CN113336830B

CN113336830B - Tumor targeting polypeptide, preparation method and application thereof

Info

Publication number: CN113336830B
Application number: CN202110610291.6A
Authority: CN
Inventors: 王富军; 赵健; 曹雪玮; 傅龙云; 张涛铸; 单含文; 杨旭中
Original assignee: Zhejiang Funuo Pharmaceutical Co ltd
Current assignee: Zhejiang Funuo Pharmaceutical Co ltd
Priority date: 2017-03-17
Filing date: 2017-03-17
Publication date: 2023-07-07
Anticipated expiration: 2037-03-17
Also published as: CN113332448B; CN113332447A; CN113332448A; CN113332447B; CN108570108B; CN113336830A; CN108570108A

Abstract

The invention relates to the field of biological medicine, in particular to a tumor targeting polypeptide, a preparation method and application thereof. The invention discloses a tumor targeting polypeptide. The tumor targeting polypeptide has better specific binding capacity, thereby reducing the influence of tumor therapeutic drugs on normal cells, reducing the occurrence of adverse reactions of the drugs and improving the therapeutic effect.

Description

Tumor targeting polypeptide, preparation method and application thereof

The application is a divisional application of the prior application (the invention name is tumor targeting polypeptide, the preparation method and the application thereof, the application date is 2017, 03, 17 and the application number is 201710161670.5).

Technical Field

The invention relates to the field of biological medicine, in particular to a tumor targeting polypeptide, a preparation method and application thereof.

Background

Tumors are one of three major diseases in the world today that seriously affect human health and life. In many common neoplastic diseases, such as breast cancer, non-small cell lung cancer, colorectal cancer, bladder cancer, ovarian cancer, gastric cancer, pancreatic cancer, epidermoid squamous carcinoma, renal cancer, head and neck malignancy, glioblastoma, etc., there is often a tumor cell surfacePhenomenon of aberrant overexpression of Epidermal Growth Factor Receptor (EGFR) ¹ And high expression of EGFR is often closely related to abnormal cell proliferation activity. The number of EGFR on the surface of each normal human cell is typically 4X 10 ⁴ To 1X 10 ⁵ About, EGFR on the surface of each tumor cell exceeds 2X 10 ⁶ 20-50 times that of normal cells ² . EGFR mediated signal transduction plays an important role in proliferation, injury repair, invasion, angiogenesis and the like of tumor cells, so EGFR naturally becomes a key target site of special attention in tumor diagnosis and treatment, and becomes an important direction for development of tumor drugs and clinical treatment application ³ 。

Members of the EGFR family include four of ErbB1/HER1/EGFR, erbB2/HER2, erbB3/HER3 and ErbB4/HER4, which belong to the tyrosine kinase class of receptors. The human EGFR protein consists of 1186 amino acid residues and has a relative molecular weight of 17 kilodaltons. The whole receptor protein consists of three parts: (1) An extracellular region consisting of 621 amino acid residues at the amino terminus, in which a ligand-binding region is present; (2) A transmembrane region, a helical hydrophobic region structure composed of 23 amino acid residues is deeply buried in a cell membrane lipid bilayer, and a receptor protein is anchored on a cell membrane; (3) An intracellular region, consisting of 542 amino acid residues, can be further subdivided into 3 subregions: a membrane proximal subregion, a tyrosine kinase subregion, and a carboxy terminal subregion.

It has now been found that a variety of Epidermal Growth Factor (EGF) class ligand molecules are capable of specifically binding and exerting biological effects on EGFR. These ligand molecules include Epidermal Growth Factor (EGF), transforming growth factor (TGF-alpha), amphiregulin (AR), B-cytokine (BTC), heparin-binding-like epidermal growth factor (HB-EGF), epiregulin (EPR), vaccinia virus-derived vaccinia Virus Growth Factor (VGF), and the like.

These ligand molecules that bind EGFR have very similar and conserved three-dimensional structures in spatial structure, i.e., the ligand molecule forms a typical tricyclic structure internally by action of disulfide bonds within the polypeptide chain (Cys 6-Cys20, cys14-Cys31, cys33-Cys 42) ⁴ 。

It was found that the specific recognition and binding process of EGF with EGFR is essentially representative of the interaction process of such ligand molecules with receptors. The three ring structures of EGF interact with EGFR in three parts of upper, lower and middle to form recognition and specific combination with EGFR, wherein the C ring structure extends into EGFR protein molecule, plays an important role in EGFR recognition and combination ^5-6 。

EGF ligand molecules are combined with EGFR, so that an epidermal growth factor receptor can form a homodimer or a heterodimer with each other to cause activation of an intracellular tyrosine kinase region, opposite tyrosine residues can be phosphorylated with each other, a series of cascade reactions are started, signals are transmitted into nuclei, a series of related genes are finally caused to be activated, proliferation and apoptosis inhibition of tumor cells are caused, metastasis of the tumor cells is promoted, chemoradiotherapy tolerance is caused, and the EGF ligand molecules play an important role in the tumorigenesis and development process. Also, polypeptide molecules which are synthesized by artificial design and mimic the specific recognition and binding of EGF-like growth factors to their receptors, since they can recognize and bind specifically similarly to EGFR, and such specific binding of these specifically designed polypeptides does not result in subsequent signaling of EGFR molecules ⁷ Therefore, can be used for the targeted drug delivery to tumor cells in vivo for the targeted treatment of tumor diseases ^8-9 The method comprises the steps of carrying out a first treatment on the surface of the Or the specific polypeptide itself is combined with the receptor binding site on the surface of the in vivo growth factor in a competitive manner, thereby inhibiting the growth of tumor cells and generating the aim of treatment ^10-11 The method comprises the steps of carrying out a first treatment on the surface of the Or radiolabeled ^12-13 Fluorescent dye-labeled polypeptides ^14-15 After being injected into the body, the polypeptide can be specifically aggregated on the surface of tumor cells, thereby being used for the imaging diagnosis, marking and treatment of solid tumors ^16-17 . Such polypeptides that specifically recognize binding to tumor cells in vitro and in vivo are simply referred to as tumor targeting peptides.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a polypeptide structure which can be efficiently and specifically identified and has stronger binding force, and can be specifically combined with an epidermal growth factor receptor which is excessively expressed on the surface of a tumor cell, so that the polypeptide structure can be used for identifying and diagnosing the marker of the tumor cell, or can be combined with EGFR in a competitive manner, or can carry tumor therapeutic drug molecules to reach the surface of the tumor cell, and the targeting therapeutic effect can be exerted.

To this end, in one aspect, the invention discloses a tumor targeting polypeptide comprising a first sequence of amino acid sequence YXGXR and another second sequence of amino acid sequence YXGXR, linked by a connecting peptide, wherein Y represents tyrosine or a derivative thereof; g represents glycine or a derivative thereof; r represents arginine or a derivative thereof; x represents an amino acid containing a fatty group or/and a hydroxyl group or a combination or derivative thereof in a side chain.

In some embodiments, the connecting peptide comprises an amino acid sequence that forms an alpha-helix, preferably the connecting peptide comprises a rigid alpha-helix structure, more preferably the connecting peptide comprises a sequence having the amino acid sequence HMAATT.

In some embodiments, the connecting peptide comprises more than 6 amino acid residues; or the linker peptide comprises less than 3 amino acid residues; or the connecting peptide consists of 1 amino acid residue; the connecting peptide consists of histidine or a derivative thereof.

In some embodiments, the tumor targeting polypeptide is a polypeptide having an amino acid sequence of one of SEQ ID nos. 1-4 or 8 or 9 or a modified derivative thereof; preferably a polypeptide having the amino acid sequence SEQ ID NO.1 or SEQ ID NO.8 or SEQ ID NO. 9.

In some embodiments, the tumor targeting polypeptide can specifically bind to any member of the EGFR family, preferably the tumor targeting polypeptide can bind to EGFR with a Kd less than 50nM.

In another aspect, the invention also discloses a conjugate comprising the tumor targeting polypeptide of claim 1 and an active ingredient, both conjugated via a linker (linker), wherein the active ingredient is a therapeutic ingredient, a diagnostic ingredient, a radioisotope, a radionuclide, a toxin, or a combination thereof.

In some embodiments, the linker comprises a covalent bond linkage or a non-covalent bond linkage, preferably the covalent bond linkage comprises a direct covalent bond, a peptide bond, an ester bond, a disulfide bond, an amide bond, an imide bond, a phosphodiester bond, a urea bond, an isocyanate bond, or a combination thereof.

In some embodiments, the therapeutic ingredient comprises a Cell Penetrating Peptide (CPP), preferably the cell penetrating peptide is a polypeptide shown in the amino acid sequence of SEQ ID No.12, an amino acid fragment of the transactivator TAT from HIV, an amino acid fragment of the EC-SOD derived Heparin Binding Domain (HBD), an amino acid fragment of the HBEGF derived Heparin Binding Domain (HBD), or derivatives thereof.

In some embodiments, the conjugate comprises a polypeptide having an amino acid sequence of one of SEQ ID NO. 13-15.

In some embodiments, the therapeutic component comprises a radiation therapeutic component, a chemotherapeutic component, an antibody, an enzyme, or a combination thereof.

In some embodiments, the radiotherapeutic component comprises a radioisotope, the radioisotope being iodine-131, lutetium-177, yttrium-90, samarium-153, phosphorus-32, cesium-131, palladium-103, radium-223, iodine-125, boron-10, actinium-225, bismuth-213, radium-225, lead-212, thorium-232, or a combination thereof.

In some embodiments, the chemotherapeutic component comprises capecitabine (capecitabine), cisplatin (cislatin), trastuzumab (trastuzumab), fulvestrant (fulvestrant), tamoxifen (tamoxifen), letrozole (letrozole), exemestane (exemestane), anastrozole (anastrozole), aminoglutethimide (aminoglutethimide), testosterone (testolactone), vorozole (vorozole), formestane (formoteratine), fadrozole (fadrozole), letrozole (letrozole), erlotinib (erlotinib), afatinib (labatinib), dasatinib (dasatinib), gefitinib (gefitinib), imatinib (imatinib), pazotinib (lapatinib), lapatinib (lapatinib), sultinib (sulatib), or a combination thereof.

In some embodiments, the diagnostic component comprises a radiodiagnostic component, a fluorescent component, a quantum dot, or a combination thereof, wherein the radiodiagnostic component comprises fluorine-18, technetium-99, molybdenum-99, rubidium-82, strontium-82, thallium-201, or a combination thereof.

In another aspect, the invention discloses a nucleic acid sequence encoding a tumor targeting polypeptide of the invention.

In another aspect, the invention discloses an expression vector comprising a polynucleotide molecule of the invention, preferably the expression vector is expressible in a cell.

In another aspect, the invention discloses a host cell comprising the expression vector of the invention, which is a prokaryotic cell or a eukaryotic cell.

In another aspect, the invention discloses a method of producing a tumor targeting polypeptide of the invention, comprising culturing a host cell of the invention to produce the tumor targeting polypeptide.

In another aspect, the invention discloses a pharmaceutical composition comprising the conjugate of the invention and a pharmaceutically acceptable carrier.

In some embodiments, the composition comprises a radioactive therapeutic component, a radioactive nucleic acid, a toxin, a therapeutic component, or a chemotherapeutic component, or a combination thereof.

In another aspect, the invention discloses a pharmaceutical composition comprising a tumor targeting polypeptide of the invention and a pharmaceutically acceptable carrier.

In another aspect, the invention discloses a method of treating a subject having a tumor comprising administering to the subject an effective amount of a conjugate of the invention; the tumor comprises cells expressing at least one EGFR family member.

In some embodiments, the tumor is one of breast cancer, colorectal cancer, pancreatic cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, non-small cell lung cancer.

In some embodiments, the method further comprises concurrently administering an effective dose of a therapeutic agent.

In some embodiments, the biological subject is a human.

In another aspect, the invention discloses a solution comprising an effective concentration of the conjugate of the invention, the solution being plasma of a biological subject.

According to the invention, through artificial mutation modification optimization of the amino acid sequence structure of the S3 mimic peptide combined with EGFR, a tumor targeting peptide with better specific binding capacity is obtained, and the tumor targeting peptide can be used for diagnostic analysis of tumors after being coupled with a molecular imaging reagent (radioisotope label, fluorescent dye label and the like); or the tumor cell targeting therapeutic drug is prepared after being coupled with the tumor therapeutic drug and is used for the targeting treatment of malignant tumor, thereby reducing the influence of the tumor therapeutic drug on normal cells, reducing the occurrence of adverse reaction of the drug and improving the treatment effect; or directly utilizes the characteristic that the polypeptide structure can be highly compatible with a target site (EGFR), competitively binds with and masks the EGFR site, and the growth of tumor cells is inhibited to achieve the aim of treating tumor diseases.

Drawings

FIG. 1 shows comparison of the transmembrane efficiency of ELBD sequences with other sequences in nature.

FIG. 2 shows comparison of the transmembrane efficiencies of ELBD and mutant fusion proteins.

FIG. 3 concentration and time dependence of EGFP-ELBD-HBD mutant recombinant protein transmembrane efficiency.

FIG. 4 is a broad spectrum schematic of the targeting peptide ELBD.

FIG. 5 shows the selectivity of EGFP-ELBD-HBD mutant recombinant proteins for human cells.

FIG. 6 is a schematic representation of the binding capacity of EGFP-S3-HBD recombinant protein and EGFP-ELBD-HBD recombinant protein, respectively, to the surface of HeLa cells.

FIG. 7 is a comparative schematic diagram of the inhibition of tumor cell growth by recombinant protein TCS-ELBD-CPP.

FIG. 8 shows more schematic diagrams of the inhibition of growth of normal cells and tumor cells by the recombinant protein MAP 30-ELBD-CPP.

Detailed Description

The present invention incorporates herein in its entirety the contents of chinese invention patent CN 201310170530.6 and the references mentioned herein.

Reference is made to:

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18. tumor cell targeting transmembrane peptide, chinese patent application No.: CN 201310170530.6

The term "VGF third loop-like artificial mimetic peptide" (also referred to herein as "S3 mimetic peptide") refers herein to a class of short peptide sequences that have the function of specifically binding to EGFR, may or may not have the function of promoting cell growth and division, and that have a spatial structure that matches that of Domain III of EGFR, and that can specifically bind thereto, resulting in or not in dimerization of EGFR. A representative VGF third loop-like artificial mimetic peptide has a structure in which 2 Cys residues are present in the molecule, the amino acid peptide chain is folded into a loop structure in the interior, the spatial conformation is similar to the third loop structure, and the structural conformation matches the Domain III binding region of EGFR, which is necessary for the biological activity. Tyr, gly, arg in this loop structure and Leu at the C-terminus are highly conserved, and any deletion of these conserved amino acids or a major deletion mutation at the C-terminus, including Leu, can result in loss of receptor binding activity.

The tumor targeting peptide is a short peptide sequence, and is obtained by artificial mutation modification optimization of the amino acid sequence structure of an EGFR-bound S3 mimetic peptide. Can be used for the marking identification diagnosis of tumor cells, or can be combined with EGFR competitively, or carries tumor therapeutic drug molecules to reach the surface of the tumor cells, thereby playing a targeted therapeutic role.

Tumor targeting peptides can be used for diagnostic analysis of tumors after coupling with molecular imaging reagents (radioisotope labels, fluorescent dye labels, etc.); or the tumor cell targeting therapeutic drug is prepared after being coupled with the tumor therapeutic drug and is used for the targeting treatment of malignant tumor, thereby reducing the influence of the tumor therapeutic drug on normal cells, reducing the occurrence of adverse reaction of the drug and improving the treatment effect; or directly utilizes the characteristic that the polypeptide structure can be highly compatible with a target site (EGFR), competitively binds with and masks the EGFR site, and the growth of tumor cells is inhibited to achieve the aim of treating tumor diseases.

"cell-penetrating peptides" (cell penetrating peptides, CPPs), also known as protein transduction domains or membrane transduction peptides, are polypeptides consisting of 30 amino acids or less capable of penetrating the cell membrane, including but not limited to sequences such as but not limited to the human immunodeficiency virus transcriptional activator TAT, the herpes simplex virus type I VP22 transcription factor, the Penetratin of Drosophila homologous antennaprotein (Antp), transportan, human-derived penetrating peptides such as ARF, bagP, cytC, hCT, hLF, hClck, TCTP, NRTN, synthetic facultative molecules consisting of large T antigen nuclear localization sequences and different hydrophobic peptide fragments MPG, MAP, pep-1, poly-arginine sequences ranging from 4 to 15 different lengths, synB1, polyomavir Vpl, bac, NF-KB, SV4OT anti, HATF3, hCT, pVEC, integrin, DPV6, S413PV, poly-P, heparin binding domain, etc., wherein preferably TAT (its amino acid sequence is YGRKKRRQRRR) or EC-SOD carboxy-terminal heparin binding domain (its amino acid sequence is GPGLWERQAREHSERKKRRRESECKAA) or variants thereof, such as cell penetrating peptides comprised in chinese patent CN201210587097.1, CN 1049111; most preferred is the heparin-binding domain of HBEGF derived from HBEGF, the amino acid sequence of which is shown in SEQ ID NO. 12.

The "connecting peptide" (linker) is a component of each functional domain in the protein, so that each functional domain forming the protein molecule maintains its active conformation, exerts its own biological function, provides synergistic, regulatory and allosteric effects for the biological activity of each functional domain, and avoids loss of biological activity of the protein molecule due to factors such as charge, space barrier, etc. In general, the linker peptide is divided into three forms of elasticity, rigidity, and cuttability. The connecting peptide may have a variety of secondary structural states to exert its biological functions such as alpha helices, beta sheet chains, curls/bends and turns. 59% of the naturally occurring connecting peptide was in coiled form (Chen et al, adv Drug Deliv Rev.2012, doi: 10.1016/j.addr.2012.09.039). The linker peptide in coiled form has a certain steric mobility, allowing the two domains that are linked to be relatively free to move.

The tumor cell targeting membrane penetrating peptide is a short peptide sequence, contains a CPPs structural domain with cell penetrating peptide activity and the tumor targeting peptide.

The polypeptides described herein, including but not limited to "tumor targeting peptides", "cell penetrating peptides", "tumor cell targeting penetrating peptides", and modified derivatives thereof, may be modified by methods conventional in the art for the tumor targeting peptides of the present invention. Such modification methods include modification of the amino terminal or carboxyl terminal end of the polypeptide, substitution of an intermediate amino acid residue, modification of a side chain, and the like. For example, the modification method of the terminal end of the peptide chain includes N-terminal acetylation and C-terminal amidation modification, thereby protecting the terminal amino group or carboxyl group. The peptide chain ends may also be linked to fatty acids of different lengths. PEG molecules are subjected to glycosylation modification, so that the relative molecular weight and steric hindrance of polypeptide molecules are increased, the stability of the polypeptide hydrolase is improved, and the residence time of a circulating system in a body is prolonged. Meanwhile, the half life of the polypeptide medicine can be prolonged or improved by replacing individual amino acid residues which are easy to be hydrolyzed or replacing L-type amino acid with D-type amino acid.

"conjugate" refers to a class of short peptide sequence conjugates of the tumor targeting polypeptide and an active ingredient, both conjugated via a linker (linker), wherein the active ingredient comprises a therapeutic ingredient, or a diagnostic ingredient, or a radioisotope, or a radionuclide, or a toxin, or a combination thereof.

Synthesis of polypeptides

The polypeptides of the invention, including but not limited to "tumor targeting peptides", "cell penetrating peptides", "tumor cell targeting penetrating peptides", can be subjected to solid phase synthesis or liquid-solid phase synthesis according to conventional polypeptide synthesis techniques. The tumor targeting peptide, tumor cell targeting transmembrane peptide, and tumor cell targeting transmembrane peptide can be synthesized by solid phase synthesis by a polypeptide synthesizer, and the tumor cell targeting transmembrane peptide and anticancer drugs comprise, but are not limited to, doxorubicin, docetaxel, mitomycin, daunorubicin, carboplatin, camptothecine, hydroxycamptothecin, vincristine, bleomycin, 5-fluorouracil, cyclophosphamide, gemcitabine, methotrexate, capecitabine, lomustine, etoposide, capecitabine, cisplatin, trastuzumab, fulvestrant, tamoxifen, letrozole, exemestane, anazocine, aminopropionate, protamine, and the like can be prepared by a linker, such as 3-maleimide propionic acid N-hydroxysuccinimide, and the like.

Taking doxorubicin as an example, the specific process is as follows: dissolving doxorubicin and 3-maleimide propionic acid N-hydroxysuccinimide in dimethylformamide respectively, adding triethylamine to adjust the pH value, stirring at room temperature for 2 hours, reacting, pouring into 50ml of diethyl ether, washing the separated precipitate with anhydrous diethyl ether for 2 times, centrifuging, separating the precipitate, and drying in vacuum; dissolving the precipitate and the tumor cell targeting membrane penetrating peptide with dimethylformamide, adding triethylamine, stirring at room temperature for 2 hours, pouring into 10ml of diethyl ether, washing the separated precipitate with anhydrous diethyl ether for 2 times, centrifuging, separating the precipitate, and drying in vacuum to obtain the target copolymer.

Expression of polypeptides or fusion proteins

The invention comprises DNA encoding the tumor targeting peptide or tumor cell targeting transmembrane peptide or fusion protein containing the same, and vectors and transformants containing the DNA.

In the present invention, the term "transformant" (transformation), i.e.a host cell carrying a heterologous DNA molecule, is used.

The invention also includes methods of producing the tumor targeting peptides "or tumor cell targeting transmembrane peptides of the invention or fusion proteins containing the same by synthetic and recombinant techniques. Polynucleotides (DNA or RNA), vectors, transformants and organisms can be isolated and purified by methods known in the art.

The vector used in the present invention may be, for example, a phage, plasmid, cosmid, minichromosome, viral or retroviral vector. Vectors useful for cloning and/or expressing polynucleotides of the invention are vectors capable of replicating and/or expressing polynucleotides in a host cell in which the polynucleotides are to be replicated and/or expressed. In general, the polynucleotides and/or vectors can be used in any eukaryotic or prokaryotic cell, including mammalian cells (e.g., human (e.g., heLa), monkey (e.g., cos), rabbit (e.g., rabbit reticulocytes), rat, hamster (e.g., CHO, NSO, and baby hamster kidney cells), or mouse cells (e.g., L cells)), plant cells, yeast cells, insect cells, or bacterial cells (e.g., e.coli). Examples of suitable vectors for use in various types of host cells can be found, for example, in Ausube et al Current Protocols in Molecular biology Greene Publishing Associates and Wiley-Interscience (1992) and Sambrook et al (1989). Host cells containing these polynucleotides can be used to express large amounts of proteins useful, for example, in pharmaceuticals, diagnostic agents, vaccines, and therapeutic agents.

Various methods have been developed for operably linking a polynucleotide to a vector via complementary cohesive ends. For example, complementary fragments of the homopolymer sequence may be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymer tails to form a recombinant DNA molecule.

Synthetic linkers containing one or more restriction sites provide another method of linking a DNA segment to a vector. The DNA segment produced by restriction endonuclease digestion is treated with phage T4DNA polymerase or e.coli DNA polymerase I, both of which remove the protruding γ -single stranded end with their 3',5' -exonuclease activity and fill in the 3' -concave end with their polymerization activity. Thus, the combination of these activities produces blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme capable of catalyzing the ligation of blunt-ended DNA molecules, such as phage T4DNA ligase. Thus, the reaction product is a DNA segment bearing a polymeric linker sequence at the end. These DNA segments are then cleaved with an appropriate restriction enzyme and ligated into an expression vector that has been cleaved with an enzyme that produces ends compatible with the DNA segments. Synthetic linkers containing multiple restriction endonuclease sites are commercially available from a variety of merchants.

The polynucleotide insert should be operably linked to an appropriate promoter compatible with the host cell in which the polynucleotide is to be expressed. The promoter may be a strong promoter and/or an inducible promoter. Examples of some of the promoters listed include phage lambda PL promoter, E.coli lac, trP, phoA, tac promoter, SV40 early and late promoters, and retroviral LTR promoter. Other suitable promoters are known to those skilled in the art. The expression recombinant vector further contains transcription initiation and termination sites, and a ribosome binding site for translation in the transcribed region. The coding portion of a transcript expressed by a recombinant vector may include a translation initiation codon at the start and a termination codon (UAA, UGA or UAG) suitably at the end of the polypeptide being translated.

As described above, the expression vector may include at least one selectable marker. The markers include dihydrofolate reductase, G418, glutamine synthase or neomycin resistance to eukaryotic cell culture; tetracycline, kanamycin, or ampicillin resistance genes for use in E.coli and other bacterial cultures. Representative examples of suitable hosts include, but are not limited to: bacterial cells such as E.coli, streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., saccharomyces cerevisiae or Pichia pastoris); insect cells, if fly S2 and noctuid SF9 cells; animal cells such as CHO, COS, NSO,293 and Bowes melanoma cells; and plant cells. Suitable media and culture conditions for the above-described host cells are known in the art.

In order to efficiently isolate and purify or secrete a target protein, a Tag protein or Tag polypeptide (Tag) which facilitates isolation and purification is often also available. Commonly used are glutathione-S-transferase (GST), hexahistidine peptide (His. Tag), protein A (protein A), and cellulose binding site (cellulose binding domain), etc. The target protein can be separated and purified by utilizing the special property of the tag protein or the tag polypeptide after expression through the form of fusion protein formed by the special protein or the polypeptide and the target protein. Tag specifically binds to Ni-Chelating Sepharose column. The tag protein or tag polypeptide can be purified and then digested by site-specific protease to remove fusion sequences, such as thrombin, enterokinase, xa factor, etc., so as to obtain target protein.

The invention also includes host cells comprising a nucleotide sequence of the invention operably linked to one or more heterologous control regions (e.g., promoters and/or enhancers) via techniques known in the art. Host strains can be selected which either modulate the expression of the inserted gene sequence or can modify and process the gene product in the particular manner desired. In the presence of certain inducers, expression from certain promoters will be elevated; thus, expression of the genetically engineered polypeptide can be controlled. In addition, different host cells have characteristic and specific mechanisms for translation, post-translational processing, and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines may be selected to ensure desirable modification and processing of the expressed exogenous protein.

The nucleic acids and nucleic acid recombinant vectors of the invention may be introduced into host cells by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. The method is described in a number of standard laboratory manuals, such as Davis et al, basic Methods In Molecular Biology (1986).

The polynucleotide encoding the fusion protein of the invention may be ligated to a vector containing a selectable marker for propagation in a host. In general, plasmid vectors can be introduced in a precipitate, such as a calcium phosphate precipitate or its complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and transduced into host cells.

The successfully transformed cells, i.e., cells containing the DNA recombinant vector of the invention, can be identified by well known techniques. For example, cells into which the recombinant vector is introduced may be cultured to produce the desired polypeptide. Cells were collected and lysed and their DNA content was assayed for the presence of DNA using methods as described in Southern, J.mol. Biol.1975, 98:503 or Berent et al, biotech.1985, 3:208. Alternatively, antibodies are used to detect the presence of proteins in the supernatant.

The fusion proteins of the invention are advantageously recovered and purified from recombinant cell culture by well known methods including sulfuric acid or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, hydrophobic charge interaction chromatography and lectin chromatography. In some embodiments, purification can be performed using High Performance Liquid Chromatography (HPLC).

In some embodiments, the fusion proteins of the invention may be purified using one or more of the chromatographic methods described above. In other embodiments, the fusion proteins of the invention may be purified using one or more of the following chromatography columns: q Sepharose FF column, SP Sepharose FF column, Q Sepharose High Performance column, blue Sepharose FF column, blue column, phenyl Sepharose FF column, DEAE Sepharose FF, ni-Chelating Sepharose FF column or Methyl column.

In addition, the fusion proteins of the present invention may be purified using the methods described in International publication No. WO00/44772 (incorporated herein by reference in its entirety). The method described therein can be easily adapted by a person skilled in the art for purification of the fusion protein of the invention. The fusion proteins of the invention may be recovered from products produced by recombinant techniques from prokaryotic or eukaryotic hosts including, for example, bacterial, yeast, higher plant, insect and mammalian cells.

Use of the same

The conjugates of the invention are useful as active ingredients in the treatment of various diseases caused by excessive cell proliferation, such as tumors, including but not limited to bone cancers, including Ewing's sarcoma, osteosarcoma, chondrosarcoma, etc.; brain and CNS tumors, including acoustic neuroma, neuroblastoma, neuroglioblastoma and other brain tumors, spinal cord tumors, breast cancer, colorectal cancer, and advanced colorectal adenocarcinoma; endocrine cancers including adrenocortical carcinoma, pancreatic cancer, pituitary cancer, thyroid cancer, parathyroid cancer, thymus cancer, and multiple endocrine tumors; gastrointestinal cancers including gastric cancer, esophageal cancer, small intestine cancer, liver cancer, extrahepatic bile duct cancer, gastrointestinal carcinoid tumor, and gallbladder cancer; genitourinary cancers including deltoid cancer, penile cancer, and prostate cancer; gynecological cancers including cervical cancer, ovarian cancer, vaginal cancer, uterus/endometrium cancer, pudendum cancer, gestational trophoblastic tumor, fallopian tube cancer, and uterine sarcoma; tumors of the head and neck, including oral cancer, lip cancer, salivary gland cancer, laryngeal cancer, hypopharyngeal cancer, orthoharyngeal cancer, nasal cancer, sinus cancer, and nasopharyngeal cancer; leukemia including childhood leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, acute promyelocytic leukemia, and plasma cell leukemia; bone marrow cancer blood disorders including bone marrow dysplasia syndrome, myeloproliferative disorders, aplastic anemia, fanconi anemia, idiopathic macroglobulinemia; lung cancer, including small cell lung cancer, non-small cell lung cancer; lymphomas including hodgkin's disease, non-hodgkin's lymphoma, cutaneous T-cell lymphoma, peripheral T-cell Lin Baliu, AIDS-related lymphoma; eye cancers including retinoblastoma and uveal melanoma; skin cancers including melanoma, non-melanoma skin cancer, merkel cell cancer; soft tissue sarcomas, such as pediatric soft tissue sarcoma, adult soft tissue sarcoma, kaposi's sarcoma; urinary system cancer including kidney cancer Wilms' tumor, skin cancer, urethra cancer or metastatic cell carcinoma.

The fusion protein disclosed by the invention can be used for treating cancers such as cervical cancer, breast cancer, colorectal cancer, bladder cancer or lung cancer.

Preferred neoplasms which may be treated by the fusion proteins of the invention are solid tumors and hematological malignancies.

The term "tumor" as used herein generally refers to a broad range of conditions characterized by uncontrolled abnormal growth of cells.

The effective dosage of the active ingredient used may vary with the mode of administration and the severity of the condition being treated. For most large mammals, the total dosage of the active ingredient is about 0.01-1000mg per day. Generally, the amount of clinical administration to an adult is in the range of 0.01-200 mg/day, preferably 0.05-100 mg/day.

An "effective dose" or "therapeutic amount" of the drug refers to an amount sufficient to produce a therapeutic effect. The effective amount may be administered in one or more divided doses. Generally, an effective amount is sufficient to alleviate, ameliorate, stabilize, slow or delay further progression of the disease.

Composition and method for producing the same

Compositions for use in the invention or comprising the fusion proteins of the invention. Generally, when the composition of the present invention is used for the above-mentioned uses, the fusion protein may be mixed with one or more pharmaceutically acceptable carriers or excipients to prepare pharmaceutical dosage forms for different administration routes, such as tablets, capsules, powders, granules, syrups, solutions, oral liquids, spirits, tinctures, aerosols, powder mists, injections, sterile powders for injection, suppositories and the like.

A "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. A "pharmaceutically acceptable carrier" is a pharmaceutically or food acceptable solvent, suspending agent or excipient for delivering the fusion protein of the invention to an animal or human. The carrier may be a liquid or a solid.

The fusion proteins of the invention may be administered via oral, intravenous, intramuscular or subcutaneous routes.

The dosage forms which can be orally administrated are as follows: tablets, capsules, powders, granules, syrups, solutions and spirits. The solid support comprises: starch, lactose, calcium hydrophosphate, microcrystalline cellulose, sucrose, kaolin, micro powder silica gel, talcum powder, low-substituted hydroxypropyl cellulose, sodium carboxymethyl starch and polyvinylpyrrolidone. And the liquid carrier comprises: sterile water, ethanol, polyethylene glycol, nonionic surfactants, and edible oils (e.g., corn, peanut, and sesame oils). Adjuvants commonly used in the preparation of pharmaceutical compositions include: flavoring agents, coloring agents, preservatives (e.g., oxybenzene alkyl butyl ester, sodium benzoate, sorbic acid) and antioxidants (e.g., vitamin E, vitamin C, sodium metabisulfite, and dibutyl hydroxytoluene).

Among the above dosage forms, those useful for administration by the injection route include: injectable, injectable sterile powders are prepared by mixing the drug with one or more pharmaceutically acceptable excipients for administration by injection. The solvent comprises: sterile water, ethanol, glycerol, propylene glycol, and polyethylene glycol. In addition, antibacterial agent (such as benzyl alcohol, butyl hydroxy benzoate, and merthiolate), isotonic regulator (such as sodium chloride and glucose), suspending agent (such as sodium carboxymethyl cellulose and methylcellulose), solubilizer (such as tween-80 and lecithin), antioxidant (such as vitamin E, vitamin C, and sodium metabisulfite), and filler (such as lactose and mannitol) can be added.

The preferred pharmaceutical compositions are solid compositions, especially lyophilized powder for injection, from the standpoint of ease of preparation and administration. Intravenous administration is preferred.

The invention is further illustrated below in conjunction with specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Proportions and percentages are by weight unless otherwise indicated

Herein, the polypeptide having the sequence shown in SEQ ID NO.1, abbreviated as ELBD or V16, represents the same polypeptide.

Example 1: construction of expression vectors

Construction of EGFP-Tn-CPP series expression vector

(1) Construction of EGFP-Tn-HBD series mutant expression vector

In chinese patent application No. CN 201310170530.6 we disclose a tumor cell targeting transmembrane peptide characterized by comprising a CPPs domain with cell transmembrane peptide activity and a VGF third loop-like artificial mimetic peptide domain with targeting tumor cells. In order to seek higher membrane penetrating efficiency and targeting, based on the polypeptide structure, a series of S3 simulated peptide-like structure mutant nucleotide sequences with BamHI and SalI enzyme cutting sites at two ends are synthesized by a DNA artificial solid phase synthesis method according to different mutation requirements, and the polypeptide sequences (SEQ ID NO. 1-11) shown in the table I are specifically listed.

TABLE 1 artificially synthesized VGF third loop sequence and partial mutant amino acid sequence

Starting from EGFP-T0-HBD expression plasmid (China patent application No. CN 201310170530.6, ESH in example 1), the original EGFP-T0-HBD-pET28a expression vector was digested simultaneously with BamHI and SalI, and ligated with the synthesized fragment using T4 DNA ligase.

E.coli DH5 alpha strain is transformed, the recombinant plasmid DNA is recovered, and sequencing verification is carried out, thus the series EGFP-Tn-HBD-pET28a expression vector is constructed, and n=1-12. Wherein for V16, specifically designated ELBD, the corresponding expression vector constructed was EGFP-ELBD-HBD-pET28a.

Meanwhile, constructing corresponding expression vectors of third loop sequences of other natural growth factors, namely EGFP-BTC-HBD-pET28a, EGFP-NGR 2-beta-HBD-pET 28a and EGFP-HGR 2-beta-HBD-pET 28a.

(2) Construction of EGFP-Tn-TAT series mutant expression vector

The DNA artificial solid phase synthesis method is adopted to synthesize the penetrating peptide TAT nucleotide sequences with Sal I and Xho I enzyme cleavage sites at the two ends, and the penetrating peptide TAT nucleotide sequences are respectively inserted into EGFP-Tn-HBD-pET28a series expression vectors which are recovered by Sal I+Xho I double enzyme cleavage in the embodiment, so as to construct corresponding EGFP-Tn-TAT-pET28a series expression vectors with TAT penetrating peptide.

(3) Construction of EGFP-ELBD-H2 expression vector

The EGFP-ELBD-HBD-pET28a plasmid is taken as a vector, DNA sequences with Sal I and Xho I enzyme cutting sites at two ends are synthesized by artificial solid phase, and the sequences are optimized by E.coli codons and code H2 membrane penetrating peptide, wherein H2 is another humanized membrane penetrating peptide and is derived from heparin growth factors. EGFP-Tn-HBD-pET28a expression vector was digested with Sal I and Xho I, and ligated with this synthesized fragment using T4 DNA ligase.

Transforming E.coli DH5 alpha strain, culturing overnight at 37 ℃ for 15 hours, purifying recombinant plasmid DNA by a plasmid DNA recovery kit, and carrying out sequencing verification by a DNA sequence sequencing company.

(4) Construction of EGFP-TAT expression vector

DNA sequences with BamHI and Xho I enzyme cutting sites at two ends are synthesized by adopting a DNA artificial solid phase synthesis method, and the sequences are subjected to codon optimization to code a membrane penetrating peptide TAT. The synthetic DNA fragment was inserted into EGFP-ELBD-HBD-pET28a expression vector which had been digested with BamHI and XhoI, transformed into E.coli DH 5. Alpha. Strain, cultured overnight at 37℃for 15h, and the plasmid DNA was purified using a plasmid DNA recovery kit and submitted to sequencing and verification by DNA sequencing company.

B. Construction of recombinant expression vectors of different antitumor protein medicines and ELBD-CPP

(1) Construction of TCS-ELBD-H2 expression vector

And (3) adopting an artificial solid phase to synthesize a DNA primer with BamHI and Xho I enzyme cutting sites, carrying out PCR amplification by taking the ELBD-H2 gene in the constructed plasmid EGFP-ELBD-H2-pET28a as a template, and adding the enzyme cutting sites BamHI and Xho I.

EGFP-ELBD-H2-pET28a is used as a template, and according to the ELBD-H2 gene sequence, the following primers are designed for PCR amplification, and the forward and reverse primers respectively introduce BamHI and Xho I restriction sites

Primer 1:5'-CGCGGATCCGGTGGTGGTGGTTCTGGTGGTGGTGGTT-3'

Primer 2:5'-CGCCTCGAGGTCTTTACCTTT-3'

PCR amplification reaction conditions: the reaction conditions for PCR amplification were: firstly, denaturation at 95 ℃ for 5min, then denaturation at 95 ℃ for 45s, renaturation at 58 ℃ for 30s, extension at 72 ℃ for 30s, circulation for 33 times, and finally, denaturation at 72 ℃ for 10min.

The amplified fragments were purified using a DNA fragment recovery kit and digested with Bam HI and Xho I to recover fragments. Meanwhile, the TCS-HBD-pET28b vector plasmid is digested with BamHI and XhoI, and the vector fragment is recovered. And ligated with T4 DNA ligase. Transforming E.coli DH5 alpha strain, culturing overnight at 37 ℃ for 15 hours, purifying recombinant plasmid DNA by a plasmid DNA recovery kit, and carrying out sequencing verification by a DNA sequence sequencing company. .

(2) Construction of MAP30-ELBD-H2 expression vector

Similar to the procedure described in the construction examples of the TCS-ELBD-H2 expression vector described above. The primers and PCR amplification conditions used are as described above.

Primers with BamHI and Xho I cleavage sites are synthesized, and PCR amplification is performed by taking the ELBD-H2 gene in the original plasmid EGFP-ELBD-H2-pET28a as a template, and the cleavage sites BamHI and Xho I are added.

The amplified fragment was purified using a DNA purification kit and digested with BamHI and Xho I to recover the fragment. Simultaneously, the MAP30-HBD-pET28b vector plasmid is digested with BamHI and Xho I, and the vector fragment is recovered. And ligated with T4 DNA ligase. Transforming E.coli DH5 alpha strain, purifying recombinant plasmid DNA by using plasmid DNA recovery kit, and carrying out sequencing verification by DNA sequence sequencing company.

Example 2: recombinant protein expression purification

Expression and purification of EGFP series recombinant proteins

(1) EGFP-Tn-HBD series recombinant protein expression purification

(1) EGFP-Tn-HBD-pET28a plasmid construction protocol is described in example 1. A single colony transformed with EGFP-Tn-HBD-pET28a plasmid was picked from a solid LB medium plate of the deposited strain, and cultured with shaking in 30ml of LB liquid medium containing kanamycin (10-50 mg/L) at 37℃until the OD600 became about 0.5-1.0.

(2) The bacterial culture solution is taken, and 1% of inoculation amount is inoculated into a certain volume of culture medium containing kanamycin (10-50 mg/L) with a certain concentration, and the expansion culture is continued until the OD600 is about 0.5-1.0.

(3) Adjusting the culture temperature to 15-20deg.C, adding appropriate amount of IPTG (0.1-10 MM) to induce target protein expression, continuously culturing for 10-20 hr, and centrifuging at 500-1000rpm to collect thallus.

(4) The collected cells were resuspended in 20mM Tris-HCl buffer (pH 8.0-8.5) and sonicated at a power (10-100 w). Centrifuging at high speed (4-10deg.C) at low temperature, discarding supernatant, and retaining inclusion body.

(5) Washing inclusion bodies with 1.0-0.5% Triton for 10-60min.

(6) The inclusion bodies were denatured with 6M-8M urea for 5-30min.

(7) And (3) dialyzing and renaturating, and sequentially reducing the urea content in the dialyzate until the urea concentration of the dialyzate is 0M.

According to the steps, the EGFP-Tn-HBD recombinant protein and the EGFP-ELBD-TAT recombinant protein are expressed and purified respectively.

(2) EGFP-ELBD-H2 recombinant protein expression purification

(1) EGFP-ELBD-H2-pET28a plasmid construction protocol is described in example 1. A single colony transformed with EGFP-ELBD-H2-pET28a plasmid was then picked from the solid LB medium plates of the deposited strain and grown in 30ml LB liquid medium containing kanamycin (10-50 mg/L) with shaking at 37℃until an OD600 of about 0.5-1.0 was reached.

(4) The collected cells were resuspended in 20mM Tris-HCl buffer (pH 8.0-8.5) and sonicated at a power (10-100 w). The supernatant containing the target protein was collected by high-speed centrifugation (8000-13400 rpm) at low temperature (4-10 ℃).

(5) And (3) performing affinity chromatography separation on the collected supernatant through a nickel chelating affinity chromatography column. Similar to most methods for purifying target proteins by nickel chelate affinity chromatography, gradient elution (20 mM imidazole elution, 200mM imidazole elution) is performed using imidazole buffers with different concentrations, and target protein fractions eluted with different concentrations of imidazole are collected.

(3) EGFP-TAT recombinant protein expression purification

(1) EGFP-TAT-pET28a plasmid construction protocol is described in example 1. A single colony transformed with EGFP-TAT-pET28a plasmid was picked from a solid LB medium plate of the deposited strain, and cultured with shaking in 30ml of LB liquid medium containing kanamycin (10-50 mg/L) at 37℃until the OD600 became about 0.5-1.0.

(3) Regulating the culture temperature to 15-20deg.C, adding appropriate amount of IPTG (0.1-10 mM) to induce target protein expression, culturing for 10-20 hr, and centrifuging at low speed to collect thallus.

(4) The collected cells were resuspended in 20mM Tris-HCl buffer (pH 8.0-8.5) and sonicated at a power (10-100 w). The supernatant containing the target protein was collected by high-speed centrifugation under low-temperature conditions.

(5) And (3) performing affinity chromatography separation on the collected supernatant through a nickel chelating affinity chromatography column. Similar to most methods for purifying target proteins by nickel chelate affinity chromatography, gradient elution (10 mM imidazole elution, 200mM imidazole elution, 1M imidazole elution) is performed with imidazole buffers having different concentrations, and target protein fractions eluted with different concentrations of imidazole are collected.

(4) EGFP-ELBD-TAT recombinant protein expression purification

(1) EGFP-ELBD-TAT-pET28a plasmid construction protocol is described in example 1. A single colony transformed with EGFP-ELBD-TAT-pET28a plasmid was picked from a solid LB medium plate of the deposited strain and cultured with shaking in 30ml of LB liquid medium containing kanamycin (10-50 mg/L) at 37℃until the OD600 was about 0.5-1.0.

(3) The culture temperature is adjusted to be in the range of 15 ℃ to 20 ℃, a proper amount of IPTG (0.1-10 mM) is added to induce the expression of target protein, the culture is continued for 10-20h, and the low-speed centrifugation at 500-1000rpm is carried out to collect the thalli.

(4) The collected cells were resuspended in 20mM Tris-HCl buffer (pH 8.0-8.5) and sonicated at a power (10-100 w). Collecting the supernatant containing the target protein by high-speed centrifugation at 4-10deg.C.

B. Expression and purification of different antitumor proteins and ELBD-CPP recombinant proteins

(1) TCS-ELBD-H2 recombinant protein expression purification

(1) The TCS-ELBD-H2-pET28b plasmid construction protocol is described in example 1. A single colony transformed with the TCS-ELBD-H2-pET28b plasmid was picked from a solid LB medium plate of the deposited strain and grown in 30ml of LB liquid medium containing kanamycin (10-50 mg/L) with shaking at 37℃until the OD600 was about 0.5-1.0.

(5) And (3) performing affinity chromatography separation on the collected supernatant through a nickel chelating affinity chromatography column. Similar to most methods for purifying target proteins by nickel chelate affinity chromatography, gradient elution (20 mM imidazole elution, 50mM imidazole elution, 200mM imidazole elution) was performed using imidazole buffers with different concentrations, and target protein fractions eluted with different concentrations of imidazole were collected.

(2) Expression and purification of MAP30-ELBD-H2 recombinant protein

(1) The MAP30-ELBD-H2-pET28b plasmid construction protocol is described in example 1. A single colony transformed with MAP30-ELBD-H2-pET28b plasmid was then picked from the solid LB medium plates of the deposited strain and grown in 30ml LB liquid medium containing kanamycin (10-50 mg/L) with shaking at 37℃until an OD600 of about 0.5-1.0 was reached.

(4) The collected cells were resuspended in 20mM Tris-HCl buffer (pH 8.0-8.5) and sonicated at a power (10-100 w). Collecting the supernatant containing the target protein by low-temperature high-speed centrifugation.

Example 3: mutant transmembrane efficiency study

A. Comparison of mutant transmembrane efficiency:

(1) Analysis of transmembrane action of EGFP-Tn recombinant protein with transmembrane peptide HBD

The recombinant protein sample concentration of 2 mu M is adopted, the sample and the tumor cells cultured in vitro are incubated for 12 hours, and the effects of various EGFP-Tn-HBD recombinant proteins on the Bcap membrane penetration of human breast cancer cells are analyzed and compared by a flow cytometry. At the same time, compared with the third loop sequence recombinant proteins (EGFP-BTC-HBD, EGFP-NRG 2-beta-HBD, EGFP-HRG-beta 2-HBD) derived from other natural growth factors

And incubating a plurality of recombinant mutant samples with human breast cancer cell Bcap cells cultured in vitro for 12 hours at a sample concentration of 2 mu M, and analyzing and observing the membrane penetrating efficiency of each mutant by adopting a laser confocal technology.

As a result, the effect of EGFP-ELBD-HBD on the membrane penetration of Bcap cells was found to be significantly higher than that of other natural growth factor-derived third loop sequence recombinant proteins (EGFP-BTC-HBD, EGFP-NGR 2-beta-HBD, EGFP-HGR 2-beta-HBD), FIG. 1A is a laser confocal result, and B is a flow cytometry detection result, wherein EGFP-ELBD-HBD is marked as E-V16-H, EGFP-BTC-HBD is marked as E-BTC-H, EGFP-NRG 2-beta-HBD is marked as E-NRG 2-beta-H, and EGFP-HRG-beta 2-HBD is marked as E-HRG-beta 2-H.

The film penetrating efficiency (figure 2) obtained by combining the sequence of Table 1 and laser confocal, flow cytometry experiments can be seen that the Cys position and the number of the putative ring have definite influence on the film penetrating result. The sequences V16-9, V16-3, V16-2, and V16-1 all had lower transmembrane effects than ELBD (V16). V16-1 is less effective than V16-3, V16-2, which is comparable to V16-9. This suggests that these three Cys have a significant impact on the tertiary structure of the mutant, and all may be involved in the loop formation process of this domain space structure, thus exerting a certain impact on the membrane-penetrating efficiency. From the results of the computer simulation analysis of the three-dimensional structure of ELBD using the Zhang Lab program, of all the simulated structures capable of looping, only two possible forms of C2-C10 and C2-C26 are available, and no form of C10-C26 is available, which indicates that C2 has a greater likelihood of participating in the looping process. Whereas the cyclization process involving only C2 is more similar to the natural third loop structure, highly conserved amino acid residues (Y-X-G-X-R) are contained within the loop.

V16-6 lacks the sequence YTGIRCSH compared with ELBD (V16), and the membrane penetration is greatly affected, which shows the importance of the sequence at the N-terminal to maintain the efficient membrane penetration synergy of ELBD (V16). It is also possible that the deletion of this sequence results in the deletion of a second Cys, thereby affecting the spatial organization of this domain into loops. Since it is unlikely that a loop will be formed between the second Cys and the third Cys as a result of computer modeling, it may be because a segment of the putatiyly formed alpha-helical sequence is located immediately between the two Cys, resulting in difficulty in disulfide bond formation therebetween.

From the results of V16-10, V16-4 and V16-5, it can be seen that the insertion sequence of the intermediate region that may form a loop directly affects the size and shape of the loop, and the orientation of the important conserved amino acid residues Y-X-G-X-R in space, resulting in a decrease in the efficiency of membrane penetration. Although Leu47 in the EGF carboxy-terminal region has been shown in the prior art to have an important effect on EGF binding to EGFR, removal of sequence V16-8 from the terminal VVL in our mutation study did not have a significant effect on the transmembrane effect of the mutant, suggesting that in the ELBD-H structure, the flexible C-terminal tail is not involved in the binding process to the receptor, while Cys, which has an important effect on the structure of the domain, and the Y-X-G-X-R sequence that binds directly to the receptor are critical factors, and the alpha-helical insert in the middle of the loop may be responsible for ensuring the correct orientation (optimal orientation) of the Y-X-G-X-R sequences on both sides thereof, making it advantageous for efficient recognition and binding to the ErbB receptor binding moiety. Meanwhile, from the above results, we thought that the sequence of ELBD could be optimized to RCSHYTGIRCSHGIYTGIRCQH

(2) Concentration and time dependence of mutant transmembrane action

Protein concentration gradients and different incubation times (0-12 h) were studied using different concentrations (0-2. Mu.M) of EGFP-ELBD-HBD recombinant protein. From the experimental results, the trend that the recombinant protein EGFP-ELBD-HBD has obviously improved membrane penetrating efficiency on human breast cancer cell Bcap cells is observed along with the extension of the incubation time and the improvement of the concentration of the recombinant protein, and the membrane penetrating efficiency of the recombinant protein is proved to have concentration and time dependence (A, B in FIG. 3 shows the concentration dependence and C shows the time dependence)

B. Broad-spectrum studies of targeting peptide ELBD:

in order to examine the universality of the recombinant protein as a tumor targeting peptide and the broad spectrum of the recombinant protein combined application of the recombinant protein and the transmembrane peptide, an ELBD sequence and a plurality of transmembrane peptides (CPP for short) such as HBD (a humanized transmembrane peptide derived from a human EC-SOD heparin binding domain, chinese patent ZL 200810044084.3), TAT (HIV-derived transmembrane peptide), HBP (heparin binding domain sequence in heparin-like growth factors, a humanized transmembrane peptide) and the like are fused, and the obtained recombinant protein sample is subjected to membrane penetration efficiency on Bcap cells by using a flow cytometer after 12h incubation by using a recombinant protein of 30 mu M EGFP-TAT,2 mu M E-ELBD-TAT,30 mu M EGFP-ELBD-HBD and 2 mu M EGFP-ELBD-HBD.

Results: the ELBD sequence as a targeting peptide can not only greatly improve the penetrating efficiency of HBD, but also improve the TAT of classical penetrating peptide, and is similar to HBP. The ELBD has the synergistic effect of improving the targeting membrane penetration effect on CPPs of different types and sources, and the ELBD sequence is proved to be capable of improving the membrane penetration efficiency of membrane penetration peptides of various sources and sequences to a great extent. (FIG. 4)

Cell selectivity of EGFP-ELBD-HBD recombinant proteins

11 cells were selected to study the tumor targeting characteristics of EGFP-ELBD-HBD recombinant proteins. The composition comprises 9 kinds of human tumor cells: heLa (human cervical cancer cells), bcap (human breast cancer cells), a549 (human lung cancer cells), a357 (human malignant melanoma), T24 (human bladder cancer cells), MGC-803 (human gastric cancer cells), 95D (human giant cell lung cancer cells), bxPC-3 (human pancreatic cancer cells), 5637 (human bladder cancer cells), and 2 normal human cells: MRC-5 (human embryonic lung fibroblasts), 293T (human kidney epithelial cells).

The concentration of the recombinant protein is 1 mu M, and the recombinant protein is incubated for 12 hours. The result shows that EGFP-ELBD-HBD recombinant proteins hardly obviously penetrate through membranes of MRC-5 and 293T normal cells; in contrast, the cells showed different degrees of membrane penetration in HeLa, bcap, A549, T24, A357 and the like (FIG. 5). This phenomenon may occur due to the different distribution of targeted receptors on the surface of different cancer cells, resulting in the recombinant protein EGFP-ELBD-HBD exhibiting different membrane penetration capabilities for different cancer cells. However, for normal cells, the membrane receptor, e.g., EGFR, is much lower than for cancer cells, and this intercellular property is critical for tumor selectivity of ELBD targeting peptides.

Example 4: ELISA method analysis of mutant and tumor cell surface binding ability

ELISA method is adopted to measure the binding strength of EGFP-S3-HBD (China patent application No. CN 201310170530.6) and EGFP-ELBD-HBD recombinant proteins with relatively high penetrating efficiency and the surface of human cervical cancer HeLa cells.

Human cervical cancer cells were grown at 1X10 ³ -1x10 ⁵ The density of individual cells/wells was inoculated into 96-well cell culture plates (with 24h of full 96-well plates as the actual operating concentration), incubated at 37℃for 24h, and washed 3 times with PBS for 15min each. Adding 50 μl/well of 0.1-0.25% glutaraldehyde precooled at 4deg.C, and fixing cells at 4deg.C for 10-45min; the fixed cells were washed 3 times with PBS for 15min each, blocked overnight at 4℃with 200. Mu.l/well of 1% BSA/PBS solution; washing with PBST buffer solution (PBS containing 0.05% Tween-20) for 3 times each for 15min; diluting the recombinant fusion protein according to a certain ratio, adding the diluted recombinant fusion protein into a 96-well plate, setting 3 parallel wells at each concentration of 50 μl/well, and incubating for 2h at 37 ℃; after 3 washes with PBST, murine anti-His-tag monoclonal antibody (l: 1000-l:3000 dilution), 50. Mu.l/well, was added and incubated for 2h at 37 ℃; plates were washed 3 times with PBST, horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (1:800-1:2500 dilution), 50 μl/well, and incubated at 37deg.C for 2h; the plate is washed 5 times by PBST, TMB substrate chromogenic solution is added, 100-200 mu l/hole is reacted for 10-30min at room temperature in a dark place. The reaction was stopped with 2mol/L sulfuric acid at 50. Mu.l/well, and the absorbance at 450nm was measured immediately on a microplate reader.

As can be seen from fig. 6, ELBD was able to bind to HeLa cell surface receptors at lower concentrations, indicating that ELBD sequences more readily bind to the cancer cell surface, which further promotes the transmembrane efficiency of recombinant proteins.

Example 5: improvement of pharmacological action of ELBD-CPP targeting transmembrane peptide on protein drugs:

TCS is a pharmaceutical protein derived from plant including tubers with antitumor activity, which has the activity of Ribosome Inactivating Protein (RIP), and which is capable of inhibiting protein synthesis in an in vitro cell-free system.

After fusion expression of the gene sequence of TCS and the ELBD mutant nucleotide sequence and the H2 transmembrane peptide sequence, the inhibition rate of TCS-ELBD-H2 on B16 (murine melanoma cells) is remarkably improved (figure 7).

MAP30 is also a protein drug with antitumor activity, which is derived from Momordica charantia seeds and is also a RIP protein. After the fusion expression with ELBD-H2, the inhibition rate of MAP30-ELBD-H2 on HeLa and B16 cells of tumor cells is obviously improved, but the inhibition rate of MAP30-ELBD-H2 on MRC-5 of normal cells is not obviously improved (figure 8).

The scope of the invention is not limited to the specific embodiments described, which are intended as single examples illustrating the various aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are also intended to fall within the scope of the appended claims. Each of the references mentioned above is incorporated by reference in its entirety.

SEQUENCE LISTING

<110> Zhejiangfuno medical Co., ltd

<120> tumor targeting polypeptide, preparation method and application thereof

<130> 20210601

<160> 15

<170> PatentIn version 3.3

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<213> artificial sequence

<400> 4

Arg Cys Ser His Tyr Thr Gly Ile Arg Cys Ser His Met Ala Ala Thr

1 5 10 15

Thr Ala Gly Ile Tyr Thr Gly Ile Arg Gly Gln His Val Val Leu

20 25 30

<210> 5

<211> 31

<212> PRT

<213> artificial sequence

<400> 5

Arg Cys Ser His Tyr Thr Gly Ile Arg Cys Ser Glu Ala Ala Ala Lys

1 5 10 15

Glu Ala Gly Ile Tyr Thr Gly Ile Arg Cys Gln His Val Val Leu

20 25 30

<210> 6

<211> 31

<212> PRT

<213> artificial sequence

<400> 6

Arg Cys Ser His Tyr Thr Gly Ile Arg Cys Ser Gly Gly Gly Gly Ser

1 5 10 15

Gly Ala Gly Ile Tyr Thr Gly Ile Arg Cys Gln His Val Val Leu

20 25 30

<210> 7

<211> 23

<212> PRT

<213> artificial sequence

<400> 7

Arg Cys Ser His Met Ala Ala Thr Thr Ala Gly Ile Tyr Thr Gly Ile

1 5 10 15

Arg Cys Gln His Val Val Leu

20

<210> 8

<211> 28

<212> PRT

<213> artificial sequence

<400> 8

Arg Cys Ser His Tyr Thr Gly Ile Arg Cys Ser His Met Ala Ala Thr

1 5 10 15

Thr Ala Gly Ile Tyr Thr Gly Ile Arg Cys Gln His

20 25

<210> 9

<211> 25

<212> PRT

<213> artificial sequence

<400> 9

Arg Cys Ser His Tyr Thr Gly Ile Arg Cys Ser His Gly Ile Tyr Thr

1 5 10 15

Gly Ile Arg Cys Gln His Val Val Leu

20 25

<210> 10

<211> 27

<212> PRT

<213> artificial sequence

<400> 10

Tyr Thr Gly Ile Arg Cys Ser His Met Ala Ala Thr Thr Ala Gly Ile

1 5 10 15

Tyr Thr Gly Ile Arg Cys Gln His Val Val Leu

20 25

<210> 11

<211> 31

<212> PRT

<213> artificial sequence

<400> 11

Arg Cys Ser His Tyr Thr Gly Ile Arg Cys Ser His Gly Ala Ala Ala

1 5 10 15

Ala Ala Gly Ile Tyr Thr Gly Ile Arg Cys Gln His Val Val Leu

20 25 30

<210> 12

<211> 21

<212> PRT

<213> artificial sequence

<400> 12

Lys Arg Lys Lys Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Cys

1 5 10 15

Leu Arg Lys Tyr Lys

20

<210> 13

<211> 56

<212> PRT

<213> artificial sequence

<400> 13

Arg Cys Ser His Tyr Thr Gly Ile Arg Cys Ser His Met Ala Ala Thr

1 5 10 15

Thr Ala Gly Ile Tyr Thr Gly Ile Arg Cys Gln His Val Val Leu Val

20 25 30

Asp Gly Gly Lys Arg Lys Lys Lys Gly Lys Gly Leu Gly Lys Lys Arg

35 40 45

Asp Pro Cys Leu Arg Lys Tyr Lys

50 55

<210> 14

<211> 53

<212> PRT

<213> artificial sequence

<400> 14

Arg Cys Ser His Tyr Thr Gly Ile Arg Cys Ser His Met Ala Ala Thr

1 5 10 15

Thr Ala Gly Ile Tyr Thr Gly Ile Arg Cys Gln His Val Asp Gly Gly

20 25 30

Lys Arg Lys Lys Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Cys

35 40 45

Leu Arg Lys Tyr Lys

50

<210> 15

<211> 50

<212> PRT

<213> artificial sequence

<400> 15

Arg Cys Ser His Tyr Thr Gly Ile Arg Cys Ser His Gly Ile Tyr Thr

1 5 10 15

Gly Ile Arg Cys Gln His Val Val Leu Val Asp Gly Gly Lys Arg Lys

20 25 30

Lys Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Cys Leu Arg Lys

35 40 45

Tyr Lys

50

Claims

1. A nucleic acid molecule encoding said tumor targeting polypeptide, wherein said tumor targeting polypeptide is a polypeptide having an amino acid sequence of one of SEQ ID No.1-4 or SEQ ID No.8-9 or RCSHYTGIRCSHGIYTGIRCQH.

2. An expression vector comprising the nucleic acid molecule of claim 1.

3. The expression vector of claim 2, wherein the expression vector is expressible in a cell.

4. A host cell comprising the nucleic acid molecule of claim 1.

5. A host cell comprising the expression vector of claim 2.

6. The host cell of claim 4, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

7. A method of producing a tumor targeting polypeptide comprising culturing the host cell of any one of claims 4-6, producing the tumor targeting polypeptide.