Alpha 1-antitrypsin monoclonal antibody and use thereof

Abstract

The application provides an alpha 1-antitrypsin monoclonal antibody and application thereof, and belongs to the field of antibodies. The alpha 1-antitrypsin antibody comprises: (1) a heavy chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3 in the heavy chain variable region as shown in SEQ ID NO. 5; and (2) a light chain variable region, wherein the light chain variable region comprises a light chain CDR1, a light chain CDR2 and a light chain CDR3 in the light chain variable region as shown in SEQ ID NO. 9. The antibody provided by the application has high specificity, solves the problem of poor experimental repeatability caused by non-specific binding of existing antibodies, can be applied to various application scenarios such as protein immunoblotting and flow cytometry, has cross-species reactivity, and can be directly applied to comparative research on pathological models of humans and rodents. The antibody provided by the application has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of antibodies, specifically relating to an α1-antitrypsin monoclonal antibody and its applications. Background Technology

[0002] α1-Antitrypsin is an acute-phase glycoprotein belonging to the serine protease inhibitor superfamily. It is primarily synthesized by hepatocytes, with smaller amounts produced by monocytes, macrophages, intestinal epithelial cells, and bronchial epithelial cells. The core function of α1-antitrypsin is to inhibit neutrophil elastase, and it also inhibits plasmin, thrombin, trypsin, and chymotrypsin. α1-antitrypsin levels are typically elevated in myocardial infarction, surgery, bacterial infections, viral infections, pancreatitis, cancer, and many other conditions. Therefore, developing a highly specific and high-affinity α1-antitrypsin antibody is of great significance for the detection of these diseases.

[0003] However, current products for detecting α1-antitrypsin have many shortcomings, such as low validation standards and incomplete application scope validation. Most products currently do not provide any specific validation data based on KO / KD knockout cell lines, leading to false positives in samples with low α1-antitrypsin expression. Flow cytometry, in particular, lacks validation with KO negative controls. The validation design of this invention precisely fills this gap. Existing commercial antibodies generally only claim to be suitable for Western blotting (WB) applications, rarely achieving specific validation at both the KO cell level and flow cytometry levels, which casts doubt on their reliability in various experimental scenarios. Summary of the Invention

[0004] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0005] In a first aspect of the present invention, an α1-antitrypsin (Serine Protease Inhibitor, SERPINA1, or Alpha-1 Antitrypsin, AAT) antibody or its antigen-binding fragment is provided, wherein the α1-antitrypsin antibody or its antigen-binding fragment comprises: (1) Heavy chain variable region: The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 as shown in SEQ ID NO.5; (2) Light chain variable region: The light chain variable region includes light chain CDR1, light chain CDR2 and light chain CDR3 in the light chain variable region as shown in SEQ ID NO.9.

[0006] When referring to antibodies defined by a specific CDR sequence as described 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 the specific CDR boundaries defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations). CDR sequences determined for different schemes all fall within the protection scope of this application.

[0007] The boundaries of the CDR of the antibody of the present invention can be determined artificially according to any method or combination thereof in the art. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers the CDR sequence determined in any of the foregoing methods.

[0008] In some embodiments, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively, and the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0009] In some embodiments, the heavy chain variable region of the α1-antitrypsin antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO. 5 or an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO. 5, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence having at least 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO. 9.

[0010] In this invention, antibodies include non-human antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies. Antibodies include monospecific and multispecific (e.g., bispecific, trispecific, and higher-order specific) antibodies. Antibodies include monovalent antibodies, single-chain antibodies, single-chain variable fragments (scFv), camelified antibodies, affinity antibodies, and disulfide-linked Fvs (sdFv). Antibodies include monoclonal populations, polyclonal populations, or recombinant antibodies.

[0011] A "humanized" antibody is an antibody whose sequence differs from that of an antibody derived from a non-human species due to substitutions, deletions, and / or additions of one or more amino acids, such that when administered to a human individual, the humanized antibody is less likely to induce an immune response and / or induces a less severe immune response compared to the non-human species antibody. In one embodiment, a humanized antibody is produced by mutations in certain amino acids in the framework regions and constant domains of the heavy and / or light chains of a non-human species antibody. In another embodiment, a constant domain from a human antibody is fused with a variable domain from a non-human species. In yet another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are altered to reduce the potential immunogenicity of the antibody when administered to a human individual, wherein the altered amino acid residues are not important for the immune-specific binding of the antibody to its antigen, or the resulting amino acid sequence change is a conserved change such that the binding of the humanized antibody to the antigen is not significantly worse than that of the non-human antibody to the antigen.

[0012] As used herein, the terms "antibody fragment," "antibody moiety," "antigen-binding fragment," or "antigen-binding moiety of an antibody," and other related terms, refer to molecules other than intact antibodies that contain a portion of the intact antibody that binds to the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; Fd; and Fv fragments, as well as dAb; bifunctional antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen binding to the polypeptide. Antigen-binding moieties of antibodies can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding moieties particularly include Fab, Fab', F(ab')2, Fv, domain-specific antibodies (dAbs) and complementarity-determining region (CDR) fragments, chimeric antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer antigen-binding properties to the antibody fragment.

[0013] In some embodiments, the antibody further includes a constant region, and according to the structure of the constant region, the antibody includes IgG antibodies, IgA antibodies, IgM antibodies, IgE antibodies, and IgD antibodies.

[0014] In some embodiments, the constant region is the human IgG constant region, which comprises four isotypes: IgG1, IgG2, IgG3, and IgG4. They share greater than 95% homology in the amino acid sequence of the Fc region, but exhibit major differences in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions such as antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to an Fc receptor (FcgR) on the surface of immune effector cells, such as natural killer cells and macrophages, leading to phagocytosis or lysis of the target cell. In CDC, the antibody kills the target cell by triggering a complement cascade reaction on the cell surface. The antibodies described herein include antibodies having the aforementioned features of variable domains combined with any IgG isotype, including those wherein the Fc sequence can be modified to achieve different effector functions.

[0015] In some implementations, the constant region is a mouse constant region, including but not limited to IgG1 and IgG2a.

[0016] In the most preferred embodiment of the present invention, the constant region is IgG1.

[0017] In a second aspect of the invention, an isolated nucleic acid molecule is provided, said nucleic acid molecule encoding the α1-antitrypsin antibody or an antigen-binding fragment thereof described in the first aspect of the invention.

[0018] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” as used herein are used interchangeably with other related terms and refer to nucleotide polymers and are not limited to any particular length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids include DNA molecules (cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and their hybrids. Nucleic acid molecules can be single-stranded or double-stranded.

[0019] In one implementation, the nucleic acid comprises one type of polynucleotide, or a mixture of two or more different types of polynucleotides.

[0020] In a third aspect of the invention, a carrier is provided, the carrier comprising the isolated nucleic acid molecules described in the second aspect of the invention.

[0021] As used herein, “vector” and related terms refer to a nucleic acid molecule (e.g., DNA or RNA) operably linked to foreign genetic material (e.g., nucleic acid transgenes). Vectors can be used as a medium for introducing foreign genetic material into cells (e.g., host cells). Vectors may include at least one restriction endonuclease recognition sequence for inserting the transgene into the vector. Vectors may also include at least one gene sequence that confers antibiotic resistance or selectivity to facilitate the selection of host cells with vector transgene constructs. Vectors can be single-stranded or double-stranded nucleic acid molecules. Vectors can be linear or circular nucleic acid molecules. Donor nucleic acids used in gene editing methods using zinc finger nucleases, TALEN, or CRISPR / Cas can be a type of vector. One type of vector is a “plasmid,” which refers to a linear or circular double-stranded extrachromosomal DNA molecule that can be linked to a transgene and is capable of replicating and transcribing and / or translating the transgene in a host cell. Viral vectors typically contain a viral RNA or DNA backbone sequence that can be linked to a transgene. The viral backbone sequence may be modified to lose infectivity but retain the ability to insert the viral backbone and co-linked transgenes into the host cell genome. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, parvovirus vectors, vaccinia virus vectors, herpes simplex virus vectors, and E.-Perot virus vectors. Some vectors can replicate autonomously in the host cells they are introduced into (e.g., bacterial vectors containing bacterial origins of replication and free-living mammalian vectors). Other vectors (e.g., non-free-living mammalian vectors) can integrate into the host cell's genome after introduction and thereby replicate along with the host genome.

[0022] An "expression vector" is a type of vector that may contain one or more regulatory sequences, such as inducible and / or constitutive promoters and enhancers. Expression vectors may include ribosome binding sites and / or polyadenylation sites. Expression vectors may include one or more origins of replication. The regulatory sequences guide the transcription or transcription and translation of a transgene linked to the expression vector, thereby transducing it into a host cell. The regulatory sequences can control the level, timing, and / or location of transgene expression. For example, regulatory sequences can exert their effect on the transgene directly or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and / or nucleic acids). The regulatory sequences may be part of the vector.

[0023] In some embodiments, the expression vector is selected from pcDNA3.1.

[0024] In a fourth aspect of the invention, hybridoma cells are provided that produce the α1-antitrypsin antibody or antigen-binding fragment thereof described in the first aspect of the invention.

[0025] In a fifth aspect of the invention, a transformed cell or a cell population comprising the transformed cell is provided, said transformed cell being obtained by introducing a host cell into a vector as described in the third aspect of the invention.

[0026] As used herein, the terms “transfection,” “transformation,” “transduction,” “introduction,” or other related terms refer to methods used to transfer or introduce exogenous nucleic acids (e.g., transgenics) into host cells. “Transfected,” “transformed,” or “transduced” cells are host cells into which exogenous nucleic acids (transgenics) have been introduced. Host cells include individual primary cells and their progeny.

[0027] As used herein, the term "transformed cell" or "population containing transformed cells" or related terms refer to cells (or populations or multiple host cells) to which a foreign (exogenous or transgenic) nucleic acid has been introduced. The foreign nucleic acid may include an expression vector operatively linked to a transgene, and the transformed cells may be used to express the nucleic acid and / or polypeptides encoded by the foreign nucleic acid (transgene). Transformed cells (or populations thereof) may be cultured cells or may be extracted from an individual. Transformed cells (or populations thereof) include primary cells of an individual and their progeny, regardless of passage number. Transformed cells (or populations thereof) include immortalized cell lines. Progeny cells may or may not contain the same genetic material as the parent cells. Transformed cells encompass progeny cells. In one embodiment, transformed cell describes any cell (including its progeny) that has been modified, transfected, transduced, transformed, and / or manipulated in any way to express an antibody, as disclosed herein. In one instance, an expression vector operatively linked to a nucleic acid, as described herein, encoding a desired antibody or an antigen-binding fragment thereof, may be introduced into a host cell (or population thereof).

[0028] In some embodiments, the host cell may be a prokaryote, such as *Escherichia coli*, or a eukaryote, such as a single-celled eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cells), a mammalian cell (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells), or a hybridoma. In one embodiment, the host cell comprises non-human cells, including CHO, BHK, NSO, SP2 / 0, and YB2 / 0. In one embodiment, the host cell comprises human cells, including HEK293, HT-1080, Huh-7, and PER.C6. Examples of host cells include the COS-7 strain of monkey kidney cells (ATCC CRL 1651), L cells, C127 cells, 3T3 cells (ATCC CCL163), Chinese hamster ovary (CHO) cells or derivatives thereof, or DHFR-deficient CHO strain DX-B 11, HeLa cells, BHK (ATCC CRL 10) cell line, CV1 / EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70); human embryonic kidney cells, such as 293, 293EBNA, or MSR 293; human epidermal A431 cells, human Colo 205 cells, transformed other primate cell lines, normal diploid cells, cell lines derived from major tissues cultured in vitro, primary explants, HL-60, U937, HaK, or Jurkat cells. In one embodiment, host cells include lymphocytes, such as Y0, NSO, or Sp20.

[0029] The vector can be introduced into the host cell using methods suitable for the host cell. Various methods for introducing nucleic acids into the host cell are known in the art, including but not limited to electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-glucan, or other substances; viral transfection; non-viral transfection; microbolite bombardment; liposome transfection; and infection (e.g., where the vector is an infectious agent).

[0030] In a sixth aspect of the invention, there is provided an α1-antitrypsin antibody or a derivative thereof of an antigen-binding fragment thereof, the derivative comprising a complex formed by directly or indirectly coupling the α1-antitrypsin antibody or the antigen-binding fragment thereof of the first aspect of the invention to a detectable marker.

[0031] In some embodiments, the detectable label or portion thereof is radioactive, colorimetric, antigenic, enzymatic, detectable beads (e.g., magnetic or electron-dense (e.g., gold) beads), biotin, streptavidin, or protein A. A variety of labels may be used, including but not limited to radionuclides, fluorescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and ligands (e.g., biotin, haptens).

[0032] In a seventh aspect of the invention, a product for detecting α1-antitrypsin is provided, the product comprising the α1-antitrypsin antibody or antigen-binding fragment thereof as described in the first aspect of the invention, the isolated nucleic acid molecule as described in the second aspect of the invention, the vector as described in the third aspect of the invention, the hybridoma cell as described in the fourth aspect of the invention, the transformed cell or cell population containing the present invention as described in the fifth aspect of the invention, and / or a derivative of the α1-antitrypsin antibody or antigen-binding fragment thereof as described in the sixth aspect of the invention.

[0033] In some implementations, the product includes one or more of a kit, test strip, reagent, and chip.

[0034] In some embodiments, the kit may also include, but is not limited to, a container for holding α1-antitrypsin antibody or its antigen-binding fragment when not in use, instructions for use, α1-antitrypsin antibody or its antigen-binding fragment attached to a solid support, known standards, and reference samples, or one or more of these.

[0035] The term "known standard" can refer to a solution containing a known amount or concentration of α1-antitrypsin, wherein such solution can be a naturally occurring solution; or such solution can be a synthetic solution, such as a buffer solution in which a known amount of α1-antitrypsin is diluted. Known standards described herein may include α1-antitrypsin isolated from subjects, recombinant or purified α1-antitrypsin protein, or α1-antitrypsin associated with disease symptoms.

[0036] The term "reference sample" is a sample that can be compared with another sample, such as a test sample, to characterize the sample being compared. A reference sample will possess certain characteristic properties that serve as the basis for comparison with the test sample. For example, a reference sample might be used as a benchmark for α1-antitrypsin levels indicating whether a subject has cancer. The reference sample does not necessarily have to be analyzed in parallel with the test sample; therefore, in some cases, the reference sample can be a previously determined value or range used to characterize a given condition, such as α1-antitrypsin levels indicating whether a subject has a disease.

[0037] In some implementations, the kits include, but are not limited to, ELISA kits, immunofluorescence kits, FACS kits, Western Blot kits, IHC kits, and ICC kits.

[0038] In some implementations, the product for detecting α1-antitrypsin can detect the full-length or functional fragments of α1-antitrypsin.

[0039] In an eighth aspect of the invention, a method for preparing the α1-antitrypsin antibody or its antigen-binding fragment as described in the first aspect of the invention is provided, the method comprising culturing hybridoma cells as described in the fourth aspect of the invention or transformed cells as described in the fifth aspect of the invention or a cell population containing the thereof.

[0040] The antibodies or antigen-binding fragments described herein can be purified using protein isolation / purification methods commonly known in the field of protein chemistry. Non-limiting examples include extraction, recrystallization, salting out (e.g., with ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal-phase chromatography, reverse-phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, or any combination of these methods. Following purification, the peptides are exchanged in different buffers and / or concentrated using any of a variety of methods known in the art, including but not limited to filtration and dialysis.

[0041] In a ninth aspect of the invention, the use of the α1-antitrypsin antibody or antigen-binding fragment thereof described in the first aspect of the invention, the isolated nucleic acid molecule described in the second aspect of the invention, the vector described in the third aspect of the invention, the hybridoma cell described in the fourth aspect of the invention, the transformed cell or cell population containing the present invention described in the fifth aspect of the invention, and / or a derivative of the α1-antitrypsin antibody or antigen-binding fragment thereof described in the sixth aspect of the invention in the preparation of a product for detecting α1-antitrypsin is provided.

[0042] In some embodiments, the product is the product described in the seventh aspect of the present invention.

[0043] In some implementations, the product can be used to diagnose α1-antitrypsin-related diseases. These α1-antitrypsin-related diseases include, but are not limited to, myocardial infarction, surgery, bacterial infections, viral infections, pancreatitis, and cancer. These cancers include, but are not limited to, liver cancer, breast cancer, rhabdomyosarcoma, and prostate cancer.

[0044] In a ninth aspect of the invention, the use of the α1-antitrypsin antibody or its antigen-binding fragment described in the first aspect of the invention, the isolated nucleic acid molecule described in the second aspect of the invention, the vector described in the third aspect of the invention, the hybridoma cell described in the fourth aspect of the invention, the transformed cell or cell population containing the present invention described in the fifth aspect of the invention, and / or a derivative of the α1-antitrypsin antibody or its antigen-binding fragment described in the sixth aspect of the invention in the preparation of products for diagnosing α1-antitrypsin-related diseases is also provided.

[0045] The tenth aspect of the present invention also provides a method for detecting α1-antitrypsin or a fragment thereof in a test sample for non-diagnostic or diagnostic purposes, the method comprising contacting the test sample with the α1-antitrypsin antibody or its antigen-binding fragment as described in the first aspect of the present invention, or contacting the test sample with an antibody derivative of the α1-antitrypsin antibody or its antigen-binding fragment as described in the sixth aspect of the present invention, or contacting the test sample with a product for detecting α1-antitrypsin protein as described in the seventh aspect of the present invention, and detecting the formation of a complex of the α1-antitrypsin antibody or its antigen-binding fragment or an antibody derivative of the α1-antitrypsin antibody or its antigen-binding fragment with α1-antitrypsin or a fragment thereof.

[0046] The eleventh aspect of the present invention provides a method for diagnosing α1-antitrypsin-related diseases, the method comprising contacting a test sample with an α1-antitrypsin antibody or its antigen-binding fragment as described in the first aspect of the present invention, or contacting the test sample with an antibody derivative of an α1-antitrypsin antibody or its antigen-binding fragment as described in the sixth aspect of the present invention, or contacting the test sample with a product for detecting α1-antitrypsin protein as described in the seventh aspect of the present invention, and detecting the formation of a complex of the α1-antitrypsin antibody or its antigen-binding fragment or an antibody derivative of an α1-antitrypsin antibody or its antigen-binding fragment with α1-antitrypsin or its fragment.

[0047] The advantages and beneficial effects of this invention are as follows: 1. Western blot analysis was performed on lysates of HepG2 (human liver cancer cells), MCF7 (human breast cancer cells), RH30 (human rhabdomyosarcoma cells), DU145 (human prostate cancer cells), H9c2 (rat cardiomyocytes), and C2C12 (mouse myoblasts) using the antibody of this invention. The results showed that the antibody of this invention clearly detected a single band at approximately 52 kDa, with no nonspecific bands and an extremely low signal-to-noise ratio. This indicates that the antibody has extremely high epitope recognition specificity, solving the problem of poor experimental reproducibility caused by nonspecific binding of existing antibodies.

[0048] 2. To confirm that the anti-α1-antitrypsin antibody of the present invention does not exhibit non-specific binding, the present invention performed the industry-recognized gold standard verification of gene knockout (KO). In wild-type cell lysates, the antibody showed a clear, single target band at approximately 52 kDa; while in SERPINA1 gene knockout KO cell lysates, this band completely disappeared without any residual signal. Therefore, the band identified by the antibody of the present invention in Western blot analysis is indeed the SERPINA1 protein itself, and not other proteins with similar molecular weights. This also demonstrates that the antibody of the present invention has absolute specificity and does not cross-react with any other cellular components in the context of complex whole-cell lysates lacking the target protein.

[0049] 3. Unlike conventional antibodies that are limited to detecting human samples, the antibody of this invention exhibits cross-species (human, rat, mouse) reactivity. This antibody stably recognizes endogenous SERPINA1 protein, avoiding antigenic epitope masking due to differences in protein modification. This characteristic allows the same antibody reagent to be directly applied to comparative studies of pathological models in humans and rodents, filling the gap in commercially available SERPINA1 antibodies' inability to simultaneously support multiplex detection in both humans and model animals. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0051] Figure 1 These are the experimental results of Western Blot detection of exogenous SERPINA1 protein; Figure 2 This is the result of Western blotting detection of intracellular SERPINA1 protein. HepG2 represents lysate of human hepatocellular carcinoma cells (55 kDa); MCF-7 represents lysate of human breast cancer cells (55 kDa); RH30 represents lysate of human rhabdomyosarcoma cells (55 kDa); DU145 represents lysate of human prostate cancer cells (55 kDa); H9c2 represents lysate of rat embryonic heart cells (55 kDa); and C2C12 represents lysate of mouse myoblast cells (55 kDa). Figure 3 The following is a graph showing the results of Western blotting analysis of SERPINA1 protein expression in wild-type / KO monoclonal cell lines. In graph A, the internal control (molecular weight 90 kDa) is represented by WT (human embryonic kidney 293T cell lysate) and KO (SERPINA1 knockout cell line lysate). In graph B, the experimental group (molecular weight 55 kDa) is represented by WT (293T cell lysate) and KO (SERPINA1 knockout cell line lysate). Figure 4 This is a graph showing the results of flow cytometry analysis of SERPINA1 protein expression in the human breast cancer cell line MCF7. Detailed Implementation

[0052] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0053] Example 1: Preparation and Preliminary Validation of Antibodies 1. Experimental materials, equipment and consumables: as shown in Table 1 and Table 2 below.

[0054] Table 1 Experimental Materials

[0055] Table 2 Equipment and Consumables

[0056] 2. Experimental Methods (1) Antigen preparation: Based on comprehensive bioinformatics analysis, spatial structure prediction, reference to homologous protein information, and combined with downstream application requirements, the [Thr]-[Lys] amino acid sequence of Uniport ID: P01009 was selected and recombinantly expressed using the E. coli expression system, with an N-His tag and signal peptide. The truncated protein expressed in prokaryotes was used as an immunogen to immunize mice.

[0057] (2) Immunity The recombinant protein (SERPINA1 antigen) was mixed with complete Freund's adjuvant (1:1) and emulsified. Five BALB / c mice were immunized subcutaneously. Two weeks later, the SERPINA1 antigen containing the recombinant protein (SEQ ID NO.1) was emulsified with incomplete Freund's adjuvant (1:1) for a second and third immunization. After the three immunizations, blood was collected and serum titers were determined by serial dilution using ELISA. Mice with the highest antibody titers against the antigen were selected for the next step of cell fusion.

[0058] (3) Cell fusion Prepare mouse-derived sp2 / 0 myeloma cells in advance, ensuring they are in the logarithmic growth phase at the time of fusion. Obtain spleens from immunized mice and prepare a single-cell suspension of lymphocytes. Mix the mouse spleen lymphocytes with the myeloma cells and perform electrofusion according to the program set on a BTX 2001 LITE cell fusion instrument. After fusion, allow the cells to stand for 4 minutes, then dilute them in 50 ml centrifuge tubes to 40 ml of HAT-containing medium and incubate at 37°C for 1 hour. Then, aliquot the diluted solution into 10 wells of 96-well plates and incubate at 37°C with 5% CO2. Observe the fused cell status in the 96-well plates 6-9 days after fusion, change the medium with HT, and continue incubation at 37°C with 5% CO2.

[0059] (4) Screening and cloning Seven to ten days after fusion, cells were coated with 1 μg / mL SERPINA1 antigen (SEQ ID NO.1) for ELISA antigen coating, and clones were tested and screened. The corresponding cell line numbers were labeled, and the positive wells were subjected to limiting dilution until the entire 96-well plate showed a positive result for ELISA. Stable monoclonal lines with high positive values ​​were selected to obtain hybridoma cell lines secreting specific monoclonal antibodies, recorded as 21-C1-E12.

[0060] (5) Perform antibody sequencing on the selected hybridoma cell lines. Total RNA was isolated from 21-C1-E12 hybridoma cells according to the TriZol reagent instructions. The total RNA was reverse transcribed into cDNA according to the Vazyme first-strand cDNA synthesis kit instructions. The nucleotide sequences of the heavy chain variable region and light chain variable region of the 21-C1-E12 anti-SERPINA1 monoclonal antibody were amplified. The nucleotide sequences of the heavy chain variable region and light chain variable region were then cloned into a eukaryotic expression vector (Thermo Fisher, pcDNA3.1) with a constant region for cell transfection.

[0061] (6) Cell transfection and screening Prepare HEK 293F cells for transfection in advance. After centrifugation and replacement with fresh culture medium, transfer the cells into 6-well plates at a density of 2 ml per well (0.5 × 10⁻⁶ cells / well). 6 Cells / ml. The eukaryotic expression vector was mixed with lipo2000 at a ratio of 1:3 and added to the prepared 293F cells, which were then cultured in a shaker at 37°C and 5% CO2. After 3-5 days of culture, the transfected cell supernatant was subjected to ELISA to screen positive wells against the corresponding antigen. The cell supernatant from the positive wells was then subjected to immunohistochemical detection. If the immunohistochemical detection was positive, the detected antibody sequence was confirmed to be correct.

[0062] (7) Preparation and purification of monoclonal antibodies on cells The confirmed positive expression vector was used to transfect large numbers of cells. After culturing for 3-5 days, the cell suspension was collected, centrifuged, and the supernatant was purified using affinity chromatography. The purified monoclonal antibody concentration was determined, aliquoted, and stored at 4-8°C.

[0063] One of the antibodies was sequenced, and the obtained base sequence was translated into an amino acid sequence. The amino acid sequences of the heavy chain and light chain variable regions of the 21-C1-E12 anti-SERPINA1 monoclonal antibody were analyzed and obtained as shown in Table 3 below.

[0064] Table 3. Antigen-antibody amino acid sequences

[0065] 3. Experimental Results (1) The results of indirect ELISA detection of supernatant titer are shown in Table 4 below. Through cell fusion and screening, 11 hybridoma cell lines that can stably secrete anti-SERPINA1 antibodies were successfully obtained, and their numbers are: 1-G4-F3, 3-G10-C10, 4-C4-G6, 7-H7-H7, 8-E1-H5, 11-D9-H7, 14-E8-F6, 15-B3-G9, 16-E12-F6, 21-C1-E12, and 26-C10-E8. The results of indirect ELISA detection showed that the supernatant of the above 11 cell lines all showed significant binding signals to the SERPINA1 antigen. Among them, the detection values ​​(OD values) of many cell lines (such as 1-G4-F3, 14-E8-F6, etc.) were higher than 3.0, which was significantly higher than the blank control (0.04), proving that the secreted antibodies have high affinity.

[0066] Table 4 Results of indirect ELISA detection of supernatant titer

[0067] (2) The subtype detection results are shown in Table 5 below. The antibody subtype detection results show that the monoclonal antibody obtained in this invention contains two IgG subtypes: IgG1 ((4-C4G6, 7-H7-H7, 21-C1-E12, 26-C10-E8) and IgG2a (the remaining 7 strains)). This provides a diverse selection basis for the subsequent application of the antibody in functional experiments (such as complement activation and binding to different Fc receptors).

[0068] Table 5 Subtype Detection Results

[0069] (3) Western Blot (WB) results are as follows Figure 1 As shown in the figure. Further verification by Western blotting revealed a single, clear band at approximately 44.3 kDa in the supernatant of all 11 cell lines, perfectly consistent with the theoretical molecular weight of SERPINA1 protein. The blank control showed no band, ruling out non-specific reactions. These results confirm that the antibody secreted by the hybridoma lines obtained in this invention can specifically recognize SERPINA1 protein in its native or denatured state.

[0070] In summary, this invention successfully prepared a group of hybridoma cell lines capable of secreting high-affinity and high-specificity anti-SERPINA1 monoclonal antibodies. The hybridoma supernatant was validated using both ELISA and Western blotting methods, demonstrating that these antibodies exhibited excellent binding activity and specificity against the target antigen.

[0071] Example 2: Endogenous Western Blot Detection of Antibody Specificity 1. The experimental materials are shown in Table 6 below.

[0072] Table 6 Experimental Materials

[0073] 2. Experimental Methods In this embodiment, the 21-C1-E12 anti-SERPINA1 monoclonal antibody was used as the primary antibody. Western blotting was used to detect the expression levels of SERPINA1 protein in six common cell lines to verify its ability to recognize SERPINA1 protein. The method is as follows: (1) Six cell lines with different SERPINA1 expression levels were selected for culture: HepG2 (human liver cancer cells), MCF7 (human breast cancer cells), RH30 (human rhabdomyosarcoma cells), DU145 (human prostate cancer cells), H9c2 (rat cardiomyocytes) and C2C12 (mouse myoblasts). Proteins were extracted, lysates were prepared, gels were prepared, samples were spotted, electrophores were performed, and membranes were transferred. PVDF membranes were activated with methanol for 1 min, washed twice with pure water, and then washed three times with TBST. Blocking: The membrane was placed in blocking solution prepared with 5% skim milk and shaken at room temperature for 1 h. (2) Primary antibody incubation: Dilute 1 mg / mL of 21-C1-E12 anti-SERPINA1 monoclonal antibody at a ratio of 1:5000 to 5 ml of antibody dilution buffer, place the blocked membrane into the corresponding diluted antibody, and incubate overnight at 4°C with shaking. (3) Take out the membrane and wash it in TBST solution 4 times (10min×4). (4) Secondary antibody incubation: Dilute HRP-anti-mouse IgG with antibody dilution buffer at a ratio of 1:5000, mix well, add to membrane strip, and shake at room temperature for 1 hour; (5) Remove the membrane strip and wash it in TBST solution 4 times (4×10min). (6) Development: Mix color developer A and B in a 1:1 ratio and develop using a developing system.

[0074] 3. Experimental Results The results are as follows Figure 2 As shown in the diagram, in the swimming lane, the 21-C1-E12 anti-SERPINA1 monoclonal antibody specifically recognized SERPINA1 protein in six cell lysates with a molecular weight of approximately 55 kDa, indicating that the 21-C1-E12 anti-SERPINA1 monoclonal antibody of this invention has good specificity and can accurately recognize SERPINA1 protein.

[0075] Example 3: Constructing a target knockout (KO) monoclonal cell line and performing specific Western blot screening. 1. The experimental materials are the same as in Example 2.

[0076] 2. Experimental methods: In this embodiment, the 21-C1-E12 anti-SERPINA1 monoclonal antibody of the present invention was used as the primary antibody. Western blotting was used to detect the expression level of SERPINA1 protein in wild-type / KO monoclonal cell lines, verifying its specificity against SERPINA1 protein. The method is as follows: (1) Prepare wild-type 293T cell line and SERPINA1 knockout cell lysate, prepare gel, spot, electrophoresis, transfer membrane. PVDF membrane needs to be activated. Activate with methanol for 1 min, wash the membrane twice with pure water and then wash it three times with TBST. Block: Place the membrane in blocking solution prepared with PBST containing 5% skim milk and shake at room temperature for 1 h. (2) Primary antibody incubation: Dilute 1 mg / mL of 21-C1-E12 antibody at a ratio of 1:5000 to 5 ml of antibody dilution solution, place the blocked membrane into the corresponding diluted antibody, and incubate overnight at 4°C with shaking. (3) Take out the membrane and wash it in TBST solution 4 times (10min×4). (4) Secondary antibody incubation: Dilute HRP-anti-mouse IgG with antibody dilution buffer at a ratio of 1:5000, mix well, add to membrane strip, and shake at room temperature for 1 hour; (5) Remove the membrane strip and wash it in TBST solution 4 times (4×10min). (6) Development: Mix color developer A and B in a 1:1 ratio and develop using a developing system.

[0077] 3. Experimental Results Experimental results are as follows Figure 3As shown in the diagram, the 21-C1-E12 anti-SERPINA1 mouse monoclonal antibody showed a signal in the WT lane (molecular weight 55 kDa) but no signal in the KO lane. This indicates that the expression level of SERPINA1 knockdown cell line protein was significantly reduced, and the signal recognized by the SERPINA1 mouse monoclonal antibody was also significantly reduced. This demonstrates that the 21-C1-E12 cloned SERPINA1 mouse monoclonal antibody of this invention can recognize SERPINA1 protein with high specificity.

[0078] Example 4 Flow Cytometry Detection 1. The experimental materials are shown in Table 7 below.

[0079] Table 7 Experimental Materials

[0080] 2. Experimental Methods (1) Cell collection Collect cells from culture dishes or flasks into centrifuge tubes (each tube should contain at least 5 × 10⁵ cells. For adherent cells, digest with trypsin first), centrifuge at 250g for 5 minutes, and remove the culture medium. Wash cells 1-2 times with 1×PBS, centrifuge at 250g for 5 minutes, and remove the PBS.

[0081] (2) Cell fixation Add 100 μL of freshly prepared fixative to a centrifuge tube and mix well to prepare a single-cell suspension. Fix at room temperature for 20 minutes. After fixation, add 1 mL of 1×PBS to wash the cells, centrifuge at 250g for 5 minutes, and remove the PBS.

[0082] (3) Cell permeability Add 100 μL of permeabilizer to a centrifuge tube, resuspend the cells, and permeabilize at room temperature for 10 minutes. After permeabilization, add 1 mL of 1×PBS to wash the cells, centrifuge at 250g for 5 minutes, and remove the PBS.

[0083] (4) Incubation of primary antibody Add 100 μL of diluted 0.5 μg / mL primary antibody to a centrifuge tube, resuspend the cells, and incubate at room temperature for 1 h or overnight at 4°C. After incubation, wash the cells with 1 mL of 1×PBS, centrifuge at 250g for 5 minutes, and remove the PBS.

[0084] (5) Incubation of secondary antibodies Add 100 μL of diluted fluorescein-conjugated secondary antibody to a centrifuge tube, resuspend the cells, and incubate at room temperature in the dark for 30 minutes. After incubation, wash the cells with 1 mL of 1×PBS, centrifuge at 250g for 5 minutes, and remove the PBS.

[0085] (6) On-machine testing Add 200 μL of 1×PBS to a centrifuge tube, resuspend the cells, and analyze them by flow cytometry.

[0086] (7) Data collection (7.1) Instrument Setup Turn on the flow cytometer, set the parameters, and select the corresponding laser and appropriate filter.

[0087] (7.2) Sample testing Load the prepared samples onto the flow chamber, ensuring they are evenly distributed within the flow chamber. Set an appropriate gating strategy, create scatter plots and histograms, load samples at a low speed, adjust the gain and threshold, and set the gate to select and analyze specific cell populations. Once the parameters are determined, run or record all samples.

[0088] (7.3) Data storage Save the original data file for later analysis.

[0089] 3. Experimental Results The expression of SERPINA1 protein in the human breast cancer cell line MCF7 was detected by flow cytometry. The specific experimental method is as follows: MCF7 cells were stained with SERPINA1 antibody (diluted at 1:2000) and the corresponding isotype control antibody, and fluorescently labeled with Alexa Fluor® 647, followed by flow cytometry analysis.

[0090] Experimental results are as follows Figure 4 As shown, the fluorescence signal of the isotype control (green dashed line in the figure) is distributed in the low fluorescence intensity region, representing the background signal of non-specific staining. However, the fluorescence intensity distribution curve of the cell population stained with SERPINA1 antibody (red solid line in the figure) shows a significant shift towards high fluorescence intensity. This result indicates that a significant SERPINA1 protein-specific fluorescence signal can be detected in MCF7 cells, thus confirming the expression of SERPINA1 protein in this cell line. The antibody used in this invention can effectively and specifically recognize endogenous SERPINA1 protein in MCF7 cells.

[0091] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An α1-antitrypsin antibody or its antigen-binding fragment, characterized in that, The α1-antitrypsin antibody or its antigen-binding fragment comprises: (1) Heavy chain variable region: The heavy chain variable region includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 as shown in SEQ ID NO.5; (2) Light chain variable region: The light chain variable region includes light chain CDR1, light chain CDR2 and light chain CDR3 in the light chain variable region as shown in SEQ ID NO.

9.

2. The α1-antitrypsin antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively, and the amino acid sequences of the light chain CDR1, light chain CDR2 and light chain CDR3 are shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

3. The α1-antitrypsin antibody or its antigen-binding fragment according to any one of claims 1-2, characterized in that, The heavy chain variable region of the α1-antitrypsin antibody or its antigen-binding fragment contains the amino acid sequence shown in SEQ ID NO. 5 or an amino acid sequence having at least 94% identity with SEQ ID NO. 5, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence having at least 94% identity with SEQ ID NO.

9.

4. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the α1-antitrypsin antibody or its antigen-binding fragment as described in any one of claims 1-3.

5. A carrier, characterized in that, The carrier comprises the isolated nucleic acid molecule as described in claim 4.

6. Hybridoma cells, characterized in that, The hybridoma cells produce the α1-antitrypsin antibody or its antigen-binding fragment as described in any one of claims 1-3.

7. A transformed cell or a cell population containing therein, characterized in that, The transformed cells are obtained by introducing the vector of claim 5 into host cells.

8. An α1-antitrypsin antibody or a derivative thereof containing an antigen-binding fragment, characterized in that, The derivatives include complexes formed by directly or indirectly conjugating the α1-antitrypsin antibody or its antigen-binding fragment as described in any one of claims 1-3 to a detectable marker.

9. A product for detecting α1-antitrypsin, characterized in that, The product comprises the α1-antitrypsin antibody or its antigen-binding fragment as described in any one of claims 1-3, the isolated nucleic acid molecule as described in claim 4, the vector as described in claim 5, the hybridoma cell as described in claim 6, the transformed cell or cell population containing the transformed cell as described in claim 7, and / or a derivative of the α1-antitrypsin antibody or its antigen-binding fragment as described in claim 8.

10. The product according to claim 9, characterized in that, The products include one or more of the following: reagent kits, test strips, reagents, and chips.

11. A method for preparing the α1-antitrypsin antibody or its antigen-binding fragment according to any one of claims 1-3, characterized in that, The method includes culturing the hybridoma cells of claim 6 or the transformed cells of claim 7 or a cell population containing the latter.

12. The use of the α1-antitrypsin antibody or its antigen-binding fragment as described in any one of claims 1-3, the isolated nucleic acid molecule as described in claim 4, the vector as described in claim 5, the hybridoma cell as described in claim 6, the transformed cell or cell population containing the thereof as described in claim 7, and / or a derivative of the α1-antitrypsin antibody or its antigen-binding fragment as described in claim 8 in the preparation of a product for detecting α1-antitrypsin.

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