was determined by dilution spot plate method. The library capacity determination results are shown in Table 3, and the library capacity of the two single-domain antibody libraries is greater than or equal to 1.00×10 8 CFU quality standard.

[0047] Table 3. Electroporation system and library capacity statistics

[0048] Library name Number of electrotransferred (individual) Single electrotransferred library capacity (CFU) Total library capacity (CFU) RA044 2 6.75 x 10 8 ]] 1.35 x 10 9 ]] RA045 1 3.75 x 10 8 ]] 3.75 x 10 8 ]]

[0049] 2.3 Single-domain antibody library sequencing result analysis

[0050] Clones were picked for sequencing, and sequencing successful results were selected for further analysis. The sequencing analysis is summarized in Table 4.

[0051] Table 4. Single-domain antibody library sequencing information statistics

[0052]

[0053] The single-domain antibody library gene sequence analysis standard is that the correct insertion rate is greater than or equal to 80%, the empty occupation ratio is less than or equal to 10%, and the restriction site accuracy is greater than or equal to 90%; the effective library capacity calculation method: total library capacity x antibody correct expression rate x unique sequence ratio = effective library capacity.

[0054] 2.4 Single-domain antibody library antibody gene analysis results

[0055] The analysis of the sequences successfully sequenced from the single-domain antibody library showed that the empty load ratio of RA044 and RA045 was less than 10%, the correct insertion rate of the antibody gene was greater than 80%, and the CDR region had a high richness in alignment, meeting the quality standards for the construction of a single-domain antibody library. In summary, the single-domain antibody library of RA044 and RA045 met the quality standards.

[0056] Table 5 Library construction index summary table

[0057]

[0058] 3. Alpaca immune library screening

[0059] The antibody sequences of the constructed alpaca immune library were displayed on the surface of phages through phage display technology. Then, specific proteins or cells were used as antigen materials, and the phage display antibodies that bound to the antigens were enriched through multiple rounds of screening. The positive clones that specifically bound to the antigens were further screened through ELISA, and the antibody sequences of the positive clones were obtained through sequencing.

[0060] 3.1 Immune library screening method

[0061] The immune library screening method comprises the following steps:

[0062] (1) Phage preparation: The alpaca immune library or the obtained output set is prepared through a series of steps such as bacteria infection, helper phage infection, phage expansion, phage precipitation and resuspension, to prepare the enriched phages and put them into the next round of screening.(2) Solid-phase screening: The antigen is coated on the surface of an immunotube with high adsorption capacity, and then the prepared phages are added to the immunotube for incubation, washing and elution. After 4 rounds of screening, the specific monoclonal antibodies against the antigen are enriched.(3) Cell screening: The prepared phages after negative screening and the antigen overexpressing cells are incubated, washed and eluted. After 4 rounds of screening, the specific monoclonal antibodies against the antigen are enriched.

[0063] The results showed that the positive phages were enriched to different degrees after the second, third and fourth rounds of solid-phase screening and cell screening. The specific data are shown in Table 6.

[0064] Table 6 Screening data statistics table

[0065]

[0066]

[0067] Note: N / A means not detected or not applicable.

[0068] 3.2 Monoclonal primary screening

[0069] The immune library open recruitment results showed that a plurality of good enrichment phage output collections (pools) were obtained by using solid phase and cell cross screening. Monoclonal antibodies were selected from the pools enriched in the four rounds of screening, and FACS detection screening was performed using Hu-18.2-HEK293 cells. A total of 270 clones were selected, and 22 positive clones that combined with Hu-18.2-HEK293 cells were obtained. Sequencing analysis was performed on the 22 positive clones, and 16 unique sequences were obtained.

[0070] The preliminary screening results are summarized in Table 7.

[0071] Table 7 FACS preliminary screening result statistical summary table

[0072]

[0073] According to the sequence diversity analysis comparison, and removing the molecules containing post-translational modification sites, 12 antibody clones were selected for full-length construction, the 12 antibody clones were C002, C003, C005, C008, C013, C022, C004, C007, C011, C012, C017 and C019, the amino acid sequences thereof were SEQ ID NO: 16-27, and the encoding nucleotide sequences thereof were SEQ ID NO: 28-39.

[0074] 4. Preparation and detection of llama immune library candidate molecules

[0075] 4.1 Construction of expression plasmid of full-length antibody protein

[0076] The coding nucleotide sequence of the nanobody was fused with the coding nucleotide sequence of the Fc segment of human IgG1 (the amino acid sequence and the encoding nucleotide sequence thereof were SEQ ID NO: 40 and SEQ ID NO: 41, respectively) for full gene synthesis, and was constructed into pcDNA3.4 vector (Invitrogen) to obtain the expression plasmid of VHH-Fc fusion protein, i.e., full-length antibody protein.

[0077] 4.2 Expression and purification of full-length antibody protein

[0078] The constructed antibody plasmid was transfected into Expi CHO cells (Gibco, A29133) for transient expression, and the antibody expression volume was 10 mL, and the expression time was 7 days. The expressed protein was purified and subpackaged, and the purified protein was detected by SDS-PAGE, SEC, FACS / ELISA, affinity kinetics, etc.

[0079] 4.3 SDS-PAGE identification of full-length antibody protein

[0080] Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to identify the molecular weight and purity of the full-length antibody protein, the method and steps are as follows: (1) Preparation of purified protein sample solution: Non-reducing sample solution preparation: Mix the purified sample solution, 4x LDS loading buffer (Zeleaves, ZY6SL1197) and iodoacetamide (Zeleaves, ZY144) according to the proportion, so that the final concentration of iodoacetamide is 40 mM, the non-reducing sample loading amount is 1 μg, and the mixed sample is placed in a 75°C dry bath instrument for heating for 10 min. Preparation of reduced sample solution: Mix the purified sample solution, 4x LDS loading buffer and dithiothreitol (DTT) (Zeleaves, ZY3483) according to the proportion, so that the final concentration of DTT is 5 mM, the reduced sample loading amount is 2 μg, and the mixed sample is placed in a 100°C dry bath instrument for heating for 10 min. (2) Electrophoresis: 140V, 75min. (3) Staining, decolorizing and scanning: Coomassie brilliant blue staining, after decolorizing, scanning with EPSON V550 color scanner. (4) Calculate purity: Calculate the purity of the reduced band by peak area normalization method, or the purity of the sum of heavy chain and light chain.

[0081] System suitability criteria: The reference product IPI (Ipilimumab) has a non-reducing band molecular weight of about 150 kDa and a purity of more than 90%; the reduced heavy chain has a molecular weight of about 50 kDa, the light chain has a molecular weight of about 25 kDa, and the heavy chain plus light chain has a purity of more than 90%. 4.4 SEC identification of full-length antibody protein

[0082] Size Exclusion Chromatography (SEC) is used to identify the purity of the full-length antibody protein, and the method and steps are as follows: (1) Mobile phase preparation: Prepare 0.15M PB+NaCl, pH adjusted to 6.0. (2) Sample treatment: Dilute the sample concentration to 0.5 mg / mL. (3) Column conditions: Use XBridge BEH SEC 3.5μm, 7.8x300mm, column temperature set to 20°C, and detection baseline stable. (4) Parameter settings: Flow rate set to 0.8 mL / min; sample injection volume set to 20 μL; detection wavelength 280 nm, bandwidth 4 nm, reference wavelength 360 nm, bandwidth 100 nm, peak width (response time) > 0.1 min (2s response time); slit 4 nm; negative absorbance baseline 100 mAU.

[0083] System suitability criteria: the reference product Herceptin (trastuzumab) monomer purity is greater than 95%, the separation degree of BSA monomer and dimer is greater than 1.5, and the baseline is smooth, which is considered to pass the system suitability.

[0084] 4.5 Affinity kinetics of full-length antibody proteins

[0085] Surface plasmon resonance (SPR) technology based on Biacore T200 (Cytiva) platform was used to detect the interaction between candidate antibody proteins and antigens. The affinity detection process includes capture, equilibrium, binding, dissociation and regeneration, and the parameter settings are shown in Table 8.

[0086] Table 8 Affinity detection parameter settings

[0087]

[0088] Note: The values of time and sample preparation concentration are for reference only, and the specific values can be optimized and adjusted according to the experimental situation.

[0089] System suitability criteria: Chi 2 ≤10%×Rmax. Chi 2 : the degree of fitting of the fitting line to the experimental line, Rmax: the highest response value of curve fitting.

[0090] 4.6 ELISA detection of full-length antibody proteins

[0091] Method and steps of ELISA detection of full-length antibody proteins: (1) plate coating: dilute the antigen Hu-18.2-VLP with 1×PBS to a concentration of 2 μg / mL, add 30 μL / well to the 96-well ELISA plate, and coat at 4°C overnight. (2) blocking: wash the plate with PBST 3 times, add blocking solution (5% PBSM), and block at room temperature for 2 h. (3) incubation: wash the plate, add 30 μL / well of 1% PBSM diluted sample, and incubate at room temperature for 60 min. (4) secondary antibody incubation: wash the plate with PBST 3 times, add secondary antibody Goat-Anti-Human-IgG-Fc-HRP (purchased from Abeam), and incubate at room temperature for 50 min. (5) color development: wash the plate with PBST 3 times, add 30 μL TMB (purchased from SurModics) per well. (6) termination: add 2M termination solution to terminate the reaction and detect OD 450 .

[0092] System suitability criteria, as shown in Table 9:

[0093] Table 9

[0094] Serial number Determination index Release standard 1 Sample name accuracy Completely consistent 2 [R 2 ]]> ≥0.90 3 Positive antibody up and down platform OD ratio ≥3 4 Blank control OD value (background value) ≤0.25

[0095] In the above detection, the positive control antibody is IMAB362 (Zolbetuximab, a monoclonal antibody targeting Claudin-18.2), the negative control antibody NC is IPI, the blank control BC is 1% PBSM; the negative control antigen is GAG-VLP (virus-like particles expressing GAG protein of human immunodeficiency virus HIV-1, purchased from Tri-Ethical Biotech Co., Ltd.).

[0096] 4.7 FACS detection of full-length antibody proteins

[0097] FACS detection was performed on the binding activity of 12 full-length expressed fusion antibody proteins to three human pancreatic cancer cells Hu-18.2-BxPC-3, human gastric cancer cells Hu-18.2-NUGC4 and Hu-18.2-HEK293 cells overexpressing human CLDN18.2, and one human CLDN18.1 overexpressing cell Hu-CLDN18.1-HEK293 (the above four cell lines were purchased from Tri-Ethical Biotech Co., Ltd.), and the method and steps are as follows:

[0098] (1) Cell plating: Transfer the cells in the culture bottle to a centrifuge tube, centrifuge to remove the supernatant, resuspend and count with culture medium, then adjust the cell density to 1 x 10 6 cells / mL. Take a 96-well round-bottom plate, add cells to the plate with a 100 μL pipette gun, 100 μL per well. Centrifuge at 300 g / min for 5 min. Remove the supernatant.

[0099] (2) Addition of antibody protein sample: Dilute the antibody protein sample with 2% FBS FACS to 8 concentration gradients: 150.000, 37.500, 9.375, 2.3438, 0.5859, 0.1465, 0.0366, 0.0092 nM. Add 100 μL of 12-way pipette to the 96-well cell plate, 100 μL per well. Mix well and incubate in a 4°C refrigerator for 1 h.

[0100] (3) Addition of secondary antibody: Dilute the secondary antibody PE labelled anti-Human Fc (Jackson) 1:200 with 2% FBS FACS Buffer, centrifuge the cell culture plate to remove the supernatant, and add 100 μL of the secondary antibody dilution to the cell culture plate using a 100 μL 12-way pipette. Incubate the cell culture plate in a 4°C refrigerator for 30 min. Centrifuge to remove the supernatant, wash the plate twice with FACS Buffer, and resuspend the cells in 120 μL of FACS Buffer per well.

[0101] (4) Data collection: Open the flow cytometer (purchased from Beckman), and after the instrument is cleaned, perform FACS combined detection.

[0102] In the above detection, the positive control antibody is IMAB362, the isotype control antibody is IgG1 (P93950-1, purchased from San Yoo), the control added only secondary antibody is marked as "cell sec", and the blank cell control without any antibody is marked as "cell only".

[0103] 4.8 Experimental results

[0104] (1) Physicochemical property detection results

[0105] 12 candidate antibody molecules fused with Fc were successfully prepared into target proteins after plasmid construction, protein expression and purification, and the physicochemical property detection results thereof are shown in Table 10:

[0106] Table 10: Physicochemical property detection results

[0107]

[0108]

[0109] The results show that: (1) the purified protein amount of the 12 target proteins is 0.03 mg to 3.19 mg, and three proteins (C007, C011 and C012) are not subjected to SDS-PAGE detection, SEC detection and subsequent affinity kinetics detection due to low purification amount. (2) After SDS-PAGE detection, the purity of the 9 tested samples is greater than 95%. (3) After SEC detection, the purity of C022 (P237142) does not reach 90%, and the purity of the remaining 8 tested samples is greater than 90%.

[0110] (2) Affinity kinetics detection results

[0111] The affinity kinetics detection results of the 9 target proteins show that the affinity with the antigen is better than that of the positive control antibody IMAB362, and all meet Chi 2 ≤10% x Rmax, meeting the system suitability standard, and the results are reliable. See Table 11.

[0112] Table 11: Affinity kinetics detection results

[0113]

[0114] Note: Chi 2 : fitting degree of the fitting line and the experimental line; Rmax: highest response value of curve fitting.

[0115] (3) ELISA detection results

[0116] ELISA results of binding of 12 candidate antibody proteins fused with Fc to antigen Hu-18.2-VLP and negative control antigen GAG-VLP are as follows: Figure 2 to Figure 3 And Table 12.

[0117] Table 12 Summary of Candidate Antibody ELISA Detection Results (EC) 50 (μg / mL)

[0118]

[0119]

[0120] Note: / indicates not tested, N indicates no binding, W indicates weak binding, Y indicates binding but EC cannot be calculated. 50 value.

[0121] ELISA results showed that among the 12 Fc fusion antibody proteins, 9 antibodies exhibited strong binding activity to the hu-18.2-VLP antigen, EC... 50 The values ​​ranged from 0.00355 μg / mL to 0.01172 μg / mL. None of the 12 antibody proteins showed nonspecific binding to GAG-VLP.

[0122] (4) Candidate antibody FACS detection results

[0123] The FACS results of the candidate antibodies are as follows Figure 4 to Figure 7 .

[0124] Table 13 Summary of EC50 results (nM) of purified candidate antibodies detected by FACS

[0125] Serial number Protein number Sample name Hu-18.2-HEK293 hu-18.1-HEK293 Hu-18.2-BxPC-3 Hu-18.2-NUGC4 1 P237129 C002 2.519 N 5.320 2.340 2 P237130 C003 6.040 N 5.320 3.850 3 P237131 C004 3.643 Y 4.350 1.100 4 P237132 C005 3.298 N 4.430 4.000 5 P237133 C007 N N N / A N / A 6 P237134 C008 3.411 N 2.170 4.360 7 P237135 C011 W Y W W 8 P237136 C012 N N N N 9 P237137 C013 3.113 N 6.190 2.360 10 P237139 C017 2.113 Y 4.550 1.130 11 P237140 C019 W Y 4.830 W 12 P237142 C022 6.179 N 6.090 6.530

[0126] Note: 1) Y represents good binding, W represents weak binding, N represents no binding, and N / A represents no detection.

[0127] FACS detection results showed that: among the 12 purified antibody samples, 9 antibody samples (C002, C003, C004, C005, C008, C013, C017, C019 and C022) had better binding on Hu-18.2-BxPC-3 cells, and the EC50 value range was 2.170nM-6.190nM; 8 antibody samples (C002, C003, C004, C005, C008, C013, C017 and C022) had better binding on Hu-18.2-HEK293 and HU-18.2-NUGC4 cells, and the EC50 value range of antibody binding to the two cells was divided into 2.113nM-6.179nM and 1.100nM-6.530nM; 4 candidate antibody samples (C004, C011, C017 and C019) had non-specific binding on Hu-CLDN18.1-HEK293 cells; 6 antibody samples (C002, C003, C005, C008, C013 and C022) had specific binding to three CLDN18.2-expressing cells Hu-18.2-BxPC-3, Hu-18.2-HEK293 and HU-18.2-NUGC4, and had no binding to CLDN18.1-expressing cell Hu-CLDN18.1-HEK293.

[0128] The amino acid sequences of the VHH of the 6 nanobody clones specifically binding to CLDN18.2 were analyzed for CDR1-3 using the Kabat numbering scheme and definition scheme, and the results are shown in Table 14.

[0129] Table 14 Amino acid sequences of complementarity determining regions (CDR1-3) of 6 candidate nanobodies

[0130]

[0131]

[0132] Although the embodiments of the present application have been shown and described above, it should be understood that the above-described embodiments are exemplary and should not be construed as limiting the present application, and those of ordinary skill in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A nanobody targeting Claudin 18.2, characterized in that, The amino acid sequence of the complementarity determining region of the nanobody targeting claudin 18.2 comprises CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 5, and CDR3 as shown in SEQ ID NO:

10.

2. The Nanobody targeting Claudin 18.2 according to claim 1, characterized in that, The amino acid sequence of the nanobody targeting claudin 18.2 is shown in SEQ ID NO:

16.

3. A nucleic acid, characterized in that, The nucleic acid sequence encoding the nanobody targeting claudin 18.2 according to any one of claims 1 to 2.

4. The nucleic acid of claim 3, wherein, The nucleotide sequence encoding the nanobody targeting claudin 18.2 is shown in SEQ ID NO:

28.

5. A fusion protein, characterized in that, The fusion protein is a recombinant protein produced by fusing the nanobody targeting claudin 18.2 according to claim 1 or 2 with the Fc fragment of immunoglobulin.

6. The fusion protein of claim 5, wherein, The amino acid sequence of the Fc fragment of immunoglobulin is shown in SEQ ID NO:

40.

7. The fusion protein of claim 5 or 6, wherein, The nucleotide sequence encoding the Fc fragment of immunoglobulin is shown in SEQ ID NO:

41.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the nanobody targeting claudin 18.2 according to any one of claims 1 to 2, or the nucleic acid according to any one of claims 3 to 4, or the fusion protein according to any one of claims 5 to 7.

9. The pharmaceutical composition of claim 8, wherein, The dosage form of the pharmaceutical composition comprises at least one of microspheres, microcapsules, and nanocapsules.

10. Use of the nanobody targeting claudin 18.2 according to any one of claims 1 to 2, or the nucleic acid according to any one of claims 3 to 4, or the fusion protein according to any one of claims 5 to 7, or the pharmaceutical composition according to any one of claims 8 to 9 in the preparation of a detection reagent or a live imaging probe targeting claudin 18.2.

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