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. An amino acid sequence of a complementary determining region of the nano antibody for targeting claudin 18.2 comprises a CDR1 (Complementary Determining Region) as shown in SEQ ID NO: 1, a CDR2 (Complementary Determining Region) as shown in SEQ ID NO: 5 and a CDR3 (Complementary Determining Region) as shown in SEQ ID NO: 12. 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

[0001] This invention is a divisional application of application CN202411580820.2, filed on November 7, 2024, entitled "A Nanobody Targeting Densin 18.2 and Its Application". Technical Field

[0002] This invention relates to the fields of immunology and molecular biology, specifically to a nanobody targeting dendritic protein 18.2 and its applications. Background Technology

[0003] Tight junctions (TJs) are important cell adhesion structures, primarily found in the junctional complexes between epithelial and endothelial cells, bringing adjacent cell membranes together to form a physical barrier surrounding the cell. TJ membrane domains contain at least three different proteins, called occludins, claudins, and junctional adhesion molecules (JAMs). Clauudins (CLDNs) are a class of transmembrane proteins first discovered in 1998, comprising at least 27 family members. CLDNs have four transmembrane domains, with both N-termini and C-termini located in the cytoplasm. CLDN18 is a widely studied tight protein within the CLDN family, first identified in 2001. The human CLDN18 gene has two splice variants, encoding two protein isoforms: CLDN18.1 and CLDN18.2 (dense protein 18.2). Both are composed of 261 amino acids and have two extracellular loops, differing only in extracellular loop 1 (8 amino acids). In humans, the expression of CLDN 18.1 and CLDN18.2 is tissue-specific. CLDN18.1 is mainly expressed in lung tissue, while CLDN18.2 is expressed only on differentiated gastric mucosal epithelial cells in normal tissues, not on gastric stem cells. However, CLDN 18.2 is highly expressed in various 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 the gastric mucosa is located in the supramolecular complex of cell tight junctions, making it difficult for the antigenic epitope to access targeted drugs. However, in malignant tumor cells, the loss of polarity leads to the exposure of the CLDN18.2 epitope, allowing it to come into contact with targeted drugs. This characteristic makes CLDN18.2 an ideal target for targeted therapy of gastrointestinal tumors.

[0004] To date, the global R&D pipeline targeting CLDN18.2 covers all major technological routes, including monoclonal antibodies, antibody-drug conjugates (ADCs), bispecific antibodies, and chimeric antigen receptor T cells (CAR-T), with monoclonal antibodies and ADCs being the most prevalent. The most advanced targeted therapy is IMAB362 (Zolbetuxi mab), a CLDN18.2 monoclonal antibody developed by Astellas Pharma, a Japanese pharmaceutical company. It was approved this year by both the Japanese Ministry of Health, Labour and Welfare (MHLW) and the US FDA for the treatment of CLDN18.2-positive gastric cancer patients, becoming the world's first CLDN18.2 targeted therapy. The most advanced CLDN18.2 CAR-T therapy is CT041, an autologous CAR-T cell candidate developed by CARsgen Therapeutics for the treatment of gastric cancer, pancreatic cancer, and other solid tumors. Its clinical trials have entered Phase II. These studies demonstrate the safety and efficacy of targeted therapy targeting CLDN18.2.

[0005] Heavy chain antibodies are naturally occurring single-heavy chain domain antibodies lacking the light chain, found in cartilaginous fish such as camels, nurse sharks, great white sharks, and rays. Cloning the variable region of heavy chain antibodies yields single-domain antibodies (sdAbs) consisting only of the heavy chain variable region, also known as nanobodies (Nbs) due to their small molecular weight. Although nanobodies have a molecular weight only 1 / 10 that of a complete conventional antibody (approximately 15 kDa), they retain the complete antigen recognition and binding capabilities of heavy chain antibodies. Compared to conventional antibodies, they possess advantages such as high specificity, high affinity, high stability, strong targeting and tissue penetration, low immunogenicity, and ease of humanization. The disadvantage of nanobodies is their short half-life, but this can be prolonged by fusing them with the Fc fragment of antiserum albumin or antibodies. Nanobodies have been widely used in research on biochemical mechanisms and structural biology, as well as in the development of diagnostic reagents and therapeutic drugs for diseases such as tumors.

[0006] Gastrointestinal tumors are among the most common solid tumors worldwide, with my country being a particularly high-incidence country for gastrointestinal cancers such as gastric and pancreatic cancer. While there are currently clinically developed antibodies and CAR-T drugs targeting CLDN18.2, only one drug has reached the market, highlighting the urgent need to continue developing antibodies targeting CLDN18.2. This invention aims to develop novel antibodies targeting CLDN18.2, particularly candidate nanobody molecules that specifically recognize CLDN18.2 but not CLDN18.1, for use in developing diagnostic reagents, targeted antibody drugs, and CAR-T / NK cell immunotherapy drugs for diseases such as gastrointestinal tumors. Therefore, this invention discloses a nanobody specifically targeting CLDN18.2 and its applications. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a nanobody that specifically targets CLDN18.2 without recognizing CLDN18.1 and its application.

[0008] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0009] In a first aspect, a nanobody targeting dentin 18.2, wherein the amino acid sequence of the complementarity-determining region of the nanobody targeting dentin 18.2 includes CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:12.

[0010] Furthermore, the amino acid sequence of the nanobody targeting dentin 18.2 is shown in SEQ ID NO:18.

[0011] The second aspect is a nucleic acid, including a nucleic acid sequence encoding a nanobody targeting dendritic protein 18.2.

[0012] Furthermore, the nucleotide sequence encoding the nanobody targeting dendritic protein 18.2 is shown in SEQ ID NO:30.

[0013] Thirdly, a fusion protein, wherein the fusion protein is a recombinant protein produced by fusing the aforementioned nanobody targeting dendritic protein 18.2 with the Fc fragment of immunoglobulins (IgG, IgA, etc.).

[0014] Furthermore, the amino acid sequence of the Fc fragment of the immunoglobulin is shown in SEQ ID NO:40.

[0015] Furthermore, the encoding nucleotide sequence of the Fc fragment of the immunoglobulin is shown in SEQ ID NO:41.

[0016] Fourthly, a pharmaceutical composition comprising the aforementioned nanobody targeting dendritic protein 18.2, or the aforementioned nucleic acid, or the aforementioned fusion protein.

[0017] Furthermore, the dosage form of the pharmaceutical composition includes at least one of microsphere formulations, microcapsule formulations, and nanocapsule formulations. It also includes pharmaceutically acceptable excipients and / or carriers.

[0018] Fifthly, the use of the nanobody targeting dentin 18.2, the nucleic acid, the fusion protein, or the pharmaceutical composition described herein in the preparation of a detection reagent, in vivo imaging probe, or therapeutic product targeting dentin 18.2.

[0019] The core innovation of this invention lies in providing a nanobody molecule that specifically targets dense protein 18.2 (CLDN18.2) without recognizing CLDN18.1. No antibody with the same CDR region sequence as the antibody of this invention has been found in the prior art, thus solving the problem of the lack of candidate nanobody molecules that specifically target CLDN18.2 in the prior art.

[0020] The beneficial effects of this invention are:

[0021] (1) The CLDN18.2 target selected in this invention is the safest and most effective therapeutic target for digestive system tumors: CLDN18.2 is expressed only at the tight junctions between gastric mucosal epithelial cells in normal tissues and cannot come into contact with surrounding targeted drugs, but 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 do not damage normally expressed tissues, but have a specific killing effect on CLDN18.2-expressing tumor tissues.

[0022] (2) This invention provides candidate nanobody molecules for targeted therapy of digestive system tumors: Compared with hematologic malignancies, the development of specific immunotargeted therapy drugs for solid tumors has been slow, and one of the limiting factors is the lack of effective and safe targets. Current research has shown that CLDN18.2 is a safe and effective therapeutic target for digestive system tumors, but research on targeted drugs targeting CLDN18.2 started late. There is only one monoclonal antibody drug that has just been launched in a few countries. The bottleneck is in the antibody discovery stage. It is difficult to develop antibody molecules that specifically target CLDN18.2 without recognizing CLDN18.1, and the nanobody involved is even rarer.

[0023] (3) The nanobodies of the present invention have a wide range of applications: (a) The present invention obtained 6 candidate nanobodies against CLDN18.2 through screening from an alpaca immune library. Their C-terminus was fused with the Fc fragment of human IgG1. The binding activity (ELISA detection) and affinity (SPR detection) of these nanobodies against the CLDN18.2 antigen were detected at the CLDN18.2-VLP level of the antigen protein. The results were better or comparable to the traditional control antibody IMAB362. At various cellular levels overexpressing CLDN18.2, FACS detection of the binding activity of the Fc fusion antibody against the CLDN18.2 antigen showed that 4 nanobodies were better than the traditional control antibody IMAB362. (b) Nanobodies have significant advantages over other antibodies, such as the ability to regulate their half-life through chemical modification or protein fusion, strong permeability, the ability to recognize hidden epitopes inaccessible to ordinary antibodies, resistance to pepsin, acid, and heat, ease of production, and, being single-chain and small in molecular weight, easy assembly with other types of antibodies to form bivalent antibodies / chimeric antigen receptors (CARs) and multivalent antibodies / CARs. Therefore, the nanobodies of this invention can be developed into immunoassay reagents, CAR-T / NK immunotherapy drugs, and antibody drugs. Considering the characteristics of the CLDN18.2 target, the candidate antibodies of this invention, if used as immunodiagnostic reagents for cancer, will have better efficacy; if developed into cell or antibody immunotherapy drugs, they will have lower toxicity and better clinical efficacy, thus providing patients with more drug options.

[0024] (4) Can be used to develop dual-target drugs: Studies have found that Claudin18.2, like CDH17, is a safe and effective therapeutic target for digestive system tumors. Both 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 nanobody of the present invention can be used in combination with CDH17 antibody to develop anti-CLDN18.2 / CDH17 bispecific antibody drugs and dual-target CAR-T / NK drugs, which are expected to enhance the efficacy of monoclonal antibodies or single-target CAR-T / NK and other immune cell drugs. Attached Figure Description

[0025] Figure 1 Flowchart for constructing the CLDN18.2 alpaca immune single-domain antibody library;

[0026] Figure 2 The results of ELISA detection show the binding of the candidate antibody protein fused with Fc to the antigen Hu-18.2-VLP.

[0027] Figure 3ELISA results showing the binding of candidate antibody proteins fused with Fc to the negative control antigen GAG-VLP;

[0028] Figure 4 FACS results for the binding of the candidate antibody protein fused with Fc to Hu-18.2-HEK293 cells overexpressing human antigen CLDN18.2;

[0029] Figure 5 FACS results for the binding of the candidate antibody protein fused with Fc to Hu-CLDN18.1-HEK293 cells overexpressing human antigen CLDN18.1;

[0030] Figure 6 FACS results for the binding of candidate antibody proteins fused with Fc to Hu-18.2-BxPC-3 cells overexpressing human antigen CLDN18.2;

[0031] Figure 7 FACS results for the binding of candidate antibody proteins fused with Fc to Hu-18.2-NUGC4 cells overexpressing human antigen CLDN18.2. Detailed Implementation

[0032] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0033] Example

[0034] 1. Alpaca Immunization

[0035] 1.1 Alpaca Immunization Methods

[0036] Two alpacas (numbered #1 and #2) were cross-immunized using full-length human CLDN18.2 antigen-containing virus-like particles (VLPs) Hu-18.2-VLP (purchased from Sanyou Biotechnology), and overexpressing full-length human CLDN18.2 cell lines Hu-18.2-HEK293 and Hu-18.2-KATOⅢ (purchased from Sanyou Biotechnology). Each alpaca was immunized with 500 μg of antigen or 1.00 × 10⁻⁶ ions of antigen per immunization. 7 Cellular immunization. The initial antigen immunization uses Freund's complete adjuvant (Sigma), subsequent immunizations use Freund's incomplete adjuvant (Sigma), and cellular immunizations do not use adjuvants. Injection is administered subcutaneously at multiple sites. The alpaca immunization schedule is shown in Table 1.

[0037] Table 1 Alpaca Immunization Program

[0038]

[0039] 1.2 ELISA serum titer detection

[0040] The plates were coated with Hu-18.2-VLP antigen (2 μg / mL, 30 μL) and incubated overnight at 4°C. Blocking solution (5% PBSM, 5% skim milk powder) was used for blocking for 2 hours at room temperature. Serum was added in 3-fold serial dilutions and incubated for 1 hour at room temperature. Secondary antibody (Goat anti-Llama IgG (H+L)) and HRP (ThermoFisher, A16060) were added and incubated for 1 hour. 30 μl of TMB (from SurModics) was added for color development and incubated for 30 minutes at room temperature. The reaction was terminated with 50 μl of TMB stop solution (from Beyotime, P0215), and the OD450 value was read from the molecular device. Serum titer was determined according to 1.65 times NC standard, with a titer greater than 32K considered acceptable. The ELISA results for the two immunized alpacas are shown in Table 2. The ELISA serum titer test results showed that the immunization titers of both alpacas were acceptable.

[0041] Table 2. Serum efficacy value against Hu-18.2-VLP.

[0042] Serum to be tested Number of immunizations Valence (k) #1 4 64 #2 4 128

[0043] 2. Construction of an alpaca immune bank

[0044] 2.1 Construction of the immune library

[0045] Blood was collected from two alpacas after four immunizations to construct separate immune libraries. 80 ml of peripheral blood was collected from each alpaca, and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque density gradient separation medium (GE, 17144003S). RNA was extracted from the isolated PBMCs and reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, 6210A) to obtain all antibody nucleotide sequences. Primers were designed at the VH and CH2 proximal ends of the germline gene VH, and PCR yielded two DNA fragments of different sizes. The smaller target fragment was recovered by gel excision. Degenerate primers containing NcoI and NotI restriction sites were designed by comparing all sequences of the V and J genes of the VHH antibody. The recovered DNA fragment products were then used as templates to amplify all VHH gene fragments. The phage display vector and VHH fragment were digested and ligated. The ligation products were recovered using a recovery kit (Omega, D6492-02) and transformed into competent Escherichia coli SS320 (Lucigen, MC1061F) using an electroporator (Bio-Rad, MicroPulser) to obtain camel-derived immune phage display libraries (RA044 and RA045). The above process is as follows: Figure 1 As shown.

[0046] 2.2 Determination of Single-Domain Antibody Library Size

[0047] The library size of the single-domain antibody libraries was determined using the dilution spotting method. The results are shown in Table 3. The library sizes of both single-domain antibody libraries were greater than or equal to 1.00 × 10⁻⁶. 8 CFU quality standards.

[0048] Table 3. Statistics on Electrostatic Transfer System and Storage Capacity

[0049] Library Name Number of electronic transfers (pieces) Single-unit transfer of storage capacity (CFU) Total storage 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 ]]>

[0050] 2.3 Analysis of Sequencing Results of Single-Domain Antibody Library

[0051] Clones were selected for sequencing, and the successfully sequenced results were selected for further analysis. The sequencing analysis summary is shown in Table 4.

[0052] Table 4. Statistical Table of Sequencing Information for Single-Domain Antibody Libraries

[0053]

[0054] The antibody gene sequence analysis criteria for single-domain antibody library construction are: correct insertion rate greater than or equal to 80%, empty sequence percentage less than or equal to 10%, and enzyme restriction site accuracy greater than or equal to 90%. The effective library capacity is calculated as: total library capacity × antibody correct expression rate × unique sequence percentage = effective library capacity.

[0055] 2.4 Results of antibody gene analysis of single-domain antibody library

[0056] Table 5 shows the analysis of successfully sequenced sequences from the single-domain antibody libraries. The analysis indicates that the empty vector ratio in RA044 and RA045 is less than 10%, the correct insertion rate of the antibody gene is greater than 80%, and the CDR region alignment shows high richness, meeting the quality standards for single-domain antibody library construction. In conclusion, the RA044 and RA045 single-domain antibody libraries are of acceptable quality.

[0057] Table 5 Summary of Library Construction Indicators

[0058]

[0059] 3. Screening of the alpaca immune bank

[0060] The antibody sequences of the constructed alpaca immune library are displayed on the surface of phages using phage display technology. Then, using specific proteins and cells as antigen materials, the phage-displayed antibodies that bind to the antigen are continuously enriched through multiple rounds of screening. Positive clones that specifically bind to the antigen are further screened by ELISA, and the antibody sequences of the positive clones are obtained by sequencing.

[0061] 3.1 Immune library screening methods

[0062] The immune library screening method includes the following steps:

[0063] (1) Phage preparation: Alpaca immune curd bacteria or the obtained output collection are inoculated, assisted phage infection, phage amplification, phage precipitation and resuspension, etc., 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 the 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 negatively screened phages and antigen-overexpressing cells are incubated, washed and eluted. After 4 rounds of screening, the specific monoclonal antibodies against the antigen are enriched.

[0064] The results showed that the positive phages were enriched to varying degrees after the second, third, and fourth rounds of solid-phase and cell-based screening. Specific data are shown in Table 6.

[0065] Table 6. Preliminary Selection Data Statistics

[0066]

[0067]

[0068] Note: N / A indicates not detected or not applicable.

[0069] 3.2 Initial screening of monoclonal antibodies

[0070] The results of the immunorepository screening showed that multiple phage output pools with good enrichment were obtained using a solid-phase, cell-cross-selection method. Single clones were selected from the enriched pools after four rounds of screening and then screened using FACS analysis on Hu-18.2-HEK293 cells. A total of 270 clones were selected, and 22 positive clones that bound to Hu-18.2-HEK293 cells were obtained. Sequencing analysis of these 22 positive clones yielded 16 molecules with unique sequences.

[0071] The summary statistics of the initial screening results are shown in Table 7.

[0072] Table 7 Summary of FACS Initial Screening Results

[0073]

[0074] Based on sequence diversity analysis and comparison, and after removing molecules containing post-translational modification sites, 12 antibody clones were selected for full-length construction. These 12 antibody clones are numbered as C002, C003, C005, C008, C013, C022, C004, C007, C011, C012, C017 and C019, and their amino acid sequences are shown in SEQ ID NO: 16-27, and their encoding nucleotide sequences are shown in SEQ ID NO: 28-39.

[0075] 4. Preparation and detection of candidate molecules for alpaca immune library

[0076] 4.1 Construction of expression plasmids for full-length antibody proteins

[0077] The nucleotide sequence encoding the nanobody was fused with the nucleotide sequence encoding the Fc segment of human IgG1 (whose amino acid sequence and encoding nucleotide sequence are shown in SEQ ID NO: 40 and SEQ ID NO: 41, respectively) to synthesize the whole gene, which was then constructed into the pcDNA3.4 vector (Invitrogen) to obtain the expression plasmid of the VHH-Fc fusion protein, i.e., the full-length antibody protein.

[0078] 4.2 Expression and purification of full-length antibody protein

[0079] The constructed antibody plasmid was transfected into Expi CHO cells (Gibco, A29133) for transient expression at a volume of 10 mL for 7 days. The expressed protein was purified and aliquoted; the purified protein was then analyzed by SDS-PAGE, SEC, FACS / ELISA, and affinity kinetics.

[0080] 4.3 SDS-PAGE identification of full-length antibody protein

[0081] The molecular weight and purity of the full-length antibody protein were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The method and steps are as follows: (1) Preparation of purified protein sample solution: Preparation of non-reduced 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 loading amount of non-reduced sample 1μg. The mixed sample was heated in a 75℃ dry bath for 10min. Preparation of reduced 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 loading amount of reduced sample 2μg. The mixed sample was heated in a 100℃ dry bath for 10min. (2) Electrophoresis: 140V, 75min. (3) Staining, destaining and scanning: Coomassie brilliant blue staining on gel, followed by destaining and scanning with an EPSON V550 color scanner. (4) Calculation of purity: The purity of the reduced bands, or the purity of the reduced heavy chain plus the light chain, was calculated using ImageJ according to the peak area normalization method.

[0082] Systemic suitability criteria: Reference IPI (ipilimumab) non-reduced band molecular weight approximately 150 kDa, purity greater than 90%; reduced heavy chain molecular weight approximately 50 kDa, light chain molecular weight approximately 25 kDa, heavy chain plus light chain purity greater than 90%. 4.4 SEC identification of full-length antibody protein

[0083] The purity of the full-length antibody protein was determined by size exclusion chromatography (SEC). 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 preparation: Dilute the sample concentration to 0.5 mg / mL. (3) 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.

[0084] Systemic fitness criteria: The reference product Herceptin (trastuzumab) has a monomer purity greater than 95%, the separation degree between BSA monomer and dimer is greater than 1.5, and the baseline is stable, then the systemic fitness is considered to be passed.

[0085] 4.5 Affinity kinetics of full-length antibody proteins

[0086] The interaction between candidate antibody proteins and antigens was detected using surface plasmon resonance (SPR) technology based on the Biacore T200 (Cytiva) platform. The affinity detection process included capture, equilibration, binding, dissociation, and regeneration; parameter settings are detailed in Table 8.

[0087] Table 8 Affinity Detection Parameter Settings

[0088]

[0089] Note: The values ​​for time and sample concentration are for reference only. Specific values ​​can be optimized and adjusted according to experimental conditions.

[0090] System adaptability standard: Chi 2 ≤10%×Rmax. Chi 2 Rmax: The degree of fit between the fitted line and the experimental line; Rmax: The highest response value of the curve fitting.

[0091] 4.6 ELISA detection of full-length antibody protein

[0092] Methods and steps for ELISA detection of full-length antibody protein: (1) Plate coating: Dilute antigen Hu-18.2-VLP to a concentration of 2 μg / mL with 1×PBS, add 30 μL / well to a 96-well ELISA plate, and coat overnight at 4℃. (2) Blocking: Wash the plate 3 times with PBST, add blocking buffer (5% PBSM), and block at room temperature for 2 h. (3) Incubation: Wash the plate, add 30 μL / well of sample diluted with 1% PBSM, and incubate at room temperature for 60 min. (4) Secondary antibody incubation: Wash the plate 3 times with PBST, add secondary antibody Goat-Anti-Human-IgG-Fc-HRP (purchased from Abcam), and incubate at room temperature for 50 min. (5) Color development: Wash the plate 3 times with PBST, add 30 μL 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 .

[0093] System adaptability criteria are shown in Table 9:

[0094] Table 9

[0095] Serial Number Judgment Indicators Release criteria 1 Sample name accuracy completely consistent 2 <![CDATA[R 2 ]]> ≥0.90 3 positive antibody upper and lower plateau OD ratio ≥3 4 Blank control OD value (background value) ≤0.25

[0096] In the above tests, 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 Bio).

[0097] 4.7 FACS detection of full-length antibody protein

[0098] The binding activity of 12 full-length expressed fusion antibody proteins to three human pancreatic cancer cells (Hu-18.2-BxPC-3, Hu-18.2-NUGC4, and Hu-18.2-HEK293) overexpressing human CLDN18.2, and one human CLDN18.1 overexpressing cell line (Hu-CLDN18.1-HEK293) was detected by FACS. The methods and procedures are as follows:

[0099] (1) Cell plating: Transfer cells from culture flasks to centrifuge tubes, centrifuge to remove supernatant, resuspend in culture medium, count, and adjust cell density to 1×10⁻⁶. 6 cells / mL. Take a 96-well round-bottom plate and add 100 μL of cells to each well using a 100 μL pipette. Centrifuge at 300 g / min for 5 min. Discard the supernatant.

[0100] (2) Addition of antibody protein samples: The antibody protein samples were diluted to eight concentration gradients using FACS containing 2% FBS: 150.000, 37.500, 9.375, 2.3438, 0.5859, 0.1465, 0.0366, and 0.0092 nM. Using a 100 μL 12-channel pipette, 100 μL of the antibody diluent was added to each well of a 96-well cell plate. After mixing, the plate was incubated at 4°C for 1 hour.

[0101] (3) Addition of secondary antibody: Dilute the PE-labeled anti-Human Fc (Jackson) secondary antibody 1:200 with FACS Buffer containing 2% FBS. Centrifuge the cell culture plate to remove the supernatant, and add 100 μL of the secondary antibody dilution buffer to the cell culture plate using a 100 μL 12-line pipette. Incubate the cell culture plate at 4°C 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 in each well.

[0102] (4) Data acquisition: Turn on the flow cytometer (purchased from Beckman), and after the instrument is cleaned, perform FACS binding detection.

[0103] In the above tests, the positive control antibody was IMAB362, the isotype control antibody was IgG1 (P93950-1, purchased from Sanyou), the control with only secondary antibody was labeled "cell sec", and the blank cell control without any antibody was labeled "cell only".

[0104] 4.8 Experimental Results

[0105] (1) Results of physicochemical property testing

[0106] Twelve candidate antibody molecules fused with Fc were successfully converted into target proteins through plasmid construction, protein expression, and purification. The results of their physicochemical property testing are shown in Table 10.

[0107] Table 10 Summary of Physicochemical Property Test Results

[0108]

[0109]

[0110] The results showed that: (1) The purified protein amounts of the 12 target proteins ranged from 0.03 mg to 3.19 mg. Three proteins (C007, C011, and C012) were not subjected to SDS-PAGE, SEC, and subsequent affinity kinetics assays due to their low purification amounts. (2) According to SDS-PAGE, the purity of all nine tested samples was greater than 95%. (3) According to SEC, the purity of C022 (P237142) did not reach 90%, while the purity of the other eight tested samples was greater than 90%.

[0111] (2) Affinity kinetics test results

[0112] Affinity kinetics analysis of the nine target proteins showed that their affinity for the antigens was superior to that of the positive control antibody IMAB362, and all met the Chi-related criteria. 2 ≤10%×Rmax meets the system adaptability criteria, and the results are reliable. See Table 11.

[0113] Table 11 Affinity kinetics test results

[0114]

[0115] Note: Chi 2 Rmax: The degree of fit between the fitted line and the experimental line; Rmax: The highest response value of the curve fitting.

[0116] (3) ELISA test results

[0117] 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: Figures 2-3 And Table 12.

[0118] Table 12 Summary of Candidate Antibody ELISA Detection Results (EC) 50 (μg / mL)

[0119]

[0120]

[0121] Note: / indicates not tested, N indicates no binding, W indicates weak binding, Y indicates binding but EC cannot be calculated. 50 value.

[0122] 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.

[0123] (4) Candidate antibody FACS detection results

[0124] The FACS results of the candidate antibodies are as follows Figures 4-7 .

[0125] Table 13 Summary of EC50 results (nM) of purified candidate antibodies detected by FACS

[0126] Serial Number Protein numbering 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

[0127] Note: 1) Y represents good binding, W represents weak binding, N represents no binding, and N / A represents no detection.

[0128] FACS analysis showed that among the 12 purified antibody samples, 9 antibody samples (C002, C003, C004, C005, C008, C013, C017, C019, and C022) showed good binding to Hu-18.2-BxPC-3 cells, with EC50 values ​​ranging from 2.170 nM to 6.190 nM; 8 antibody samples (C002, C003, C004, C005, C008, C013, C017, and C022) showed good binding to both Hu-18.2-HEK293 and HU-18.2-NUGC4 cells, with EC50 values ​​for antibody binding to both cell types ranging from [missing value range]. The effective molecular weight ranges were 2.113 nM to 6.179 nM and 1.100 nM to 6.530 nM. Four candidate antibody samples (C004, C011, C017, and C019) showed non-specific binding to Hu-CLDN18.1-HEK293 cells. Six antibody samples (C002, C003, C005, C008, C013, and C022) showed specific binding to three CLDN18.2-expressing cell lines: Hu-18.2-BxPC-3, Hu-18.2-HEK293, and HU-18.2-NUGC4, but not to Hu-CLDN18.1-HEK293 cells.

[0129] The amino acid sequences of VHH from the six nanobody clones that specifically bind to CLDN18.2 were analyzed using the Kabat numbering and definition schemes, and the results are shown in Table 14.

[0130] Table 14. Amino acid sequences of the complementarity-determining regions (CDR1–3) of the six candidate nanobodies.

[0131]

[0132]

[0133] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

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

12.

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

18.

3. A nucleic acid, characterized in that, Includes a nucleic acid sequence encoding a nanobody targeting dendritic protein 18.2 as described in any one of claims 1 to 2.

4. The nucleic acid according to claim 3, characterized in that, The nucleotide sequence encoding a nanobody targeting dendritic protein 18.2 is shown in SEQ ID NO:

30.

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

6. The 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 dentin 18.2 as described in any one of claims 1 to 2, 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. The pharmaceutical composition according to claim 8, characterized in that, The dosage form of the pharmaceutical composition includes at least one of microsphere formulations, microcapsule formulations, and nanocapsule formulations.

10. The use of a nanobody targeting dentin 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 dentin 18.2.

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