CN115197326A - Fusion protein, encoding gene and recombinant vector thereof, and application of fusion protein and encoding gene and recombinant vector - Google Patents

Abstract

The invention relates to the field of biomedicine, and discloses a fusion protein, a coding gene and a recombinant vector thereof, and application of the fusion protein and the coding gene and the recombinant vector. The fusion protein provided by the invention can specifically bind to in vivo NK cells and guide the NK cells to efficiently kill cancer cells expressing IL13R alpha 2, and has the advantages of outstanding effect of killing cancer cells, low treatment cost, low side effect risk and the like.

Description

Fusion protein, encoding gene and recombinant vector thereof, and application of fusion protein and encoding gene and recombinant vector

Technical Field

The invention relates to the field of biomedicine, in particular to a fusion protein, a coding gene and a recombinant vector thereof, and application of the fusion protein and the coding gene and the recombinant vector.

Background

In recent years, a great breakthrough is made in the field of treatment of malignant tumors by biomacromolecule drugs, and the development of drugs such as monoclonal antibodies, double/multi-target antibodies or proteins becomes one of the hot researches in the field. Interleukin 13 receptor alpha 2 (IL 13R alpha 2) is specifically and highly expressed on the surface of malignant tumor tissue cells such as human glioma, melanoma and the like, so that the interleukin 13 receptor alpha 2 becomes a potential tumor treatment target. Previous studies have shown that IL13 variants (E13Y) are capable of specifically binding IL13 ra 2.

One example of a technique that utilizes T Cell loading to target tumor receptors was reported in the new england journal of medicine in 2016, i.e., a clinical trial of Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T), in which tumor regresses for up to 7 months after glioblastoma patients are treated with CAR-T cells loaded with IL-13 variants. However, the method is based on CAR-T therapy to treat patients, so that the problems of long preparation period of treatment cells, high cost, severe adverse reaction risk and the like caused by the CAR-T therapy cannot be avoided.

To avoid the above-mentioned drawbacks of CAR-T therapy, researchers have developed bispecific antibodies that can direct immune cells (e.g., T cells, etc.) to kill cancer cells with high efficiency. The CD3 xIL 13R alpha 2 bispecific antibody based on T cells is reported to be in preclinical research, so that the bispecific antibody is expected to become a novel antitumor drug. However, the bispecific antibody based on the T cell (CD 3) overcomes the defects of long preparation period, high cost, high specificity (the prepared CAR-T cell can only be returned to a patient for use) and the like in CAR-T therapy, but still has a large risk of adverse reaction. For example, clinical trials have shown that bispecific antibodies based on T cells (CD 3) induce cytokine storm in patients after administration, i.e., induce a large amount of pro-inflammatory cytokine production such as IL-6, TNF- α, etc. for a short period of time, and in severe cases even threaten life.

Disclosure of Invention

The invention aims to solve the problems that in the prior art, when a bispecific antibody based on T cells is used, a cytokine storm is caused in a patient body, so that a greater risk of adverse reaction exists, and the like, and provides a fusion protein, a coding gene and a recombinant vector thereof, and application thereof. The fusion protein can bind to NK cells in vivo and guide the NK cells to efficiently kill cancer cells (such as IL13R alpha 2-expressing cancer cells). The fusion protein can realize quantitative production, is not limited by individuals during use, and reduces or avoids risks of adverse reactions such as cytokine storm and the like.

In order to exert NK cell anticancer function while avoiding the problem that T cell-based cell therapy, bispecific antibody therapy, and the like are liable to cause adverse reactions such as cytokine storm, the present inventors have studied and invented bispecific proteins based on an anti-NKG 2D antibody and an IL-13 variant (E13Y). The bispecific protein (fusion protein) can be produced in large quantities under GMP standard and can be used by patients at reasonable price, and simultaneously, compared with the existing bispecific antibody based on T cells, the bispecific protein provided by the invention can effectively reduce or avoid the risk of side effects such as cytokine storm and the like, and is safer to use.

In order to achieve the above object, one aspect of the present invention provides a fusion protein comprising the following fragments:

fragment a: the amino acid sequence is shown as SEQ ID NO. 1; and

fragment b: the amino acid sequence is shown as SEQ ID NO. 2.

In a second aspect, the invention provides a gene encoding a fusion protein as described above.

In a third aspect, the present invention provides a recombinant vector containing the gene as described above.

In a fourth aspect, the present invention provides the use of the fusion protein, gene or recombinant vector as described above in the preparation of a medicament for the treatment and/or prevention of a tumor.

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

alternatively, the pharmaceutical composition contains only the fusion protein, gene or recombinant vector as described above as an active ingredient.

Through the technical scheme, the fusion protein provided by the invention has the effects of accurately and efficiently killing various different types of cancer cells, having an outstanding cancer cell killing effect, being low in treatment cost and the like. Meanwhile, the fusion protein also has the advantages of low side effect, especially low risk of triggering cell storm and secondary side effect thereof.

Drawings

FIG. 1 is a schematic diagram of the structure of a fusion protein provided by the present invention;

FIG. 2 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;

FIG. 3 is a graph showing the results of the binding experiment of the fusion protein provided by the present invention to CHO-IL13R α 2 cells in example 2;

FIG. 4 is a graph showing the results of the binding experiment of the fusion protein of the present invention to melanoma A375 cells in example 3;

FIG. 5 is a graph showing the results of the binding experiment of the fusion protein of the present invention to glioma U251 cells in example 3;

FIG. 6 is a graph showing the effect of the fusion protein of the present invention in promoting NK cell killing of cancer cells such as melanoma A375 in example 4;

FIG. 7 is a graph showing the effect of the fusion protein of the present invention in promoting NK cell killing of cancer cells such as glioblastoma U251 in example 4;

FIG. 8 is a graph showing the results of the fusion protein of the present invention promoting the production of interleukin 6 (IL-6) in example 5;

FIG. 9 is a graph showing the results of the fusion protein of the present invention promoting the production of tumor necrosis factor alpha (TNF-. Alpha.) in example 5.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The fusion protein provided by the invention is a bispecific antibody (protein), which can be specifically bound to NK cells on one hand, and can be used for targeting and guiding the NK cells bound with the bispecific antibody to kill tumor cells expressing IL-13R alpha 2 on the other hand. Therefore, unless otherwise specified, the concept of "fusion protein" and "bispecific antibody" provided by the present invention are consistent and may be used interchangeably.

Since the interleukin 13 (IL-13) receptor IL13R alpha 2 is highly expressed in some tumor cells, in contrast to IL13R alpha 2 which is not normally expressed or is low expressed in normal human tissue cells, IL13R alpha 2 can be used as a potential tumor treatment target. Human IL-13 variant E13Y is a protein that selectively binds IL13R α 2 and does not bind IL13R α 1, thus giving it the potential for precise tumor control by fusion protein technology. NKG2D is an important activating receptor of NK cells, and NKG2D signals can promote NK cell activation and kill target cells. The inventor prepares the heterodimer fusion protein capable of targeting and positioning the tumor cells highly expressing IL13R alpha 2 by fusing E13Y and NKG2D antibody protein, thereby promoting NK cells to kill the tumor cells in a targeted manner.

In one aspect, the present invention provides a fusion protein (schematically shown in FIG. 1) comprising the following fragments:

fragment a: the amino acid sequence is shown as SEQ ID NO. 1; and

fragment b: the amino acid sequence is shown as SEQ ID NO. 2.

SPGPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFNGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:1)

QSALTQPASVSGSPGQSITISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSAFLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVLGGGGSGGGGSGGGGSQVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRGLGDGTYFDYWGQGTTVTVSSGGGGSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:2)

In the fusion protein provided by the invention, the fragment a and the fragment b can be connected by any existing mode in the fusion protein technology. For the purpose of facilitating production, according to a preferred embodiment of the present invention, the fragments a and b are covalently linked by disulfide bonds of the hinge region of the Fc region (IgG 1-Fc) of human IgG1, i.e., disulfide bonds formed between C129 (fragment a) -C264 (fragment b), C132 (fragment a) -C267 (fragment b).

In order to realize that the fragment a and the fragment b form heterodimer in the expression process and avoid the generation of homodimer of the fragment a or the fragment b, the invention utilizes a Knob-into-hole (KIH) technology and realizes the purpose by mutating amino acids in a human IgG1-Fc CH3 domain (T366S, L368A and Y407V mutation in a chain, namely 'hole' and T366W mutation in a b chain, namely 'Knob'). Specific mutation sites in the sequences of the present invention are S269, A271 and V310 in fragment a (SEQ ID NO: 1) and W404 in fragment b (SEQ ID NO: 2).

In a second aspect, the invention provides a gene encoding a fusion protein as described above.

The present inventors have found that when the genes expressing the fragment a and the fragment b are the genes having the nucleotide sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively, the expression amount and the activity of the expression product are superior to those of the genes having other sequences.

According to a preferred embodiment of the present invention, wherein the gene comprises:

(A) The nucleotide sequence of the coding segment a is shown as SEQ ID NO. 3;

(B) The nucleotide sequence of the coding segment b is shown as SEQ ID NO. 4.

TCACCAGGACCAGTTCCTCCTTCTACCGCACTTAGATACTTGATTGAAGAGCTGGTCAATATCACACAGAACCAGAAGGCTCCCCTGTGCAACGGTAGTATGGTGTGGTCTATCAACTTGACAGCAGGAATGTATTGTGCCGCCCTGGAGTCCCTGATCAATGTCTCCGGCTGTTCCGCTATTGAGAAGACCCAGCGGATGCTGTCCGGGTTTTGTCCCCATAAAGTGAGTGCTGGGCAGTTCTCTAGTCTCCATGTCAGGGATACCAAGATCGAGGTGGCACAGTTTGTCAAGGACCTGCTGCTGCATCTGAAGAAGCTGTTTCGGGAAGGACGTTTCAACGGAGGAGGTGGCAGCCCTAAGTCCTGTGACAAGACCCACACATGTCCACCTTGCCCTGCTCCTGAACTGCTCGGTGGACCTAGTGTTTTCTTGTTTCCTCCAAAGCCCAAAGATACTCTCATGATTTCCAGAACACCTGAAGTGACTTGTGTTGTCGTGGCAGTGTCTCACGAGGATCCCGAGGTCAAGTTTAATTGGTACGTCGACGGCGTGGAAGTGCACAACGCCAAGACCAAACCCCGAGAAGAGCAGTATGCATCTACCTACAGAGTCGTGAGTGTGCTCACTGTGTTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAATGTAAGGTGTCAAACAAAGCCTTGCCCGCACCAATCGAAAAGACTATCTCCAAGGCAAAGGGACAACCTAGAGAACCCCAGGTATACACACTGCCTCCCTCTCGGGAAGAAATGACTAAGAACCAGGTGTCCTTGTCATGCGCTGTAAAGGGCTTTTACCCTAGTGACATTGCTGTCGAATGGGAGTCAAATGGTCAGCCAGAGAACAATTATAAGACTACCCCACCAGTCCTCGACTCCGATGGCAGCTTTTTTCTGGTGTCCAAGCTCACCGTAGACAAGAGCCGGTGGCAGCAGGGCAATGTATTTAGTTGTAGCGTTATGCACGAGGCTCTGCATAACCACTATACTCAGAAGAGCCTCAGTCTCTCTCCTGGCAAA(SEQ ID NO:3)

CAATCAGCTCTGACACAACCAGCAAGCGTGTCAGGTAGTCCTGGACAATCCATTACAATCAGCTGTTCTGGATCCTCCTCTAATATAGGCAACAACGCAGTGAATTGGTATCAGCAGCTCCCAGGGAAAGCTCCAAAGTTGCTGATCTACTATGACGACCTGTTGCCAAGTGGAGTGTCCGACCGGTTCTCAGGGTCCAAATCAGGGACCAGCGCTTTTCTGGCCATTTCTGGGCTGCAGTCAGAAGACGAGGCCGACTACTACTGCGCCGCTTGGGACGATAGTCTGAACGGACCTGTGTTCGGCGGTGGTACCAAACTGACTGTTCTGGGCGGAGGTGGATCAGGTGGTGGAGGTAGTGGCGGTGGGGGTTCTCAGGTGCAGCTTGTTGAAAGTGGAGGAGGCCTCGTAAAGCCTGGAGGCTCTTTGAGACTGAGTTGTGCTGCATCAGGCTTTACCTTCTCCTCATACGGAATGCATTGGGTGAGGCAGGCACCAGGGAAAGGACTGGAGTGGGTCGCTTTTATACGCTATGACGGTTCAAACAAATACTATGCCGACTCCGTGAAGGGCCGATTTACAATCTCTAGAGACAATTCAAAGAACACCCTGTACCTGCAGATGAACTCACTGAGGGCTGAGGATACCGCCGTCTATTACTGCGCTAAGGACCGAGGGCTGGGAGACGGTACCTATTTCGATTACTGGGGACAGGGCACAACAGTGACCGTTAGCTCAGGCGGCGGAGGAAGCCCTAAATCTTGTGACAAAACACATACTTGTCCACCATGCCCCGCTCCAGAATTGTTGGGCGGGCCTTCAGTTTTCCTCTTCCCCCCTAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCAGAGGTGACCTGCGTGGTGGTAGCCGTCTCTCACGAAGACCCCGAAGTGAAGTTCAACTGGTACGTCGACGGGGTGGAGGTGCATAATGCCAAAACCAAGCCCCGTGAGGAACAATATGCTAGTACTTACCGAGTGGTGTCCGTACTTACCGTGCTGCACCAGGACTGGTTGAACGGGAAAGAATACAAGTGTAAAGTATCTAATAAAGCACTGCCAGCTCCAATCGAAAAGACCATCTCTAAGGCAAAGGGCCAGCCCAGAGAACCCCAGGTCTACACTCTGCCTCCCTCTCGTGAAGAGATGACTAAGAACCAGGTCAGTCTGTGGTGTTTGGTGAAAGGATTTTACCCAAGCGACATCGCAGTGGAATGGGAGAGTAACGGTCAGCCAGAGAATAATTATAAGACAACTCCTCCAGTGCTCGATTCTGATGGTTCCTTTTTCTTGTATTCTAAACTGACAGTGGATAAATCACGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCTGTGATGCATGAGGCTCTGCACAACCACTACACTCAGAAGAGCCTGAGCCTGAGTCCAGGGAAG(SEQ ID NO:4)

It will be understood by those skilled in the art that the gene expressing the fusion protein provided by the present invention may further comprise nucleotide sequences such as promoter, enhancer, non-coding region, etc. in addition to the above two fragments (a) and (B), so as to achieve the purpose of improving the performance of the gene in terms of expression amount, expression efficiency, product activity, etc. In addition, the gene may include a nucleotide sequence having a tag for the purpose of facilitating detection of the expression of the product or the like, for example, a nucleotide sequence expressing glutamine synthetase or the like.

In a third aspect, the present invention provides a recombinant vector comprising the gene as described above.

The recombinant vector provided by the invention can be a recombinant vector prepared by connecting any conventional vector in the prior art with the gene. According to a preferred embodiment of the present invention, the vector may be at least one selected from the group consisting of plasmids (e.g., commercially available plasmids pSeTag2, PEE14, pMH3, etc., or plasmids that can be used to prepare recombinant vectors, which are prepared by themselves according to the prior art), prokaryotic expression vectors (e.g., escherichia coli, bacillus subtilis, streptomyces, proteus mirabilis, etc.), eukaryotic expression vectors (e.g., fungi such as pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma, etc., insect cells such as spodoptera frugiperda, etc., plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.), and phages.

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

According to a preferred embodiment of the present invention, the recombinant vector may further comprise a marker gene for screening and detecting the expression of the product, such as a gene expressing glutamine synthetase, and the like.

In a fourth aspect, the present invention provides the use of the fusion protein, gene or recombinant vector as described above in the preparation of a medicament for the treatment and/or prevention of a tumor.

The fusion protein, the gene or the recombinant vector provided by the invention can be used for preparing a medicine for treating and/or preventing any tumor which can be targeted and identified by the fusion protein. Since the fusion protein provided by the present invention contains the fragment (i.e., fragment a) of IL13 variant (E13Y) capable of efficiently targeting and recognizing interleukin 13 receptor α 2 (IL 13 ra 2), according to a preferred embodiment of the present invention, the tumor is selected from tumors expressing IL13 ra 2.

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

According to a preferred embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The carrier is auxiliary materials which have no influence or little influence on the activity of the fusion protein in the process of preparing the medicament and are used for enabling the medicament to form a specified dosage form.

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

alternatively, the pharmaceutical composition contains only the fusion protein, gene or recombinant vector as described above as an active ingredient.

According to a preferred embodiment of the present invention, the pharmaceutical composition may also be administered in combination with other drugs to enhance the therapeutic effect of the pharmaceutical composition. For example, the other drug may be selected from a drug that modulates IL13 ra 2, and the like.

The pharmaceutical composition provided by the invention can be any pharmaceutical dosage form for treating and/or preventing tumors in the field. The dosage form of the pharmaceutical composition can be selected by those skilled in the art according to actual needs without destroying the therapeutic effect of the pharmaceutical composition.

The pharmaceutical compositions provided herein can be administered by any administration means known in the art. For the purpose of convenient administration, according to a preferred embodiment of the present invention, wherein the administration mode of the pharmaceutical composition may be selected from at least one of oral administration, nasal administration, intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, intraperitoneal administration.

The present invention will be described in detail below by way of examples. It should be understood that the following examples are only for the purpose of further explaining and illustrating the contents of the present invention, and are not intended to limit the present invention.

In the following examples, the a-chain and b-chain encoding genes (shown in SEQ ID NO:3 and SEQ ID NO:4, respectively) in the fusion protein were synthesized by Cinzhi Sazhou, and the reagents used were commercially available products from the normal biological or chemical reagent suppliers without specific reference, and were analytically pure.

Preparation example 1

This preparation example is intended to illustrate the preparation of the fusion protein provided by the present invention.

The test method comprises the following steps: culturing ExpicHO cells (Thermo Fisher) in ExpicHO Expression Medium (Thermo Fisher) at 6 × 10 concentration ⁶ mL, obtain expichho cell suspension. pTT5 vector (Kinzhi, suzhou) containing a-chain and b-chain encoding genes (shown in SEQ ID NO:3 and SEQ ID NO:4, respectively) was added to 2mL of OptiSFM medium (Thermo Fisher) to obtain solution A. 160 μ L Expifeactinamine CHO transfection reagent (Thermo Fisher) was added to 2mL OptiSFM medium to obtain solution B. Then, the solution a and the solution B were mixed to obtain a transfection mixture, and the transfection mixture was added to 50mL of expichho cell suspension in total over 5 minutes. At 37 ℃ C, 8% CO ₂ After culturing under the conditions for 1 day, 8mL of Feed, 300. Mu.L of Enhancer (Thermo Fisher) was added, and the mixture was heated at 32 ℃ and 5% CO ₂ Culture supernatants were harvested after 9 days of culture under conditions, with 8mL of Feed added on day 5. Protein A purification column (GE) is used for affinity purification of fusion Protein from culture supernatant, and the amino acid sequences of the fragment a and the fragment b in the fusion Protein are respectively shown as SEQ ID NO 1 and SEQ ID NO 2. Among them, disulfide bonds are formed between C129 (fragment a) -C264 (fragment b) and C132 (fragment a) -C267 (fragment b), so that the fragment a and the fragment b form heterodimers (the structural schematic diagram is shown in fig. 1).

Example 1

This example is presented to illustrate the results of targeted recognition and binding of human peripheral blood NK cells by the fusion proteins provided by the present invention. The peripheral blood of the person used in this example was obtained from the first subsidiary hospital of medical university of Anhui, and informed consent of the relevant persons was obtained.

The test method comprises the following steps: human Peripheral Blood Mononuclear Cells (PBMC) were obtained by Ficoll gradient density centrifugation and diluted to 1X 10 with complete RPMI-1640 medium ⁷ /mL, 100U/mL IL-2 was added and incubated for 24 hours.

PBMC were diluted 5X 10 with 1 XPBS ⁶ To the flask, 90. Mu.L of the serum was added to a 1.5mL EP tube, and 10. Mu.L of the mouse serum was added thereto, followed by standing and blocking at 4 ℃ for 30 minutes. The fusion protein was added to the EP tube according to different concentration gradients and incubated for 30min at 4 ℃.

After the incubation is finished, the PBMC are washed twice by PBS, and the specific operation is as follows: to the EP tube was added 1mL of 1 XPBS, centrifuged at 4 ℃ and 250g for 5min, and the supernatant was discarded. After washing, 100. Mu.L of 1 XPBS-resuspended cells were added to each EP tube, and then 1. Mu.L of APC-labeled mouse anti-human IgG-Fc antibody (BioLegend) and 0.5. Mu.L of FITC-labeled mouse anti-human CD4 antibody (BD Co.) were added thereto, and the mixture was incubated at 4 ℃ for 30min in the dark. After incubation, PBMCs were washed twice with PBS. After washing, 200. Mu.L of 1 XPBS was added to each EP tube to resuspend the cells, and the binding ratio of the fusion protein to the antibody was measured at different concentrations by flow cytometry (model FACS Cantosys, BD Co.). Human IgG was used as a negative control for treatment and detection as described above, and the results are shown in FIG. 2.

Example 2

This example illustrates the results of the targeting recognition and binding of the fusion proteins provided by the invention to CHO-IL13R α 2 cells.

The specific test method was the same as in example 1, except that the NK cells were replaced with CHO-IL-13R α 2 cells (puromycin-resistant). The results are shown in FIG. 3.

The CHO-IL13R alpha 2 cells used in this example were obtained by the laboratory. The construction method comprises the following steps: the pLVX-EF1a-IRES-puro vector containing the IL13R α 2 coding sequence (synthesized by Kirgiz, suzhou) was co-transfected with the packaging plasmids pMD2G, psPAX2 (both from Youbao) into 293T cells (from ATCC). After 48h, culture supernatants containing the fulminant virus were harvested, and CHO-K1 cells (purchased from ATCC) were infected with the supernatants to obtain CHO-IL13 R.alpha.2 cells. Then, puromycin-resistant CHO-IL13R alpha 2 cells were obtained by puromycin (Invivogen) screening.

Example 3

This example illustrates the results of targeting, recognizing and binding a375 cells (melanoma cell line) and U251 cells (glioma cell line) by the fusion protein provided by the present invention.

The specific test method was the same as in example 1, except that the NK cells were replaced with A375 cells and U251 cells, respectively (both purchased from Shanghai cell Bank of Chinese academy of sciences). The results are shown in FIG. 4 (A375) and FIG. 5 (U251).

Example 4

This example illustrates the effect of the fusion proteins provided by the present invention on T cell anti-cancer activity. The peripheral blood of the person used in this example was obtained from the first subsidiary hospital of medical university of Anhui, and informed consent of the relevant persons was obtained.

The test method comprises the following steps: human Peripheral Blood Mononuclear Cells (PBMC) were obtained by Ficoll gradient density centrifugation and diluted to 1X 10 with complete RPMI-1640 medium ⁷ /mL, 100U/mL IL-2 was added and incubated for 24 hours.

(1) Tumor cells (A375 and U251, respectively, were obtained from Shanghai cell Bank of Chinese academy of sciences) were washed 2 times with complete RPMI-1640 medium, in the same manner as in example 1. Adding into real-time unmarked dynamic cell assay (RTCA) chip (from XCELLIGENCE), placing 10000 cells per well on RTCA instrument (from XCELLIGENCE, model: RTCA TP), and reacting at 37 deg.C and 5% CO ₂ The culture was carried out in an incubator for 24 hours.

(2) The PBMC were resuspended in complete RPMI-1640 medium and loaded onto RTCA chips at 100000 cells per well.

(3) The fusion protein was diluted with complete RPMI-1640 medium according to concentration gradient and added to the chip.

(4) Placing the chip on an RTCA apparatus at 37 deg.C, 5% ₂ The incubator was incubated for 40 hours.

(5) Data were collected, analyzed and the ratio of specific killing mediated by fusion protein at various concentrations at hour 40 was calculated. The calculation formula is as follows: specific killing ratio = (fusion protein group killing ratio-no fusion protein group killing ratio)/(100% -no fusion protein group killing ratio)

The results are shown in fig. 6 (a 375) and 7 (U251).

Example 5

This example illustrates the effect of the fusion protein provided by the present invention on the production of interleukin (IL-6) and tumor necrosis factor alpha (TNF-alpha) by PBMC cells. The peripheral blood of the person used in this example was obtained from the first subsidiary hospital of medical university of Anhui, and informed consent of the relevant persons was obtained.

The test method comprises the following steps: human Peripheral Blood Mononuclear Cells (PBMC) were obtained by Ficoll gradient density centrifugation and diluted to 1X 10 with complete RPMI-1640 medium ⁷ /mL, 100U/mL IL-2 was added and incubated for 24 hours.

(1) Tumor cells (A375 and U251, respectively, both from Shanghai cell Bank of Chinese academy of sciences) were washed 2 times with complete RPMI-1640 medium, in the same manner as in example 1. Then, 10000 cells per well were added to a 96-well plate.

(2) The PBMC were resuspended in complete RPMI-1640 medium and added to 96-well plates at 100000 cells per well.

(3) The% of CO in 96-well plates at 37 ℃ 5% ₂ The culture was carried out in an incubator for 48 hours.

(4) Culture supernatants were collected and assayed for IL-6 and TNF-. Alpha.content using CBA kit (BD).

(5) IL-6, TNF-alpha detection Beads were mixed in equal volumes and added to flow tubes at 20. Mu.L per tube.

(6) The standard, culture supernatant was added to flow tubes at 20. Mu.L per tube.

(7) The detection antibody was added to the flow tube at 20. Mu.L per tube.

(8) Vortex and mix well, then stand 3 hours at room temperature in the dark.

(9) The samples were examined by flow cytometry (model FACS Cantosys, BD) and the cytokine content of the supernatant was calculated.

Meanwhile, E13Y and T cell (CD 3) antibody fusion protein (the preparation method is 202110391048. X) is adopted to carry out detection according to the method to be used as a positive control, and human IgG is adopted to carry out detection according to the method to be used as a negative control. The results are shown in FIG. 8 (IL-6) and FIG. 9 (TNF-. Alpha.).

As can be seen from the figure, with the fusion protein provided by the present invention, the production amount of IL-6 was substantially equivalent to that of human IgG when the concentration of the fusion protein was 0.1. Mu.g/mL or less, and when the amount of the fusion protein was increased to 1. Mu.g/mL, the production amount of IL-6 was more than that of human IgG but still lower than that of IL-6 (about half or less) with the fusion protein using E13Y and T cell antibody. When the concentration of the fusion protein provided by the invention is 1 mu g/mL or less, the production amount of TNF-alpha is basically equal to that of human IgG, and when the concentration of the fusion protein reaches 10 mu g/mL, the production amount of TNF-alpha exceeds that of human IgG, but is still far lower than that of the fusion protein using E13Y and T cell antibody. Therefore, the fusion protein provided by the invention has a greatly reduced risk of generating cytokine storm when being used compared with the fusion protein of E13Y and T cell antibody, thereby having higher safety.

The results of the above examples show that the fusion protein provided by the invention can be used for identifying and combining various different tumor cells expressing IL13R alpha 2 in a targeted manner, and shows higher combination efficiency. Moreover, the fusion protein can promote NK cells to kill tumor cells expressing IL13R alpha 2 such as A375 and U251. Meanwhile, the fusion protein provided by the invention is not easy to cause side effects such as cytokine storm and the like, which shows that the fusion protein provided by the invention has the potential for preparing broad-spectrum, efficient and safe anticancer drugs.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

SEQUENCE LISTING

<110> first subsidiary hospital of medical university of Anhui

<120> fusion protein, encoding gene and recombinant vector thereof, and applications thereof

<130> I75775AYFY

<160> 4

<170> PatentIn version 3.5

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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-encoding 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 acggaggagg tggcagccct 360

aagtcctgtg acaagaccca 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 agaaccccag gtatacacac tgcctccctc tcgggaagaa 780

atgactaaga accaggtgtc cttgtcatgc gctgtaaagg 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-encoding 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 600

aagaacaccc tgtacctgca gatgaactca ctgagggctg aggataccgc cgtctattac 660

tgcgctaagg accgagggct gggagacggt acctatttcg attactgggg acagggcaca 720

acagtgaccg ttagctcagg cggcggagga agccctaaat cttgtgacaa aacacatact 780

tgtccaccat gccccgctcc agaattgttg ggcgggcctt cagttttcct cttcccccct 840

aagcctaagg acaccctgat gatcagcaga accccagagg tgacctgcgt ggtggtagcc 900

gtctctcacg aagaccccga agtgaagttc aactggtacg tcgacggggt ggaggtgcat 960

aatgccaaaa ccaagccccg tgaggaacaa tatgctagta cttaccgagt ggtgtccgta 1020

cttaccgtgc tgcaccagga ctggttgaac gggaaagaat acaagtgtaa agtatctaat 1080

aaagcactgc cagctccaat cgaaaagacc atctctaagg caaagggcca gcccagagaa 1140

ccccaggtct acactctgcc tccctctcgt gaagagatga ctaagaacca ggtcagtctg 1200

tggtgtttgg tgaaaggatt ttacccaagc gacatcgcag tggaatggga gagtaacggt 1260

cagccagaga ataattataa gacaactcct ccagtgctcg attctgatgg ttcctttttc 1320

ttgtattcta aactgacagt ggataaatca cggtggcagc agggcaacgt gttctcctgc 1380

tctgtgatgc atgaggctct gcacaaccac tacactcaga agagcctgag cctgagtcca 1440

gggaag 1446

Claims (10)

1. A fusion protein comprising the following fragments:

fragment a: the amino acid sequence is shown as SEQ ID NO. 1; and

fragment b: the amino acid sequence is shown as SEQ ID NO. 2.

2. The fusion protein of claim 1, wherein the fragment a and the fragment b are covalently linked by disulfide bonds of a hinge region of an Fc segment of human IgG;

and/or, the fusion protein further comprises an amino acid tag for purification.

3. A gene encoding a fusion protein according to claim 1 or 2.

4. The gene of claim 3, wherein the gene comprises:

(A) The nucleotide sequence of the coding segment a is shown as SEQ ID NO. 3;

(B) The nucleotide sequence of the coding segment b is shown as SEQ ID NO. 4.

5. A recombinant vector comprising the gene of claim 3 or 4.

6. Use of the fusion protein of claim 1 or 2, the gene of claim 3 or 4, or the recombinant vector of claim 5 for the preparation of a medicament for the treatment and/or prevention of a tumor.

7. The use according to claim 6, wherein the tumor is selected from tumors expressing interleukin 13 receptor alpha 2.

8. Use according to claim 6 or 7, wherein the tumour is selected from at least one of malignant glioma, melanoma, adrenocortical carcinoma, pancreatic carcinoma, squamous head and neck cell carcinoma, renal cell carcinoma and pancreatic ductal adenocarcinoma.

9. The use of claim 6, wherein the medicament further comprises a pharmaceutically acceptable carrier.

10. A pharmaceutical composition for treating and/or preventing tumor, comprising the fusion protein of claim 1 or 2, the gene of claim 3 or 4, or the recombinant vector of claim 5;

alternatively, the pharmaceutical composition contains only the fusion protein of claim 1 or 2, the gene of claim 3 or 4, or the recombinant vector of claim 5 as an active ingredient.

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