An all-human antagonistic antibody taking kiaa1199 protein as a target and application thereof

Abstract

The present application belongs to the technical field of genetic engineering and protein engineering, and particularly relates to a full human source antagonistic antibody with KIAA1199 protein as a target and application thereof; the present application screens a full human source antagonistic antibody (SEQ ID NO:1 or SEQ ID NO:2) with KIAA1199 protein as a target from a human scFv phage antibody library, and respectively determines nucleotide sequences thereof to obtain coding genes thereof; the full human source antagonistic antibody obtained through the method has specificity of targeting KIAA1199 protein, and has application prospects of an inhibitor of targeting KIAA1199 protein or preparation of a drug for treating or relieving inflammatory diseases and / or autoimmune diseases.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and protein engineering technology, specifically relating to a fully human antagonistic antibody targeting KIAA1199 protein and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic, progressive autoimmune disease with extremely complex etiologies. Early symptoms include migratory joint pain and dysfunction, while later stages often present with joint stiffness and severe deformities. Its pathogenesis is not yet fully understood, but it has been found to primarily involve the excessive proliferation and activation of synovial fibroblasts (FLS), forming erosive pannus. This pannus further promotes chronic inflammation and abnormal bone metabolism, leading to irreversible damage to joint tissues and joint dysfunction. Pannus is a granulation tissue composed of proliferating fibroblasts, numerous small blood vessels, and various inflammatory cells. On the one hand, pannus prevents bone from receiving nutrients through the synovium; on the other hand, it releases various inflammatory mediators and proteolytic enzymes, eroding articular cartilage, ligaments, tendons, and other tissues, resulting in articular cartilage destruction. On the other hand, the formation of pannus leads to the infiltration of leukocytes, lymphocytes, macrophages, etc., and enhances the expression of chemokines and adhesion molecules, including integrins, selectins, and members of the immunoglobulin superfamily. This causes changes in the internal microenvironment of the synovium, and together with the activation of synovial fibroblasts, leads to the occurrence of synovial inflammation.

[0003] KIAA1199 protein is a biomarker recently discovered to be highly expressed in overproliferating tissues. KIAA1199 protein has four domains and an N-terminal secretory signaling peptide: the G8 domain contains eight conserved glycine residues and five repeating β-sheet pairs, participating in extracellular ligand binding and catalytic processes; the GG domain consists of seven β-sheets and two α-helices, containing approximately 100 amino acids, and is associated with disease development due to gene mutations; the PbH1 domain consists of parallel β-helical repeats and participates in the hydrolysis of polysaccharides. KIAA1199 protein can participate in the degradation of hyaluronic acid (HA), an important polysaccharide constituting the articular cartilage matrix and synovial fluid, crucial for maintaining normal joint physiological function. In rheumatoid arthritis patients, the concentration and molecular weight of hyaluronic acid in the synovial fluid are reduced, thus losing its lubricating and protective effects on the joints and exacerbating the progression of RA. On the one hand, KIAA1199 protein is involved in the degradation of HA by excessively proliferating FLS. Enhanced HA degradation is positively correlated with the upregulation of KIAA1199 expression in FLS of RA patients, and the N-terminal sequence of KIAA1199 protein is essential for its intracellular transport and HA depolymerization. On the other hand, the inventors found that KIAA1199 protein is highly expressed in synovium and is also involved in the formation of synovial pannus. Studies have shown that KIAA1199 protein can promote the proliferation and migration of FLS cells. In addition, three-dimensional angiogenesis, chicken embryo allantoic membrane experiments, and CIA model mouse intervention experiments have all confirmed the mechanism by which KIAA1199 protein accelerates the progression of RA by promoting angiogenesis.

[0004] Abnormally high expression of KIAA1199 protein in vivo is closely related to various pathologies, including inflammation. Therefore, antagonists targeting KIAA1199 have therapeutic potential for these diseases. However, these intramembrane small molecule antagonists generally act on the highly conserved ATP binding site, a common site for growth factors, resulting in drawbacks such as high toxicity and poor specificity. Extramembrane antagonists are less reported. While peptide antagonists offer advantages such as high activity, low dosage, low toxicity, and amino acids as metabolic end products, small peptides are unstable in vivo due to their susceptibility to peptidase hydrolysis. Furthermore, the flexible molecular structure of small peptides can accommodate different conformations required for recognition by various receptor molecules, leading to reduced selectivity, poor efficacy, and potential side effects.

[0005] Existing research reports that screening antibody antagonists from human combined antibody libraries using techniques such as phage display offers advantages not found in small organic molecule antagonists or peptide antagonists. Furthermore, antibodies produced using this method are not only structurally stable (preventing premature degradation in vivo before reaching the lesion) and exhibit good targeting (significantly reducing toxic side effects), but more importantly, they overcome the drawbacks of hybridoma antibody production, such as rejection reactions, complex preparation processes, low fusion efficiency, low antibody yield, instability, and weak penetration. This method also enables high-throughput, rapid antibody screening at a low cost. Additionally, antibodies screened in this way sometimes possess physiological functions not found in natural antibodies. However, there are currently no reports of antibody antagonists specifically targeting KIAA1199. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a fully human antagonistic antibody targeting KIAA1199 protein and its application. The technical solution adopted by this invention is as follows: a fully human antagonistic antibody targeting KIAA1199 protein, wherein the antibody is a single-chain antibody and its amino acid sequence is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0007] It also provides genes encoding fully human antagonistic antibodies targeting KIAA1199 protein as described above, the nucleotide sequence of the gene encoding the amino acid shown in SEQ ID NO:1 is shown in SEQ ID NO:3, and the nucleotide sequence of the gene encoding the amino acid shown in SEQ ID NO:2 is shown in SEQ ID NO:4.

[0008] And an expression vector containing the genes described above.

[0009] And, a recombinant cell containing the expression vector described above.

[0010] And, an enzymatic digestion fragment of a fully human antagonistic antibody targeting KIAA1199 protein as described above, characterized in that: the enzymatic digestion fragment is a Fab, Fab′, F(ab′)2 or Fv fragment.

[0011] And, a gene encoding an enzymatically digested fragment as described above.

[0012] The application of the fully human antagonistic antibody targeting KIAA1199 protein as described above in the preparation of inhibitors targeting KIAA1199 protein.

[0013] The application of fully human antagonistic antibodies targeting KIAA1199 protein as described above in the preparation of drugs for treating or alleviating inflammatory diseases and / or autoimmune diseases, especially in the preparation of drugs for treating or alleviating rheumatoid arthritis.

[0014] The beneficial effects of this invention are as follows: This invention screens fully human antagonistic antibodies targeting KIAA1199 protein from a human scFv phage antibody library, and determines their nucleotide sequences to obtain their encoding genes. The fully human antagonistic antibodies obtained by this method have specificity in targeting KIAA1199 protein and have the potential for application as inhibitors targeting KIAA1199 protein or in the preparation of drugs for treating or alleviating inflammatory diseases and / or autoimmune diseases. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0016] Figure 1 Image of ELISA results for monoclonal bacteriophages;

[0017] Figure 2 Figure 1 shows the experimental results of KIAA1199 scFv inhibiting FLS proliferation.

[0018] Figure 3 Experimental results of KIAA1199 scFv inhibiting FLS scratch repair;

[0019] Figure 4 Figure showing the results of KIAA1199 scFv inhibiting FLS degradation of hyaluronic acid. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to embodiments, test examples, and accompanying drawings.

[0021] This invention provides a fully human antagonistic antibody targeting the KIAA1199 protein, wherein the antibody is a single-chain antibody and its amino acid sequence is shown in SEQ ID NO:1 and SEQ ID NO:2. It also provides an enzymatic digestion fragment of the fully human antagonistic antibody targeting the KIAA1199 protein as described above, wherein the enzymatic digestion fragment is a Fab, Fab′, F(ab′)2, or Fv fragment.

[0022] The fully human antagonistic antibody targeting KIAA1199 protein provided by this invention can specifically target and bind to KIAA1199 protein and inhibit the activity of KIAA1199 protein, and can be used as an antagonist of KIAA1199.

[0023] The fully human antagonistic antibody targeting KIAA1199 protein provided by this invention can be prepared by bioengineering. Therefore, this invention further provides a gene encoding the fully human antagonistic antibody targeting KIAA1199 protein as described above.

[0024] The nucleotide sequences of the gene are SEQ ID NO:3 and SEQ ID NO:4, which correspond to fully human antagonistic antibodies with amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0025] The DNA of the aforementioned single-chain antibody can be obtained using conventional gene recombination techniques. After obtaining the DNA sequence encoding the single-chain antibody via PCR, it is cloned into vectors, which can be plasmids, viruses, or gene fragments commonly used in molecular biology. A protein secretion signal peptide sequence is added to the front end of the DNA sequence encoding the antibody to ensure that the antibody can be secreted from the cell. The vector sequence includes a promoter for gene expression, protein translation initiation and termination signals, and a polyadenylate (PolyA) sequence. The vector contains an antibiotic resistance gene to facilitate replication and expression in host cells such as bacteria and eukaryotic cells. Additionally, the vector includes a eukaryotic cell selectivity gene for the stable selection of host cell lines for transfection.

[0026] After constructing the plasmid containing the DNA sequence encoding the aforementioned antibody, the recombinant vector can be used to transfect or transform host cells to express the corresponding protein. Various expression systems can be used to express antibodies, including eukaryotic and prokaryotic cells, such as mammalian cells, insect cells, yeast, and bacteria. Because prokaryotic cells readily form inclusion bodies when expressing antibodies, mammalian cells are the preferred system for expressing this protein. Various mammalian cells can be used for large-scale antibody expression, such as CHO cells, 293 cells, NSO cells, and COS cells, all of which are included in the cell types applicable to this invention. The recombinant plasmid containing the gene encoding the aforementioned antibody can be transfected into host cells. Various transfection methods exist, including electroporation, liposome transfection, and calcium phosphate transfection.

[0027] A preferred method for protein expression is to utilize stably transfected host cells. For example, after stably transfecting host cells without neomycin resistance with a recombinant vector containing a neomycin resistance gene, the concentration of neomycin in the cell culture medium can be increased to screen for stable cell lines with high expression. Similarly, after stably transfecting host cells lacking DHFR with a recombinant vector containing a dihydrofolate reductase (DHFR) gene, the concentration of methotrexate (MTX) in the cell culture medium can be increased to screen for stable cell lines with high expression.

[0028] Other expression systems besides mammalian cells, such as insect cells, yeast, and bacteria, can also be used to express the antibodies of this invention, and they are also included in the category of host cells that can be used in this invention. These expression systems have higher protein yields than mammalian cells, but they tend to form inclusion bodies, thus requiring further protein refolding.

[0029] The antibodies of the present invention can also be delivered and expressed using viral vectors, including but not limited to adenovirus vectors, adeno-associated viral vectors, retroviral vectors, herpes simplex virus-based vectors, and lentiviral vectors.

[0030] The application of the fully human antagonistic antibody targeting KIAA1199 protein, as described above, in the preparation of inhibitors targeting KIAA1199 protein. The single-chain antibody of this invention can be further fused with other effector molecules to achieve additional effects without affecting its targeting; all of these fall within the scope of this invention.

[0031] The antibodies of the present invention can be prepared into various forms of pharmaceutical formulations according to conventional pharmaceutical techniques, with injections being more preferred and freeze-dried injections being the most preferred.

[0032] The antibodies of the present invention can form pharmaceutical compositions with other drugs, and the compositions can be used in conjunction with other treatment methods to treat diseases, including chemotherapy, radiotherapy, and biological therapy.

[0033] The application of fully human antagonistic antibodies targeting KIAA1199 protein as described above in the preparation of drugs for the treatment or relief of inflammatory diseases and / or autoimmune diseases (such as rheumatoid arthritis).

[0034] Example 1

[0035] The fully human antagonistic antibody targeting the KIAA1199 protein described in this invention was screened from a human ScFv phage antibody library. The specific process is as follows:

[0036] 1. Explanation of Experimental Case Information

[0037] Antigen: Human KIAA1199 protein;

[0038] Phage library used for screening: human scFv phage antibody library;

[0039] Objective: To screen antagonistic antibodies targeting the KIAA1199 protein from a human scFv phage antibody library;

[0040] 2. Materials

[0041] The main reagents used in this invention are shown in Table 1:

[0042] Table 1 Main reagents used

[0043]

[0044] 3. Screening of KIAA1199 high-affinity single-chain antibodies

[0045] 3.1 Biological Screening

[0046] Streptavidin magnetic beads and phage libraries (1×10⁻⁶) were blocked with 3% MILK-PBS blocking buffer, respectively. 12 Block the streptavidin magnetic beads at room temperature and 20 rpm for 1 hour using a rotary mixer (PFU). Discard the blocking solution, add 1 mL of 0.1% PBST, vortex to resuspend for 20 seconds, and magnetically separate for 2 minutes to obtain the blocked streptavidin magnetic beads.

[0047] (1) Add 10 μg of biotinylated antigen protein to each of the blocked streptavidin magnetic beads;

[0048] (KIAA1199 protein), and then bound on a shaker at 20 rpm for 1 hour.

[0049] (2) Discard the antigen and PBS, wash the magnetic beads three times with 0.1% PBST, and then wash once with PBS. Obtain the antigen-coated magnetic beads;

[0050] (3) Add the blocked phage library to the antigen-coated magnetic beads, and then allow them to bind for 1 hour on a shaker at 20 rpm at room temperature. Perform magnetic separation for 2 minutes and remove the supernatant.

[0051] (4) Add 1 mL of 0.1% PBST, vortex to resuspend the magnetic beads for 20 s, magnetically separate for 2 min, and remove the supernatant. Repeat this step 15 times;

[0052] (5) Wash the tube once with 0.05% PBST, add 400 μL of glycine hydrochloric acid solution (Glycine-HCl, pH=2.2), mix thoroughly by inverting, and place on a rotary mixer at room temperature at 20 rpm for 10 min;

[0053] (6) Magnetic separation for 2 min, aspirate the supernatant and add it to a new 1.5 mL EP tube (with 200 μL Tris-HCl neutralization buffer added, pH = 8.0);

[0054] (7) In order to increase the number of high affinity phages, more stringent washing conditions will be implemented in the subsequent washing process, such as reducing the amount of coating antigen, increasing the number of washes and the concentration of Tween-20, or using competitive elution methods. See Table 2 for details.

[0055] Table 2. Selection criteria for each round of washing

[0056]

[0057] 3.2 Detection of eluted phage titers

[0058] (1) Culture Escherichia coli TG1 to the logarithmic growth phase;

[0059] (2) Dilute the elution buffer, then mix 10 μL with 200 μL of Escherichia coli and incubate at 37°C for 10 min;

[0060] (3) Transfer the above mixture to an LB-Amp culture plate, then invert the culture plate and incubate overnight at 37°C;

[0061] (4) The titers of the eluted products in the first, second and third rounds of screening were calculated respectively, and the results are shown in Table 2.

[0062] 3.3 Amplification of eluted bacteriophages

[0063] (1) Culture Escherichia coli TG1 to the logarithmic growth phase, add the eluted phage library to 10 mL of Escherichia coli TG1 culture medium, incubate at 37°C for 30 min, and then shake at 220 rpm for 30 min.

[0064] (2) Add the mixed Escherichia coli TG1 culture medium to 30 mL of 2YT-Amp medium and incubate at 37℃ and 220 rpm for 1 h;

[0065] (3) Add 160 μL of M13KO7 (concentration 1×10⁻⁶) 12 Add pfu / mL helper phage to the culture medium, incubate at 37°C without shaking for 30 min, then incubate with shaking at 220 rpm for 1 h;

[0066] (4) Centrifuge at 5000 rpm for 10 min, discard the supernatant, and resuspend TG1 in 50 mL of 2YT (containing 100 μg / mL ampicillin and 50 μg / mL kanamycin). Incubate overnight at 220 rpm and 30 °C.

[0067] (5) Centrifuge, transfer the supernatant to a centrifuge tube, and add 12.5 mL of PEG / NaCl to the supernatant. Mix thoroughly and place the mixture on ice for 4 hours;

[0068] (6) After centrifugation at 13000 rpm, resuspend the precipitate with 1 mL of PBS, centrifuge again at 13000 rpm, and transfer the supernatant to an EP tube to amplify the phage for further selection. The titer is checked as described in 3.1.

[0069] 3.4 The third round of elution products infected TG1 Escherichia coli

[0070] Add 10 to 200 μL of log-phase TG1 -3 / 10 -4 / 10 -5 The third round of elution products at dilution were incubated at 37°C for 30 min, spread on 2YTAG plates, and incubated overnight at 37°C. The colony count was calculated the next day.

[0071] 3.5 ELISA detection of monoclonal phages

[0072] (1) Select 96 single clones from 2-YT plates and inoculate them into 96-well deep-well plates containing 600 μL of 2YTAG medium (containing 2% glucose) per well. Incubate at 37℃ with shaking at 700 rpm until the logarithmic growth phase.

[0073] (2) Add ten times the number of K07M13 helper phages to each well and shake to mix.

[0074] (3) Incubate at 37℃ for 30 min, then incubate at 37℃ and 600 rpm for 60 min, and centrifuge at 3200g for 10 min. Quickly discard the supernatant and resuspend the precipitate at the bottom of the plate with 300 μL of 2TY culture medium (containing 100 ug / mL ampicillin and 50 ug / mL kanamycin), and incubate overnight at 30℃ and 700 rpm.

[0075] (4) The next day, centrifuge the deep well plate at 4000 rpm for 10 min, transfer the supernatant to a new 96-well plate, and store at 4°C.

[0076] (5) Dilute the biotinylated antigen to 1 μg / mL with 0.05% PBST solution, add it to the streptavidin ELISA plate, 100 μL / well, and mix at 37°C for 1 h.

[0077] (6) Wash each well with 300 μL PBST three times;

[0078] (7) Block the wells with 250 μL of blocking buffer (PBS containing 5% skim milk powder) at 37°C for 1.5 h. Block the wells with 200 μL of blocking buffer (PBS containing 3% skim milk powder) at room temperature for 1 h.

[0079] (8) Discard the blocking solution and wash the plate 3 times with washing buffer (PBST), tapping the plate wells on a clean paper towel each time with the wells facing down.

[0080] (9) Add 50 μL of diluted amplified phage and 50 μL of blocking buffer (PBS containing 1% skim milk powder) to the wells and incubate at 37°C for 1 h;

[0081] (10) Wash each well with 300 μL PBST three times;

[0082] (11) Dilute M13-HRP with blocking buffer (1:5000) and incubate at room temperature for 1 h;

[0083] (12) Wash 6 times with 0.1% PBST, add 100 μL TMB (prepared by mixing solution A and solution B in a 1:1 ratio) to each well, incubate at room temperature for 10 min, and then add 100 μL 2M HCl to each well to terminate the reaction;

[0084] (13) The OD value was measured using a 450nm microplate reader, see [link to ELISA reader]. Figure 1 Clones marked with a background color (i.e., samples with an absorbance greater than 3 at 450 nm) were selected for sequencing, ultimately yielding two antibody protein sequences (denoted as SEQ ID NO: 1 and 2, respectively) and nucleotide sequences with KIAA1199 targeting specificity, denoted as SEQ ID NO: 3 and 4, respectively. The absorbances of the single-chain antibodies shown in SEQ ID NO: 1 and 2 are 4.686 and 4.5156, respectively.

[0085] Example 2: Expression of KIAA1199 high-affinity single-chain antibody

[0086] 1. Construction of recombinant expression vectors

[0087] (1) Based on the sequencing results, the antibody gene of SEQ ID NO 3 was amplified using primers;

[0088] (2) The antibody gene fragment was inserted into the expression vector PET28A by homologous recombination.

[0089] (3) Melt a tube of 100 μL of DH5α competent cells on ice, gently tap the tube wall to resuspend the cells, add 10 μg of plasmid to the competent cells, tap a few times, and incubate on ice for 30 min.

[0090] (4) Heat shock in a 42℃ water bath for 90 seconds, then quickly place on ice for 5 minutes;

[0091] (5) Add 500 μL of SOC medium and incubate at 37°C for 60 min;

[0092] (6) Centrifuge at 5000 rpm for 3 min, and as needed, spread a certain amount of bacterial cells on LB / Amp (50 μg / mL) plates to screen for positive clones. PCR is used to identify whether the transformation was successful.

[0093] (7) The sample was sent to Qingke Biotechnology for sequencing. After confirming that the sequencing results were correct, the plasmid was extracted using a plasmid extraction kit. The plasmid was then transformed into BL-21(DE3) Escherichia coli.

[0094] 2. Expression of KIAA1199-scFv protein

[0095] (1) Inoculate BL-21(DE3) into 3 mL LB / Amp (50 μg / mL) liquid medium and incubate at 37 °C with shaking;

[0096] (2) The next day, take the overnight culture medium and inoculate it into 1L LB / Amp (50μg / mL) liquid medium at a ratio of 1 / 100. When the cells grow to OD600 of about 0.6, incubate at 15℃ for 15min. Then add an appropriate amount of 100mmol / L LPTG to the final concentration of 1mmol / L and induce culture at 15℃ for 22h.

[0097] (3) After centrifuging the bacterial culture, suspend the bacteria in 40 mL of PBS buffer and then sonicate them on ice. Then centrifuge the lysate (4000 rpm / min, 40 min).

[0098] (4) Take 8 μL of each extraction solution (total protein, supernatant, precipitate), add 2 μL of 5×SDS Loading Buffer, and heat at 95℃ for 10 min;

[0099] (5) SDS-PAGE electrophoresis was performed. It was found that the protein was mainly expressed in the supernatant;

[0100] (6) Pass the protein supernatant through a 0.45 μm sieve for further purification.

[0101] 3. Purification of KIAA1199-scFv protein

[0102] (1) Wash 2 mL of Ni column (G&E) with at least 5 column volumes of binding buffer;

[0103] (2) Add the filtered protein supernatant and let it flow slowly through the column, while retaining the column-penetrating liquid for detecting the binding efficiency;

[0104] (3) Wash the column with 5 column volumes of binding buffer, repeating three times;

[0105] (4) Elute with 5 times the volume of 200 nM imidazole solution;

[0106] (5) The imidazole eluent was replaced and concentrated using an ultrafiltration tube, and the concentration was calculated. The purity was then analyzed by SDS-PAGE.

[0107] Experimental verification of the antagonistic ability of the KIAA1199 single-chain antibody.

[0108] KIAA1199 has been shown to promote the proliferation and migration of synovial fibroblasts (FLS), therefore this invention verifies the ability of KIAA1199 scFv to inhibit FLS proliferation and migration.

[0109] 1. Detection experiment on inhibition of synovial fibroblast proliferation

[0110] The MTT assay is a commonly used method for detecting cell viability and growth, and is widely used in detecting cell proliferation and cytotoxicity. Its detection principle lies in the fact that exogenous MTT can be reduced to water-insoluble blue formazan by succinate dehydrogenase in the mitochondria of living cells, and deposited in the cell aggregates. In dead cells, the enzyme is inactive. In the presence of DMSO (dimethyl sulfoxide), the insoluble formazan deposit is completely dissolved. The absorbance at 570 nm is measured using a microplate reader. Higher cell viability and activity result in higher absorbance; conversely, inhibited cell activity and increased cell death result in lower absorbance.

[0111] (1) Cell plating: Logarithmic growth phase synovial fibroblasts (FLS) were digested with trypsin to prepare a cell suspension. 100 μL was added to each well, and unused wells were filled with sterile PBS. The cells were cultured in a 5% CO2, 37°C incubator until they adhered and covered the bottom of the well.

[0112] (2) Perform drug treatment according to the experimental protocol. Use KIAA1199 antibody at a concentration of 1 μg / mL and PBS to treat the cells. Set up 4 to 6 replicates for each sample.

[0113] (3) MTT reaction: Add 20 μL of MTT solution (5 mg / mL, i.e. 0.5% MTT) to each well and incubate in an incubator for 4 h;

[0114] (4) Dissolving formazan with DMSO: Carefully remove the supernatant, add 150 μL of DMSO (dimethyl sulfoxide) to each well, and shake at low speed (200 rpm / min) for 15 min in a microplate shaker to fully dissolve the crystals.

[0115] (5) Absorbance measurement: The absorbance at 570 nm was measured using an ELISA reader, and the experimental results were analyzed. Statistical results are shown below. Figure 2 KIAA1199-scFv can inhibit the proliferation of FLS cells.

[0116] 2. Experiment on inhibiting synovial fibroblast scratch repair

[0117] Wound healing is a simple and cost-effective in vitro method for studying cell migration. The principle is that when cells grow to a fused monolayer, a blank area, called a "scratch," is artificially created on the fused monolayer. Cells at the edge of the scratch gradually move into the blank area, healing the "scratch." This process, to some extent, simulates the in vivo cell migration process and is a simple in vitro method for studying cell migration. The specific steps of the wound healing assay are as follows:

[0118] (1) Preparation: All instruments that can be sterilized must be sterilized. Rulers and marker pens should be irradiated with ultraviolet light for 30 minutes before operation (inside a laminar flow hood).

[0119] (2)(2) First, use a marker pen to draw horizontal lines evenly on the back of the 6-hole plate, using a ruler as a guide. Draw one line approximately every 0.5 to 1 cm, passing through the holes. Each hole should have at least 5 lines passing through it.

[0120] (3) Add approximately 5×10 to the hole 5 FLS cells

[0121] (4) On the second day, use the tip of the gun to compare with the ruler and try to make a horizontal line mark on the back. The tip of the gun should be vertical and not tilted.

[0122] (5) Wash the cells three times with PBS to remove the cells that have been cut off, add serum-free culture medium, and add PBS and 1 μg / mL KIAA1199 antibody respectively.

[0123] (6) Incubate at 37℃ in a 5% CO2 incubator. Take samples at 0 and 48 h intervals, photograph them, and analyze the experimental results. See results below. Figure 3 KIAA1199-scFv can inhibit the proliferation of FLS cells.

[0124] 3. Experiment on inhibiting synovial fibroblast-like degradation of hyaluronic acid

[0125] (1) RA FLS cells were seeded into T25 cell culture flasks and cultured until they were in good condition.

[0126] (2) Add KIAA1199-scFv at 1 μg / mL to RA FLS and culture until 95% density.

[0127] (3) FITC-labeled high molecular weight hyaluronic acid (HMW-HA, 1200–1600kDa) with a final concentration of 10μg / mL was added to the RA FLS and KIAA1199-scFv+RA FLS culture media, respectively, and incubated at 37℃ in a 5% CO2 incubator.

[0128] (4) After 24 hours, the supernatant was collected by centrifugation at 3000 rpm / min. The supernatant was equilibrated with phosphate-buffered saline and analyzed by size exclusion chromatography on a Sepharose CL-2B column. 2 mL of the fraction was collected at a rate of 0.5 mL / min and then detected using a Varioskan Lux (Thermo Fisher Scientific) spectrometer. The results are shown in […]. Figure 4 .

[0129] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A fully human antagonistic antibody targeting the KIAA1199 protein, characterized in that: The antibody is a single-chain antibody, and its amino acid sequence is shown in SEQ ID NO:1 or SEQ ID NO:

2.

2. The gene encoding the fully human antagonistic antibody targeting the KIAA1199 protein as described in claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the gene encoding the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 3; The nucleotide sequence of the gene encoding the amino acid sequence shown in SEQ ID NO:2 is shown in SEQ ID NO:

4.

4. An expression vector containing the gene as described in claim 2 or 3.

5. A recombinant cell containing the expression vector as described in claim 4.

6. An inhibitor targeting KIAA1199 protein, comprising the fully human antagonistic antibody targeting KIAA1199 protein as described in claim 1.

7. The use of the fully human antagonistic antibody targeting KIAA1199 protein as described in claim 1 in the preparation of a medicament for treating or alleviating rheumatoid arthritis.

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