Targeted interferon fusion protein and use thereof

Abstract

The application discloses a kind of targeted interferon fusion protein and its application.Belong to fusion protein technical field.The fusion protein is connected into hetero-tetramer by two class heavy chains and two class light chains by disulfide bond, and the fusion protein realizes multiple functions by single molecule: by targeting tumor endothelial cell marker 8 (TEM8) binding tumor vascular endothelial cell, inhibiting neovascularization;By targeting TEM8 positive tumor stromal cell, destroy tumor immune barrier and remove immunosuppression;By targeting TEM8 positive tumor cell, directly inhibit its invasion and metastasis;By I, III type interferon regulation innate immunity and adaptive immunity, reshape tumor local microenvironment.The application realizes multi-dimensional synergistic precise targeted tumor treatment, with the advantages of high efficiency and low toxicity, and can be used for preparing the drug for treating TEM8 positive solid tumor and other related diseases.

Description

Technical Field

[0001] This invention relates to the field of fusion protein technology, and more specifically to a targeted interferon fusion protein and its applications. Background Technology

[0002] Tumor endothelial marker 8 (TEM8) is a cell surface protein that plays a crucial role in tumor angiogenesis. Its specific high expression in tumor vascular endothelial cells, tumor stromal cells, and some tumor cells makes it a highly promising target for anti-tumor drugs. TEM8 was initially discovered in colorectal cancer tumor vascular endothelial cells. Subsequent studies have confirmed that it is significantly upregulated in tumor vascular endothelial cells of various tumor types, tumor-associated fibroblasts, and some tumor cells, while its expression is extremely low in normal adult tissues and physiological angiogenesis (such as wound healing). This specificity makes TEM8 an ideal "tumor homing" signal, laying the molecular foundation for the development of highly selective anti-tumor drugs.

[0003] Interferons are a class of cytokines with antiviral, antiproliferative, and immunomodulatory activities, among which type I and type III interferons have attracted much attention in tumor immunotherapy. However, the direct application of type I interferons (such as IFNα) in current technologies has significant drawbacks: although they have shown some efficacy in diseases such as hairy cell leukemia and melanoma, their pleiotropic effects lead to severe systemic toxicity, which seriously limits their widespread clinical application. Although type III interferons exhibit better local action characteristics through different receptor systems, their antitumor efficacy when used alone is limited. To overcome these bottlenecks, some studies have attempted to fuse interferons with targeting antibodies using genetic engineering techniques, aiming to precisely deliver interferons to the tumor microenvironment. However, existing fusion protein designs mostly carry only a single type of interferon, and the combination of the targeting unit and the interferon functional unit is limited, making it difficult to achieve synergistic effects of multiple immunomodulatory and antitumor effects. In addition, there is currently a lack of fusion proteins that can simultaneously target TEM8 and integrate the dual functions of type I and type III interferons.

[0004] Therefore, how to develop a fusion protein that can precisely target TEM8, synergistically exert the anti-tumor and immunomodulatory functions of type I and type III interferon, and effectively reduce systemic toxic side effects is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a targeted interferon fusion protein and its application, achieving multiple synergistic effects through a single molecule, offering new ideas and tools to overcome current challenges in the treatment of solid tumors. Specifically, it employs a multiple mechanism of "TEM8 targeting tumor endothelium, tumor stroma, and tumor cells + interferon induction," making it suitable for patients with TEM8-positive solid tumors while protecting them from the toxic side effects of interferon.

[0006] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0007] The primary objective of this application is to provide: a targeted interferon fusion protein, wherein the fusion protein comprises a heterotetrameric structure consisting of two heavy-like chains and two light-like chains linked by disulfide bonds, wherein: The heavy chain-like structure is a polypeptide chain with an antibody heavy chain structure and a type I interferon functional domain fused together. It includes: the heavy chain variable region of the tumor endothelial cell marker 8 targeting antibody, the human IgG1 antibody constant region, and the type I interferon co-sequence. The light chain is a polypeptide chain with an antibody light chain structure and a type III interferon functional domain, comprising: a light chain variable region of the tumor endothelial cell marker 8 targeting antibody, a human antibody light chain constant region, and a type III interferon λ1 sequence.

[0008] As a preferred technical solution, the amino acid sequences of the complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region of the tumor endothelial cell marker 8 targeting antibody are shown below: SYAIS, SEQ ID NO. 3; TTNYAGIIQKQGFPIFG, SEQ ID NO. 4; DTDYMFDY, SEQ ID NO. 5.

[0009] As a preferred technical solution, the amino acid sequences of the complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region of the tumor endothelial cell marker 8 targeting antibody are shown below: QGDNLRDFYAS, SEQ ID NO. 6; GKNRRPS, SEQ ID NO. 7; SSNKHRDSVV, SEQ ID NO. 8.

[0010] As a preferred technical solution, the heavy chain variable region of the tumor endothelial cell marker 8 targeting antibody further includes FR-H1, FR-H2, FR-H3, and FR-H4 framework regions, whose amino acid sequences are shown below: QVQLVQSGAEVKKPGTSVKVSCKVPGYTFS, SEQ ID NO. 9; WVRQAPGQGLEWMG, SEQ ID NO. 10; RVTITGDESTSTVYMELSSLRSEDTAVYYCAR, SEQ ID NO. 11; WGQGTLVTVSS, SEQ ID NO. 12.

[0011] The light chain variable region of the tumor endothelial cell marker 8 targeting antibody also includes FR-L1, FR-L2, FR-L3, and FR-L4 framework regions, whose amino acid sequences are shown below: SSELTQDPVVSVALGETVSITC, SEQ ID NO. 13; WYQQKPGQAPLLVMY, SEQ ID NO. 14; GIPDRFSGSTSGNTLSLTITGAQAEDEADYYC, SEQ ID NO. 15.

[0012] FGGGTKVTVL, SEQ ID NO. 16.

[0013] As a preferred technical solution, the amino acid sequence of the heavy chain variable region of the tumor endothelial cell marker 8 targeting antibody is as follows: SEQ ID NO. 17.

[0014] The amino acid sequence of the light chain variable region of the tumor endothelial cell marker 8 targeting antibody is shown below: SSELTQDPVVSVALGETVSITCQGDNLRDFYASWYQQKPGQAPLLVMYGKNRRPSGIPDRFSGSTSGNTLSLTITGAQAEDEADYYCSSNKHRDSVVFGGGTKVTVL, SEQ ID NO. 18.

[0015] As a preferred technical solution, the amino acid sequence of the constant region of the human IgG1 antibody is shown below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV SEQ ID NO. 19.

[0016] The amino acid sequence of the type I interferon co-sequence is shown below: SEQ ID NO. 20.

[0017] The amino acid sequence of the constant region of the human antibody light chain is shown below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, SEQ ID NO. 21.

[0018] The amino acid sequence of the type III interferon λ1 is shown below: KPTTTGKGCHIGRFKSLSPQELASFKKARDALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAELALTLKVLEAAAGPALEDVLDQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQEAPKKESAGCLEASVTFNLFRLLTRDLKYVADGNLCLRTSTHPES, SEQ ID NO. 22.

[0019] The constant region of the human IgG1 antibody and the co-sequence of type I interferon are linked by a flexible linker peptide 1, the amino acid sequence of which is shown below: GGGGSGGGGSGGGGS, SEQ ID NO. 23.

[0020] The human antibody light chain constant region is linked to type III interferon λ1 via a flexible linker peptide 2, the amino acid sequence of which is shown below: GGGGSGGGGSGGGGSGGGGS, SEQ ID NO. 24.

[0021] As a preferred technical solution, the polypeptide chain of the fusion protein, from the N-terminus to the C-terminus, consists of: the variable region of the heavy chain of the tumor endothelial cell marker 8 targeting antibody - the constant region of the human IgG1 antibody - the flexible linker peptide 1 - the type I interferon co-sequence, and its amino acid sequence is shown below: , SEQ ID NO.1.

[0022] The polypeptide chain of the light chain-like structure, from N-terminus to C-terminus, consists of: the variable region of the light chain of the tumor endothelial cell marker 8 targeting antibody, the constant region of the human antibody light chain, and the flexible linker peptide 2-type III interferon λ1, with the amino acid sequence shown below: SSELTQDPVVSVALGETVSITCQGDNLRDFYASWYQQKPGQAPLLVMYGKNRRPSGIPDRFSGSTSGNTLSLTITGAQAEDEADYYCSSNKHRDSVVFGGGTK VTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGECGGGGSGGGGSGGGGSGGGGSKPTTTGKGCHIGRFKSLSPQELASFKKARDALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAELALTLKVLEAAAGPALEDVLDQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQEAPKKESAGCLEASVTFNLFRLLTRDLKYVADGNLCLRTSTHPES, SEQ ID NO. 2.

[0023] Another object of this application is to provide: a nucleic acid molecule encoding the said fusion protein.

[0024] Another object of this application is to provide: an expression vector or host cell comprising the nucleic acid molecule.

[0025] Another object of this application is to provide a method for preparing the fusion protein, comprising culturing the host cells under suitable expression conditions and recovering the fusion protein from the culture.

[0026] Another object of this application is to provide the use of the fusion protein in the preparation of a medicament for treating tumor endothelial cell marker 8 positive solid tumors.

[0027] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: The technical solution of this application integrates the functions of inhibiting tumor cell proliferation, survival, migration, inhibiting angiogenesis, and targeted delivery of interferon to kill tumor cells into a single molecule, achieving multiple synergistic effects: (1) Blocking the uPA-TEM8 signaling pathway directly inhibits the invasion and metastasis of tumor cells; (2) It inhibits TEM8-mediated cell-matrix adhesion and angiogenesis. Normal blood vessels are minimally affected because TEM8 expression is extremely low. (3) Dual activation of interferons: Type I interferons activate innate immunity, promote antigen presentation, and activate natural killer cells and CD8+ T cells. Type III interferons play a key role in the immune defense of the epithelial barrier, and work synergistically with type I interferons to enhance antigen presentation and T cell activation. Attached Figure Description

[0028] 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 embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0029] Figure 1 Here is a schematic diagram of the structure of the fusion protein 259 / 260 of this invention.

[0030] Figure 2 The image shows the enzyme digestion identification results of the fusion protein expression plasmid in Example 1 of this invention. The left side is a schematic diagram of the nucleic acid marker bands. Lanes 1 and 4 are markers, lane 2 is the pcDNA3.4-259 band, lane 3 is the XbaI and EcoRV double digestion band of pcDNA3.4-259, lane 5 is the pcDNA3.4-260 band, and lane 6 is the XbaI and EcoRV double digestion band of pcDNA3.4-260.

[0031] Figure 3 The diagram shows the expression and purification results of the fusion protein prepared in Example 1 of this invention. In the diagram, A is the correspondence between elution volume and OD280 and pH; B is the SDS-PAGE result, with the left side showing the protein ladder diagram, lane 1 showing the protein ladder band, lane 2 showing the denatured and reduced sample bands of fusion protein 259 / 260, where the upper side is the heavy chain-like band and the lower side is the light chain-like band, and lane 3 showing the non-denatured and non-reduced sample bands of fusion protein 259 / 260.

[0032] Figure 4 The image shows the ELISA results of the binding of fusion protein 259 / 260 to recombinant human TEM8, IFNAR2, and IFNLR1 proteins in Example 2 of this invention.

[0033] Figure 5 Figure 3 shows the results of the fusion protein inhibiting HeLa cell proliferation in Example 3 of this invention.

[0034] Figure 6Figure A shows the results of transcription of antiviral-related genes in HepG2 cells stimulated by the fusion protein in Example 4 of this invention; Figure A is a bar chart of the mean optical density of RT-PCR, and the optical density is derived from Figure B; Figure B is a nucleic acid gel image after RT-PCR.

[0035] Figure 7 Here is a flow cytometry result of the binding of the fusion protein to tumor cells A549 and SK-BR-3 in Example 5 of this invention.

[0036] Figure 8 The image shows the ADCC results of NK92 cell-mediated killing of A549 and SK-BR-3 cells dependent on the fusion protein in Example 6 of this invention. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] In the examples, all raw materials not mentioned are commercially available, and all experimental methods not mentioned are conventional experimental methods, which will not be described in detail here.

[0039] Example 1 Plasmid construction, protein expression and purification (its structural diagram is shown in Figure 1) Figure 1 (As shown) (1) The aforementioned SEQ ID No.1 (number 259) and SEQ ID No.2 (number 260) sequences were synthesized by gene (with restriction endonuclease XbaI and EcoRV restriction sites added to both ends of the sequences), and then constructed into the pcDNA3.4 plasmid expressing the fusion protein using the pcDNA3.4-TOPOTAcloning kit (purchased from Invitrogen (Shanghai) Trading Co., Ltd.), that is, pcDNA3.4-259 and pcDNA3.4-260.

[0040] (2) The plasmid obtained in the previous step was transformed into Escherichia coli TOP10 (purchased from Beijing Qingke Biotechnology Co., Ltd., DLC112), amplified by shaking on LB medium, extracted using a plasmid large-scale extraction kit (purchased from Beijing Jumei Biotechnology Co., Ltd., MF-985-01), and then digested with restriction endonucleases XbaI and EcoRV. The results were then analyzed by gel electrophoresis. See below for details. Figure 2From left to right, lanes 1 and 4 are marker bands, lane 2 is the pcDNA3.4-259 plasmid band, lane 3 is the XbaI and EcoRV double digestion band of pcDNA3.4-259 plasmid, lane 5 is the pcDNA3.4-260 plasmid band, and lane 6 is the XbaI and EcoRV double digestion band of pcDNA3.4-260 plasmid. The upper side of the double digestion bands in lanes 3 and 6 is the linearized band of the empty plasmid, and the lower side is the position of the target band, which is consistent with the designed size and position.

[0041] (3) The expression plasmid was transfected into 293F cells using PEI transfection reagent for protein expression.

[0042] (4) 120 h after transfection, the supernatant was harvested, centrifuged and filtered. The protein A affinity column (purchased from Cytiva) was treated with 5 column volumes of equilibration buffer (5.6 mM NaH2PO4, 14.4 mM Na2HPO4, 0.15 M NaCl, pH 7.2). The supernatant was loaded onto the column. After washing, contaminating proteins with buffer (5.6 mM NaH2PO4, 14.4 mM Na2HPO4, 0.5 M NaCl, pH 7.2) to baseline. Proteins were then eluted with 50 mM citrate / sodium citrate buffer (pH 3.2). Samples with a concentration of 80 mAu or higher were collected. The pH was adjusted to 7.0 with 1 M Tris-Cl (pH 8.0), concentrated in a concentration tube, filtered for sterilization, and stored at 4°C. 3 μg of the sample was then subjected to SDS-PAGE staining. Results are shown below. Figure 3 : Where A is the chromatography diagram, which is a graph showing the correspondence between elution volume and OD280 and pH; Figure 3 Lane B shows the SDS-PAGE results, where lane 1 is the protein ladder plot and lane 2 shows the bands of purified protein after denaturation and reduction. The protein band positions are consistent with the designed size and position (after expression in cells, they form a Y-shaped heterotetramer structure, becoming a protein; under denaturation and reduction conditions, they form monomers). Figure 3 (As shown in section B), lane 3 represents the non-denatured, non-reduced sample band of the purified protein. The protein obtained in this step is the fusion protein 259 / 260.

[0043] Example 2 Antigen-antibody binding ELISA assay (1) Commercial recombinant human TEM8, IFNAR2 (purchased from MedChemExpress, HY-P7521A, HY-P72613), and IFNLR1 (purchased from Beijing Baipusaisi Biotechnology Co., Ltd., ILR-H52H9) proteins were diluted with pH 9.6 NaHCO3 coating buffer, plated on ELISA plates, 100 ng / well, and incubated overnight at 4°C.

[0044] (2) Wash the ELISA plate coated yesterday three times with PBS.

[0045] (3) Block with PBST containing 3% BSA, room temperature, 30 min.

[0046] (4) Dilute the purified fusion protein 259 / 260 and control antibody (control antibody Erbitux (Ctrl Ab, purchased from Merck Serono Ltd., Erbitux®) with PBST containing 3% BSA. The initial concentration was 100 μg / mL, and the concentration was serially diluted 10 times to 0.001 μg / mL for a total of 6 concentrations. The solutions were added to ELISA plates and incubated at room temperature for 30 min.

[0047] (5) Wash 3 times with PBST, add 100 μL of mouse anti-human HRP secondary antibody (1:5000, purchased from Nanjing Genscript Biotech Co., Ltd., A01854) to each well, and incubate at room temperature for 30 min.

[0048] (6) Wash 3 times with PBST, add 50 μL of TMB colorimetric solution to each well, and incubate for 5 min.

[0049] (7) Add 50 μL of 1 M HCl to each well and detect OD using a microplate reader. 450 Absorbance. Results are as follows: Figure 4 As shown.

[0050] The results show that the fusion protein 259 / 260 has binding activity with the three target proteins TEM8, IFNAR2, and IFNLR1 in a dose-dependent manner, indicating that the expressed and purified fusion protein has the TEM8 targeting antibody and type I and type III interferon domains; while the control protein does not have binding activity with TEM8, IFNAR2, and IFNLR1.

[0051] Example 3 HeLa proliferation inhibition experiment (1) Cell plating HeLa cells in logarithmic growth phase were digested, resuspended in culture medium, and their density was adjusted to 5 × 10⁶ cells / mL. 4Add 100 μL of resuspended cell suspension to each well of a 96-well plate (i.e., 5000 cells / well; add PBS to the blank group and surrounding wells to prevent evaporation), and incubate overnight at 37°C with 5% CO2.

[0052] (2) Processing Fusion proteins 259 / 260 and IFNα2a (purchased from 3SBio Co., Ltd., Interfen®) were serially diluted with serum-free medium to set four concentrations (0.01, 0.1, 1, 10 μg / mL). The original liquid in the well plate was aspirated, and 100 μL of protein dilution was added to each well (as shown in Table 1 below; serum-free medium was added to the blank group and the control group). The plates were incubated at 37°C for 72 h.

[0053] Table 1. Sample Layout for 96-well Plate

[0054] In the table, 259 / 260-10 represents adding 10 μg / mL of 259 / 260 diluent, and so on; IFNα2a-10 represents adding 10 μg / mL of IFNα2a diluent, and so on.

[0055] (3) CCK8 detection Add 10 μL of CCK-8 reagent to each well (protect from light), incubate at 37°C for 1 h in the dark, and measure the absorbance (OD) at 450 nm using a microplate reader. 450 Cell viability is calculated using the following formula: Cell viability (%) = [OD (experimental) - OD (blank)] / [OD (control) - OD (blank)] × 100.

[0056] The results are as follows Figure 5 As shown, all four concentrations of 259 / 260 inhibited HeLa cell proliferation in a concentration-dependent manner; the four concentrations of the IFNα2a positive control group showed positive characteristics. Interferon is known to have antitumor activity and can inhibit the proliferation of cervical cancer HeLa cells. The results indicate that 259 / 260 significantly inhibits HeLa cell proliferation, demonstrating the presence of an interferon-active functional domain.

[0057] Example 4 HepG2 induction experiment (1) Cell plating HepG2 cells in logarithmic growth phase were digested, cell counting was performed using trypan blue, and the density was adjusted to 23. 10^5 cells / mL were seeded into plates, and 1 mL of cell suspension was added to each well of a 12-well plate. The plates were then incubated overnight at 37°C with 5% CO2.

[0058] (2) Cell treatment The fusion proteins 259 / 260 were diluted to 1000 and 100 ng / mL using serum-free medium, and IFNα2a was diluted to 100 and 10 ng / mL. The original liquid in the well plate was discarded, and 1.5 mL of dilution solution was added to each well (as shown in Table 2 below; serum-free medium was added to the control group). The plates were incubated at 37°C for 1 h.

[0059] Table 2. Layout of 12-well plate samples

[0060] In the table, 259 / 260-1000 represents adding 1000 ng / mL of 259 / 260 diluent, and so on; IFNα2a-100 represents adding 100 ng / mL of IFNα2a diluent, and so on.

[0061] (3) RNA was extracted, reverse transcribed, and then RT-PCR was performed to detect the transcription levels of MXA, OAS, and GAPDH (internal reference gene) mRNA. The primer sequences are shown in Table 3 below.

[0062] Table 3 RT-PCR Primer Sequences

[0063] The results are as follows Figure 6 As shown, both concentrations of 259 / 260 induced upregulation of MXA and OAS gene mRNA transcription levels; the IFNα2a positive control showed positive characteristics at both concentrations. The results indicate that 259 / 260 can effectively induce the transcriptional expression of antiviral genes in HepG2 cells and possesses interferon functional activity.

[0064] Example 5 Combined with cell flow cytometry experiments (1) Take 1×10⁻⁶ A549 (human lung cancer cells) and SK-BR-3 (human breast cancer cells) in the logarithmic growth phase respectively. 6 Centrifuge at 300 g for 5 min and discard the supernatant.

[0065] (2) Resuspend in 500 μL of physiological saline, centrifuge, and wash twice.

[0066] (3) Add 50 μL of fusion protein 259 / 260 with a concentration of 40 μg / mL and incubate at room temperature for 30 min.

[0067] (4) Centrifuge and discard the supernatant, resuspend in 500 μL of physiological saline, centrifuge, and wash twice.

[0068] (5) Add 50 μL of diluted goat anti-human AF488 secondary antibody (1:1000, goat anti-human secondary antibody coupled with fluorescent dye AF488, purchased from Startech Biotechnology Co., Ltd., S0B4057), and incubate at room temperature for 30 min.

[0069] (6) Resuspend in 500 μL of physiological saline, centrifuge, and wash twice.

[0070] (7) Resuspend in 500 μL of physiological saline and analyze by flow cytometry. Results are as follows: Figure 7 As shown.

[0071] Results analysis: Figure 7 The left side of the image shows the results for A549 cells, and the right side shows the results for SK-BR-3 cells. In the image, Ctrl represents the control signal peak (cells without primary antibody), and 259 / 260 represents the fusion protein's 259 / 260 binding signal peak. A rightward shift indicates a positive result, indicating binding. The results show that this fusion protein can bind to A549 and SK-BR-3 cells that express the corresponding antigen in situ, exhibiting binding activity under physiological conditions.

[0072] Example 6 Antibody-dependent cell-mediated cytotoxicity (ADCC) activity assay (1) A549 and SK-BR-3 (target cells) in the logarithmic growth phase were digested, counted, and their density was adjusted to 1×10⁻⁶. 5 Cells / mL, seeded into 96-well plates, 50 μL / well, i.e. 5000 cells / well, and incubated overnight in an incubator.

[0073] (2) Dilute the purified fusion proteins 259 / 260 and control proteins 37 / 38 (control antibodies constructed and expressed by our company, which mediate the ADCC effect of NK cells against SK-BR-3 and A549 cells, and have been patented, CN121930366A) with culture medium. The initial concentration is 10 μg / mL, and the concentration is serially diluted 10 times to 0.1 μg / mL, for a total of 3 concentrations, namely 10, 1 and 0.1 μg / mL.

[0074] (3) Discard the culture medium in the 96-well plate, and add 50 μL of the antibody diluted in the previous step, blank culture medium, etc. according to the sample well settings (see Table 4 for details). Incubate in an incubator for 30 min to form a protein-target cell complex.

[0075] (4) Dilute effector cells (NK92) to 1×10 6 cells / mL (experimental setting effector-to-target ratio of 10:1), add 50 μL of effector cells, blank culture medium, etc. according to the sample well settings, and incubate in an incubator for 5 h.

[0076] Table 4. Experimental layout of 96-well plate for NK92 against A549 and SK-BR-3.

[0077] Note: Background blank: Contains culture medium; used to subtract low background absorbance values ​​from control and sample wells. Spontaneous effect: contains culture medium and effector cells; Lysis buffer control: containing culture medium, add 10 μL Lysis Solution to each well; used to subtract high background absorbance values ​​from the control. Spontaneous target cell release: Contains target cells and culture medium, without lysis treatment; used to measure spontaneous LDH release from untreated normal cells. Maximum target cell concentration: 10 μL Lysis Solution is added to each well containing target cells and culture medium; this is used to determine the maximum release of LDH from cells.

[0078] (5) After culturing for 4.5 h, add 10 μL of Lysis Solution from the LDH detection kit (purchased from MedChemExpress, HY-K1090) according to the sample well settings, and continue culturing for 5 h.

[0079] (6) Take out the 96-well plate, aspirate 50 μL of culture medium into each well and add 50 μL of Working Solution from the LDH detection kit to each well. Incubate in the dark on a shaker for 30 min.

[0080] (7) Add Stop Solution from the LDH detection kit to each well of the aforementioned 96-well plate, and use a microplate reader to detect ABS: OD. 490 .

[0081] (8) Calculate cytotoxicity according to the cytotoxicity calculation formula.

[0082] ADCC experimental group = ABS (experimental group) - ABS (background blank); Effector cell spontaneous release = ABS (spontaneous effector release) - ABS (background blank); Spontaneous release from target cells = ABS (spontaneous release from target cells) - ABS (background blank); Maximum release from target cells = ABS (maximum target cell release) - ABS (lysate control); Cytotoxicity (%) = (ADCC experimental group - spontaneous release from effector cells - spontaneous release from target cells) / (maximum release from target cells - spontaneous release from target cells) × 100%.

[0083] The results are as follows Figure 8 As shown.

[0084] The results show that the fusion proteins 259 / 260 and the positive controls 37 / 38 can induce antibody-dependent NK cell-mediated cytotoxicity, i.e., they have ADCC activity.

[0085] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0086] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A targeted interferon fusion protein, characterized in that, The fusion protein is a heterotetrameric structure composed of two heavy-like chains and two light-like chains linked by disulfide bonds, wherein: The heavy chain-like structure is a polypeptide chain with an antibody heavy chain structure and a type I interferon functional domain fused together. It includes: the heavy chain variable region of the tumor endothelial cell marker 8 targeting antibody, the human IgG1 antibody constant region, and the type I interferon co-sequence. The light chain is a polypeptide chain with an antibody light chain structure and a type III interferon functional domain, comprising: a light chain variable region of the tumor endothelial cell marker 8 targeting antibody, a human antibody light chain constant region, and a type III interferon λ1 sequence.

2. The fusion protein of claim 1, wherein, The amino acid sequences of the complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region of the tumor endothelial cell marker 8 targeting antibody are shown in SEQ ID NO. 3-5, respectively. The amino acid sequences of the complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region of the tumor endothelial cell marker 8 targeting antibody are shown in SEQ ID NO. 6-8, respectively.

3. The fusion protein of claim 1, wherein, The heavy chain variable region of the tumor endothelial cell marker 8 targeting antibody also includes FR-H1, FR-H2, FR-H3, and FR-H4 framework regions, whose amino acid sequences are shown in SEQ ID NO. 9-12, respectively. The light chain variable region of the tumor endothelial cell marker 8 targeting antibody also includes FR-L1, FR-L2, FR-L3, and FR-L4 framework regions, whose amino acid sequences are shown in SEQ ID NO. 13-16, respectively.

4. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the tumor endothelial cell marker 8 targeting antibody is shown in SEQ ID NO. 17; The amino acid sequence of the light chain variable region of the tumor endothelial cell marker 8 targeting antibody is shown in SEQ ID NO.

18.

5. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the constant region of the human IgG1 antibody is shown in SEQ ID NO. 19; The amino acid sequence of the type I interferon co-sequence is shown in SEQ ID NO. 20; The amino acid sequence of the constant region of the human antibody light chain is shown in SEQ ID NO. 21; The amino acid sequence of the type III interferon λ1 is shown in SEQ ID NO. 22; The constant region of the human IgG1 antibody and the co-sequence of type I interferon are linked by a flexible linker peptide 1, the amino acid sequence of which is shown in SEQ ID NO. 23; The human antibody light chain constant region is linked to type III interferon λ1 via a flexible linker peptide 2, the amino acid sequence of which is shown in SEQ ID NO.

24.

6. The fusion protein according to any one of claims 1-5, characterized in that, The polypeptide chain of the heavy chain, from N-terminus to C-terminus, consists of: the variable region of the heavy chain of the tumor endothelial cell marker 8 targeting antibody - the constant region of the human IgG1 antibody - the flexible linker peptide 1 - the type I interferon cosequence, and its amino acid sequence is shown in SEQ ID NO. 1; The polypeptide chain of the light chain-like structure, from N-terminus to C-terminus, consists of: the variable region of the light chain of the tumor endothelial cell marker 8 targeting antibody, the constant region of the human antibody light chain, and the flexible linker peptide 2-type III interferon λ1, with the amino acid sequence shown in SEQ ID NO.

2.

7. A nucleic acid molecule encoding the fusion protein as described in any one of claims 1-6.

8. An expression vector or host cell comprising the nucleic acid molecule as described in claim 7.

9. A method for preparing the fusion protein according to any one of claims 1-6, comprising culturing the host cell as described in claim 8 under conditions suitable for expression, and recovering the fusion protein from the culture.

10. Use of the fusion protein according to any one of claims 1-6 in the preparation of a medicament for treating tumor endothelial cell marker 8 positive solid tumors.

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