Anti-msln antibodies and uses thereof

Abstract

This invention discloses an anti-MSLN antibody and its applications. The anti-MSLN antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3. The amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO:7, 8, and 9, respectively; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:4, 5, and 6, respectively. The antibody provided by this invention has good affinity and specificity for MSLN antigens, effectively detecting target proteins in cells overexpressing MSLN. It can also be widely used as a core ingredient in the development of high-sensitivity MSLN detection kits such as ELISA, Western blotting, and immunohistochemistry.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an anti-MSLN antibody and its applications. Background Technology

[0002] Mesothelin (MSLN) is a cell surface glycoprotein belonging to the glycosylphosphatidylinositol (GPI) anchoring protein family. It is mainly expressed on the surface of mesothelial cells in the pleura, peritoneum, and pericardium. Its expression level is extremely low in normal tissues, but it exhibits high specificity and high expression in various malignant tumors, making it an important therapeutic target for solid tumors (such as ovarian cancer, pancreatic cancer, and malignant mesothelioma). However, existing anti-MSLN antibodies suffer from insufficient affinity, leading to low sensitivity and a high risk of missed detection in low-abundance tumor antigens. Furthermore, antibodies prepared using traditional hybridoma techniques exhibit significant batch-to-batch variability and poor stability. Summary of the Invention

[0003] To overcome the technical shortcomings of existing anti-MSLN antibodies, such as low sensitivity and easy missed detection of low-abundance tumor antigens due to insufficient affinity, and the large batch-to-batch variability and poor stability of antibodies prepared by traditional hybridoma technology, this invention provides an anti-MSLN antibody and its application.

[0004] In one aspect, this invention provides an antibody against MSLN or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3.

[0005] The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 7, 8 and 9, respectively; and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 4, 5 and 6, respectively.

[0006] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 3.

[0007] In some embodiments, the light chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2.

[0008] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 3; and / or, the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2.

[0009] In some embodiments, the antibody or its antigen-binding fragment is a full-length antibody, Fab, Fab', F(ab')2, Fv, or scFv.

[0010] In some embodiments, the antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, or a humanized antibody.

[0011] In some embodiments, the antibody or its antigen-binding fragment is scFv, and the heavy chain variable region and the light chain variable region are connected by a linker.

[0012] In some embodiments, the antibody or its antigen-binding fragment includes scFv and a heavy chain constant region; the heavy chain constant region is a heavy chain constant region derived from IgG1, IgG2, IgG3 or IgG4; and / or, the heavy chain constant region includes a hinge region, CH2 and CH3.

[0013] In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0014] In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 12.

[0015] In another aspect, the present invention provides an isolated nucleic acid that encodes an antibody or an antigen-binding fragment thereof as described above.

[0016] Another aspect of the present invention provides a recombinant expression vector comprising the nucleic acid described above.

[0017] In some implementations, the recombinant expression vector used is pCDNA3.1.

[0018] In another aspect, the present invention provides a transformant comprising the nucleic acid as described above or the recombinant expression vector as described above, or expressing the antibody or its antigen-binding fragment as described above; the transformant is not an animal variety or a plant cell variety.

[0019] In some embodiments, the transformant is a human embryonic kidney cell (HEK293 cell), HeLa cell, or CHO cell.

[0020] Another aspect of the present invention provides an MSLN antigen detection reagent, which comprises an antibody or its antigen-binding fragment as described above and a PBS buffer with a pH of 7.4-7.6.

[0021] In another aspect, the present invention provides a kit comprising one or more of the following: an antibody or antigen-binding fragment thereof as described above, a nucleic acid as described above, a recombinant expression vector as described above, or a transformant as described above.

[0022] In some embodiments, the kit includes the antibody or its antigen-binding fragment as described above, or the MSLN antigen detection reagent as described above.

[0023] In some implementations, the kit is an immunohistochemical detection kit, an immunoprecipitation detection kit, an enzyme-linked immunosorbent assay (ELISA) kit, or an immunoblotting detection kit.

[0024] In some embodiments, the kit includes a primary antibody and a secondary antibody, wherein the primary antibody is an antibody as described above or an antigen-binding fragment thereof, and the secondary antibody is a horseradish peroxidase-labeled goat anti-mouse antibody.

[0025] Another aspect of the present invention provides a method for detecting MSLN, the method comprising contacting a sample to be tested with an antibody or antigen-binding fragment thereof as described above or a kit as described above; the method is for non-diagnostic and / or therapeutic purposes.

[0026] In another aspect, the present invention provides the use of one or more of the antibody or antigen-binding fragment thereof as described above, the nucleic acid as described above, the recombinant expression vector as described above, the transformant as described above, or the kit as described above in the preparation of a product for detecting the content or concentration of MSLN.

[0027] In some implementations, the product is a product for diagnosing diseases related to abnormal MSLN expression.

[0028] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0029] The reagents and raw materials used in this invention are all commercially available.

[0030] The positive and progressive effects of this invention are as follows:

[0031] Experimental verification shows that the antibody of this invention has a significantly better affinity for MSLN antigen than existing products and possesses excellent specificity, effectively detecting target proteins in cells overexpressing MSLN. Furthermore, based on a well-defined genetic engineering sequence and a stable mammalian cell expression system, the antibody of this invention exhibits homogeneous composition and good batch-to-batch stability, making it a widely applicable core ingredient in the development of high-sensitivity MSLN detection kits such as ELISA, Western blotting, and immunohistochemistry, providing a superior option for precision tumor diagnosis. Attached Figure Description

[0032] Figure 1 Electrophoresis images of the scFv fragments after library splicing are displayed.

[0033] Figure 2 The purified antibody was shown in an SDS-PAGE electrophoresis image.

[0034] Figure 3 The affinity curve of MSLN-H4 is shown.

[0035] Figure 4 The standard curve of MSLN concentration (μg / mL) is shown.

[0036] Figure 5 The Western Blot results of MSLN protein expression in different cell lines are shown. Detailed Implementation

[0037] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0038] As used herein, the expressions “comprising,” “including,” “containing,” and “having” are open-ended, meaning they include the listed elements, steps, or components but do not exclude other unlisted elements, steps, or components. The expression “composed of” excludes any unspecified elements, steps, or components. The expression “essentially composed of” means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the essential and novel nature of the claimed subject matter. It should be understood that the expressions “essentially composed of” and “composed of” are encompassed within the meaning of the expression “comprising.”

[0039] As used herein, the connecting term “and / or” between multiple referred elements should be understood to include both individual and combined options.

[0040] Unless otherwise stated, any numerical value or range of numerical values, such as concentration or concentration range, shall in any case be understood to be modified by the term “about”. Thus, numerical values ​​generally include at least ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values ​​within that range, including integers and fractions within that range, unless the context clearly indicates otherwise.

[0041] In this invention, the letters in the amino acid sequence represent single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0042] As used herein, “antibody” refers to an immunoglobulin or a fragment thereof that specifically binds to an antigenic epitope through at least one antigen-binding site. In this document, the definition of antibody encompasses antigen-binding fragments. The term “antibody” includes multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, single-domain antibodies, and antigen-binding fragments. Antibodies can be synthetic (e.g., produced by chemical or biological conjugation), enzymatically derived, or recombinant. Antibodies as used herein include any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b). Antibodies can be “monovalent,” “bivalent,” “trivalent,” or “quadrivalent” or more, meaning they contain one, two, three, four, or more antigen-binding sites.

[0043] As used herein, an "antigen-binding fragment" refers to a portion of a full-length antibody that is less than the full-length but contains at least a portion of the variable region of the full-length antibody (e.g., containing one or more CDRs and / or one or more antigen-binding sites), and thus retains at least a portion of the full-length antibody's ability to specifically bind antigens. Antigen-binding fragments may include, for example, antibody derivatives produced by enzymatic treatment of a full-length antibody, synthetically produced derivatives, and recombinant derivatives. Examples of antigen-binding fragments include, but are not limited to, sdAb (e.g., variable domains of heavy chain antibodies), Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', F(ab')2, biantibodies, Fd and Fd' fragments, and other fragments (e.g., fragments containing modifications).

[0044] As used herein, a “full-length antibody” typically comprises four polypeptides: two heavy chains (HC) and two light chains (LC). Each light chain contains a “light chain variable region (VL)” and a “light chain constant region (CL)” from the N-terminus (amino terminus) to the C-terminus (carboxyl terminus). Each heavy chain contains a “heavy chain variable region (VH)” and a “heavy chain constant region (CH)” from the N-terminus to the C-terminus. Generally, the heavy chain constant region of a full-length antibody may contain a CH1-hinge region – CH2-CH3 from the N-terminus to the C-terminus. In some immunoglobulin types (e.g., IgM and IgE), the heavy chain constant region may contain a CH1-hinge region – CH2-CH3-CH4 from the N-terminus to the C-terminus.

[0045] Both the light chain and heavy chain variable regions can contain three highly variable complementary determinant regions (CDRs) and four relatively conservative frame regions (FRs), connected from the N-end to the C-end in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In this paper, the CDRs (CDRL or LCDR) of the light chain variable region can be referred to as LCDR1, LCDR2, and LCDR3, and the CDRs (CDRH or HCDR) of the heavy chain variable region can be referred to as HCDR1, HCDR2, and HCDR3.

[0046] In this invention, when the antibody or its antigen-binding fragment includes "scFv and heavy chain constant region", the heavy chain constant region is a heavy chain constant region derived from IgG1, IgG2, IgG3 or IgG4; the heavy chain constant region includes a hinge region, CH2 and CH3, and does not contain CH1 and CH4.

[0047] Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulins IgA, IgD, IgE, IgG, and IgM. Complete antibodies form a “Y” shape. The stem of the Y consists of the second and third constant regions of the two heavy chains (and, for IgE and IgM, a fourth constant region) linked together, and disulfide bonds (interchain) are formed in the hinge. Heavy chains γ, α, and δ have constant regions consisting of three tandem (in a row) Ig domains, and hinge regions for added flexibility; heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the “CH2 domain” and the “CH3 domain,” respectively. Each arm of the Y includes a variable region and a first constant region of a single heavy chain that bind to a variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0048] In this invention, the amino acid sequences of CDRs are all in accordance with the Kabat definition (Chemical Computing Group) rules (the sequences in the claims of this invention are also in accordance with the Kabat definition rules). However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia (see, for example, Chothia, C. et al., Nature, 342, 877-883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)) based on the antibody's three-dimensional structure and the topology of the CDR ring; Kabat (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-4242) based on antibody sequence variability; and AbM (Martin, ACR and J. Allen (2007) “Bioinformatics tools for antibody engineering,” in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH). Verlag (pp.95–118), Contact (MacCallum, RM et al., (1996) J. Mol. Biol. 262:732-745), IMGT (Lefranc, M.-P., 2011 (6), IMGT, the International ImMunoGeneTicsInformation System Cold Spring Harb Protoc. and Lefranc, M.-P. et al., Dev.Comp. Immunol., 27, 55-77 (2003)), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.Those skilled in the art will understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined as described in any of the known schemes described herein. While the scope of protection claimed in the claims of this invention is based on the sequence shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0049] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0050] In this invention, a "Fab fragment" consists of a light chain and a heavy chain, comprising the CH1 domain and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain. A "Fab' fragment" contains a portion of a light chain and a heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of the two Fab' fragments to form the F(ab')2 molecule. An "F(ab')2 fragment" contains two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody, but lacking the constant region.

[0051] In this invention, scFv refers to a single-chain antibody fragment, which includes a heavy chain variable region, a light chain variable region, and a linker peptide of 15-20 amino acids. The VL and VH domains enable the linker peptides to pair and form monovalent molecules as single polypeptide chains [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules may have a general structure: NH2-VL-linker peptide-VH-COOH or NH2-VH-linker peptide-VL-COOH.

[0052] In this invention, "nucleic acid" refers to a nucleotide chain of any length and includes DNA and RNA. A nucleotide can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or its analogues, or any substrate capable of being incorporated into the chain by DNA or RNA polymerase.

[0053] In this invention, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, permits the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors of this invention are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of this invention can contain any type of nucleotide, including but not limited to DNA and RNA that can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors can contain naturally occurring or non-naturally occurring nucleotide linkages, or both. In an exemplary aspect, modified nucleotides or non-naturally occurring nucleotide linkages do not impede transcription or replication of the vector.

[0054] The recombinant expression vector of the present invention can be any suitable recombinant expression vector capable of being used for transformation or transfection to deliver one or more genes or sequences of interest into any suitable host cell and preferably to express the genes or sequences in the host cell. Suitable vectors include those designed for amplification and expansion or for expression or both of the above, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0055] In this invention, the term "host cell" refers to any type of cell that may contain the nucleic acids or vectors described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, animal, fungus, or algae; or it may be a prokaryotic cell, such as bacteria or protozoa. Prokaryotic cells include, for example, *Escherichia coli* (…). E. coli ) or Bacillus subtilis ( Bacillus subtilis Fungal cells such as yeast cells or Aspergillus, insect cells (such as S2 Drosophila cells or Sf9), and animal cells (such as fibroblasts, CHO cells, COS cells, HeLa cells, NSO cells, or HEK293 cells).

[0056] In this invention, applications for "non-diagnostic purposes" include, but are not limited to: for example, detecting the presence or absence of antigens (proteins containing the extracellular region of MSLN) in vitro in a laboratory; screening other antibodies targeting MSLN as positive antibodies; or competing for binding with other antibodies targeting MSLN to detect whether there is competition between antibodies, i.e., whether the antigenic epitopes are the same or similar. The term "treatment" refers to the improvement of a disease / symptom, such as reducing or eliminating the disease / symptom, preventing or slowing the occurrence, progression, and / or worsening of the disease / symptom. Therefore, treatment includes prevention, treatment, and / or cure.

[0057] The "kit" described in this application refers to a set of separate components provided for performing a specific detection method. The kit includes at least a container and an effective amount of the antibody or antigen-binding fragment, antibody-drug conjugate, or pharmaceutical composition contained in one or more of said containers. The antibody can be used as a detection antibody, capture antibody, or control. The kit may also optionally include other essential or auxiliary components for performing the detection, such as, but not limited to: labeling systems (e.g., enzymes, fluorescent dyes, biotin), substrates, buffers, standards, control samples (positive or negative), instructions, or reagents for sample processing (e.g., diluents, lysis buffers, blocking solutions).

[0058] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0059] Example 1: Preparation of anti-MSLN monoclonal antibody

[0060] 1. Antigen Preparation

[0061] The human MSLN gene sequence (GenBank accession number: Q13421) was obtained from the Uniprot database, and the recombinant human MSLN protein 296-606AA fragment was prepared using a eukaryotic expression system. The gene sequence encoding MSLN 296-606AA was cloned into a eukaryotic expression vector, transfected into HEK293 cells, and the cell supernatant was collected after 7 days of culture. The recombinant MSLN antigen was obtained by affinity chromatography purification.

[0062] 2. Animal Immunization

[0063] Two healthy female Balb / c mice aged 6-8 weeks were selected for the immunization experiment.

[0064] 2.1 First immunization

[0065] Take 50 μg of MSLN antigen, resuspend it in 50 μL of PBS buffer, and add 50 μL of Freund's complete adjuvant (Sigma, F5881) to emulsify. Inject the fully emulsified mixture subcutaneously into the left and right sides of the mouse dorsal region, 2-3 injections per side, with approximately 20 μL injected at each injection point.

[0066] 2.2 Second immunization

[0067] Two weeks after the first immunization, 50 μg of MSLN antigen was taken, resuspended in 50 μL of PBS buffer, and emulsified with 50 μL of Freund's incomplete adjuvant (Sigma, F5506). The emulsified mixture was then injected according to the same procedure as the first immunization.

[0068] 2.3 Third immunization

[0069] Two weeks after the second immunization (i.e., four weeks after the first immunization), 50 μg of MSLN antigen was taken, resuspended in 50 μL of PBS buffer, emulsified with 50 μL of Freund's incomplete adjuvant, and injected in the same manner. Seven days after the third immunization, blood was collected from the tail vein of mice, and serum was separated for antibody titer detection.

[0070] 2.4 Fourth immunization

[0071] Two weeks after the third immunization (i.e., six weeks after the first immunization), 50 μg of MSLN antigen was taken, resuspended in 50 μL of PBS buffer, emulsified with 50 μL of Freund's incomplete adjuvant, and injected in the same manner. Seven days after the fourth immunization, blood was collected from the tail vein of mice, and serum was separated for subsequent antibody titer testing and antibody library construction.

[0072] 3. Serum titer detection

[0073] The serum antibody titer of immunized mice was detected by indirect ELISA.

[0074] 3.1 Wrapping

[0075] Dilute MSLN antigen to 1 μg / mL with CBS buffer, add 100 μL / well to the microplate, and incubate overnight at 4°C. Wash three times with PBST the next day.

[0076] 3.2 Enclosure

[0077] Add 300 μL of 3% skim milk powder blocking solution to each well, incubate at room temperature for 1 hour, and wash 3 times with PBST.

[0078] 3.3 Sample Addition

[0079] The post-immunization serum and negative serum were diluted 1000 times, 3000 times, 9000 times, 27000 times, 81000 times and 243000 times respectively. 100 μL was added to each well and incubated at room temperature for 1 hour. The solution was washed 3 times with PBST.

[0080] 3.4 Secondary Antibiotic Incubation

[0081] Add 100 μL of HRP-labeled goat anti-mouse antibody (HRP-goat anti-mouse, Wuhan Sanying Biotechnology Co., Ltd., SA00001-1, dilution ratio 1:5000) to each well, incubate at room temperature for 1 hour, and wash 3 times with PBST.

[0082] 3.5 Color Development and Reading

[0083] Add 100 μL of TMB (Beyotime, P0209) chromogenic solution to each well, incubate at room temperature in the dark for 15 minutes, then add 50 μL of stop solution (2M H2SO4) and read the OD using a microplate reader. 450 value.

[0084] 3.6 Test Results

[0085] The test results showed that the serum titer of mice was >81000 (OD) after the fourth immunization. 450 A value higher than that of blank serum (more than 0.2) indicates that a high-titer antibody has been produced and can be used for antibody library construction.

[0086] 4. Total RNA extraction

[0087] Seven days after the fourth immunization, the mice were sacrificed, and the spleen and bone marrow were aseptically removed, mixed, and homogenized with Trizol reagent for total RNA extraction.

[0088] 5 cDNA Synthesis

[0089] Take the total RNA extracted above and reverse transcribe it into the first strand of cDNA according to the instructions of the reverse transcription kit (HiScript III 1st Strand cDNASynthesis Kit, Novizan, R312-02).

[0090] 6. PCR amplification of antibody heavy and light chain genes

[0091] Using cDNA obtained by reverse transcription as a template, antibody heavy chain and light chain variable region specific primers were designed for PCR amplification.

[0092] 7 splicing of scFv single-chain antibodies

[0093] The amplified heavy and light chain gene fragments were purified by gel extraction, and the purified products were used as templates for overlap extension PCR to splice the heavy and light chains into scFv single-chain antibody fragments.

[0094] The amplification products were identified by agarose gel electrophoresis (e.g.) Figure 1 As shown in the image, from left to right are the Marker and the spliced ​​scFv fragment, with the scFv fragment size being approximately 750 bp.

[0095] 8. Construction of Phage Display Library

[0096] 8.1 Enzyme digestion

[0097] The gel-purified scFv fragment and pComb3X phage vector were digested with SfiI restriction enzyme (NEB, R0123L).

[0098] The enzyme-digested scFv fragments were directly purified using a purification column, and the pComb3X vector was recovered by gel extraction after agarose gel electrophoresis.

[0099] 8.2 Connection

[0100] The digested scFv fragments were mixed with the linearized pComb3X vector in an appropriate ratio and ligated using T4 ligase.

[0101] Connect to 4℃ overnight.

[0102] 8.3 Electroconversion

[0103] The ligation product was mixed with XL1-Blue competent cells in an ice bath, dispensed into 80 μL electroporation cuvettes, and electroporated at 1800 V to obtain an antibody bacterial library.

[0104] 8.4 Determination of Library Capacity

[0105] Take 100 μL of bacterial suspension and serially dilute it 10-fold to 1000-fold with 2YT medium. Spread 100 μL of the diluted bacterial suspension onto 2YT-ATG semi-solid medium plates and incubate overnight at 37°C. Count single colonies the next day and calculate the number of transformants.

[0106] Library capacity = Single colony count × Dilution factor × Total library volume

[0107] The counting results showed that there were 489 single-clone colonies, and the calculated number of transformants was 9.78 × 10⁻⁶. 8 Twenty single clones were randomly selected for sequencing, with an accuracy of 90%, and the final effective library concentration was 8.8 × 10⁻⁶. 8 .

[0108] 9. Amplification of phage libraries

[0109] After incubating the antibody-containing bacterial library at 37°C and 250 rpm for 1 hour, VCSM13 helper phage was added, and the culture was allowed to stand for 30 minutes before incubating at 37°C and 200 rpm for another hour. The bacterial pellet was collected by centrifugation and resuspended in 100 mL of 2YT-ATK medium (containing ampicillin, tetracycline, and kanamycin), and incubated overnight at 30°C and 225 rpm. The next day, the supernatant was collected, and 4% PEG8000 and 3% NaCl were added. After centrifugation on ice, the phage pellet was obtained. The pellet was resuspended in 8 mL of PBS (pH 7.4), filtered through a 0.22 μm filter for sterilization, and stored at -80°C after adding 7% DMSO.

[0110] Take 10 μL of the filtered library and dilute it with 2YT medium. 8 After doubling, take 10 μL to infect 100 μL of XL1-Blue bacteria, spread it on 2YT-ATG semi-solid culture medium plates after 30 minutes, and count single colonies the next day.

[0111] The counting results showed that there were 169 single-clone colonies, and the calculated concentration of the amplified phage library was 1.69 × 10⁻⁶. 12 PFU / mL.

[0112] 10 Antibody Library Screening

[0113] The phage antibody library was enriched and screened using a solid-phase screening method.

[0114] 10.1 First Round of Screening

[0115] (1) Coating: Dilute the MSLN antigen to 5 μg / mL with coating buffer, add 100 μL / well to a high affinity microplate, and incubate overnight at 4°C.

[0116] (2) Sealing: Discard the coating solution, add 300 μL of 3% skim milk powder to each well, and seal at room temperature for 1 hour.

[0117] (3) Library incubation: Discard the blocking solution and add approximately 5 × 10⁻⁶ m³ / h. 12 PFU phage library, incubated at 37°C for 2 hours.

[0118] (4) Washing: Wash 3-5 times with 0.1% PBST to remove unbound phages.

[0119] (5) Elution: Add glycine-HCl buffer to elute the bound phages, and neutralize with Tris-HCl to pH 7.4.

[0120] (6) Amplification: Infect the elution buffer with XL1-Blue Escherichia coli, amplify it, and then perform the next round of screening.

[0121] 10.2 Second and Third Rounds of Screening

[0122] Repeat the above steps for the second and third rounds of screening, increasing the number of washes in each round to improve the screening rigor.

[0123] 10.3 Third round of library ELISA testing

[0124] The third-round amplified library was used for ELISA testing, following the same method as described in section 3, except that the third-round amplified library sample was added during the sample loading step. The secondary antibody was HRP-labeled M13 antibody (MA5-36125, Invitrogen, Thermo Fisher Scientific) (0.2 μg / mL). The test results showed that the third-round library was positive, indicating that phages specifically binding to MSLNs had been enriched.

[0125] 11. Monoclonal Screening and Identification

[0126] Ninety-six single-clone colonies were randomly selected from the library after the third round of screening. Phage expression was performed on each colony, and the binding activity of each colony to the MSLN antigen was detected by ELISA. The ELISA detection method was the same as in step 3, except that phage expression supernatant diluted 10-fold was added during the sample loading step, and the secondary antibody was HRP-labeled M13 antibody (0.2 μg / mL).

[0127] Select OD 450 Positive clones with a value greater than 2.0 were sequenced to obtain their antibody variable region gene sequences. Among them, the clone numbered MSLN-H4 showed the highest binding activity.

[0128] 12. Antibody Expression and Purification

[0129] 12.1 Construction of Expression Vector

[0130] The selected MSLN-H4 antibody gene sequence was constructed into the mammalian expression vector pCDNA3.1 to form the pCDNA3.1-MSLN-H4 recombinant plasmid.

[0131] 12.2 Cell Transfection and Expression

[0132] Transient transfection expression was performed using HEK293 cells: the recombinant plasmid was mixed with transfection reagent TF2 and added to HEK293 cells, which were then cultured in a shaker at 37°C and 175 rpm with 5% CO2. Serum-free TF2 was added on days 1, 3, and 5 post-transfection, and the cell supernatant was collected after 7 days of culture.

[0133] 12.3 Antibody Purification

[0134] The cell supernatant was centrifuged (3000 rpm, 10 min), filtered through a 0.22 μm filter membrane, and then purified using a Protein G affinity chromatography column. The purification steps are as follows:

[0135] (1) Wash the chromatography column with 5 column volumes of deionized water to remove air and 20% ethanol;

[0136] (2) Equilibrate the chromatography column with 5-10 column volumes of PBS buffer (pH 7.4);

[0137] (3) Flow the filtered cell supernatant through the chromatography column at a flow rate of 0.5 mL / min;

[0138] (4) Wash the chromatography column with PBS buffer until the eluent is free of protein;

[0139] (5) Elute the target protein with glycine elution buffer (pH 2.7), collect the eluent and neutralize it with Tris-HCl (pH 9.0).

[0140] 12.4 Identification of Purified Products

[0141] SDS-PAGE analysis (e.g.) Figure 2 As shown in the figure, the purified anti-MSLN antibody (MSLN-H4) has a purity of >95% and a molecular weight of approximately 150 kDa (intact IgG).

[0142] Experimental results

[0143] Using the above method, a monoclonal antibody against MSLN, named MSLN-H4, was successfully obtained. The antibody's sequence was confirmed by sequencing. After purification, the antibody purity was >95%, making it suitable for subsequent affinity testing and applied research.

[0144] Example 2: Detection of anti-MSLN antibody affinity

[0145] 1. Purpose of Detection

[0146] The binding affinity of the anti-MSLN antibody (MSLN-H4) of this invention to recombinant human MSLN protein was determined by indirect ELISA.

[0147] 2. Experimental Materials

[0148] (1) Antigen: Recombinant human MSLN protein (UniProt accession number: Q13421, 296-606AA fragment, molecular weight approximately 35 kDa), prepared by a eukaryotic expression system.

[0149] (2) Antibody: The anti-MSLN antibody (MSLN-H4) of the present invention has its heavy chain variable region amino acid sequence linked with the light chain variable region amino acid sequence to form scFv, which is fused with mouse IgG2a Fc (SEQ ID NO: 11) and transfected into HEK293F cells (Aibosen (Jiangsu) Biotechnology Co., Ltd.) for expression.

[0150] MSLN-H4 full length sequence: DVLMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIKSSGGGGS GGGGGGSSRSSEVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGGFDPETGGSAYNQKFKGRATLTADKSSSTAYMELRSLTSEDSAVYYCTAYYGSRHYWGQGTTLTVSS (SEQ ID NO: 1)

[0151] MSLN-H4 VL: DVLMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIK (SEQ ID NO: 2)

[0152] MSLN-H4 VH:EVQLQQSGAELVRPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLEWIGGFDPETGGSAYNQKFKGRATLTADKSSSTAYMELRSLTSEDSAVYYCTAYYGSRHYWGQGTTLTVSS (SEQ ID NO: 3)

[0153] MSLN-H4 LCDR1: KSSQSLLDSDGKTYLN (SEQ ID NO: 4)

[0154] MSLN-H4 LCDR2: LVSKLDS (SEQ ID NO: 5)

[0155] MSLN-H4 LCDR3: WQGTHFPWT (SEQ ID NO: 6)

[0156] MSLN-H4 HCDR1:DYEMH (SEQ ID NO: 7)

[0157] MSLN-H4 HCDR2: GFDPETGGSAYNQKFKG (SEQ ID NO: 8)

[0158] MSLN-H4 HCDR3: YYGSRHY (SEQ ID NO: 9)

[0159] Linker: SSGGGGSGGGGGGSSRSS (SEQ ID NO: 10)

[0160] Mouse IgG2a Fc and hinge region: AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 11)

[0161] MSLN-H4 and mouse IgG2a Fc fusion protein: (SEQ ID NO: 12)

[0162] The CDR definition rules are Kabat definition rules.

[0163] (3) Secondary antibody: Horseradish peroxidase-labeled goat anti-mouse antibody (HRP-goat anti-mouse, Wuhan Sanying Biotechnology Co., Ltd., SA00001-1).

[0164] (4) Other reagents: CBS coating buffer, PBST washing buffer (PBS buffer containing 0.05% Tween-20), 5% skim milk powder blocking solution, TMB colorimetric solution, and stop solution (2M H2SO4).

[0165] 3. Detection Method

[0166] (1) Coating antigen: The recombinant human MSLN protein was diluted to 1 μg / mL with CBS buffer and added to a 96-well microplate at a rate of 100 μL / well. The wells were sealed with plastic wrap and incubated overnight at 4°C. The next day, the coating solution was discarded, and the plate was washed three times with PBST washing buffer at a rate of 300 μL / well each time. The plate was then patted dry.

[0167] (2) Blocking: Add 300 μL / well of 5% skim milk powder blocking solution to each reaction well, seal with plastic wrap, and incubate at room temperature for 1 hour. Discard the blocking solution, wash the plate 3 times with PBST washing solution, 300 μL / well each time, and pat dry.

[0168] (3) Adding primary antibody: The anti-MSLN antibody (MSLN-H4) of this invention was serially diluted with PBS buffer to concentrations of 1000 ng / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL, and 0.01 ng / mL. Blank control wells (with PBS buffer added) were also prepared, with 100 μL added to each well. Three replicates were prepared for each concentration. The plates were sealed with plastic wrap and incubated at room temperature for 1 hour. The reaction solution was discarded, and the plates were washed four times with PBST washing buffer, 300 μL / well each time, and then patted dry.

[0169] (4) Add secondary antibody: Dilute HRP-labeled goat anti-mouse antibody with PBS buffer at a ratio of 1:5000, add 100 μL to each well, seal with plastic wrap, and incubate at room temperature for 1 hour. Discard the reaction solution, wash the plate 5 times with PBST washing buffer, 300 μL / well each time, and pat dry.

[0170] (5) Color development: Add 100 μL of TMB color development solution to each well and develop the color at room temperature in the dark for 10 minutes.

[0171] (6) Termination: Add 50 μL of termination solution (2M H2SO4) to each well and gently shake to mix.

[0172] (7) Reading: Place the microplate in the microplate reader and read the absorbance (OD) at a wavelength of 450 nm. 450 (630 nm as the reference wavelength).

[0173] 4. Data Processing and Results

[0174] Plotting the logarithm of antibody concentration on the x-axis, OD 450 The values ​​are plotted on the ordinate, and a four-parameter logistic regression model is used for curve fitting to calculate the half-maximal effect concentration (EC50) value. Experimental results are shown in Table 1 and... Figure 3 As shown.

[0175] Table 1 MSLN-H4 antibody concentration and OD 450 Correspondence table

[0176]

[0177] Based on four-parameter fitting, the EC50 value of the anti-MSLN antibody (MSLN-H4) of this invention binding to the MSLN antigen is 9.532 ng / mL.

[0178] 5. Conclusion

[0179] The above results demonstrate that the anti-MSLN antibody (MSLN-H4) of this invention has extremely high binding affinity to recombinant human MSLN antigen (EC50 = 9.532 ng / mL), laying a solid performance foundation for its application in MSLN antigen immunoassay.

[0180] Example 3: Kit and detection method for detecting MSLN antigen in serum based on indirect competitive ELISA.

[0181] 1. Kit Components

[0182] This embodiment provides an indirect competitive ELISA kit for detecting MSLN antigen in serum, the composition of which is as follows:

[0183]

[0184] Note: The anti-MSLN antibody (primary antibody) is the anti-MSLN antibody (MSLN-H4) described in this invention. After purification, the antibody was diluted to a suitable concentration (recommended 0.5 μg / mL) with sample diluent as the primary antibody working solution. HRP-labeled goat anti-mouse antibody (enzyme-labeled secondary antibody) was purchased from Wuhan Sanying Biotechnology Co., Ltd., SA00001-1. Sample diluent was purchased from Beyotime Biotechnology, C0221A.

[0185] 2. Solution preparation

[0186] (1) Washing solution: Dilute concentrated washing solution PBST (10×) with deionized water at a volume ratio of 1:9, mix well and store for one month at 2~8℃.

[0187] (2) Enzyme-labeled secondary antibody working solution: Dilute the HRP-labeled goat anti-mouse antibody 10,000 times with sample diluent and use immediately.

[0188] (3) Primary antibody working solution: Dilute the anti-MSLN antibody of the present invention to 0.5 μg / mL with sample diluent and use immediately.

[0189] 3. Sample pretreatment

[0190] Collected serum samples must be diluted with sample diluent (recommended dilution ratio: 1:10) and used for testing immediately after dilution. Disposable pipette tips must be used during the procedure to avoid cross-contamination.

[0191] 4. Testing Procedures

[0192] (1) Reagent warming: Take the required reagent out of the refrigerated environment and place it at room temperature (20~25℃) for more than 30 minutes to equilibrate. Shake well before use.

[0193] (2) Preparation of microplates: Take out the required number of MSLN pre-coated microplates (coated with MSLN protein, 0.1 μg / mL, 100 μL / well), and seal the remaining strips with desiccant and store them at 2~8℃.

[0194] (3) Standard dilution: The high concentration of MSLN standard (1 mg / mL) was diluted to 5 μg / mL with sample diluent, and then serially diluted 5 times to obtain a series of standard concentrations: 5 μg / mL, 1 μg / mL, 0.2 μg / mL, 0.04 μg / mL, 0.008 μg / mL, and 0 μg / mL.

[0195] (4) Sample addition: Add 50 μL of standard or diluted serum sample to be tested and 50 μL of primary antibody working solution (anti-MSLN antibody of this invention, 0.5 μg / mL) to each well. Each standard concentration and each sample to be tested should be replicated in two wells. Gently shake to mix and incubate at 25°C in the dark for 1 hour.

[0196] (5) Washing: Discard the liquid in the wells, add 300 μL of washing solution to each well, let stand for 10 seconds and then discard. Repeat washing 3 to 5 times, and pat dry on absorbent paper for the last time.

[0197] (6) Add enzyme-labeled secondary antibody: Add 100 μL of HRP-labeled goat anti-mouse antibody working solution (diluted 10,000 times) to each well and incubate at 25°C in the dark for 30 minutes.

[0198] (7) Washing: Repeat step (5) and wash 3 to 5 times.

[0199] (8) Color development: Add 100 μL of TMB substrate color development solution to each well, gently shake to mix, and incubate at 25°C in the dark for 15 minutes.

[0200] (9) Termination and measurement: Add 50 μL of stop solution (2M H2SO4) to each well and gently shake to mix. Read the OD value of each well at 450 nm wavelength (reference wavelength 630 nm) using a microplate reader within 5 minutes.

[0201] 5. Result Determination

[0202] A standard curve was plotted with the logarithm of the standard concentration on the x-axis and the OD value on the y-axis. A four-parameter logistic regression model was used for fitting the curve. The results are as follows: Figure 4 As shown.

[0203] In this embodiment, the test results of the standard are as follows:

[0204]

[0205] After fitting with four parameters, the regression equation is obtained:

[0206]

[0207] Fit quality R 2 = 0.998.

[0208] The MSLN concentration of the sample to be tested can be calculated by substituting its OD value into the above equation, and then multiplying it by the sample dilution factor to obtain the MSLN content in the original serum. The results of this method show a good negative correlation between MSLN antigen concentration and OD value, with good linearity in the range of 0.008–5 μg / mL, and it can be used for qualitative and semi-quantitative detection of MSLN in serum samples.

[0209] 6. Explanation of Method Principles

[0210] This embodiment employs the principle of indirect competitive ELISA: the ELISA plate is pre-coated with MSLN antigen, and the MSLN in the sample competes with the coating antigen for a limited amount of anti-MSLN antibody (primary antibody). The higher the MSLN content in the sample, the less primary antibody binds to the coating antigen, and subsequently, less HRP-labeled goat anti-mouse secondary antibody binds, resulting in a lower colorimetric signal. Therefore, the OD value is negatively correlated with the MSLN content in the sample.

[0211] Example 4: Detection of MSLN protein expression in different cell lines using Western blotting.

[0212] 1. Experimental Objective

[0213] The endogenous expression level of MSLN protein in different cell lines was detected by Western blotting using the anti-MSLN antibody (MSLN-H4) of this invention as the primary antibody.

[0214] 2. Experimental Materials

[0215] (1) Antibody: The anti-MSLN antibody (MSLN-H4) of this invention.

[0216] (2) Cell samples:

[0217] Negative control cells: Human embryonic kidney cell line 293T (does not express or expresses low levels of MSLN). Positive cells: Human ovarian cancer cell line OVCAR-3 (highly expresses MSLN).

[0218] (3) Secondary antibody: Horseradish peroxidase-labeled goat anti-mouse antibody (HRP-goat anti-mouse).

[0219] (4) Main reagents: RIPA lysis buffer (containing protease inhibitor, Beyotime Biotechnology, P0013B), BCA protein concentration assay kit (Beyotime Biotechnology, P0010), 5×SDS-PAGE loading buffer, SDS-PAGE gel (separating gel concentration 10% or 12%), protein molecular weight marker, electrophoresis buffer, transfer buffer, PVDF membrane, blocking buffer (5% skim milk powder dissolved in TBST), TBST washing buffer, ECL chemiluminescent substrate.

[0220] (5) Main instruments: electrophoresis apparatus, transfer apparatus, decolorization shaker, chemiluminescence imaging system.

[0221] 3. Experimental Methods

[0222] (1) Cell lysis and protein quantification

[0223] 293T cells and OVCAR-3 cells were collected separately, and each was lysed with an appropriate amount of RIPA lysis buffer (containing protease inhibitors) for 30 minutes on ice. After centrifugation at 12,000 rpm for 15 minutes at 4°C, the supernatant was collected. Protein concentration was determined using the BCA method, and the protein concentration of each sample was adjusted to 2 μg / μL with lysis buffer.

[0224] (2) SDS-PAGE electrophoresis

[0225] Take 20 μg of each of the above protein samples, add 5× SDS-PAGE loading buffer (final concentration 1×), and boil at 100℃ for 10 minutes to denature the proteins. Add the denatured protein samples and protein markers to the SDS-PAGE gel wells, and perform electrophoresis at 80V until the bromophenol blue front enters the separating gel, then adjust to 120V until the bromophenol blue reaches the bottom of the gel.

[0226] (3) Transfer membrane

[0227] After electrophoresis, the gel containing the target protein region is cut off, and the transfer membrane sandwich is assembled in the order of "cathode-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-anode". The PVDF membrane needs to be activated with methanol beforehand. Transfer the membrane at a constant voltage of 100V for 60-90 minutes under ice bath conditions.

[0228] (4) Closed

[0229] After the transfer was completed, the PVDF membrane was placed in a 5% skim milk powder blocking solution and sealed on a shaker at room temperature for 1 hour.

[0230] (5) Primary antibody incubation

[0231] Discard the blocking buffer, add the anti-MSLN antibody of this invention (MSLN-H4, dilution ratio 1:1000) diluted with the blocking buffer, and incubate overnight on a shaker at 4°C. The next day, wash the membrane three times with TBST for 10 minutes each time.

[0232] (6) Secondary antibody incubation

[0233] Add HRP-labeled goat anti-mouse secondary antibody diluted with blocking buffer (1:5000), and incubate on a shaker at room temperature for 1 hour. Wash the membrane three times with TBST for 10 minutes each time.

[0234] (7) Chemiluminescence detection

[0235] Mix equal volumes of ECL chemiluminescent substrate solutions A and B, and uniformly drop the mixture onto a PVDF membrane. Incubate in the dark for 1-2 minutes. Acquire images using a chemiluminescence imaging system.

[0236] 4. Western Blot Detection Results

[0237] Negative control cells (293T): No obvious specific bands were observed, indicating that the MSLN expression level in 293T cells was extremely low, consistent with the expectation of a negative control. Positive cells (OVCAR-3): Two obvious specific bands appeared at approximately 40 kDa and approximately 70 kDa. Figure 5 This is because MSLN proteins undergo varying degrees of glycosylation modifications during post-translational processing, resulting in protein forms with different molecular weights.

[0238] 5. Conclusion

[0239] The above results demonstrate that the anti-MSLN antibody (MSLN-H4) of this invention can specifically recognize endogenously expressed MSLN protein in different cell lines, exhibiting high sensitivity and specificity in Western blotting. This antibody can clearly distinguish between MSLN-overexpressing cells (OVCAR-3) and MSLN-negative cells (293T), and can detect different molecular weight forms of MSLN produced by glycosylation modification (approximately 40 kDa and 70 kDa), making it suitable for qualitative and semi-quantitative analysis of MSLN protein expression.

Claims

1. An antibody against MSLN or an antigen-binding fragment thereof, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3. The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 7, 8 and 9, respectively; and the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 4, 5 and 6, respectively.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, (1) The heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; and / or, the light chain variable region comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; or, (2) The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO: 3; and / or, the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

2.

3. The antibody or its antigen-binding fragment as described in claim 2, characterized in that, The antibody or its antigen-binding fragment is a full-length antibody, Fab, Fab', F(ab')2, Fv or scFv; and / or, the antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody or a humanized antibody.

4. The antibody or its antigen-binding fragment as described in claim 3, characterized in that, The antibody or its antigen-binding fragment is scFv, and the heavy chain variable region and the light chain variable region are connected by a linker; or, The antibody or its antigen-binding fragment includes scFv and a heavy chain constant region; the heavy chain constant region is a heavy chain constant region derived from IgG1, IgG2, IgG3 or IgG4; and / or, the heavy chain constant region includes a hinge region, CH2 and CH3.

5. The antibody or its antigen-binding fragment as described in claim 4, characterized in that, The amino acid sequence of the antibody or its antigen-binding fragment has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; or, The amino acid sequence of the antibody or its antigen-binding fragment has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

12.

6. The antibody or its antigen-binding fragment as described in claim 5, characterized in that, The amino acid sequence of the antibody or its antigen-binding fragment is shown in SEQ ID NO: 1; or, The amino acid sequence of the antibody or its antigen-binding fragment is shown in SEQ ID NO:

12.

7. An isolated nucleic acid, characterized in that, The nucleic acid encodes the antibody or its antigen-binding fragment as described in any one of claims 1-6.

8. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 7.

9. A transformant, characterized in that, The transformant comprises the nucleic acid as described in claim 7 or the recombinant expression vector as described in claim 8, or expresses the antibody or its antigen-binding fragment as described in any one of claims 1-6; the transformant is not an animal variety or a plant cell variety.

10. A reagent kit, characterized in that, The kit comprises one or more of the antibody or antigen-binding fragment thereof as described in any one of claims 1-6, the nucleic acid as described in claim 7, the recombinant expression vector as described in claim 8, or the transformant as described in claim 9.

11. A method for detecting MSLN, characterized in that, The method comprises contacting the sample to be tested with an antibody or antigen-binding fragment thereof as described in any one of claims 1-6 or a kit as described in claim 10; the method is for non-diagnostic and / or therapeutic purposes.

12. The use of one or more of the antibody or antigen-binding fragment thereof as described in any one of claims 1-6, the nucleic acid as described in claim 7, the recombinant expression vector as described in claim 8, the transformant as described in claim 9, or the kit as described in claim 10 in the preparation of a product for detecting the content or concentration of MSLN.

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