Bispecific antibodies against il-8 and vegf and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a bispecific antibody against IL-8 and VEGF and application thereof. The bispecific antibody comprises a first antigen binding region and a second antigen binding region, wherein the first antigen binding region has IL-8 binding activity; and the second antigen binding region has VEGF binding activity. The application adopts a DVD-Ig mode design, connects variable regions of an anti-VEGF antibody and an anti-IL-8 antibody through a connecting peptide, and forms a novel bispecific antibody. The bispecific antibody can significantly block the biological activity of IL-8 and VEGF, and shows a tumor inhibition effect better than that of an anti-VEGF antibody or an anti-IL-8 antibody alone in a mouse glioma model. The application provides a new drug candidate molecule with higher efficiency and higher potential for cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a bispecific antibody against IL-8 and VEGF and its applications. Background Technology

[0002] Malignant tumors are a serious global public health problem threatening human health. Despite significant progress in targeted therapy and immunotherapy, tumor resistance, recurrence, and metastasis remain major challenges in clinical treatment. The occurrence, development, and resistance to treatment of tumors are not isolated events, but are driven by dynamic, bidirectional interactions between tumor cells and the complex tumor microenvironment (TME). Within the TME, pro-angiogenic factors and inflammatory chemokines constitute two core regulatory networks. They not only play crucial roles individually but also engage in profound interactions, jointly shaping a pathological environment conducive to tumor growth, immune escape, and distant dissemination. Among these, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) are among the most important pivotal molecules in these two networks.

[0003] VEGF and IL-8 form a tightly interconnected and mutually reinforcing synergistic network in the tumor immune microenvironment. Studies have shown that hypoxia (HIF-1α) is the most important common upstream signal inducing VEGF and IL-8 expression. In various tumor models, the vascular collapse and hypoxia induced by treatments such as androgen signaling synchronously and continuously upregulate the expression of VEGF-A and IL-8. A positive feedback loop exists between these two pathways. For example, IL-8 can promote the secretion of more VEGF by tumor cells or stromal cells by activating its receptor; conversely, VEGF may also indirectly regulate IL-8 levels by influencing the inflammatory environment. They play complementary roles in mediating treatment resistance. When the VEGF pathway is blocked by drugs, tumors become highly dependent on alternative pathways such as IL-8 to maintain angiogenesis and survival. For example, in an ovarian cancer model, tumor cells that have acquired resistance to bevacizumab have significantly higher IL-8 expression levels than sensitive cells; simultaneously, inhibiting IL-8 can resensitize these resistant cells to bevacizumab. For example, in a prostate cancer model, enzalutamide-induced drug resistance is closely related to hypoxia-mediated adaptive activation of VEGF-A and IL-8 signaling. Simultaneous inhibition of these two factors can restore and maintain long-term drug control of the tumor. They synergistically shape an immunosuppressive ecosystem: VEGF not only promotes angiogenesis but is also an immunosuppressive factor itself, inhibiting dendritic cell maturation and T cell activation. When VEGF and IL-8 act together, they synergistically shape a deeply immunosuppressive microenvironment from different dimensions: VEGF leads to abnormal vascular structure and dysfunction, hindering the infiltration of immune cells into the tumor parenchyma; while IL-8 actively recruits and activates immunosuppressive cells, directly disarming infiltrating immune cells locally in the tumor. This dual role of vascular barrier and cell inhibition is a significant reason for the low response rate of immunotherapies such as immune checkpoint inhibitors in many tumors. Therefore, simultaneously targeting these two key nodes, VEGF and IL-8, to synergistically block angiogenesis and inflammatory immunosuppressive signals is a promising new strategy to overcome current treatment bottlenecks and achieve more durable tumor control.

[0004] Currently, there are no reports in the literature or patents regarding bispecific antibodies that simultaneously target IL-8 and VEGF, two key synergistic factors. More reports focus on IL-8 monoclonal antibodies or VEGF monoclonal antibodies. For example, patent CN115850474A discloses a monoclonal antibody YX40 that binds to IL-8, screened from a phage antibody library. Its heavy chain CDR sequence includes GFSLNNYA, IGSDGIP, and ASGYVGDDRYNI; its light chain CDR sequence includes PSVYNNNY, AAS, and AGAYSNDSDDG. Another example is patent CN120504739A, which discloses an anti-VEGF antibody. This monoclonal antibody, after binding to VEGF, inhibits the binding of VEGF to its receptor, blocking downstream signal transduction pathways of VEGF; animal studies show that this antibody can inhibit the growth of glioblastoma. However, therapies targeting a single pathway (such as anti-VEGF monotherapy) often have limited efficacy or lead to acquired resistance due to tumor compensation and adaptation mechanisms.

[0005] Therefore, developing a novel, highly efficient bispecific antibody that can simultaneously block IL-8 and VEGF is of great significance for the treatment of diseases related to IL-8 / VEGF abnormalities. Summary of the Invention

[0006] To overcome the limitations of existing single-target therapies, this invention, based on a deep understanding of the synergistic mechanism of VEGF and IL-8 in the tumor immune microenvironment, provides a novel bispecific antibody that can efficiently block both IL-8 and VEGF simultaneously, offering a more effective and promising new drug candidate molecule for cancer treatment.

[0007] One of the objectives of this invention is to provide a bispecific antibody against IL-8 and VEGF.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A bispecific antibody against IL-8 and VEGF, comprising:

[0010] A. First antigen-binding region, which has IL-8 binding activity;

[0011] The first antigen-binding region includes a first heavy chain variable region and a first light chain variable region. The first light chain variable region includes amino acid sequences VL1-CDR1, VL1-CDR2, and VL1-CDR3 as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively. The first heavy chain variable region includes amino acid sequences VH1-CDR1, VH1-CDR2, and VH1-CDR3 as shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0012] B. Second antigen-binding region, which has VEGF binding activity;

[0013] The second antigen-binding region includes a second heavy chain variable region and a second light chain variable region. The second light chain variable region includes amino acid sequences VL2-CDR1, VL2-CDR2, and VL2-CDR3 as shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively. The second heavy chain variable region includes amino acid sequences VH2-CDR1, VH2-CDR2, and VH2-CDR3 as shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively.

[0014] Preferably, the first heavy chain variable region is connected to the second heavy chain variable region via linker 1; the first light chain variable region is connected to the second light chain variable region via linker 2.

[0015] Preferably, the amino acid sequence of linker1 is shown in SEQ ID NO.21, and the amino acid sequence of linker2 is shown in SEQ ID NO.22.

[0016] Preferably, the heavy chain variable region of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO.13, and the light chain variable region of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO.14.

[0017] Preferably, the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 15, and the light chain amino acid sequence is shown in SEQ ID NO: 16.

[0018] The second objective of this invention is to provide a nucleic acid molecule.

[0019] To achieve the above objectives, the present invention adopts the following technical solution:

[0020] A nucleic acid molecule that encodes the aforementioned bispecific antibody.

[0021] Preferably, the nucleotide sequence encoding the heavy chain variable region of the bispecific antibody is shown in SEQ ID NO.17, and the nucleotide sequence encoding the light chain variable region of the bispecific antibody is shown in SEQ ID NO.18.

[0022] Preferably, the nucleotide sequence encoding the heavy chain of the bispecific antibody is shown in SEQ ID NO.19, and the nucleotide sequence encoding the light chain of the bispecific antibody is shown in SEQ ID NO.20.

[0023] A third objective of this invention is to provide an expression vector comprising the aforementioned nucleic acid molecules.

[0024] A fourth objective of this invention is to provide a recombinant cell comprising the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, and / or the aforementioned expression vector.

[0025] A fifth objective of this invention is to provide a pharmaceutical composition comprising the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector and / or the aforementioned recombinant cell, and pharmaceutically acceptable excipients.

[0026] The sixth objective of this invention is to provide a kit comprising the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, and / or the aforementioned recombinant cells.

[0027] The seventh objective of this invention is to provide the use of the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector and / or the aforementioned recombinant cells in the preparation of reagents and / or kits for detecting IL-8 and / or VEGF.

[0028] The eighth objective of this invention is to provide the use of the aforementioned bispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell and / or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases related to abnormal expression or activity of IL-8 and / or VEGF.

[0029] Preferably, the diseases associated with abnormal expression or activity of IL-8 and / or VEGF include malignant tumors.

[0030] Preferably, the malignant tumor includes lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, kidney tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, and / or leukemia.

[0031] Preferably, the gastrointestinal cancer includes colon cancer and / or rectal cancer.

[0032] Preferably, the lung cancer includes non-small cell lung cancer.

[0033] Preferably, the glioma includes glioblastoma.

[0034] Preferably, the treatment includes inhibiting tumor cell growth.

[0035] The beneficial effects of this invention are as follows:

[0036] 1. VEGF and IL-8 form a tightly interconnected and mutually reinforcing synergistic network in the tumor microenvironment. VEGF promotes angiogenesis, while IL-8 promotes inflammation and immunosuppression. Based on the synergistic mechanism of IL-8 and VEGF in the tumor immune microenvironment, this invention provides a bispecific antibody (IN-12) capable of simultaneously and specifically binding to both IL-8 and VEGF. This bispecific antibody can simultaneously block these two key factors, thereby synergistically disrupting the tumor's angiogenesis support and immunosuppressive barriers, providing a more effective and promising novel drug candidate molecule for cancer treatment.

[0037] 2. The IN-12 bispecific antibody provided by this invention can simultaneously target VEGF and IL-8, effectively overcoming the limitations of single-target therapy and providing more durable tumor control. Animal experimental results show that in a mouse glioma model, the IN-12 bispecific antibody can effectively inhibit tumor growth, and its tumor-inhibiting effect is superior to that of monoclonal antibodies using either anti-VEGF antibody (Avastin) or anti-IL-8 antibody (10F8) alone. This indicates that bispecific antibodies have a significant synergistic effect in vivo and can more effectively control tumor growth.

[0038] 3. The IN-12 bispecific antibody of the present invention can simultaneously block the biological activities of IL-8 and VEGF. Experimental results show that the IN-12 bispecific antibody can significantly reduce the number of migrating cells, verifying its ability to block the biological function of IL-8; at the same time, the IN-12 bispecific antibody can block the binding of VEGF to the VEGF receptor on the surface of the 293T-VEGFR2-Luc cell line and produce a dose-dependent concentration-dependent effect, with an IC50 of 88.94 ng / mL, indicating that it has a significant VEGF blocking effect. Attached Figure Description

[0039] Figure 1 The image shows the SDS-PAGE results after the IN-12 antibody expression and purification.

[0040] Figure 2 The image shows the SEC-HPLC detection results after the IN-12 antibody expression and purification.

[0041] Figure 3 The image shows the results of ELISA detection of the binding of IN-12 bispecific antibody to human VEGF.

[0042] Figure 4 The image shows the results of ELISA detection of the binding of IN-12 double antibody to human IL-8.

[0043] Figure 5 The graph shows the fitted curve of the affinity constant KD between the IN-12 antibody and IL-8.

[0044] Figure 6 The fitted curve for the affinity constant KD of IN-12 antibody and VEGF is shown.

[0045] Figure 7 The graph shows the results of detection related to the blocking of IL-8 biological function by IN-12 (DVD) antibody.

[0046] Figure 8 The image shows the results of the detection of the biological activity of IN-12 bispecific antibody blocking VEGF.

[0047] Figure 9 The figure shows the results of the IN-12 antibody treatment experiment in a mouse glioma model. Detailed Implementation

[0048] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0049] To enhance understanding of the present invention, certain key technical and scientific terms will be clearly defined below. Unless otherwise specified herein, all other technical and scientific terms shall follow their generally accepted and understood meanings within the art to which this invention pertains. It should be noted that the terminology used herein is intended to describe specific embodiments and not to be construed as limiting.

[0050] Vascular endothelial growth factor (VEGF): The VEGF family, especially VEGF-A, is the most important driver of physiological and pathological angiogenesis. In most solid tumors, due to internal hypoxia caused by rapid proliferation, tumor cells highly express hypoxia-inducible factor-1α (HIF-1α), which in turn strongly upregulates the transcription and secretion of VEGF. VEGF binds to its main functional receptor VEGFR2 (KDR), activating a series of downstream signaling pathways such as PI3K-AKT and RAS-MAPK, promoting the proliferation, migration, and survival of vascular endothelial cells and increasing vascular permeability, thereby providing the necessary oxygen and nutrients for tumor growth. Based on its core role, targeting the VEGF / VEGFR pathway has become an important pillar of anti-tumor therapy. Bevacizumab, as the first humanized anti-VEGF-A monoclonal antibody, has been approved for the treatment of various malignant tumors, including colorectal cancer, non-small cell lung cancer, and glioblastoma. However, clinical practice has shown that anti-VEGF monotherapy has significant limitations: First, the duration of efficacy is short. In many tumor types, such as glioblastoma, although bevacizumab can "normalize" tumor angiogenesis, reduce edema, and prolong progression-free survival in the short term, it has little effect on improving overall survival, suggesting that it fails to achieve durable disease control. Second, it induces an aggressive phenotype. Inhibiting VEGF signaling can exacerbate hypoxia within the tumor, potentially activating the invasion and metastasis programs of tumor cells. For example, in glioblastoma, VEGF inhibition can undo its negative regulation of the hepatocyte growth factor receptor (MET) pathway, leading to increased MET phosphorylation levels, thereby enhancing the migration and invasion capabilities of tumor cells. This phenomenon may be related to the tumor's use of existing blood vessels for spread after treatment through "vascular co-selection." Finally, it activates compensatory resistance mechanisms, allowing tumor cells to evade treatment by initiating VEGF-independent angiogenesis pathways.

[0051] Interleukin-8 (IL-8 / CXCL8): IL-8 is an important member of the CXC chemokine family, exerting a wide range of biological functions primarily through binding to G protein-coupled receptors CXCR1 and CXCR2. Traditionally, IL-8 has been considered a neutrophil chemokine, playing a central role in acute inflammation. However, recent studies have revealed the complex and crucial role of IL-8 in tumor progression and immune regulation, making it an emerging therapeutic target. In the tumor microenvironment, IL-8 originates from a wide range of sources, not limited to tumor cells, but also including tumor-associated endothelial cells, macrophages, and fibroblasts. Its main functions are, firstly, to directly promote malignant tumor progression. IL-8 can act directly on tumor cells through autocrine or paracrine mechanisms, activating signaling pathways such as PI3K / AKT and ERK / MAPK, thereby promoting tumor cell proliferation, survival, epithelial-mesenchymal transition (EMT), and invasion and metastasis. In models such as prostate cancer, IL-8 signaling is even associated with androgen receptor expression and functional activation, participating in the development of castration-resistant prostate cancer. Secondly, it drives the formation of an immunosuppressive microenvironment, which is one of the most crucial tumor-promoting mechanisms of IL-8. IL-8 is a chemotactic agent for key "immunosuppressive cells" in the tumor microenvironment. It can efficiently recruit neutrophils, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs) with the M2 phenotype into the tumor site. These cells strongly inhibit the activation and function of effector T cells by secreting inhibitory cytokines such as TGF-β and IL-10, consuming essential amino acids such as arginine, and expressing immune checkpoint molecules such as PD-L1, leading to an "immunely cold" phenotype in the tumor. Clinical analysis confirms that tumors with high IL-8 expression are usually accompanied by less T cell infiltration and more MDSC aggregation. Furthermore, it can also act as a key compensatory factor for angiogenesis; IL-8 itself is a potent pro-angiogenic factor. It can directly stimulate endothelial cell proliferation, migration, and the formation of tubular structures. More importantly, under hypoxic stress caused by VEGF pathway inhibition, IL-8 expression is often significantly upregulated, serving as a core compensatory mechanism to maintain angiogenesis. For example, in ovarian cancer research, when tumor cells upregulate the TCEB2 gene, leading to HIF-1α degradation and VEGF expression inhibition, IL-8 expression compensatorily increases, thereby maintaining endothelial cell survival and angiogenesis. In colon cancer cells, even with HIF-1α deficiency, IL-8 can still be induced to maintain angiogenesis.

[0052] Malignant tumor: A malignant tumor is a type of tumor in which cells grow uncontrollably in the body, are invasive, can invade surrounding tissues, and may spread to other parts of the body through the blood or lymphatic system. Malignant tumors include many types, such as glioblastoma, non-small cell lung cancer, colorectal cancer, ovarian cancer, and prostate cancer. The cells of these tumors have a high proliferative and metastatic capacity.

[0053] Bispecific antibodies are antibodies containing two independent antigen-binding sites, enabling them to simultaneously bind to two different antigens or two different epitopes of the same antigen. These antibodies typically consist of two heavy chain variable regions and two light chain variable regions, providing multiple therapeutic effects by simultaneously targeting two different antigens, thus enabling more complex and effective treatment strategies.

[0054] Complementarity-Determining Regions (CDRs): These are amino acid sequences in the variable regions of an antibody responsible for recognizing and binding antigens. They are divided into CDR1, CDR2, and CDR3 on the light and heavy chains. They are key parts for the antibody to specifically bind to antigens.

[0055] Heavy Chain: Antibody heavy chains are large peptide chains in immunoglobulins, consisting of 450 to 550 amino acid residues. Based on the antigenicity differences in constant regions, they are divided into five classes: μ, γ, α, δ, and ε, which correspond to immunoglobulins IgM, IgG, IgA, IgD, and IgE, respectively.

[0056] Light chain: The light chain of an antibody is one of the two peptide chains that make up the antibody molecule. It combines with the heavy chain to form the complete antibody molecule. The combination of the heavy and light chains determines the specificity of the antibody. Light chains are mainly divided into two types: κ (kappa) and λ (lambda).

[0057] Linker: A linker is a short peptide sequence used in an antibody structure to connect different regions. In bispecific antibodies, linkers are typically used to connect two different variable regions to maintain structural stability and functional integrity.

[0058] Example 1

[0059] 1. Antibody sequence selection and structural design

[0060] This invention provides a bifunctional antibody against IL-8 and VEGF. The anti-VEGF monoclonal antibody sequence uses the already marketed VEGF monoclonal antibody Avastin (Bevacizumab) sequence, while the anti-IL-8 monoclonal antibody sequence uses the expired patented IL-8 monoclonal antibody 10F8 (HuMax IL8) sequence. The bifunctional antibody molecule is structurally designed using a DVD-Ig model, where the variable region of the heavy chain of one antibody is linked to the variable region of the other antibody via linker 1, and then connected to the constant region of the heavy chain to form the heavy chain of the bifunctional antibody molecule. Similarly, the variable region of the light chain of one antibody is linked to the variable region of the light chain of the other antibody via linker 2, and then connected to the constant region of the light chain to form the light chain of the bifunctional antibody molecule.

[0061] The dual-resistance design is shown in Table 1.

[0062] Table 1. Composition Design of IN-12 Heavy and Light Chains

[0063]

[0064] The sequences of the bispecific antibodies are shown in Table 2.

[0065] Table 2. IN-12 Bispecific Antibody Sequence

[0066]

[0067] 2. Antibody preparation

[0068] 2.1 Plasmid Construction and Transient Transformation

[0069] The above gene sequence was synthesized, homologous recombination, transformed, identified, sequenced, aligned, and extracted. The sequence was then constructed into the pcDNA3.4 vector to obtain a protein expression plasmid. The constructed plasmid was transiently transfected into Expi CHO cells. One day before transfection, the Expi CHO cells were cultured to a concentration of (4-5) × 10⁻⁶ cells / mL. 6 Cells / mL, cultured overnight. On the day of transfection, the cell density was (8-10) × 10⁻⁶ cells / mL. 6 Approximately 100 viable cells / mL, with a cell viability of >98%; preheat MetaCell™ CHO-310 medium at 37°C; add plasmid DNA to a 1.5 mL EP tube; add 1 L of medium to a 3 L cell culture flask and incubate at 37°C with 7% CO2; add equal volumes of Electroporation Buffer Part A and Electroporation Buffer Part B to a centrifuge tube and mix by pipetting; take 1.00 × 10⁻⁶ cells / mL. 10Centrifuge cells at 300 g for 5 min. Discard the supernatant after centrifugation, add the prepared electroporation buffer and plasmid, gently pipette to mix, and transfer to an electroporation tube for electroporation. After electroporation, add cells to culture medium and incubate at 37°C with shaking in a 7% CO2 incubator. On the first day after transfection (18-22 h), slowly add the appropriate proportions of MetaCell™ CHO TransFeed and MetaCell™ Titer Enhancer to the cell culture medium while gently shaking the cell culture flask. Transfer the flask to a shaker at 32°C with 7% CO2 and continue culturing. On the fifth day after transfection, add the appropriate proportions of MetaCell™ CHO TransFeed while gently shaking the cell culture flask. Transfer the flask to a shaker at 32°C with 7% CO2 and continue culturing. Collect the supernatant after expression is complete: the viable cell density should be no less than 50% of the maximum cell density, and the cell viability should be no less than 60% to ensure a good protein yield.

[0070] 2.2 Purification

[0071] ① Preparation of chromatography column: Fill an empty Thermo centrifuge column (Pierce™ Centrifuge Columns, 10 mL) with 2 mL of MabSelect SuRe LX packing material, and equilibrate the column with 5 times its volume of equilibration buffer.

[0072] ② Sample loading: Add the sample to the equilibrated chromatography column and collect the eluent. The sample can be loaded repeatedly to increase the binding efficiency.

[0073] ③ Washing: After sample loading, add 5 times the volume of equilibration buffer to wash away loosely bound proteins. Then add another 5 times the volume of washing buffer to remove more proteins and collect the eluent.

[0074] ④ Elution: After washing, add 5 times the volume of eluent and let it stand to flow through the packing material by gravity, then collect the eluent.

[0075] ⑤ Neutralization: After elution, the pH of the sample was adjusted to 5.0 with 0.5M Arg.

[0076] ⑥ Centrifugation: After neutralization, the sample is centrifuged at 3500×g for 3 min using a horizontal rotor, filtered through a 0.22 μm filter, and the sample is collected.

[0077] ⑦ Concentration: Add the neutralized sample to a 50 KD ultrafiltration tube and centrifuge at 3,500 × g for 10 min using a horizontal rotor. After concentration, take 2 μL of the replaced sample and quantify it using a Nano-300.

[0078] ⑧ Sample recovery: After concentration, transfer the sample from the ultrafiltration tube to a centrifuge tube, centrifuge at 3,500×g for 3 min using a horizontal rotor, filter using a 0.22 μm filter, and collect the sample.

[0079] 2.3 Identification

[0080] 2.3.1 SDS-PAGE identification

[0081] ① Preparation of purified sample solution: According to the A280 determination, 1 μg of non-reduced sample was added to 4×LDS loading buffer and iodoacetamide (final concentration 40 mM), and heated in a dry bath at 75℃ for 10 min. 2 μg of reduced sample was added to 4×LDS loading buffer and DTT (final concentration 5 mM), and heated in a dry bath at 100℃ for 10 min.

[0082] ② Electrophoresis: 140 V, 75 min.

[0083] ③ Staining, destaining, and scanning: Gel Coomassie Brilliant Blue staining, followed by destaining and scanning with an EPSON V550 color scanner.

[0084] ④ Calculate purity: Use Image J to calculate the purity of the reduced band, or the purity of the reduced heavy chain plus light chain, according to the peak area normalization method.

[0085] 2.3.2 SEC Authentication

[0086] ① Preparation of mobile phase: Prepare 0.15 M PB + NaCl and adjust the pH to 6.0.

[0087] ② Sample preparation: Dilute the sample concentration to 0.5 mg / mL.

[0088] ③ Column conditions: XBridge BEH 200Å, SEC 3.5 μm, 7.8×300 mm, column temperature set at 20℃, and the detection baseline was stable.

[0089] ④ Parameter settings: Flow rate set to 0.8 mL / min; sample injection volume set to 20 μL; detection wavelength 280 nm, bandwidth 4 nm; reference wavelength 360 nm, bandwidth 100 nm; peak width (response time) > 0.1 min (2 s response time); slit width 4 nm; negative absorbance baseline 100 mAU.

[0090] ⑤ System adaptability criteria: If the purity of the reference Herceptin monomer is greater than 95%, the separation degree between the BSA monomer and dimer is greater than 1.5, and the baseline is stable, then the system adaptability is considered to be passed.

[0091] 2.4 Experimental Results

[0092] The experimental results are shown in Figures 1-2 After one-step purification of the IN-12 antibody expression, SDS-PAGE analysis showed a purity of over 95%, meeting the requirements; SEC-HPLC analysis also showed a purity of over 95%, meeting the expected requirements.

[0093] Example 2. Detection of the binding of IN-12 bispecific antibody molecules to human IL-8 and VEGF

[0094] Enzyme-linked immunosorbent assay (ELISA) was used to detect the binding of IN-12 double antibody molecules to human IL-8 and VEGF. 96-well ELISA plates were coated with recombinant human IL-8-his / VEGF protein (2 μg / ml, diluted in PBS) and incubated overnight at 4°C. After washing, 5% skim milk was added, and the plates were blocked at 37°C for 2 hours. After washing with PBS-0.1% Tween 20, the test sample IN-12 (60 nM initial serial dilution) and positive control were added, and the plates were incubated at 37°C for 1 hour. After washing with PBS-0.1% Tween 20, horseradish peroxidase-labeled goat anti-human IgG (Sigma-Aldrich) was added, and the plates were incubated at 37°C for 1 hour. After thorough washing with PBS-0.1% Tween 20, the plates were developed using a commercial ELISA kit. OD values ​​were then read using a microplate reader (Thermo Scientific).

[0095] Test results as follows Figures 3-4 As shown, IN-12 bispecific antibodies can bind well to human VEGF and IL-8.

[0096] Example 3. Detection of IN-12 antibody affinity constant

[0097] Using a Biacore T200 (Cytiva) affinity assay instrument, the probe Protein A was selected. The antibody test sample was diluted to 80-120 nM with HBS-EP (pH 7.4) at a flow rate of 10 μL / min, and then bound to the Protein A probe. Antigen IL-8 was diluted to 20 nM, 6.6 nM, and 2.2 nM with HBS-EP (pH 7.4), and VEGF was diluted to 50 nM, 10 nM, and 2 nM, with a flow rate of 30 μL / min. The antibody was then bound for 30 seconds, followed by binding with a VEGF probe for 180 seconds, and dissociation for 1800 seconds. The entire reaction was controlled at 25℃. The obtained data were fitted using software to calculate the affinity constant KD value.

[0098] The test results are shown in Tables 3 and 4. Figures 5-6As shown, the affinity constant of the IN-12 antibody with IL-8 is 7.1E-11 M, and the affinity constant with VEGF is 9.6E-10 M.

[0099] Table 3. Results of IN-12 antibody-IL-8 affinity assay

[0100]

[0101] Table 4. Results of IN-12 antibody-VEGF affinity assay

[0102]

[0103] Example 4. IN-12 bispecific antibody can block IL-8 biological activity.

[0104] Before the experiment, Corning Transwell chambers (5µm pore size) were coated with Retrolectin (TAKARA, catalog number: T100A). Retrolectin was added to PBS at a concentration of 20 µg / ml; after mixing, 100 µl was added to each chamber and incubated at 37°C for 2 hours or 4°C overnight. After coating, the chambers were washed once with PBS and then once with complete 1640 medium. They were then allowed to air dry slightly before use (care should be taken not to damage the membrane). Jurkat-CXCR1 / CXCR2 cells were counted, and a cell suspension was prepared using complete 1640 medium at a cell density of 4 × 10⁻⁶ cells / mL. 5 Add 200 μl of cells to each chamber, then add 200 μl to each chamber. Next, add 600 μl of pre-mixed complete 1640 medium containing cytokines or a mixture of cytokines and antibodies to the lower chamber through the gaps in the chamber. Be careful not to generate air bubbles at the bottom of the chamber. If air bubbles are generated, remove the chamber, remove the air bubbles, and then reinsert the chamber. After incubating at 37°C in a CO2 incubator for 4 hours, gently remove the upper chamber, centrifuge the cells in the lower chamber (wash the lower chamber once with PBS to ensure that all cells are aspirated), resuspend in 50 μl of PBS, and count the cells.

[0105] Experimental results are as follows Figure 7 As shown, after the IN-12(DVD) antibody binds to IL-8, the number of cells migrating is significantly reduced, which is significantly different from the control group, thus verifying the biological function of IL-12(DVD) at the cellular level.

[0106] Example 5. IN-12 bispecific antibody can block the biological activity of VEGF.

[0107] 1. Experimental Methods

[0108] ① Cell resuscitation: First, prepare the growth medium required for cell resuscitation (add 56 mL of FBS to 500 mL of DMEM medium and mix well), and preheat it in a 37°C water bath. After verifying that the cell information is correct, quickly thaw the cells in a 37°C water bath. In a biosafety cabinet, add the thawed cells to a 15 mL centrifuge tube, add 9 mL of culture medium, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells in 5 mL of preheated culture medium, and then transfer them to a 10 cm culture dish and incubate in a 37°C, 5% CO2 incubator.

[0109] ② Preparation: Irradiate the biosafety cabinet with ultraviolet light for at least 30 minutes before operation. Wipe your hands and the UV-irradiated biosafety cabinet with 75% alcohol. Preheat the cell culture medium and PBS required for cell passage in a 37°C water bath. Remove the cell culture dish from the 37°C incubator and examine the cells under a microscope for contamination, while simultaneously observing the cell production status.

[0110] ③ Cell digestion: Carefully aspirate the old culture medium from the culture dish, add an appropriate amount of pre-warmed PBS to the culture dish, gently shake to wash the cell surface, and then aspirate the PBS. Add 1 mL of pre-warmed EDTA-Trypsin, and place the culture dish back in a 37℃, 5% CO2 incubator for 30 sl min. After the cells have digested, add 4 mL of pre-warmed growth medium, and transfer the entire cell suspension to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min.

[0111] ④ Cell counting: Dilute the cell pellet with culture medium, and use a pipette to blow the pellet up and down to separate the cell clusters. Separate an appropriate volume for cell counting.

[0112] ⑤ Cell passage: Then, passage the cells to cell culture dishes according to the counting density, following the cell passage requirements. Unless otherwise specified, generally culture for 48 hours at a density of 4 × 10⁶ cells / mL. 5 cells / mL; cultured for 72 hours at a density of 2×10⁻⁶ cells / mL; 5 cells / mL.

[0113] ⑥ Activity Assay: In a biosafety cabinet, add 5.05 mL of FBS to 500 mL of DMEM medium and mix thoroughly. Adjust the target cell density to 2 × 10⁻⁵ cells using Assaymedium via cell passage. 5Cells / mL, 50 μL / well for plating. First, control sample IN-11 and test sample IN-12 were diluted with Assay Medium to obtain final concentrations of 0.4 mg / mL and 0.3 mg / mL, respectively. Then, serial dilutions were performed as required, for a total of 11 dilutions. VEGF was diluted to 2 ng / mL, and the two were mixed and incubated at room temperature for 1 hour. Then, the mixture was combined with 293T-VEGFR2-Luc cells, with two replicates for each concentration gradient. The 96-well plate was incubated for 6 hours. After incubation, the 96-well plate was placed at room temperature for 20-30 minutes to equilibrate. All test reagents were equilibrated to room temperature first. 100 μL of Bio-Light Luciferase Assay System was added to each well, and the plate was incubated at room temperature in the dark for 10 minutes. After 10 minutes of color development, the Luminescence signal value was read using a microplate reader, and the data were processed using a four-parameter fitting method.

[0114] 2. Experimental Results

[0115] The results are as follows Figure 8 As shown, the IN-12 bispecific antibody molecule blocks the binding of VEGF to the VEGF receptor on the surface of the 293T-VEGFR2-Luc cell line and can produce a dose-dependent concentration-dependent effect, with an IC50 of 88.94 ng / mL.

[0116] Example 6. Drug efficacy detection in IN-12 bispecific antibody mouse model

[0117] 1. Experimental Methods

[0118] ① Remove the frozen tumor cells (GL261-luc-IL8-mcherry) from liquid nitrogen, thaw them in a 37°C water bath, add 1 mL of fresh DMEM complete medium, then transfer the cells to a 15 mL centrifuge tube, centrifuge at 500 g for 3 min, discard the supernatant, add 1 mL of fresh DMEM complete medium to resuspend the cells, seed them into 10 cm cell culture dishes, and culture at 37°C.

[0119] ② Once the cell density reached approximately 90%, the cells were passaged, and well-grown tumor cells (GL261-luc-IL8-mcherry) were digested. The cell concentration was measured to be 1.73 × 10⁻⁶. 7 cells / mL, add PBS to adjust cell concentration to 1×10⁻⁶. 6 100 μL of cell suspension was injected into each mouse after anesthesia. The right abdominal area was prepared and disinfected with povidone-iodine. The needle was inserted into the lower abdomen of the mouse, and 100 μL of cell suspension was injected into each mouse. The size of the tumor was measured at approximately day 12, and the tumor formation was observed.

[0120] ③ Based on the size of the tumors in the mice, the mice were divided into 4 groups: control group, Avastin group, 10F8 group, and IN-12 group. The mice were administered 10 mg / kg once every 3 days. The tumor size and mouse weight were measured before each administration.

[0121] 2. Experimental Results

[0122] The results are as follows Figure 9 As shown, the IN-12 bispecific antibody effectively inhibited tumor production in mice, and its tumor-inhibiting effect was superior to that of the individual monoclonal antibody groups.

Claims

1. A bispecific antibody against IL-8 and VEGF, characterized in that, include: A. First antigen-binding region, which has IL-8 binding activity; The first antigen-binding region includes a first heavy chain variable region and a first light chain variable region. The first light chain variable region includes amino acid sequences VL1-CDR1, VL1-CDR2, and VL1-CDR3 as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively. The first heavy chain variable region includes amino acid sequences VH1-CDR1, VH1-CDR2, and VH1-CDR3 as shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively. B. Second antigen-binding region, which has VEGF binding activity; The second antigen-binding region includes a second heavy chain variable region and a second light chain variable region. The second light chain variable region includes amino acid sequences VL2-CDR1, VL2-CDR2, and VL2-CDR3 as shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9, respectively. The second heavy chain variable region includes amino acid sequences VH2-CDR1, VH2-CDR2, and VH2-CDR3 as shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively.

2. The bispecific antibody according to claim 1, characterized in that, The first heavy chain variable region is connected to the second heavy chain variable region through linker1; the first light chain variable region is connected to the second light chain variable region through linker2.

3. The bispecific antibody according to claim 2, characterized in that, The amino acid sequence of linker1 is shown in SEQ ID NO.21, and the amino acid sequence of linker2 is shown in SEQ ID NO.

22.

4. The bispecific antibody according to claim 1, characterized in that, The heavy chain variable region of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO.13, and the light chain variable region of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO.

14.

5. The bispecific antibody according to claim 1, characterized in that, The heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 15, and the light chain amino acid sequence is shown in SEQ ID NO:

16.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the bispecific antibody as described in any one of claims 1 to 5.

7. The nucleic acid molecule according to claim 6, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the bispecific antibody is shown in SEQ ID NO.17, and the nucleotide sequence encoding the light chain variable region of the bispecific antibody is shown in SEQ ID NO.

18.

8. The nucleic acid molecule according to claim 6, characterized in that, The nucleotide sequence encoding the heavy chain of the bispecific antibody is shown in SEQ ID NO.19, and the nucleotide sequence encoding the light chain of the bispecific antibody is shown in SEQ ID NO.

20.

9. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule according to any one of claims 6 to 8.

10. A recombinant cell, characterized in that, The recombinant cells comprise the bispecific antibody as described in any one of claims 1 to 5, the nucleic acid molecule as described in any one of claims 6 to 8, and / or the expression vector as described in claim 9.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the bispecific antibody according to any one of claims 1 to 5, the nucleic acid molecule according to any one of claims 6 to 8, the expression vector according to claim 9, and / or the recombinant cell according to claim 10, as well as pharmaceutically acceptable excipients.

12. A reagent kit, characterized in that, The kit comprises the bispecific antibody according to any one of claims 1 to 5, the nucleic acid molecule according to any one of claims 6 to 8, the expression vector according to claim 9, and / or the recombinant cells according to claim 10.

13. The use of the bispecific antibody according to any one of claims 1 to 5, the nucleic acid molecule according to any one of claims 6 to 8, the expression vector according to claim 9, and / or the recombinant cell according to claim 10 in the preparation of reagents and / or kits for detecting IL-8 and / or VEGF.

14. The use of the bispecific antibody according to any one of claims 1 to 5, the nucleic acid molecule according to any one of claims 6 to 8, the expression vector according to claim 9, and / or the recombinant cell according to claim 10, and / or the pharmaceutical composition according to claim 11 in the preparation of a medicament for the prevention and / or treatment of diseases associated with abnormal expression or activity of IL-8 and / or VEGF.

15. The application according to claim 14, characterized in that, The diseases associated with abnormal expression or activity of IL-8 and / or VEGF include malignant tumors; the malignant tumors include lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, kidney tumors, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer and / or leukemia.

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