A new fusion protein of Trichosanthes kirilowii and its application

Abstract

The present invention discloses a novel trichosanthes fusion protein. Specifically, the present invention modifies the trichosanthes protein with poor intracellular performance, fuses the transmembrane protein and the matrix metalloproteinase substrate domain, and utilizes the self-cleavage mediated by the intein to obtain a high-specificity low-immune prototype tumor-suppressive trichosanthes fusion protein that is easy to PEGylate and very conducive to intracellular entry. The present invention not only provides a new method for mediating the intracellular entry of trichosanthes protein, but also opens up new ideas for the new use of traditional Chinese medicine.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more specifically, to a new trichosanthin fusion protein. Background Art

[0002] Malignant tumors are one of the biggest threats to human life and safety. According to the World Health Organization, the incidence of cancer is increasing year by year, and is showing a trend of younger age. At present, the treatment of malignant tumors is still mainly surgical resection combined with chemotherapy and radiotherapy. Small molecule chemotherapy drugs are prone to drug resistance and have huge toxic side effects, so they cannot effectively kill tumor cells and cause great pain to patients. With the development of biotechnology, biomacromolecule drugs, especially protein drugs, have received more and more attention due to their strong biological activity and good biocompatibility. Protein drugs have become one of the most cutting-edge directions in new drug research and development in the 21st century.

[0003] Trichosanthesin (TCS) is a protein extracted from the root of the cucurbitaceae plant Trichosanthes kirilowii. It has been used as an abortion drug for terminating mid-term pregnancy in traditional Chinese medicine for centuries. TCS is a type I ribosome inactivating protein with a molecular weight of 27 kDa. It has N-glycosidase activity and can recognize the large ribosome subunit of mammalian cells and depurinate it, thereby inhibiting cell protein synthesis and leading to cell death. At the same time, studies have shown that TCS can induce cell apoptosis through multiple pathways. It is precisely because of these biological activities of TCS that it has been proven to have a broad spectrum of anti-tumor activity. However, TCS does not have tumor targeting in vivo and lacks a certain ability to enter cells. In addition, it is reported that TCS has strong immunogenicity. These have become restrictions on the clinical application of TCS as an anti-tumor drug.

[0004] At present, chemical modification has become an important method to improve the drugability of trichosanthes protein. The traditional chemical modification sites of protein drugs are mainly limited to the side chain amino groups of lysine residues in proteins and the side chain thiol groups of cysteine ​​introduced by means of genetic engineering. The disadvantages of these two modification methods are obvious. The former will lead to uneven modification products due to the large number of lysine residues in the protein, which increases the difficulty and cost of separation and purification of the modified products. Although the latter can achieve site-specific modification of proteins, due to the instability of free thiol groups, protein drugs are prone to oxidation of thiol groups during preparation and purification, forming intermolecular dimers.

[0005] Therefore, there is an urgent need in this field to develop a new protein modification method to achieve efficient, specific, and site-specific modification of protein drugs, so as to obtain low-toxic, efficient trichosanthin with tumor inhibitory activity. Summary of the invention

[0006] The purpose of the present invention is to provide a new trichosanthin fusion protein, specifically, a tumor-targeted drug delivery system based on recombinant trichosanthin protein.

[0007] In the first aspect of the present invention, a fusion protein is provided, wherein the fusion protein has a structure shown in Formula Ia or Formula Ib:

[0008] A-L1-B-L2-C Formula Ia or

[0009] B-L1-A-L2-C Formula Ib

[0010] in,

[0011] A is the trichosanthin element,

[0012] B is the membrane-penetrating element.

[0013] C is the matrix metalloproteinase 2 substrate peptide element,

[0014] L1 and L2 are each free or connected peptide elements,

[0015] “-” is the peptide bond connecting each element,

[0016] And the fusion protein has tumor suppressor activity.

[0017] In another preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO.:3.

[0018] In another preferred embodiment, the trichosanthin element includes wild-type or mutant trichosanthin, and preferably, the trichosanthin includes the full-length protein or a fragment thereof.

[0019] In another preferred embodiment, the trichosanthin protein does not contain Cys.

[0020] In another preferred example, the amino acid sequence of the wild-type trichosanthin is shown as SEQ ID NO.:1, and the polynucleotide encoding it is shown as SEQ ID NO.:2.

[0021] In another preferred embodiment, the transmembrane element comprises any one of the polypeptides shown in SEQ ID NO.: 5-7 (VSRRRRRRGGRRRR, YGRKKRRGGQRRR, RRRRRRRR).

[0022] In another preferred embodiment, the matrix metalloproteinase 2 substrate peptide element includes any one of the polypeptides shown in SEQ ID NO.: 8-9 (PLGLAG, PLGVR).

[0023] In another preferred embodiment, the connecting peptide element comprises 0-10 identical or different amino acid residues, preferably 3-5.

[0024] In another preferred embodiment, the connecting peptide element is 3 Gly.

[0025] In another preferred embodiment, the tumor comprises a solid tumor, preferably, a matrix metalloproteinase 2 (MMP-2) positive tumor.

[0026] In another preferred embodiment, the tumor includes fibrosarcoma, glioma, or liver cancer tumor.

[0027] The second aspect of the present invention provides a precursor protein of the fusion protein described in the first aspect of the present invention, characterized in that the precursor protein has a structure shown in Formula IIa or Formula IIb:

[0028] A-L1-B-L2-CDE Formula IIa or

[0029] B-L1-A-L2-CDE Formula IIb;

[0030] in,

[0031] A is the trichosanthin element,

[0032] B is the membrane-penetrating element.

[0033] C is a matrix metalloproteinase 2 (MMP-2) substrate peptide element,

[0034] D is none or 1-3 Cys,

[0035] E is an intein element,

[0036] L1 and L2 are each free or connected peptide elements,

[0037] “-” is the peptide bond connecting each element,

[0038] The precursor protein is cleaved by a thiol-containing reagent to produce the fusion protein described in the first aspect of the present invention.

[0039] In another preferred embodiment, the intein element further contains a tag sequence at its C-terminus.

[0040] In another preferred example, the tag sequence includes a chitin binding domain (CBD) tag sequence, a 6His tag sequence, a GST tag sequence, or an MBP tag sequence.

[0041] In another preferred embodiment, the thiol-containing reagent includes L-cysteine, DTT, mercaptoethanol or sodium 2-mercaptoethane sulfonate.

[0042] The third aspect of the present invention provides a polynucleotide sequence, wherein the polynucleotide sequence encodes the fusion protein described in the first aspect of the present invention or encodes the precursor protein described in the second aspect of the present invention.

[0043] In another preferred embodiment, the polynucleotide sequence is shown as SEQ ID NO.:4.

[0044] The fourth aspect of the present invention provides a vector, wherein the vector contains the polynucleotide described in the third aspect of the present invention.

[0045] The fifth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the fourth aspect of the present invention or the polynucleotide described in the second aspect of the present invention is integrated into the gene of the host cell.

[0046] The sixth aspect of the present invention provides a modified product of the fusion protein described in the first aspect of the present invention, wherein the modified product is formed by site-specific PEGylation of the fusion protein described in the first aspect of the present invention.

[0047] In another preferred embodiment, the site-specific PEGylation is to perform PEG modification on the C-terminus of the fusion protein.

[0048] In another preferred embodiment, the fusion protein modifier contains PEG with a molecular weight of 3-40KD, preferably 5KD.

[0049] The seventh aspect of the present invention provides use of the fusion protein described in the first aspect of the present invention or the modified substance described in the sixth aspect of the present invention for preparing a pharmaceutical composition for treating tumors.

[0050] In an eighth aspect, the present invention provides a pharmaceutical composition, which contains the fusion protein described in the first aspect of the present invention or the modified substance described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier.

[0051] The ninth aspect of the present invention provides a method for producing the fusion protein or its precursor protein according to the first aspect of the present invention, comprising the steps of:

[0052] (i) Cultivating the host cell according to the fifth aspect of the present invention under appropriate conditions to obtain the expression of the fusion protein or its precursor protein.

[0053] In another preferred embodiment, the method may further include the steps of:

[0054] (ii) separating and purifying the obtained fusion protein or its precursor protein.

[0055] In another preferred embodiment, the separation and purification include direct separation and purification mediated by intein.

[0056] The tenth aspect of the present invention provides a method for non-therapeutic inhibition of tumor cells in vitro, comprising the steps of: adding the fusion protein described in the first aspect of the present invention, the modified substance described in the sixth aspect of the present invention, or the pharmaceutical composition described in the eighth aspect of the present invention to a tumor cell culture, thereby inhibiting tumor cells.

[0057] In the eleventh aspect of the present invention, a method for treating tumors is provided, comprising the steps of administering a safe amount of the fusion protein described in the first aspect of the present invention, the modified substance described in the sixth aspect of the present invention, or the pharmaceutical composition described in the eighth aspect of the present invention to a subject in need, thereby treating the tumor.

[0058] In another preferred embodiment, the subject in need is a mammal, including a mouse, a rat, or a human, preferably a human.

[0059] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The synthetic route of the TCS-cell-penetrating peptide and TCS-cell-penetrating peptide-MMP-2 substrate peptide-PEG linker mediated by the intein in Preparation Example 1 and Preparation Example 2 according to the present invention.

[0061] Figure 2 FIG. 1 is a diagram showing the anti-tumor mechanism of the TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG conjugate according to the present invention.

[0062] Figure 3 This is an electrophoresis diagram of the prokaryotic expression and purification of the intein-mediated recombinant TCS protein prepared in Example 1 according to the present invention.

[0063] Figure 4 This is an electrophoresis diagram of the prokaryotic expression and purification of the intein-mediated recombinant TCS-membrane-penetrating peptide-MMP-2 substrate peptide fusion protein prepared in Example 2 according to the present invention.

[0064] Figure 5 This is the electrophoresis diagram of the synthesis of the intein-mediated TCS-transmembrane peptide conjugate prepared in Example 1 according to the present invention.

[0065] Figure 6 This is a purification chromatogram of the TCS-membrane-penetrating peptide conjugate prepared in Example 1 according to the present invention.

[0066] Figure 7 This is the electrophoresis diagram of the synthesis of the intein-mediated TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG conjugate prepared in Example 2 according to the present invention.

[0067] Figure 8 This is a purification chromatogram of the TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG conjugate prepared in Example 2 according to the present invention.

[0068] Fig. 9 The electrophoretic diagrams of the purified TCS, TCS-cell-penetrating peptide conjugate and TCS-cell-penetrating peptide-MMP-2 substrate peptide-PEG conjugate prepared in Preparation Example 1 and Preparation Example 2 of the present invention are shown.

[0069] Fig.10 This is a Western Blot diagram of the detection of MMP-2 enzyme content in HT1080 and HUVEC cells and their culture medium in Experimental Example 1 of the present invention.

[0070] Fig.11 This is the electrophoresis diagram of the TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG connector before and after being cleaved by MMP-2 in Experimental Example 2 of the present invention.

[0071] Fig.12 This is the inhibitory effect of TCS, TCS-cell-penetrating peptide linker and TCS-cell-penetrating peptide-MMP-2 substrate peptide-PEG linker in Experimental Example 3 of the present invention on the proliferation of tumor cells HT1080 with high expression of MMP-2 enzyme.

[0072] Fig.13 This is the inhibitory effect of TCS, TCS-cell-penetrating peptide linker and TCS-cell-penetrating peptide-MMP-2 substrate peptide-PEG linker in Experimental Example 3 of the present invention on the proliferation of normal HUVEC cells with low expression of MMP-2 enzyme.

[0073] Fig.14 This is an in vivo imaging diagram of tumor targeting and tissue distribution of the TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG conjugate in HT1080 tumor-bearing nude mice according to Experimental Example 4 of the present invention.

[0074] Fig.15 The figures show the tumor inhibition effects of TCS, TCS-cell-penetrating peptide conjugate and TCS-cell-penetrating peptide-MMP-2 substrate peptide-PEG conjugate on HT1080 tumor-bearing nude mice according to Experimental Example 5 of the present invention.

[0075] Fig.16 These are photos of tumors after treatment with TCS, TCS-cell-penetrating peptide conjugate, and TCS-cell-penetrating peptide-MMP-2 substrate peptide-PEG conjugate in Experimental Example 5 of the present invention.

[0076] Fig.17This is a curve diagram of the survival rate of each group of animals during the administration of TCS, TCS-cell-penetrating peptide conjugate and TCS-cell-penetrating peptide-MMP-2 substrate peptide-PEG conjugate in Experimental Example 5 of the present invention.

[0077] Fig.18 The figure shows the changes in body weight of animals in each group during the administration of TCS, TCS-cell-penetrating peptide conjugate and TCS-cell-penetrating peptide-MMP-2 substrate peptide-PEG conjugate in Experimental Example 5 of the present invention.

[0078] Fig.19 Pathological sections of organs (lung, spleen, kidney) of each group of animals after treatment with TCS, TCS-cell-penetrating peptide conjugate and TCS-cell-penetrating peptide-MMP-2 substrate peptide-PEG conjugate in Experimental Example 5 of the present invention.

[0079] Fig. 20 This is the immunogenicity determination of TCS and TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG conjugates according to Experimental Example 6 of the present invention. DETAILED DESCRIPTION

[0080] After extensive and in-depth research, the inventor unexpectedly prepared a new fusion protein for the first time. The fusion protein modified the trichosanthin protein with poor intracellular performance, fused the transmembrane protein and the matrix metalloproteinase substrate domain, and used the intein-mediated self-cleavage to obtain a high-specificity low-immune prototype tumor-suppressive trichosanthin fusion protein that is easy to PEGylate and very conducive to intracellular entry. The present invention not only provides a new method for mediating the intracellular entry of trichosanthin protein, but also opens up new ideas for the new use of traditional Chinese medicine. On this basis, the present invention was completed.

[0081] Fusion protein and its preparation

[0082] In the present invention, "recombinant fusion protein", "protein of the present invention", "fusion protein of the present invention", and "fusion protein" are used interchangeably, and refer to a fusion protein having a structure described in Formula Ia or Formula Ib, that is, a fusion protein containing a trichosanthin element, a transmembrane element, and a matrix metalloproteinase 2 substrate peptide element. The protein of the present invention can be a monomer or a multimer (such as a dimer) formed by a monomer. In addition, it should be understood that the term also includes active fragments and derivatives of the fusion protein.

[0083] The present invention also includes active fragments, derivatives and analogs of the fusion protein according to the present invention. As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially maintain the tumor suppressor function or activity of human trichosanthin. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a fusion protein with another compound (such as a compound that prolongs the half-life of a polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to this polypeptide sequence (fusion proteins formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His). According to the teachings of this article, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

[0084] A preferred class of active derivatives refers to polypeptides formed by replacing at most 3, preferably at most 2, and more preferably at most 1 amino acid with similar or similar properties compared to the amino acid sequence of Formula Ia or Formula Ib. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1. Preferably, the derivative does not contain Cys.

[0085] Table 1

[0086] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0087] The present invention also provides analogs of the fusion protein of the present invention. The difference between these analogs and the polypeptide shown in SEQ ID NO:.3 may be a difference in amino acid sequence, or a difference in modification form that does not affect the sequence, or both. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-natural or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0088] Modifications (usually without changing the primary structure) include: chemical derivatization of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the polypeptide or in further processing steps. This modification can be accomplished by exposing the polypeptide to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylase). Modifications also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to improve their resistance to proteolysis or optimize their solubility.

[0089] The polypeptides of the present invention can also be used in the form of salts derived from pharmaceutically or physiologically acceptable acids or bases. These salts include (but are not limited to) salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid. Other salts include: salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium or magnesium), as well as in the form of esters, carbamates or other conventional "prodrugs".

[0090] As used herein, "isolated" means that a substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in their natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist with them in their natural state.

[0091] As used herein, "isolated recombinant fusion protein" means that the recombinant fusion protein is substantially free of other proteins, lipids, carbohydrates or other substances naturally associated therewith. Those skilled in the art can purify the recombinant fusion protein using standard protein purification techniques. Substantially pure protein can produce a single major band on a non-reducing polyacrylamide gel.

[0092] The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0093] The present invention also relates to variants of the above-mentioned polynucleotides, which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention. The variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the encoded polypeptide.

[0094] As used herein, the term "primer" refers to a general term for oligonucleotides that can be paired with a template and used as a starting point to synthesize a DNA chain complementary to the template under the action of a DNA polymerase. Primers can be natural RNA, DNA, or any form of natural nucleotides. Primers can even be non-natural nucleotides such as LNA or ZNA. The primer is "substantially" (or "essentially") complementary to a specific sequence on a chain on the template. The primer must be fully complementary to a chain on the template to begin extension, but the sequence of the primer does not have to be completely complementary to the sequence of the template. For example, a sequence that is not complementary to the template is added to the 5' end of a primer whose 3' end is complementary to the template, and such a primer is still substantially complementary to the template. As long as there is a primer that is long enough to fully bind to the template, a primer that is not completely complementary can also form a primer-template complex with the template to perform amplification.

[0095] The full-length nucleotide sequence or fragments of the fusion protein of the present invention or its elements can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the published relevant nucleotide sequences, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order.

[0096] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0097] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0098] The method of using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The DNA / RNA fragments amplified can be separated and purified by conventional methods such as by gel electrophoresis.

[0099] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the fusion protein coding sequence, and a method for producing the protein of the present invention by recombinant technology.

[0100] The polynucleotide sequences of the present invention can be used to express or produce recombinant proteins by conventional recombinant DNA techniques. Generally, the following steps are involved:

[0101] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding the protein of the present invention, or a recombinant expression vector containing the polynucleotide;

[0102] (2) Host cells cultured in a suitable culture medium;

[0103] (3) Isolate and purify proteins from culture medium or cells.

[0104] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the protein of the present invention and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0105] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0106] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0107] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: bacterial cells of Escherichia coli and Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, NS0, COS7, or 293 cells, etc.

[0108] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0109] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0110] The protein in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0111] The present invention also provides a precursor protein of the fusion protein, which utilizes an intein-mediated fusion protein acquisition method, adds an intein encoding gene to the gene construct of the fusion protein, and directly obtains a high-purity fusion protein by tag shearing after the protein is expressed, thereby eliminating the need for further separation or purification steps.

[0112] Cell-penetrating peptides

[0113] The fusion protein provided by the present invention preferably contains a cell-penetrating peptide. Trichosanthin TCS belongs to type I ribosome inactivating protein, contains only A chain with N-glycosidase activity, lacks lectin-like B chain mediated cell entry than type II ribosome inactivating protein, therefore, this type of protein toxin has low cell entry efficiency, and its anti-tumor cell toxicity is far inferior to type II ribosome inactivating protein. Cell-penetrating peptides are a class of polypeptides with cell membrane penetration activity found in nature or obtained by artificial screening. There is no special limitation on the cell-penetrating peptides that can be used in the present invention, and can be any cell-penetrating peptide rich in basic amino acids and cell-penetrating peptides with amphipathicity, as long as it can promote the cell to take up TCS that lacks cell entry ability. Preferably, the cell-penetrating peptides of the present invention include low molecular weight protamine LMWP (VSRRRRRRGGRRRR (SEQ ID NO.: 5)), TAT (YGRKKRRQRRR (SEQ ID NO.: 6)), R8 (RRRRRRRR (SEQ ID NO.: 7)) The cell-penetrating peptides of the present invention can not only enter the cell by themselves, but also carry the TCS of the present invention into the cell. In addition, the cell-penetrating peptide of the present invention can be directly obtained by introducing the gene of the cell-penetrating peptide into the expression vector expressing the fusion protein to obtain a fusion protein containing the cell-penetrating peptide, or can also be obtained by a method mediated by an intein to obtain a cell-penetrating peptide-modified method. The preferred method includes:

[0114] (a) transforming the recombinant plasmid into Escherichia coli BL21 (DE3) competent cells;

[0115] (b) culturing the strain containing the recombinant plasmid in LB medium to the logarithmic growth phase, and adding isopropyl-β-D-thiogalactopyranoside (IPTG) to induce the expression of the target protein;

[0116] (c) collecting the cells by centrifugation and disrupting them by ultrasonication;

[0117] (d) The supernatant containing the target protein with C-terminal fusion intein and CBD tag is affinity purified using a chitin column, after washing away the impurities, the column is cleaved overnight with a buffer containing sodium 2-mercaptoethanesulfonate (MESNA), and the eluate is collected;

[0118] (e) After ultrafiltration and concentration, the above-mentioned cell-penetrating peptide is added and reacted overnight to form a TCS-cell-penetrating peptide conjugate, wherein the molar ratio of protein to polypeptide is 1:20; excess polypeptide in the solution is removed by a desalting column, and the yield of the TCS-cell-penetrating peptide conjugate obtained in step (e) is 90%-95%.

[0119] Intein

[0120] Intein-mediated protein prokaryotic expression, purification and modification is a new type of protein site-specific modification technology. Intein is a polypeptide with self-cleavage function. The intein sequence and chitin binding domain (CBD) affinity tag are fused to the C-terminus of the target protein. This technology uses the E. coli prokaryotic expression system to express foreign proteins. When the chitin column is used for affinity purification of the target protein, a thiol-containing reagent is used for on-column cleavage. Under the mediation of the intein, the target protein can be cut off from the intein, and a responsive active group is introduced at the C-terminus of the protein for downstream protein modification.

[0121] The affinity tag that can be used in the present invention is not particularly limited, and can be any affinity tag that is suitable for self-cleavage of intein, such as 6His.

[0122] In addition, the self-cleavage cleavage reagent that can be used for the inclusion-mediated self-cleavage of the present invention is not particularly limited, and is preferably a thiol-containing cleavage reagent. In a preferred embodiment, sodium 2-mercaptoethane sulfonate is selected for cleavage, and a thioester bond is introduced at the end of the protein. At this time, the protein can react efficiently with a polypeptide with cysteine ​​at the N-terminus; and if L-cysteine ​​is used as a cleavage reagent, an additional cysteine ​​is introduced at the end of the protein, which can react with PEG with maleimide as the terminal group, thereby achieving site-specific PEG modification of the protein.

[0123] PEGylated site-directed modification

[0124] PEG modification is the use of polyethylene glycol with functional groups for protein drug modification, which usually includes random modification and site-specific modification. The present invention is preferably PEG site-specific modification, for example, a fusion protein fused with an intein and a CBD tag at the end is purified using a chitin affinity column, and the target protein is cleaved on the column using L-cysteine. Under the mediation of the intein, the target protein is cut from the intein, the CBD tag and the chitin affinity column, and a cysteine ​​residue is introduced at the C-terminus of the target protein. The side chain thiol of the cysteine ​​residue can react with the PEG of the terminal maleimide to achieve site-specific PEGylation modification of the protein C-terminus.

[0125] A preferred method for PEG site-directed modification is as follows:

[0126] (a) transforming the recombinant plasmid into Escherichia coli BL21 (DE3) competent cells;

[0127] (b) culturing the strain containing the recombinant plasmid in LB medium to the logarithmic growth phase, and adding isopropyl-β-D-thiogalactopyranoside (IPTG) to induce the expression of the target protein;

[0128] (c) collecting the cells by centrifugation and disrupting them by ultrasonication;

[0129] (d) the supernatant containing the target protein with C-terminal fusion intein and CBD tag is affinity purified using a chitin column, after washing away the impurities, the column is cleaved overnight with a buffer containing L-cysteine, and the eluate is collected;

[0130] (e) ultrafiltration concentration to remove free cysteine ​​in the solution, adding maleimide-PEG (Mal-PEG), reacting overnight to form a fusion protein-PEG conjugate, wherein the molar ratio of protein to Mal-PEG is 1:10;

[0131] (f) separating and purifying the fusion protein-PEG conjugate obtained in step (e) using a cation exchange column.

[0132] Connector Peptide

[0133] The present invention provides a fusion protein, which may optionally contain a connecting peptide (peptide linker). The size and complexity of the connecting peptide may affect the activity of the protein. Generally, the connecting peptide should have sufficient length and flexibility to ensure that the two connected proteins have sufficient spatial freedom to exert their functions. At the same time, the formation of α helix or β fold in the connecting peptide is avoided to affect the stability of the fusion protein.

[0134] The length of the connecting peptide is generally 0-10 amino acids, preferably 1-5 amino acids.

[0135] Pharmaceutical compositions and methods of administration

[0136] The present invention also provides a composition comprising an effective amount of the fusion protein of the present invention and a pharmaceutically acceptable carrier. Typically, the fusion protein of the present invention can be formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably, the pH is about 6-8.

[0137] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce function or activity in humans and / or animals and can be accepted by humans and / or animals, such as 0.001-99wt%; preferably 0.01-95wt%; more preferably, 0.1-90wt%.

[0138] As used herein, "pharmaceutically acceptable" ingredients are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

[0139] The pharmaceutical composition of the present invention contains a safe and effective amount of the fusion protein of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be prepared into a sustained-release preparation.

[0140] The effective amount of the fusion protein of the present invention may vary with the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the fusion protein of the present invention, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's body weight, the patient's immune status, the route of administration, etc. For tumor patients, generally, when the fusion protein of the present invention is administered at a dose of about 0.5 mg-5 mg / kg animal body weight (preferably 2 mg-4 mg / kg animal body weight) per day, satisfactory results can be obtained. For example, depending on the urgency of the treatment situation, several divided doses may be administered per day, or the dose may be reduced proportionally.

[0141] Beneficial effects of the present invention

[0142] The present invention obtains a high-specificity, low-immunity prototype tumor-suppressive trichosanthin fusion protein that is easy to PEGylate and very conducive to cell entry by structural modification and transformation of wild trichosanthin protein. The present invention not only provides a new method for mediating the cell entry of trichosanthin protein, but also opens up new ideas for the new use of traditional Chinese medicine.

[0143] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0144] Reagents and medicines

[0145] The prokaryotic expression vector pTXB1 and chitin affinity column were purchased from NEB. The recombinant expression plasmids of TCS and TCS-transmembrane peptide-MMP-2 substrate peptide fusion protein were constructed by Shanghai Jierui Biotechnology Co., Ltd. Yeast powder and peptone were purchased from Oas. Ampicillin (Amp) and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from Amresco. HEPES and thiazolyl blue (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) were purchased from Sigma-Aldrich. L-cysteine, sodium chloride (NaCl), Tween 20, and EDTA were purchased from Sinopharm Group (Shanghai) Chemical Reagent Company. Maleimide polyethylene glycol (Mal-PEG) was purchased from Beijing Jiankai Technology Co., Ltd. Sodium 2-mercaptoethane sulfonate (MENSA) was purchased from J&K Technology Group. Peptides were purchased from Shanghai Lion Chemical Co., Ltd. Succinimidyl ester Cy5 dye was purchased from Dalian Meilun Biotechnology Co., Ltd. Bradford and BCA protein concentration assay kits were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Human fibrosarcoma cells (HT1080) and human umbilical vein epithelial cells (HUVEC) were purchased from the Cell Bank of the Chinese Academy of Sciences. DMEM cell culture medium powder, trypsin, fetal bovine serum (FBS), and double antibody (penicillin-streptomycin) were purchased from Gibco and Invitrogen, respectively.

[0146] Preparation Example 1 Synthesis of TCS-transmembrane peptide

[0147] Prokaryotic expression and purification of recombinant TCS protein

[0148] a: Transform the TCS recombinant expression plasmid (TCS gene sequence shown in SEQ ID NO.: 2) into Escherichia coli BL21 (DE3) competent cells.

[0149] b: The strain containing the recombinant plasmid was cultured in LB medium containing 100 μg / ml Amp at 250 rpm in a 37°C constant temperature shaker until the logarithmic growth phase (absorbance at 600 nm was 0.6-0.8), IPTG was added at a final concentration of 1 mM, and expression was carried out overnight (14 h) at 25°C and 150 rpm.

[0150] c: Collect the cells by centrifugation at 6,000 rpm and 4°C for 20 min.

[0151] d: Resuspend the cells in HEPES buffer (containing 20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.5‰ Tween 20, pH 8.5).

[0152] e: Use a probe ultrasonic disruptor at 400 W power for 30 min.

[0153] f: Centrifuge at 12,000 rpm, 4°C for 20 min and collect the supernatant.

[0154] g: The supernatant containing the target protein is passed through a chitin column pre-equilibrated with HEPES buffer at a flow rate of 1 ml / min. After loading, 25 column volumes of HEPES buffer are used to wash away non-specifically bound proteins.

[0155] h: 3 column volumes of HEPES buffer containing 50 mM MESNA were passed through the column, and a small amount of buffer was retained. The column outlet was closed and the column was cut overnight (16 h).

[0156] i: Open the column outlet, collect the buffer containing the target protein that flows out, and continue to add 3 times the column volume of HEPES buffer to continue eluting the target protein.

[0157] Synthesis of TCS-transmembrane peptide

[0158] The collected protein eluate was concentrated using an ultrafiltration tube with a molecular weight cutoff of 10,000, and the protein concentration was determined using a Bradford protein concentration assay kit. Diluted to 100 μM with HEPES buffer, added with a cell-penetrating peptide (CVSRRRRRRGGRRRR (C+SEQ ID NO.: 6)) to a final concentration of 2 mM, and reacted overnight (16 h) in a 4°C flat shaker. After the reaction was completed, the reaction efficiency was detected by 12% polyacrylamide (SDS-PAGE) electrophoresis, reaching 90%-95% ( Figure 5 ). Use a desalting column to remove excess cell-penetrating peptide in the solution to obtain TCS-cell-penetrating peptide conjugate ( Figure 6 ).

[0159] Preparation Example 2 Synthesis of PEGylated TCS-Cell-penetrating Peptide-MMP-2 Substrate Peptide

[0160] Expression and purification of TCS-transmembrane-MMP-2 substrate peptide fusion protein

[0161] a: The recombinant expression plasmid of the fusion protein (SEQ ID NO.: 5) was transformed into Escherichia coli BL21 (DE3) competent cells.

[0162] b: The strain containing the recombinant plasmid was cultured in LB medium containing 100 μg / ml Amp at 250 rpm in a 37°C constant temperature shaker until the logarithmic growth phase (absorbance at 600 nm was 0.6-0.8), IPTG was added at a final concentration of 1 mM, and expression was carried out overnight (14 h) at 25°C and 150 rpm.

[0163] c: Collect the cells by centrifugation at 6,000 rpm and 4°C for 20 min.

[0164] d: Resuspend the cells in HEPES buffer (containing 20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.5‰ Tween 20, pH 8.5).

[0165] e: Use a probe ultrasonic disruptor at 400 W power for 30 min.

[0166] f: Centrifuge at 12,000 rpm, 4°C for 20 min and collect the supernatant.

[0167] g: The supernatant containing the target protein is passed through a chitin column pre-equilibrated with HEPES buffer at a flow rate of 1 ml / min. After loading, 25 column volumes of HEPES buffer are used to wash away non-specifically bound proteins.

[0168] h: 3 column volumes of HEPES buffer containing 50 mM L-cysteine ​​were passed through the column, and a small amount of buffer was retained. The column outlet was closed and the column was cut overnight (16 h).

[0169] i: Open the column outlet, collect the buffer containing the target protein that flows out, and continue to add 3 times the column volume of HEPES buffer to continue eluting the target protein.

[0170] PEGylation modification of TCS-cell-penetrating peptide-MMP-2 substrate peptide fusion protein

[0171] The collected protein eluate was concentrated using an ultrafiltration tube with a molecular weight cutoff of 10,000, and free L-cysteine ​​in the solution was removed using a desalting column. The protein concentration was determined using the BCA method, and Mal-PEG was added at a molar ratio of 1:10 and reacted at 4°C overnight (16h). The reaction efficiency was detected by SDS-PAGE electrophoresis at a concentration of 12%, and reached 80%-85% ( Figure 7 The reaction product was separated and purified using a cation exchange column SPFF and a fast protein purification chromatograph FPLC. The sample was pushed into the column using an injector, and gradient eluted with a pH 7.2 phosphate buffer containing 0.15-1M NaCl at a flow rate of 0.02M / min. The first elution peak was collected to obtain a fusion protein-PEG connection product ( Figure 8 ).

[0172] Experimental Example 1 Determination of MMP-2 enzyme content in HT1080 and HUVEC cells and their culture medium

[0173] The MMP-2 enzyme content in HT1080 and HUVEC cells and their culture medium was detected by Western Blot, which is a common method in the art. The specific method is as follows: HT1080 and HUVEC cells in the logarithmic growth phase were digested with trypsin and diluted to 1.5×10 5Cell suspension of 10 cells / ml was transferred to a 6-well cell culture plate of Nunc, 2 ml of cell suspension was added to each well, and cultured for 4 hours (37°C, 5% CO2) with DMEM complete medium containing 10% calf serum. After the cells adhered to the wall, the medium was discarded, the cells were washed twice with PBS, and replaced with serum-free DMEM medium, 1 ml per well, and cultured for 24 hours. The supernatant of the culture medium was collected, and the cells were lysed with cell lysis buffer for 30 minutes, centrifuged at 4°C for 15 minutes, the supernatant was collected, the protein concentration was determined by BCA method, and diluted to the same concentration with buffer. The loading buffer was added to the culture medium and cell lysate samples and boiled in boiling water for 5 minutes. The gel was run on a 10% polyacrylamide gel and electrotransferred to a 0.45μm PVDF membrane, and blocked with 5% skim milk powder for 2 hours. The blocking solution was used to prepare 1,000-fold diluted anti-MMP-2 rabbit antibody (purchased from Cell Signaling Technology) and 20,000-fold diluted anti-β-actin mouse antibody (purchased from Sigma) and incubated at 4°C overnight. After washing the primary antibody three times, the secondary antibody (purchased from Bio-Technology Co., Ltd.) of the corresponding species was incubated at room temperature for 1 hour. After washing the secondary antibody three times, ECL color development substrate (purchased from Thermo Peirce) was added to the membrane and imaged in a multifunctional gel imager (Biorad).

[0174] The results are as follows:

[0175] from Fig.10 It can be seen that both the HT1080 cells and the cell culture medium have significant overexpression of MMP-2 enzyme compared with HUVEC cells. This shows that HT1080 cells can be used as a cell model for subsequent MMP-2 enzyme sensitivity experiments.

[0176] Experimental Example 2 In vitro enzymatic cleavage experiment of TCS-cell-penetrating peptide-MMP-2 substrate peptide-PEG

[0177] (1) Preparation of enzyme digestion medium

[0178] HT1080 cells in the logarithmic growth phase were digested with trypsin and diluted to 1.5×10 5 Cell suspension of 10 cells / ml was transferred to a 6-well cell culture plate of Nunc, 2 ml of cell suspension was added to each well, and cultured for 4 hours (37°C, 5% CO2) with DMEM complete medium containing 10% calf serum. After the cells adhered to the wall, the medium was discarded, the cells were washed twice with PBS, and replaced with serum-free DMEM medium, 1 ml per well, and cultured for another 24 hours. The medium was aspirated, centrifuged at 12,000 rpm and 4°C for 10 minutes to remove cell debris, and the supernatant was used for in vitro enzyme digestion experiments.

[0179] (2) In vitro enzyme digestion experiment

[0180] The TCS-penetrating peptide-MMP-2 substrate peptide-PEG conjugate was diluted to a final concentration of 0.1 mg / ml using the enzyme cleavage medium prepared by the above method, and 0.2‰ NaN3 was added at the same time. The enzyme cleavage was carried out at 250 rpm in a constant temperature shaker at 37°C for 48 hours. The enzyme cleavage efficiency was detected by SDS-PAGE electrophoresis. The results are shown in the figure. The enzyme cleavage product is a band of about 30 kDa, which is slightly smaller than the protein band before PEG modification ( Fig.11 ).

[0181] Experimental Example 3 MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromine blue, trade name: thiazolidine blue) method was used to determine the in vitro anti-tumor effects of TCS, TCS-cell-penetrating peptide conjugate, and TCS-cell-penetrating peptide-MMP-2 substrate peptide-PEG conjugate

[0182] HT1080 and HUVEC cells in the logarithmic growth phase were digested and diluted to a density of 1.5×10 4 The cell suspension of 10 cells / mL was transferred to a Nunc 96-well cell culture plate, 200 μL of the cell suspension was added to each well, and the cells were cultured for 24 h (37° C., 5% CO 2 ) in DMEM complete medium containing 10% calf serum.

[0183] The optimal drug concentration range was determined by preliminary experiments, and solutions of different concentrations were added, with 6 replicates for each concentration. After 48 hours of culture, 20 μL of MTT (5 mg / mL, purchased from Sigma-Aldrich, USA) was added, cultured for 4 hours, 200 μL of DMSO was added, and the crystals were fully dissolved and mixed by shaking. The OD value of each group was measured by an ELISA reader (model: 511 19300FI, Fisher), with the main wavelength of 570 nm and the reference wavelength of 490 nm. The cell proliferation inhibition rate of each group was calculated: Proliferation inhibition rate (%) = (OD value of the control group - OD value of the experimental group) / OD value of the control group × 100%.

[0184] The results are as follows:

[0185] (1) Effects of TCS-penetrating peptide on the proliferation rate of normal cells HUVEC and tumor cells HT1080: Compared with TCS, TCS-penetrating peptide has a significant increase in toxicity to both cells ( Fig.12 , Fig.13 ). This should be because the TCS protein alone has limited ability to enter cells and is difficult to enter cells to exert its toxicity. The cell-penetrating peptide modification increases the ability of TCS to enter cells, indicating that the cell-penetrating peptide can carry TCS into cells and exert its cytotoxic effect. At the same time, it shows that the ability of the cell-penetrating peptide to carry TCS into cells is not cell-selective.

[0186] (2) Effects of TCS-transmembrane peptide-MMP-2 substrate peptide-PEG conjugate on the proliferation rate of normal cells HUVEC and tumor cells HT1080: In the tumor cells HT1080 with high expression of MMP-2, the cytotoxicity of this conjugate was significantly increased compared with that of TCS-transmembrane peptide conjugate ( Fig.12 ); however, in HUVEC cells with low expression of MMP-2, the expression of MMP-2 in HUVEC cells was significantly weaker than that in TCS-transmembrane peptide conjugates ( Fig.13 ). This should be because the MMP-2 enzyme secreted and expressed by HT1080 specifically cuts the PEG chain in the above-mentioned connector, thereby exposing the cell-penetrating peptide, thereby mediating TCS into the cell and exerting its cytotoxicity; while HUVEC lacks the expression of MMP-2 and cannot cut off PEG, resulting in PEG blocking the cell-penetrating peptide and losing its cell-penetrating ability. This further proves the MMP-2 tumor enzyme responsiveness of the TCS-cell-penetrating peptide-MMP-2 substrate peptide-PEG connector.

[0187] Experimental Example 4 In vivo tumor targeting and tissue distribution experiment of TCS-cell-penetrating peptide-MMP-2 substrate peptide-PEG conjugate

[0188] (1) Cy5 fluorescence labeling of TCS-cell-penetrating peptide-MMP-2 substrate peptide-PEG conjugate

[0189] The TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG conjugate prepared in Preparation Example 2 was labeled with NHS-Cy5 at a molar excess of 3 times, and the reaction was carried out overnight at 4° C. in the dark. The excess Cy5 dye was removed using a desalting column.

[0190] (2) Establishment of HT1080 human fibrosarcoma tumor-bearing nude mouse model

[0191] HT1080 cells in the logarithmic growth phase were digested and dispersed with 0.25% trypsin, and the cell count was adjusted to prepare 5×10 6 Four Balb / c nude mice (purchased from Shanghai Sleike Experimental Animal Co., Ltd.) with a body mass of 18-22 g were taken and 100 μl of the cell suspension was injected subcutaneously into the back of the nude mice. The growth and tumor formation of HT1080 cells in Balb / c mice were observed.

[0192] (3) In vivo tumor targeting and tissue distribution experiments of the conjugate

[0193] When the tumor volume is 400 mm 3At about 1:10 p.m., the Cy5-labeled conjugate was injected into the tail vein at 100 μl / mouse. At 1, 2, 4, 8, 12, and 24 hours after injection, the nude mice were placed in a small animal in vivo imaging device for observation and photography. 24 hours after injection, the animals were killed by dislocation, and the main organs (heart, liver, spleen, lung, kidney) and tumors were removed and observed and photographed in a in vivo imaging device.

[0194] The results are as follows:

[0195] from Fig.14 As can be seen in a, TCS-transmembrane peptide-MMP-2 substrate peptide-PEG conjugate can quickly reach the tumor site after tail vein injection and accumulate significantly in the tumor site within 12 hours. After 24 hours, with the metabolism of the drug, the accumulation in the tumor site decreased significantly. However, from the tissue distribution diagram ( Fig.14 As can be seen in b), there is still a large amount of drug accumulation in the tumor site, more than in the heart and spleen, while the liver and kidneys, the main metabolic organs of PEGylated protein drugs, have the most drug distribution.

[0196] Experimental Example 5 In vivo anti-tumor experiments of TCS, TCS-cell-penetrating peptide conjugates, and TCS-cell-penetrating peptide-MMP-2 substrate peptide-PEG conjugates

[0197] (1) Establishment of HT1080 human fibrosarcoma tumor-bearing nude mouse model

[0198] HT1080 cells in the logarithmic growth phase were digested and dispersed with 0.25% trypsin, and the cell count was adjusted to prepare 5×10 6 24 Balb / c nude mice (purchased from Shanghai Sleike Experimental Animal Co., Ltd.) with a body mass of 18-22 g were taken and 100 μl of the cell suspension was injected subcutaneously into the back of the nude mice. The growth and tumor formation of HT1080 cells in Balb / c mice were observed.

[0199] (2) Antitumor effect in tumor-bearing nude mice

[0200] When the tumor volume is 100 mm 3 The mice were randomly divided into 4 groups: TCS group, TCS-penetrating peptide group, TCS-penetrating peptide-MMP-2 substrate peptide-PEG group, and the saline group was used as a negative control. Each group was injected with the tail vein, and the dose was 2.5 mg / kg, once every two days. During the administration period, the weight of the nude mice was monitored every day, and the long diameter (L) and short diameter (S) of the tumor were measured, and the tumor volume was calculated: V = L × S 2 / 2. After 18 days of administration, the nude mice were killed by dislocation, the tumors were removed, the blood stains and capsule on the tumor surface were carefully removed, and the tumors were weighed.

[0201] Fig.15 The figure shows the growth of tumor volume during drug administration. As can be seen from the figure, compared with the saline group, the TCS group had almost no inhibitory effect on tumor growth. The TCS-transmembrane peptide group and the TCS-transmembrane peptide-MMP-2 substrate peptide-PEG group both had a more significant tumor inhibition effect. The tumor inhibition effect was: the TCS-transmembrane peptide-MMP-2 substrate peptide-PEG group was greater than the TCS-transmembrane peptide group and the TCS group. Fig.16 The tumor photos of each group after the administration were intuitively displayed. It can be seen from the figure that the tumors of mice injected with normal saline and TCS were significantly larger, while the tumors of mice injected with TCS-transmembrane peptide and TCS-transmembrane peptide-MMP-2 substrate peptide-PEG were significantly reduced to varying degrees.

[0202] Fig.17 and Fig.18 The figure shows the survival curve of mice in each group and the weight change of mice during the administration process. It can be seen from the figure that the weight of mice in the TCS group and the TCS-transmembrane peptide group decreased significantly to varying degrees during the administration process, and the experimental mice died near the end of the experiment. The weight of mice in the TCS-transmembrane peptide-MMP-2 substrate peptide-PEG group did not decrease significantly compared with the saline control group, and no animal deaths occurred. This to a certain extent reflects that the systemic toxicity of TCS and TCS-transmembrane peptide is greater, while the toxicity of TCS-transmembrane peptide-MMP-2 substrate peptide-PEG is lower.

[0203] Fig.19 The results of pathological section analysis of the organs of mice in each group at the end of the experiment. As can be seen from the figure, TCS and TCS-transmembrane peptide caused certain toxicity to the lungs, spleen and kidneys of mice, which were manifested as: thickening of alveolar walls, interstitial hemorrhage, disordered epithelial arrangement; destruction of spleen follicle structure, giant cell hyperplasia; swelling and deformity of glomeruli and renal convoluted tubules, and disappearance of Bowman's cysts. However, the toxicity of TCS-transmembrane peptide-MMP-2 substrate peptide-PEG to various organs was much milder.

[0204] Experimental Example 6 Immunogenicity Experiment of TCS, TCS-Cell-penetrating Peptide-MMP-2 Substrate Peptide-PEG

[0205] (1) Mouse Immunization Experiment

[0206] Ten Balb / c mice (purchased from Shanghai Sleike Experimental Animal Co., Ltd.) with a body mass of 18-22 g were randomly divided into two groups, the TCS group and the TCS-transmembrane peptide-MMP-2 substrate peptide-PEG group. Both groups were immunized by subcutaneous injection, with an injection dose of 10 μg / mouse, once every 7 days, for three consecutive immunizations. Blood samples were collected by orbital blood sampling before the first immunization and 7 days after the third immunization, and serum was obtained by centrifugation.

[0207] (2) Determination of anti-TCS mouse IgG titer

[0208] The anti-TCS mouse IgG titer in the collected serum samples was determined by the conventional ELISA method in the art. The specific method is as follows:

[0209] TCS protein was diluted with coating buffer (50mM sodium carbonate pH 9.6) to a concentration of 10μl / ml solution, and 100μl of the above solution was added to each well of a 96-well ELISA microplate, and the microplate was placed at 4°C overnight. After washing three times with 300μl PBST, each well was blocked with PBST containing 1% goat blocking serum at 37°C for 1.5 hours. Serum samples were diluted 1,000-1,000,000 times with PBST containing 0.01% goat blocking serum, and 100μl of the diluted serum samples were added to the wells. At the same time, negative serum before immunization was used as a negative control, mouse anti-TCS IgG antibody (purchased from Santa Cruz) was used as a positive control, and primary antibody dilution was used as a blank control. Incubate at 37°C for 2 hours. After washing three times with PBST, 100μl of 1,000-fold diluted HRP-labeled goat anti-mouse secondary antibody was added to each well and incubated at 37°C for 1 hour. After washing three times with PBST, 200 μl TMB color substrate was added to each well, reacted at 37°C for 15 minutes, 50 μl 20% sulfuric acid was added to each well to terminate the reaction, and the OD value of each group was measured with an ELISA reader (model: 51119300FI, Fisher) at a wavelength of 450 nm to draw a titer curve.

[0210] The results are as follows:

[0211] from Fig. 20 It can be seen that the unmodified TCS protein has a strong immunogenicity, while the immunogenicity of TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG is significantly lower than that of TCS. This shows that PEG modification can significantly reduce the immunogenicity of TCS and increase its safety in clinical use.

[0212] In summary, the TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG conjugate of the present invention can accumulate at the tumor site through the EPR effect and has a high bioavailability. At the same time, it can be activated by MMP-2 enzyme cleavage highly expressed in the tumor and its microenvironment, thereby mediating TCS entry into cells to kill tumor cells, having significant tumor targeting and tumor enzyme sensitivity, and showing a significant growth inhibitory effect on tumor cells both in vivo and in vitro. At the same time, the toxicity and immunogenicity of the TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG conjugate of the present invention are also significantly reduced.

Claims

1. A fusion protein, characterized in that The fusion protein has the structure shown in Formula Ia: A-L1-B-L2-C Formula Ia in, A is a trichosanthin element, and the trichosanthin does not contain Cys, B is the membrane-penetrating element. C is the matrix metalloproteinase 2 substrate peptide element, L1 and L2 are each connecting peptide elements, "-" is the peptide bond connecting each element, The fusion protein has tumor suppressor activity, and the tumor is a matrix metalloproteinase 2 (MMP-2) positive tumor. Wherein, the transmembrane element is represented by any polypeptide shown in SEQ ID NO.: 5-7; The matrix metalloproteinase 2 substrate peptide element is shown in any one of the polypeptides shown in SEQ ID NO.: 8-9; The connecting peptide element comprises 3-5 identical or different amino acid residues.

2. The fusion protein according to claim 1, characterized in that The trichosanthin element is wild-type trichosanthin.

3. The fusion protein according to claim 2, characterized in that The amino acid sequence of the wild-type trichosanthin is shown in SEQ ID NO.:1, and the polynucleotide encoding it is shown in SEQ ID NO.:

2.

4. The fusion protein according to claim 1, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO.:

3.

5. The fusion protein according to claim 1, characterized in that The connecting peptide element is 3 Gly.

6. The precursor protein of the fusion protein according to claim 1, characterized in that The precursor protein has a structure shown in Formula IIa: A-L1-B-L2-CDE Formula IIa in, A is the trichosanthin element, B is the membrane-penetrating element. C is a matrix metalloproteinase 2 (MMP-2) substrate peptide element, D is none or 1-3 Cys, E is an intein element, L1 and L2 are each free or connected peptide elements, "-" is the peptide bond connecting each element, The precursor protein is cleaved by a thiol-containing reagent to produce the fusion protein of claim 1, Furthermore, the transmembrane element is represented by any polypeptide shown in SEQ ID NO.: 5-7; The matrix metalloproteinase 2 substrate peptide element is shown in any one of the polypeptides shown in SEQ ID NOs.: 8-9.

7. A polynucleotide, characterized in that The polynucleotide encodes the fusion protein according to claim 1 or encodes the precursor protein according to claim 6.

8. A carrier, characterized in that The vector contains the polynucleotide according to claim 7.

9. A host cell, characterized in that The host cell contains the vector according to claim 8 or the polynucleotide according to claim 7 is integrated into the gene of the host cell.

10. The modified product of the fusion protein according to claim 1, characterized in that: The modified substance is formed by subjecting the fusion protein according to claim 1 to site-specific PEGylation to form a fusion protein modified substance.

11. The modified article according to claim 10, wherein The site-specific PEGylation is to perform PEG modification on the C-terminus of the fusion protein.

12. Use of the fusion protein according to claim 1 or the modified product according to claim 10, characterized in that: The invention is used for preparing a pharmaceutical composition for treating tumors, wherein the tumors are matrix metalloproteinase 2 (MMP-2) positive tumors.

13. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the fusion protein according to claim 1 or the modified substance according to claim 10, and a pharmaceutically acceptable carrier.

14. A method for producing the fusion protein or its precursor protein according to claim 1, comprising the steps of: (i) Cultivating the host cell according to claim 9 under appropriate conditions to obtain the expression of the fusion protein or its precursor protein.

15. The method according to claim 14, characterized in that The method may further comprise the steps of: (ii) separating and purifying the obtained fusion protein or its precursor protein.

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