A bispecific antibody that simultaneously targets TNF-α and IL-6R, its preparation method and application

Abstract

This invention belongs to the technical field of molecular biology and discloses a bispecific antibody that simultaneously targets TNF-α and IL-6R, its preparation method, and its applications. The bispecific antibody provided by this invention can simultaneously target TNF-α and IL-6R, with its TNF-α affinity being significantly superior to its IL-6R affinity (approximately 6-fold). This results in excellent local targeting of inflammation, preferentially anchoring to inflammatory foci, achieving higher concentrations at inflammatory sites, and stably binding to IL-6R. Consequently, this bispecific antibody possesses excellent overall neutralization efficiency. Its application in the treatment of refractory autoimmune diseases such as rheumatoid arthritis (RA) demonstrates excellent immunosuppressive, anti-inflammatory, and systemic safety properties, making it a highly promising, safe, and effective anti-inflammatory drug with excellent application prospects.

Description

Technical Field

[0001] This invention belongs to the technical field of molecular biology, and in particular relates to a bispecific antibody that simultaneously targets TNF-α and IL-6R, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovitis, synovial hyperplasia, pannus formation, and progressive destruction of articular cartilage and bone. A complex network of immune cell infiltration and the various pro-inflammatory factors secreted by these cells is considered a key driver of RA's development and progression, with some core factors becoming important targets for clinical treatment.

[0003] Among these core factors, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are two key hubs driving the inflammatory cascade. TNF-α, as the upstream dominant factor, binds to its receptor, directly promoting synovial cell proliferation and inhibiting apoptosis. It also induces synovial fibroblasts and monocytes / macrophages to secrete large amounts of other pro-inflammatory factors, further exacerbating joint damage. IL-6 is another multifunctional pro-inflammatory factor that plays a decisive role in rheumatoid arthritis (RA). On the one hand, IL-6 promotes osteoclast differentiation and activation by upregulating RANKL expression, leading to bone erosion. On the other hand, IL-6 disrupts the balance of the immune system, promoting the differentiation of naive T cells into pathogenic Th17 cells while inhibiting the generation of Treg cells. Furthermore, TNF-α and IL-6 exhibit a high degree of functional overlap and synergistic amplification in the pathological microenvironment of RA. The intracellular signaling pathways they activate significantly overlap, meaning they can compensate for each other in driving the expression of downstream pathogenic genes. This overlap and synergistic interweaving of signaling pathways constitutes a complex inflammatory network, resulting in the continued transmission or even compensatory upregulation of inflammatory signals through another pathway even after the inhibition of one pathway. Clinical data show that approximately 20% of patients develop primary or secondary resistance to a single TNF-α inhibitor, and 30%–40% of patients do not respond to anti-IL-6R monotherapy.

[0004] Therefore, obtaining a bispecific antibody that can simultaneously target TNF-α and IL-6R to simultaneously sever the TNF-α and IL-6-mediated synergistic inflammatory network is of great significance for the development of drugs for the treatment of refractory autoimmune diseases such as RA. Summary of the Invention

[0005] The primary objective of this invention is to provide a bispecific antibody that simultaneously targets TNF-α and IL-6R, with a significantly higher affinity for TNF-α than for IL-6R (approximately 6 times greater). This results in excellent local targeting of the bispecific antibody, preferentially anchoring to inflammatory foci and achieving higher concentrations at the inflammatory site. Furthermore, it enables stable binding to IL-6R, thereby granting the bispecific antibody excellent overall neutralization efficiency. When applied to the treatment of refractory autoimmune diseases such as rheumatoid arthritis (RA), it combines excellent immunosuppressive, anti-inflammatory, and systemic safety effects, making it a highly promising, safe, and effective anti-inflammatory drug with excellent application prospects.

[0006] A second objective of this invention is to provide nucleic acid molecules.

[0007] A third objective of this invention is to provide a cell line.

[0008] The fourth objective of this invention is to provide a method for preparing bispecific antibodies.

[0009] The fifth objective of this invention is to provide the use of the above-mentioned bispecific antibody in the preparation of a medicament for treating refractory autoimmune diseases.

[0010] Specifically, the bispecific antibody provided by the present invention comprises: a heavy chain, which includes VH, CH and TNFR connected in sequence, wherein VH includes an amino acid fragment with the sequence shown in SEQ ID NO:1, CH includes an amino acid fragment with the sequence shown in SEQ ID NO:2, and TNFR includes an amino acid fragment with the sequence shown in SEQ ID NO:3; and a light chain, which includes VL and CL, wherein VL includes an amino acid fragment with the sequence shown in SEQ ID NO:5, and CL includes an amino acid fragment with the sequence shown in SEQ ID NO:6.

[0011] Furthermore, the amino acid sequence of the heavy chain is shown in SEQ ID NO:4.

[0012] Furthermore, the amino acid sequence of the light chain is shown in SEQ ID NO:7.

[0013] The nucleic acid molecule provided by this invention includes a nucleic acid fragment encoding the above-mentioned bispecific antibody.

[0014] Furthermore, the nucleic acid fragment encoding the bispecific antibody includes a heavy chain encoding gene with a sequence as shown in SEQ ID NO:8.

[0015] Furthermore, the nucleic acid fragment encoding the bispecific antibody includes a light chain encoding gene with a sequence as shown in SEQ ID NO:9.

[0016] The cell line provided by this invention includes a nucleic acid fragment encoding the above-mentioned bispecific antibody.

[0017] Furthermore, the chassis cells of the cell line are CHO cells and / or HEK293 cells.

[0018] The method for preparing the bispecific antibody provided by the present invention includes: taking the above-mentioned cell line for protein expression to obtain the bispecific antibody.

[0019] This invention provides the use of the above-mentioned bispecific antibody in the preparation of a drug for treating refractory autoimmune diseases. Attached Figure Description

[0020] Figure 1 The diagram below shows the structure of a bispecific antibody that simultaneously targets TNF-α and IL-6R, as provided in an embodiment of the present invention. Figure 2 A schematic diagram of the structure of the bispecific antibody that simultaneously targets TNF-α and IL-6R, provided for Comparative Example 1; Figure 3 A schematic diagram of the structure of the bispecific antibody that simultaneously targets TNF-α and IL-6R, provided for Comparative Example 2; Figure 4 The figure shows the experimental results of ELISA detection of the affinity of the bispecific antibody for TNF-α provided in Example 1 of the present invention; Figure 5 The figure shows the experimental results of ELISA detection of the affinity of the bispecific antibody for TNF-α provided in Comparative Example 1 of Test Example 1 of this invention; Figure 6 The figure shows the experimental results of ELISA detection of the affinity of the bispecific antibody for TNF-α provided in Comparative Example 2 of Test Example 1 of this invention; Figure 7 The figure shows the experimental results of ELISA detection of the affinity of the positive control Etanercept for TNF-α in Test Example 1 of this invention; Figure 8 The figure shows the experimental results of ELISA detection of the affinity of the bispecific antibody for IL-6R provided in Example 1 of the present invention; Figure 9 The figure shows the experimental results of ELISA detection of the affinity of the bispecific antibody for IL-6R provided in Comparative Example 1 of Test Example 1 of this invention. Figure 10 The figure shows the experimental results of ELISA detection of the affinity of the bispecific antibody for IL-6R provided in Comparative Example 2 of Test Example 1 of this invention; Figure 11This is a graph showing the ELISA results of the positive control Tocilizumab for IL-6R affinity in Test Example 1 of this invention. Figure 12 The figure shows the experimental results of SPR detection of the bispecific antibody for TNF-α affinity provided in Example 1 of the present invention; Figure 13 The figure shows the experimental results of SPR detection of the bispecific antibody for TNF-α affinity provided in Comparative Example 1 of Test Example 1 of this invention; Figure 14 The figure shows the experimental results of SPR detection of the bispecific antibody for TNF-α affinity provided in Comparative Example 2 of Test Example 1 of this invention; Figure 15 The figure shows the experimental results of SPR detection of TNF-α affinity of the positive control Etanercept in Test Example 1 of this invention; Figure 16 This is a graph showing the experimental results of the apoptosis inhibition experiment in Test Example 2 of the present invention; Figure 17 The figure shows the experimental results of clinical scores of mice in each group after the first and booster immunizations in Test Example 3 of this invention; Figure 18 This is a graph showing the difference in clinical scores between mice in each group and the model control group on day 50 after the first immunization in Test Example 3 of this invention. Figure 19 These are the gross and pathological staining results of the toe joints of mice in each group after sacrifice on day 50 following the completion of the first immunization in Test Example 3 of this invention. Figure 20 The figure shows the experimental results of HE staining pathological scores of the toe joints of mice in each group after sacrifice on day 50 after the first immunization in Test Example 3 of this invention. Figure 21 The figure shows the experimental results of toluidine blue staining intensity on the toe joints of mice in each group after sacrifice on day 50 following the completion of the first immunization in Test Example 3 of this invention. Detailed Implementation

[0021] The bispecific antibody provided by this invention includes TNFR and an anti-IL-6R antibody with a typical Y-shaped structure. By fusing TNFR to an anti-IL-6R antibody with a specific structure, a bispecific antibody that can simultaneously target TNF-α and IL-6R can be obtained. Furthermore, the affinity of TNFR for TNF-α is significantly enhanced. The resulting bispecific antibody has a certain difference in affinity for TNF-α and IL-6R, which allows the bispecific antibody to be well targeted and enriched at the site of inflammation, exhibiting good therapeutic targeting and systemic safety.

[0022] In this invention, the bispecific antibody specifically comprises a heavy chain and a light chain, and the number of both heavy and light chains is two. The two heavy chains are covalently linked through a hinge region, and the CH1 of each heavy chain is connected to the corresponding light chain through a disulfide bond to form a complete Fab arm. More specifically, the heavy chain comprises VH, CH, and TNFR connected in sequence. The VH comprises an amino acid fragment with the sequence shown in SEQ ID NO:1, the CH comprises an amino acid fragment with the sequence shown in SEQ ID NO:2, and the TNFR comprises an amino acid fragment with the sequence shown in SEQ ID NO:3. The light chain comprises VL and CL. The VL comprises an amino acid fragment with the sequence shown in SEQ ID NO:5, and the CL comprises an amino acid fragment with the sequence shown in SEQ ID NO:6. More specifically, the amino acid sequence of the heavy chain is shown in SEQ ID NO:4, and the amino acid sequence of the light chain is shown in SEQ ID NO:7.

[0023] The present invention also provides a nucleic acid molecule comprising a nucleic acid fragment encoding the aforementioned bispecific antibody. More specifically, the nucleic acid molecule is limited to those capable of achieving the expression and secretion of the aforementioned bispecific antibody, and is a conventionally used technique in the field of existing biotechnology. Specific examples include, but are not limited to, one or more of DNA molecules, RNA molecules, and recombinant plasmid vectors.

[0024] In this invention, the nucleic acid molecule preferably includes a heavy chain coding gene with a sequence as shown in SEQ ID NO:8. In this case, the heavy chain coding gene is obtained through human cell codon optimization and contains a nucleotide sequence encoding a signal peptide, exhibiting good protein secretion expression in cells such as CHO cells and HEK293 cells.

[0025] In this invention, the nucleic acid molecule preferably includes a light chain coding gene with a sequence as shown in SEQ ID NO:9. In this case, the light chain coding gene is obtained through human cell codon optimization and contains a nucleotide sequence encoding a signal peptide, exhibiting good protein secretion expression in cells such as CHO cells and HEK293 cells.

[0026] The present invention also provides a cell line comprising a nucleic acid fragment encoding the aforementioned bispecific antibody. More specifically, the cell line is limited to the ability to express and secrete the aforementioned bispecific antibody, and is a conventional technique in the field of existing biotechnology. Specific examples of the chassis cells used include, but are not limited to, CHO cells and / or HEK293 cells.

[0027] This invention also provides a method for preparing a bispecific antibody, which specifically includes: performing protein expression on the above-mentioned cell line to obtain the bispecific antibody. More specifically, performing protein expression on the above-mentioned cell line is a conventional technique in the field of existing biotechnology, and this invention does not impose any particular limitation on it.

[0028] This invention also provides the application of the above-mentioned bispecific antibody in the preparation of a drug for treating refractory autoimmune diseases. More specifically, the bispecific antibody, utilizing its excellent affinity for TNF-α and the poor affinity between TNF-α and IL-6R, can efficiently target inflammatory sites and effectively block the synergistic effect of the TNF-α and IL-6 inflammatory pathways, exhibiting remarkable in vivo therapeutic effects.

[0029] In this invention, specific examples of the refractory autoimmune diseases include, but are not limited to, one or more of rheumatoid arthritis, ankylosing spondylitis, and systemic lupus erythematosus.

[0030] In this invention, when the bispecific antibody is used as a drug for treating rheumatoid arthritis, it can fundamentally intervene in the pathological process of deep joint tissues—effectively inhibiting synovial hyperplasia and inflammatory cell infiltration, reducing joint lesions, effectively preventing the loss of proteoglycans in the cartilage matrix, and promoting the recovery of osteoblast function. It has excellent in vivo therapeutic effects on arthritis and protective effects against articular cartilage and bone damage.

[0031] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0032] Example This embodiment illustrates a bispecific antibody that simultaneously targets TNF-α and IL-6R, referring to... Figure 1 The bispecific antibody comprises two heavy chains and two light chains; the heavy chains consist of VH, CH and TNFR linked sequentially, and the light chains consist of VL and CL. The specific amino acid sequences of each fragment are shown in Table 1.

[0033] Table 1.

[0034] The preparation of this bispecific antibody specifically includes the following steps: (1) Based on the amino acid sequence of the bispecific antibody, the codon of human cells was optimized to obtain the encoding gene of the bispecific antibody (heavy chain encoding gene: SEQ ID NO:8, light chain encoding gene: SEQ ID NO:9), which was synthesized by the external biotechnology company and carried on the pcDNA3.4 plasmid vector to obtain the expression construct expressing the bispecific antibody.

[0035] (2) Based on the constructed expression construct, the biosynthesis company was sent to the biosynthesis company. CHO cells were used as the protein expression system to express bispecific antibodies and purify them by Protein A affinity chromatography to obtain bispecific antibodies.

[0036] The bispecific antibody provided in this embodiment has a molecular weight of 196 kDa.

[0037] Comparative Example 1 This comparative example uses the method provided in Example 1 to prepare a bispecific antibody. The amino acid sequences of the fragments contained in the bispecific antibody provided in this comparative example are consistent with those in Example 1. The difference lies in the structure of the bispecific antibody obtained by combining the fragments. Figure 2 The bispecific antibody comprises one heavy chain-I, one heavy chain-II, and one light chain; wherein, heavy chain-I comprises VH, CH1, CH2, and CH3 connected in sequence, heavy chain-II comprises TNFR, CH2, and CH3 connected in sequence, and the light chain comprises VL and CL. The specific amino acid sequences of heavy chain-I, heavy chain-II, and light chain are shown in Table 2. Under the same conditions, the bispecific antibody was prepared.

[0038] Table 2.

[0039] The bispecific antibody provided in this comparative example has a molecular weight of 123.622 kDa.

[0040] Comparative Example 2 This comparative example uses the method provided in Example 1 to prepare a bispecific antibody. The amino acid sequences of the fragments contained in the bispecific antibody provided in this comparative example are consistent with those in Example 1. The difference lies in the structure of the bispecific antibody obtained by combining the fragments. Figure 3 The bispecific antibody comprises two recombinant heavy chains; wherein the recombinant heavy chain comprises VH, VL, TNFR, CH2 and CH3 connected in sequence, and the specific amino acid sequence of the recombinant heavy chain is shown in Table 3. Under the same conditions, the bispecific antibody was prepared.

[0041] Table 3.

[0042] The bispecific antibody provided in this comparative example has a molecular weight of 154.27 kDa.

[0043] Test Example 1 This test example illustrates the binding affinity of the bispecific antibodies provided in the above examples and comparative examples for TNF-α and IL-6R. The test specifically includes: 1. ELISA detection: (1) Add 100 μL / well to the coating solution-1 containing 2 μg / mL recombinant human IL-6R (R&D Systems, catalog number 227-SR) or the coating solution-2 containing 2 μg / mL human TNF-α protein (R&D Systems, catalog number NBP2-35076) to the 96-well microplate. After the protein coating is completed, the plate is washed 3 times and patted dry.

[0044] (2) Add PBS buffer (10mM, pH=7.4) containing 5% skim milk powder to the 96-well microplate after protein coating at a dosage of 200μL / well. After incubation at 37℃ for 60min to complete the blocking process, wash the plate 3 times and pat dry.

[0045] (3) The bispecific antibodies provided in the examples and comparative examples were serially diluted with PBS buffer (10mM, pH=7.4) to obtain a series of antibody diluents with concentrations ranging from 200nM to 3.125nM.

[0046] (4) Add each antibody dilution solution to a 96-well microplate that has been blocked, according to the addition amount of 100 μL / well. After incubating at 37°C for 60 min to complete the primary antibody binding, wash the plate 3 times and pat dry.

[0047] (5) Add 100 μL of HRP-labeled anti-human secondary antibody (Southernbiotech, catalog number 2040-05) diluted 1:16000 to the 96-well microplate containing the primary antibody at a concentration of 100 μL / well. After incubation at 37°C for 30 min to complete the secondary antibody binding, wash the plate three times and pat dry. Then, use TMB chromogenic solution (Biopanda, catalog number TMB-S-004) and stop solution (Solarbio, catalog number C1058) according to the instructions to perform chromogenic treatment on the 96-well microplate containing the secondary antibody. Immediately afterward, use a microplate reader to read the OD of each well. 450 The EC50 values ​​were calculated by fitting the results, with Etanercept and Tocilizumab used as positive controls. All testing conditions were kept consistent across groups. The results are as follows: Figures 4-7 As shown in Table 4.

[0048] Table 4.

[0049] Depend on Figures 4-7 As shown in Table 4, compared with the bispecific antibodies provided in the positive control and Comparative Examples 1 and 2, the bispecific antibody provided in this embodiment of the invention has a very good binding affinity for human TNF-α protein, and its affinity for human TNF-α protein and IL-6R differs by about 6 times.

[0050] 2. SPR detection: (1) The SPR instrument (cytiva, catalog number BIAcore8K) was used for testing. The SPR instrument was cleaned and balanced according to the instruction manual. The sensor chip was surface chemically activated, and the recombinant TNF-α protein (Sino Biological, catalog number 10602-HNAE) was fixed and blocked according to the instruction manual. The flow channel that did not bind the recombinant TNF-α protein was used as the reference channel.

[0051] (2) The bispecific antibodies provided in the examples and comparative examples were serially diluted using the PBS buffer prepared by the SPR instrument to obtain a series of antibody diluents with concentrations ranging from 200 nM to 3.125 nM.

[0052] (3) At a flow rate of 30 μL / min, each antibody dilution was continuously injected into the detection channel for 2 min. During this period, the signal changes during the binding phase were recorded in real time until the binding phase ended. Then, PBS buffer was injected and the signal changes during the dissociation phase were recorded.

[0053] (4) The reference channel signal was used as the background for subtraction, and the obtained sensor image was analyzed. The binding and dissociation curves were fitted using the instrument's matching analysis software, and the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) were calculated. Etanercept was used as a control, and the test conditions used in each group were kept consistent. Each group was tested in parallel three times and the average value was taken. The results are as follows: Figures 12-15 As shown in Table 5.

[0054] Table 5.

[0055] Depend on Figures 12-15 As shown in Table 5, compared to Comparative Examples 1-2 and the positive control, the bispecific antibody provided in this invention has a KD value of 18.88 nM with TNF-α, exhibiting excellent affinity. That is, the introduction of specific VH, CH, VL, and CL in the bispecific antibody, arranged in a specific configuration, effectively optimizes the structure and spatial conformation of the contained TNFR, significantly improving its affinity for TNF-α.

[0056] Test Example 2 This test case illustrates the ability of the bispecific antibody provided in the examples to inhibit TNF-α and induce apoptosis. The test specifically includes: 1. According to 5×10 5 For cell / well seeding, take L929 cells (ATCC, catalog number CCL-1) in logarithmic growth phase and seed them into 6-well cell culture plates containing DMEM medium. Incubate overnight at 37°C and 5% CO2 to allow the cells to adhere fully. The next day, discard the old medium and proceed with the following grouping: (1) Negative control group: Add DMEM medium to a 6-well cell culture plate at a rate of 3 mL / well, mix gently, and incubate at 37°C and 5% CO2 for 24 h.

[0057] (2) Positive control group: Add DMEM medium containing 100 ng / mL TNF-α to a 6-well cell culture plate at a dosage of 3 mL / well, mix gently, and incubate at 37℃ and 5% CO2 for 24 h.

[0058] (3) Etanercept group: Add DMEM medium containing 100ng / mL TNF-α and 50nM Etanercept to a 6-well cell culture plate at a dosage of 3mL / well, mix gently, and incubate at 37℃ and 5% CO2 for 24h.

[0059] (4) Antibody group: Add DMEM medium containing 100ng / mL TNF-α and 50nM bispecific antibody to a 6-well cell culture plate at a rate of 3mL / well. After gently mixing, incubate at 37℃ and 5% CO2 for 24h.

[0060] 2. After incubation, collect all cells from each well (including dead cells suspended in the supernatant and adherent cells detached by trypsin digestion). Wash with PBS buffer (0.2 mM, pH=7.2), centrifuge, and resuspend the cell pellet in 100 μL of PBS buffer (0.2 mM, pH=7.2). Stain with 5 μL of PI (BD, catalog number 556463) for 5 min, then add 400 μL of PBS buffer (0.2 mM, pH=7.2) and place in a flow cytometer for detection. Analyze the proportion of viable cells in each group. Calculate the inhibition rate of TNF-α-induced apoptosis using the following formula. The test conditions used in each group were kept consistent. Each group was tested in triplicate, and the average value was taken. The results are shown below. Figure 16 As shown.

[0061] Inhibition rate of TNF-α-induced apoptosis = (X1-X2) / (X3-X2)×100% In the above formula, X1 is the proportion of live cells in the experimental group (in %), X2 is the proportion of live cells in the positive control group (in %), and X3 is the proportion of live cells in the negative control group (in %).

[0062] Depend on Figure 16 The results show that, compared with comparative examples 1-2, the positive control, and Etanercept, the bispecific antibody provided in this embodiment of the invention can significantly inhibit TNF-α-induced apoptosis, with an inhibition rate 18 times higher than that of Etanercept.

[0063] Test Example 3 This test case illustrates the ability of the bispecific antibody provided in the embodiments to treat arthritis. The test specifically includes: 1. Construction and administration of CIA model mice Male DBA / 1J mice, aged 6-8 weeks and weighing approximately 20g, were used as experimental animals. After acclimatization under standard feeding conditions for 7 days, they were randomly divided into groups for the following procedures: (1) Negative control group (n=5): Mice were injected intradermally at multiple points on the tail and back of mice with an emulsion prepared by PBS buffer (10mM, pH=7.4) and complete Freund's adjuvant (the volume ratio of PBS buffer and complete Freund's adjuvant was 1:1) at a dose of 100 μL / mouse (referred to as day 0). The mice were then fed for 21 days. On day 21, mice were injected intradermally at multiple points on the tail and back of mice with an emulsion prepared by PBS buffer (10mM, pH=7.4) and incomplete Freund's adjuvant (the volume ratio of PBS buffer and incomplete Freund's adjuvant was 1:1) at a dose of 100 μL / mouse. On day 28, mice were injected intraperitoneally with PBS buffer (10mM, pH=7.4) at a dose of 100 μL / mouse. The first administration was completed. The mice were then administered the medication twice a week at a dose of 100 μL / mouse each time until day 50.

[0064] (2) Model control group (n=5): Mice were given a dose of 100 μL / mouse, and a complete Freund's adjuvant emulsion containing 1 mg / mL bovine type II collagen (prepared by emulsifying PBS buffer (10 mM, pH=7.4) containing bovine type II collagen with complete Freund's adjuvant at a 1:1 volume ratio) was injected intradermally into the tail and back of multiple sites to complete the first immunization (recorded as day 0). Mice were then fed for 21 days. On day 21, a dose of 100 μL / mouse was administered, and an incomplete Freund's adjuvant emulsion containing 1 mg / mL bovine type II collagen was injected intradermally into the tail and back of multiple sites to complete the first immunization (recorded as day 0). Mice were then fed for another 21 days. On day 21, mice were given a dose of 100 μL / mouse, and an incomplete Freund's adjuvant emulsion containing 1 mg / mL bovine type II collagen was injected intradermally into the tail and back of multiple sites to complete the first immunization (recorded as day 0). Full Freund's adjuvant emulsion (prepared by emulsifying PBS buffer (10 mM, pH=7.4) containing bovine type II collagen with incomplete Freund's adjuvant at a 1:1 volume ratio) was administered to mice via multiple intradermal injections into the tail and back to complete booster immunization. On day 28, mice were given PBS buffer (10 mM, pH=7.4) via intraperitoneal injection at a dose of 100 μL / mouse to complete the first administration. Subsequently, the mice were given 100 μL / mouse twice a week until day 50.

[0065] (3) Etanercept treatment group (n=5): The mice were given the first and booster immunizations according to the method provided for the model control group. On day 28, mice were given PBS buffer (10mM, pH=7.4) containing 0.4mg / mL Etanercept by intraperitoneal injection at a dose of 2mg / kg (dose volume of 100μL). The mice were then given 0.5mg / kg twice a week until day 50.

[0066] (4) Tocilizumab treatment group (n=5): The mice were given the first and booster immunizations according to the method provided for the model control group. On day 28, the mice were given PBS buffer (10mM, pH=7.4) containing 0.4mg / mL of Tocilizumab at a dose of 2mg / kg via intraperitoneal injection (dose volume of 100μL) to complete the first administration. The mice were then given 0.5mg / kg twice a week until day 50.

[0067] (5) Bispecific antibody treatment group - Example (n=5): The first immunization and booster immunization were performed according to the method provided for the model control group; on day 28, mice were given PBS buffer (10mM, pH=7.4) containing 0.4mg / mL of the bispecific antibody provided in the example via intraperitoneal injection at a dose of 2mg / kg (dose volume was 100μL) to complete the first administration; then the mice were given 0.5mg / kg twice a week until day 50.

[0068] (6) Bispecific antibody treatment group - Comparative Example 1 (n=5): The mice were given the first and booster immunizations according to the method provided by the model control group. On day 28, the mice were given PBS buffer (10mM, pH=7.4) containing 0.4mg / mL of the bispecific antibody provided by Comparative Example 1 via intraperitoneal injection at a dose of 2mg / kg (dose volume was 100μL). The mice were then given 0.5mg / kg twice a week until day 50.

[0069] (7) Bispecific antibody treatment group - Comparative Example 2 (n=5): The mice were given the first and booster immunizations according to the method provided by the model control group. On day 28, the mice were given PBS buffer (10mM, pH=7.4) containing 0.4mg / mL of the bispecific antibody provided by Comparative Example 2 via intraperitoneal injection at a dose of 2mg / kg (dose volume of 100μL). The mice were then given 0.5mg / kg twice a week until day 50.

[0070] 2. Detection of toe joint-related indices in CIA model mice (1) Starting from day 25, the swelling of the mouse's toe joints was measured twice a week using calipers and scored (0 points [normal], 1 point [swelling of the paw or one toe], 2 points [involving two joints], 3 points [erythema and severe edema of the entire paw], 4 points [severe arthritis or limb disability], with a maximum total score of 16 points for all four limbs). The score difference (Δ score) between the mouse and the model control group was calculated at day 50. The results are as follows: Figure 17 and 18 As shown.

[0071] Depend on Figure 17 and 18 The results show that, compared with the bispecific antibodies provided in Comparative Examples 1 and 2, as well as the single TNF-α inhibitor Etanercept and the anti-IL-6R monotherapy Tocilizumab, the bispecific antibody provided in this embodiment of the invention can extremely effectively inhibit the progression of arthritis in mice and significantly reduce their clinical scores.

[0072] (2) After euthanizing the mice on day 50, the toe joints were immediately excised, fixed, dehydrated, embedded, and sectioned to obtain toe joint sections with a thickness of approximately 3.5 μm. The toe joint sections were then stained with hematoxylin and eosin (HE), toluidine blue, and safranin-fast green. The reagents and conditions used for each group of sections were kept consistent during these procedures. The results are as follows: Figure 19 As shown.

[0073] (3) The pathological scores of HE-stained toe joint sections were determined based on four dimensions: synovial endothelium, cell infiltration, cartilage damage, and pannus. Specifically, these included: i. Synovial intima: 1 point indicates localized single-layer cubic degeneration, 2 points indicate localized multilayer cubic degeneration, and 3 points indicate multilayer synovial hyperplasia accompanied by extensive necrosis; ii. Cell infiltration: 1 point indicates a small amount of focal infiltration, 2 points indicate extensive focal infiltration, and 3 points indicate extensive infiltration that invades the joint capsule and forms cell aggregates; iii. Cartilage damage: 1 point indicates superficial and localized cartilage degradation in multiple areas, 2 points indicates localized deep cartilage degradation, and 3 points indicates extensive deep cartilage degradation in multiple locations. iv. Pannus: 1 point indicates pannus formation in a maximum of two locations, 2 points indicates pannus formation in a maximum of four locations with infiltration or excessive flattening of the articular surface, and 3 points indicates pannus formation in more than four locations or extensive pannus formation in two locations. Furthermore, when conducting the final pathological scoring, the highest scores for each of the mouse's limbs in each of the above categories were summed to obtain the mouse's total pathological score, ranging from 0 (no lesions) to 12 (full marks). Statistical analysis and graphing were performed using GraphPad Prism software, and the results are as follows: Figure 20 As shown.

[0074] (4) ImageJ software was used to extract the integrated optical density of the articular cartilage region from the toe joint slice images stained with toluidine blue, and to obtain the toluidine blue staining intensity data. The methods and conditions used for the toluidine blue staining intensity data analysis of each group of slices were consistent. The results are as follows: Figure 21 As shown.

[0075] Depend on Figures 19-21 The results show that, compared with the bispecific antibodies provided in Comparative Examples 1 and 2, as well as the single TNF-α inhibitor Etanercept and the single anti-IL-6R drug Tocilizumab, the bispecific antibody provided in this embodiment of the invention can effectively reduce synovial hyperplasia and inflammatory cell infiltration, and prevent cartilage matrix degeneration and bone destruction, thereby effectively alleviating the progression of joint disease.

[0076] 3. Safety assessment (1) After the mice were sacrificed on day 50, whole blood was collected immediately by cardiac blood collection. The whole blood was coagulated and separated into solid and liquid to obtain serum samples to be tested. The serum samples to be tested were used to detect related substances of liver function indicators and kidney function indicators. The processing and testing conditions of whole blood and serum samples to be tested in each group were kept consistent. The results are shown in Table 6.

[0077] Table 6.

[0078] Note: " / " indicates that the value is too low and the instrument did not detect it.

[0079] As shown in Table 6, after long-term administration of the bispecific antibody provided in this embodiment to mice, all core liver and kidney function indicators of the mice were within the normal range of healthy physiology, with no obvious liver and kidney toxicity, demonstrating excellent safety.

[0080] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

[0081] The specific sequences involved in this invention are shown in Table 7.

[0082] Table 7.

[0083]

[0084]

[0085]

Claims

1. A bispecific antibody that simultaneously targets TNF-α and IL-6R, characterized in that, The bispecific antibody includes: The heavy chain comprises VH, CH and TNFR connected in sequence, wherein VH comprises an amino acid fragment with the sequence shown in SEQ ID NO:1, CH comprises an amino acid fragment with the sequence shown in SEQ ID NO:2, and TNFR comprises an amino acid fragment with the sequence shown in SEQ ID NO:3; A light chain comprising VL and CL, wherein VL comprises an amino acid fragment with the sequence shown in SEQ ID NO:5, and CL comprises an amino acid fragment with the sequence shown in SEQ ID NO:

6.

2. The bispecific antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO:

4.

3. The bispecific antibody according to claim 1, characterized in that, The amino acid sequence of the light chain is shown in SEQ ID NO:

7.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule includes a nucleic acid fragment encoding a bispecific antibody as described in any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that, The nucleic acid fragment encoding the bispecific antibody includes a heavy chain encoding gene with a sequence as shown in SEQ ID NO:

8.

6. The nucleic acid molecule according to claim 4, characterized in that, The nucleic acid fragment encoding the bispecific antibody includes a light chain encoding gene with a sequence as shown in SEQ ID NO:

9.

7. A cell line, characterized in that, The cell line comprises a nucleic acid fragment encoding the bispecific antibody as described in any one of claims 1 to 3.

8. The cell line according to claim 7, characterized in that, The chassis cells of the cell line are CHO cells and / or HEK293 cells.

9. A method for preparing a bispecific antibody, characterized in that, The preparation method includes: taking the cell line described in claim 7 or 8 and expressing the protein to obtain the bispecific antibody.

10. The use of the bispecific antibody according to any one of claims 1 to 3 in the preparation of a medicament for treating refractory autoimmune diseases.

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