CN106674353A - Novel radix trichosanthis fusion protein and application thereof - Google Patents

Abstract

The invention discloses a novel radix trichosanthis fusion protein. Particularly, the novel radix trichosanthis fusion protein disclosed by the invention is characterized in that radix trichosanthis protein with poorer endocytosis performance is modified and is fused with a transmembrane protein and a matrix metalloproteinase substrate structural domain; intein-mediated self cleavage is used, so that a high-specificity hypoimmunity prototype tumor inhibitory radix trichosanthis fusion protein which is easier for PEG formation and is very conductive to endocytosis is obtained. The invention provides a novel method of mediated radix trichosanthis protein and opens up a new idea for novel application of traditional Chinese medicines and old medicines.

Description

A new trichosanthin fusion protein and its application

technical field

The present invention relates to the field of biomedicine, more specifically, relates to a novel trichosanthin fusion protein.

Background technique

Malignant tumors are one of the greatest threats to human life. According to the report of the World Health Organization, the incidence of cancer is increasing year by year, and it is showing a younger trend. At present, the treatment methods for malignant tumors are still based on surgical resection combined with chemotherapy and radiotherapy. Small molecule chemotherapeutic drugs are prone to drug resistance and have huge toxic and side effects, so they cannot effectively kill tumor cells, and at the same time bring great pain to patients. With the development of biotechnology, biomacromolecular drugs, especially protein drugs, have attracted more and more attention due to their strong biological activity and good biocompatibility. Protein drugs have become one of the most cutting-edge directions of new drug research and development in the 21st century.

Trichosanthin (TCS) is a protein extracted from the root of Trichosanthes trichosanthes in the family Cucurbitaceae, Trichosanthes trichosanthis, which has been used as a labor-inducing drug to terminate second-trimester pregnancy in traditional Chinese medicine for centuries. TCS is a type I ribosome inactivating protein with a molecular weight of 27kDa. It has N-glycosidase activity and can recognize the large ribosomal subunit of mammalian cells and make it depurinate, thereby inhibiting protein synthesis of cells and leading to cell death. At the same time, some studies have shown that TCS can induce apoptosis through various ways. It is because of these biological activities of TCS that it has been confirmed to have broad-spectrum antitumor activity. However, TCS does not have tumor targeting in vivo and lacks a certain ability to enter cells. In addition, it has been reported that TCS has strong immunogenicity. These have become restrictions on the clinical application of TCS as an antitumor drug.

At present, chemical modification has become an important method to improve the druggability of trichosanthin. The traditional chemical modification sites for protein drugs are mainly limited to the side chain amino groups of lysine residues in proteins and the side chain sulfhydryl groups of cysteine introduced by means of genetic engineering. The disadvantages of these two modification methods are obvious. The former, due to the large number of lysine residues in the protein, will lead to inhomogeneity of the modified product, which increases the difficulty and cost of separation and purification of the modified product. Although the latter can realize site-specific modification of proteins, due to the instability of free sulfhydryl groups, protein drugs are prone to oxidation of sulfhydryl groups during the preparation and purification process to form intermolecular dimers.

Therefore, there is an urgent need in this field to develop a novel protein modification method to achieve efficient, specific, and site-specific modification of protein drugs, so as to obtain low-toxicity, high-efficiency trichosanthin with tumor suppressive activity.

Contents of the invention

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.

In the first aspect of the present invention, a fusion protein is provided, and the fusion protein has a structure shown in formula Ia or formula Ib:

A-L1-B-L2-C Formula Ia or

B-L1-A-L2-C Formula Ib

in,

A is trichosanthin element,

B is the transmembrane element,

C is a matrix metalloproteinase 2 substrate peptide element,

L1, L2 are each none or linked peptide elements,

"-" is the peptide bond connecting each element,

And the fusion protein has tumor suppressing activity.

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

In another preferred example, the trichosanthin element includes wild-type or mutant trichosanthin, preferably, the trichosanthin includes its full-length protein or a fragment thereof.

In another preferred example, the trichosanthin does not contain Cys.

In another preferred example, 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.

In another preferred example, the transmembrane element includes any polypeptide shown in SEQ ID NO.: 5-7 (VSRRRRRGGRRRR, YGRKKRRGGQRRR, RRRRRRRR).

In another preferred example, the matrix metalloproteinase 2 substrate peptide element includes any polypeptide (PLGLAG, PLGVR) shown in SEQ ID NO.: 8-9.

In another preferred example, the connecting peptide element includes 0-10 identical or different amino acid residues, preferably 3-5.

In another preferred example, the linking peptide element is 3 Gly.

In another preferred example, the tumor includes a solid tumor, preferably, a matrix metalloproteinase 2MMP-2 positive tumor.

In another preferred example, the tumor includes fibrosarcoma, glioma, or liver cancer.

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

A-L1-B-L2-C-D-E Formula IIa or

B-L1-A-L2-C-D-E Formula IIb;

in,

A is trichosanthin element,

B is the transmembrane 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, L2 are each none or linking peptide elements,

"-" is the peptide bond connecting each element,

The fusion protein described in the first aspect of the present invention is produced after the precursor protein is cleaved by a thiol-containing reagent.

In another preferred example, the intein element further contains a tag sequence at its C-terminus.

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.

In another preferred example, the thiol-containing reagent includes L-cysteine, DTT, mercaptoethanol or sodium 2-mercaptoethanesulfonate.

The third aspect of the present invention provides a polynucleotide sequence, which 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.

In another preferred example, the polynucleotide sequence is shown in SEQ ID NO.:4.

The fourth aspect of the present invention provides a vector containing the polynucleotide described in the third aspect of the present invention.

The fifth aspect of the present invention provides a host cell containing the vector according to the fourth aspect of the present invention or the polynucleotide described in the second aspect of the present invention integrated into the gene of the host cell.

The sixth aspect of the present invention provides a modification of the fusion protein according to the first aspect of the present invention, wherein the modification is formed by site-directed PEGylation of the fusion protein according to the first aspect of the present invention to form a modification of the fusion protein.

In another preferred example, the site-specific PEGylation is PEG modification at the C-terminus of the fusion protein.

In another preferred example, the fusion protein modification contains PEG with a molecular weight of 3-40KD, preferably 5KD.

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

The eighth aspect of the present invention provides a pharmaceutical composition, which contains the fusion protein described in the first aspect of the present invention or the modification described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier.

The ninth aspect of the present invention provides a method for producing the fusion protein or its precursor protein described in the first aspect of the present invention, comprising the steps of:

(i) Under suitable conditions, cultivate the host cell described in the fifth aspect of the present invention, so as to obtain and express the fusion protein or its precursor protein.

In another preferred example, the method may also include the steps of:

(ii) Separating and purifying the obtained fusion protein or its precursor protein.

In another preferred example, the separation and purification include directly using intein-mediated separation and purification.

The tenth aspect of the present invention provides an in vitro non-therapeutic method for inhibiting tumor cells, comprising the steps of: adding the fusion protein described in the first aspect of the present invention and the modified product described in the sixth aspect of the present invention to the tumor cell culture Or the pharmaceutical composition described in the eighth aspect of the present invention, thereby inhibiting tumor cells.

In the eleventh aspect of the present invention, there is provided a method for treating tumors, comprising the step of: administering a safe amount of the fusion protein described in the first aspect of the present invention, the modification described in the sixth aspect of the present invention, or The pharmaceutical composition described in the eighth aspect of the present invention, so as to treat tumors.

In another preferred example, the desired subject is a mammal, including a mouse, a rat, or a human, preferably a human.

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 in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Fig. 1 is a synthetic route diagram of TCS-penetrating peptide and TCS-penetrating peptide-MMP-2 substrate peptide-PEG linker mediated by intein in Preparation Example 1 and Preparation Example 2 according to the present invention.

Fig. 2 is a schematic diagram of the anti-tumor mechanism of the TCS-penetrating peptide-MMP-2 substrate peptide-PEG linker according to the present invention.

Fig. 3 is an electrophoresis diagram of the prokaryotic expression and purification of the recombinant TCS protein mediated by intein in Preparation Example 1 according to the present invention.

Fig. 4 is an electrophoretic graph of prokaryotic expression and purification of the recombinant TCS-penetrating peptide-MMP-2 substrate peptide fusion protein mediated by intein in Example 2 of the preparation according to the present invention.

Fig. 5 is a synthetic electrophoresis diagram of the intein-mediated TCS-penetrating peptide linker prepared in Example 1 according to the present invention.

Fig. 6 is a chromatogram of the purification of the TCS-penetrating peptide linker prepared in Example 1 according to the present invention.

Fig. 7 is a synthetic electrophoresis diagram of the intein-mediated TCS-penetrating peptide-MMP-2 substrate peptide-PEG linker prepared in Example 2 according to the present invention.

Fig. 8 is a chromatogram of the purification of the TCS-penetrating peptide-MMP-2 substrate peptide-PEG linker prepared in Example 2 according to the present invention.

Fig. 9 is an electrophoretic diagram of purified TCS, TCS-penetrating peptide linker and TCS-penetrating peptide-MMP-2 substrate peptide-PEG linker prepared in Preparation Example 1 and Preparation Example 2 according to the present invention .

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

Fig. 11 is an electrophoresis diagram of the TCS-penetrating peptide-MMP-2 substrate peptide-PEG linker before and after being digested by MMP-2 in Experimental Example 2 of the present invention.

Figure 12 shows the proliferation of tumor cell HT1080 cells with high expression of MMP-2 enzymes by TCS, TCS-penetrating peptide linker and TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG linker in Experimental Example 3 of the present invention inhibitory effect.

Fig. 13 is according to TCS, TCS-penetrating peptide connector and TCS-penetrating peptide-MMP-2 substrate peptide-PEG connector in Experimental Example 3 of the present invention to normal cell HUVEC cell proliferation with low expression of MMP-2 enzyme inhibitory effect.

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

Fig. 15 shows the tumor inhibition of HT1080 tumor-bearing nude mice by TCS, TCS-penetrating peptide linker and TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG linker in Experimental Example 5 of the present invention.

Fig. 16 is a photograph of tumors treated with TCS, TCS-penetrating peptide linker and TCS-penetrative peptide-MMP-2 substrate peptide-PEG linker in Experimental Example 5 of the present invention.

Fig. 17 is a graph showing the survival rate of animals in each group during the administration of TCS, TCS-penetrating peptide linker and TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG linker in Experimental Example 5 of the present invention.

Fig. 18 shows the body weight changes of animals in each group during the administration of TCS, TCS-penetrating peptide linker and TCS-membrane penetrating peptide-MMP-2 substrate peptide-PEG linker in Experimental Example 5 of the present invention.

Figure 19 shows the animal organs (lung, spleen, kidney) of each group after treatment with TCS, TCS-penetrating peptide linker and TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG linker in Experimental Example 5 of the present invention ) pathological sections.

Fig. 20 is the immunogenicity determination of TCS and TCS-penetrating peptide-MMP-2 substrate peptide-PEG linker in Experimental Example 6 according to the present invention.

detailed description

After extensive and in-depth research, the inventors unexpectedly prepared a new fusion protein for the first time. Using intein-mediated self-cleavage, a high-specificity, low-immunity type tumor suppressor trichosanthin fusion protein that is easy to PEGylate and very helpful for cell entry was obtained. The invention not only provides a new method for mediating trichosanthin into cells, but also opens up new ideas for the new use of traditional Chinese medicines. On this basis, the present invention has been accomplished.

Fusion protein and its preparation

In the present invention, "recombinant fusion protein", "protein of the present invention", "fusion protein of the present invention", and "fusion protein" can be used interchangeably, referring to the structure described in formula Ia or formula Ib, that is, containing trichosanthin elements , a transmembrane element, and a fusion protein of a matrix metalloproteinase 2 substrate peptide element. The protein of the present invention may be a monomer or a multimer (such as a dimer) formed from the monomer. Furthermore, it is to be understood that the term also includes active fragments and derivatives of fusion proteins.

The invention also includes active fragments, derivatives and analogs of the fusion proteins according to the invention. As used herein, the terms "fragment", "derivative" and "analogue" refer to a polypeptide that substantially retains the tumor suppressor function or activity of human trichosanthin. The polypeptide fragments, derivatives or analogs of the present invention can be (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more A polypeptide with substituent groups in amino acid residues, or (iii) a polypeptide formed by fusion of a fusion protein with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide fused to this polypeptide sequence (a fusion protein fused to a leader sequence, a secretory sequence, or a tag sequence such as 6His). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

A preferred class of active derivatives refers to the formation of at most 3, preferably at most 2, and more preferably at most 1 amino acids replaced by amino acids with similar or similar properties compared with the amino acid sequence of formula Ia or formula Ib peptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1. Preferably, the derivatives do not contain Cys.

Table 1

initial residue representative replacement preferred substitution 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 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; Leu

The invention also provides analogs of the fusion proteins of the invention. The difference between these analogs and the polypeptide shown in SEQ ID NO: 3 may be the difference in the amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

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, those formed with the following acids: hydrochloric, hydrobromic, sulfuric, citric, tartaric, phosphoric, lactic, pyruvic, acetic, succinic, oxalic, fumaric, maleic, acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid. Other salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, as well as in the form of esters, carbamates or other conventional "prodrugs".

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the 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 together in the natural state.

As used herein, "isolated recombinant fusion protein" means that the recombinant fusion protein is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify recombinant fusion proteins using standard protein purification techniques. Essentially pure proteins yield a single major band on non-reducing polyacrylamide gels.

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand.

The present invention also relates to variants of the above polynucleotides, which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention. Variants of this polynucleotide may 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 does not substantially change the function of the encoded polypeptide.

As used herein, the term "primer" refers to a general term for oligonucleotides that can be used as a starting point to synthesize a DNA chain complementary to a template under the action of a DNA polymerase when paired with a template. Primers can be natural RNA, DNA, or any form of natural nucleotides. Primers can even be non-natural nucleotides such as LNA or ZNA, etc. A primer is "substantially" (or "essentially") complementary to a particular sequence on one strand of the template. A primer must be sufficiently complementary to one strand of the template to initiate extension, but the sequence of the primer does not have to be perfectly complementary to that of the template. For example, adding a non-complementary sequence to the 5' end of a primer whose 3' end is complementary to the template, such a primer is still substantially complementary to the template. As long as there is a sufficiently long primer that can fully bind to the template, non-completely complementary primers can also form a primer-template complex with the template, thereby performing amplification.

The full-length nucleotide sequence or fragments of the fusion protein or its elements of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the published relevant nucleotide sequences, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art can be used as Template, amplified to obtain related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

The method of amplifying DNA/RNA using PCR technique is preferably used to obtain the gene of the present invention. Primers for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

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

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

(1). Use the polynucleotide (or variant) encoding the protein of the present invention of the present invention, or transform or transduce a suitable host cell with a recombinant expression vector containing the polynucleotide;

(2). Host cells cultured in a suitable medium;

(3). Isolate and purify protein from culture medium or cells.

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 and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

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

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: bacterial cells of Escherichia coli, Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CHO, NSO, COS7, or 293 cells, etc.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

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

The protein in the above method may be expressed inside the cell, or on the cell membrane, or secreted outside the cell. Proteins can be isolated and purified by various separation methods by taking advantage of their physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

The present invention also provides the precursor protein of the fusion protein, the precursor protein utilizes the method of obtaining the fusion protein mediated by intein, the gene encoding the intein is added to the gene construct of the fusion protein, and By cleavage of the tag after expressing the protein, a high-purity fusion protein is obtained directly, eliminating the need for further isolation or purification steps.

penetrating peptide

The fusion protein provided by the present invention preferably contains a membrane-penetrating peptide. Trichosanthin TCS belongs to type I ribosome inactivating protein, which only contains the A chain with N-glycosidase activity, and lacks the lectin-like B chain that mediates transcytosis than type II ribosome inactivating protein. Therefore, this type of protein toxin The cell entry efficiency is low, and the anti-tumor cytotoxicity is far inferior to type II ribosome inactivating protein. Membrane-penetrating peptides are a class of polypeptides found in nature or artificially screened that have the ability to penetrate cell membranes. The membrane-penetrating peptides that can be used in the present invention are not particularly limited, and can be any membrane-penetrating peptides rich in basic amino acids and amphipathic, as long as they can promote the uptake of TCS that lacks cell-entrying ability by cells. Preferably, the membrane-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 membrane-penetrating peptide of the present invention can not only enter cells by itself, but also carry the TCS of the present invention into cells. In addition, the penetrating peptide of the present invention can directly obtain the fusion protein containing the penetrating peptide by introducing the gene of the penetrating peptide into the expression vector expressing the fusion protein, or can also obtain the penetrating peptide-modified protein through the mediation of intein. methods, preferred methods include:

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

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

(c) collecting the thalline by centrifugation and sonicating;

(d) Affinity-purify the supernatant of the target protein containing the C-terminal fusion intein and CBD tag with a chitin column, wash away the impurity protein, and wash it with a buffer containing 2-mercaptoethanesulfonic acid sodium (MESNA) The solution was cut overnight on the column, and the eluate was collected;

(e) After ultrafiltration and concentration, add the above-mentioned penetrating peptide and react overnight to form a TCS-penetrating peptide linker, wherein the molar ratio of protein to polypeptide is 1:20; use a desalting column to remove excess polypeptide in the solution, from step The yield of the TCS-penetrating peptide linker obtained in (e) is 90%-95%.

Intein

Intein-mediated protein prokaryotic expression, purification and modification method is a new protein site-specific modification technology. Intein is a polypeptide with self-cleavage function. Fusion intein sequence and chitin binding domain (CBD) affinity tag at the C-terminus of the target protein. This technology uses Escherichia coli prokaryotic expression system for exogenous protein expression. When using chitin column for affinity purification of the target protein, use reagents containing sulfhydryl groups for on-column cleavage. Under the mediation of intein, the target protein can be extracted from Cut off the intein, and at the same time introduce a corresponding active group at the C-terminus of the protein for downstream protein modification.

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

In addition, there is no particular limitation on the cleavage reagents that can be used in the present invention to mediate self-cleavage, and thiol-containing cleavage reagents are preferred. In a preferred embodiment, sodium 2-mercaptoethanesulfonate is selected for cleavage, and a thioester bond will be introduced at the end of the protein. At this time, the protein can react efficiently with the polypeptide whose N-terminal is cysteine; and If L-cysteine is used as the cleavage reagent, an additional cysteine will be introduced at the end of the protein, which can react with PEG with maleimide as the end group, thereby achieving site-specific PEG modification of the protein.

PEG modification

PEG modification refers to the use of polyethylene glycol with functional groups for protein drug modification, usually with random modification and site-specific modification. The present invention is preferably PEG site-directed modification, for example, the fusion protein with intein and CBD tags fused at the end is purified with a chitin affinity column, and the target protein is cleaved on the column with L-cysteine. Under the mediation of intein, the target protein is excised from the intein, CBD tag and chitin affinity column, and a cysteine residue is introduced at the C-terminus of the target protein. The side chain sulfhydryl group of the cysteine residue can be used to react with the PEG of the terminal maleimide to realize site-directed PEGylation of the C-terminus of the protein.

A preferred PEG-directed modification method is as follows:

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

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

(c) collecting the thalline by centrifugation and sonicating;

(d) Affinity-purify the supernatant of the target protein containing the C-terminal fusion intein and CBD tag with a chitin column, wash away the impurity protein, and perform on-column cleavage with a buffer containing L-cysteine Overnight, collect the eluate;

(e) Concentrate by ultrafiltration and remove free cysteine in the solution, add maleimide-PEG (Mal-PEG), and react overnight to form a fusion protein-PEG linker, wherein the mole of protein and Mal-PEG The ratio is 1:10;

(f) Using a cation exchange column to separate and purify the fusion protein-PEG conjugate obtained in step (e).

connecting peptide

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

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

Pharmaceutical composition and method of administration

The present invention also provides a composition, which contains an effective amount of the fusion protein of the present invention and a pharmaceutically acceptable carrier. Generally, the fusion protein of the present invention can be prepared in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably, the pH is about 6-8.

As used herein, the term "effective amount" or "effective dose" refers to the amount that can produce functions or activities on 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%.

As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation and allergic reactions), ie, has a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.

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, dextrose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should match the mode of administration, and the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably produced under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparations of the present invention can also be made into sustained-release preparations.

The effective amount of the fusion protein of the present invention may vary with the mode of administration, the severity of the disease to be treated, and the like. The selection of a preferred effective amount can be determined by those of ordinary skill in the art based on various factors (eg, 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 body weight of the patient, the immune status of the patient, the dose way etc. For tumor patients, usually, 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, several divided doses may be administered daily or the dose may be proportionally reduced as the exigencies of the therapeutic situation dictate.

Beneficial effect of the present invention

The invention obtains the high specificity and low immunotype tumor suppressive trichosanthemi fusion protein which is easier to PEGylate and is very helpful for cell entry by modifying and transforming the structure of the wild trichosanthin protein. The invention not only provides a new method for mediating trichosanthin into cells, but also opens up new ideas for the new use of traditional Chinese medicines.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the following examples is generally according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory handbook (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacture conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Reagents and Medicines

The prokaryotic expression vector pTXB1 and chitin affinity column were purchased from NEB. The recombinant expression plasmid of TCS and TCS-penetrating peptide-MMP-2 substrate peptide fusion protein was 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 thiazolium 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 (Shanghai) Chemical Reagent Company. Maleimide polyethylene glycol (Mal-PEG) was purchased from Beijing Jiankai Technology Co., Ltd. Sodium 2-mercaptoethanesulfonate (MENSA) was purchased from Bailingwei Technology Group. Peptides were purchased from Shanghai Lyon Chemical Co., Ltd. Succinimide 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. Dry powder of DMEM cell culture medium, trypsin (Trypsin), fetal bovine serum (FBS), double antibody (penicillin-streptomycin) were purchased from Gibco and Invitrogen, respectively.

Preparation Example 1 Synthesis of TCS-penetrating peptide

Prokaryotic expression and purification of recombinant TCS protein

a: The TCS recombinant expression plasmid (TCS gene sequence shown in SEQ ID NO.: 2) was transformed into Escherichia coli BL21 (DE3) competent cells.

b: Culture the strain containing the recombinant plasmid with LB medium containing 100 μg/ml Amp in a 37°C constant temperature shaker at 250 rpm to the logarithmic growth phase (absorbance value at 600 nm is 0.6-0.8), and add IPTG with a final concentration of 1 mM , expressed overnight (14h) at 25°C, 150rpm.

c: Collect the bacterial cells by centrifuging at 6,000 rpm at 4°C for 20 minutes.

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

e: Use a probe sonicator with a power of 400 W to sonicate the cells for 30 min.

f: centrifuge at 12,000 rpm for 20 min at 4°C, and collect the supernatant.

g: Pass the supernatant containing the target protein through a chitin column pre-equilibrated with HEPES buffer at a flow rate of 1 ml/min. After loading the sample, wash away non-specifically bound impurities with 25 times the column volume of HEPES buffer.

h: Use 3 times the column volume of HEPES buffer containing 50 mM MESNA to flow through the column, and keep a small amount of buffer, close the outlet of the column, and cut on the column overnight (16h).

i: Open the outlet of the column, collect the effluent buffer containing the target protein, and continue to add 3 times the column volume of HEPES buffer to continue to elute the target protein.

Synthesis of TCS-penetrating peptide

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

Preparation Example 2 Synthesis of PEGylated TCS-penetrating peptide-MMP-2 substrate peptide

Expression and purification of TCS-penetrating peptide-MMP-2 substrate peptide fusion protein

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

b: Culture the strain containing the recombinant plasmid with LB medium containing 100 μg/ml Amp in a 37°C constant temperature shaker at 250 rpm to the logarithmic growth phase (absorbance value at 600 nm is 0.6-0.8), and add IPTG with a final concentration of 1 mM , expressed overnight (14h) at 25°C, 150rpm.

c: Collect the bacterial cells by centrifuging at 6,000 rpm at 4°C for 20 minutes.

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

e: Use a probe sonicator with a power of 400 W to sonicate the cells for 30 min.

f: centrifuge at 12,000 rpm for 20 min at 4°C, and collect the supernatant.

g: Pass the supernatant containing the target protein through a chitin column pre-equilibrated with HEPES buffer at a flow rate of 1 ml/min. After loading the sample, wash away non-specifically bound impurities with 25 times the column volume of HEPES buffer.

h: Use 3 times the column volume of HEPES buffer containing 50mM L-cysteine to flow through the column, and keep a small amount of buffer, close the outlet of the column, and cut on the column overnight (16h).

i: Open the outlet of the column, collect the effluent buffer containing the target protein, and continue to add 3 times the column volume of HEPES buffer to continue to elute the target protein.

PEGylation of TCS-penetrating peptide-MMP-2 substrate peptide fusion protein

Concentrate the collected protein eluate with an ultrafiltration tube with a molecular weight cut-off of 10,000, remove free L-cysteine in the solution with a desalting column, determine the protein concentration by the BCA method, and add Mal -PEG, react overnight (16h) at 4°C. The reaction efficiency was detected by SDS-PAGE electrophoresis at a concentration of 12%, reaching 80%-85% (Fig. 7). Use cation exchange column SPFF and fast protein purification chromatograph FPLC to separate and purify the reaction product, push the sample into the column with a sample injector, use pH 7.2 phosphate buffer containing 0.15-1M NaCl at 0.02M/min Gradient elution was performed at a flow rate, and the first elution peak was collected to obtain a fusion protein-PEG ligation product (Figure 8).

Experimental example 1 Determination of MMP-2 enzyme content in HT1080 and HUVEC cells and their culture medium

The MMP-2 enzyme content in HT1080 and HUVEC cells and their culture medium was detected by conventional Western Blot in the field. The specific method is as follows: HT1080 and HUVEC cells in the logarithmic growth phase were digested with trypsin and diluted to a cell suspension of ^1.5 ×105 cells/ml, transferred to a Nunc 6-well cell culture plate, and 2 ml of cells were added to each well. The suspension was cultured with DMEM complete medium containing 10% calf serum for 4 hours (37° C., 5% CO ₂ ). 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, 1ml per well, and continued to culture for 24h. The medium supernatant was collected, and the cells were lysed with cell lysate for 30 min, centrifuged at 4°C for 15 min, the supernatant was collected, the protein concentration was determined by the BCA method, and diluted to the same concentration with buffer. Add loading buffer to the medium and cell lysate samples and cook in boiling water for 5 minutes. Run the gel with 10% polyacrylamide gel, transfer to 0.45 μm PVDF membrane by electroporation, and block with 5% skimmed milk powder for 2 hours. A 1,000-fold dilution of anti-MMP-2 rabbit antibody (purchased from Cell Signaling Technology) and a 20,000-fold dilution of anti-β-actin mouse antibody (purchased from Sigma) were prepared in blocking solution and incubated overnight at 4°C. After washing the primary antibody three times, incubate with a 1,000-fold diluted secondary antibody from the corresponding species (purchased from Beyond Biotechnology Co., Ltd.), and incubate at room temperature for 1 hour. After washing the secondary antibody three times, ECL chromogenic substrate (purchased from Thermo Peirce) was added dropwise on the membrane, and imaged in a multifunctional gel imager (Biorad).

The result is as follows:

It can be seen from Figure 10 that both in the cells of HT1080 cells and in the cell culture medium, compared with HUVEC cells, there is a significant overexpression of MMP-2 enzyme. This shows that HT1080 cells can be used as a cell model for subsequent MMP-2 enzyme sensitivity experiments.

Experimental example 2 In vitro enzyme digestion experiment of TCS-penetrating peptide-MMP-2 substrate peptide-PEG

(1) Preparation of Enzyme Digestion Medium

The HT1080 cells in the logarithmic growth phase were digested with trypsin and diluted to a cell suspension of ^1.5 ×105 cells/ml, transferred to a Nunc 6-well cell culture plate, and 2ml of the cell suspension was added to each well, and used with 10 % calf serum DMEM complete culture medium for 4h (37 ° C, 5% CO ₂ ). 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, 1ml per well, and continued to culture for 24h. Aspirate the medium, centrifuge at 12,000rpm at 4°C for 10min to remove cell debris, and use the supernatant for in vitro enzyme digestion experiments.

(2) In vitro enzyme digestion experiment

Dilute the TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG linker with the enzyme digestion medium prepared by the above method to a final concentration of 0.1mg/ml, add 0.2‰ NaN3 at the same time, and place it in a constant temperature shaker at 37°C at 250rpm Enzyme digestion 48h. The digestion efficiency was detected by SDS-PAGE electrophoresis. The result is shown in the figure. The digested product was a band of about 30kDa, which was slightly smaller than the protein band before PEG modification (Figure 11).

Experimental example 3 MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyl bromotetrazolium blue, trade name: thiazolium blue) method for the determination of TCS and TCS-penetrating peptide Linkers, and in vitro antitumor effects of TCS-penetrating peptide-MMP-2 substrate peptide-PEG linkers

The HT1080 and HUVEC cells in the logarithmic growth phase ^were digested and diluted to a cell suspension with a density of 1.5×104 cells/mL, and transferred to a Nunc 96 cell culture well plate, and 200 μL of the cell suspension was added to each well , cultured in DMEM complete medium containing 10% calf serum for 24 hours (37°C, 5% CO ₂ ).

Determine the optimal drug concentration range through preliminary experiments, add solutions with different concentrations, and make 6 replicate holes for each concentration. After culturing for 48 hours, 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 with a microplate reader (model: 51119300FI, Fisher), the main wavelength was 570nm, and the reference wavelength was 490nm. 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%.

The result is as follows:

(1) The effect of TCS-penetrating peptide on the proliferation rate of normal cells HUVEC and tumor cell HT1080: Compared with TCS, the toxicity of TCS-penetrating peptide to both cells was significantly increased (Figure 12, Figure 13). This should be due to the limited ability of TCS protein to enter the cell alone, and it is difficult to enter the cell to exert its toxicity. The penetrating peptide modification increased the cell-entry ability of TCS, indicating that the penetrating peptide could carry TCS into cells and exert its cytotoxic effect. At the same time, it shows that the ability of penetrating peptides to carry TCS into cells has no cell selectivity.

(2) Effects of TCS-penetrating peptide-MMP-2 substrate peptide-PEG linker on the proliferation rate of common cell HUVEC and tumor cell HT1080: In the tumor cell HT1080 with high expression of MMP-2, the linker compared with The cytotoxicity of TCS was significantly increased, which was comparable to that of the TCS-penetrating peptide linker (Fig. 12); while in HUVEC, a common cell with low MMP-2 expression, it was significantly weaker than that of the TCS-penetrating peptide linker (Fig. 13). This should be due to the fact that the MMP-2 enzyme secreted and expressed by HT1080 specifically cuts the PEG chain in the above-mentioned linker, thereby exposing the membrane-penetrating peptide, thereby mediating TCS into the cell and exerting its cytotoxicity; while HUVEC lacks MMP-2 The expression of PEG cannot be cut off, causing PEG to block the membrane-penetrating peptide, making it lose its ability to enter the cell. This further demonstrates the MMP-2 tumor enzyme responsiveness of the TCS-penetrating peptide-MMP-2 substrate peptide-PEG linker.

Experimental example 4 In vivo tumor targeting and tissue distribution experiment of TCS-penetrating peptide-MMP-2 substrate peptide-PEG linker

(1) Cy5 fluorescent labeling of TCS-penetrating peptide-MMP-2 substrate peptide-PEG linker

The TCS-penetrating peptide-MMP-2 substrate peptide-PEG conjugate prepared according to Preparation Example 2 was labeled with NHS-Cy5 at a molar ratio of 3 times, and reacted overnight at 4°C in the dark. Remove excess Cy5 dye with a desalting column.

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

After the HT1080 cells in the logarithmic growth phase were digested and dispersed with 0.25% trypsin, the cell count was adjusted to prepare a cell suspension of 5×10 ⁶ cells/m1. Four Balb/c nude mice with a body weight of 18-22 g (purchased from Shanghai Sleike Experimental Animal Co., Ltd.) were taken, and 100 μl of cell suspension was subcutaneously injected into the back of the nude mice. The growth and tumorigenesis of HT1080 cells in Balb/c were observed.

(3) In vivo tumor targeting and tissue distribution experiments of the linker

When the tumor volume was about 400 mm ³ , the Cy5-labeled linker was injected through the tail vein, 100 μl/mouse. At 1, 2, 4, 8, 12, and 24 hours after the injection, the nude mice were placed in a small animal live imager for observation and photographing. After 24 hours of injection, the animals were killed by dislocation, and the main organs (heart, liver, spleen, lung, kidney) and tumors were taken out, observed and photographed in an in vivo imager.

The result is as follows:

It can be seen from Figure 14a that the TCS-penetrating 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, it can be seen from the tissue distribution map (Fig. 14b) that there is still a lot of drug accumulation in the tumor site, and the heart and spleen are redundant, while the main metabolic organs of PEGylated protein drugs-liver and kidney have the most drug distribution.

Experimental example 5 In vivo anti-tumor experiment of TCS, TCS-penetrating peptide linker and TCS-membrane-penetrating peptide-MMP-2 substrate peptide-PEG linker

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

After the HT1080 cells in the logarithmic growth phase were digested and dispersed with 0.25% trypsin, the cell count was adjusted to prepare a cell suspension of 5×10 ⁶ cells/m1. 24 Balb/c nude mice with a body weight of 18-22 g (purchased from Shanghai Sleike Experimental Animal Co., Ltd.) were taken, and 100 μl of cell suspension was subcutaneously injected into the back of the nude mice. The growth and tumorigenesis of HT1080 cells in Balb/c were observed.

(2) Anti-tumor effect in tumor-bearing nude mice

When the tumor volume was about 100 mm ³ , they were randomly divided into 4 groups: TCS group, TCS-penetrating peptide group, TCS-penetrating peptide-MMP-2 substrate peptide-PEG group, and normal saline group as negative control. Each group was administered by tail vein injection, and the dosage was 2.5 mg/kg, once every two days. During the administration period, the weight change 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 sacrificed by dislocation, the tumor was removed, the blood and capsule on the surface of the tumor were carefully removed, and weighed.

Figure 15 is a graph of tumor volume growth during administration. It can be seen from the figure that, compared with the normal saline group, the TCS group has almost no inhibitory effect on tumor growth. However, both the TCS-penetrating peptide group and the TCS-penetrating peptide-MMP-2 substrate peptide-PEG group had significant tumor inhibitory effects. The antitumor effect is: TCS-penetrating peptide-MMP-2 substrate peptide-PEG group is greater than TCS-penetrating peptide group is greater than TCS group. Figure 16 visually shows the pictures of tumors in each group after administration. It can be seen from the figure that the tumors of mice injected with normal saline and TCS were significantly larger, while those injected with TCS-penetrating peptide and TCS-penetrating peptide -MMP-2 substrate peptide-PEG mouse tumors were significantly reduced in different degrees.

Figure 17 and Figure 18 are the survival curves of mice in each group and the change of body weight of mice during the administration process. It can be seen from the figure that the body weight of the mice in the TCS group and the TCS-penetrating peptide group decreased significantly in varying degrees during the administration process, and the experimental mice died near the end of the experiment. However, the body weight of the mice in the TCS-penetrating peptide-MMP-2 substrate peptide-PEG group did not decrease significantly compared with the normal saline control group, and no animal death occurred. This partly reflects the greater systemic toxicity of TCS and TCS-penetrating peptide, while the lower toxicity of TCS-penetrating peptide-MMP-2 substrate peptide-PEG.

Fig. 19 is the result of taking out the organs of mice in each group for pathological section analysis at the end of the experiment. It can be seen from the figure that TCS and TCS-penetrating peptide have caused certain toxicity to the lung, spleen and kidney of mice. Destroyed, giant cells proliferated; glomeruli and renal convoluted ducts were swollen and deformed, and Bowman's cyst cavity disappeared. The toxicity of TCS-penetrating peptide-MMP-2 substrate peptide-PEG to various organs is much milder.

Experimental example 6 Immunogenicity experiment of TCS, TCS-penetrating peptide-MMP-2 substrate peptide-PEG

(1) Mouse immunization experiment

Take 10 Balb/c mice with a body weight of 18-22 g (purchased from Shanghai Slayco Experimental Animal Co., Ltd.), and randomly divide them into two groups, the TCS group and the TCS-penetrating peptide-MMP-2 substrate peptide- PEG group. Both groups were immunized by subcutaneous injection at a dosage of 10 μg per mouse every 7 days for three consecutive times. Before the first immunization and 7 days after the third immunization, blood samples were collected by orbital blood collection, and the serum was obtained by centrifugation.

(2) Determination of anti-TCS mouse IgG titer

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

Dilute TCS protein with coating buffer (50mM sodium carbonate, pH 9.6) to a solution with a concentration of 10 μl/ml, add 100 μl of the above solution to each well of a 96-well ELISA microplate, and place the microplate at 4°C overnight. After washing three times by adding 300 μl PBST to each well, block with PBST containing 1% goat blocking serum at 37° C. for 1.5 hours. The serum samples were diluted 1,000-1,000,000 times with PBST containing 0.01% goat blocking serum, and 100 μl of the diluted serum samples were added to the wells. At the same time, the negative serum before immunization was used as a negative control, and the mouse-derived anti-TCS IgG antibody (purchased (from Santa Cruz Company) was used as a positive control, and the primary antibody dilution was used as a blank control. Incubate at 37°C for 2 hours. After washing three times with PBST, 100 μl of HRP-labeled goat anti-mouse secondary antibody diluted 1,000 times was added to each well, and incubated at 37°C for 1 hour. After washing three times with PBST, add 200 μl TMB chromogenic substrate to each well, react at 37°C for 15 minutes, add 50 μl 20% sulfuric acid to each well to terminate the reaction, and measure the OD of each group with a microplate reader (model: 511 19300FI, Fisher) value, the wavelength is 450nm. Draw a titer curve.

The result is as follows:

It can be seen from Figure 20 that the unmodified TCS protein has strong immunogenicity, while the immunogenicity of TCS-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 the safety of its clinical application.

In summary, the TCS-penetrating peptide-MMP-2 substrate peptide-PEG linker of the present invention can accumulate in tumor sites through the EPR effect, and has high bioavailability. At the same time, it can be digested and activated by MMP-2, which is highly expressed in the tumor and its microenvironment, and then mediate TCS into the cell to kill tumor cells. exhibited significant growth inhibition. At the same time, the toxicity and immunogenicity of the TCS-penetrating peptide-MMP-2 substrate peptide-PEG linker of the present invention are also significantly reduced.

Claims (11)

1. A fusion protein having a structure according to formula Ia or Ib:

A-L1-B-L2-C is of formula Ia or

B-L1-A-L2-C formula Ib

Wherein,

a is a trichosanthin component and is a new active ingredient,

b is a membrane penetrating element which is provided with a plurality of membrane penetrating elements,

c is a substrate peptide element of matrix metalloproteinase 2,

l1, L2 are each nothing or a linker peptide element,

"-" is a peptide bond linking the elements,

and the fusion protein has tumor inhibition activity.

2. The fusion protein of claim 1, wherein the trichosanthin element comprises wild-type or mutant trichosanthin.

3. A precursor protein of the fusion protein of claim 1, wherein the precursor protein has a structure according to formula IIa or formula IIb:

A-L1-B-L2-C-D-E of formula IIa or

B-L1-A-L2-C-D-E formula IIb;

wherein,

a is a trichosanthin component and is a new active ingredient,

b is a membrane penetrating element which is provided with a plurality of membrane penetrating elements,

c is a matrix metalloproteinase 2(MMP-2) substrate peptide element,

d is none or 1-3 Cys,

e is an intein element and E is a peptide element,

l1, L2 are each nothing or a linker peptide element,

"-" is a peptide bond linking the elements,

cleaving the precursor protein with a thiol-containing reagent to produce the fusion protein of claim 1.

4. A polynucleotide sequence encoding the fusion protein of claim 1 or encoding the precursor protein of claim 3.

5. A vector comprising the polynucleotide of claim 4.

6. A host cell comprising the vector of claim 5 or a gene of said host cell into which has been integrated the polynucleotide of claim 3.

7. The modified fusion protein of claim 1, wherein the modified fusion protein is formed by site-directed PEGylation of the fusion protein of claim 1.

8. Use of the fusion protein according to claim 1 or the modification according to claim 7 for the preparation of a pharmaceutical composition for the treatment of tumors.

9. A pharmaceutical composition comprising the fusion protein of claim 1 or the modification of claim 7, and a pharmaceutically acceptable carrier.

10. A method of producing the fusion protein of claim 1 or a precursor protein thereof, comprising the steps of:

(i) culturing the host cell of claim 6 under suitable conditions to obtain expression of the fusion protein or its precursor protein.

11. A method of non-therapeutically inhibiting tumor cells in vitro comprising the steps of: adding the fusion protein of claim 1, the modification of claim 7, or the pharmaceutical composition of claim 9 to a tumor cell culture, thereby inhibiting the tumor cell.