CN111378049B

CN111378049B - Anti-tumor targeting fusion protein, conjugate and application thereof

Info

Publication number: CN111378049B
Application number: CN201811632324.1A
Authority: CN
Inventors: 高向东; 尹骏; 刘定康; 姚文兵; 陈烨
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2022-03-22
Anticipated expiration: 2038-12-29
Also published as: CN111378049A

Abstract

The invention relates to an anti-tumor targeting fusion protein, a conjugate and application thereof. The anti-tumor targeting fusion protein is composed of a sequence which is constructed according to formula I or formula II from N end to C end. Formula I: PsTag-Linker-CPP-KLAC; formula II: KLAC-CPP-Linker-PsTag. The conjugate is formed by coupling an anti-tumor targeting fusion protein with an anti-tumor compound through a coupling site of the anti-tumor targeting fusion protein, wherein the coupling site is cysteine in KLAC. The anti-tumor targeting fusion protein not only has obviously improved pharmacokinetic properties, but also has obvious tumor targeting; the conjugate can realize the synergistic killing of tumor cells by two different anti-tumor mechanisms, and reduce the efflux of the tumor cells to the drug by inhibiting the generation of ATP to form the effect of reversing drug resistance, thereby obviously improving the drug effect; the conjugates also reduce or eliminate the myocardial cytotoxicity of the conjugated anti-tumor compounds.

Description

Anti-tumor targeting fusion protein, conjugate and application thereof

Technical Field

The invention relates to an anti-tumor targeted fusion protein, a conjugate and application thereof, belonging to the field of research on long-acting targeted anti-tumor drugs.

Background

To the best of the applicant's knowledge, drug therapy is still one of the main means in cancer therapy, but the treatment failure is often caused by the generation of drug resistance, so in addition to further enhancing the drug efficacy and reducing the toxicity, the drug resistance of antitumor drugs is one of the most important research problems in the current antitumor field.

One of the major causes of drug resistance in tumor cells is the overexpression of ABC transporters (i.e., ATP-dependent transporters), and thus a more direct and effective method to reverse multiple drug resistance by directly reducing cellular ATP production. Mitochondria are important organelles for eukaryotic cells to generate ATP, and can effectively block the generation of ATP in cells by directly destroying the mitochondria of drug-resistant tumor cells.

In recent years, therapeutic polypeptides have shown great application prospects in antimicrobial, antitumor and biomedical materials and are of great interest; among them, KLA is a bioactive peptide that has been extensively studied to be able to break down mitochondria. KLA acts by disrupting the outer mitochondrial membrane, resulting in dysfunction of ATP synthesis and enabling cytochrome C to leak out into the cytoplasm, further activating death signaling proteins to promote apoptosis. Although KLA can effectively destroy and apoptosis tumor cell mitochondria, its further development is limited due to problems such as lack of targeting and extremely short in vivo half-life.

PEG modification (PEGylation) has been widely used to extend the half-life of therapeutic polypeptides. PEG modifications alter the physicochemical properties of biomedical molecules, such as their conformation, electrostatic binding and hydrophobicity, leading to an improvement in the pharmacokinetic behavior of the drug. In general, PEG modifications can improve drug solubility, reducing immunogenicity. Moreover, the PEG modification can prolong the half-life of the drug, and can also passively target to tumor tissues through the EPR effect, thereby increasing the distribution of the drug at the tumor part. In addition, the PEG modification site needs to be carefully determined to avoid interfering with the active site of the therapeutic fusion polypeptide. Furthermore, PEG is a heterogeneous mixture of related polymers, which conjugation to antitumor polypeptides or proteins results in many different chemical species with similar molecular size and chemical properties. This complicates purification and increases the cost of production of the PEG-modified product. A more serious problem is the finding that after long-term use of PEG, PEG accumulates in the tissues and, in severe cases, can cause damage to the kidneys. Therefore, development of new technical means for overcoming the above problems is urgently needed.

The inventor has already filed 2015.10.15 for a Chinese patent application (application No. 201510675827.7, application publication No. CN105524147A), and a series of recombinant polypeptides obtained by a library-building screening method can be expressed by fusion with bioactive proteins to become a PEG-modified alternative method, and the polypeptide fusion protein has the advantages of enhancing stability, prolonging half-life period, reducing immunogenicity, not changing bioactivity, and the like. On the basis of this, the inventors have further studied the results.

Disclosure of Invention

The main purposes of the invention are: the problems in the prior art are overcome, and the anti-tumor targeting fusion protein is provided, so that the tumor targeting property and the pharmacokinetic property of the anti-tumor medicament can be improved; meanwhile, a conjugate based on the fusion protein is also provided, and has an anti-tumor effect; also provides the application of the fusion protein and the conjugate.

The technical scheme for solving the technical problems of the invention is as follows:

an anti-tumor targeting fusion protein, characterized by consisting of a sequence constructed according to formula I or formula II from N-terminus to C-terminus:

formula I: PsTag-Linker-CPP-KLAC;

formula II: KLAC-CPP-Linker-PsTag;

in formula I or formula II, PsTag is a recombinant polypeptide consisting of 100 to 5000 amino acid residues, wherein the total amount of glycine, alanine, serine, threonine, proline and lysine in total accounts for more than 95% of the total number of amino acid residues; at least 95% of the structure of the recombinant polypeptide is random coil, as determined by GOR algorithm; determined according to Chou-Fasman algorithm, in the structure of the recombinant polypeptide, the sum of the proportion of alpha helix and beta sheet is less than 2%; the sequence of the recombinant polypeptide contains at most 1 methionine;

the Linker is a connecting peptide, and the connecting peptide has an MMP2 enzyme cutting site sequence; CPP is cell penetrating peptide; KLAC is an antitumor polypeptide.

Preferably, the MMP2 enzyme cutting site sequence is PLGLAG; the sequence of the connecting peptide is as follows: EEEEEEEEPLGLAG, PLGLAGEEEEEEEE, or PLGLAG.

Preferably, the cell-penetrating peptide is selected from one of R8, TAT, MPG, Transportan, Pep-1, EB1, TAT-DRBD, or the cell-penetrating peptide contains at least one of R8, TAT, MPG, Transportan, Pep-1, EB1, TAT-DRBD;

the sequence of R8 is: RRRRRRRRRR;

the TAT sequence is: GRKKRRQRRRPPQ;

the MPG sequence is: GLAFLGFLGAAGSTMGAWSQPKSKRKV, respectively;

the Transportan sequence is: AGYLLGKINLKALAALAKKIL, respectively;

the Pep-1 sequence is: KETWWETWWTEWSQPKKKRKV;

the EB1 sequence is: LIRLWSHLIHIWFQNRRLKWKKK, respectively;

the Tat-DRBD sequence is: YGRKKRRQRRR-AGDLSAGFFMEELNTYRQKQGVVLKYQELPNSGPPHDRRFTFQVI IDGREFPEGEGRSKKEAKNAAAKLAVEILNKEKKA.

Preferably, the sequence of the recombinant polypeptide is shown in SEQ ID No.2 to SEQ ID No. 20.

The invention also proposes:

nucleic acid encoding the anti-tumor targeting fusion protein described hereinbefore.

An expression vector comprising a nucleic acid as described hereinbefore.

A host cell comprising an expression vector as described above.

The invention also proposes:

an anti-tumor targeting fusion protein conjugate, which is characterized in that the anti-tumor targeting fusion protein is conjugated with an anti-tumor compound through a coupling site of the anti-tumor targeting fusion protein, wherein the coupling site is cysteine in KLAC.

Preferably, the anti-tumor compound is doxorubicin or a derivative thereof, paclitaxel or a derivative thereof, maytansine or a derivative thereof, or dolastatin or a derivative thereof.

More preferably, the structure of the adriamycin derivative is shown as a formula A or a formula B or a formula C:

the structure of the taxol derivative is shown as a formula D or a formula E:

the structure of the maytansine derivative is shown as a formula F or a formula G:

the structure of the dolastatin derivative is shown as a formula H or a formula J:

the invention also proposes:

the use of the anti-tumor targeting fusion protein as described above, characterized in that the use is for the preparation of a medicament or a pharmaceutical composition for the treatment or prevention of tumors, or for the preparation of an anti-tumor targeting fusion protein conjugate, or for the preparation of an anti-tumor targeting fusion protein-doxorubicin conjugate by conjugation with doxorubicin, which has a lower myocardial cytotoxicity than doxorubicin.

The use of the anti-tumor targeting fusion protein conjugate is characterized in that the use is used for preparing a medicament or a pharmaceutical composition for treating or preventing tumors, or is used for preparing a medicament or a pharmaceutical composition containing the anti-tumor targeting fusion protein-adriamycin conjugate, and the myocardial cytotoxicity of the medicament or the pharmaceutical composition is lower than that of adriamycin.

Preferably, the tumor is a drug-resistant tumor caused by overexpression of ABC transporters.

In practical research, the inventor knows that there is a significant difference between the microenvironment of tumor cells and normal cells, such as abnormal blood vessels, oxidation, perfusion, pH and metabolic state, so that the tumor microenvironment can be used to develop a drug targeting delivery system for releasing drugs at the tumor site. MMP2 is a protease over-expressed in tumor tissues, and the inventor determines to adopt MMP2 enzyme cutting site sequence (especially 'PLGLAG' sequence) after research, and combines the MMP2 enzyme cutting site sequence with CPP, so that the whole fusion protein can be expected to be cut in the tumor microenvironment to release an active component, namely an anti-tumor polypeptide KLAC. Meanwhile, PsTag (recombinant polypeptide) was a result of previous studies by the inventors of a group of subjects who put it into the structure of the above-mentioned fusion protein in order to expect improvement of the pharmacokinetic properties of the antitumor polypeptide KLAC.

Experiments prove that compared with the single administration of the antitumor polypeptide KLAC, the antitumor targeted fusion protein not only has obviously improved pharmacokinetic properties (such as the improvement of the half-life period in vivo), but also has obvious tumor targeting property, so that the antitumor curative effect and the safety can be greatly improved.

On the basis of the anti-tumor target fusion protein, the inventor further researches and discovers that the only cysteine of the anti-tumor polypeptide KLAC can be used as a coupling site to be coupled with other anti-tumor compounds, and the obtained conjugate can realize the synergistic killing of tumor cells by two different anti-tumor mechanisms on one hand, and can reduce the efflux action of the tumor cells on the medicament by inhibiting the generation of ATP on the other hand to form the effect of reversing the medicament resistance, so that the medicament effect is obviously improved under the combined action of the two aspects finally.

Drawings

FIG. 1 is a graph showing the in vivo half-life results of example 2 of the present invention.

FIG. 2 is a graph showing the results of tissue distribution in vivo in example 2 of the present invention.

FIG. 3 is a graph showing the in vitro anti-tumor results of example 2 of the present invention.

Detailed Description

Some terms to which the present invention relates are defined as follows.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses modified amino acid polymers, such as by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation, e.g., conjugation to a labeling component.

The term "recombinant polypeptide" is a flexible nonstructural polypeptide, or a flexible nonstructural polypeptide sequence. The flexible nonstructural polypeptide sequence contains 1 to 5000 amino acid residues, wherein the total number of glycine (G), alanine (a), serine (S), threonine (T), proline (P), and lysine (K) together account for more than 95% of the total number of amino acid residues; and the flexible unstructured polypeptidic sequence has at least 95% unstructured random coil formation as determined by GOR algorithm. The sum of the proportions of alpha helices and beta sheets is less than 2%, as determined by the Chou-Fasman algorithm. The sequence contains a maximum of 1 methionine.

The term "passively targeted" refers to a drug that selectively reaches a specific physiological site, organ, tissue or cell and exerts a therapeutic effect on the drug at the target site. Since the tumor area does not have an intact lymphatic return system of filterable particles, the lymphatic system passively accumulates macromolecules and increases the concentration of macromolecules in the tumor. The activity of the medicine at a target position can be enhanced, the toxic and side effect of the medicine at a non-target position can be reduced, and the therapeutic index of the medicine can be improved.

The term "MMP 2 enzyme" refers to a matrix metalloproteinase highly expressed by tumor tissues, which is an important enzyme system for degrading extracellular matrix, and matrix metalloproteinase 2 (MMP-2) gene is an important member of MMPs, is a key enzyme in the process of tumor cell invasion and metastasis, is widely present in various cells, and participates in the physiological and pathological processes of tumor infiltration and metastasis. The term "MMP 2 cleavage site sequence" refers to a polypeptide sequence capable of being cleaved by MMP2 enzyme.

The term "CPP" refers to a class of cationic or amphiphilic polypeptides consisting of 30 or fewer amino acid structures that can penetrate the plasma membrane into the cytoplasm. CPP can enter cells without toxic reactions, can effectively penetrate cell membranes, and carry substances into living cells.

"conjugation", "linking", "connecting" and "fusion" are used interchangeably herein. These terms refer to the joining together of two or more chemical elements or components in any manner, including chemical conjugation or recombinant means. For example, two different proteins can be joined together by "in-frame fusion," which refers to the joining of two or more Open Reading Frames (ORFs) in a manner that preserves the correct reading frame of the original ORFs, to form a longer contiguous ORF. Thus, the resulting recombinant fusion protein is a single protein containing two or more segments corresponding to the additional polypeptides encoded by the original ORFs (which segments would not normally be so linked in nature). As another example, the two proteins can also be linked together by a trial chemical cross-linking agent to obtain a protein conjugate containing two separate polypeptides linked by the cross-linking agent.

In the context of describing a polypeptide, a "sequence" is an amino acid sequence in a polypeptide from the carboxy-terminal direction of the amino acids in which residues in the sequence that are adjacent to each other are linked in the primary structure of the polypeptide.

The terms "DNA," "polynucleotide," "nucleic acid," "nucleotide," and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The polynucleotide may have any three-dimensional structure and may exhibit any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci defined by linkage analysis, exons, introns, messenger RNA (mrna), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched nucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure may be modified before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling element.

The term "functional variant" of a protein refers to a modified version of a native protein which comprises substitution, deletion and/or addition of one or several amino acids, for example less than 15 amino acids, or preferably less than 10 or 5 amino acids, and which substantially retains the biological activity of the native protein. Generally, conservative substitutions of amino acids are preferred, the substitutions being within the knowledge of the skilled person. The deletion is preferably a deletion of amino acids from regions not involved in the biological function of the protein.

The present invention will be described in further detail with reference to examples. The invention is not limited to the examples given.

Example 1 construction, expression and purification of fusion proteins

After each amino acid is singly polymerized, the tendency of forming a certain protein secondary structure is predicted, and the tendency of forming the protein secondary structure by the amino acids of Ala, Pro, Thr, Gly, Ser and Lys is predicted:

using GOR algorithm (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.plIs there a page — npsa _ gor4.html) determines the random curl content in the secondary structure; using Chou&Fasman algorithm (http://cho- fas.sourceforge.net/) The alpha helix and beta sheet in the secondary structure were determined. The hydrophobic profile, hydrophilic profile and mRNA secondary structure of the polyamines were determined using DNAMAN V6 software.

The oligomeric polypeptide chain is designed according to the polypeptide motif design principle. Selecting the preferred codon of colibacillus, designing the corresponding DNA primer, and composing the polypeptide motif library. Taking 20 DNA fragments with 5' ends added with phosphoric acid, carrying out self-ligation reaction, uniformly mixing each tube, incubating at 4 ℃ for 5min, incubating at 70 ℃ for 10min, denaturing and inactivating, and carrying out 2% TBE agarose gel electrophoresis on the product for 90V and 90 min. The conditions for the self-ligation reaction are affected by: template concentration, amount of DNA ligase, ligation reaction temperature, ligation reaction time, choice of agarose gel electrophoresis buffer, choice of gel concentration, and electrophoresis time. By changing these parameters, the degree of progress of the self-ligation reaction can be changed, and thus the most suitable size of the target gene fragment can be selected as desired.

The gene fragment is adopted to construct an escherichia coli engineering strain. Then, the seed solution of the engineered strain was inoculated into LB medium and cultured overnight at 37 ℃ and 220 rpm. The next day, the cells were transferred and cultured at 37 ℃ until OD₆₀₀Reaching 0.8, IPTG is added to induce the expression of the three fusion proteins. After overnight fermentation, the fermentation liquor is centrifuged, thallus is collected, ultrasonication is carried out to obtain semitransparent thallus, and ultrasonic supernatant is taken to pass through ammonium sulfate precipitation, Q ion exchange column and Ni²⁺Purifying with affinity chromatography column to obtain pure fusion protein.

According to the above construction method, the following fusion proteins were constructed, expressed and purified in this example:

PAK1：PsTag01-Linker01-CPP01-KLAC

PAK2：KLAC-CPP02-Linker02-PsTag02

PAK3：PsTag03-Linker03-CPP03-KLAC

PAK4：KLAC-CPP04-Linker01-PsTag04

PAK5：PsTag05-Linker02-CPP05-KLAC

PAK6：KLAC-CPP06-Linker03-PsTag06

PAK7：PsTag07-Linker01-CPP07-KLAC

PAK8：KLAC-CPP04-Linker02-PsTag08

PAK9：PsTag09-Linker03-CPP03-KLAC

PAK10：KLAC-CPP05-Linker01-PsTag10

PAK11：PsTag11-Linker02-CPP02-KLAC

wherein, the sequence of KLAC is shown in SEQ ID No. 1.

Specific sequences of PsTag01 through PsTag11 are as follows.

The sequence of PsTag01 is SEQ ID No.2, i.e.:

SSPSASTPPSSGPSASSGSAPGSGSGTGAGSAPGGSGSPGAGSAPGASGSSPSASPPAPAGPGASPPAPAGAPAPTAPAPGPPAAAGSPGSAGSPPAGSG。

the number of amino acids contained: 100 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 96.00 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 0 percent.

The sequence of PsTag02 is SEQ ID No.3, i.e.:

SPTPSGAGAGTGASPGTGSGAGSAAGSAGASGPGPAATPPAGPSGGGGTGATAPSSAGPAGGGPTGAATSPSPPTGPSPPSGTPSSAPGSSGGASGPGPT。

The sequence of PsTag03 is SEQ ID No.4, i.e.:

SASTGPGGGGSTAGPPGGSGPPSSGGPGGGPTGGGTSAGAPTGSSPAAPGGGGSPSAASGGTATPAGGASSPGTTPPGGPGSPTATSPSTGSGSGPPSSS。

The sequence of PsTag04 is SEQ ID No.5, i.e.:

SAGPPPAPGSPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTG。

the number of amino acids contained: 100 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 95.00 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 0 percent.

The sequence of PsTag05 is SEQ ID No.6, i.e.:

GPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPTSGPSTASGPSTASGPTSGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPTSGPSTASGPSTASGPTSGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPTSGPSTASGPSTASGPTS。

the number of amino acids contained: 400 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 98.84 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 0 percent.

The sequence of PsTag06 is SEQ ID No.7, i.e.:

PSPTSSPTGTGGGGGGGSGGSSSPGSPGAPPPGPGSTSSTGSAPPGGSSPAAGGGGSGGAPGGGSGPGPASSPGPSSGGGGGGGGGTGGSGGATPSSASGGTASSAPTPASGPPAGSSPAAPGSAGSSSTGSPGGPGGGPSASGTAGPSAGSTGSSGGGSTTAPSSPTPSTGAPSSGSTGAPPSSAAGTGAGPSTPSSSATGASASGAPSGPAPGAAPTAPTSASTSGSGAAPASPTPSGSGGAPGPSGGPTTSGSATPTPGSGGGTGGPPGTPASPTGATPSGGGSGGPGTPSTAPPSAAAPASPTPSAASGPPGPSGGPTSPPGGGGSTPPGTPASPTGAGAGGGGGSATTSAAGGSGSAASGSATPPATPSSPTASPAGSTSGASGSSATSPGGGGA。

the number of amino acids contained: 400 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 96.25 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 0.5 percent.

The sequence of PsTag07 is SEQ ID No.8, i.e.:

SAGPPPAPGSPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPTSGPTASGPSAGSGGGPTASGTPGAGSPGGTAGGTGPGGSGGGPTGSPP STATPAGPTSGGSAPTAGPSSGSSPPTSSTGSSTSSPGAGPPPTGGGGGGGGGTTGPSATPSATPGSAGPPPAPGSPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPTSGPTASGPSAGSGGGPTASGTPGAGSPGGTAGGTGPGGSGGGPTGSPPSTATPAGPTSGGSAPTAGPSSGSSPPTSSTGSSTSSPGAGPPPTGGGGGGGGGTTGPSATPSATPG。

the number of amino acids contained: 432 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 99.07 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 0 percent.

The sequence of PsTag08 is SEQ ID No.9, i.e.:

GPSATPGSTGPTSAPGSTGPATPSGTSGPSTAGPTSGPSATPGTSGPSGTSPTAGPTSAPGTSGPSTPGTSAGPAGSTPSTGPSAPTGSTGPSGTPASTGPATPSGTSGPSATPGSTGPTSAPGSTGPSATPGTSGPSGTSAPTGPSATPGTSGPSGTSAPTGPTSAPGTSGPSTPGTSAGPTSAPGSTGPSAGTPSTGPTSAPGSTGPSAGTPSTGPSATPGSTGPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPTSGPTASGPSATPGSTGPTSAPGSTGPSATPGTSGPSGTSAPTGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPSATPGSTGPTSAPGSTGPSTGSPATGPSTPAGST。

the number of amino acids contained: 432 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 98.84 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 0 percent.

The sequence of PsTag09 is SEQ ID No.10, i.e.:

PTSGSPGTPPGGAPGGSAPGGSSPSPPGAAGSGGGGGGGGSPSSAPSPGGSSAAGPTASSAGGSAGGGTSPAAGGGTSPSSPTSTGTGSSPPPSSSGTTAGPGGGSTGTSPAPPGGGGPSSSTPPPGSGGSGPAGGPGTSTTGSPPPGPGPSSTAPSAGASASSPPGSGGGGSSTSAGPPAATPSPSSATSGPSGGGAAPSPSTSTSAAPGGSSSSTGSSPSASPGASPSGSASGGSSPPTGASPTSTPAGGSPPTSGATGAPPSGPSGGGGGGSPTTPPPGAPSAPSSPAPGSAGAGGGSGGASPSGGSGGSPGATPPGGTTAPSPGGSAGAPAGPSTTGAASGPSPPSAPSTASPSGGGTAGGSSPSSAGSTPPPAAGAPPGSPGGAGGSASSATGTSGSPPPATSAPSSGGGGGGGGPSGSGPGPSAGAGPSGSGGGSPGPSASPTSTSGPPTTPSSSTGSAPASPSSSSAPTGSSPPTAAPSAPPSGGSGGAPSSGGGSGGSTGAPPAGSTPASSGGSGSSGGTPSPSGGSGPPSGGSSPSPGAAGPSATGPAASTGATSPGGGGGAGSGGGGGSPASGGTSTPSSAGPPSGGGTPAAPSGGGAGTSGSATPPSPGGSGTAGTPPGSTASSSSGPPPPPPPTSASGGGSGGSASGGAGSTSGSGGGGGGGSGPPPTASGPGGSGGGPSGGGGGSTSSGASPPAGSSTSGGGTGAAGSPASTSGGPGAGGGGTAGGSAGPSGASTSSPPAASTPPGTSAGTGAPASGGSGGSPAGSGASSGASPSGGGAPPTTGASGGSGSSGGGAAPSGSSGGGSSGGTTPASSGSAASSAPSTSTPSSTTSPSAAPGSSGGSGATPPSSSSPTPPPGSGSSGGTSPGGSPSASPGGSTPSGGGGGGGGGGSGPAPPSAAPSAGGTSGGAGGGGGGTSSGSTSPPSGGSPGGGAASSSPTGTPSTPASSATSGSAGSSSSGSPAPSGGPSGAAPSSGPTPAGGGGG。

the number of amino acids contained: 1000 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 98.00 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 1.3 percent.

The sequence of PsTag10 is SEQ ID No.11, i.e.:

AGGPPPTGPPGAAGAPPTSGGSSASPGPGGTSGGSGGTGGAAGTAPPTTGASTPSSSGSTSPATSSSGTPGPGPPPAGPSGGPGPAGPSSPTSASPGPGGTGGGAGGPGPATPPSGGSTGASTPGAPTSPAGTAGPSASPSGPSPPSPAGASTTAPPAASGGAGSPPPTTGPGSGGGSPSGGGPTAPSGSGGASTPGPPPAGATSPGPTGPGAPASGGSGTSGGGSGGAPPTSSSGSGGGPATTGGASAGPGTTPGGPAPPPASGSGGTAPPPGPPTSTSPGGSATGGSGTSSAPGSSSGGGSPGGGSAGPASGGPTSSSGTSGPTPSTSGSASGGSSSGPPTGPPTSPPPSATSSPPPPGSSAPGSSSGGGGSTGGSGPGAGSPSPSASSGASAPTPASSPSASTASTTAPSTGPAPGAPPSAGAGPGPPASGPGTGGGSSPPGAGSGGSGGGPPPGSGSSTSAPTPASSPSASSTGPSAGSGGSGPTGGGSPTSSPASPTSPPSAPPATSPGGPATPSPPPGPSPGGPAASGPTAPAGTGGPATGAAGPSPTTSGGTTGPSGGPSATTSGGSTPPAGTSPASGSGGGSTTPSAPGGGAAPGGPPGPAGGAASSGSSTPPTAPSPGSGGSAGPTPSASGGGGSAPAGTPSGGPSPSSGPTTPSAPGGSGGGPPGTPGSGTAPPSSASPSGGGTSGGSTSAGPGPTSSPGTGGSSAGGPPPPTSAGSAGTGGGTPAASGSGSPAGGGGSGGSGGSPGAAGGTSPPPSTAPSPGASSPGPAPPSAGGASAAPSGPSSGGAPGSPSSAGGGGSASGGPPGTGPSSSTSSTASPTAGSGGSGPPPSTTSGTGGPTGASGGGPTPGAGPPAAPAGPTASSASSGGPGGGGSTAPSTPPSPPPATSAPSTGGGGGGGGPSGSGPGPSAGAGPSGSGSGGPGPSASPSGGSGPPTTPSGGSGGAPASPSSGGAPSGGSPPTAAPSAPPSGGSGGAPSSGGGGGGGT。

the number of amino acids contained: 1000 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 97.70 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 1.8 percent.

The sequence of PsTag11 is SEQ ID No.12, i.e.:

PTSGSPGTPPGGAPGGSAPGGSSPSPPGAAGSGGGGGGGGSPSSAPSPGGSSAAGPTASSAGGSAGGGTSPAAGGGTSPSSPTSTGTGSSPPPSSSGTTAGPGGGSTGTSPAPPGGGGPSSSTPPPGSGGSGPAGGPGTSTTGSPPPGPGPSSTAPSAGASASSPPGSGGGGSSTSAGPPAATPSPSSATSGPSGGGAAPSPSTSTSAAPGGSSSSTGSSPSASPGASPSGSASGGSSPPTGASPTSTPAGGSPPTSGATGAPPSGPSGGGGGGSPTTPPPGAPSAPSSPAPGSAGAGGGSGGASPSGGSGGSPGATPPGGTTAPSPGGSAGAPAGPSTTGAASGPSPPSAPSTASPSGGGTAGGSSPSSAGSTPPPAAGAPPGSPGGAGGSASSATGTSGSPPPATSAPSSGGGGGGGGPSGSGPGPSAGAGPSGSGGGSPGPSASPTSTSGPPTTPSSSTGSAPASPSSSSAPTGSSPPTAAPSAPPSGGSGGAPSSGGGSGGSTGAPPAGSTPASSGGSGSSGGTPSPSGGSGPPSGGSSPSPGAAGPSATGPAASTGATSPGGGGGAGSGGGGGSPASGGTSTPSSAGPPSGGGTPAAPSGGGAGTSGSATPPSPGGSGTAGTPPGSTASSSSGPPPPPPPTSASGGGSGGSASGGAGSTSGSGGGGGGGSGPPPTASGPGGSGGGPSGGGGGSTSSGASPPAGSSTSGGGTGAAGSPASTSGGPGAGGGGTAGGSAGPSGASTSSPPAASTPPGTSAGTGAPASGGSGGSPAGSGASSGASPSGGGAPPTTGASGGSGSSGGGAAPSGSSGGGSSGGTTPASSGSAASSAPSTSTPSSTTSPSAAPGSSGGSGATPPSSSSPTPPPGSGSSGGTSPGGSPSASPGGSTPSGGGGGGGGGGSGPAPPSAAPSAGGT SGGAGGGGGGTSSGSTSPPSGGSPGGGAASSSPTGTPSTPASSATSGSAGSSSSGSPAPSGGPSGAAPSSGPTPAGGGGGPTSGSPGTPPGGAPGGSAPGGSSPSPPGAAGSGGGGGGGGSPSSAPSPGGSSAAGPTASSAGGSAGGGTSPAAGGGTSPSSPTSTGTGSSPPPSSSGTTAGPGGGSTGTSPAPPGGGGPSSSTPPPGSGGSGPAGGPGTSTTGSPPPGPGPSSTAPSAGASASSPPGSGGGGSSTSAGPPAATPSPSSATSGPSGGGAAPSPSTSTSAAPGGSSSSTGSSPSASPGASPSGSASGGSSPPTGASPTSTPAGGSPPTSGATGAPPSGPSGGGGGGSPTTPPPGAPSAPSSPAPGSAGAGGGSGGASPSGGSGGSPGATPPGGTTAPSPGGSAGAPAGPSTTGAASGPSPPSAPSTASPSGGGTAGGSSPSSAGSTPPPAAGAPPGSPGGAGGSASSATGTSGSPPPATSAPSSGGGGGGGGPSGSGPGPSAGAGPSGSGGGSPGPSASPTSTSGPPTTPSSSTGSAPASPSSSSAPTGSSPPTAAPSAPPSGGSGGAPSSGGGSGGSTGAPPAGSTPASSGGSGSSGGTPSPSGGSGPPSGGSSPSPGAAGPSATGPAASTGATSPGGGGGAGSGGGGGSPASGGTSTPSSAGPPSGGGTPAAPSGGGAGTSGSATPPSPGGSGTAGTPPGSTASSSSGPPPPPPPTSASGGGSGGSASGGAGSTSGSGGGGGGGSGPPPTASGPGGSGGGPSGGGGGSTSSGASPPAGSSTSGGGTGAAGSPASTSGGPGAGGGGTAGGSAGPSGASTSSPPAASTPPGTSAGTGAPASGGSGGSPAGSGASSGASPSGGGAPPTTGASGGSGSSGGGAAPSGSSGGGSSGGTTPASSGSAASSAPSTSTPSSTTSPSAAPGSSGGSGATPPSSSSPTPPPGSGSSGGTSPGGSPSASPGGSTPSGGGGGGGGGGSGPAPPSAAPSAGGTSGGAGGGGGGTSSGSTSPPSGGSPGGGAASSSPTGTPSTPASSATSGSAGSSSSGSPAPSGGPSGAAPSSGPTPAGGGGGPTSGSPGTPPGGAPGGSAPGGSSPSPPGAAGSGGGGGGGGSPSSAPSPGGSSAAGPTASSAGGSAGGGTSPAAGGGTSPSSPTSTGTGSSPPPSSSGTTAGPGGGSTGTSPAPPGGGGPSSSTPPPGSGGSGPAGGPGTSTTGSPPPGPGPSSTAPSAGASASSPPGSGGGGSSTSAGPPAATPSPSSATSGPSGGGAAPSPSTSTSAAPGGSSSSTGSSPSASPGASPSGSASGGSSPPTGASPTSTPAGGSPPTSGATGAPPSGPSGGGGGGSPTTPPPGAPSAPSSPAPGSAGAGGGSGGASPSGGSGGSPGATPPGGTTAPSPGGSAGAPAGPSTTGAASGPSPPSAPSTASPSGGGTAGGSSPSSAGSTPPPAAGAPPGSPGGAGGSASSATGTSGSPPPATSAPSSGGGGGGGGPSGSGPGPSAGAGPSGSGGGSPGPSASPTSTSGPPTTPSSSTGSAPASPSSSSAPTGSSPPTAAPSAPPSGGSGGAPSSGGGSGGSTGAPPAGSTPASSGGSGSSGGTPSPSGGSGPPSGGSSPSPGAAGPSATGPAASTGATSPGGGGGAGSGGGGGSPASGGTSTPSSAGPPSGGGTPAAPSGGGAGTSGSATPPSPGGSGTAGTPPGSTASSSSGPPPPPPPTSASGGGSGGSASGGAGSTSGSGGGGGGGSGPPPTASGPGGSGGGPSGGGGGSTSSGASPPAGSSTSGGGTGAAGSPASTSGGPGAGGGGTAGGSAGPSGASTSSPPAASTPPGTSAGTGAPASGGSGGSPAGSGASSGASPSGGGAPPTTGASGGSGSSGGGAAPSGSSGGGSSGGTTPASSGSAASSAPSTSTPSSTTSPSAAPGSSGGSGATPPSSSSPTPPPGSGSSGGTSPGGSPSASPGGSTPSGGGGGGGGGGSGPAPPSAAPSAGGTSGGAGGGGGGTSSGSTSPPSGGSPGGGAASSSPTGTPSTPASSATSGSAGSSSSGSPAPSGGPSGAAPSSGPTPAGGGGGPTSGSPGTPPGGAPGGSAPGGSSPSPPGAAGSGGGGGGGGSPSSAPSPGGSSAAGPTASSAGGSAGGGTSPAAGGGTSPSSPTSTGTGSSPPPSSSGTTAGPGGGSTGTSPAPPGGGGPSSSTPPPGSGGSGPAGGPGTSTTGSPPPGPGPSSTAPSAGASASSPPGSGGGGSSTSAGPPAATPSPSSATSGPSGGGAAPSPSTSTSAAPGGSSSSTGSSPSASPGASPSGSASGGSSPPTGASPTSTPAGGSPPTSGATGAPPSGPSGGGGGGSPTTPPPGAPSAPSSPAPGSAGAGGGSGGASPS GGSGGSPGATPPGGTTAPSPGGSAGAPAGPSTTGAASGPSPPSAPSTASPSGGGTAGGSSPSSAGSTPPPAAGAPPGSPGGAGGSASSATGTSGSPPPATSAPSSGGGGGGGGPSGSGPGPSAGAGPSGSGGGSPGPSASPTSTSGPPTTPSSSTGSAPASPSSSSAPTGSSPPTAAPSAPPSGGSGGAPSSGGGSGGSTGAPPAGSTPASSGGSGSSGGTPSPSGGSGPPSGGSSPSPGAAGPSATGPAASTGATSPGGGGGAGSGGGGGSPASGGTSTPSSAGPPSGGGTPAAPSGGGAGTSGSATPPSPGGSGTAGTPPGSTASSSSGPPPPPPPTSASGGGSGGSASGGAGSTSGSGGGGGGGSGPPPTASGPGGSGGGPSGGGGGSTSSGASPPAGSSTSGGGTGAAGSPASTSGGPGAGGGGTAGGSAGPSGASTSSPPAASTPPGTSAGTGAPASGGSGGSPAGSGASSGASPSGGGAPPTTGASGGSGSSGGGAAPSGSSGGGSSGGTTPASSGSAASSAPSTSTPSSTTSPSAAPGSSGGSGATPPSSSSPTPPPGSGSSGGTSPGGSPSASPGGSTPSGGGGGGGGGGSGPAPPSAAPSAGGTSGGAGGGGGGTSSGSTSPPSGGSPGGGAASSSPTGTPSTPASSATSGSAGSSSSGSPAPSGGPSGAAPSSGPTPAGGGGGPTSGSPGTPPGGAPGGSAPGGSSPSPPGAAGSGGGGGGGGSPSSAPSPGGSSAAGPTASSAGGSAGGGTSPAAGGGTSPSSPTSTGTGSSPPPSSSGTTAGPGGGSTGTSPAPPGGGGPSSSTPPPGSGGSGPAGGPGTSTTGSPPPGPGPSSTAPSAGASASSPPGSGGGGSSTSAGPPAATPSPSSATSGPSGGGAAPSPSTSTSAAPGGSSSSTGSSPSASPGASPSGSASGGSSPPTGASPTSTPAGGSPPTSGATGAPPSGPSGGGGGGSPTTPPPGAPSAPSSPAPGSAGAGGGSGGASPSGGSGGSPGATPPGGTTAPSPGGSAGAPAGPSTTGAASGPSPPSAPSTASPSGGGTAGGSSPSSAGSTPPPAAGAPPGSPGGAGGSASSATGTSGSPPPATSAPSSGGGGGGGGPSGSGPGPSAGAGPSGSGGGSPGPSASPTSTSGPPTTPSSSTGSAPASPSSSSAPTGSSPPTAAPSAPPSGGSGGAPSSGGGSGGSTGAPPAGSTPASSGGSGSSGGTPSPSGGSGPPSGGSSPSPGAAGPSATGPAASTGATSPGGGGGAGSGGGGGSPASGGTSTPSSAGPPSGGGTPAAPSGGGAGTSGSATPPSPGGSGTAGTPPGSTASSSSGPPPPPPPTSASGGGSGGSASGGAGSTSGSGGGGGGGSGPPPTASGPGGSGGGPSGGGGGSTSSGASPPAGSSTSGGGTGAAGSPASTSGGPGAGGGGTAGGSAGPSGASTSSPPAASTPPGTSAGTGAPASGGSGGSPAGSGASSGASPSGGGAPPTTGASGGSGSSGGGAAPSGSSGGGSSGGTTPASSGSAASSAPSTSTPSSTTSPSAAPGSSGGSGATPPSSSSPTPPPGSGSSGGTSPGGSPSASPGGSTPSGGGGGGGGGGSGPAPPSAAPSAGGTSGGAGGGGGGTSSGSTSPPSGGSPGGGAASSSPTGTPSTPASSATSGSAGSSSSGSPAPSGGPSGAAPSSGPTPAGGGGG。

the number of amino acids contained: 5000 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 98.40 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 1.3 percent.

The relevant parameters of PsTag01 to PsTag11 are summarized in the following table.

GOR value: and determining the proportion of random coil in the polypeptide sequence by GOR algorithm.

Chou-Fasman values: the sum of the proportions of alpha helices and beta sheets in the polypeptide sequence was determined by the Chou-Fasman algorithm.

In addition, the sequence of Linker01 is: EEEEEEEEPLGLAG, respectively; the sequence of Linker02 is: PLGLAGEEEEEEEE, respectively; the sequence of Linker03 is: PLGLAG.

CPP01 is R8; CPP02 is TAT; CPP03 is MPG; CPP04 is Transportan; CPP05 is Pep-1; CPP06 is EB 1; CPP07 is Tat-DRBD.

Wherein, the specific sequence of each cell-penetrating peptide is as follows:

the sequence of R8 is: RRRRRRRRRR;

the TAT sequence is: GRKKRRQRRRPPQ;

the MPG sequence is: GLAFLGFLGAAGSTMGAWSQPKSKRKV, respectively;

the Transportan sequence is: AGYLLGKINLKALAALAKKIL, respectively;

the Pep-1 sequence is: KETWWETWWTEWSQPKKKRKV;

the EB1 sequence is: LIRLWSHLIHIWFQNRRLKWKKK, respectively;

Example 2 in vivo half-Life, in vivo tissue distribution and in vitro antitumor Activity of the drug

(I) in vivo half-life assay

BALB/c mice weighing about 20g and 6-8 weeks old were randomly grouped into 5 mice per group and administered to the tail vein separately. Select 9 time points: 0h, 0.5h, 1h, 4h, 12h, 24h, 48h, 72h and 96h, collecting blood samples of each group of mice by taking blood from retroorbital venous plexus at each time point, standing at room temperature for 30min, centrifuging at 3000rpm for 15min, and carefully sucking serum by using a pipette. Then, the content of the fusion protein in each group of serum was detected by using a His-Tag ELISA kit (L00436, Nanjing Kinsrui) according to the instruction. Establishing a curve equation of the concentration and the OD value of the standard substance by using Microsoft Excel 2013, substituting the OD value of the detected sample into the curve equation to calculate the content of the detected sample, calculating the numerical value of the calculated sample by using PKSolve software to calculate a main pharmacokinetic parameter-half life, and fitting a blood concentration-time curve by using Sigmplot.

(II) in vivo tissue distribution determination

About 20g of tumor-bearing BALB/c mice with the age of 6-8 weeks are taken, Cy5 fluorescence labeled protein is injected into tail veins, tumors and main tissues and organs (heart, liver, spleen, lung and kidney) are taken out about 8 hours after administration, the fluorescence distribution of each tissue is observed under an IVIS imaging Spectrum fluorescence imaging instrument, and the fluorescence intensity of each tissue is quantitatively analyzed.

(III) in vitro determination of antitumor Activity

Three tumor cells, A375, A549 and MCF7, were individually suspended in culture medium containing 10% fetal bovine serum to prepare single cell suspensions, and 5000 cells per well were inoculated into 96-well plates in a volume of 200. mu.L per well. The administration is carried out after the overnight adherence, and after 3-5 days of culture, the incubation is continued for 4h by adding MTT solution to each well. Terminating the culture, removing culture supernatant in each well, adding 150. mu.L DMSO into each well, and shaking for 10min to fully melt the crystals. Selecting 490nm wavelength, measuring the light absorption value of each well on an enzyme linked immunosorbent assay, recording the result, and drawing a cell growth curve by taking time as an abscissa and the light absorption value as an ordinate.

The in vivo half-life, in vivo tissue distribution and in vitro antitumor activity of each fusion protein of example 1 were determined by the above three methods, respectively.

The results of in vivo half-life, in vivo primary and secondary tissue distribution for each fusion protein of example 1 are shown in the table below. Compared with KLAC, the half-life period in vivo of each fusion protein is obviously prolonged; each fusion protein was distributed mainly in tumor tissues, with a small distribution in the kidney and little distribution in other tissues.

Note: the half-life of KLAC is currently not a clear value. According to the relevant literature: most polypeptides have a half-life of only 2-30 minutes due to enzymatic degradation by blood proteases and rapid clearance by the kidney (<30kDa molecules are rapidly excreted by glomerular filtration). From this, the half-life of KLAC was at most 0.5 hour. The relevant documents are: s.c. pencala, m.r.miller, a.pal, j.dong, n.r.madadi, j.xie, h.joo, j.tsai, p.baton, v.samoshin, a.franz, t.cox, j.mills, w.k.chan, m.s.park, m.m.ahamadsheh, a biometric approach for enhancing the in vivo hall of the peptides of peptide, nat.chem.biol.11(2015) 793-.

In the aspect of in vitro anti-tumor activity, each fusion protein in example 1 has good anti-tumor activity on three tumor cells, namely A375, A549 and MCF 7.

FIGS. 1 to 3 are schematic diagrams of the above results, respectively, and the results of the above three aspects of each fusion protein in example 1 are similar to the schematic diagrams, which is limited by the space and will not be repeated herein.

Based on the above results, the following conclusions were drawn:

the protein constructed according to formula I or formula II from N-terminal to C-terminal has a targeted anti-tumor function, and has a longer half-life in vivo.

Formula I: PsTag-Linker-CPP-KLAC;

formula II: KLAC-CPP-Linker-PsTag;

wherein, PsTag is a recombinant polypeptide, which is composed of 100 to 5000 amino acid residues, wherein the total amount of glycine, alanine, serine, threonine, proline and lysine accounts for more than 95 percent of the total number of the amino acid residues; at least 95% of the structure of the recombinant polypeptide is random coil, as determined by GOR algorithm; determined according to Chou-Fasman algorithm, in the structure of the recombinant polypeptide, the sum of the proportion of alpha helix and beta sheet is less than 2%; the sequence of the recombinant polypeptide contains at most 1 methionine;

linker is connecting peptide and has MMP2 enzyme cutting site sequence; CPP is cell penetrating peptide; KLAC is an antitumor polypeptide and has a cysteine that serves as a conjugation site.

Specifically, the MMP2 enzyme cutting site sequence is PLGLAG; the sequence of the linker peptide is: EEEEEEEEPLGLAG, PLGLAGEEEEEEEE, or PLGLAG.

The cell-penetrating peptide is selected from one of R8, Steamyl-R8, TAT, MPG, Transportan, Pep-1, EB1, TAT-DRBD and PF6, or the cell-penetrating peptide contains at least one of R8, Steamyl-R8, TAT, MPG, Transportan, Pep-1, EB1, TAT-DRBD and PF 6.

Example 3, verification

The results obtained in example 2 were verified by constructing 9 fusion proteins according to the construction method of example 1 and then measuring the half-life in vivo, tissue distribution in vivo and antitumor activity in vitro according to the method of example 2.

The fusion protein constructed in this example was:

PAK12：PsTag12-Linker01-CPP01-KLAC

PAK13：KLAC-CPP02-Linker02-PsTag13

PAK14：PsTag14-Linker03-CPP03-KLAC

PAK15：KLAC-CPP04-Linker01-PsTag15

PAK16：PsTag16-Linker02-CPP05-KLAC

PAK17：KLAC-CPP06-Linker03-PsTag17

PAK18：PsTag18-Linker01-CPP07-KLAC

PAK19：KLAC-CPP06-Linker02-PsTag19

PAK20：PsTag20-Linker03-CPP05-KLAC

specific sequences of PsTag 12-PsTag 20 are as follows.

The sequence of PsTag12 is SEQ ID No.13, i.e.:

GPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPTSGPSTASGPSTASGPTSGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPTSGPSTASGPSTASGPTS。

the number of amino acids contained: 200 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 97.00 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 0 percent.

The sequence of PsTag13 is SEQ ID No.14, i.e.:

PSPGTGGGPGGSPTGPAAPSAPTPPPSGAGAGSPSSSAPGGGSTSSPTGTGSGGPTPGSTPAPTPSGGGGSSPPGPSAPATTPAGPAAPSTSGGSPASPPPGGGGSSAPTSGASSATPSPPTSGASTPPPSSGTSGPSSSPGTASPGTGPAPSAPASPGSTSGGTAGPSPSASTAASAPTSGASSATPAAPATSTPPTPS。

the number of amino acids contained: 200 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 98.00 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 1 percent.

The sequence of PsTag14 is SEQ ID No.15, i.e.:

SAGPPPAPGSPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPTSGPTASGPSAGSGGGPTASGTPGAGSPGGTAGGTGPGGSGGGPTGSPPSTATPAGPTSGGSAPTAGPSSGSSPPTSSTGSSTSSPGAGPPPTGGGGGGGGGTTGPSATPSATPG。

the number of amino acids contained: 216 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 98.15 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 0 percent.

The sequence of PsTag15 is SEQ ID No.16, i.e.:

GPSATPGSTGPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPTSGPTASGPSATPGSTGPTSAPGSTGPSATPGTSGPSGTSAPTGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPSATPGSTGPTSAPGSTGPSTGSPATGPSTPAGST。

the number of amino acids contained: 216 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 97.69 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 0 percent.

The sequence of PsTag16 is SEQ ID No.17, i.e.:

GPTSGPSTASGPSTASGPTSGPASTGSPSTGPTSGPSTASGPTSGPSTASGPSTASGPTSGPTSGPSTASGPSTASGPTSGPTSGPSTASGPSTASGPTSGPTSGPSTASGPSTASGPTSGPTSGPSTASGPSTASGPTSGPASTGSPSTGPTSGPSTASGPTSGPSTASGPSTASGPTSGPTSGPSTASGPSTASGPTSGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPTSGPSTASGPSTASGPTSGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPTSGPSTASGPSTASGPTSGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPASTGSPSTGPTSGPSTASGPTSGPSTASGPSTASGPTS。

the number of amino acids contained: 600 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 99.00 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 0 percent.

The sequence of PsTag17 is SEQ ID No.18, i.e.:

SAASATGPTPGTGGPPAPGGTSGAPGGTSGPPAGATGSGPSTSGSAAGGPGGGSGTASATSAPPTSASAAGGAPGSTPAGSSTPSSSPGGTTGSGPSSSPPSAATSASSPPPPAPAGAPTSSAGTAGPTAAPTPPATSAAPGGGGPGPGSGSSSPPGPTSAASPAAPSSGGTAPPTPSGSGGAPAPGPGAAAGGPPAPTASPPAGGGTPGGTPGAGGGAPPGSTTGSPSSSGTPGPASGSAATTPPGSTAGTGAASASTAPPGSGAAPASAGPASPGGTTGTGAASGGGTSSPGAAAPSSSAPASASPPSPTSTAGTPSPGTPGPAAGTAPPGSPPASTSAPSPPSGPSAPAPTTPTGPAPTGPSTPSGGGGTGTGGPTSGAGGGGPPSPGTPGPAPTGGPAGTSGSTGPSASSTSATGTSASAAAPPPPTPTAPSSAGSAGPTPAPAAGGPTPTSTGSPGTGATSASGATAGGTSAGGGSGGGSSPGSGAAPSAGSAGGGAPAASSPGASGGSTSSTPGGTSTGGGSPAGAATSGATGTAATPASGASTPSGGGGSAGAGTTPATGSTGSPGTSSSPGAAGSGAGAPSPPTAGPPGGGG。

the number of amino acids contained: 600 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 95.00 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 1.0 percent.

The sequence of PsTag18 is SEQ ID No.19, i.e.:

SAGPPPAPGSPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPTSGPTASGPSAGSGGGPTASGTPGAGSPGGTAGGTGPGGSGGGPTGSPPSTATPAGPTSGGSAPTAGPSSGSSPPTSSTGSSTSSPGAGPPPTGGGGGGGGGTTGPSATPSATPGSAGPPPAPGSPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPTSGPTASGPSAGSGGGPTASGTPGAGSPGGTAGGTGPGGSGGGPTGSPPSTATPAGPTSGGSAPTAGPSSGSSPPTSSTGSSTSSPGAGPPPTGGGGGGGGGTTGPSATPSATPGSAGPPPAPGSPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPTSGPTASGPSAGSGGGPTASGTPGAGSPGGTAGGTGPGGSGGGPTGSPPSTATPAGPTSGGSAPTAGPSSGSSPPTSSTGSSTSSPGAGPPPTGGGGGGGGGTTGPSATPSATPG。

the number of amino acids contained: 648 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 99.38 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 0 percent.

The sequence of PsTag19 is SEQ ID No.20, i.e.:

GPSGTPASTGPATPSGTSGPSGTPASTGPATPSGTSGPAGSTPSTGPSAPTGSTGPSGTPASTGPATPSGTSGPATPSGTSGPSTAGPTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPSAPTGSTGPSATPGSTGPTSAPGSTGPTSAPGTSGPSTPGTSAGPTSAPGSTGPSAGTPSTGPATPSGTSGPSTAGPTSGPSATPGSTGPTSAPGSTGPATPSGTSGPSTAGPTSGPSATPGTSGPSGTSPTAGPTSAPGTSGPSTPGTSAGPAGSTPSTGPSAPTGSTGPSGTPASTGPATPSGTSGPSATPGSTGPTSAPGSTGPSATPGTSGPSGTSAPTGPSATPGTSGPSGTSAPTGPTSAPGTSGPSTPGTSAGPTSAPGSTGPSAGTPSTGPTSAPGSTGPSAGTPSTGPSATPGSTGPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPTSGPTASGPSATPGSTGPTSAPGSTGPSATPGTSGPSGTSAPTGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPSATPGSTGPTSAPGSTGPSTGSPATGPSTPAGST。

the number of amino acids contained: 648 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 99.23 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 0 percent.

The sequence of PsTag20 is SEQ ID No.21, i.e.:

SAASATGPTPGTGGPPAPGGTSGAPGGTSGPPAGATGSGPSTSGSAAGGPGGGSGTASATSAPPTSASAAGGAPGSTPAGSSTPSSSPGGTTGSGPSSSPPSAATSASSPPPPAPAGAPTSSAGTAGPTAAPTPPATSAAPGGGGPGPGSGSSSPPGPTSAASPAAPSSGGTAPPTPSGSGGAPAPGPGAAAGGPPAPTASPPAGGGTPGGTPGAGGGAPPGSTTGSPSSSGTPGPASGSAATTPPGSTAGTGAASASTAPPGSGAAPASAGPASPGGTTGTGAASGGGTSSPGAAAPSSSAPASASPPSPTSTAGTPSPGTPGPAAGTAPPGSPPASTSAPSPPSGPSAPAPTTPTGPAPTGPSTPSGGGGTGTGGPTSGAGGGGPPSPGTPGPAPTGGPAGTSGSTGPSASSTSATGTSASAAAPPPPTPTAPSSAGSAGPTPAPAAGGPTPTSTGSPGTGATSASGATAGGTSAGGGSGGGSSPGSGAAPSAGSAGGGAPAASSPGASGGSTSSTPGGTSTGGGSPAGAATSGATGTAATPASGASTPSGGGGSAGAGTTPATGSTGSPGTSSSPGAAGSGAGAPSPPTAGPPGGGGSAASATGPTPGTGGPPAPGGTSGAPGGTSGPPAGATGSGPSTSGSAAGGPGGGSGTASATSAPPTSASAAGGAPGSTPAGSSTPSSSPGGTTGSGPSSSPPSAATSASSPPPPAPAGAPTSSAGTAGPTAAPTPPATSAAPGGGGPGPGSGSSSPPGPTSAASPAAPSSGGTAPPTPSGSGGAPAPGPGAAAGGPPAPTASPPAGGGTPGGTPGAGGGAPPGSTTGSPSSSGTPGPASGSAATTPPGSTAGTGAASASTAPPGSGAAPASAGPASPGGTTGTGAASGGGTSSPGAAAPSSSAPASASPPSPTSTAGTPSPGTPGPAAGTAPPGSPPASTSAPSPPSGPSAPAPTTPTGPAPTGPSTPSGGGGTGTGGPTSGAGGGGPPSPGTPGPAPTGGPAGTSGSTGPSASSTSATGTSASAAAPPPPTPTAPSSAGSAGPTPAPAAGGPTPTSTGSPGTGATSASGATAGGTSAGGGSGGGSSPGSGAAPSAGSAGGGAPAASSPGASGGSTSSTPGGTSTGGGSPAGAATSGATGTAATPASGASTPSGGGGSAGAGTTPATGSTGSPGTSSSPGAAGSGAGAPSPPTAGPPGGGG。

the number of amino acids contained: 1200 AA. The proportion of the irregular curls calculated by the GOR method is as follows: 95.08 percent. The Chou-Fasman method calculates the ratio of the sum of the alpha helix and the beta sheet: 1 percent.

The relevant parameters of PsTag12 to PsTag20 are summarized in the following table.

CPP01 is R8; CPP02 is TAT; CPP03 is MPG; CPP04 is Transportan; CPP05 is Pep-1; CPP06 is EB 1; CPP07 is Tat-DRBD. The specific sequence is the same as in example 1.

The results of the in vivo half-life, in vivo primary and secondary tissue distribution of each fusion protein of this example are shown in the table below. Compared with KLAC, the half-life period in vivo of each fusion protein is obviously prolonged; each fusion protein was distributed mainly in tumor tissues, with a small distribution in the kidney and little distribution in other tissues.

In the aspect of in vitro anti-tumor activity, each fusion protein of the embodiment has good anti-tumor activity on three tumor cells, namely A375, A549 and MCF 7.

The above three-aspect result graphs of each fusion protein of this embodiment are similar to those of the example graph of embodiment 2, which is limited by space and will not be repeated herein.

Based on the above results, it can be assumed that the conclusion obtained in example 2 is correct, i.e., the verification is successful.

Example 4 in vitro anti-tumor Effect and in vivo tissue distribution of fusion protein conjugates

Some of the proteins selected from the fusion proteins of examples 1 and 2 were conjugated to the cysteine of KLAC with an anti-tumor compound according to the prior art to obtain the following conjugates:

PAKD1：PAK14-DOX1，

PAKD2：PAK7-DOX2，

PAKD3：PAK18-DOX3，

PAKP1：PAK14-PTX1，

PAKP2：PAK7-PTX2，

PAKP3：PAK18-PTX1，

PAKDMA：PAK7-DMA，

PAKDMB：PAK7-DMB，

PAKMMA1：PAK7-MMA1，

PAKMMA2：PAK7-MMA2。

wherein DOX1 to DOX3 are doxorubicin or a derivative thereof; DOX1 is adriamycin, and DOX2 has the structure:

the structure of DOX3 is:

in addition, the structure of the adriamycin derivative can also be as follows:

PTX1 to PTX3 are paclitaxel or derivatives thereof; PTX1 is paclitaxel; PTX2 has the structure:

PTX3 has the structure:

DMA to DMB is maytansine or its derivatives; DMA is maytansine; the DMB structure is as follows:

furthermore, the maytansine derivatives may also be:

MMA1 to MMA2 are dolastatin or derivatives thereof; MMA1 is dolastatin, MMA2 has the structure:

furthermore, dolastatin derivatives may also be:

using the method of example 2, the conjugates were tested for anti-tumor activity against three tumor cells, A375, A549 and MCF7, and IC was calculated using GraphPad₅₀(ii) a The distribution of major and minor tissues in vivo was determined. PAK14 was used as a control group in the measurement. The results are shown in the following table.

From the above results, it can be seen that each conjugate of this example has good anti-tumor activity against three tumor cells, a375, a549 and MCF 7; each conjugate was distributed predominantly within the tumor tissue, with a small distribution in the kidney and little distribution in other tissues. This shows that the conjugate of the anti-tumor targeting fusion protein and the anti-tumor compound still has a targeting function and realizes the synergistic killing of tumor cells by two different anti-tumor mechanisms.

Example 5 Activity of fusion protein conjugates against drug-resistant tumors

The conjugates of example 4, PAKD1, PAKD2, PAKP1 and PAKP2, were selected and tested for their antitumor activity against MCF7 tumor cells and MCF7/R resistant tumor cells. Meanwhile, adriamycin and paclitaxel are used as control groups. Note: the drug resistance mechanism of MCF7/R resistant tumor cells is ABC transporter overexpression. The results are shown in the following table.

The results show that the adriamycin and the taxol only have good killing effect on the non-drug-resistant MCF7 tumor cells, but have greatly reduced effect on the MCF7/R drug-resistant tumor cells; in contrast, the conjugates PAKD1, PAKD2, PAKP1 and PAKP2 have the same level of killing effect on MCF7 tumor cells and MCF7/R drug-resistant tumor cells. This indicates that the conjugate of the anti-tumor targeting fusion protein and the anti-tumor compound of the invention has the effect of reversing drug resistance.

Example 6 myocardial cytotoxicity of fusion protein conjugates

The conjugates of example 4, PAKD1 and PAKD2, were selected and tested for toxicity to rat cardiomyocytes. Meanwhile, doxorubicin was used as a control group.

55 clean-grade SD rats were randomly divided into four groups according to the random number table method: group a, normal control group (n ═ 10), was raised in parallel; group B, doxorubicin model group (n ═ 15), administered i.p. doxorubicin; group C, conjugate administration group (n ═ 15), i.p. PAKD 1; group D, conjugate administration group (n ═ 15), i.p. PAKD 2; B. c, D the three groups are given at a 5. mu. mol/kg weekly dose for 6 weeks, with a cumulative dose of 30. mu. mol/kg; after the experiment is finished, the heart of a fresh experimental rat is taken, and the local ROS content of the myocardium and the Caspase-3 gene expression are respectively detected by an ELISA method and real-time fluorescence quantitative PCR.

The results are shown in the following table.

	ROS(IU/mL)	Caspase-3 (relative expression quantity)
			Doxorubicin model group	76.80±3.89	6.46±0.54
PAKD1 administration group	59.81±4.46	1.03±0.06
			PAKD2 administration group	60.09±4.13	1.11±0.08
Control group	58.32±4.28	1

Wherein, in terms of ROS content, the adriamycin model group is higher than the control group and the fusion protein conjugate administration group, and the difference has statistical significance (P < 0.05); in the aspect of the expression condition of Caspase3, the doxorubicin model group is obviously improved, and the fusion protein conjugate administration group has no obvious difference from a control group (P > 0.05).

The results show that the adriamycin can obviously cause the ROS of rat myocardial cells to be increased and cause the myocardial cell apoptosis, and the PAKD1 and PAKD2 have no obvious toxicity to the rat myocardial cells.

In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the claims of the present invention.

Sequence listing

<120> anti-tumor targeting fusion protein, conjugate and use thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Lys Leu Ala Cys

1

Claims

1. An anti-tumor targeting fusion protein, characterized by consisting of a sequence constructed according to formula I or formula II from N-terminus to C-terminus:

formula I: PsTag-Linker-CPP-KLAC;

formula II: KLAC-CPP-Linker-PsTag;

in the formula I or the formula II, PsTag is recombinant polypeptide, and the sequence of the recombinant polypeptide is shown in one of SEQ ID No.2 to SEQ ID No. 16;

the Linker is a connecting peptide, and the connecting peptide has an MMP2 enzyme cutting site sequence; CPP is cell penetrating peptide; KLAC is an anti-tumor polypeptide;

the MMP2 enzyme cutting site sequence is PLGLAG; the sequence of the connecting peptide is as follows: EEEEEEEEPLGLAG or PLGLAGEEEEEEEE;

the cell-penetrating peptide is R8, and has the sequence: RRRRRRRR.

2. A nucleic acid encoding the anti-tumor targeting fusion protein of claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. A host cell comprising the expression vector of claim 3.

5. An anti-tumor targeting fusion protein conjugate, which is prepared by coupling the anti-tumor targeting fusion protein of claim 1 with an anti-tumor compound via a coupling site thereof, wherein the coupling site is cysteine in KLAC;

the anti-tumor compound is adriamycin or derivatives thereof, or paclitaxel or derivatives thereof;

the structure of the adriamycin derivative is shown as a formula A:

formula A:

the structure of the taxol derivative is shown as a formula D:

formula D:

6. use of the anti-tumor targeted fusion protein according to claim 1, characterized in that the use is for the preparation of a medicament or a pharmaceutical composition for the treatment or prevention of tumors that are resistant to drug caused by overexpression of ABC transporters, or for the preparation of a conjugate of an anti-tumor targeted fusion protein that is resistant to drug caused by overexpression of ABC transporters, or for the preparation of a conjugate of an anti-tumor targeted fusion protein-doxorubicin with lower myocardial cytotoxicity than doxorubicin by conjugation to doxorubicin, and that are resistant to drug caused by overexpression of ABC transporters.

7. The use of the anti-tumor targeting fusion protein conjugate according to claim 5, wherein the use is for the preparation of a medicament or a pharmaceutical composition for the treatment or prevention of tumors, and the tumors are drug-resistant tumors resulting from the overexpression of ABC transporters.