TWI486446B - Method for targeted chemotherapeutic drugs formation in the treatment of infectious and malignant diseases - Google Patents

Description

Method for forming target chemotherapeutic drugs for treating infectious and malignant diseases

The present invention relates to a fusion protein for use in combination with a prodrug to treat cancer.

The main feature of human epidermal cancer is the functional activation of growth factors and receptors in the epidermal growth factor receptor (EGFR) family. The signaling pathway dominated by the EGF-EGFR axis plays an important role in the proliferation, survival, metastasis and angiogenesis of cancer cells (Ciardiello, F., and Tortora, G. EGFR antagonists in cancer treatment, N Engl J Med 358 , 1160-1174, 2008.).

The relatively safe prodrug, 5-fluorocytosine (5-FC), can be converted to the commonly used chemotherapeutic drug 5-fluorouracil (5-FU) by cytosine deaminase. . Compared to 5-FC, 5-FU is 1000 times more toxic (Miller, CR, Williams, CR, Buchsbaum, DJ, and Gillespie, GYIntratumoral 5-fluorouracil produced by cytosine deaminase/5-fluorocytosine gene therapy is effective for experimental Human glioblastomas, Cancer Res 62 , 773-780, 2002; Hamstra, DA, Rice, DJ, Fahmy, S., Ross, BD, and Rehemtulla, A. Enzyme/prodrug therapy for head and neck cancer using a catalytically superior cytosine deaminase , Hum Gene Ther 10 , 1993-2003, 1999.). Many cancers with overexpressed EGFR, such as head and neck cancer, pancreatic cancer, colorectal cancer, etc., are often treated with 5-FU. However, the high systemic toxicity of 5-FU is a problem.

Therefore, a targeted prodrug/drug system is needed to concentrate the production of chemotherapeutic drugs at the location of the tumor.

The present invention provides a fusion protein for use in combination with a prodrug for treating cancer, wherein the fusion protein comprises: (i) a ligand selected from the group consisting of angiopoietin (Angiopoietin), a brain-derived neurotrophic factor ( Brain Derived Neurotrophic Factor, Ciliary Neurotrophic Factor, Epidermal Growth Factor, Fibroblast Growth Factor, Glial Derived Neurotrophic Factor, Hepatocyte Growth Factor, Heregulin, Insulin-like Growth Factor, Interleukin, Keratinocyte Growth Factor, Macrophage Inflammatory Protein (Macrophage Inflammatory Protein), Macrophage Chemoattractant Protein, Nerve growth factor, Neurotrophin, Platelet Derived Growth Factor, Pigment Epithelial-derived Factor Pigment Epithelium D Erived Factor), Platelet Factor, Stromal Cell Derived Factor, Stem Cell Factor, Tissue inhibitor of metalloproteinase, Transforming Growth Factor Factor), Tumor Necrosis Factor, and Vascular Endothelial Growth Factor group of growth factor receptor-specific binding; (ii) prodrug drug enzyme, which can be used as a precursor drug Converted to an active pharmaceutical ingredient; and (iii) a link between the ligand and the precursor drug enzyme.

In one aspect, the invention provides the use of a fusion protein as described herein in combination with a prodrug to prepare a pharmaceutical composition for treating cancer.

In another aspect, the invention provides a DNA construct comprising: (i) a nucleotide sequence encoding a ligand that specifically binds to a growth factor receptor, wherein the ligand is selected from the group consisting of angiopoietin, the brain Derived neurotrophic factor , ciliary neurotrophic factor, epidermal growth factor, fibroblast growth factor, glial-derived neurotrophic factor, hepatocyte growth factor, modulin, insulin-like growth factor, interleukin, keratinocyte growth factor, macrophage Inflammatory proteins, macrophage chemotactic proteins, nerve growth factors, neurotrophic factors, platelet-derived growth factors, pigment epithelium-derived factors, platelet factors, stromal cell-derived factors, stem cell factors, matrix metalloproteinase tissue inhibitors, transforming growth factors a group consisting of tumor necrosis factor and vascular endothelial growth factor; and (ii) a nucleotide sequence encoding a precursor drug enzyme.

Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Unless otherwise defined, the following terms have the meanings indicated when used herein.

The articles "a" or "the" as used herein mean that the grammatical term is one or greater than one (ie, at least one). For example, an element represents an element or is greater than an element.

An "individual" as used herein is any animal that has cancer or may have cancer, such as mammals and especially humans.

As used herein, the term "polypeptide" refers to a molecule or polymer consisting of amino acid residues linked by a peptide bond. The polypeptide can be synthesized using, for example, an automated polypeptide synthesizer. The "protein" used herein refers to a large-scale polypeptide. "Peptide" typically represents a shorter polypeptide.

As used herein, a "fusion protein" is a protein created by binding to a gene encoding two or more original proteins.

As used herein, "precursor drug" refers to a compound that must undergo chemical conversion of a precursor drug enzyme to become an active agent at the time of administration.

The "precursor drug enzyme" used herein is an enzyme capable of converting a precursor drug into an active drug.

The "bonder" used herein is a short fragment of a polypeptide.

The term "polynucleotide" or "nucleic acid" as used herein refers to a polymer composed of nucleotide units. Polynucleotides comprise naturally occurring nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as nucleic acid analogs, comprising non-naturally synthesized nucleic acids, such as recombinant polynucleotides. Polynucleotides can be synthesized using, for example, an automated DNA synthesizer. "Nucleic acid" B represents a large-scale polynucleotide. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), it also contains an RNA sequence (i.e., A, U, C, G), wherein U replaces T. The word "cDNA" refers to DNA that is complementary or identical to the mRNA, either in single or double stranded form.

As used herein, the term "encoding" refers to the intrinsic property of a particular nucleotide sequence (eg, gene, cDNA, or mRNA) in a polynucleotide as a model for other polymers and macromolecules in a biological process. A macromolecule has a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of an amino acid, and the resulting biological properties. Thus, if a gene produced by transcription of a gene is translated in a cell or other biological system to produce a protein, the gene encodes the protein. It will be understood by those skilled in the art that many different polynucleotides and nucleic acids can encode the same polypeptide as a result of degeneracy of the genetic code. It will also be appreciated that those skilled in the art can use conventionally used techniques to replace nucleotides without affecting the polypeptide sequence compiled by the polynucleotide, which reflects any particularity of the polypeptide to be expressed. Codon usage preferences of the host organism. Thus, unless otherwise specifically indicated, a "polynucleotide encoding an amino acid sequence" comprises any degenerate polynucleotide sequence that encodes the same amino acid sequence. The polynucleotide encoding the protein and RNA may comprise an intron.

As used herein, the term "recombinant polynucleotide" refers to a polynucleotide having non-naturally binding sequences. The recombinant polynucleotide may be in the form of a vector. A "vector" can comprise a nucleotide sequence of interest as well as a regulatory sequence. Vectors can be used to represent a given nucleotide sequence or to maintain a given nucleotide sequence to replicate, manipulate, or transfer between different positions (eg, between different organisms). The vector can be introduced into a suitable host cell for the above purposes.

Examples of vectors include, but are not limited to, plasmid, cosmid, YAC, or PAC. Typically, a given nucleotide sequence in a vector is operably linked to a regulatory sequence such that when the vector is introduced into a host cell, the given nucleotide sequence can be expressed in the host cell under the control of the regulatory sequence. . For example, a regulatory sequence can include a promoter, a start codon, a replication region sequence, an enhancer, an operator sequence, a sequestration signal. Sequences (eg, IL2 signal peptides) and other regulatory sequences. Preferably, the vector may further comprise a marker sequence (e.g., an antibiotic resistance marker sequence) to facilitate screening.

Amino acids can be expressed in three letters or one letter. Table 1 lists the standard amino acid abbreviations.

The invention features a fusion protein that binds to a prodrug to treat cancer, wherein the fusion protein comprises (i) a ligand that specifically binds to a growth factor receptor, which is selected from the group consisting of Angiopoietin, brain-derived neurotrophic factor, ciliary neurotrophic factor, epidermal growth factor, fibroblast growth factor, glial-derived neurotrophic factor, hepatocyte growth factor, modulating protein, insulin-like growth factor, interleukin, Keratinocyte growth factor, macrophage inflammatory protein, macrophage chemotactic protein, nerve growth factor, neurotrophic factor, platelet-derived growth factor, pigment epithelium-derived factor, platelet factor, stromal cell-derived factor, stem cell factor, matrix metalloprotein a group consisting of a tissue inhibitor of growth factor, a transforming growth factor, a tumor necrosis factor, and a vascular endothelial growth factor; (ii) a precursor drug enzyme that converts the precursor drug into an active pharmaceutical ingredient; and (iii) a chain, It is located between the ligand and the precursor drug enzyme.

According to an embodiment of the present invention, the fusion protein comprises a precursor drug enzyme selected from the group consisting of alcohol dehydrogenation hydrazine, alkaline strontium phosphate, beta amidoxime, beta glucuronide, carboxylesterase, carboxypeptide脢A, carboxypeptide 脢G2, cytosine deaminase, cytosine deaminase uracil phosphoribosyl transfer 脢, glycosides quinone, nitro-reductive quinone, penicillin amidoxime, thymidine group.

In an embodiment of the invention, the precursor drug enzyme is located at the amino terminus of the fusion protein, and the ligand is located at the carboxy terminus of the fusion protein.

In a particular embodiment of the invention, the system is epidermal growth factor (EGF) or vascular endothelial growth factor (VEGF). Specifically, the "ligand" used herein has an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (EGF) and SEQ ID NO: 2 to 5 (VEGF).

According to an embodiment, the prodrug used to treat cancer is 5-FC and the precursor The pharmaceutical enzyme is cytosine deaminase or cytosine deaminase fused to uracil phosphoribosyltransferase. Specifically, the "precursor drug enzyme" used herein has SEQ ID NO: 6 ("cytosine deaminase" or "Fcy") or SEQ ID NO: 7 ("cytosine deaminase fused to uracil phosphoribosyltransferase" Or the amino acid sequence of "Fcy-Fur".

In an embodiment of the invention, the fusion protein has a structure selected from the group consisting of SEQ ID NO: 8 (Fcy-hEGF), SEQ ID NO: 9 (Fcy-Fur-hEGF), SEQ ID NO: 10 (Fcy-(VPGVG) ₂ -hEGF-cMyc- KKKRKV), SEQ ID NO: 11 (Fcy-hVEGFa), SEQ ID NO: 12 (Fcy-Fur-hVEGFa), SEQ ID NO: 13 (Fcy-hEGF), SEQ ID NO: 14 (Fcy-Fur-hEGF), SEQ ID NO: 15 (Fcy-hVEGFa) ), SEQ ID NO: 16 (Fcy-Fur-hVEGFa), SEQ ID NO: 17 (Fcy-Fur-hVEGFc), SEQ ID NO: 18 (Fcy-mVEGFa), SEQ ID NO: 19 (Fcy-Fur-mVEGFa), and SEQ ID NO: 20 (Fcy) -Fur-mVEGFc) The amino acid sequence of the group consisting of.

In another aspect, the invention provides the use of a fusion protein described herein in combination with a prodrug to prepare a pharmaceutical composition for treating cancer.

In still another aspect, the present invention provides a DNA construct comprising: (i) a nucleotide sequence encoding a ligand that specifically binds to a growth factor receptor, wherein the ligand system is selected from the group consisting of angiopoietin, a brain Derived neurotrophic factor, ciliary neurotrophic factor, epidermal growth factor, fibroblast growth factor, glial-derived neurotrophic factor, hepatocyte growth factor, modulating protein, insulin-like growth factor, interleukin, keratinocyte growth factor, Macrophage inflammatory protein, macrophage chemotactic protein, nerve growth factor, neurotrophic factor, platelet-derived growth factor, pigment epithelium-derived factor, platelet factor, stromal cell-derived factor, stem cell factor, matrix metalloproteinase tissue inhibitory factor, a group consisting of a transforming growth factor, a tumor necrosis factor, and a vascular endothelial growth factor; and (ii) a nucleotide sequence encoding a precursor drug enzyme.

According to another embodiment of the present invention, the DNA construct comprises a nucleotide sequence encoding a precursor drug enzyme selected from the group consisting of alcohol dehydrogenation hydrazine, alkaline strontium phosphate, beta-indole oxime, and bis-glucuronide gluconate. Carboxylesterase, carboxypeptide 脢A, carboxypeptide 脢G2, cytosine deaminase, cytosine deaminase uracil phosphoribosyl transfer 脢, glycosides quinone, nitro-reductive quinone, penicillin amidoxime, thymidine group.

In a particular embodiment of the invention, the nucleotide sequence encoding a ligand that specifically binds to a growth factor receptor is selected from the group consisting of SEQ ID NO: 21 (EGF) and SEQ ID NO: 22 to 25 (VEGF).

In a particular embodiment of the invention, the nucleotide sequence encoding the prodrug pharmacological enzyme is SEQ ID NO: 26 (Fcy) or SEQ ID NO: 27 (Fcy-Fur).

In an embodiment of the invention, the DNA construct of the invention has a sequence selected from SEQ ID NO: 28 (Fcy-hEGF), SEQ ID NO: 29 (Fcy-Fur-hEGF), SEQ ID NO: 30 (Fcy-(VPGVG) ₂ -hEGF- cMyc-KKKRKV), SEQ ID NO: 31 (Fcy-hVEGFa), SEQ ID NO: 32 (Fcy-Fur-hVEGFa), SEQ ID NO: 33 (Fcy-hEGF), SEQ ID NO: 34 (Fcy-Fur-hEGF), SEQ ID NO: 35 (Fcy -hVEGFa), SEQ ID NO: 36 (Fcy-Fur-hVEGFa), SEQ ID NO: 37 (Fcy-Fur-hVEGFc), SEQ ID NO: 38 (Fcy-mVEGFa), SEQ ID NO: 39 (Fcy-Fur-mVEGFa), and SEQ ID NO: 40 The nucleotide sequence of the group consisting of (Fcy-Fur-mVEGFc).

The invention will be further illustrated by the following examples, which are provided for purposes of illustration and not limitation.

Example 1: Fcy-hEGF-myc-his in yeast expression vector ₆ , Fcy-Fur-hEGF-myc-his ₆ , hEGF-myc-his ₆ And Fcy-myc-his ₆ DNA selection

The gene sequences expressing Fcy and Fur were amplified using PCR using a cDNA gene library derived from yeast, and the human EGF and VEGF lines were used as a template using a cDNA gene library derived from the human cancer cell line SKOV3-ip1 and subjected to PCR. amplification. The PCR product was treated with restriction 脢BamHI and EcoRI, and the target sequence was introduced by PCR primer and ligated to the protein expression vector pPICZ-α cleaved with the same restriction 脢BamHH and EcoRI. Fcy and hEGF are individually selected into this vector, which is located after the α-secreting signal peptide at the amino terminus, wherein the carboxy terminus is c-myc and six which have been built into the pPICZ-α vector. The histidine (myc-his ₆ ) tag facilitates protein identification and purification. Another PCR product of Fcy has a BamHI recognition sequence at both the 5' and 3' ends, and this product was cleaved with BamHI and ligated with the pPIC-α-hEGF-myc-his ₆ construct also cleaved with BamHI, pPIC- The α-hEGF-myc-his ₆ was treated with calf intestinal alkaline strontium phosphate (CIAP) after cutting with BamHI to avoid re-engagement of the vector. Schematic diagrams of Fcy-hEGF-myc-his ₆ , Fcy-Fur-hEGF-myc-his ₆ , hEGF-myc-his ₆ and Fcy-myc-his ₆ in the yeast expression vector pPICZ-α are shown in Fig. 1. Figure 2 shows (a) pPICZ-α-Fcy-hEGF-myc-his ₆ (4198 bp), (b) pPICZ-α-Fcy-Fur-hEGF-myc-his ₆ (4951 bp), (c) against yeast The performance-optimized construct pPICZ-α-Fcy-(VPGVG) ₂ -hEGF-cMyc-KKKRKV-6His (4213 bp), (d) pPICZ-α-Fcy-hVEGF-myc-his ₆ (4384 bp), and (e) A map of the nucleotide and amino acid sequences obtained by pPICZ-α-Fcy-Fur-hVEGF-myc-his6 (5137 bp).

Example 2: Fcy-hEGF-myc-his in a bacterial expression vector ₆ , Fcy-Fur-hEGF-myc-his ₆ , hEGF-myc-his ₆ And Fcy-myc-his ₆ DNA selection

To investigate protein expression in hosts other than yeast, we constructed growth factors such as EGF or VEGF that fuse with Fcy or Fcy-Fur. Figure 3 shows Fcy-hEGF (or VEGF)-his ₆ , Fcy-Fur-hEGF (or VEGF)-his ₆ , hEGF (or VEGF)-his ₆ , and Fcy-his ₆ fusion in the E. coli expression vector pET56 Schematic representation of the gene. Similar to the selection strategy of the yeast expression construct, the desired fusion gene was amplified by PCR and cleaved with different sets of restriction 脢NcoI and XhoI. The cleaved fragments were colonized into vector pET56 cut with the same restriction. The resulting construct is shown in Figure 4.

Example 3: Fcy-hEGF-myc-his ₆ , hEGF-myc-his ₆ And Fcy-myc-his ₆ Performance and purification

Fcy-hEGF-myc-his ₆ , hEGF-myc-his ₆ , or Fcy-myc-his ₆ lines in the pPICZ-α construct were transformed into wild-type X-33 P. pastoris (Pichia) In pastoris), it was cultured for 3 to 4 days on a dish containing zeocin antibiotics (200 μg/mL) until colonies appeared. Each colony with high protein expression was screened by anti-c-myc antibody detection. For the performance of a large number of the selected colonies was inoculated in a medium containing 0.5 liters of BMD shake flasks, grown to an OD ₆₀₀ of 8-10, and the daily addition of methanol to induce protein expression. After 3 days of induction, the medium containing the protein was collected and filtered prior to injection into a nickel-resin column (Qiagen). The column was washed with 10 column volumes and 5 mM imidazole in PBS, and the concentration of imidazole was increased using ÄKTAprime plus purification system to wash out the protein. This protein was identified by Coomassie blue stained SDS-PAGE gel (Figure 5, right) and western blot analysis using c-myc specific antibodies (Figure 5, left).

Example 4: Fcy-hEGF-myc-his ₆ , hEGF-myc-his ₆ And Fcy-myc-his ₆ In vitro binding to purified EGFR

Figure 6 shows a schematic diagram measuring the binding of Fcy-hEGF-myc-his ₆ to purified EGFR immobilized on an ELISA plate in vitro. Fcy-hEGF-myc-his ₆ , hEGF-myc-his ₆ , and Fcy-myc-his ₆ in vitro binding to purified EGFR immobilized on ELISA plates using anti-His labeled HRP (horseradish peroxide) ₆ antibody measurements. The binding affinities of Fcy-hEGF-myc-his ₆ and hEGF-myc-his ₆ to EGFR were 5 nM and 9 nM, respectively, while Fcy-myc-his ₆ and EGFR were not recognized for binding (Fig. 6, right). .

Example 5: Fcy-hEGF-myc-his ₆ , hEGF-myc-his ₆ And Fcy-myc-his ₆ Binding cells with different EGFR expression levels

The amount of EGFR expression in A431, MDA-468, MDA-231, and MCF-7 cells was analyzed by fluorescence activated cell sorter (FACS). The cell surface was stained with the anti-EGFR antibody cetuximab (erbitux), which was then bound to an anti-human IgG antibody labeled with FITC. In order to test the binding ability of purified Fcy-hEGF-myc-his ₆ , hEGF-myc-his ₆ , and Fcy-myc-his ₆ to EGFR, A431, MCF-7, MDA-468, and MDA-231 were respectively It was co-cultured with the protein labeled with his ₆ for 1 hour. The cells were later co-cultured with a His ₆ specific antibody labeled with FITC, followed by FACS analysis. Fluorescence intensity represents the amount of EGFR detected by cetuximab or the amount of protein labeled with his _{6 in} cancer cells (see Figure 7).

Example 6: Fcy-hEGF-myc-his ₆ With Fcy-myc-his ₆ In vitro enzyme activity

The enzyme activity of Fcy-hEGF and Fcy was measured by measuring the yield of 5-FU. Add 50 nanomole of Fcy-hEGF-myc-his ₆ (1.25 mg) or Fcy-myc-his ₆ (0.75 mg) to 37 ° C with increasing 5-FC concentrations (0, 0.03, 0.1, 0.3, 1, and 3 mM) In a 0.3 ml PBS, the 5-FC was converted to 5-FU. 2 μl of the reaction was taken every 3 minutes and the fluorescence intensity of 5-FC and 5-FU was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific). The concentration of 5-FU was calculated using absorbance values at 255 nm and 290 nm using the formula deduced by Senter et al.: [5-FU] mM = 0.185 x A255-0.049 x A290 (Senter, PD, Su, PC, Katsuragi T., Sakai, T., Cosand, WL, Hellstrom, I., and Hellstrom, KE (1991) Generation of 5-fluorouracil from 5-fluorocytosine by monoclonal antibody-cytosine deaminase conjugates, Bioconjug Chem 2, 447-451). The 5-FU production rate (V) at different concentrations of 5-FC was adapted to the Michaelis-Menten equation using Graphpad prism 5 software (San Diego, USA), as shown in FIG. The km values of purified Fcy-hEGF and Fcy protein to 5-FC were 0.25 and 0.49 mM, respectively. The Vmax generated by 5-FU was 177 and 173 min ^-1 for Fcy-hEGF and Fcy protein, respectively.

Example 7: MTT assay for measuring cell viability of A431, MDA-468, HUVEC, MDA-231, and MCF-7 treated with Fcy-hEGF and 5-FC

To verify the effect of 5-FC/Fcy-hEGF-myc-his ₆ on cell viability, A431, MDA-468, HUVEC, MDA-231, and MCF-7 were present in the presence of 0.1-mg/ml 5-FC. Increasing concentrations of Fcy-hEGF-myc-his ₆ co-culture (Figures 9a and 9d), while another experiment used a fixed amount of Fcy-hEGF-myc-his ₆ (0.2 μg/ml) with increasing concentrations of 5-FC (0-1 mg/ml) (Fig. 9b and Fig. 9c). After adding the protein to 5-FC for 3 days, the cells cultured in the 96-well culture plate were analyzed for survival rate by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). 25 μl of MTT solution (5 mg/ml in PBS) was added to a 96-well plate. After 2 hours of action, the MTT solution was removed, the cells were washed with PBS, and then 0.1 ml of extraction buffer (20% sodium dodecyl sulfate in 50% dimethylformamide) was added. After 4 hours at 37 ° C, the absorbance was measured at 570 nm using a disc reader (Bio-Rad) and the extraction buffer was used as a control. Compared to the low EGFR expression HUVEC, MDA-231, and MCF-7,5-FC lower and Fcy-hEGF-myc-his ₆ for EGFR overexpression of the MDA-468 and A431 IC ₅₀ (see FIG. 10 ).

Example 8: MTT assay for measuring cell viability of EGFR-causing colorectal cancer cells LS174T and HCT116 inhibited by Fcy-hEGF and 5-FC

Similar to Example 7, the cell viability of EGFR colorectal cancer cells LS174T and HCT116, which were affected by Fcy-hEGF (0.2 μg/ml) and increasing concentrations of 5-FC, was measured by the MTT assay. When the IC _{50 of} 5-FC was 2.5 μg/ml (10 μM) and 6.0 μg/ml (24 μM), a significant effect was observed for LS174T and HCT116 cells.

Example 9: Fcy-hEGF/5-FC inhibits the growth of HCT116 in vivo

Animal experiments were performed using colorectal cancer cell HCT116 overexpressing EGFR. The cells were subcutaneously inoculated into the balb/c nude mice and then bilaterally dorsal. When tumors reached a length of 3-5 mm after 2 weeks, mice were treated with 20 μg of Fcy or Fcy-hEGF every 3 days. Mice were injected with 10 mg of 5-FC (500 mg/kg) daily. The tumor sizes of the two treatment groups are shown in Fig. 12. Fcy-hEGF showed better antitumor effect compared to Fcy ( p = 0.03).

Example 10: Schematic diagram of cancer target precursor drug-growth factor fusion protein

In addition to the constructs shown in Figures 1 to 4, a DNA construct comprising a growth factor and a precursor drug enzyme and a protein thereof as shown in Figure 13 can also be constructed. The DNA sequence of the "growth factor" portion can be selected from the group consisting of angiopoietin, brain-derived neurotrophic factor, ciliary neurotrophic factor, epidermal growth factor, fibroblast growth factor, glial-derived neurotrophic factor, hepatocyte growth factor. , protein, insulin-like growth factor, interleukin, keratin Cell growth factor, macrophage inflammatory protein, macrophage chemotactic protein, nerve growth factor, neurotrophic factor, platelet-derived growth factor, pigment epithelium-derived factor, platelet factor, stromal cell-derived factor, stem cell factor, matrix metalloproteinase Any of a group consisting of tissue inhibitory factor, transforming growth factor, tumor necrosis factor, and vascular endothelial growth factor. The DNA sequence of the "precursor drug enzyme" portion may be selected from the group consisting of dehydrogenated alcohol, alkaline strontium phosphate, beta-ammonium glucoside, bismuth glucoside, carboxylesterase, carboxypeptide 脢A, carboxypeptide 脢G2, cytosine deaminase, cytosine deaminase uracil phosphoribosyl transfer 脢 (Fcy-Fur in yeast), glycosides, nitro-reductive quinone, penicillin amidoxime, and thymidine Any of the groups.

The embodiments are shown in the drawings to illustrate the invention. However, it should be understood that the invention is not limited to the preferred embodiments shown herein. In the schema:

Figure 1 is a schematic representation of the yeast expression constructs of the present invention.

Figure 2a shows the DNA construct and sequence (4198 bp) of pPICZ-α-Fcy-hEGF-myc-his6.

Figure 2b shows the DNA construct and sequence (4951 bp) of pPICZ-α-Fcy-Fur-hEGF-myc-his6.

Figure 2c is a DNA construct and sequence of pPICZ-α-Fcy-(VPGVG)2-hEGF-cMyc-KKKRKV-6His (4213 bp) optimized for yeast expression hosts.

Figure 2d shows the DNA construct and sequence (4384 bp) of pPICZ-α-Fcy-hVEGFa-myc-his6.

Figure 2e is the DNA construct and sequence (5137 bp) of pPICZ-α-Fcy-Fur-hVEGFa-myc-his6.

Figure 3 is a schematic representation of the E. coli expression constructs of the present invention.

Figure 4a shows the DNA construct and sequence of pET56-Fcy-hEGF-His6 (5785 bp).

Figure 4b shows the DNA construct and sequence (6538 bp) of pET56-Fcy-Fur-hEGF-His6.

Figure 4c shows the DNA construct and sequence of pET56-Fcy-hVEGFa-His6 (5971 bp).

Figure 4d shows the DNA construct and sequence of pET56-Fcy-Fur-hVEGFa-His6 (6742 bp).

Figure 4e shows the DNA construct and sequence (6685 bp) of pET56-Fcy-Fur-hVEGFc-His6.

Figure 4f is the DNA construct and sequence (5977 bp) of pET56-Fcy-mVEGFa-His6.

Figure 4g shows the DNA construct and sequence (6730 bp) of pET56-Fcy-Fur-mVEGFa-His6.

Figure 4h is the DNA construct of pET56-Fcy-Fur-mVEGFc-His6 and the amino acid sequence (6685 bp).

Figure 5 shows the purification of the his ₆ tag and the result of Fcy stained gel of the western blot, hEGF Fcy-hEGF Coomassie blue (Coomassie blue).

Figure 6 shows (a) schematic protein was purified by immobilized epidermal growth factor receptor (EGFR) in the in vitro binding, and (b) his Fcy _6-tagged, of hEGF, and Fcy-hEGF binding to the EGFR in vitro Saturation curves and affinity.

Figure 7 shows the results of flow cytometry for evaluation of anti-EGFR antibodies, purified his _6- tagged Fcy, hEGF, and Fcy-hEGF with A431 (a), MCF-7 (b), MDA-468 (c And the degree of binding of MDA-231(d) cells.

Figure 8 shows the results of enzyme activity for converting 5-FC to 5-FU in vitro by purified Fcy and Fcy-hEGF.

Figure 9 shows the results of the MTT assay for assessing A431, MDA-468, MDA-231, MCF-7, and the effects of Fcy-hEGF and 5-FC. Cell viability of HUVEC.

Figure 10 shows the results of enhancing 5-FC cytotoxicity by Fcy-EGF.

Figure 11 shows the results of MTT assays to assess cell viability of HCT116 and LS174T affected by Fcy-hEGF in the presence of increasing concentrations of 5-FC.

Figure 12 shows the inhibitory effect of 5-FC-bound Fcy-hEGF on the growth of HCT116 colorectal cancer in vivo.

Figure 13 is a selection strategy for a cancer target prodrug drug-growth factor fusion construct, wherein the DNA sequence of the "growth factor" portion can encode any one selected from the group consisting of angiopoietin ( Angiopoietin), Brain Derived Neurotrophic Factor, Ciliary Neurotrophic Factor, Epidermal Growth Factor, Fibroblast Growth Factor, Glial-derived Neurons Glial Derived Neurotrophic Factor, Hepatocyte Growth Factor, Heregulin, Insulin-like Growth Factor, Interleukin, Keratinocyte Growth Factor), Macrophage Inflammatory Protein, Macrophage Chemoattractant Protein, Nerve growth factor, Neurotrophin, Platelet Derived Growth Factor) Pigment Epithelium Derived Factor, Platelet Factor, Stromal Cell Derived Factor, Stem Cell Factor, Tissue inhibitor of metalloproteinase, Transformation The transforming growth factor, the tumor necrosis factor (Tumor Necrosis Factor), and the Vascular Endothelial Growth Factor; and the DNA sequence of the "prodrug drug" moiety can be selected from the group consisting of the following proteins. Either: Alcohol dehydrogenase, alkaline phosphoric acid Alkaline phosphatase, beta-lactamase, beta-glucoronidase, carboxyesterases, Carboxypeptidase A, carboxypeptide G2 (Carboxypeptidase G2), Cytosine deaminase, Cytosine deaminase-uracil phosphoribosyltransferase, Glycosidases, Nitroreductase, Penicillin Penicillin amidase, thymidine kinase.

<110> Blue 耿立<120> Method for the formation of target chemotherapeutic drugs for the treatment of infectious and malignant diseases <130> LKL0003CN <150> 61/492,649 <151> 2011-06-02 <160> 40 <170> PatentIn Version 3.5 <210> 1 <211> 56 <212> PRT <213> Homo sapiens <400> 1 <210> 2 <211> 121 <212> PRT <213> Homo sapiens <400> 2 <210> 3 <211> 105 <212> PRT <213> Homo sapiens <400> 3 <210> 4 <211> 120 <212> PRT <213> Home Mole <400> 4 <210> 5 <211> 105 <212> PRT <213> Home Mole <400> 5 <210> 6 <211> 158 <212> PRT <213> S. cerevisiae <400> 6 <210> 7 <211> 409 <212> PRT <213> S. cerevisiae <400> 7 <210> 8 <211> 219 <212> PRT <213> Artificial sequence <220><223> Fcy-hEGF <400> 8 <210> 9 <211> 470 <212> PRT <213> Artificial sequence <220><223> Fcy-Fur-hEGF <400> 9 <210> 10 <211> 250 <212> PRT <213> Artificial sequence <220><223> Fcy-(VPGVG)2-hEGF-cMyc-KKKRKV <400> 10 <210> 11 <211> 284 <212> PRT <213> Artificial sequence <220><223> Fcy-hVEGFa <400> 11 <210> 12 <211> 535 <212> PRT <213> Artificial sequence <220><223> Fcy-Fur-hVEGFa <400> 12 <210> 13 <211> 219 <212> PRT <213> Artificial sequence <220><223> Fcy-hEGF <400> 13 <210> 14 <211> 470 <212> PRT <213> Artificial sequence <220><223> Fcy-Fur-hEGF <400> 14 <210> 15 <211> 284 <212> PRT <213> Artificial sequence <220><223> Fcy-hVEGFa <400> 15 <210> 16 <211> 535 <212> PRT <213> Artificial sequence <220><223> Fcy-Fur-hVEGFa <400> 16 <210> 17 <211> 519 <212> PRT <213> Artificial sequence <220><223> Fcy-Fur-hVEGFc <400> 17 <210> 18 <211> 283 <212> PRT <213> Artificial sequence <220><223> Fcy-mVEGFa <400> 18 <210> 19 <211> 534 <212> PRT <213> Artificial sequence <220><223> Fcy-Fur-mVEGFa <400> 19 <210> 20 <211> 519 <212> PRT <213> Artificial sequence <220><223> Fcy-Fur-mVEGFc <400> 20 <210> 21 <211> 168 <212> DNA <213> Homo sapiens <400> 21 <210> 22 <211> 363 <212> DNA <213> Homo sapiens <400> 22 <210> 23 <211> 315 <212> DNA <213> Homo sapiens <400> 23 <210> 24 <211> 360 <212> DNA <213> House Mole <400> 24 <210> 25 <211> 315 <212> DNA <213> House Mole <400> 25 <210> 26 <211> 474 <212> DNA <213> Saccharomyces Cerevisiae <400> 26 <210> 27 <211> 1227 <212> DNA <213> S. cerevisiae <400> 27 <210> 28 <211> 657 <212> DNA <213> Artificial sequence <220><223> Fcy-hEGF <400> 28 <210> 29 <211> 1410 <212> DNA <213> Artificial sequence <220><223> Fcy-Fur-hEGF <400> 29 <210> 30 <211> 750 <212> DNA <213> Artificial sequence <220><223> Fcy-(VPGVG)2-hEGF-cMyc-KKKRKV <400> 30 <210> 31 <211> 852 <212> DNA <213> Artificial sequence <220><223> Fcy-hVEGFa <400> 31 <210> 32 <211> 1605 <212> DNA <213> Artificial sequence <220><223> Fcy-Fur-hVEGFa <400> 32 <210> 33 <211> 657 <212> DNA <213> Artificial sequence <220><223> Fcy-hEGF <400> 33 <210> 34 <211> 1410 <212> DNA <213> Artificial sequence <220><223> Fcy-Fur-hEGF <400> 34 <210> 35 <211> 852 <212> DNA <213> Artificial sequence <220><223> Fcy-hVEGFa <400> 35 <210> 36 <211> 1605 <212> DNA <213> Artificial sequence <220><223> Fcy-Fur-hVEGFa <400> 36 <210> 37 <211> 1557 <212> DNA <213> Artificial sequence <220><223> Fcy-Fur-hVEGFc <400> 37 <210> 38 <211> 849 <212> DNA <213> Artificial sequence <220><223> Fcy-mVEGFa <400> 38 <210> 39 <211> 1602 <212> DNA <213> Artificial sequence <220><223> Fcy-Fur-mVEGFa <400> 39 <210> 40 <211> 1557 <212> DNA <213> Artificial sequence <220><223> Fcy-Fur-mVEGFc <400> 40

Claims (16)

A fusion protein for treating cancer, which is used in combination with a cancer treatment pre-drug, wherein the fusion protein comprises: (i) a ligand selected from the group consisting of Epidermal Growth Factor, fibroblast growth factor (Fibroblast Growth Factor), Hepatocyte Growth Factor, Insulin-like Growth Factor, Macrophage Inflammatory Protein, Platelet Derived Growth Factor, Growth factor receptor specificity of the group consisting of Stromal Cell Derived Factor, Stem Cell Factor, Transforming Growth Factor, and Vascular Endothelial Growth Factor Sexual binding; (ii) a precursor drug enzyme that converts the precursor drug into an active pharmaceutical ingredient; and (iii) a linker between the ligand and the precursor drug enzyme. The fusion protein according to claim 1, wherein the precursor drug enzyme is selected from the group consisting of Alcohol dehydrogenase, Alkaline phosphatase, β-lactamase, and B. Β-glucoronidase, Carboxyesterases, Carboxypeptidase A, Carboxypeptidase G2, Cytosine deaminase, Cytosine Cytosine deaminase-uracil phosphoribosyltransferase, Glycosidases, Nitroreductase, Penicillin amidase, thymidine kinase Group. The fusion protein according to claim 1, wherein the precursor drug enzyme is located at the amino terminus of the fusion protein, and the ligand is located at the carboxy terminus of the fusion protein. A fusion protein according to the first aspect of the patent application, wherein the ligand system is epidermal growth factor (EGF). The fusion protein according to claim 1, wherein the ligand is vascular endothelial growth factor (VEGF). The fusion protein according to claim 1, wherein the drug for the treatment of cancer is 5-fluorocytosine, and the precursor drug enzyme is cytosine deaminase or Cytosine deaminase fused with uracil phosphoribosyltransferase. A fusion protein according to claim 4, wherein the ligand has the amino acid sequence of SEQ ID NO: 1. The fusion protein according to claim 5, wherein the ligand has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5. A fusion protein according to claim 6 wherein the precursor drug enzyme has the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7. The fusion protein according to claim 3, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 8 to 20. A use of a fusion protein of claim 1 in combination with a prodrug for the preparation of a pharmaceutical composition for treating cancer. A DNA construct comprising: (i) a nucleotide sequence encoding a ligand that specifically binds to a growth factor receptor, wherein the ligand is selected from the group consisting of epidermal growth factor, fibroblast growth factor, hepatocyte growth factor a group consisting of insulin-like growth factor, macrophage inflammatory protein, platelet-derived growth factor, stromal cell-derived factor, stem cell factor, transforming growth factor, and vascular endothelial growth factor; and (ii) a prodrug-enhancing drug enzyme Nucleotide sequence. The DNA construct according to claim 12, wherein the precursor drug enzyme is selected from the group consisting of alcohol dehydrogenation hydrazine, alkaline strontium phosphate, beta amidoxime, beta glucuronide, carboxylesterase, carboxy Peptide 脢A, carboxypeptide 脢G2, cytosine deamination A group consisting of an enzyme, a cytosine deaminase, a uracil phosphoribosyltransferase, a glycoside glucoside, a nitro-reductive quinone, a penicillin amidoxime, and a thymidine. The DNA construct according to claim 12, wherein the nucleotide sequence encoding the ligand which specifically binds to the growth factor receptor is selected from the group consisting of SEQ ID NOs: 21 to 25. A DNA construct according to claim 13 wherein the nucleotide sequence encoding the precursor drug enzyme is SEQ ID NO: 26 or SEQ ID NO: 27. The DNA construct according to claim 12, wherein the DNA construct has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 28 to 40.