Fusion protein, its encoding gene, recombinant vector and their application

Abstract

The present invention relates to the field of biomedicine, and discloses a fusion protein, a gene encoding the fusion protein, a recombinant vector, and their applications. The fusion protein provided by the present invention can specifically bind to NK cells in the body and guide them to efficiently kill cancer cells expressing IL13Rα2, and has the advantages of outstanding killing effect on cancer cells, low treatment cost, low risk of side effects, etc.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a fusion protein and its encoding gene and a recombinant vector as well as their application. Background Art

[0002] In recent years, biomacromolecule drugs have made great breakthroughs in the treatment of malignant tumors. The development of drugs such as monoclonal antibodies, dual / multi-target antibodies or proteins has become one of the hot research topics in this field. Interleukin 13 receptor α2 (IL13Rα2) is highly expressed on the surface of malignant tumor tissue cells such as human glioma and melanoma, making it a potential target for tumor treatment. Previous studies have shown that the IL13 variant (E13Y) can specifically bind to IL13Rα2.

[0003] In 2016, the New England Journal of Medicine reported a clinical trial of a technology that uses T cells to load tumor-targeting receptors, namely chimeric antigen receptor T-cell immunotherapy (CAR-T), which used CAR-T cells loaded with IL-13 variants to treat glioblastoma patients, and the tumors regressed for up to 7 months. However, because this method treats patients based on CAR-T therapy, it still cannot avoid the problems brought by CAR-T therapy itself, such as long preparation cycle of therapeutic cells, high cost, and risk of severe adverse reactions.

[0004] In order to avoid the above-mentioned defects of CAR-T therapy, researchers have developed bispecific antibodies that can guide immune cells (such as T cells, etc.) to efficiently kill cancer cells. It is reported that the CD3×IL13Rα2 bispecific antibody based on T cells is currently in preclinical research, making this bispecific antibody promising to become a new anti-tumor drug. However, although this bispecific antibody based on T cells (CD3) solves the defects of long preparation cycle, high cost and high specificity (the prepared CAR-T cells can only be returned to the patient for use) in CAR-T therapy, there is still a large risk of adverse reactions. For example, clinical trial results show that bispecific antibodies based on T cells (CD3) will induce cytokine storms in patients after administration, that is, a large amount of pro-inflammatory cytokines such as IL-6 and TNF-α are produced in a short period of time, and severe cases may even be life-threatening. Summary of the invention

[0005] The purpose of the present invention is to overcome the problem that bispecific antibodies based on T cells in the prior art will cause cytokine storms in patients when used, thereby having a large risk of adverse reactions, and provide a fusion protein and its encoding gene and recombinant vector and their applications. The fusion protein can bind to NK cells in the body and guide them to efficiently kill cancer cells (such as cancer cells expressing IL13Rα2). On the one hand, the fusion protein can achieve quantitative production and is not subject to individual restrictions when used. On the other hand, the risk of causing adverse reactions such as cytokine storms is also reduced or avoided.

[0006] In order to exert the anti-cancer function of NK cells and avoid the problem of adverse reactions such as cytokine storms caused by cell therapy based on T cells and bispecific antibody therapy, the inventors of the present invention have invented a bispecific protein based on anti-NKG2D antibody and IL-13 variant (E13Y) after research. The bispecific protein (fusion protein) can be mass-produced under GMP standards and used by patients at a reasonable price. At the same time, compared with the existing bispecific antibodies based on T cells, the bispecific protein provided by the present invention can effectively reduce or avoid the risk of side effects such as cytokine storms, and is safer to use.

[0007] In order to achieve the above object, the present invention provides a fusion protein, which comprises the following fragments:

[0008] Fragment a: the amino acid sequence is shown in SEQ ID NO: 1; and

[0009] Fragment b: the amino acid sequence is shown in SEQ ID NO:2.

[0010] The second aspect of the present invention provides a gene encoding and expressing the fusion protein described above.

[0011] The third aspect of the present invention provides a recombinant vector, wherein the recombinant vector contains the gene as described above.

[0012] The fourth aspect of the present invention provides the use of the fusion protein, gene or recombinant vector as described above in the preparation of a drug for treating and / or preventing tumors.

[0013] The fifth aspect of the present invention provides a pharmaceutical composition for treating and / or preventing tumors, wherein the pharmaceutical composition contains the fusion protein, gene or recombinant vector as described above;

[0014] Alternatively, the pharmaceutical composition contains only the aforementioned fusion protein, gene or recombinant vector as an active component.

[0015] Through the above technical scheme, the fusion protein provided by the present invention has the advantages of being able to accurately and efficiently kill a variety of different types of cancer cells, having an outstanding killing effect on cancer cells, and having low treatment costs. At the same time, the fusion protein also has the advantages of low side effects, especially low risk of inducing cell storms and their secondary side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a structural schematic diagram of the fusion protein provided by the present invention;

[0017] Figure 2 This is a graph showing the results of an experiment on the binding of the fusion protein provided by the present invention to peripheral blood NK cells in Example 1;

[0018] Figure 3 This is a graph showing the results of the experiment on binding of the fusion protein provided by the present invention to CHO-IL13Rα2 cells in Example 2;

[0019] Figure 4 This is a graph showing the results of an experiment in which the fusion protein provided by the present invention binds to melanoma A375 cells in Example 3;

[0020] Figure 5 This is a result diagram of the binding experiment between the fusion protein provided by the present invention and glioma U251 cells in Example 3;

[0021] Figure 6 This is a diagram showing the effect of the fusion protein provided by the present invention in Example 4 on promoting NK cells to kill cancer cells, taking melanoma A375 as an example;

[0022] Figure 7 This is a diagram showing the effect of the fusion protein provided by the present invention in Example 4 on promoting NK cells to kill cancer cells using glioblastoma U251 as an example;

[0023] Figure 8 This is a result diagram of Example 5, showing that the fusion protein provided by the present invention promotes the production of interleukin 6 (IL-6);

[0024] Fig. 9 This is a graph showing the result of Example 5, in which the fusion protein provided by the present invention promotes the production of tumor necrosis factor α (TNF-α). DETAILED DESCRIPTION

[0025] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0026] The fusion protein provided in the present invention is a bispecific antibody (protein), which can specifically bind to NK cells on the one hand, and can target and guide the NK cells bound thereto to kill tumor cells expressing IL13Rα2 on the other hand. Therefore, unless otherwise specified, the concepts of "fusion protein" and "bispecific antibody" provided in the present invention are consistent and can be used interchangeably.

[0027] Since the interleukin 13 (IL-13) receptor IL13Rα2 is highly expressed in some tumor cells, and in contrast, IL13Rα2 is usually not expressed or lowly expressed in normal human tissue cells, IL13Rα2 can be used as a potential target for tumor treatment. The human IL-13 variant E13Y is a protein that can selectively bind to IL13Rα2 and does not bind to IL13Rα1, which makes it have the potential to achieve precise tumor prevention and treatment through fusion protein technology. NKG2D is an important activating receptor for NK cells, and the NKG2D signal can promote NK cell activation and kill target cells. The inventors of the present invention fused E13Y with NKG2D antibody protein to produce a heterodimeric fusion protein that can target tumor cells that highly express IL13Rα2, thereby promoting NK cells to target and kill tumor cells.

[0028] On the one hand, the present invention provides a fusion protein (its schematic diagram is as follows Figure 1 The fusion protein contains the following fragments:

[0029] Fragment a: the amino acid sequence is shown in SEQ ID NO: 1; and

[0030] Fragment b: the amino acid sequence is shown in SEQ ID NO:2.

[0031] SPGPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFNGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:1)

[0032] QSALTQPASVSGSPGQSITISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSAFLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVLGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGLGDGTYFDYWGQGTTVTVSSGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:2)

[0033] In the fusion protein provided by the present invention, fragment a and fragment b can be connected by any existing method in the fusion protein technology. For the purpose of facilitating production, according to a preferred embodiment of the present invention, fragment a and fragment b are covalently connected via a disulfide bond in the hinge region of the Fc segment (IgG1-Fc) of human IgG1, i.e., a disulfide bond formed between C129 (fragment a)-C264 (fragment b) and C132 (fragment a)-C267 (fragment b).

[0034] In order to achieve the formation of heterodimers between fragment a and fragment b during expression and avoid the generation of homodimers of fragment a or fragment b, the present invention utilizes the Knob-into-hole (KIH) technology, which is achieved by mutating the amino acids in the CH3 domain of human IgG1-Fc (T366S, L368A, Y407V mutations in the a chain, i.e., "hole"; T366W mutations in the b chain, i.e., "knob"). Specifically in the sequence of the present invention, the specific mutation sites are S269, A271, V310 in fragment a (SEQ ID NO: 1) and W404 in fragment b (SEQ ID NO: 2).

[0035] The second aspect of the present invention provides a gene encoding and expressing the fusion protein described above.

[0036] Any gene capable of encoding and expressing the fusion protein as described above belongs to the content of the present invention. The inventors of the present invention cleverly discovered during the research process that when the genes expressing fragment a and fragment b are genes with nucleotide sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, they are superior to genes with other sequences in terms of expression amount and activity of expression products.

[0037] According to a preferred embodiment of the present invention, the gene comprises:

[0038] (A) the nucleotide sequence encoding fragment a, as shown in SEQ ID NO: 3;

[0039] (B) The nucleotide sequence encoding fragment b is shown in SEQ ID NO:4.

[0040]

[0041]

[0042] Those skilled in the art should know that, in addition to the two fragments (A) and (B) above, the gene expressing the fusion protein provided by the present invention may also include nucleotide sequences such as promoters, enhancers, and non-coding regions to achieve the purpose of improving the expression amount, expression efficiency, product activity, and other aspects of the gene. In addition, the gene may also include a nucleotide sequence with a tag for the purpose of conveniently detecting the expression of the product, such as a nucleotide sequence expressing glutamine synthetase.

[0043] The third aspect of the present invention provides a recombinant vector, wherein the recombinant vector contains the gene as described above.

[0044] The recombinant vector provided by the present invention can be a recombinant vector obtained by connecting any existing vector in the prior art of the art to the gene as described above. According to a preferred embodiment of the present invention, the vector can be selected from at least one of a plasmid (e.g., commercially available plasmids pSeTag2, PEE14, pMH3, etc., or a plasmid prepared by the prior art that can be used to prepare a recombinant vector), a prokaryotic expression vector (e.g., Escherichia coli, Bacillus subtilis, Streptomyces, Proteus mirabilis, etc.), a eukaryotic expression vector (e.g., fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworms, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.), and a bacteriophage.

[0045] According to a preferred embodiment of the present invention, in order to increase the expression level of the fusion protein provided by the present invention, the recombinant vector may contain, in addition to the gene as described above, a promoter and a gene sequence encoding a secretion signal peptide.

[0046] According to a preferred embodiment of the present invention, the recombinant vector may further contain a tag gene for screening and detecting product expression, such as a gene expressing glutamine synthetase.

[0047] The fourth aspect of the present invention provides the use of the fusion protein, gene or recombinant vector as described above in the preparation of a drug for treating and / or preventing tumors.

[0048] The above-mentioned fusion protein, gene or recombinant vector provided by the present invention can be used to prepare a drug for treating and / or preventing any tumor that can be targeted and recognized by the fusion protein. Since the fusion protein provided by the present invention contains an IL13 variant (E13Y) fragment (i.e., fragment a) that can efficiently target and recognize interleukin 13 receptor α2 (IL13Rα2), according to a preferred embodiment of the present invention, the tumor is selected from a tumor expressing IL13Rα2.

[0049] According to a preferred embodiment of the present invention, the tumor is selected from at least one of malignant glioma, melanoma, adrenocortical carcinoma, pancreatic cancer, squamous head and neck cell carcinoma, renal cell carcinoma, and pancreatic ductal adenocarcinoma.

[0050] According to a preferred embodiment of the present invention, the drug further contains a pharmaceutically acceptable carrier, which refers to an auxiliary material that has no effect or little effect on the activity of the fusion protein during the preparation of the drug and is used to form the drug into a specified dosage form.

[0051] The fifth aspect of the present invention provides a pharmaceutical composition for treating and / or preventing tumors, wherein the pharmaceutical composition contains the fusion protein, gene or recombinant vector as described above;

[0052] Alternatively, the pharmaceutical composition contains only the aforementioned fusion protein, gene or recombinant vector as an active component.

[0053] According to a preferred embodiment of the present invention, the pharmaceutical composition can also be administered in combination with other drugs to improve the therapeutic effect of the pharmaceutical composition. For example, the other drugs can be selected from drugs that regulate IL13Rα2.

[0054] The pharmaceutical composition provided by the present invention can be any pharmaceutical dosage form currently used in the art for treating and / or preventing tumors. Without destroying the therapeutic effect of the pharmaceutical composition, those skilled in the art can select the dosage form of the pharmaceutical composition according to actual needs.

[0055] The pharmaceutical composition provided by the present invention can be administered by any existing administration method in the art. For the purpose of convenient administration, according to a preferred embodiment of the present invention, the administration method of the pharmaceutical composition can be selected from at least one of oral administration, nasal administration, intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, and intraperitoneal administration.

[0056] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0057] In the following examples, the a-chain and b-chain encoding genes in the fusion protein (shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively) were commissioned to Suzhou GeneWeizhi Co., Ltd. for synthesis, and the reagents used were commercially available products purchased from regular biological or chemical reagent suppliers, and the purity was analytical grade unless otherwise specified.

[0058] Preparation Example 1

[0059] This preparation example is used to illustrate the preparation of the fusion protein provided by the present invention.

[0060] Experimental method: ExpiCHO cells (Thermo Fisher) were cultured using ExpiCHO Expression Medium (Thermo Fisher) and the cell concentration was adjusted to 6×10 6 / mL to obtain an ExpiCHO cell suspension. The pTT5 vector (Suzhou Jinweizhi Company) containing the a-chain and b-chain encoding genes (as shown in SEQ ID NO:3 and SEQ ID NO:4, respectively) was added to 2mL OptiSFM medium (Thermo Fisher) to obtain solution A. 160μL ExpiFectamineCHO transfection reagent (Thermo Fisher) was added to 2mL OptiSFM medium to obtain solution B. Solution A and solution B were then mixed to obtain a transfection mixture, and the transfection mixture was added to 50mL ExpiCHO cell suspension within 5 minutes. After culturing for 1 day at 37°C and 8% CO2, 8mL Feed and 300μL Enhancer (Thermo Fisher) were added, and the culture supernatant was harvested after 9 days of culture at 32°C and 5% CO2, with 8mL Feed added on the 5th day. The fusion protein was affinity purified from the culture supernatant using a Protein A purification column (GE). The amino acid sequences of fragment a and fragment b in the fusion protein are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The disulfide bonds formed between C129 (fragment a)-C264 (fragment b) and C132 (fragment a)-C267 (fragment b) enable fragment a and fragment b to form a heterodimer (the structural schematic diagram is shown in Figure 1 shown).

[0061] Example 1

[0062] This example is used to illustrate the results of the fusion protein provided by the present invention targeting and binding to human peripheral blood NK cells. The human peripheral blood used in this example was obtained from the First Affiliated Hospital of Anhui Medical University, and informed consent was obtained from the relevant personnel.

[0063] Experimental method: Human peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll gradient density centrifugation and diluted to 1×10 7 / mL, add 100U / mL IL-2 and culture for 24 hours.

[0064] Dilute PBMC to 5 × 10 6 / mL, take 90μL and add it to a 1.5mL EP tube, add 10μL mouse serum to it, and incubate at 4℃ for 30min. Add the fusion protein to the EP tube according to different concentration gradients and incubate at 4℃ for 30min.

[0065] After the incubation, the PBMCs were washed twice with PBS. The specific operation was as follows: 1 mL of 1×PBS was added to the EP tube, centrifuged at 4°C and 250g for 5 minutes, and the supernatant was discarded. After the washing, 100 μL of 1×PBS was added to each EP tube to resuspend the cells, and then 1 μL APC-labeled mouse anti-human IgG-Fc antibody (BioLegend) and 0.5 μL FITC-labeled mouse anti-human CD4 antibody (BD Company) were added, and incubated at 4°C in the dark for 30 minutes. After the incubation, the PBMCs were washed twice with PBS. After the washing, 200 μL of 1×PBS was added to each EP tube to resuspend the cells, and the binding ratio of different concentrations of fusion protein and antibody was detected using a flow cytometer (BD Company, FACS Cantosys model). Human IgG was treated and detected according to the above method as a negative control, and the results are as shown in the figure. Figure 2 shown.

[0066] Example 2

[0067] This example is used to illustrate the results of the fusion protein provided by the present invention targeting and binding to CHO-IL13Rα2 cells.

[0068] The specific test method is the same as that in Example 1, except that the NK cells therein are replaced with CHO-IL13Rα2 cells (having puromycin resistance). Figure 3 shown.

[0069] The CHO-IL13 Rα2 cells used in this example were constructed by this laboratory. The construction method is as follows: The pLVX-EF1a-IRES-puro vector containing the IL13 Rα2 coding sequence (synthesized by Suzhou Jinweizhi) and the packaging plasmids pMD2G and psPAX2 (the above vectors and packaging plasmids were purchased from Youbao Bio) were co-transfected into 293T cells (purchased from ATCC). After 48 hours, the culture supernatant containing full virus was harvested and used to infect CHO-K1 cells (purchased from ATCC) to obtain CHO-IL13Rα2 cells. Then, puromycin (Invivogen) was used to screen and obtain CHO-IL13Rα2 cells with puromycin resistance.

[0070] Example 3

[0071] This example is used to illustrate the results of the fusion protein provided by the present invention targeting, recognizing and binding to A375 cells (melanoma cell line) and U251 cells (glioma cell line).

[0072] The specific test method is the same as that in Example 1, except that the NK cells therein are replaced by A375 cells and U251 cells (both purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences). Figure 4 (A375) and Figure 5 (U251) as shown.

[0073] Example 4

[0074] This example is used to illustrate the effect of the fusion protein provided by the present invention on the anti-cancer activity of T cells. The human peripheral blood used in this example was obtained from the First Affiliated Hospital of Anhui Medical University, and informed consent was obtained from the relevant personnel.

[0075] Experimental method: Human peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll gradient density centrifugation and diluted to 1×10 7 / mL, add 100U / mL IL-2 and culture for 24 hours.

[0076] (1) Tumor cells (A375 and U251, both purchased from Shanghai Cell Bank of Chinese Academy of Sciences) were washed twice with complete RPMI-1640 medium, and the washing method was the same as in Example 1. Then, they were added to a real-time label-free dynamic cell analysis (RTCA) chip (purchased from XCELLIGENCE), 10,000 cells per well, placed on a RTCA instrument (purchased from XCELLIGENCE, model RTCATP), and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0077] (2) PBMCs were resuspended in complete RPMI-1640 medium and added to the RTCA chip, with 100,000 cells per well.

[0078] (3) Dilute the fusion protein according to a concentration gradient using complete RPMI-1640 medium and add it to the chip.

[0079] (4) Place the chip on the RTCA instrument and incubate in a 37°C, 5% CO2 incubator for 40 hours.

[0080] (5) Collect data, analyze and calculate the specific killing ratio mediated by different concentrations of fusion protein at the 40th hour. The calculation formula is: specific killing ratio = (killing ratio of fusion protein group - killing ratio of non-fusion protein group) / (100% - killing ratio of non-fusion protein group)

[0081] The results are as follows Figure 6 (A375) and Figure 7 (U251) as shown.

[0082] Example 5

[0083] This example is used to illustrate the effect of the fusion protein provided by the present invention on the production of interleukin (IL-6) and tumor necrosis factor α (TNF-α) by PBMC cells. The human peripheral blood used in this example was obtained from the First Affiliated Hospital of Anhui Medical University, and informed consent was obtained from the relevant personnel.

[0084] Experimental method: Human peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll gradient density centrifugation and diluted to 1×10 7 / mL, add 100U / mL IL-2 and culture for 24 hours.

[0085] (1) Tumor cells (A375 and U251, both purchased from Shanghai Cell Bank of Chinese Academy of Sciences) were washed twice with complete RPMI-1640 medium, using the same washing method as in Example 1. Then, they were added to a 96-well plate, with 10,000 cells per well.

[0086] (2) PBMCs were resuspended in complete RPMI-1640 medium and added to a 96-well plate, with 100,000 cells per well.

[0087] (3) The 96-well plate was incubated in a 37°C, 5% CO2 incubator for 48 hours.

[0088] (4) The culture supernatant was collected and the IL-6 and TNF-α contents therein were detected using a CBA kit (BD).

[0089] (5) Mix equal volumes of IL-6 and TNF-α detection beads and add them to flow cytometry tubes, 20 μL per tube.

[0090] (6) Add the standard sample and culture supernatant into the flow cytometry tubes, 20 μL per tube.

[0091] (7) Add the detection antibody to the flow cytometry tube, 20 μL per tube.

[0092] (8) Vortex to mix, and then incubate at room temperature in the dark for 3 hours.

[0093] (9) The samples were tested using a flow cytometer (BD, FACS Cantosys model) and the cytokine content in the supernatant was calculated.

[0094] At the same time, E13Y and T cell (CD3) antibody fusion protein (preparation method reference 202110391048.X) was used as a positive control and human IgG was used as a negative control. Figure 8 (IL-6) and Fig. 9 (TNF-α) as shown.

[0095] As can be seen from the figure, using the fusion protein provided by the present invention, when the concentration of the fusion protein is 0.1 μg / mL and below, the production of IL-6 is basically the same as that of human IgG, and when the fusion protein dosage is increased to 1 μg / mL, the production of IL-6 exceeds that of human IgG, but it is still lower than the production of IL-6 using E13Y and T cell antibody fusion protein (about half or less). When the concentration of the fusion protein provided by the present invention is 1 μg / mL and below, the production of TNF-α is basically the same as that of human IgG, although when the fusion protein concentration reaches 10 μg / mL, the production of TNF-α exceeds that of human IgG, but it is still far lower than the production of TNF-α using E13Y and T cell antibody fusion protein. It can be seen from this that the risk of cytokine storm generated by the fusion protein provided by the present invention when used is greatly reduced compared with that when E13Y and T cell antibody fusion protein are used, thereby having higher safety.

[0096] It can be seen from the results of the above examples that the fusion protein provided by the present invention can target and recognize a variety of different tumor cells expressing IL13Rα2, and all show a high binding efficiency. Moreover, the fusion protein can also promote NK cells to kill tumor cells expressing IL13Rα2, such as A375 and U251. At the same time, the fusion protein provided by the present invention is not easy to cause side effects such as cytokine storms, indicating that the fusion protein provided by the present invention has the potential to be used for the preparation of broad-spectrum, efficient and safe anticancer drugs.

[0097] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention. SEQUENCE LISTING <110> The First Affiliated Hospital of Anhui Medical University <120> Fusion protein, its encoding gene, recombinant vector and their application <130> I75775AYFY <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 350 <212> PRT <213> Fragment a <400> 1 Ser Pro Gly Pro Val Pro Pro Ser Thr Ala Leu Arg Tyr Leu Ile Glu 1 5 10 15 Glu Leu Val Asn Ile Thr Gln Asn Gln Lys Ala Pro Leu Cys Asn Gly 20 25 30 Ser Met Val Trp Ser Ile Asn Leu Thr Ala Gly Met Tyr Cys Ala Ala 35 40 45 Leu Glu Ser Leu Ile Asn Val Ser Gly Cys Ser Ala Ile Glu Lys Thr 50 55 60 Gln Arg Met Leu Ser Gly Phe Cys Pro His Lys Val Ser Ala Gly Gln 65 70 75 80 Phe Ser Ser Leu His Val Arg Asp Thr Lys Ile Glu Val Ala Gln Phe 85 90 95 Val Lys Asp Leu Leu Leu His Leu Lys Lys Leu Phe Arg Glu Gly Arg 100 105 110 Phe Asn Gly Gly Gly Gly Ser Pro Lys Ser Cys Asp Lys Thr His Thr 115 120 125 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 130 135 140 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 145 150 155 160 Glu Val Thr Cys Val Val Val Ala Val Ser His Glu Asp Pro Glu Val 165 170 175 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 180 185 190 Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val 195 200 205 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 210 215 220 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 225 230 235 240 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 245 250 255 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 260 265 270 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 275 280 285 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 290 295 300 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 305 310 315 320 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 325 330 335 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 <210> 2 <211> 482 <212> PRT <213> Fragment b <400> 2 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val Ser Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Phe Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly 100 105 110 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln 115 120 125 Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg 130 135 140 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His 145 150 155 160 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Phe Ile 165 170 175 Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg 180 185 190 Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met 195 200 205 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp 210 215 220 Arg Gly Leu Gly Asp Gly Thr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Pro Lys Ser Cys Asp 245 250 255 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 260 265 270 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 275 280 285 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala Val Ser His Glu 290 295 300 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 305 310 315 320 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg 325 330 335 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 340 345 350 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 355 360 365 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 370 375 380 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 385 390 395 400 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 405 410 415 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 420 425 430 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 435 440 445 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 450 455 460 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 465 470 475 480 Gly Lys <210> 3 <211> 1050 <212> DNA <213> Fragment a coding gene <400> 3 tcaccaggac cagttcctcc ttctaccgca cttagatact tgattgaaga gctggtcaat 60 atcacacaga accagaaggc tcccctgtgc aacggtagta tggtgtggtc tatcaacttg 120 acagcaggaa tgtattgtgc cgccctggag tccctgatca atgtctccgg ctgttccgct 180 attgagaaga cccagcggat gctgtccggg ttttgtcccc ataaagtgag tgctgggcag 240 ttctctagtc tccatgtcag ggataccaag atcgaggtgg cacagtttgt caaggacctg 300 ctgctgcatc tgaagaagct gtttcgggaa ggacgtttca acggagagg tggcagccct 360 aagtcctgtg aaagaccca cacatgtcca ccttgccctg ctcctgaact gctcggtgga 420 cctagtgttt tcttgtttcc tccaaagccc aaagatactc tcatgatttc cagaacacct 480 gaagtgactt gtgttgtcgt ggcagtgtct cacgaggatc ccgaggtcaa gtttaattgg 540 tacgtcgacg gcgtggaagt gcacaacgcc aagaccaaac cccgagaaga gcagtatgca 600 tctacctaca gagtcgtgag tgtgctcact gtgttgcacc aggattggct gaacggcaag 660 gagtacaaat gtaaggtgtc aaacaaagcc ttgcccgcac caatcgaaaa gactatctcc 720 aaggcaaagg gacaacctag agaacccag gtatacacac tgcctccctc tcgggaagaa 780 atgactaaga accaggtgtc cttgtcatgc gctttaaagg gcttttaccc tagtgacatt 840 gctgtcgaat gggagtcaaa tggtcagcca gagaacaatt ataagactac cccaccagtc 900 ctcgactccg atggcagctt ttttctggtg tccaagctca ccgtagacaa gagccggtgg 960 cagcagggca atgtatttag ttgtagcgtt atgcacgagg ctctgcataa ccactatact 1020 cagaagagcc tcagtctctc tcctggcaaa 1050 <210> 4 <211> 1446 <212> DNA <213> Fragment b coding gene <400> 4 caatcagctc tgacacaacc agcaagcgtg tcaggtagtc ctggacaatc cattacaatc 60 agctgttctg gatcctcctc taatataggc aacaacgcag tgaattggta tcagcagctc 120 ccagggaaag ctccaaagtt gctgatctac tatgacgacc tgttgccaag tggagtgtcc 180 gaccggttct cagggtccaa atcagggacc agcgcttttc tggccatttc tgggctgcag 240 tcagaagacg aggccgacta ctactgcgcc gcttgggacg atagtctgaa cggacctgtg 300 ttcggcggtg gtaccaaact gactgttctg ggcggaggtg gatcaggtgg tggaggtagt 360 ggcggtgggg gttctcaggt gcagcttgtt gaaagtggag gaggcctcgt aaagcctgga 420 ggctctttga gactgagttg tgctgcatca ggctttacct tctcctcata cggaatgcat 480 tgggtgaggc aggcaccagg gaaaggactg gagtgggtcg cttttatacg ctatgacggt 540 tcaaacaaat actatgccga ctccgtgaag ggccgattta caatctctag agacaattca aagaacaccc tgtacctgca gatgaactca ctgagggctg aggataccgc cgtctattac tgcgctaagg accgagggct gggagacggt acctatttcg attactgggg acagggcaca 720 acagtgaccg ttagctcagg cggcggagga agccctaaat cttgtgacaa aacacatact tgtccaccat gccccgctcc agaattgttg ggcgggcctt cagttttcct cttcccccct 840 aagcctaagg acaccctgat gatcagcaga accccagagg tgacctgcgt ggtggtagcc gtctctcacg aagaccccga agtgaagttc aactggtacg tcgacggggt ggaggtgcat 960 aatgccaaaa ccaagccccg tgaggaaca tatgctagta cttaccgagt ggtgtccgta cttaccgtgc tgcaccagga ctggttgaac gggaagaat acaagtgtaa agtatctaat aaagcactgc cagctccaat cgaaaagacc atctctaagg caagggcca gcccagagaa ccccaggtct acactctgcc tccctctcgt gagagatga ctaagaacca ggtcagtctg tggtgtttgg tgaaaggatt ttacccaagc gacatcgcag tggaatggga gagtaacggt cagccagaga ataattataa gacaactcct ccagtgctcg attctgatgg ttcctttttc 1320 ttgtattcta aactgacagt ggataaatca cggtggcagc agggcaacgt gttctcctgc 1380 tctgtgatgc atgaggctct gcacaaccac tacactcaga agagcctgag cctgagtcca 1440 gggaag 1446

Claims

1. A fusion protein, characterized in that The fusion protein consists of the following fragments: Fragment a: the amino acid sequence is shown in SEQ ID NO: 1; and Fragment b: the amino acid sequence is shown in SEQ ID NO:

2.

2. The fusion protein according to claim 1, wherein The fragment a and the fragment b are covalently linked via a disulfide bond in the hinge region of the Fc segment of human IgG.

3. A gene encoding and expressing the fusion protein according to claim 1 or 2.

4. The gene according to claim 3, wherein The genes include: (A) the nucleotide sequence encoding fragment a, as shown in SEQ ID NO: 3; (B) The nucleotide sequence encoding fragment b is shown in SEQ ID NO:

4.

5. A recombinant vector, characterized in that: The recombinant vector contains the gene described in claim 3 or 4.

6. Use of the fusion protein according to claim 1 or 2, the gene according to claim 3 or 4, or the recombinant vector according to claim 5 in the preparation of a medicament for treating malignant glioma and / or melanoma.

7. The use according to claim 6, wherein: The medicine also contains a pharmaceutically acceptable carrier.

8. A pharmaceutical composition for treating tumors, characterized in that: The pharmaceutical composition contains the fusion protein according to claim 1 or 2, the gene according to claim 3 or 4, or the recombinant vector according to claim 5.

9. The pharmaceutical composition according to claim 8, wherein The pharmaceutical composition contains only the fusion protein according to claim 1 or 2, the gene according to claim 3 or 4, or the recombinant vector according to claim 5 as an active component.

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