Preparation and application of anti-human TNF alpha protein monoclonal antibody

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to preparation and application of an anti-human TNF alpha protein monoclonal antibody. The 1F1 monoclonal antibody and the 1H2 monoclonal antibody are successfully prepared and screened through a series of steps of mouse immunization, plasma B cell enrichment, droplet generation, high-throughput detection and sorting, single cell BCR sequencing and the like. Wherein the amino acid sequence of the 1F1 heavy chain variable region is SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is SEQ ID NO: 3; the 1H2 heavy chain variable region is SEQ ID NO: 5, and the light chain variable region is SEQ ID NO: 7. ELISA verifies that the antibody pair is high in specificity, and compared with a commercially available kit, the antibody pair is better in detection sensitivity and free of cross reaction. The antibody can be used for preparing products for detecting TNF alpha and medicines for treating related diseases, and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the preparation and application of anti-human TNFα protein monoclonal antibodies. Background Technology

[0002] Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine that plays crucial roles in cell metabolism, antiviral responses, blood coagulation, cell growth regulation, insulin response, immune cell regulation, and inflammation. TNF was discovered in 1975 as an endotoxin found in serum that promotes hemorrhagic necrosis of tumors. In 1985, TNFα was sequenced to be 157 amino acids with a molecular weight of 17 kDa and an isoelectric point of 5.3. In addition to TNFα, another protein with a molecular weight of 25 kDa was isolated and sequenced, later showing 50% sequence homology, and was named TNFβ. The short arm of human chromosome 6 contains the TNFα-generating gene within the major histocompatibility complex (MHC) genome. This gene is 3 kb in length and contains four exons and three introns. TNF-α exists in two forms: transmembrane 17 kDa soluble TNFα (sTNFα) and 26 kDa membrane-bound TNFα (tmTNFα). TNFα is synthesized in cells as the precursor TNF, which is expressed on the cell membrane as a type II polypeptide containing 233 amino acid residues and a molar mass of 26 kDa. Once it reaches the cell surface, it trimers with two other precursor TNF proteins to form a transmembrane TNFα molecule. This transmembrane TNFα molecule participates in cell-cell contact-dependent signaling, also known as drugamine signaling, and its effects vary depending on the cell type. For example, it produces cytotoxicity in various tumor cells; it causes cell death in HIV-infected lymphocytes; and it induces growth inhibition in macrophages infected with intracellular parasites.

[0003] TNFα plays a role in the pathogenesis of various chronic autoimmune and inflammatory diseases and bears a significant burden on global health and the economy. Under normal physiological conditions, TNFα participates in fighting infection and tumor surveillance; however, its overexpression can lead to a variety of pathological states, including: autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis; chronic inflammatory diseases such as Crohn's disease and ulcerative colitis; septic shock, a systemic inflammatory response syndrome caused by infection; and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Accurate detection of TNFα concentration is of significant clinical value for early disease diagnosis, monitoring of treatment efficacy, and prognostic assessment.

[0004] The antibody pairs used in existing ELISA diagnostic kits suffer from problems such as insufficient affinity, poor specificity, or cross-reactivity, resulting in low detection sensitivity and failing to meet the needs of accurate clinical diagnosis. Therefore, the development of high-performance TNFα antibodies is of great significance for disease diagnosis. Summary of the Invention

[0005] Existing ELISA diagnostic kits use antibody pairs that suffer from insufficient affinity, poor specificity, or cross-reactivity, resulting in low detection sensitivity and failing to meet the needs of precise clinical diagnosis. This invention aims to develop highly sensitive and specific TNFα antibody pairs for use in diagnostic reagent development.

[0006] The objectives of this invention include: The first aspect of the present invention is to provide a monoclonal antibody or an antigen-binding fragment thereof.

[0007] A second aspect of the present invention is to provide a recombinant protein.

[0008] A third aspect of the present invention aims to provide biological materials related to the monoclonal antibody or its antigen-binding fragment of the first aspect of the present invention or the recombinant protein of the second aspect of the present invention.

[0009] A fourth aspect of the present invention is to provide a coupling agent.

[0010] The fifth aspect of this invention aims to provide the use of the monoclonal antibody or antigen-binding fragment thereof of the first aspect, the recombinant protein of the second aspect, the biomaterial of the third aspect, and / or the conjugate of the fourth aspect in the preparation of products.

[0011] The sixth aspect of this invention aims to provide a product.

[0012] The seventh aspect of this invention is to provide a medicine.

[0013] The object of the eighth aspect of the present invention is to provide a method for preparing the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present invention.

[0014] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof targeting TNFα protein, said monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain.

[0015] In some embodiments of the present invention, the monoclonal antibody includes one or both of A) or B); A) 1F1 The heavy chain includes: Heavy chain variable region, which includes CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1; The light chain comprises: The light chain variable region, which includes CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region, The light chain variable region has the amino acid sequence shown in SEQ ID NO: 3; B) 1H2 The heavy chain includes: Heavy chain variable region, which includes CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 5; The light chain comprises: Light chain variable region, which includes CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region; The light chain variable region has the amino acid sequence shown in SEQ ID NO: 7.

[0016] In some embodiments of the present invention, when IMGT is used as the definition scheme: A) 1F1 The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region are shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively. The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region are shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively. B) 1H2 The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region are shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively. The amino acid sequences of the CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region are shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

[0017] In some embodiments of the present invention, those skilled in the art may adopt other definition schemes recognized in the art, such as Kabat, Chothia, and Contact. Other CDR sequences obtained based on the heavy chain and light chain variable regions of the present invention are still within the protection scope of the present invention.

[0018] In some embodiments of the present invention, specifically, A) 1F1 The amino acid sequence of the heavy chain variable region is SEQ ID NO: 1; The amino acid sequence of the light chain variable region is SEQ ID NO: 3; B) 1H2 The amino acid sequence of the heavy chain variable region is SEQ ID NO: 5; The amino acid sequence of the variable region of the light chain is SEQ ID NO: 7.

[0019] In some embodiments of the present invention, based on the amino acid sequence of the constant region of the antibody heavy chain, immunoglobulin molecules can be classified into five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. Based on the amino acid sequence of the light chain, the antibody light chain can be classified as a lambda (λ) chain or a kappa (κ) chain. The antibodies disclosed in this invention can be any of the above types or subtypes.

[0020] In some embodiments of the present invention, the monoclonal antibody or its antigen-binding fragment may be an isotype selected from IgG, IgA, IgM, IgE and IgD.

[0021] In some embodiments of the invention, the antigen-binding fragment comprises (i) a Fab fragment, i.e., a monovalent fragment consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain; (ii) an F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments connected by disulfide bonds at the hinge region; (iii) a Fab' fragment, which is essentially a Fab fragment having a portion of the hinge region (see Fundamental Immunology (edited by Paul, Supplement 3, 1993); (iv) an Fd fragment consisting of a VH domain and a CH1 domain; (v) an Fd' fragment having a VH domain and a CH1 domain and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (vi) an Fv fragment consisting of the VL domain and the VH domain of an antibody arm; (vii) a dAb fragment (Ward et al. (1989), Nature, Vol. 341: 544). (546 pages) It consists of (viii) a separate complementarity-determining region (CDR); and (ix) a nanobody, i.e., a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains (VL and VH) of the Fv fragment are encoded by separate genes, these domains can still be linked via synthetic linkers using recombination methods, allowing these domains to be fabricated into a single protein chain where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al. (1988), Science, Vol. 242: p. 423). 426 pages; and Huston et al. (1988), Proc. Natl. Acad. Sci. USA, Vol. 85: 5879 (Page 5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of antibodies. Furthermore, antigen-binding fragments also include "linear antibodies," which contain a pair of tandem Fd segments (VH...). CH1 VH CH1) and any of the aforementioned segments in modified form, this pair of tandem Fd segments together with the complementary light chain polypeptide form an antigen-binding region, and the modified forms of these segments retain antigen-binding activity.

[0022] These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and the use of the fragments can be screened in the same manner as for intact antibodies.

[0023] In some embodiments of the present invention, the monoclonal antibody or its antigen-binding fragment comprises at least one of a full-length antibody, Fab, Fab', F(ab')2, Fv or scFv.

[0024] A second aspect of the invention provides a recombinant protein comprising: a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect of the invention; and optionally a tag sequence for assisting expression and / or purification.

[0025] In some embodiments of the present invention, the tag sequence is selected from at least one of the following groups: His tag, GGGS sequence, FLAG tag, HA tag, GST tag, and SumO tag.

[0026] A third aspect of the invention provides biological materials relating to the monoclonal antibody or its antigen-binding fragment of the first aspect of the invention or the recombinant protein of the second aspect; said biological material comprising at least one of 1) to 12): 1) A nucleic acid molecule encoding a monoclonal antibody or its antigen-binding fragment of the first aspect of the present invention, or a recombinant protein of the second aspect of the present invention; 2) An expression cassette containing the nucleic acid molecule described in 1); 3) A carrier containing the nucleic acid molecule described in 1); 4) A carrier containing the expression box described in 2); 5) Transgenic cell lines containing the nucleic acid molecules described in 1); 6) A transgenic cell line containing the expression cassette described in 2); 7) Transgenic cell lines containing the vector described in 3); 8) A transgenic cell line containing the vector described in 4); 9) Microorganisms containing the nucleic acid molecules described in 1); 10) Microorganisms containing the expression cassette described in 2); 11) Microorganisms containing the carrier described in 3); 12) Microorganisms containing the carrier described in 4).

[0027] In some embodiments of the present invention, the transgenic cell line does not contain propagation material.

[0028] In some embodiments of the present invention, the nucleic acid molecule (bases) includes: A) 1F1 The nucleic acid sequence of the heavy chain variable region is SEQ ID NO: 2; The nucleic acid sequence of the light chain variable region is SEQ ID NO: 4; B) 1H2 The nucleic acid sequence of the heavy chain variable region is SEQ ID NO: 6; The nucleic acid sequence of the light chain variable region is SEQ ID NO: 8.

[0029] In some embodiments of the present invention, the vector may be any suitable recombinant expression vector, including plasmids for amplifying nucleic acid molecules, or viral vectors for transfecting cells, including but not limited to retroviral vectors, DNA vectors, murine leukemia virus vectors, SFG vectors, plasmids, RNA vectors, adenovirus vectors, baculovirus vectors, Epstein-Barr virus vectors, papillomavirus vectors, vaccinia virus vectors, herpes simplex virus vectors, adenovirus-associated vectors, lentiviral vectors, or any combination thereof. In some embodiments of the present invention, the transgenic cell line is a host cell that produces the monoclonal antibody or antigen-binding fragment of the present invention, including but not limited to prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells.

[0030] In some embodiments, the transgenic cell line includes human cells, such as CHO cells (including but not limited to CHOS cells, CHO-K1 cells) and HEK293 cells (including but not limited to HEK293A, HEK293T and HEK293F).

[0031] A fourth aspect of the present invention provides a conjugate comprising at least one of a monoclonal antibody or an antigen-binding fragment thereof from the first aspect of the present invention or a recombinant protein from the second aspect; and a conjugation portion comprising a detectable marker.

[0032] In some embodiments of the present invention, the detectable marker is selected from radioactive isotopes, enzymes, fluorescent compounds, or any combination thereof; In some embodiments of the present invention, the fluorescent compounds include fluorescein, fluorescein isothiocyanate, rhodamine, and 5... dimethylamine l Naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), etc.

[0033] In some embodiments of the present invention, the enzymes include horseradish peroxidase, β-carotene... Galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When an antibody or antigen-binding fragment conjugates with a detectable enzyme, detection can be achieved by adding additional reagents. The enzyme uses these additional reagents to produce a distinguishable reaction product. For example, when horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine produces a visually detectable colored reaction product.

[0034] In some embodiments of the present invention, the radioactive labeling includes, but is not limited to, the following radioactive isotopes or radioactive nucleotides: 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 111 In、 125 I, 131 I.

[0035] In some embodiments of the present invention, monoclonal antibodies or antigen-binding fragments may also be conjugated with biotin and detected by indirect measurement of binding to avidin or streptavidin.

[0036] A fifth aspect of the invention provides the use of the monoclonal antibody or antigen-binding fragment thereof of the first aspect, the recombinant protein of the second aspect, the biomaterial of the third aspect, and / or the conjugate of the fourth aspect in the preparation of a product.

[0037] In some embodiments of the present invention, the product includes one of 1) to 2): The product includes one of 1) to 2): 1) Products for detecting TNFα; 2) Medications for treating diseases related to TNFα; In some embodiments of the present invention, the TNFα-related diseases include, but are not limited to, autoimmune diseases, chronic inflammatory diseases, septic shock, and neurodegenerative diseases.

[0038] In some embodiments of the present invention, the autoimmune diseases include, but are not limited to, rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis.

[0039] In some embodiments of the present invention, the chronic inflammatory disease includes inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.

[0040] In some embodiments of the present invention, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, etc.

[0041] A sixth aspect of the present invention provides a product comprising a monoclonal antibody or antigen-binding fragment thereof of the first aspect of the invention, a recombinant protein of the second aspect, and / or a conjugate of the fourth aspect.

[0042] In some embodiments of the present invention, the product includes at least one of reagents, detection plates, reagent kits, and detection chips.

[0043] In some embodiments of the present invention, the product includes supporting auxiliary reagents and consumables for testing.

[0044] A seventh aspect of the present invention provides a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof of the first aspect of the invention, a recombinant protein of the second aspect and / or a conjugate of the fourth aspect, and pharmaceutically acceptable excipients.

[0045] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: propellants, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and release inhibitors.

[0046] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the pharmaceutical preparation. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994) and the *Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients*.

[0047] In some embodiments of the present invention, the dosage form of the product includes one of the following: powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0048] In some embodiments of the present invention, the product is administered via the gastrointestinal tract or non-gastrointestinal route.

[0049] The eighth aspect of the present invention provides a method for preparing a monoclonal antibody or antigen-binding fragment thereof according to the first aspect of the present invention, obtained by culturing the transgenic cell line or microorganism of the third aspect of the present invention.

[0050] The beneficial effects of this invention are: This invention successfully prepared and screened two monoclonal antibodies, 1F1 and 1H2, through a series of steps including mouse immunization, plasma B cell enrichment, droplet generation, high-throughput detection and sorting, and single-cell BCR sequencing. The amino acid sequence of the heavy chain variable region of 1F1 is SEQ ID NO: 1, and the light chain variable region is SEQ ID NO: 3; the heavy chain variable region of 1H2 is SEQ ID NO: 5, and the light chain variable region is SEQ ID NO: 7. ELISA validation showed that these antibodies have high specificity, superior detection sensitivity compared to commercially available kits, and no cross-reactivity. These antibodies can be used to prepare products for detecting TNF-α and drugs for treating related diseases, possessing significant clinical application value. Attached Figure Description

[0051] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is the logic diagram for the droplet sorting gate.

[0052] Figure 2 The results of the phylogenetic analysis of the heavy and light chains of the candidate antibodies are shown. Detailed Implementation

[0053] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0054] The conceptual process of this invention is as follows: S1: Mouse immunization Mice were immunized by multiple injections of Freund's adjuvant, and after 3-4 immunizations, the serum titers of the mice were measured to evaluate the immunization effect.

[0055] S2: Enrichment of plasma B cells in immunized mice PBMCs were isolated from the spleens of immunized mice. The mice were then positively screened using a mouse B cell isolation kit to initially identify B cells. Further negative screening was performed using a CD138 kit to obtain mouse plasma B cells.

[0056] S3: Droplet formation The plasma B cell suspension enriched in step S2 was adjusted to a suitable cell density and then blocked with mouse Fc inhibitor. After washing, the cell density was adjusted to 4 × 10⁶ cells / mL using droplet generation buffer. 6 The first aqueous phase consisted of cells / mL. Biotinylated antigen was coated onto streptavidin magnetic beads. Live and dead cell labeling dyes and mouse secondary antibody fluorescent antibodies were added to the magnetic bead phase, forming the second aqueous phase. This aqueous and oil phases were simultaneously pumped into a microfluidic droplet generation chip. The pumping pressures of the aqueous and oil phases were adjusted to generate droplets with a diameter of 50 ± 5 μm. The collected droplets were incubated at 37°C to induce antibody secretion by individual B cells within the droplets. The secreted antibodies formed complexes with the antigen-coated magnetic beads and fluorescent secondary antibody, causing the magnetic beads to carry fluorescence. Further, through a toroidal magnetic field, the magnetic beads aggregated into a line within the droplets, facilitating fluorescence detection.

[0057] S4: High-throughput droplet detection and sorting A microfluidic droplet sorting system was used to detect the incubated droplet clusters. Fluorescent dyes in the droplets were excited by 488nm and 647nm excitation light, and the droplets were clustered using a scatter plot based on the collected fluorescence values. A positive gate was established based on the clustering data, triggering a signal only for droplets with strong fluorescence signals from magnetic beads (indicating that B cells secreted antibodies with high affinity for the target antigen). Based on the trigger signals, the target droplets were precisely sorted into collection tubes using dielectrophoresis.

[0058] S5: Droplet demulsification and single-cell recovery Add a demulsifier (such as perfluorooctanoic acid) and cell buffer reagent to the collected target droplets to disrupt the oil-water interface of the droplets and release individual B cells from the droplets.

[0059] S6: Single-cell BCR library preparation and sequencing The recovered cells were labeled using the mouse high-throughput single-cell VDJ kit, and BCR libraries were constructed. Sequencing yielded 20G of data. The BCR sequencing data were assembled and compared with the IMGT reference database to identify the FR and CDR regions in the BCR sequences. Cell typing was performed based on the CDR3 region sequence, and sequence analysis and screening were conducted.

[0060] S7: Antibody Expression The antibody sequences obtained in step S5 were analyzed and screened, and gene synthesis was performed. An expression vector was constructed and transfected into mammalian 293F cells for transient expression of recombinant antibodies. The antibodies were then purified by affinity chromatography using protein A.

[0061] S8: Antibody Screening The antibodies obtained in step S5 are screened for specific antibodies using an indirect ELISA method. The selected antibodies are then further screened for antibody pairing using a sandwich ELISA method.

[0062] S9: Antibody Performance Verification The antibody pairs obtained in step S8 were compared with commercially available products using ELISA to evaluate the performance of the screened antibody pairs.

[0063] S10: Cross-reactivity test The antibody pairs obtained in step S8 were subjected to cross-reactivity tests with homologous cytokine proteins to evaluate the specificity of the antibody pairs.

[0064] The experimental results of this invention are as follows.

[0065] Example 1: Preparation and Screening of Antibodies 1. Animal immunization Transmembrane-soluble recombinant TNFα protein was used as the immunogen. Five-week-old female Balb / c mice were purchased from the Guangdong Provincial Laboratory Animal Center. Immunization strategy: The initial immunization dose was 100 μg / mouse, using Freund's complete adjuvant, administered via multiple injection sites. The second booster immunization was administered 14 days later, with the antigen dose adjusted to 50 μg / mL, using Freund's incomplete adjuvant, administered via multiple injection sites. Subsequent booster immunizations were given 14 days apart, for a total of four immunizations.

[0066] One week after 4-week immunization, blood was collected from the mandible of mice, and serum was separated. Serum titers were determined using an indirect ELISA method (serum dilution gradient: 10). 4 10 5 10 6 The valence reaches 10. 6 This satisfies the needs of downstream research and development. After the required immunogenicity is achieved, the mice are given a booster immunization 3 days before sacrifice (French incomplete adjuvant + 50 μg antigen / mouse).

[0067] 2. Sorting of mouse plasma cells Three days after the initial immunization, the spleens of mice were harvested, minced, and ground. PBMCs were separated using a 40μm filter, centrifuged at 300g for 10 min, and the supernatant was removed. Cells were resuspended and counted. B cells were sorted using a mouse B cell isolation kit, and plasma cells were then obtained from the isolated B cells using a CD138 kit and counted.

[0068] 3. Droplet formation Droplet generation was performed using a two-phase droplet generation chip. Before droplet generation, mouse Fc inhibitor was added to the plasma cells obtained from the previous sorting step and the cells were blocked at room temperature for 30 min. The cells were washed twice with 5 mL of DPBS, and then resuspended in DMEM / F12 medium containing 16 vol% OptiPrep, 0.1% Pluronic F68, 25 mM HEPES (pH 7.4), 5% HyClone (ultra-low IgG fetal bovine serum), and 1% Pen / Strep to obtain a cell concentration of 4 × 10⁶ cells / year. 6 The cells / mL ratio, based on Poisson distribution, resulted in a final average droplet occupancy of approximately 0.3 cells per 40 μL droplet. Paramagnetic nanoparticles were coated with biotinylated TNFα recombinant antigen, resuspended in working buffer, and then 75 nM Alexa Fluor® 647 Rabbit Anti-Mouse IgG was added. The dead cell labeling dye SYTOX was added at a 1:100 ratio. TMGreen. Droplet generation and sorting were performed using microfluidic equipment. A 50 μm two-phase aqueous separation chip was used, and 5% Drop-surf droplets were used to generate oil in the oil phase, preparing droplets with a diameter of 50±5 μm.

[0069] 4. Droplet sorting The droplets generated in step 3 were placed in a 37°C incubator for 2 hours of static incubation, then magnetically concentrated in a toroidal magnetic field for 5 minutes. The temperature of the sample loading tank was adjusted to 4°C, and the sample was then assembled onto the plunger pump loading valve. A 50 μm sorting chip was used to connect the sheath fluid flow, the interstitial flow, the sample, and the collection tubing. Both the sheath fluid flow and the interstitial flow used 2% Drop-surf as the fluid source. The pressures for the sheath fluid flow and the interstitial flow were set to 100 MPa and 150 MPa, respectively. The sample loading rate was 2.5 μL / min. The 488nm laser power was set to 2500 ohms, and the Alexa Fluor 488 channel gain and Alexa Fluor 647 channel gain were both set to 1700 ohms. A scatter plot of the droplet signals was generated and displayed, as shown below. Figure 1 As shown, gate P1 encloses droplets based on their width, gate P2 distinguishes between live and dead cells, enclosing live cell populations, and gate P3 encloses droplets secreting specific antibodies based on the fluorescence signal from the magnetic bead pull wire. A sorting voltage of 500V and an application time of 1500ms were set for continuous sorting and collection, resulting in the collection of 4177 droplets.

[0070] 5. Single-cell BCR sequencing Add 100 μL of 5% serum-containing DMEM / F12 medium to the continuously collected droplets, then add 100 μL of 1H,1H,2H,2H-perfluorooctanol, mix gently, and centrifuge at 700g for 10 minutes at 4°C to promote complete phase separation. After centrifugation, slowly remove the bottom oil phase with a syringe for cell counting, obtaining 3876 cells with 85% viability.

[0071] The recovered cells were labeled using the mouse high-throughput single-cell VDJ kit, and a BCR library was constructed. Sequencing yielded 20G of data. The BCR sequencing data were assembled and compared with the IMGT reference database to identify the FR and CDR regions in the BCR sequence. The data analysis results are shown in Table 1. Cell typing was performed based on the CDR3 region sequence, and phylogenetic analysis was conducted. The results are shown below. Figure 2 As shown.

[0072] Table 1. BCR sequencing analysis results

[0073] 6. Gene synthesis and construction Based on phylogenetic analysis, 23 pairs of antibody genes were selected from different branches for gene synthesis. Previously, the mouse heavy chain (IgG1) gene was cloned into the pcDNA3.1 vector using SalI and HindIII, and the light chain (kappa) constant region was cloned into the pcDNA3.1 vector using BsiwI and HindIII. The synthesized heavy chain variable region gene was then cloned into the expression vector using AgeI and SalI, and the light chain variable region gene was cloned into the expression vector using AgeI and BsiwI.

[0074] 7. Antibody expression The day before transfection, 293F cells were passaged. On the day of transfection, cell viability was required to be >90% and cell density to be 3-4 x 10⁻⁴. 6 cells / mL. Adjust the cell density to 1.0*10⁻⁶ cells / mL during transfection. 6 Cells / mL were transfected with PEI (1 mg / mL) and 80 μg plasmid DNA (H:L = 1:1) and incubated at room temperature for 10 min to form a cationic polymer (PEI:DNA = 3:1, mass ratio). This polymer was then slowly added to 293F cells, with each antibody transfecting 80 mL of cells. After 6 days, the cell supernatant was collected for purification. The supernatant from cells after expression was purified using Protein A packing material. The cells were washed with 10 mL of equilibration buffer, followed by antibody elution with 5 mL of 0.1 M glycine. The eluted antibody solution was immediately neutralized with 500 μL of 1 M Tris-HCl and dialyzed against PBS at 4 °C. The dialysate was changed every 2 h, and the dialysate was repeated 3 times.

[0075] 8. ELISA verification of antibody expression supernatant Dilute transmembrane-soluble recombinant TNFα protein to a final concentration of 1 μg / mL with carbonate coating buffer. Add 100 μL to each well of a 96-well microplate and incubate overnight at 4°C. After incubation, remove the supernatant, add 100 μL of PBST and incubate for 5 min. Wash the plate three times. Then add 150 μL of 5% skim milk to each well and block at 37°C for 1 h. After blocking, remove the supernatant, add 150 μL of PBST and incubate for 5 min. Wash the plate three times. Then add 100 μL of purified antibody (1 μg / mL) to each well and incubate at 37°C for 1 h. After incubation, remove the supernatant, add 100 μL of PBST and incubate for 5 min. Wash the plate three times. Add 100 μL of goat anti-mouse secondary antibody (diluted 1:10000 with 5% skim milk) to each well and incubate at 37°C for 1 h. After incubation, the supernatant was removed, and 100 μL of PBST was added and allowed to stand for 5 min. The plate was washed three times, and then 50 μL of TMB substrate was added to each well in the dark and incubated at room temperature for 10 min. 25 μL of 2M sulfuric acid was added to each well to stop the colorimetric reaction. The plate was then placed on a microplate reader to measure the OD value at 450 nm. A total of 15 specific antibodies were ultimately screened from the 23 purified antibodies.

[0076] Example 2: Validation of the antibody 1. ELISA sandwich assay to verify paired antibodies Take 50 μg of each of the 15 specific antibodies obtained from the above screening, add 10 times the volume of starter solution and 50 μg of HRP sequentially, and react at 37℃ for 2 h. Add 1 / 10 volume of stop solution and react at room temperature for 1 h. Dilute the 15 purified antibodies to a final concentration of 1 μg / mL, and add 100 μL to each well for coating. Incubate overnight at 4℃ or 2 h at 37℃. Wash the plate three times with 300 μL PBST for 3 min each time, add 280 μL of 5% skim milk to each well, and block at 37℃ for 1 h. Wash the plate three times with 300 μL PBST for 3 min each time, then dilute the recombinant TNFα protein to a final concentration of 1 μg / mL, add 100 μL of the diluted recombinant TNFα protein to each well, and incubate at 37℃ for 40 min. Wash the plate three times with 300 μL PBST for 3 min each time. Add 100 μL of HRP-labeled antibody (diluted 1:10000 with 5% skim milk) to each well and block at 37°C for 40 min. Wash the plate three times with 300 μL PBST for 3 min each time, then add 100 μL of TMB substrate to each well in the dark and incubate at room temperature for 10 min. Add 50 μL of 2M sulfuric acid to each well and then place the plate on a microplate reader to detect the OD value at 450 nm. The 1F1 and 1H2 antibodies showed good pairing performance.

[0077] The amino acid sequence of the heavy chain variable region of antibody 1F1 is as follows: QVQLQQSGPELVKPGASVKISCKASGYAFSRSWMNWVKQRPGQGLEWIGRIYPGDGDDKYNGKFKDKASLTADRSSSTAYMQLSSLTSVDSAVYFCAIREFTTVVGGFAYWGQGTTLTVSS (SEQ ID NO: 1); The corresponding base sequence is: CAGGTCCAACTGCAGCAGTCTGGACCTGAGTTGGTGAAGCCTGGGGCCTCAGTGAAGATTTCCTGCAAAGCTTCTGGCTACGCATTCAGTAGGTCTTGGATGAACTGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGACGGATTTATCCTGGAGATGGAGATGATAAGTACAAT GGGAAATTCAAGGACAAGGCCTCACTGACTGCAGACAGATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGTGGACTCTGCGGTCTATTTCTGTGCAATAAGGGAGTTTACTACGGTAGTGGGGGGCTTTGCCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ IDNO: 2).

[0078] The amino acid sequence of the light chain variable region of antibody 1F1 is as follows: DIVMTQSPSSLTVTAGEKVTMTCKSSQSLLNDGVQKNYLTWYQQKPEQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSFPPTFGAGTKLELK (SEQ ID NO: 3); The corresponding base sequence is: GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGACCTGCAAGTCCAGTCAGAGTCTGTTAAACGATGGAGTTCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCGGAACAGCCTCCTAAACTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGCGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAAAATGATTATAGTTTTCCTCCTACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 4).

[0079] The amino acid sequence of the heavy chain variable region of antibody 1H2 is as follows: EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQSNGKSLEWIGNIAPYYGGTSYNQKFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARGGYGNPYFDYWGQGTTLTVSS (SEQ ID NO: 5); The corresponding base sequence is: GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGAGAAGCCTGGCGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACAACATGAACTGGGTGAAGCAGAGCAATGGAAAGAGCCTTGAGTGGATTGGAAATATTGCTCCTTACTATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAAGAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGGGGGTATGGTAACCCGTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 6).

[0080] The amino acid sequence of the light chain variable region of antibody 1H2 is as follows: DVVMTQTPLSLPVSLGDQASISCRSSQSLLHSNGDTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPRTFGGGTKLEIK (SEQ ID NO: 7); The corresponding base sequence is: GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGGGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTCTACACAGTAATGGAGACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTC CAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 8).

[0081] The variable region sequence of the above antibody was analyzed using the IMGT definition scheme, and the results are shown in Table 2.

[0082] Table 2

[0083] 2. Comparison with existing test kits Antibodies 1F1 and 1H2 were paired and detected using a sandwich ELISA kit, and the results were compared with those of a commercially available human TNFα ELISA kit. The results are shown in Table 3.

[0084] Table 3

[0085] 3. Cross-reactivity test The paired antibodies screened in this invention can recognize both natural and recombinant human TNFα. To evaluate the specificity of the 1F1 and 1H2 antibodies for detecting TNFα, antibodies against various cytokine proteins (IL-1, ... and IL-1 were also tested.) 1α, IL 1β, IL 2R, IL 3. IL 4. IL 5. IL 6. IL 8. IL 10. IL Cross-reactivity tests were performed on 12p70, IL17, IFNα, and IFNγ. No cross-reactivity was observed in any of the tested cytokine proteins.

[0086] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A monoclonal antibody targeting human TNFα or its antigen-binding fragment, characterized in that: The monoclonal antibody or its antigen-binding fragment comprises a heavy chain and a light chain; The monoclonal antibody includes one or both of A) or B); A) 1F1 The heavy chain includes: Heavy chain variable region, which includes CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1; The light chain comprises: The light chain variable region, which includes CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region, The light chain variable region has the amino acid sequence shown in SEQ ID NO: 3; B) 1H2 The heavy chain includes: Heavy chain variable region, which includes CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region; The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 5; The light chain comprises: Light chain variable region, which includes CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region; The light chain variable region has the amino acid sequence shown in SEQ ID NO:

7.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that: When the CDR is a scheme defined by IMGT: A) 1F1 The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region are shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively. The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region are shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively. B) 1H2 The amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable region are shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively. The amino acid sequences of the CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region are shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

3. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that: A) 1F1 The amino acid sequence of the heavy chain variable region is SEQ ID NO: 1; The amino acid sequence of the light chain variable region is SEQ ID NO: 3; B) 1H2 The amino acid sequence of the heavy chain variable region is SEQ ID NO: 5; The amino acid sequence of the variable region of the light chain is SEQ ID NO:

7.

4. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that: The monoclonal antibody or its antigen-binding fragment comprises at least one of full-length antibody, Fab, Fab', F(ab')2, Fv or scFv.

5. A recombinant protein comprising: a monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4, and a tag sequence.

6. A biological material relating to the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4, or the recombinant protein as described in claim 5, wherein the biological material comprises at least one of 1) to 12): 1) A nucleic acid molecule encoding a monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4, or the recombinant protein as described in claim 5; 2) An expression cassette containing the nucleic acid molecule described in 1); 3) A carrier containing the nucleic acid molecule described in 1); 4) A carrier containing the expression box described in 2); 5) Transgenic cell lines containing the nucleic acid molecules described in 1); 6) Transgenic cell lines containing the expression cassette described in 2); 7) Transgenic cell lines containing the vector described in 3); 8) A transgenic cell line containing the vector described in 4); 9) Microorganisms containing the nucleic acid molecules described in 1); 10) Microorganisms containing the expression cassette described in 2); 11) Microorganisms containing the carrier described in 3); 12) Microorganisms containing the carrier described in 4).

7. A conjugate comprising: at least one of the monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 4, or the recombinant protein according to claim 5; And a coupling portion, which includes a detectable marker.

8. The use of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, the recombinant protein as described in claim 5, the biomaterial as described in claim 6, and the conjugate as described in claim 7 in the preparation of the product; The product includes one of 1) to 2): 1) Products for detecting TNFα; 2) Medications for treating diseases related to TNFα; Preferably, the TNFα-related diseases include, but are not limited to, autoimmune diseases, chronic inflammatory diseases, septic shock, and neurodegenerative diseases.

9. A product for detecting TNFα, comprising at least one of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, the recombinant protein as described in claim 5, or the conjugate as described in claim 7; The product includes at least one of reagents, test plates, reagent kits, and test chips.

10. A pharmaceutical composition comprising at least one of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, the recombinant protein as described in claim 5, or the conjugate as described in claim 7, and a pharmaceutically acceptable excipient.

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