A MSLN single-domain antibody and its application

Abstract

The present invention relates to the field of biomedicine technology and provides a MSLN single-domain antibody and its application. The amino acid sequence of the MSLN single-domain antibody is shown in SEQ ID No. 1. The MSLN single-domain antibody provided by the present invention has high activity, strong binding force with the antigen, and high affinity, with an affinity constant of 2.52×10 ‑8 M, provides a new antibody and treatment option for ovarian cancer, etc.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an MSLN single-domain antibody and applications thereof. Background Art

[0002] Mesothelin (MSLN) is a cell surface glycoprotein and a splice variant of a precursor protein. It was first discovered in mesothelioma cells, hence its name. MSLN expression is upregulated in various tumors, including pancreatic and ovarian cancers, while its expression in normal tissues is relatively low. This specific high expression in tumor tissues makes MSLN an attractive target for cancer therapy.

[0003] Single-domain antibodies (sdAbs) are small antibody fragments of heavy-chain antibodies that possess characteristics such as small molecular weight, strong tissue penetration, high stability, and low production costs. Compared with traditional monoclonal antibodies, sdAbs have shown great potential in targeted tumor therapy, especially in achieving deep penetration and rapid clearance of tumor tissues.

[0004] Given the high expression of MSLN in various tumors and the unique advantages of single-domain antibodies, the development of single-domain antibodies targeting MSLN has important clinical significance. This new antibody can specifically recognize and bind to MSLN and is expected to play an important role in the diagnosis and treatment of ovarian cancer, providing patients with more effective and precise treatment options. Summary of the Invention

[0005] In view of this, the present invention proposes a MSLN single-domain antibody with high affinity and its application.

[0006] The technical solution of the present invention is achieved as follows: On the one hand, the present invention provides an MSLN monoclonal antibody, and the amino acid sequence of the MSLN single domain antibody is shown in SEQ ID No.1.

[0007] On the other hand, the present invention also provides a use of an MSLN single domain antibody in the preparation of a drug for treating ovarian cancer.

[0008] On the other hand, a pharmaceutical composition contains the MSLN single-domain antibody shown in SEQ ID No.1.

[0009] In another aspect, a kit comprises the MSLN single domain antibody shown in SEQ ID No. 1.

[0010] The MSLN single domain antibody and its application of the present invention have the following advantages over the prior art: the MSLN single domain antibody provided by the present invention has high activity, strong binding force with the antigen, and high affinity, with an affinity constant of 2.52×10 -8 M, provides a new antibody and treatment option for ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0012] Figure 1 This is the electrophoresis diagram of the VHH gene fragment of the alpaca-derived Anti-MSLN nanobody;

[0013] Figure 2 This is the SDS-PAGE electrophoresis diagram of Anti-MSLN single domain antibody;

[0014] Figure 3 Figure 1 is a graph showing the binding activity of Anti-MSLN single domain antibody to tumor cells. Figure A is for comparison, and Figure B is for MSLN-230 single domain antibody.

[0015] Figure 4 EC50 value of antibody protein binding to MSLN antigen;

[0016] Figure 5 is the binding affinity between the antibody protein and the MSLN antigen protein. DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0018] Example 1 Design and Construction of Phage Display Immunized Alpaca Nanobody Gene Library

[0019] (1) Isolation of alpaca peripheral blood lymphocytes and extraction and purification of RNA

[0020] Whole blood was collected from alpacas, peripheral blood lymphocytes were separated, and total RNA was extracted from the separated lymphocytes using an RNA extraction kit (QIAGEN).

[0021] (2) Amplification

[0022] Using the nested PCR amplification method, two rounds of amplification were performed to obtain the highly specific alpaca heavy chain VH and light chain VL. Then, using the Overlap-PCR method, the heavy chain VH obtained above was linked to the light chain VL by Overlap-PCR to obtain the alpaca nanobody VHH gene fragment. The results are shown in Figure 1 .

[0023] The PCR amplification primer sequences are as follows:

[0024] Light Chain Fd 5'primers:

[0025] YTL-F:GGTGGTCCTGGCTGCNCTN;

[0026] YTL-R:GGGGTACCTGTCATCCACGGACCAGCTGA.

[0027] Heavy Chain Fd 3'primers:

[0028] YTVHH-F:GATCGCCGGCCAGKTGCAGCTCGTGGAGTCNGGNGG;

[0029] YTVHH-R:CATGTGTAGATTCCTGGCCGGCCTGGCCTGAGGAGACGGT GACCTGG.

[0030] (3) The VHH fragment and phage display vector were digested with sfiI (NEB), ligated with T4 ligase (NEB), and then electroporated into TG1 competent cells.

[0031] (4) Identification and preservation of the capacity and diversity of the VHH antibody gene library

[0032] The library capacity was calculated by multiplying the titer measured after transformation by the total amount transformed: 120 single colonies were grown on the plate, 40 of which were randomly selected for electroporation titer testing, followed by PCR amplification and sequencing. Fifty colonies were PCR amplified, 49 of which were positive, with a transformation efficiency of 98.5%. The library capacity was calculated to be 1.63×10 11 The size of the amplified fragment was the same as the size of the inserted VHH; the sequencing results of 49 clones showed that there were no repeated VHH sequences, and the library capacity and diversity of the antibody gene library met the design requirements.

[0033] Example 2 Preparation of MSLN Single Domain Antibody

[0034] (1) Screening of MSLN single-domain antibodies

[0035] Immunotubes were coated with MSLN antigen protein at 12.5 μg / mL, 25 μg / mL, and 50 μg / mL for three rounds of screening. The tubes were blocked with 4% skim milk in PBST, and the phage library described above was added for binding time. Nonspecifically bound phage were removed by washing, and specifically bound phage were eluted with TEA. Amplification was then performed for three rounds of screening.

[0036] Table 1 Affinity screening enrichment effect on phage antibodies

[0037] Number of screening rounds Antigen concentration Input phage titer Output phage titer Amplification 1 50 <![CDATA[1×10 12 ]]> <![CDATA[2.76×10 6 ]]> <![CDATA[1.7×10 10 ]]> 2 25 <![CDATA[1×10 12 ]]> <![CDATA[3.1×10 6 ]]> <![CDATA[2.4×10 11 ]]> 3 12.5 <![CDATA[1×10 12 ]]> <![CDATA[1.87×10 5 ]]> -

[0038] (2) ELISA method was used to determine the culture supernatant of a single clone and screen for positive clones

[0039] Single colonies were randomly picked from the agar plates with good single colony growth and separation, inoculated into 96-well culture plates containing 2YT liquid culture medium containing Amp, cultured overnight, centrifuged, and the supernatant separated. The MSLN antigen protein was used as the antigen to coat a 96-well ELISA plate for phage ELISA assay. The positive well clones of the MSLN antigen protein were selected, and the gene sequences of the MSLN antigen protein-specific nanoantibody clones were identified by DNA sequencing. Among them, MSLN-230 had the best effect, and its amino acid sequence is shown in SEQ ID No. 1.

[0040] SEQ ID No. 1: EVQLQASGGGLVQPGGSLRLSCAASGASYTNSKTAWFR QAPGKGLEFVSAISEHSYPRRYYADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARFRGSTVTTPSRVPAYWGQGTQVTVSS.

[0041] Table 2 Absorbance values ​​of culture plates

[0042] 1 2 3 4 5 6 7 8 9 10 11 12 A 0.3884 0.3044 1.5344 2.6113 0.8078 3.7761 3.3305 0.656 0.334 0.4122 0.3979 0.252 B 1.8102 0.3728 0.8176 0.4274 2.6721 0.4548 1.3424 0.4225 3.5269 0.629 0.8056 0.6388 C 0.3271 1.2843 1.7047 0.7425 0.4751 1.5201 0.8556 0.3156 2.2836 1.2605 0.2974 3.3825 D 1.3607 1.3294 0.1964 0.9677 0.6578 1.8803 2.2366 0.5398 0.8798 0.9813 0.4773 1.2636 E 0.3932 1.2828 0.3595 1.1164 0.2973 1.5616 1.1826 0.8612 0.8781 0.8429 1.8095 0.8739 F 0.6894 1.1427 0.5733 1.6583 0.8391 0.6967 0.6965 0.1454 0.2566 3.4604 1.0648 0.8805 G 0.3408 0.4575 0.3476 0.925 1.0029 1.6153 1.4306 2.2261 0.3625 0.2455 0.846 0.1073 H 0.4005 2.2403 0.7202 0.3608 2.3884 0.1022 0.716 0.879 1.0031 0.5543 0.3749 1.9176

[0043] Example 3 Construction of MSLN single domain antibody expression plasmid

[0044] The specific MSLN-230 gene obtained in Example 2 was amplified by PCR to obtain a PCR product with restriction endonucleases BbsI and BamHI sites. The PCR product and vector (pSJF2 vector) (Kim Is. Biosic Biochem. 2002, 66(5):1148-51) were treated with restriction endonucleases BbsI and BamHI, respectively. The two products were ligated and recombined by T4 ligase to obtain a plasmid VHH-pSJF2 that can be efficiently expressed in Escherichia coli. The gene sequence was then determined to confirm the correctness of the sequence.

[0045] Example 4 Expression, purification and identification of specific MSLN single domain antibodies

[0046] (I) Expression and purification of specific MSLN single-domain antibodies

[0047] (1) The strain containing the plasmid VHH-pSJF2 described in Example 3 was inoculated onto an LB culture plate containing ampicillin and incubated at 37°C overnight.

[0048] (2) Select a single colony and inoculate it into 5 ml of LB culture medium containing ampicillin. Incubate at 37°C in a shaking incubator overnight.

[0049] (3) Transfer the culture to 100 ml of 2YT culture medium containing ampicillin and culture at 37°C with a shaker at 220 rpm. When the OD value reaches 0.6-1.0, add 0.1-0.5 M IPTG and continue to culture overnight.

[0050] (4) Centrifuge at 5000 rpm for 20 minutes to collect the bacteria.

[0051] (5) The bacteria were washed twice with 0.05 M Tris buffer, and the soluble nanobody expressed in the bacterial periplasm was extracted with hypertonic sucrose. The soluble nanobody protein in the supernatant was collected by centrifugation.

[0052] (6) Nanobody protein with a purity of more than 90% was obtained by separation using Ni+ ion affinity chromatography magnetic beads (BeaverBeadsTM His-tag Protein Purification, Suzhou Beaver Biomedical Engineering Co., Ltd.).

[0053] Results Figure 2 SDS-PAGE electrophoresis diagram. Figure 2 As shown, MSLN-230-pSJF2 carries c-myc and His6 tags, and the expressed protein molecule is approximately 25 kd.

[0054] (II) Activity determination of expressed MSLN single-domain antibody

[0055] 1. Test materials: ELISA plate (Thermofisher), CD99 antigen protein, Anti-Myc tagantibody-HRP (Beijing Sino Biological), TMB colorimetric solution (Beijing Meikewande, Cat: 1001), coating solution pH 9.6, BSA (Sigma).

[0056] 2. Experimental process:

[0057] 2.1 Coat the cells with MSLN antigen protein at a concentration of 2 μg / ml, 100 μl / well, and incubate at 4°C overnight.

[0058] 2.2 Add 2% skim milk in PBS to block the cells, 300 μl / well. Incubate at 37°C for 1.5 h.

[0059] 2.3 Dilute the MSLN antigen protein nanobodies with different numbers to a final concentration of 10.0 μg / mL and 1.0 μg / mL, 100 μL / well.

[0060] 2.4 Dilute Anti-Myc tag antibody (HRP) (1:5000), 100 μl / well, incubate at 37°C for 1 h.

[0061] 2.5 Add TMB colorimetric solution, 100 μL / well, and react for 10 minutes in the dark.

[0062] 2.6 Add 50 μl / well 2M H2SO4 to terminate the reaction and measure the OD value at a wavelength of 450 nm.

[0063] Figure 3 The results show that the purified MSLN-230 single domain antibody has a high binding activity with the MSLN antigen protein.

[0064] Example 5: Detection of Cell Binding Activity of MSLN Single Domain Antibody

[0065] The screened antibodies were tested for binding to human MSLN protein expressed on CHOK1 cells. CHO-K1 cells stably expressing human MSLN were harvested from a flask. 100 μL of 1 × 10 6 Cells at 100 cells / mL were incubated with diluted mouse antibodies on ice for 30 minutes. After washing twice with 200 μL FACS buffer, the cells were incubated with secondary antibodies on ice for 30 minutes. The cells were washed twice with 200 μL FACS buffer, transferred to BD Falcon 5mL tubes, and analyzed by FACS. The results showed that mouse antibodies can bind to human MSLN with a high EC50 ( Figure 4). EC50 of antibody protein binding to antigen = 2.35.

[0066] Example 6 Affinity determination of MSLN single domain antibody

[0067] First, a 1 nM biotinylated single-domain heavy chain antibody targeting an ovarian cancer-specific membrane antigen was incubated with five different concentrations of MSLN antigen (100 nM to 1500 μM) in an EP tube for 30 minutes. Subsequently, 90 μL of the mixture was added to an MSLN protein-coated ELISA plate blocked with 3% BSA-PBST. After incubation for 10 minutes, the reaction solution was aspirated and washed with PBST. Next, 100 μL of HRP-labeled streptavidin at a dilution of 1:2000 was added, incubated for 1 hour, and then washed five times with PBST. TMB was used for color development, and the OD450 value was measured. Nonlinear regression analysis was used to determine the MSLN concentration corresponding to half the OD450 value. Based on the principles of antigen-antibody competitive binding assays, the affinity constant of the biotinylated nanobody targeting an ovarian cancer-specific membrane antigen was determined to be 2.52 × 10 -8 M, see the results Figure 5 .

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A MSLN single domain antibody, characterized in that: The amino acid sequence of the MSLN single-domain antibody is shown in SEQ ID No.

1.

2. A kit, characterized in that: Contains the MSLN single-domain antibody according to claim 1.

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