Targeting claudin 18.2 nano antibody and application thereof

Abstract

The invention relates to a nano antibody 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 nano antibody for targeting claudin 18.2 comprises a CDR1 as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 4, a CDR2 as shown in any one of SEQ ID NO: 5 to SEQ ID NO: 9 and a CDR3 as shown in any one of SEQ ID NO: 10 to SEQ ID NO: 15. According to the invention, an alpaca immune bacteriophage display library is constructed, a series of nano antibody molecules specifically targeting claudin 18.2 are screened from the alpaca immune bacteriophage display library, and functions and applications of the nano antibody are verified at CLDN18.2 antigen protein and CLDN18.2 target cell levels.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology and molecular biology, and in particular to a nano antibody targeting claudin 18.2 and an application thereof. Background Art

[0002] Tight junctions (TJs) are an important cell adhesion structure, mainly existing in the junction complex between epithelial cells and endothelial cells, which makes the adjacent cell membranes close together to form a physical barrier structure surrounding the cells. The tight junction membrane domain contains at least three different proteins, called occludins, claudins and junctio nal adhesion molecules (JAMs). 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 both the N-terminus and C-terminus are located in the cytoplasm. CLDN18 is a widely studied claudin in the CLDNs protein family and was first identified in 2001. The human CLDN18 gene has two splice variants, encoding two protein subtypes, CLDN18.1 and CLDN18.2 (claudin 18.2), both of which are composed of 261 amino acids and have two extracellular loops, with only 8 amino acids different in extracellular loop 1. In the human body, the expression of CLDN 18.1 and CLDN18.2 is tissue-specific. CLDN18.1 is mainly expressed in lung tissue, and CLDN18.2 is only expressed in differentiated gastric mucosal epithelial cells in normal tissues, not in gastric stem cells. However, CLDN 18.2 is highly expressed in a variety of tumor tissues, 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 gastric mucosa exists in the cell tight junction supramolecular complex, and the antigen epitope is difficult to contact with targeted drugs. However, the loss of polarity in malignant tumor cells leads to the exposure of CLDN18.2 epitopes, which can contact with targeted drugs. This characteristic makes CLDN18.2 an ideal target for targeted therapy of digestive tract tumors.

[0003] So far, the global R&D pipeline for CLDN18.2 targets covers all mainstream technology routes, including monoclonal antibodies, antibody-drug conjugates (ADCs), bispecific antibodies and chimeric antigen receptor T cells (CAR-T), among which monoclonal antibodies and ADC routes have the most layout. The fastest-progressing targeted drug is the CLDN18.2 monoclonal antibody drug IMAB362 (Zolbetuxi mab) developed by the Japanese pharmaceutical company Astellas, which has been approved by the Ministry of Health, Labor and Welfare of Japan (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 CLDN18.2 CAR-T is CT041, an autologous CAR-T cell candidate product developed by Coherus Pharmaceuticals for the treatment of gastric cancer, pancreatic cancer and other solid tumors. The relevant clinical trials have entered Phase II. These studies have shown the safety of the CLDN18.2 target and the effectiveness of targeted therapy.

[0004] Heavy chain antibodies are single heavy chain domain antibodies that are naturally missing light chains and are found in cartilaginous fish such as camelids, nurse sharks, star sharks and rays. By cloning the variable region of heavy chain antibodies, single domain antibodies (sdAb) consisting only of the heavy chain variable region can be obtained. Because of their small molecular weight, they are also called nanobodies (Nbs). Although the molecular weight of nanobodies is only 1 / 10 of that of complete traditional antibodies (about 15kDa), they retain the complete antigen recognition and binding ability of heavy chain antibodies. Compared with traditional antibodies, they have the advantages of strong specificity, strong affinity, high stability, strong targeting and tissue penetration, low immunogenicity, and easy humanization. The disadvantage of nanobodies is that they have a short half-life, but they can be fused with anti-serum albumin or the Fc segment of antibodies to prolong their half-life in the blood. Nanobodies have been widely used in the study of biochemical mechanisms and structural biology, as well as the development of diagnostic reagents and therapeutic drugs for diseases such as tumors.

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

[0006] The technical problem to be solved by the present invention is to provide a nano antibody that specifically targets claudin 18.2 but does not recognize CLDN18.1 and its application.

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

[0008] In the first aspect, a nanobody targeting claudin 18.2, the amino acid sequence of the complementary determining region of the nanobody targeting claudin 18.2 includes a CDR1 as shown in any one of SEQ ID NOs: 1 to 4, a CDR2 as shown in any one of SEQ ID NOs: 5 to 9, and a CDR3 with an amino acid sequence as shown in any one of SEQ ID NOs: 10 to 15.

[0009] Furthermore, the amino acid sequence of the nanobody targeting claudin 18.2 is shown in any one of SEQ ID NOs: 16 to 21.

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

[0011] Furthermore, the nucleotide sequence encoding the nanobody targeting claudin 18.2 is shown in any one of SEQ ID NOs: 28 to 33.

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

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

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

[0015] In a fourth aspect, a pharmaceutical composition comprises the nano-antibody targeting claudin 18.2, or the nucleic acid, or the fusion protein.

[0016] Furthermore, the dosage form of the pharmaceutical composition includes at least one of microsphere preparation, microcapsule preparation, and nanocapsule preparation, and also includes pharmaceutically acceptable excipients and / or carriers.

[0017] In the fifth aspect, a nano-antibody targeting claudin 18.2, or a nucleic acid, or a fusion protein, or a pharmaceutical composition is used in the preparation of a detection reagent, in vivo imaging probe or therapeutic product targeting claudin 18.2.

[0018] The core innovation of the present invention is that it provides a group of 6 nano antibody molecules that specifically target claudin 18.2 (CLDN18.2) but do not recognize CLDN18.1. No antibodies with the same CDR region sequence as the antibody of the present invention have been found in the prior art, which solves the problem of the lack of candidate nano antibody molecules that specifically target CLDN18.2 in the prior art.

[0019] The beneficial effects of the present invention are:

[0020] (1) The CLDN18.2 target selected by the present invention is the potential safest and most effective target for the treatment of digestive system tumors: CLDN18.2 is only expressed in the tight junctions between gastric mucosal epithelial tissue cells in normal tissues and is not exposed to 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; existing studies have shown that CAR-T or antibody drugs targeting CLDN18.2 do not damage normal expressing tissues, but have a specific killing effect on CLDN18.2-expressing tumor tissues.

[0021] (2) The present invention provides candidate nano-antibody molecules for the targeted treatment of digestive system tumors: Compared with blood tumors, the research and development of specific immune targeted therapeutic drugs for solid tumors has been slow, and one of the limiting factors is the lack of effective and safe targets. Current studies have shown that CLDN18.2 is a safe and effective therapeutic target for digestive system tumors, but the research on targeted drugs for CLDN18.2 started relatively late, and only one monoclonal antibody drug has just been launched in a few countries worldwide. The bottleneck is in the antibody discovery stage. It is difficult to develop antibody molecules that specifically target CLDN18.2 but do not recognize CLDN18.1, and the nano-antibodies involved are even rarer.

[0022] (3) The nanobodies of the present invention have a wide range of applications: (a) The present invention screened 6 candidate nanobodies against CLDN18.2 by alpaca immune library, fused their C-termini with the Fc fragment of human IgG1, and tested their binding activity (ELISA test) and affinity (SPR test) with the CLDN18.2 antigen at the level of antigen protein CLDN18.2-VLP. The results were better or equivalent to those of the traditional control antibody IMAB362. At the level of various cells overexpressing CLDN18.2, FACS detected the binding activity of Fc fusion antibodies with the CLDN18.2 antigen, and 4 nanobodies were better than the traditional control antibody IMAB362. (b) Nanobodies have significant advantages over other antibodies, such as their half-life can be adjusted by chemical modification or protein fusion modification, strong permeability, can recognize hidden epitopes that are inaccessible to ordinary antibodies, are resistant to pepsin, acid, heat, and easy to produce, and because they are single-chain and have a small molecular weight, they are 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 invention can be developed into immune detection reagents, CAR-T / NK immune cell drugs, and antibody drugs. Combined with the characteristics of the CLDN18.2 target, the candidate antibodies of the present invention, if used as immune diagnostic reagents for cancer, will have better effects; if developed into cell or antibody immunotherapy drugs, they have lower toxic side effects and better clinical efficacy, thereby providing patients with more drug options.

[0023] (4) Can be used to develop dual-target drugs: Studies have found that claudin 18.2, like CDH17, is a safe and effective target for the treatment of digestive system tumors. The two are co-expressed in digestive system tumors such as esophageal cancer, gastric cancer, pancreatic cancer, and colorectal cancer. Clinical or preclinical trials have shown that targeting these two targets can specifically kill tumor cells without harming normal tissues. Therefore, the anti-CLDN18.2 candidate nanoantibody of the present invention can be used in combination with the CDH17 antibody to develop anti-CLDN18.2 / CDH17 dual-antibody drugs and dual-target CAR-T / NK drugs, which is expected to enhance the efficacy of monoclonal antibodies or single-target CAR-T / NK and other immune cell drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Flowchart for the construction of a CLDN18.2 alpaca immune single domain antibody library;

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

[0026] Figure 3ELISA results of the candidate antibody protein expressed by fusion with Fc and binding to the negative control antigen GAG-VLP;

[0027] Figure 4 The results of FACS detection of the candidate antibody protein expressed by fusion with Fc and the cell Hu-18.2-HEK293 overexpressing the human antigen CLDN18.2;

[0028] Figure 5 The FACS detection results of the candidate antibody protein expressed by fusion with Fc and the cell Hu-CLDN18.1-HEK293 overexpressing the human antigen CLDN18.1;

[0029] Figure 6 The results of FACS detection of the candidate antibody protein expressed by fusion with Fc and the cell Hu-18.2-BxPC-3 overexpressing the human antigen CLDN18.2;

[0030] Figure 7 The figure shows the FACS detection result of the candidate antibody protein expressed by fusion with Fc and binding to the cell Hu-18.2-NUGC4 overexpressing the human antigen CLDN18.2. DETAILED DESCRIPTION

[0031] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0032] Example

[0033] 1. Alpaca Immunity

[0034] 1.1 Alpaca immunization method

[0035] Two alpacas (Alpaca) (number: #1, #2) were cross-immunized with virus-like particles (VLP) Hu-18.2-VLP (purchased from Sanyou Biotechnology) expressing full-length human CLDN18.2 antigen, and full-length human CLDN18.2 overexpressing cell lines Hu-18.2-HEK293 and Hu-18.2-KATOⅢ (purchased from Sanyou Biotechnology). Each alpaca was immunized with 500 μg antigen or 1.00×10 7 Cells. The first antigen immunization was Freund's complete adjuvant (Sigma), and the subsequent immunization was Freund's incomplete adjuvant (Sigma). No adjuvant was added to the cell immunization. The injection method was subcutaneous multiple injections. The immunization scheme for alpacas is shown in Table 1.

[0036] Table 1 Alpaca immunization scheme

[0037]

[0038] 1.2ELISA serum titer test

[0039] The plate was coated with Hu-18.2-VLP antigen (2μg / mL, 30μL) and incubated overnight at 4℃; the blocking solution was 5% PBSM (PBS, 5% skim milk powder) at room temperature for 2h; 3-fold gradient dilution of serum was added and incubated at room temperature for 1h; the secondary antibody Goat anti-LlamaIgG ​​(H+L) Secondary Antibody, HRP (ThermoFisher, A16060) was added and incubated for 1h; 30μl TMB (purchased from SurModics) was added for color development at room temperature for 30min; 50μl TMB color development stop solution (purchased from Biyuntian, P0215) was added to terminate the reaction, and the OD450 value of the microplate reader (Molecular Devices) was read. The serum titer was calibrated according to 1.65 times NC, and the titer was greater than 32K for qualified. The ELISA test results of the two immunized alpacas are shown in Table 2. After ELISA serum titer detection, the results showed that the immune titers of both alpacas were qualified.

[0040] Table 2 Efficacy of serum against Hu-18.2-VLP

[0041] Serum to be tested Immunization times Potency (k) #1 4 64 #2 4 128

[0042] 2. Construction of alpaca immune library

[0043] 2.1 Immune library construction

[0044] Blood was collected from two alpacas after four immunizations, and immune libraries were constructed. 80 ml of peripheral blood was collected from each alpaca, and peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Paque density gradient separation solution (GE, 17144003S). RNA was extracted from the separated PBMCs, and reverse transcribed into cDNA by reverse transcription kits (TaKaRa, 6210A) to obtain all antibody nucleotide sequences. Primers were designed on the front end of the germline gene VH and CH2, and PCR obtained two DNA fragments of different sizes. The smaller target fragment was recovered by cutting the gel; by comparing all the sequences of all V genes and J genes of the VHH antibody, degenerate primers containing NcoI and NotI restriction sites were designed, and then all VHH gene fragments were amplified using the recovered DNA fragment products as templates. The phage display vector and VHH fragment were digested and ligated, and the ligation product was recovered by a recovery kit (Omega, D6492-02) and transformed into competent Escherichia coli SS320 (Lucigen, MC1061F) using an electroporator (Bio-Rad, MicroPulser), thereby obtaining camel-derived immune phage display libraries (RA044 and RA045). Figure 1 shown.

[0045] 2.2 Determination of single domain antibody library capacity

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

[0047] Table 3 Statistics of electroporation system and storage capacity

[0048] Library Name Number of electroporations Single electroporation capacity (CFU) Total reservoir capacity (CFU) RA044 2 <![CDATA[6.75×10 8 ]]> <![CDATA[1.35×10 9 ]]> RA045 1 <![CDATA[3.75×10 8 ]]> <![CDATA[3.75×10 8 ]]>

[0049] 2.3 Analysis of single domain antibody library sequencing results

[0050] The clones were picked for sequencing, and the successful sequencing results were selected for further analysis. The sequencing analysis summary is shown in Table 4.

[0051] Table 4 Statistics of single domain antibody library sequencing information

[0052]

[0053] The antibody gene sequence analysis standards for the construction of the single-domain antibody library are that the correct insertion rate is greater than or equal to 80%, the empty ratio is less than or equal to 10%, and the correct rate of restriction sites is greater than or equal to 90%; the effective library capacity is calculated as follows: total library capacity × antibody correct expression rate × unique sequence ratio = effective library capacity.

[0054] 2.4 Analysis results of antibody genes in single domain antibody library

[0055] The analysis of the sequences successfully sequenced in the single domain antibody library is shown in Table 5. The analysis shows that the empty load ratio of RA044 and RA045 is less than 10%, the correct insertion rate of the antibody gene is greater than 80%, and the CDR region alignment has a high richness, which meets the quality standards for the construction of the single domain antibody library. In summary, the quality of the construction of the RA044 and RA045 single domain antibody libraries is qualified.

[0056] Table 5 Summary of library construction indicators

[0057]

[0058] 3. Screening of Alpaca Immune Library

[0059] The constructed alpaca immune library antibody sequence is displayed on the phage surface through phage display technology, and then specific proteins and cells are used as antigen materials. After multiple rounds of screening, the phage-displayed antibodies binding to the antigen are continuously enriched, and the positive clones that specifically bind to the antigen are further screened through ELISA, and the positive clone antibody sequence is obtained by 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 bacteria or the obtained output collection is subjected to a series of steps such as inoculation, auxiliary phage infection, phage expansion, phage precipitation and resuspension to prepare enriched phages and put them into the next step of screening. (2) Solid phase screening: The antigen is coated on the surface of an immunotube with high adsorption capacity, and then the prepared phage is added to the immunotube for incubation, washing and elution. After 4 rounds of panning, the specific monoclonal antibodies against the antigen are enriched. (3) Cell screening: The prepared negatively screened phages and antigen-overexpressing cells are incubated, washed and eluted. After 4 rounds of panning, the specific monoclonal antibodies against the antigen are enriched.

[0063] The results showed that the positive phages were enriched to varying 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 Statistics of audition data

[0065]

[0066]

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

[0068] 3.2 Monoclonal screening

[0069] The results of the immune library screening showed that multiple sets of well-enriched phage output sets (Pools) were obtained by solid phase and cell cross-screening. Single clones were selected from the enriched Pools in the four rounds of screening and screened by FACS detection using Hu-18.2-HEK293 cells. A total of 270 clones were picked, and 22 positive clones that bound to Hu-18.2-HEK293 cells were obtained. The 22 positive clones were selected for sequencing analysis, and 16 molecules with unique sequences were obtained.

[0070] The summary statistics of the initial screening results are shown in Table 7.

[0071] Table 7 Statistical summary of FACS initial screening results

[0072]

[0073] Based on sequence diversity analysis and comparison, and removing molecules containing post-translational modification sites, 12 antibody clones were selected for full-length construction. The 12 antibody clones were numbered: C002, C003, C005, C008, C013, C022, C004, C007, C011, C012, C017 and C019, whose amino acid sequences are shown in SEQ ID NOs: 16-27, and their encoding nucleotide sequences are shown in SEQ ID NOs: 28-39.

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

[0075] 4.1 Construction of expression plasmid for 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 (whose amino acid sequence and coding nucleotide sequence are shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively) for whole gene synthesis and constructed into a pcDNA3.4 vector (Invitrogen) to obtain an expression plasmid for 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. The antibody expression volume was 10 mL and the expression time was 7 days. The expressed protein was purified and packaged; the purified protein was tested 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) was used 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: Preparation of non-reducing sample solution: The purified sample solution, 4×LDS loading buffer (Zeye Biotechnology, ZY6SL1197) and iodoacetamide (Zeye Biotechnology, ZY144) were mixed in proportion to make the final concentration of iodoacetamide 40mM and the non-reducing sample loading amount 1μg. The mixed sample was placed in a 75℃ dry bath and heated for 10min. Preparation of reducing sample solution: The purified sample solution, 4×LDS loading buffer and dithiothreitol (DTT) (Zeye Biotechnology, ZY3483) were mixed in proportion to make the final concentration of DTT 5mM and the reducing sample loading amount 2μg. The mixed sample was placed in a 100℃ dry bath and heated for 10min. (2) Electrophoresis: 140V, 75min. (3) Staining, destaining, and scanning: The gel was stained with Coomassie Brilliant Blue and scanned with an EPSON V550 color scanner after destaining. (4) Purity calculation: ImageJ was used to calculate the purity of the reduced bands or the purity of the reduced heavy chain plus the light chain according to the peak area normalization method.

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

[0082] Size Exclusion Chromatography (SEC) was used to identify the purity of the full-length antibody protein. The method and steps are as follows: (1) Mobile phase preparation: Prepare 0.15M PB+NaCl and adjust the pH to 6.0. (2) Sample treatment: Dilute the sample concentration to 0.5 mg / mL. (3) Chromatographic column conditions: Use XBridge BEH SEC 3.5μm, 7.8×300mm, column temperature set at 20℃, detection baseline stable. (4) Parameter settings: flow rate set at 0.8mL / min; sample injection volume set at 20μL; detection wavelength 280nm, bandwidth 4nm, reference wavelength 360nm, bandwidth 100nm, peak width (response time) > 0.1min (2s response time); slit 4nm; negative absorbance baseline 100mAU.

[0083] System suitability criteria: If the purity of the reference product Herceptin (trastuzumab) monomer is greater than 95%, the separation between BSA monomer and dimer is greater than 1.5, and the baseline is stable, the system is considered to have passed the suitability test.

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

[0085] The interaction between the candidate antibody protein and the antigen was detected using the surface plasmon resonance (SPR) technology based on the Biacore T200 (Cytiva) platform. The affinity detection process includes capture, equilibrium, binding, dissociation and regeneration. The parameter settings are detailed in Table 8.

[0086] Table 8 Affinity detection parameter settings

[0087]

[0088] Table Notes: The time and sample concentration values ​​are for reference only. The specific values ​​can be optimized and adjusted according to the experimental conditions.

[0089] System suitability standard: Chi 2 ≤10%×Rmax. 2 : The degree of fit between the fitting line and the experimental line, Rmax: The maximum response value of the curve fitting.

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

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

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

[0093] Table 9

[0094] Serial number Judgment index Release criteria 1 Sample name accuracy completely consistent 2 <![CDATA[R 2 ]]> ≥0.90 3 Positive antibody upper and lower platform OD ratio ≥3 4 Blank control OD value (background value) ≤0.25

[0095] In the above test, the positive control antibody was IMAB362 (Zolbetuximab, a monoclonal antibody targeting Claudin-18.2), the negative control antibody NC was IPI, and the blank control BC was 1% PBSM; the negative control antigen was GAG-VLP (virus-like particles expressing the GAG ​​protein of human immunodeficiency virus HIV-1, purchased from Sanyou Biotechnology).

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

[0097] 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 all purchased from Sanyou Biotechnology) was detected by FACS, and the method and steps are as follows:

[0098] (1) Cell plating: Transfer cells from the culture flask to a centrifuge tube, centrifuge to remove the supernatant, resuspend in culture medium, count, and adjust the cell density to 1×10 6 cells / mL. Take a 96-well round-bottom plate and use a 100μL pipette to add the cells to each well. Centrifuge at 300g / min for 5min. Discard the supernatant.

[0099] (2) Addition of antibody protein samples: Dilute the antibody protein samples into 8 dilutions with a concentration gradient using FACS containing 2% FBS: 150.000, 37.500, 9.375, 2.3438, 0.5859, 0.1465, 0.0366, 0.0092 nM. Use a 100 μL 12-channel pipette to add the antibody dilution to the 96-well cell plate, 100 μL per well. Mix well and incubate in a 4°C refrigerator for 1 hour.

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

[0101] (4) Data acquisition: Turn on the flow cytometer (purchased from Beckman), and after the instrument is cleaned, perform FACS binding detection.

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

[0103] 4.8 Experimental Results

[0104] (1) Physical and chemical properties test results

[0105] The 12 candidate antibody molecules fused with Fc were successfully prepared through plasmid construction, protein expression and purification. The results of the physical and chemical property tests are shown in Table 10:

[0106] Table 10 Summary of physical and chemical properties test results

[0107]

[0108]

[0109] The results showed that: (1) The amount of protein purified from the 12 target proteins was 0.03 mg to 3.19 mg. Three proteins (C007, C011 and C012) were not subjected to SDS-PAGE, SEC and subsequent affinity kinetics tests due to their low purification amount. (2) The purity of the nine tested samples was greater than 95% after SDS-PAGE. (3) The purity of C022 (P237142) did not reach 90% after SEC, while the purity of the remaining eight tested samples was greater than 90%.

[0110] (2) Affinity kinetics test results

[0111] The affinity kinetics of the 9 target proteins showed that their affinity to the antigen was better than that of the positive control antibody IMAB362, and all met the Chi 2 ≤10%×Rmax, meets the system suitability standard and the result is reliable. See Table 11.

[0112] Table 11 Affinity kinetics test results

[0113]

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

[0115] (3) ELISA test results

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

[0117] Table 12 Summary of candidate antibody ELISA test results EC 50 (μg / mL)

[0118]

[0119]

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

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

[0122] (4) FACS test results of candidate antibodies

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

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

[0125] Serial number Protein No. 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] Notes: 1) Y represents good binding, W represents weak binding, N represents no binding, and N / A represents not detected.

[0127] The results of FACS test showed that among the 12 purified antibody samples, 9 antibody samples (C002, C003, C004, C005, C008, C013, C017, C019 and C022) had good binding on Hu-18.2-BxPC-3 cells, with EC50 values ​​ranging from 2.170nM to 6.190nM; 8 antibody samples (C002, C003, C004, C005, C008, C013, C017 and C022) had good binding on Hu-18.2-HEK293 and HU-18.2-NUGC4 cells, with EC50 values ​​ranging from 2.170nM to 6.190nM. 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, but had no binding to CLDN18.1-expressing cells Hu-CLDN18.1-HEK293.

[0128] The amino acid sequences of the VHHs of the six Nanobody clones that specifically bind to CLDN18.2 were analyzed using the Kabat numbering scheme and definition scheme. The results are shown in Table 14.

[0129] Table 14 Amino acid sequences of the complementary determining regions (CDR1-3) of the 6 candidate Nanobodies

[0130]

[0131]

[0132] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A nanobody targeting claudin 18.2, characterized in that: The amino acid sequence of the complementary determining region of the nanobody targeting claudin 18.2 includes CDR1 as shown in any one of SEQ ID NOs: 1 to 4, CDR2 as shown in any one of SEQ ID NOs: 5 to 9, and CDR3 as shown in any one of SEQ ID NOs: 10 to 15.

2. A 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 any one of SEQ ID NOs: 16 to 21.

3. A nucleic acid, characterized in that It comprises a nucleic acid sequence encoding a nanobody targeting claudin 18.2 as described in any one of claims 1 to 2 or its complementary sequence.

4. A nucleic acid according to claim 3, characterized in that The nucleotide sequence encoding the nanobody targeting claudin 18.2 is shown in any one of SEQ ID NOs: 28 to 33.

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

6. A fusion protein according to claim 5, characterized in that: The amino acid sequence of the Fc fragment of the immunoglobulin is shown in SEQ ID NO:

40.

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

41.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a nanobody targeting claudin 18.2 as described in any one of claims 1 to 2, or a nucleic acid as described in any one of claims 3 to 4, or a fusion protein as described in any one of claims 5 to 7.

9. A pharmaceutical composition according to claim 8, characterized in that: The dosage form of the pharmaceutical composition includes at least one of a microsphere preparation, a microcapsule preparation, and a nanocapsule preparation.

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

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