WO2016138610A1 - Albumin conjugated tumor necrosis factor related apoptosis-inducing ligand variant, preparation method therefor and use thereof - Google Patents

Abstract

Provided is an albumin conjugated tumor necrosis factor related apoptosis-inducing ligand variant, and the variant is a fusion protein of tumor necrosis factor related apoptosis-inducing ligand and albumin binding domain. Also provided are a nucleotide sequence encoding the variant, a recombinant vector and a recombinant bacteria comprising the nucleotide sequence, and a preparation method for the variant and a use of the variant.

Description

Albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant, preparation method and use thereof Technical field

The invention relates to the field of genetic engineering, in particular to an albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant, a preparation method thereof and use thereof.

Background technique

In recent years, the number of new malignant tumors worldwide has been rising. Due to limited treatment, malignant tumors have become the number one killer of human life. Surgery combined with traditional radiotherapy and chemotherapy methods is still the main means of current cancer treatment. The biggest drawback of traditional chemoradiotherapy is the lack of selectivity. While killing tumor cells, it also harms a large number of normal cells, and long-term use of toxic side effects. Therefore, there is an urgent need to develop selective and highly potent anti-tumor drugs (Corti A et al., Medicinal Research Reviews, 2012, 32: 1078-1091).

TNF related apoptosis-inducing ligand (TRAIL) is a protein with high selectivity to kill tumor cells. It transmits death signals through the death receptors DR4 and DR5 on the surface of tumor cells, and induces tumor cell apoptosis. The death receptors DR4 and DR5 are highly expressed on the surface of tumor cells and are lowly expressed on the surface of normal cells. Moreover, the normal cell surface also highly expresses TRAIL decoy receptors DcR1 and DcR2. TRAIL binds to decoy receptors but does not transmit death signals. Therefore, TRAIL is highly toxic or low-toxic to most normal cells while killing tumor cells efficiently. It has strong tumor cell killing selectivity and is a promising new anti-tumor drug (Stuckey D W, et al., Trends in Molecular Medicine, 2013, 19(11): 685-694; Ashkenazi A., Nature Reviews Drug Discovery, 2008, 7(12): 1001-1012.).

As a novel candidate anti-tumor drug, genetically engineered human soluble TRAIL (human TRAIL, hTRAIL) has entered the clinical phase I-II trial (Soria J C et al., J Clin Oncol 2011, 29:4442-4451) . The results showed that hTRAIL has good safety and many tumor patients respond to the treatment of hTRAIL. However, hTRAIL has a small molecular weight (about 20 KD per monomer) and is easily cleared by the kidneys in the body, and its half-life in plasma is less than 30 minutes (Xiang H, et al. Drug metabolism and disposition, 2004, 32(11): 1230-1238.). The "slow half-life" characteristic greatly impairs the in vivo anti-tumor effect of hTRAIL. Therefore, prolonging the half-life of hTRAIL and increasing its anti-tumor effect has become a new research hotspot.

Previous studies have extended hTRAIL half-life by polyethylene glycol (PEG) modification and nanoparticle encapsulation (Chae S Y, et al. Molecular cancer therapeutics, 2010, 9(6): 1719-1729; Lim S M, et al. Biomaterials, 2011, 32(13): 3538-3546). More and more research has found that the use Serum albumin as an antitumor drug carrier may increase the half-life of the tumor and may increase the drug absorption of the tumor. Serum albumin is the most abundant protein in mammalian serum that binds to the FcRn receptor. In addition, albumin can also be enriched in tumors and sites of inflammation. Therefore, after the drug forms a complex with albumin, it may not only prolong the half-life of the drug, but also enrich the tumor site, thereby enhancing the anti-tumor effect (Kratz F. Journal of Controlled Release, 2014).

Methods for preparing hTRAIL-albumin complexes include: 1) direct coupling of hTRAIL to serum albumin; 2) recombinant expression of hTRAIL and albumin encoding gene (Müller N, et al. Biochemical and Biophysical Research Communications, 2010) , 396(4): 793-799). The direct coupling method consumes a large amount of albumin, and the recombinant expression of TRAIL and albumin gene is limited. Moreover, the introduction of exogenous serum albumin increases the risk of serious infection.

Recent studies have found that the fusion of an albumin-binding domain with a protein/peptide molecule may allow protein/peptide molecules to bind to endogenous albumin, thereby extending half-life (Sleep D et al., Biochimica et Biophysica Acta 1830 (2013) 5526–5534). To date, there have been no reports of using this method to extend the half-life of TRAIL to enhance its anti-tumor effect.

Summary of the invention

In order to solve the above problems, the present invention provides an albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant, a preparation method thereof and use thereof.

The albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variant of the invention is a fusion protein of a tumor necrosis factor-related apoptosis-inducing ligand and an albumin binding domain, and the albumin binding domain is linked to the tumor necrosis factor via a linker The N-terminus of the related apoptosis-inducing ligand.

Wherein the amino acid sequence of the tumor necrosis factor-related apoptosis inducing ligand is as shown in SEQ ID NO: 2 or 4.

Wherein the amino acid sequence of the albumin binding domain is as shown in SEQ ID NO: 6.

Wherein the albumin binding domain is encoded by the nucleotide sequence set forth in SEQ ID NO: 5.

Wherein the linker consists of 2-20 amino acids. Preferably, the linker is a (G4S) ₃ linker, the amino acid sequence of which is set forth in SEQ ID NO: 8.

Wherein the albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant is encoded by the nucleotide sequence set forth in SEQ ID NO: 9 or 11. Its amino acid sequence is shown as SEQ ID NO: 10 or 12.

The present invention also provides a nucleotide sequence comprising a coding sequence of a tumor necrosis factor-related apoptosis inducing ligand and a coding sequence of an albumin binding domain, which are linked by a coding sequence of a linker.

Wherein, the coding sequence of the tumor necrosis factor-related apoptosis inducing ligand is as shown in SEQ ID NO: 1 or 3.

Wherein, the albumin binding domain coding sequence is as shown in SEQ ID NO: 5.

Wherein the linker is a (G4S) ₃ linker, and the nucleotide sequence thereof is shown in SEQ ID NO: 7.

Wherein the nucleotide sequence is as shown in SEQ ID NO: 9 or 11.

The present invention also provides a recombinant vector or recombinant strain of the aforementioned nucleotide sequence.

The present invention also provides a method for preparing the aforementioned albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variant, which is prepared by genetic engineering using the aforementioned nucleotide sequence as a target fragment.

The present invention also provides the use of the aforementioned albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant in the preparation of a medicament for treating a cell proliferative disorder.

Among them, the drug for treating a cell proliferative disease is a drug for treating a tumor or an autoimmune disease.

The present invention also provides an antitumor drug which is prepared by using the aforementioned albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variant as an active ingredient, together with a pharmaceutically acceptable adjuvant.

At present, studies have extended the TRAIL half-life to enhance the anti-tumor effect by covalently linking hTRAIL to human albumin HSA to form a complex. However, this method consumes a lot of HSA, and the introduction of HSA from other people's blood sources may increase the risk of infection. The method utilizes the method of adding albumin binding domain to make TRAIL enter the body and use endogenous albumin to prolong the half-life and enhance the anti-tumor effect, and the cost is low, and there is no risk of infection.

Although the preparation of fusion protein by ligation with albumin binding domain is one of the methods to improve the half-life of target protein drugs in the field, it is not easy to construct a fusion protein with prolonged half-life and enhanced pharmacodynamic activity. The key difficulty lies in: After the protein is ligated, the albumin binding domain itself may affect the activity of the target protein. At the same time, the modified target protein may also affect its activity after binding to albumin, and the structure of the protein is complex, and the interaction between protein and protein is also It is unclear, therefore, which albumin binding domain is selected, the fusion protein which is obtained by extending the half-life of the target protein and having excellent pharmacodynamic activity is uncertain, and it is highly probable that an inactive fusion protein is obtained. For example, the present inventors screened an albumin binding domain SA21 in the early stage, and did not significantly prolong the half-life of hTRAIL after ligation with hTRAIL; in addition, the albumin binding domain ABD screened by the present invention was ligated to the C-terminus of hTRAIL, not only Significantly prolonged half-life also reduced the in vivo anti-tumor activity of hTRAIL. These facts indicate that it is not easy to construct a fusion protein with increased half-life and enhanced anti-tumor activity by adding an albumin binding domain.

However, the present invention obtains a specific albumin binding domain and optimizes its nucleotide sequence, and at the same time, the fusion protein prepared by fusion with TRAIL under a specific linkage mode has a long half-life and excellent antitumor activity, and has achieved unexpected results. Less technical effects.

The invention prepares pure albumin-bound tumor necrosis factor by genetic engineering method Sub-related apoptosis-inducing ligand variants: ABD-hTRAIL and ABD-mmTRAIL, compared with hTRAIL and mmTRAIL, their half-life is significantly prolonged, and the anti-tumor activity in vivo is also significantly enhanced, and the pharmacodynamic and pharmacokinetic properties are excellent. The clinical application prospects are good.

DRAWINGS

Figure 1 ABD-hTRAIL purified SDS-PAGE gel electrophoresis. M: protein molecular weight standard; 1: hTRAIL; 2: ABD-hTRAIL

Figure 2 ABD-mmTRAIL purified SDS-PAGE gel electrophoresis. M: protein molecular weight standard; 1: ABD-mmTRAIL; 2: mmTRAIL

Figure 3 ABD-hTRAIL and hTRAIL are compared to albumin binding ability. A: gel filtration method; B: ELISA plate method

Figure 4 is a comparison of the binding ability of ABD-mmTRAIL and mmTRAIL to albumin. A: Gel filtration method B: ELISA plate method

Figure 5 Effect of albumin binding on the killing activity of ABD-hTRAIL cells

Figure 6 Effect of albumin binding on the killing activity of ABD-mmTRAIL cells

Figure 7 Comparison of in vivo metabolic rates of ABD-hTRAIL and hTRAIL

Figure 8 Comparison of in vivo metabolic rates between ABD-mmTRAIL and mmTRAIL

Figure 9 ABD-hTRAIL and hTRAIL tumor uptake and tissue distribution comparison. 1: heart, 2: liver, 3: spleen, 4: lung, 5: kidney, 6: small intestine, 7: colon, 8: muscle, 9: brain, 10: tumor

Figure 10 ABD-mmTRAIL and mmTRAIL tumor uptake and tissue distribution comparison. 1: heart, 2: liver, 3: spleen, 4: lung, 5: kidney, 6: small intestine, 7: colon, 8: brain, 9: muscle, 10: tumor

Figure 11 Comparison of anti-tumor effects of ABD-hTRAIL and hTRAIL in vivo (arrow indicates administration time). A: tumor growth curve after treatment; B: treatment end tumor size

Figure 12 Comparison of in vivo antitumor effects of ABD-mmTRAIL and mmTRAIL (arrows indicate administration time). A: tumor growth curve after treatment; B: treatment end tumor size

detailed description

The above content of the present invention will be further described in detail below by way of specific embodiments in the form of embodiments. However, the scope of the above-mentioned subject matter of the present invention should not be construed as being limited to the following embodiments. Any technique implemented based on the above description of the present invention is within the scope of the present invention.

Example 1 Preparation of Albumin-bound TRAIL Variant of the Invention

1. Design and gene cloning of albumin-binding TRAIL variants

The albumin-binding domain (ABD) was ligated to hTRAIL or mmTRAIL to prepare albumin-bound TRAIL variants, ABD-hTRAIL and ABD-mmTRAIL.

hTRAIL and mmTRAIL are fragments of human and monkey full-length TRAIL 114-281 amino acids. The ABD was ligated to the N-terminus of hTRAIL or mmTRAIL via (G4S)3, and the resulting fusion proteins were named ABD-hTRAIL and ABD-mmTRAIL, respectively. Fusion protein for cloning BamHI and NotI restriction sites were added to both ends of the coding gene. The gene was commissioned by Nanjing Jinsirui Company for synthesis. After the recombinant gene was digested with BamH/NotI, the DNA fragment was recovered by agarose gel, and then ligated with the pQE30 vector which was also digested and recovered by gel, and the ligated product was transformed into TOP10. Positive monoclonal strains were initially identified by ampicillin screening (100 μg/ml) and double restriction enzyme digestion, and positive clones were further verified by DNA sequence analysis. The plasmid containing the ABD-hTRAIL or ABD-mmTRAIL coding gene was designated as pQE30-ABD-hTRAIL or pQE30-ABD-mmTRAIL, respectively.

Table 1 The present invention relates to amino acid and nucleic acid sequences

2. Induced expression, separation and purification of albumin-bound TRAIL variants

The expression plasmid was extracted, the expression strain M15 was transformed, and a positive clone strain was obtained by screening with LB plate medium containing ampicillin (100 μg/ml) and kanamycin (30 μg/ml). The positive clone strain was inoculated into LB liquid medium containing the same antibiotic, and cultured at 37 ° C until the bacterial solution A ₆₀₀ reached 0.5 to 1, and 0.05-1 mM isopropylthiogalactoside (IPTG) was added to induce expression. After 4 hours, the cells were collected by centrifugation. The obtained cells were resuspended in phosphate buffer (50 m M, pH 8.0). After ultrasonic disruption, the supernatant was collected by centrifugation (20000 g, 15 min). Ni-NTA Super Flow gel was added to the supernatant and slowly shaken at 4 ° C for 3 h. The gel was packed, the heteroprotein was washed with 40 mM imidazole, and eluted with 300 mM imidazole to obtain the protein of interest. The purified protein was endotoxin removed using a de-endotoxin kit (Nanjing Kingsray).

As shown in Fig. 1 and Fig. 2, the molecular weights of hTRAIL and mmTRAIL were about 20 KD, and the albumin-bound TRAIL variants formed by fusion of ABD, that is, ABD-hTRAIL and ABD-mmTRAIL had molecular weights of about 25 KD.

The experimental results show that the albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variants ABD-hTRAIL and ABD-mmTRAIL were prepared by the present invention.

The beneficial effects of the present invention will be described below by way of experimental examples:

Experimental Example 1 Binding of albumin-bound TRAIL variants to human serum albumin

1. Test method

1) Gel filtration

ABD-hTRAIL or ABD-mmTRAIL and human serum albumin (HSA) were diluted to the same level with phosphate buffer (10 mM Na ₂ HPO ₄ , 2.68 mM KCl, 2 mM KH ₂ PO ₄ ) containing 500 mM NaCl. The molar concentration is then mixed in equal volumes. After 10 to 30 min of binding at room temperature, the mixture sample was analyzed on a gel filtration column Superdex 75 (GE Healthcare). hTRAIL and mmTRAIL were used as controls for the fusion proteins ABD-hTRAIL and ABD-mmTRAIL, respectively.

As a result, as shown in Fig. 3A, after hTRAIL was mixed with HSA (hTRAIL + HSA), gel filtration analysis revealed that no complex having a molecular weight significantly larger than HSA was formed, suggesting that hTRAIL did not bind to HSA. When ABD-hTRAIL was mixed with HSA (ABD-hTRAIL+HSA), the molecular weight of the complex formed was much larger than that of HSA, suggesting that ABD-hTRAIL binds to HSA.

Under the same conditions, as shown in Figure 4A, the mixture of mmTRAIL and HSA showed a double peak on the gel filtration column with molecular weight corresponding to HSA and mmTRAIL, suggesting that mmTRAIL did not bind to HSA. The molecular weight of the main component of ABD-mmTRAIL and HSA mixture is much larger than that of HSA, suggesting that ABD-mmTRAIL binds to HSA.

2) ELISA plate method

The HSA was dissolved to 20 mg/ml with phosphate buffer PBS (137 mM NaCl, 10 mM Na ₂ HPO ₄ , 2.68 mM KCl, 2 mM KH ₂ PO ₄ ). HSA (100 μl/well) was added to the plate and coated overnight at 4 °C. The plate was washed twice with PBS (200 μl/well), and 100 μl of different concentrations of fusion protein were added. After incubating for 2 h at 37 ° C, the plate was washed 4 times with PBST (PBS + 0.075% Tween 20), and then incubated with mouse anti-TRAIL antibody for 1 h. After washing the plate with PBST, the horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody was added for incubation. Finally TMP chromogenic substrate, 2M phosphate color reaction was stopped, measuring A _450nm microplate reader. hTRAIL and mmTRAIL were used as controls.

2, test results

As a result, as shown in Figs. 3B and 4B, hTRAIL and mmTRAIL were incubated with HSA and detected with an anti-TRAIL antibody, and the color reaction did not change with an increase in protein concentration. However, as the concentration of ABD-hTRAIL and ABD-mmTRAIL increased, the A _{450 nm} absorption value also increased accordingly. This result indicates that hTRAIL and mmTRAIL hardly bind to HSA. ABD-hTRAIL and ABD-mmTRAIL can be combined with HSA.

The experimental results indicate that the albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variants prepared by the present invention: ABD-hTRAIL and ABD-mmTRAIL all have albumin binding ability.

Experimental Example 2 In vitro cell killing activity of albumin-bound TRAIL variants

1. Test method

Colon cancer Colo205 cells were seeded in RPMI 1640 and cultured at 37 ° C, 5% CO ₂ . ABD-hTRAIL and ABD-mmTRAIL were mixed with has 1:1, respectively, and added to the cells (20000 cells/well) after 30-60 min at room temperature. After overnight action, 10 ul of CCK8 was added to the wells to determine cell viability.

2, test results

As shown in Fig. 5 and Fig. 6, ABD-hTRAIL and ABD-mmTRAIL bind to proteins and have a killing effect on tumor cells.

The experimental results show that the albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variants prepared by the present invention: ABD-hTRAIL and ABD-mmTRAIL have tumor cell killing activity after binding to albumin, and can be used for anti-tumor in vivo. .

Experimental Example 3 In vivo metabolism of albumin-bound TRAIL variants

1. Test method

BALB/c mice were divided into two groups. One group was injected with hTRAIL or mmTRAIL protein at a tail vein of 10 mg/kg. The other group injected the same amount of ABD-hTRAIL or ABD-mmTRAIL. Blood was taken at different time points after injection, heparin was anticoagulated, and mouse plasma was obtained by centrifugation. After the mouse plasma was diluted 25, 50, 100 and 250 times with RMPI1640 medium, the killing activity was measured using Colo205 cells. The rate of protein metabolism in vivo is monitored by changes in residual protein activity in plasma.

2, test results

Results As shown in Figures 7A and 8A, the metabolic rates of hTRAIL and mmTRAIL were similar. After intravenous injection, the protein content in the plasma drops rapidly. After 4 hours of intravenous injection, even if the serum was only diluted 25-fold, the tumor cell killing activity was not detected, suggesting that the residual amount of hTRAIL and mmTRAIL protein in the plasma was small. Figures 7B and 8B show that ABD-hTRAIL and ABD-mmTRAIL are much more slowly metabolized in plasma. Even after 48 hours, diluted 250 times, the killing of tumor cells by residual proteins in plasma can be detected.

The above results indicate that the albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variants prepared by the present invention, ABD-hTRAIL and ABD-mmTRAIL, have slow metabolism and long half-life in vivo compared with hTRAIL and mmTRAIL. The retention time in the body is long.

Experimental Example 4 Tumor absorption and tissue distribution of albumin-bound TRAIL variants

1. Test method

ABD-hTRAIL and hTRAIL were labeled with CF750 fluorescent dye, respectively. 5×10 ⁵ Colo205 cells (100 μl) were inoculated subcutaneously into the right hind limb of nude mice. When the tumor grew to a size of 100-200 mm ³ , 50 ug of CF750-labeled protein was administered by tail vein injection, and then at 2, 4, 8 , 24h in vivo imaging scan to observe the protein intake by the tumor. At the end of the last scan (24h), the mice were sacrificed and the organs and tissues were removed for scanning to examine the tissue distribution of the protein.

2, test results

As shown in Figure 9, hTRAIL and ABD-hTRAIL were detected in the tumor 2 hours after intravenous injection. At 4-8h, the amount of hTRAIL uptake by the tumor increased compared with 2h. However, at 24h, the amount of hTRAIL in the tumor was significantly reduced. Unlike hTRAIL, ABD-hTRAIL uptake reached the peak during 4-8h, but the amount of protein did not decrease significantly after 24h. Moreover, at the same time point, the tumors ingested more ABD-hTRAIL than hTRAIL. After 24 hours, the mouse tissue was removed and found to have more ABD-hTRAIL content than hTRAIL. In addition, the two proteins are mainly distributed in the liver and kidney of the metabolic organs. Consistent with in vivo imaging observations, tissue-organ scans also demonstrated that ABD-hTRAIL is more readily enriched in tumor tissue than hTRAIL.

ABD-mmTRAIL and mmTRAIL show the same pattern by tumor absorption and tissue distribution. As a result, as shown in FIG. 10, both in vivo imaging and tissue distribution showed that ABD-mmTRAIL was more easily enriched in tumor tissues than mmTRAIL.

The test results indicated that the albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variants: ABD-hTRAIL and ABD-mmTRAIL prepared by the present invention were more targeted than hTRAIL and mmTRAIL.

Experimental Example 5 Anti-tumor effect of albumin-binding TRAIL variant in vivo

1. Test method

5 × 10 ⁵ Colo205 cells (100 μl) were inoculated subcutaneously into the right hind limb of nude mice and randomly divided into groups. At a certain time after inoculation, 5 mg/kg protein was administered by tail vein injection, and the control group was given the same volume of PBS. Tumor size was recorded daily and mice were sacrificed at 24 days and tumors were taken.

2, test results

As shown in Fig. 11, once on the 6th day and the 11th day after inoculation, the tumor growth trend of the ABD-hTRAIL group was significantly delayed compared with the hTRAIL group. By day 24, the sizes of the PBS control group, hTRAIL group and ABD-hTRAIL group were: 674.3 ± 194.1 mm ³ , 546.1 ± 265.1 mm ³ and 65.6 + 41.7 mm ³ (Fig. 11A). Tumor size observations were consistent with growth curve results (Figure 11B). In the PBS control group, the tumor weights of the hTRAIL group and the ABD-hTRAIL group were 0.367±0.06 g, 0.32±0.09 g, and 0.016±0.02 g, respectively.

Figure 12 shows that ABD-mmTRAIL is similar to ABD-hTRAIL treatment. On the 5th day and the 9th day after the inoculation, the tumor growth was significantly slower than that of the mmTRAIL treatment group in the ABD-mmTRAIL group. At the end of the 24 day observation, the tumor sizes of the PBS control, mmTRAIL group and ABD-mmTRAIL group were 925.7 ± 222.7 mm ³ , 642.4 ± 194.5 mm ³ and 152.3 ± 44.7 mm ^{3 , respectively} (Fig. 12A). Tumor size was consistent with tumor growth curve results (Figure 12B). The average weight of the tumor in the PBS control group, mmTRAIL group and ABD-mmTRAIL group was 0.539±0.069 g, 0.341±0.089 g, and 0.088+0.028 g, respectively.

The above results indicate that the albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variants prepared by the present invention: ABD-hTRAIL and ABD-mmTRAIL have stronger in vivo antitumor activity than hTRAIL and mmTRAIL.

The invention provides a pure albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variant: ABD-hTRAIL and ABD-mmTRAIL by genetic engineering, and their half-life is significantly prolonged compared with hTRAIL and mmTRAIL. At the same time, the anti-tumor activity in the body is also obviously enhanced, the pharmacodynamics and pharmacokinetic performance are excellent, and the clinical application prospect is good.

Claims (18)

1. An albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variant characterized by a fusion protein of a tumor necrosis factor-related apoptosis-inducing ligand and an albumin binding domain, and an albumin binding domain is linked by a linker The N-terminus of the tumor necrosis factor-related apoptosis-inducing ligand.
2. The albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant according to claim 1, wherein the amino acid sequence of the tumor necrosis factor-related apoptosis inducing ligand is as shown in SEQ ID NO: 2 or 4. Show.
3. The albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant according to claim 1, wherein the amino acid sequence of the albumin binding domain is as shown in SEQ ID NO: 6.
4. The albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant according to claim 3, wherein the albumin binding domain is encoded by the nucleotide sequence shown in SEQ ID NO: 5.
5. The albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant according to claim 1, wherein the linker is composed of 2 to 20 amino acids.
6. The albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant according to claim 5, wherein the linker is a (G4S) ₃ linker, and the amino acid sequence thereof is as shown in SEQ ID NO: 8. .
7. The albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant according to claim 1, wherein the amino acid sequence is as shown in SEQ ID NO: 10 or 12.
8. The albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant according to claim 7, which is encoded by the nucleotide sequence shown in SEQ ID NO: 9 or 11.
9. A nucleotide sequence characterized in that it comprises a coding sequence for a tumor necrosis factor-related apoptosis-inducing ligand and a coding sequence for an albumin binding domain, which are linked by a coding sequence of a linker.
10. The coding sequence according to claim 9, wherein the coding sequence of the tumor necrosis factor-related apoptosis inducing ligand is as shown in SEQ ID NO: 1 or 3.
11. The coding sequence according to claim 9, wherein the coding sequence of the albumin binding domain is as shown in SEQ ID NO: 5.
12. The nucleotide sequence according to claim 9, wherein the linker is a (G4S) ₃ linker, and the nucleotide sequence thereof is shown in SEQ ID NO: 7.
13. The nucleotide sequence according to claim 9, which is represented by SEQ ID NO: 9 or 11.
14. A recombinant vector or recombinant strain comprising the nucleotide sequence of any one of claims 9 to 13.
15. A method for producing an albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant according to any one of claims 1 to 8, which is characterized in that it is a nucleoside according to any one of claims 9 to 13. The acid sequence is the target fragment and is prepared by genetic engineering.
16. Use of an albumin-binding tumor necrosis factor-related apoptosis inducing ligand variant according to any one of claims 1 to 8 for the preparation of a medicament for treating a cell proliferative disorder.
17. The use according to claim 16, wherein the drug for treating a cell proliferative disease is a drug for treating a tumor or an autoimmune disease.
18. An antitumor drug characterized by comprising an albumin-binding tumor necrosis factor-related apoptosis-inducing ligand variant according to any one of claims 1 to 8 as an active ingredient, together with a pharmaceutically acceptable excipient Prepared preparation.

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