阅读说明：本技术 纳米抗体靶向性泛素化降解细胞内源性Survivin的组合物及其应用 (Composition for degrading endogenous Survivin of cells through targeted ubiquitination of nano antibody and application of composition ) 是由 马兴元 刘畅 郑文云 胡发彪 郭伟 刘秋丽 付云辉 于 2020-05-18 设计创作，主要内容包括：本发明提供纳米抗体靶向性泛素化降解细胞内源性Survivin的组合物,包括Survivin纳米抗体、Fc结构域、T2A多肽和TRIM21,所述组合物优选为Nb4A-Fc-T2A-TRIM21。本发明采用纳米抗体和TRIM21共表达的方式,一方面可以提供纳米抗体以结合Survivin抗原,Fc片段以结合TRIM21；另一方面如果细胞内源性TRIM21水平不足以进行蛋白质的泛素化降解,则TRIM21可得以补充,维持泛素化的正常进行。此方式达到了靶向细胞内源性Survivin的泛素化降解的目的,为下游研究Survivin降解后对细胞产生的影响打下了坚实的基础,提示具有较强的药物应用前景。(The invention provides a composition for degrading endogenous Survivin of cells through targeted ubiquitination of nano antibodies, which comprises Survivin nano antibodies, an Fc domain, a T2A polypeptide and TRIM21, wherein the composition is preferably Nb4A-Fc-T2A-TRIM 21. According to the invention, a mode of co-expression of a nano antibody and TRIM21 is adopted, on one hand, the nano antibody can be provided to be combined with Survivin antigen, and the Fc fragment is combined with TRIM 21; on the other hand, if the level of cellular endogenous TRIM21 is not sufficient for ubiquitination degradation of proteins, TRIM21 can be supplemented to maintain normal ubiquitination. The method achieves the purpose of target cell endogenous Survivin ubiquitination degradation, lays a solid foundation for downstream research on the influence of Survivin degradation on cells, and prompts a strong medicine application prospect.)

1. The composition of the endogenous Survivin of the cells degraded through targeted ubiquitination of the nano-antibody is characterized by comprising the Survivin nano-antibody, an Fc domain, a T2A polypeptide and TRIM21, wherein the sequence of the Survivin nano-antibody is shown in any one of SEQ ID No.1, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8.

2. The composition for the targeted ubiquitination degradation of endogenous Survivin in cells of claim 1, wherein the composition is Nb4A-Fc-T2A-TRIM21, the sequence of Nb4A is shown in SEQ ID No.1, and GSG is added before the T2A polypeptide.

3. A recombinant plasmid comprising the composition of any one of claims 1-2.

4. The recombinant plasmid of claim 3, which is pcDNA3.1(+) -Nb4A-Fc-T2A-TRIM 21.

5. The preparation method of the composition of the nano-antibody targeted ubiquitination degradation of the endogenous Survivin of the cells of the claims 1-2, wherein the nano-antibody is fused with the gene sequence of the Fc region of the antibody, and then is connected with the T2A small peptide to realize the co-expression with the TRIM21, and three amino acids of GSG are added in front of the T2A polypeptide.

6. The use of the composition of the nano-antibody targeting ubiquitination degradation of cell endogenous Survivin or the recombinant plasmid thereof as claimed in any one of claims 1 to 2 in the preparation of a gene medicament for treating Survivin over-expressed tumors.

7. The use of claim 6, wherein the Survivin overexpressing tumor comprises lung cancer, pancreatic cancer, liver cancer, breast cancer, and bladder cancer.

Technical Field

The invention relates to the field of biotechnology tumor treatment, in particular to a composition for degrading endogenous Survivin of cells through targeted ubiquitination of a nano antibody and application thereof.

Background

Survivin (Survivin) is the smallest member of the Inhibitor of Apoptosis Protein (IAP) family and is also the most potent IAP family molecule, Survivin is structurally very unique compared to other inhibitors of apoptosis of the IAP family, contains only a single BIR domain and a carboxy-terminal helix structure, and has no other identifiable protein domains. Survivin protein is encoded by BIRC5 gene, consists of 4 exons and 3 introns, and is located in the telomere region of chromosome 17. Survivin is highly expressed in most human cancer cells, but hardly expressed in normal, terminally differentiated adult tissues, and Survivin protein has the functions of inhibiting tumor cell apoptosis, inhibiting autophagy, promoting tumor cell proliferation, promoting tumor chemotherapy resistance and the like, thereby becoming a new target for tumor therapy. Research shows that reduction of the expression level of cell endogenous Survivin can inhibit cell proliferation and promote apoptosis, so that research on degradation of Survivin protein can provide a new idea for development of novel antitumor drugs.

Today there are many strategies to inhibit Survivin expression, which can be summarized in the following four categories: (1) survivin transcription inhibitors: a variety of Survivin antisense oligonucleotides have been designed and successfully delivered to cells via vectors, which significantly inhibit the expression of Survivin protein and ultimately promote apoptosis and inhibit cell proliferation; ribozymes are a class of small RNA molecules that are capable of cleaving a target RNA by the activity of endonucleases, however, due to their limitations (e.g., susceptibility to degradation and abnormal cellular trafficking), none of the ribozymes currently enter clinical trials; further, siRNA is included. (2) Post-translational levels of Survivin inhibitors: CDK inhibitors and Hsp90 inhibitors, many CDK inhibitors such as pyriproxyfen and pravastatin are able to inhibit mitotic phosphorylation of Survivin at the Thr34 site, thereby accelerating the degradation of Survivin. (3) Survivin protein based vaccines: activation of apoptosis in tumor cells transfected with Survivin-containing vectors; expression of cytotoxic genes in tumor cells driven by Survivin promoters. (4) Gene therapy and Survivin mutants: based on the specific recognition of T lymphocytes to specific tumor-associated antigens, early studies show that a vaccine based on Survivin has significant cytotoxic activity to hepatoma cells; a negative dominant mutant of Survivin may also inhibit the functional activity of Survivin by forming a heterodimer with the endogenous Survivin of the cell.

Based on the methods, a method for rapidly degrading Survivin at a protein level is lacked at present, and a real post-translational protein consumption method based on nano antibody protein targeting is developed, so that endogenous Survivin protein of a target cell is rapidly degraded.

Antibodies bind proteins with high affinity and specificity, and thus, are the basis for targeting proteins of interest. The use of antibodies to interfere with protein function has been previously investigated, however, this requires that the antibody bind to an epitope that prevents protein function and effectively compete stoichiometrically with endogenous ligands, and therefore this method of inhibition is only applicable to a very limited number of proteins. Since antibody-bound proteins are recognized by the cytosolic antibody receptor TRIM21, TRIM21 is E3 ubiquitin ligase, which binds with high affinity to the Fc domain of the antibody, TRIM21 will recruit the ubiquitin-proteasome system to the antibody-bound proteins, causing them to be degraded. As early as 2007, the literature suggests that IAP-XAFl complexes degrade the cellular endogenous Survivin proteins at the protein level, mediated by E3 ligase and proteasome; recent studies have also shown that cytoplasmic E3 ubiquitin ligase CRL9 can promote ubiquitination degradation of Survivin protein to maintain cellular microtubule and genomic stability, thereby inhibiting tumor progression.

In the early stage of the laboratory, human Survivin is used as an antigen, a series of nano antibodies are screened from a random heptapeptide and dodecapeptide phage display library, the affinity and specificity of the nano antibodies to Survivin are strong, 10 chimeric nano antibodies are obtained in total, and 5 nano antibodies with higher affinity to Survivin are finally obtained through affinity determination, wherein the nano antibodies are Nb1A, Nb2A, Nb4A, Nb1B and Nb4B respectively, the sequences of the nano antibodies are shown as SEQ ID No.5, SEQ ID No.6, SEQ ID No.1, SEQ ID No.7 and SEQ ID No.8 respectively, and Nb4A is the optimal nano antibody for targeting Survivin. [1 ] the article-Zhang N, Guo H, ZHEN W, et al, design and screening of a polymeric survivin-specific oligonucleotide and its antagonist activities in vitro [ J ] Anti Cancer Drugs,2016,27(9): 839-847; 2. a patent-targeted survivin nano antibody and a preparation method and application thereof, Maxingyuan, Zhang Na, Wudong, Zhengwenyun, Guoshua and Hufa Biao; 3. academic paper-zhang na university of eastern china: development of Survivin targeting chimeric nano-antibody based on camel antibody structure ].

Disclosure of Invention

The first purpose of the invention is to provide a composition for degrading cell endogenous Survivin through nano-antibody targeting ubiquitination.

The second purpose of the invention is to provide a recombinant plasmid containing the composition of the nano-antibody targeting ubiquitination degradation of the endogenous Survivin of the cell.

The third purpose of the invention is to provide a preparation method of the composition for degrading the endogenous Survivin of the cells by the targeting ubiquitination of the nano antibody.

The fourth purpose of the invention is to provide the application of the composition of the nano antibody targeting ubiquitination degradation of the cell endogenous Survivin or the recombinant plasmid thereof in the preparation of the gene medicine for treating Survivin over-expressed tumors.

In order to achieve the first object, the invention provides a composition of a nano-antibody targeted ubiquitination degradation of endogenous Survivin of cells, which comprises a Survivin nano-antibody, an Fc domain, a T2A polypeptide and TRIM21, wherein the Survivin nano-antibody has a sequence shown in any one of SEQ ID No.1, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8.

As a preferable scheme, the composition is Nb4A-Fc-T2A-TRIM21, the sequence of Nb4A is shown as SEQ ID NO.1, and three amino acids of GSG are added in front of the T2A polypeptide.

The Fc is a constant region of the antibody, can be an Fc fragment of any antibody, and the sequence used by the invention is derived from the Fc region of the anti-PD-L1 antibody, and the sequence is shown as SEQ ID NO. 2.

In order to achieve the second object, the invention provides a recombinant plasmid containing a composition of the nano-antibody targeting ubiquitination degradation of endogenous Survivin of cells.

As a preferred scheme, the recombinant plasmid is pcDNA3.1(+) -Nb4A-Fc-T2A-TRIM 21.

In order to achieve the third purpose, the invention provides a preparation method of the composition of the nanometer antibody targeting ubiquitination degradation of endogenous Survivin of cells, which utilizes the targeting property of the nanometer antibody of Survivin on Survivin and the specific binding capacity of the Fc region of the antibody and the PRYSPRY domain at the carboxyl terminal of ubiquitin ligase TRIM21 to fuse the gene sequence of the nanometer antibody and the Fc region of the antibody, and then uses T2A small peptide for connection to realize the co-expression with TRIM 21. To ensure high cleavage efficiency of T2A and correct positional expression of downstream fragments, three amino acids of GSG were added before T2A.

Wherein the T2A small peptide is from a beta-tetrasome virus capsid protein of the Ophiopogon glaucens, the sequence of T2A is shown as SEQ ID NO.3, and the sequence of TRIM21 is shown as SEQ ID NO. 4.

In order to realize the fourth purpose, the invention provides an application of the composition of the nano antibody targeting ubiquitination degradation of the endogenous Survivin of the cells or the recombinant plasmid thereof in the preparation of gene drugs for treating Survivin over-expressed tumors.

As a preferred embodiment, the Survivin-overexpressing tumor includes lung cancer, pancreatic cancer, liver cancer, breast cancer, and bladder cancer. Because Survivin is abnormally expressed in most tumor cells and plays roles in resisting apoptosis and promoting proliferation, the composition Nb4A-Fc-T2A-TRIM21 in the invention targets Survivin in all cells to degrade endogenous Survivin of the cells, has no cell specificity, has the effect of reducing the expression level of endogenous Survivin protein for the tumor cells with Survivin overexpression, and can induce apoptosis when Survivin in the tumor cells is degraded.

Western-Blot analysis shows that Nb4A-Fc-T2A-TRIM21 can degrade endogenous Survivin protein of cells in a large amount, and the capability of degrading Survivin is 3.72 times that of Nb4A and 2.84 times that of TRIM 21; and the proteasome inhibitor MG132 is utilized to verify that the degradation of Survivin by the method is mediated by ubiquitin-proteasome pathway. The apoptosis rate of Nb4A-Fc-T2A-TIRM21 in flow detection is found to reach 58.52% of MCF-7 cells, which is more than twice of that of Nb4A and TRIM21 alone, but the apoptosis rate of L-02 cells with low Survivin expression is only 14.56%, which shows that Nb4A-Fc-T2A-TIRM21 can promote apoptosis by degrading Survivin cancer cells.

The invention selects the Survivin-targeted nano antibody screened by using the phage display library and aiming at Survivin, and the antibody is combined with protein with high affinity and specificity, particularly Nb4A has the advantages of small molecular weight, better specificity and stronger capability of identifying antigen epitope. In the embodiment of the invention, the Nb4A nano antibody is taken as an example, Nb4A is fused with an Fc region, and T2A and TRIM21 are used for co-expression. T2A is an 18 amino acid polypeptide encoded by 54 bases from the capsid protein of the Neisseria lanuginosa beta-tetrad virus (Thosaasigna virus), which is capable of forming two separate polypeptide chains because the ribosomes of eukaryotes are unable to form a peptide bond between two amino acids, glycine (Gly) and proline (Pro). In order to ensure high cutting efficiency of T2A and correct positioning expression of downstream fragments, three amino acids of GSG are added in front of T2A, and the three amino acids are applied to the design to realize co-expression of Nb4A-Fc and TRIM21 in the same eukaryotic cell, so that the purpose of cell endogenous Survivin degradation mediated by ubiquitin ligase TRIM21 can be achieved.

TRIM21 selected in the present invention is E3 ubiquitin ligase whose PRYSPRY domain binds with high affinity to the Fc domain of an antibody, which is the heavy chain constant region of an antibody, where the overlapping-CH 2-and-CH 3-domains can bind to TRIM 21. TRIM21 recruits ubiquitin-proteasome system to Survivin combined with nano antibody Nb4A, so that Survivin is degraded sharply, the effect of Survivin in resisting apoptosis is inhibited, and the occurrence of tumor cell apoptosis is promoted.

The invention has the advantages that: we used the mode of nanobody and TRIM21 co-expression, and on one hand nanobodies can be provided to bind to Survivin antigen, Fc fragment to bind to TRIM 21; on the other hand, if the level of endogenous TRIM21 in the cell is not enough to perform ubiquitination degradation of the protein, TRIM21 can be supplemented to maintain the normal proceeding of ubiquitination. The method achieves the aim of ubiquitination degradation of target cell endogenous Survivin, and lays a solid foundation for the downstream research of the influence of Survivin degradation on cells. The invention establishes a platform for directly degrading endogenous Survivin at the protein level, and provides a new strategy for clinically targeting Survivin tumor treatment.

Drawings

Fig. 1 is a schematic flow chart of a process for degrading endogenous Survivin of cells through targeting ubiquitination of a nano antibody.

FIG. 2 shows the analysis of the identity between the amino acid sequence of the Fc fragment of an antibody and the 3D structure, and Panel A shows the analysis of the identity between the amino acid sequence of the Fc region of an anti-PD-L1 antibody and the amino acid sequence of the Fc region of human IgG using Muscle software, in which 1: fc region amino acid sequence of human IgG, 2: an Fc region amino acid sequence of an anti-PD-L1 antibody; panel B shows the results of protein 3D structure prediction of two Fc fragments using I-TASSER.

FIG. 3 shows the 3D structure of the protein before and after the fusion of Nb4A with Fc fragment, and Panel A shows the predicted result of the 3D structure of Nb4A protein; panel B is the 3D structural prediction of Fc fragment; panel C is the 3D structure prediction result for the Nb4A-Fc fusion protein.

FIG. 4 is a schematic diagram of the construction of an expression plasmid, wherein the A diagram is a schematic diagram of the construction of a recombinant plasmid pcDNA3.1(+) -Nb4A, the B diagram is a schematic diagram of the construction of a recombinant plasmid pcDNA3.1(+) -Nb4A-Fc, the C diagram is a schematic diagram of the construction of a recombinant plasmid pcDNA3.1(+) -TRIM21, and the D diagram is a schematic diagram of the construction of a recombinant plasmid pcDNA3.1(+) -Nb4A-Fc-T2A-TRIM 21.

Fig. 5 is an agarose gel electrophoresis of the PCR amplification of the nanobody Nb4A gene, lane M: DL 1000 Marker; lane 1: PCR amplified Nb4A gene fragment.

FIG. 6 is an agarose gel electrophoresis of the PCR amplification of the Nb4A-Fc gene, lane M: DL2000 Marker; lane 1: and (3) overlapping and extending the Nb4A-Fc gene fragment amplified by the PCR.

FIG. 7 is an agarose gel electrophoresis of TRIM21 gene overlap extension PCR amplification, lane M: DL2000 Marker; lane 1: PCR amplified TRIM21 gene fragment.

FIG. 8 is an agarose gel electrophoresis of the overlap extension PCR amplification of the Nb4A-Fc-T2A-TRIM21 gene, lane M: DL 5000 Marker; lane 1: and (3) overlapping and extending the Nb4A-Fc-T2A-TRIM21 gene fragment amplified by the PCR.

FIG. 9 is an agarose gel electrophoresis of plasmid pcDNA3.1(+) before and after double digestion, lane M: DL 1000 Marker; lane 1: pcDNA3.1(+) plasmid; lane 2: after double digestion of pcDNA3.1(+) plasmid.

FIG. 10 is a Western-Blot analysis of changes in cell endogenous Survivin, which includes transfecting recombinant plasmids pcDNA3.1(+) -Nb4A, pcDNA3.1(+) -Nb4A-Fc, pcDNA3.1(+) -TRIM21, pcDNA3.1(+) -Nb4A-Fc-T2A-TRIM21 and pcDNA3.1(+) plasmid into MCF-7 cells, setting blank control and Lipofectamine3000 negative control groups, and detecting the degradation of Survivin in cells by Western Blot 48h later. The A diagram is a Western-blot result diagram, and the B diagram is a bar chart of the expression quantity of endogenous Survivin for each group of cells (P)<0.01，***P<0.001 was compared with the control group,^#P<0.05 mean ± SD, n ═ 3). Wherein, Control is blank Control; pcNDA3.1(+) represents a negative control group of the transfection plasmid pcNDA3.1 (+); nb4A represents the experimental group for transfection of recombinant plasmid pcNDA3.1(+) -Nb 4A; nb4A-Fc represents the experimental group for transfection of recombinant plasmid pcNDA3.1(+) -Nb 4A-Fc; TRIM21 represents the experimental group transfected with the recombinant plasmid pcNDA3.1(+) -TRIM 21; nb4A-Fc-T2A-TRIM21 represents the experimental group for transfection of the recombinant plasmid pcNDA3.1(+) -Nb4A-Fc-T2A-TRIM 21; lipofectamine3000 represents a control group with only transfection reagent without DNA. (in the following, the appearance of the above-mentioned group names represents the same asMeaning).

FIG. 11 shows the change of nuclear morphology observed by Hoechst33258 nuclear staining, and A shows the result of nuclear staining after different recombinant plasmids are transfected into MCF-7 cells for 24 h; b, shows the result of nuclear staining 48 hours after different recombinant plasmids are transfected into MCF-7 cells; c, the result of nuclear staining after different recombinant plasmids are transfected into the L-02 cells for 24 hours; FIG. D shows the result of nuclear staining 48h after different recombinant plasmids were transfected into L-02 cells. (Bar 100 μm).

FIG. 12 is an Annexin V/PI double staining method for detecting the apoptosis-promoting capacity of different plasmids on MCF-7 cells and L-02 cells, and A is a graph for flow-detecting the apoptosis-promoting capacity of different plasmids on MCF-7 cells after 48h transfection; panel B is a statistic of the rate of MCF-7 apoptosis in each group (×) P <0.001 compared to control, mean ± SD, n ═ 3); and the C picture shows that the pro-apoptotic capacity of the Nb4A-Fc-T2A-TRIM21 on L-02 cells is detected by flow. Fig. 13 is a graph of proteasome inhibitor MG132 demonstrating degradation of survivin mediated through the ubiquitin-proteasome pathway.

FIG. 13 shows a Western Blot analysis of the intervention of MG132 on the ability of recombinant plasmid to degrade Survivin, MCF-7 cells were pretreated with 0.5. mu.M of MG132 for 3h, transfected recombinant plasmids pcDNA3.1(+) -Nb4A, pcDNA3.1(+) -TRIM21, and pcDNA3.1(+) -Nb4A-Fc-T2A-TRIM21 were transfected into MCF-7 cells, respectively, a blank control group was set (treated with 0.5. mu.M of MG132 but not transfected), and after 48h, Western Blot was used to detect the expression level of Survivin in cells.

FIG. 14 shows the effect of MG132 on the apoptosis-promoting ability of recombinant plasmids, wherein A shows the apoptosis rate of MCF-7 cells transfected with different plasmids for 48h by flow cytometry with or without intervention of MG132 (the upper layer shows the detection result of flow cytometry with 0.5. mu.M of MG132, and the lower layer shows the detection result of flow cytometry without MG 132); panel B is a comparison of the rate of apoptosis of MCF-7 cells in each group with and without intervention of MG132 (× P <0.00, mean ± SD compared to the + MG132 group, n ═ 3).

Detailed Description

The invention will be further illustrated with reference to the following specific examples. The following examples are given by way of illustration of the conventional methods unless otherwise specified. The test materials, reagents and the like used in the following examples are commercially available unless otherwise specified. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.