WO2008064540A1

WO2008064540A1 - Compositions comprising adeno-associated virus-mediated anti-angiogenic factor-hgfk1 and uses thereof

Info

Publication number: WO2008064540A1
Application number: PCT/CN2007/001343
Authority: WO
Inventors: Marie C. M. Lin; Zan Shen; Hsiang Fu Kung
Original assignee: Lin Marie C M; Zan Shen; Hsiang Fu Kung
Priority date: 2006-11-27
Filing date: 2007-04-23
Publication date: 2008-06-05
Also published as: CN1958075A

Abstract

Constructs AVV-HGFK1 and Adv-HGFK1 expressing anti-angiogenic factor-HGFK1 mediated by adeno-associated virus and adenovirus. Compositions, comprising AVV-HGFK1 and the adenovirus-mediated construct expressing antioncogene, tumor suppressor gene or cytokine. Those constructs or compositions can be used to treat, control or prevent primary carcinoma and metastatic carcinoma. The uses of those constructs or compositions for preparation of the medicines for preventing or treating cancers.

Description

Composition containing adeno-associated virus-mediated anti-angiogenic factor HGFK1 and application thereof

The present invention relates to a construct or composition for preventing or treating cancer, in particular to an adeno-associated virus vector or an adenovirus vector-mediated expression of the anti-angiogenic factor HGFK1 construct AAV-HGFK1 or Adv-HGFK1, and a construct containing the same A composition of AAV-HGFK1 with an adenoviral vector or a construct of an adenoviral vector expressing an anti-oncogene, tumor suppressor gene or cytokine gene. technical background

In the growth, infiltration, and metastasis of solid tumors, sustained angiogenesis is one of the key factors influencing this process, as neovascularization provides the nutrient shield, oxygen, and various growth factors necessary for tumor cells to proliferate.

Accordingly, Folkman first proposed in 1971 to inhibit tumor angiogenesis as a means of treating tumors (Folkman J. et al., J Exp Med, 1971, 133: 275-88.). Preclinical studies have shown systematic systemic application of purified recombinant anti-angiogenic factors, such as angiostatin and endostatin, to inhibit tumor growth and metastasis (Hoffmann S. et al J Cell Biochem, 2006; 98: 954-65. / Kurup A et Al., Ann Oncol, 2006; 17: 97-103).

HGFK1 is the Kringle 1 domain of HGF (HGFK1) of human hepatocyte growth factor and contains amino acid sequences 127 to 214 of HGF. HGFK1 is a recently discovered anti-angiogenic factor. In vitro experiments have shown that the anti-angiogenic effect of recombinant HGFK1 protein is significantly stronger than that of angiostatin (Xin L. et al., Biochem Biophys Res Commun, 2000; 277: 186-190). ).

However, there are certain limitations in the clinical application of recombinant anti-angiogenic factors. For example, after intravenous administration, the anti-angiogenic factor is stable for several hours in the body, and to achieve anti-tumor effect, it is necessary to maintain an effective concentration in the body for a long time. Anti-angiogenic factor. Therefore, how to maintain the effective concentration of anti-angiogenic factors in vivo for a long time is an urgent problem to be solved before entering clinical application. Gene therapy is an effective way to solve this problem. Since the first gene therapy trials for human diseases in 1990, gene therapy has attracted attention as a new medical treatment, especially for diseases that cannot be completely cured by traditional medical methods, such as diabetes, cancer and AIDS. . Currently used gene therapy vectors mainly include both viral and non-viral vectors. In general, viral vectors are superior to non-viral vectors in terms of expression intensity and temporal and spatial properties.

Currently, the most commonly used viral vector for gene therapy is an adenoviral vector. Adenovirus (Adv) is a DNA double-stranded, non-enveloped virus with approximately 36 kb of genomic DNA encoding 14 proteins. The adenoviral vector is also an ideal viral vector. The virus strain used at the beginning has strong immunogenicity and may cause a strong allergic reaction. Encouragingly, Adv has recently been improved and its immunogenicity has been greatly reduced. In general, the main advantages of current adenoviral vectors are: (1) stable sexual shields, high expression, relatively safe for humans; (2) a wide range of infected host cells that can infect both mitotic and quiescent cells; 3) Adenovirus infection cells do not need to integrate into the host cell genome, there is no risk of activating oncogenes or insertion mutations; ( ⁴ ) are defective viruses that cannot replicate autonomously; ( ⁵ ) are easy to prepare, recombinant viruses can be injected intravenously , spray, intratracheal instillation or preparation into capsules orally and enter the body by intestinal absorption, etc., to obtain a high-priced viral vector.

Recently, adeno associated virus (AAV) has been used as an expression vector for the treatment of cancer, leukemia and AIDS, and satisfactory results have been obtained (Buning H et al., Curr Opin Mol Ther, 2003; 5 :367-75). However, AAV has shortcomings such as low yield, helper virus contamination, and wild-type AAV contamination. Although the above defects have been improved at present, compared with Adv, there are still problems such as the inefficient transmission and expression of AAV foreign genes.

Faced with so many expression vectors, we can successfully construct a construct that can be successfully recombined with HGFK1 gene, which can stably and efficiently express and effectively mediate HGFK1's anti-angiogenic effect in tumor tissues, thereby achieving prevention or treatment of primary disease. The purpose of cancer, metastatic cancer, and recurrent cancer is in urgent need of in-depth research and exploration.

In addition, there are many known tumor suppressor genes, anti-oncogenes and cytokines, which genes or factors can be combined with anti-angiogenic factors in gene therapy of cancer, and which carrier is selected to express these factors, making them Achieve synergistic prevention or treatment of cancer The role of the disease, there is currently no relevant 4 rumors.

P53 is a protein product of tumor suppressor gene expression, which inhibits cell division, induces apoptosis, up-regulates multiple tumor suppressor genes and down-regulates multiple oncogenes, and inhibits vascular endothelial growth factor (VEGF) gene and The role of drug resistance (MDR) gene expression (Sherr CJ, Cell, 2004; 1 16: 235-46.). Adv-mediated p53 (Adv-p53) has been used in a large number of clinical cases, and its safety and efficacy have been tested (Peng Z, Hum Gene Ther, 2005, 16(9): 1016-27).

hTERTC27 is an anti-cancer gene discovered by our research team. It encodes a 27 kDa amino acid sequence at the C-terminus of human telomerase, which can be specifically expressed in tumor cells and has tumor cell-specific cytotoxicity. It can cause tumor cell growth inhibition and apoptosis, but does not inhibit and apoptosis in normal cells. In vivo test-face confirmed that hTERTC27 can inhibit tumor formation and growth (Huang J. et al" Biochem Biophys Res Commun, 2003 Feb 14; 301: 627-32. 1 Huang J. et al., Cancer Res, 2002 Jun 1 ; 62: 3226-32.) The results of the hTERTC27 study have been filed for US patents (US Patent Application No. 10/449,565 filed 30 May 2003).

The cytokine IL-2 can significantly enhance the immune function of T cells, sputum cells, macrophages, and sputum cells, and also induce killer LAK cells and activate tumor infiltrating lymphocytes (TIL). IL-2 synergizes with many other cytokines to enhance immune function, so IL-2 has significant immune enhancement and anti-tumor effects (Waldmann TA, Annu Rev Med, 2006; 57: 65-81).

The present inventors have selected a tumor suppressor, an anticancer or a cytokine which can be used in combination with HGFK1 through creative labor, and have utilized different expression vectors to enable them to fully exert synergistic effects in preventing or treating cancer. Summary of the invention

In order to prevent or treat cancer more effectively, the invention overcomes the shortcomings of the tumor angiogenesis inhibitor in the prior art, and the effect is not satisfactory. The present invention provides a method capable of inhibiting tumor blood vessel growth and inhibiting cancer cell growth and metastasis. Recombinant constructs. In particular, the inventors selected a tumor suppressor gene such as p53, such as an anti-cancer gene of hTERT C27 or a cytokine gene such as IL-2, and expressed by creative labor. The construct of the anti-angiogenic factor constitutes a composition for synergistic prevention or treatment of cancer. The constructs and compositions of the present invention are not easily degraded, have a long action time, and are stable in effect, thereby being capable of effectively preventing and/or treating cancer growth, infiltration, metastasis, and recurrence.

In one aspect of the invention, a construct for preventing or treating cancer comprising a nucleic acid sequence of an anti-angiogenic factor HGFK1 and an adeno-associated virus expression vector or an adenovirus expression vector, and referred to as AAV-HGFK1 or Adv-HGFK1, is provided. The nucleic acid sequence of HGFK1 is SEQ ID NO: 1 or a homologous sequence thereof.

In a second aspect of the invention, there is provided a composition for preventing or treating cancer, which comprises an effective dose:

a first construct comprising an anti-angiogenic factor gene and an adeno-associated virus expression vector; an adenovirus expression vector or a second construct comprising a tumor suppressor gene, an anti-oncogene, and a cytokine or Combination and adenoviral expression vectors.

In a third aspect of the invention, there is provided the use of a construct or composition of the invention for the manufacture of a medicament for the prophylaxis or treatment of cancer, wherein said medicament comprises a prophylactically or therapeutically effective amount of said construct or composition.

In a fourth aspect of the invention, a specific antibody to the HGFK1 protein is provided. This antibody can be used for immunohistochemical staining and Western Blot detection of HGFK1 protein. Thus, the antibodies can also be used in the preparation of related kits.

In a fifth aspect of the invention, there is provided a kit for detecting a HGFK1 protein or gene, the kit comprising a specific antibody of the HGFK1 protein of the present invention, or a probe or primer for detecting the HGFK1 gene.

A further aspect of the invention provides a method of preventing or treating cancer in a subject comprising administering to the individual a prophylactically or therapeutically effective amount of a construct or composition of the invention, the individual in need of administration. In an embodiment of the invention, the administering comprises administering by intravenous or intratumoral injection. In a further embodiment, the individual is a mammal, preferably a human.

In another aspect of the invention, there is also provided a method of inhibiting tumor or cancer cell growth comprising contacting an effective amount of a construct or composition of the invention with a tumor or cancer cell. The method of inhibiting tumor or cancer cell growth of the present invention can be carried out in vivo or in vitro. Contact in the method comprises transfecting or transforming a vector or composition of the invention into a tumor or cancer Cell.

The anti-angiogenic factor gene used in the present invention may be selected from the group consisting of angiostatin, an endostatin, and a human HGFK1 gene. Preferably, the anti-angiogenic factor gene comprises a human HGFK1 gene encoding the amino acid sequence of positions 127 to 214 of HGF, and the nucleic acid sequence is SEQ ID NO: 1.

In one embodiment of the invention, the anti-angiogenic factor HGFK1 gene preferably encodes the amino acid sequence of SEQ ID NO: 5 or a homologous sequence thereof.

In a preferred embodiment of the present invention, the amino acid sequence of the anti-angiogenic factor HGFK1, in addition to SEQ ID NO: 5, further comprises six histidine sequences, i.e., the amino acid sequence of SEQ ID NO: 6.

The tumor suppressor gene used in the present invention is preferably p53; the anticancer gene is preferably TERTC27; and the cytokine is preferably a gene encoding IL-2.

In one embodiment of the invention, the composition may contain any pharmaceutically acceptable carrier.

In one embodiment of the invention, the cancer is a solid tumor, preferably hepatocellular carcinoma, colorectal cancer, and glial cell carcinoma, preferably primary and metastatic liver cancer.

The construct or composition of the present invention can be used for the prevention or treatment of primary cancer, metastatic cancer. It is preferred to treat metastatic cancer.

In the present invention, the HGFK1-expressing construct constructed using an adenovirus or an adeno-associated virus vector can stably and stably express the anti-angiogenic gene HGFK1 protein in vivo as compared with the expression of a conventional vector in the prior art. In particular, the anti-angiogenic construct of the composition of the present invention can exert a synergistic effect with an adenovirus expression vector or a construct containing an adenoviral vector inhibiting a tumor cell growth gene, and a construct containing anti-angiogenesis is used alone. The body can significantly enhance the effect of inhibiting tumors. DRAWINGS

Figure 1A shows the construct AAV-HGFK 1 of an adeno-associated virus vector containing the HGFK1 gene. Figure IB shows a schematic representation of the construct pAd-X of an adenoviral vector (Adv) comprising the p53, hTERTC27, HGF 1 or IL-2 gene, wherein X is any of the p53, hTERTC27, HGFK1 or IL-2 genes. .

Figure 2 is a bar graph showing the transfection efficiency of the adenoviral vector Adv significantly enhancing the construct of the adeno-associated virus AAV vector. Adenovirus Adv significantly enhanced the transfection efficiency of adeno-associated virus AAV recombinant vector by more than 10 times. The abbreviation Adv used in the figure is Adeno virus; LD-AAV-EGFP is low dose AAV-EGFP (MOI is lxlO ³ ); HD-AAV-EGFP is high dose AAV-EGFP (MOI is lx lO ⁴ ); EGFP is an enhanced green fluorescent protein.

Figure 3 shows the results of gene therapy for AAV-HGFK1 that significantly inhibited tumor growth, metastasis, tumor cell apoptosis, and significantly prolonged survival in experimental animals. Abbreviations in the figure: PBS is PBS buffer; EGFP is enhanced green fluorescent protein. Figure 3A shows the tumor volume of each group of hepatocellular carcinoma animal models at different time points after treatment. Figure 3B shows the pathological anatomical features of ascites, intrahepatic metastasis, lung metastasis, abdominal metastasis, and primary tumor volume at death in each group of hepatocellular carcinoma model animals. Figure 3C shows the apoptosis of tumor cells in each group of hepatocellular carcinoma models by TUNEL staining at 21 days after treatment. The arrows indicate apoptotic cells. Figure 3D shows the survival of each group of hepatocellular carcinoma models. ** indicates a value < 0.01 compared with the control group.

Figure 4 shows the survival curve of gene therapy in a nude mouse model of subcutaneously implanted human malignant glioma. Divided into seven groups: Adv-hTERTC27 Adv-HGFK1 or AAV-HGFK1 alone treatment group; AAV-HGFKl+Adv-hTERT C27 composition or AAV-HGFKl+Adv-IL-2 composition treatment group; control group was PBS or AAV-EGFP injection group. The graph shows the change in the percentage of animal survival at different times (days) after treatment. * indicates that the survival time of Adv-hTERTC27, Adv-HGFK1 or AAV-HGFK1 alone treatment group is significantly longer than that of the control group, P value < 0.01; ** means AAV-HGFKl+Adv-hTERTC27 or AAV-HGFKl+Adv-IL-2 The survival time of the treatment group was significantly longer than that of the Adv-hTERTC27, Adv-HGFK1 or AAV-HGFK1 treatment groups alone. Value <0.01. Abbreviations in the figure: PBS is PBS buffer; Adv-C27 is Adv-hTERTC27.

Figure 5 shows the therapeutic end of a gene therapy composition for an animal model of liver metastases from colorectal cancer. fruit. Figure 5A shows the percentage of survival of the AAV-HGFK1 group, the AAV-EGFP group, the AAV-EGFP+Adv-p53 group, and the AAV-HGFK1+Adv-p53 group at different times; Figure 5B shows: AAV-HGFK1 group Survival time, mean survival time, and number of animals with liver metastasis and primary tumorigenesis, AAV-EGFP group, AAV-EGFP+Adv-p53 group, and AAV-HGFK1+Adv-p53 group. ** indicates that the survival time of the AAV-HGFKl+Adv-p53 treatment group was significantly longer than that of the AAV-EGFP control group. Value < 0.01. The abbreviation EGFP in the figure is an enhanced green fluorescent protein.

Figure 6 shows the survival curve treatment results of the gene therapy composition of Buffalo rat primary hepatocellular carcinoma. The graph shows changes in the percentage of animal survival at different times after treatment (days:). Abbreviations in the figure: PBS is PBS buffer; HD-AAV-HGFK1 is high dose AAV-HGFKL

Figure 7 shows the results of the toxicity test. Abbreviations in the figure: ALT is alanine aminotransferase; AST is aspartate aminotransferase; TB is total bilirubin; LDH is lactate dehydrogenase; CK is creatine kinase; PBS is PBS buffer. Detailed description of the invention

The invention thus uses the term "human hepatocyte growth factor Kringle 1 domain (HGFK1)" to include alternative variants of its conserved amino acids that do not alter its ability to inhibit cell growth and angiogenesis.

As used herein, the term "stringent hybridization conditions" refers to hybridization and washing conditions of a gene having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% of the core with the gene. When the nucleotide sequence is complementary, it can still hybridize to the gene. Such hybridization conditions are well known in the art, for example, see Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6; Molecular Cloning, Cold Spring Harbor Laboratory, NY (1982), pp. 387. -389.

As used herein, the term "homologous sequence" refers to a nucleotide sequence identity of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the gene. And does not substantially affect the function of the encoded protein. Gene same The source sequence may also be a fragment of the gene as long as the function of the encoded protein is substantially unchanged. The term "homologous sequence" of the amino acid sequence as used herein means having at least

75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least

99% amino acid sequence identity, and does not substantially affect the function of the protein having the amino acid sequence.

The term "preventing or treating cancer" as used in the present invention means that the construct or composition used can prevent the metastasis or recurrence of cancer, alleviate, alleviate, control, ameliorate or cure the primary cancer or metastatic cancer or its associated symptoms; It can prolong the life span or survival time of cancer patients and reduce mortality.

The term "control group" as used in the present invention refers to a administration group of a construct consisting of green fluorescent protein (EGFP) and an adeno-associated virus vector (AAV) and/or a PBS buffer group.

1. A first construct comprising an anti-angiogenic factor gene and an adeno-associated virus vector

A variety of anti-angiogenic factor genes, for example, angiostatin and endostatin (Hoffmann S. et al., J Cell Biochem. 2006; 98: 954-65; Kurup A et al., Ann Oncol, 2006; 17: 97-103) And the nucleic acid sequence of the HGFK1 (Xin L. et al., Biochem Biophys Res Commun, 2000; 277: 186-190) gene can be used in the construct of the present invention. A preferred nucleic acid sequence of the invention is the HGFK1 gene of SEQ ID NO: 1 and homologous sequences thereof. The SEQ ID NO: 1 nucleic acid sequence encodes the amino acid sequence of positions 127 to 214 of HGF (SEQ ID NO: 5). The nucleic acid sequence of HGFK1 further comprises a nucleic acid sequence which hybridizes to SEQ ID NO: 1 under stringent hybridization conditions or a homologous sequence thereof. The adeno associated virus (AAV) used in the present invention includes all types of AAV, such as AAV1, AAV2, AAV5, AAV8, AAVl, preferably AAV2 (Choi VW et al. Curr. Gene Titer. 2005; 5 : 299-310.). The AAV selected by the present invention can efficiently transmit the HGFK1 gene and improve the expression efficiency. Meanwhile, the construct expressing the HGFK1 gene of the present invention constructed from the AAV, particularly AAV2, can synergize with the construct constructed by the adenovirus expression vector in the composition of the present invention, and significantly enhance the treatment or The effect of preventing cancer.

According to a conventional method of molecular biology and a DNA recombination technique, an anti-angiogenic factor gene sequence can be obtained and recombined with an adeno-associated virus vector to constitute a construct of the present invention. Phase For more information, see Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, NY; Marston, F (1987) DNA Cloning Techniques: A Practical Approach Vol III IRL Press, Oxford UK; DNA Cloning : FM Ausubel et al, Current Protocols in - Molecular Biology, John Wiley & Sons, Inc. (1994).

2. The nucleic acid sequence of HGFK1 gene and the construct of adenovirus vector

In addition to replacing the adeno-associated viral vector with an adenoviral vector, a construct comprising the HGFK1 gene and an adenoviral vector was constructed according to conventional methods well known in the art and methods of the first construct.

Adenoviral vector comprising a packaging system used in the construction, preferably pAd Adeno-X ^TM packaging systems, packaging systems AdEasyTM, AdMax ™ packaging system, Ad5 / F35Max ^TM packaging system, pAd / CMV / V5-DEST ™ packaging system / CMV /V5-DESTTM packaging system. The adenovirus expression vector selected by the present invention, particularly the pAd/CMV/V5-DESTTM packaging system, can efficiently transmit HGFK1 factor and has the effect of preventing and treating cancer when used alone or in combination. For example, see the embodiments of the present invention.

3. A nucleic acid sequence comprising a tumor suppressor gene, an anti-oncogene or a cytokine and a second construct of an adenoviral vector

A foreign gene that can be used to construct a second construct, including a tumor suppressor gene such as p53, such as an anti-oncogene of hTERT C27 or a nucleic acid sequence of a cytokine gene such as IL-2. The genes of these factors can be combined with adenoviral vectors to form a second construct. In gene therapy, these second constructs containing a tumor suppressor gene, an anti-oncogene or a cytokine and an adenoviral vector are combined with a first construct comprising an anti-angiogenic factor gene and an adeno-associated virus vector, resulting in an unexpected The effect of preventing or treating cancer. The foreign gene for use in the second construct of the present invention is preferably a nucleic acid sequence of p53, hTERT C27 or IL-2. The genes encoding p53, hTERT C27 and IL-2 have the nucleic acid sequences of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

Adenoviral vector comprising a packaging system used in the construction, Adeno-X ^TM packaging systems, packaging systems AdEasyTM, AdMax ™ packaging system, Ad5 / F35Max ^TM packaging system, pAd / CMV / V5-DEST ^TM packaging system, preferably pAd / CMV / V5-DEST ™ packaging system. A construct containing a tumor suppressor gene, an anti-oncogene gene or a cytokine gene and an adenoviral vector can be constructed by the method of the above 2.

Techniques for constructing constructs comprising a foreign gene and an adenoviral vector are well known in the art, for example, see the construction of a construct of the HGFK1 gene and an adenoviral vector described above.

4. Composition for the prevention and treatment of cancer

The composition for preventing and treating cancer of the present invention comprises a prophylactically or therapeutically effective amount of a first construct comprising an anti-angiogenic factor gene and an adeno-associated virus vector; an adenoviral vector, or a tumor suppressor gene, an anti-oncogene or a second construct of a cytokine and an adenoviral vector; and optionally, a pharmaceutically acceptable carrier.

Conventional pharmaceutical methods can be used to prepare suitable formulations for administration to a cancer patient for the construct or composition of the invention. Any suitable, for example parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ocular, intraventricular, intraorbital, intraspinal, intracranial, intraperitoneal, nasal Internal, aerosol or oral administration methods. The dosage form can be a liquid solution or suspension; a tablet or capsule; a powder, a nasal drop or an aerosol.

Compositions suitable for parenteral administration include sterile aqueous or non-aqueous formulations of the compositions of the invention which are preferably isotonic with the blood of the recipient. Formulations are prepared according to known methods using suitable dispersing or wetting agents and suspending agents as pharmaceutical carriers. The sterile injectable preparation may also be a sterile injection solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, non-volatile oils can be used conventionally as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Biocompatible, biodegradable lactide polymers, lactide/glycolide polymers or polyoxyethylene-polyoxypropylene copolymers can also be employed to control the release of the compounds. Other potential systems for regulating parenteral delivery of the constructs or compositions of the present invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable perfusion systems, and liposomes. Formulations suitable for inhalation may include a pharmaceutical carrier such as lactose, or may be an aqueous solution containing, for example, polyoxyethylene-9-laurate, glycocholate, and deoxycholate, or may be administered as a nasal drop. Oily solution or as a gel.

Groups suitable for oral, subcutaneous, intravenous, intramuscular, etc. administration can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

An "effective amount" based on a construct or composition of the invention includes a therapeutically effective dose or a prophylactically effective dose. By "therapeutically effective dose" is meant an effective dose at the dose, and within the necessary time period, the desired therapeutic outcome is achieved, such as inhibition of cancer cell growth, reduction of lesions, reduction in relapse rate, prolonged survival, and the like. The therapeutically effective dose of the construct or composition will also vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the construct or composition to elicit the desired response in the patient. The dosage regimen can be adjusted to provide an optimal therapeutic response. The therapeutically effective dose can also be an amount that the therapeutically effective effect exceeds any toxic or detrimental effects of the compound. By "prophylactically effective dose" is meant an effective dose at the dose, and within the necessary time period, the desired prophylactic result is achieved, for example to prevent metastasis and recurrence of cancer. Preferably construct or composition for treating or prophylactically effective dosage range may be effective dosage is any integer of ⁶ lx lO ³ to lx lO, e.g., a first prophylactic or therapeutic body constructed from a MOI (multiplicity of infection) of the present invention. 4x l0 ⁿ vg/kg to lx l0 ¹⁴ vg/kg; effective dose for prevention and treatment of the second construct is: lx l0 ^1Q vp/kg to lx lO ¹¹ vp/kg; prevention and treatment of Adv-HGFK1 construct The effective dose for treatment is also; lx l0 ^1G vp/kg to lx lO ¹¹ vp/kg. A preferred effective dose of the composition of the present invention is AAV-HGFK1: 4χ 10 ^π vg/kg to l l0 ¹³ vg/kg; Adv-X: lx l0 ¹⁽⁾ vp/kg to lx lO ¹¹ vp/kg.

It should be noted that the dosage may vary depending on the severity of the cancer to be prevented or treated. For any particular patient, the specific dosage can be configured according to the individual's needs and adjusted according to the judgment of the professional who is administering or supervising the administration. The dosage ranges set forth herein are merely exemplary and are not intended to limit the dosage range selected by the medical personnel. The amount of construct in the composition can vary depending on factors such as the disease state, age, sex, and weight of the individual. The dosage form can be adjusted to provide optimal therapeutic results. 5. Prevention and treatment

The invention further relates to a method of preventing or treating cancer in a subject, the method comprising administering to the individual an effective amount of a construct or composition of the invention.

In a method of preventing or treating cancer in an individual, the individual has cancer, is suspected of having cancer, or has a susceptibility (risk) of cancer, and thus the individual needs to be prevented or treated for cancer. The judgment of the above individual state is well known to those skilled in the art or clinical practitioners and physicians. In an embodiment of the invention, the individual is a mammal, preferably a human.

The cancer may be a primary cancer or a metastatic cancer, preferably a solid tumor, more preferably a hepatocellular carcinoma, a colon cancer, and a sphincter cell carcinoma.

In the prophylactic or therapeutic method, the route of administration to the individual includes intravenous, intraperitoneal, intralesional or intratumoral, sustained release of the implant, intramuscular, intrathecal and oral administration. It is preferably administered intravenously or intratumorally. The vein is preferably administered intravenously from a vein near the tumor, for example, for liver cancer, the portal vein is selected for administration.

The invention further relates to a method of inhibiting tumor or cancer cell growth, the method comprising contacting an effective amount of a construct or composition of the invention with said tumor or cancer cell. The method of inhibiting tumor or cancer cell growth may comprise transfecting or transforming the tumor or cancer cell in vivo or in vitro using the construct or composition of the present invention, thereby inhibiting growth of the cell. In one embodiment of the invention, the cancer or tumor cell is selected from the group consisting of liver cancer, colorectal cancer, and glial cancer cells.

The construct or composition of the invention may be administered to an individual or to a tumor cell in a single dose or in multiple doses. A single dose can be, for example, the above effective dose. The multiple dose may be from about 1/2 to about 1/100, preferably from about 1/2 to about 1/10, more preferably from about 1/2 to about 1/4 of the above effective amount. One skilled in the art can determine or adjust the dosage to be administered based on the specific circumstances of the patient or tumor cell growth and the toxicity of the drug. It is also permissible to administer doses above or below the effective dose.

6. The utility of the construct or composition of the invention

The inventors found through research that the simultaneous application of the Adv vector system and the AAV vector system can significantly enhance the utility of the AAV vector system by more than ten times (Fig. 2, Fig. 4). To this end, the inventors designed AAV-HGFK1 and Adv (eg Adv-p53, Adv-hTERTC27, Adv-IL-2) Or a composition of Adv itself, and applying the composition to a primary and metastatic cancer model, has achieved encouraging results. The therapeutic effect of the composition is significantly better than the therapeutic effect of any of AAV-HGFK1, Adv-p53, Adv-hTERTC27 or Adv-IL-2 alone.

In orthotopic hepatocellular carcinoma in rats, prevention and treatment with AAV-HGFK1 is carried out by an injection route including portal vein and intratumoral injection. The results showed that AAV-HGFK1 can cause apoptosis and necrosis of tumor cells, significantly prolong the survival time of animal models of hepatocellular carcinoma (average 30 days from the control group to 49 days in the treatment group), and completely inhibit the metastasis of cancer (Fig. 3).

In the liver metastasis of colorectal cancer, the therapeutic effect of AAV-HGFK1 and AAV-EGFP+Adv-p53 was significantly better than that of the control treatment group; while the average survival time of the AAV-HGFK1+Adv-p53 composition treatment group was significantly longer than AAV-HGFK1. The group and the AAV-EGFP+Adv-p53 group were directly smaller than 0.001; the treatment of the AAV-HGFKl+ Adv-p53 composition even reached a complete cure, resulting in long-term tumor-free survival of the experimental animals (Fig. 5).

In a mouse model of human malignant glioma, prevention and treatment are carried out by a route of administration including a para-tumor injection and a tail vein injection. Adv-hTERTC27, Adv-HGFK1 and AAV-HGFK1 alone extended the mean survival time from 28.3 days to 42.8, 47.5 and 60.6 days, respectively; and AAV-HGFKl+Adv-hTERTC27 composition and AAV-HGFK1+ Adv-IL-2 The efficacy of the composition was more pronounced: up to 80 days after treatment, none of the treatment groups administered with the composition of AAV-HGFK1 + Adv-hTERTC27 died, and only one treatment group of the composition administered with AAV-HGFK1 + Adv-IL-2 Death (Figure 4).

In view of the above findings, it is understood that the present invention may encompass other cancers. The basis is as follows: 1. Several primary and metastatic cancer models are selected as the more common types of cancer, which are representative; 2. All cancers invade and metastasize depend on angiogenesis, and The invention can provide long-term, high-level prevention and treatment of anti-angiogenesis; 3. The tumor suppressor gene selected in the invention has broad-spectrum anti-cancer properties; 4. The composition of the invention can prevent cancer recurrence and metastasis .

Thus, the constructs or compositions of the invention can be used to prevent or treat the growth, infiltration, metastasis, and recurrence of solid tumors. 7. Toxicity study

The present invention provides an evaluation of the toxicity of a composition comprising a recombinant expression construct of AAV-HGFK1 and a composition of an Adv-p53 and/or Adv-hTERTC27 expression construct. We administered high doses of the composition to experimental animals and then tested for indicators of liver function in serum: alanine aminotransferase, aspartate aminotransferase, and total bilirubin; indicators that reflect myocardial damage: lactate dehydrogenase, Creatine kinase and Tn-T did not detect signs of liver function and myocardial damage. In addition, no significant damage was found in the Η & Ε staining of myocardial tissue.

8. Preparation of HGFK1 protein antibody

Standard techniques of preparation are used (Harlow, Ε., and Lane, D. (1988) wo es ; a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory) or well known in the art. Methods, the constructs of the invention or proteins expressed thereby are used to prepare antibodies against the proteins of the invention.

For example, the constructs of the invention can be purified to the extent necessary to immunize an animal, such as a rabbit. In order to prevent potential problems of low affinity or low specificity of antiserum, one or three constructs can be prepared against angiogenic factors, and each construct is injected into at least two animals. The antiserum can be produced by a series of injections, preferably including at least three booster injections. Primary immunization can be performed with Freund's complete adjuvant, followed by booster immunization with Freund's incomplete adjuvant. Antibody titers can be monitored using Western Blot and immunoprecipitation analysis using the purified protein. The immune serum can be affinity purified using a C Br-dextran coupled protein. Anti-serum specificity can be determined using an unrelated protein plate. Alternatively, or in addition, a polypeptide corresponding to a relatively unique immunogenic region of an anti-angiogenic factor of the invention can be produced and introduced by C-terminal lysine and keyhole velocin (KLH) Coupling. Antisera against each of these polypeptides can be affinity purified by polypeptide conjugated to BSA, using polypeptide conjugates in ELISA and Western Blot and assayed for specificity by Western Blot and immunoprecipitation.

Alternatively, monoclonal antibodies that specifically bind to the anti-angiogenic factors of the invention can be prepared according to standard hybridoma methods. Once produced, specific recognition of monoclonal antibodies can be determined by Western blotting or immunoprecipitation. Antibodies which bind to the factors described herein are believed to be useful; such antibodies can be used, for example, as immunoassays.

In some embodiments, antibodies can be prepared using immunogenic polypeptide fragments. For example, at least 75%, at least 50%, at least 25%, or at least 5% of the amino acid sequence of SEQ ID NO: 5 of the present invention is used as an immunogenic polypeptide fragment from the end of several bases to the amino terminal to produce an immunogenic polypeptide fragment to produce antibody. The amino acid sequence RSK KGT VSI TKS GIKC (SEQ ID NO: 7) is preferred as the immunogen of HGFK1 in the present invention.

9. Kit

The present invention also relates to a kit for detecting an anti-angiogenic factor HGFK1 protein or a gene thereof, the kit comprising the anti-angiogenic factor HGFK1 protein of the present invention, or according to an anti-angiogenic factor HGFK1 gene sequence, preferably A primer or probe designed according to SEQ ID NO: 1 or a homologous sequence thereof.

In one embodiment of the invention, the kit is for detection analysis of ELISA, Western blot, co-immunoprecipitation, and the like. In another embodiment, the kit is for detection analysis of Northern Blot, Southern Blot, PCR, and the like. Thus, in one embodiment of the invention, the kit further includes instructions for directing the assay and other commonly used reagents for the assay, such as labeling reagents.

Accordingly, the present invention also relates to the use of the antibody against the angiogenic factor HGFK1 protein for the preparation of the kit. Example

Example 1 Construct of AAV expression vector encoding human HGFK1 polypeptide

The human HGFK1 cDNA (SEQ ID NO: 1 nucleic acid sequence) was inserted into the AAV packaging vector of pAM/CAG/EGR-1-pL-WPRE-BGH-polyA which had been previously digested with the same endonuclease to constitute AAV. -HGFK1 recombinant vector. The three-vector helper virus-deficient packaging system (AAV, H22 and pFD6) was used for packaging, that is, the recombinant vector AAV-HGFK1 and the helper plasmids H22 and pFD6 were co-transfected into HEK-293 cells by calcium carbonate method, and transfected for 60-72 hours. Thereafter, the cells were harvested, and the isolated recombinant virus particles were purified by HiTrap Heparin affinity chromatography, and the final titer was determined by real-time PCR.

After infecting mouse microvascular endothelial cells and rat hepatocyte cancer cells with AAV-HGFK1 in vitro, the infected cells were stained by immunohistochemistry. The results showed that HGFK1 was highly expressed in the cytoplasm. After culturing the culture medium of the infected cells, use Western The Blot method for the detection of the concentrate showed that HGFK1 was present in the culture, indicating that HGFK1 could be secreted outside the cell.

After the AAV-HGFK1 recombinant virus was injected into the tail vein of Balb/C mice, the serum of the mouse was collected and concentrated. Detection of concentrated serum by Western Blot method revealed the presence of HGFK1 in serum, indicating that HGFK1 was expressed and secreted into the blood. After the AAV-HGFK1 recombinant virus was injected into the portal vein of Buffalo rats, the expression of HGFK1 was also detected in the concentrated serum of the rats by Western Blot. High expression of HGFK1 was also detected in rat hepatocytes by immunohistochemistry. Example 2 Construction of adenoviral vector (Adv) encoding p53, hTERTC27, IL-2 or HGFK1 gene pAd-X

The present invention provides a method for preparing and packaging a construct of a recombinant defective adenovirus expression vector encoding human p53, hTERTC27 IL-2 or HGFK1 gene, comprising the steps of: human p53 gene, hTERTC27 gene, IL-2 gene or HGFK1 gene are respectively connected to the shuttle plasmid pENTR ^TM 2B, and then the recombinant shuttle plasmid pENTR-p53, pENTR-hTERTC27, pENTR-IL-2 or pENTR-HGFKl and pAd / CMV / V5-DEST expression vectors obtained by homologous recombination pAd- P53, pAd-hTERTC27, pAd-IL-2 or pAD-HGFK1, the resulting expression vector was transfected into 293 A cells with LipofectamineTM 2000 Reagent (Invitrogen), lysed, concentrated and purified by cesium chloride column. The purified recombinant adenovirus carrying human p53, hTERTC27, IL-2 or HGFK1 gene (Adv-p53, Adv-hTERTC27, Adv-IL-2 or Adv-HGFK1) was transfected into the rabbit carotid artery and used to carry the LacZ reporter gene. Recombinant adenovirus (Adv-LacZ) was used as a control. After 3 days, the expression of mRNA and protein of human p53, hTERTC27 or IL-2 gene was detected by RT-PCR and ELISA. As a result, recombinant adenoviruses Adv-p53, Adv-hTERTC27 and Adv-IL-2 carrying a human p53 gene, hTERTC27 gene and IL-2 gene, respectively, having a titer higher than 3x10 ^1G pfu/ml were obtained (Fig. 1B). Example 3 Establishment of test model

(1) Animal model of hepatocellular carcinoma

Rat hepatocyte cancer cell line McA-RH-7777 cells were surgically injected Buffalo rats under the left hepatic subcapsular (lxlO ⁶ cells / only, one-time injection). After 7 days, the rat abdominal cavity was opened by surgery, and it was found that there was a 3x3nmi ² tumor tissue on the left lobe surface of the liver, which confirmed the establishment of the model.

(2) Colorectal cancer liver metastasis model

Mouse colorectal cancer cell line CT26 cells were injected into the spleen of BALB/c mice ( ⁵ × ^{10 5} /only, one-time injection). Twelve mice were taken after ten days. It was found through surgery that each mouse had multiple tumor lesions of different sizes, that is, liver metastasis was confirmed. Therefore, it can be stated that the success rate of this method for establishing a liver metastasis model is 100%.

(3) glioma cell model

Human glioma cell line U87 cells (5χ 10 ⁶ /po, one-time injection) were implanted under the skin of rats. After 7 days, it can be seen that there is a bulge of about 5 x ⁵ mm ² at the injection site, that is, tumor formation is confirmed. Example 4 Therapeutic effect on primary hepatocellular carcinoma

A primary hepatocellular carcinoma model was established as described in Example 3.

After the rat hepatocellular carcinoma model was established, the treatment was started immediately after the tumor formation was confirmed. They were randomly divided into three groups, six in each group: 1. PBS buffer group; 2. AAV-EGFP group (1.2 x ^{10 12} vg) /only); 3. AAV-HGFK1 group (1.2x10 ¹² vg/only). The PBS group and the AAV-EGFP group were used as a control group, and AAV-HGFK1 was used as a treatment group. The injection route included two types of portal vein and intratumoral injection, in which the injected dose in the tumor was 0.2×10 ¹² vg/only, and the dose in the portal vein was lx l0 ¹² vg/only, which was confirmed to be a one-time injection immediately after the tumor model was established. .

The test results are shown in Figure 3.

Figure 3A shows the tumor volume of each group of hepatocellular carcinoma animal models at different time points after treatment. There was no significant difference in tumor volume between the treatment group (AAV-HGFK1 injection) and the control group (PBS or AAV-EGFP injection) on day 7 and day 14 (P>0.05), and the tumor volume of the treatment group was significantly higher on the 21st day after treatment. Less than the control group (P <0.01).

Figure 3B shows the pathological anatomical features of each group of hepatocellular carcinoma model animals at death, in which ascites, intrahepatic metastasis, lung metastasis, abdominal metastasis, and primary tumor volume were observed. The results showed that ascites: 6 animals in each of the two control groups had ascites (100%), and treatment Only 2 of the group had ascites (33%); intrahepatic metastasis: 6 animals in both control groups had intrahepatic metastasis (100%), but no intrahepatic metastasis was observed in all animals in the treatment group; lung metastasis: PBS control There were 5 lung metastases in the group (83.3%) and 6 lung metastases in the AAV-EGFP control group (100%), but no lung metastasis in all animals in the treatment group; abdominal metastasis: 6 animals in both control groups There was abdominal metastasis (100%), and no abdominal metastases were seen in all animals in the treatment group. Primary tumor volume: The treatment group was significantly smaller than the control group (P<0.05).

Fig. 3C shows the apoptosis of tumor cells in each group of hepatocellular carcinoma models by TUNEL staining at 21 days after treatment. The arrow shows apoptotic cells. The tumor cell apoptosis index of the treatment group was significantly lower than that of the two control groups (P<0.01).

Figure 3D shows the survival of each group of hepatocellular carcinoma models. The mean survival time (49 days) in the AAV-HGFK1 treatment group was significantly longer than the mean survival time of both controls (both 30 days).

The above results show that treatment with AAV-HGFK1 can cause apoptosis and necrosis of tumor cells, and prolong the survival time of animal models of hepatocellular carcinoma (average 30 days from the control group to 49 days in the treatment group), and complete inhibition of cancer. Transfer. This indicates that AAV-HGFK1 significantly inhibits tumor growth, metastasis, tumor cell apoptosis, and significantly prolongs the survival time of experimental animals. Example 5 Prevention or treatment of glioma

A human malignant glioma nude mouse model was established as described in Example 3.

After the model is established, it is randomly divided into seven groups of six:

1. PBS buffer group;

2. AAV-EGFP group (l xloHyg/only);

3. AAV-HGFK1 group (UxloUyg/only);

4, Adv-HGFKl group (2.5x 10 ⁹ vp / only;);

5, Adv-IL-2+AAV-HGFKl group (l xloUyg / only +2.5xl0 ⁹ vp / only);

6, Adv-hTERTC27 group (2·5χ10 ⁹ vp/only);

7. Adv-hTERTC27+AAV-HGFKl group (l.SxlO' g/only +2.5xl0 ⁹ vp/,).

It is confirmed that the tumor is administered immediately after the tumor is formed, and the administration route is a tumor injection and a tail vein injection. All are one-time injections.

The test results are shown in Figure 4. Adv-hTERTC27, Adv-HGFK1, AAV-HGFK1 alone extended the mean survival time from 28.3 days to 42.8, 47. 5 and 60.6 days (* indicates Adv-hTERTC27, Adv-HGFKl compared to the AAV-EGFP control group). The survival time of the three treatment groups of AAV-HGFKl was significantly prolonged, corpse <0.01); while the composition of AAV-HGF 1+ Adv-hTERTC27 and AAV-HGFK1 + Adv-IL-2 were more effective (** indicates The composition of the treatment group of AAV-HGFKl + Adv-hTERTC27 and AAV-HGFKl+Adv-IL-2 was significantly longer than the treatment group of Adv-hTERTC27, Adv-HGFK1, AAV-HGFK1 alone, and the corpse was prolonged. <0.01), at 80 days after the treatment, none of the treatment groups administered with the composition of AAV-HGFK1 + Adv-hTERTC27 died, and only one death group was treated in the treatment group administered with the composition of AAV-HGFK1 + Adv-IL-2. Example 6 Prevention or treatment of liver metastases from colorectal cancer

A model of colorectal cancer liver metastasis was established as described in Example 3.

After the liver metastasis of colorectal cancer in mice, they were randomly divided into four groups, six in each group:

1. AAV-EGFP group (Ux loUyg / only);

2, AAV-HGFKl group (1.5xlO ^u vg / only);

3, AAV-EGFP+Ad-PSS lx loUyg/only +2.5 x 10 ⁹ vp/only);

4. AAV-HGFK1+Ad-P53 (1.5x l0 ^u vg/only +2.5 x 10 ⁹ vp/only).

The treatment is given immediately, and the method of administration of the animal is a one-time injection in the tail vein. The test results are shown in Figure 5.

Figure 5A shows that in the animal model of liver metastasis of colorectal cancer, the AAV-HGFK1 group and the AAV-EGFP+Adv-p53 group were significantly better than the AAV-EGFP treatment group; and the AAV-HGFKl+Adv-p53 composition treatment The mean survival time of the group was significantly longer than that of the AAV-HGFK1 group and the AAV-EGFP+Adv-p53 group, which was less than 0.001. Figure 5B shows that the mean survival time of the AAV-HGFKl+Adv-p53 treatment group was over 61 days, while the average survival time of the other groups was 21 days in the AAV-EGFP group and 30 days in the AAV-HGFK1 group, AAV-EGFP+Adv. -p53 group for 30 days. In this animal model, one of the six animals in the AAV-HGFKl+Adv-p53 treatment group achieved long-term disease-free survival; The tumors were all eliminated and some of the primary tumors were eliminated.

The above results indicated that the therapeutic effects of the AAV-HGFK1 group and the AAV-EGFP+Adv-p53 group were significantly better than those of the AAV-EGFP treatment group; while the average survival time of the AAV-HGFK1+Adv-p53 composition treatment group was significantly longer than AAV- In the HGFK1 group and the AAV-EGFP+Adv-p53 group, the P value was less than 0.001; the treatment of the AAV-HGFK1+ Adv-p53 composition even reached a complete cure, which made the experimental animals have a long-term tumor-free survival effect. Example 7 Therapeutic effect of the composition on primary hepatocellular carcinoma

An in situ hepatocellular carcinoma model was established on the liver of Buffalo rats, as in the animal model of hepatocellular carcinoma in Example 3. After the model is established, it is randomly divided into five groups:

1. PBS buffer as a control group;

2, Adv- p53 (2,5x l0 ⁹ vp/only);

3. AAV-HGFK1 (3χ 10 ^η vg/only);

4, high dose AAV-HGFK1 (3x l0 ¹² vg / only);

5, AAV-HGFKl + Adv- p53 composition (AAV-HGFK1 of 3x 10 "vg / only; Adv-p53 is 2.5x l0 ⁹ vp / only).

It was confirmed that the tumor was administered immediately after the tumor formation. The route of administration includes portal vein and intratumoral injection, both of which are single injections. The test results are shown in Figure 6.

The results showed that the composition of AAV-HGFKl+Adv-p53 was able to extend the mean survival time from 30 days to 61 days, even more than 10 times higher doses of AAV-HGFK1 ( ³ < 10 ¹² vg/) compared with the control group. Only) 53 days to treat. The mean survival time of the Adv-p5 ³ treatment group alone was 37 days. The mean survival time of the AAV-HGFK1 (3x10" vg/only) treatment group was 39 days. Example 8 Toxicity test

The present invention provides an evaluation test for detecting the toxicity of the AAV-HGFK1 recombinant expression construct and the composition composed of Adv-p53 and Adv-hTERTC27 to the body. High doses of the composition were administered to the experimental animals.

Twelve adult male Buffalo rats were randomly divided into four groups, 3 in each group:

1. PBS buffer injection group, as a control; 2. AAV-HGFK1 (4.8x l0 ¹² vg/only) injection group;

3. AAV-HGFKl+Adv-p53 (4.8xl0 ¹² vg/only +1χ10 ¹⁽⁾ νρ/only) injection group;

4. AAV-HGFKl+Adv-hTERTC27 (4.8x l0 ¹² vg/only +1χ10 ¹⁰ νρ/only) injection group.

The administration methods are portal vein injection and intrahepatic injection, all of which are one-time injections, and the intrahepatic injection dose is one-third of the dose of the portal vein. The test results are shown in Figure 7.

On the 7th and 60th day after administration, the indicators reflecting liver function in the serum were detected: alanine aminotransferase, aspartate aminotransferase and total bilirubin, and no signs of liver function damage were found (Fig. Ί.

Recently, it has been reported that anti-angiogenic therapy of tumors can cause myocardial damage. Therefore, we have also used conventional methods to detect indicators that reflect myocardial damage: lactate dehydrogenase, creatine kinase, and Τη-Τ. The first two are within the normal range, and ΤηΤ is also normal (trace, not measured). These all indicate signs of no damage to the myocardium (Fig. 7Β). Myocardial tissue was taken 120 days after administration, and no significant damage was observed by Η & Ε staining (Fig. 7C:).

Moreover, no deaths due to immune response were observed in all animal models tested. Example 9 Preparation of HGFK1 Protein Antibody

Materials and reagents

1) . Synthetic peptide: RSYKGT VSI TKS GIKC, whose sequence is identical to the amino acid sequence at the end of HGFK1 ^^

2) . Freund's complete adjuvant

3) . Penicillin and streptomycin

4) . Experimental animals: Rabbit

5) . Other materials and reagents

Method of operation

1). Immunization method

Lymph node injection method: 1 subcutaneous (or intradermal) injection of live BCG 50 mg (about 0.30 ml per side) in the two hind paws of the rabbit. After 7-10 days, the rabbit iliac crest and diaphragmatic lymph nodes are swollen; the synthetic polypeptide emulsion with complete adjuvant is injected into the swollen bilateral lymph nodes. 0.50ml (containing synthetic peptide 5mg/ml, penicillin 1 000U/ml, streptomycin 1 OOO ig/ml); 3 if necessary, after 14 days, repeat step 2; 4 after 7 days, in both lymph nodes Injection of 0.50 ml of the synthetic polypeptide emulsion with complete adjuvant (containing synthetic peptide 5 mg/ml, penicillin 1 000 U/ml, streptomycin 1 OOO^g/ml); 5 5-7 days later, ear vein blood collection . Serum titer was determined.

2). Potency measurement Agar diffusion method was used.

(1) The serum was diluted and added to the peripheral wells.

(2) The intermediate well is added with the synthetic polypeptide described.

(3) The wet box at 37 °C was expanded for 24 hours, and the results were observed.

Result determination

Potency determination of anti-HGFK1 antibodies Agar diffusion titers of 1 : 16 or 1 : 32 were acceptable.

While the invention has been described by the foregoing detailed description and specific embodiments, the invention is not limited thereto. Equivalent modifications and variations of the embodiments of the present invention can be made by those skilled in the art in light of the disclosure of the present disclosure.

Claims

Claim

A construct for preventing or treating cancer, the construct comprising a gene encoding human anti-angiogenic factor HGFK1 and an adenovirus expression vector, wherein the nucleic acid sequence of the anti-angiogenic factor HGFK1 gene is SEQ ID:1 Nucleic acid sequence or homologous sequence thereof.

A construct for preventing or treating cancer, the construct comprising a human anti-angiogenic factor HGFK1 gene and an adeno-associated virus expression vector, wherein the nucleic acid sequence of the anti-angiogenic factor HGFK1 gene is SEQ ID: 1 Nucleic acid sequences and homologous sequences thereof.

3. A composition for preventing or treating cancer, the composition comprising an effective amount: a first construct comprising a gene encoding an anti-angiogenic factor and an adeno-associated virus expression vector;

An adenovirus expression vector or a second construct comprising a gene selected from the group consisting of a tumor suppressor gene, an anti-oncogene, and a cytokine, or a combination thereof, and an adenovirus expression vector.

The composition according to claim 3, wherein the anti-angiogenic factor gene is selected from the human anti-angiogenic factor HGFK1 nucleic acid sequence having the nucleic acid sequence of SEQ ID NO: 1, or a homologous sequence thereof.

The composition according to claim 3 or 4, wherein the anti-angiogenic factor gene encodes a human anti-human antibody selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6 and homologous sequences thereof Angiogenic factor HGFK1.

6. The composition of any of claims 3-5, wherein the gland associated with the viral expression vector is AAV.

The composition according to any one of claims 3 to 6, wherein the cancer suppressant The gene is selected from the p53 gene having the nucleic acid sequence of SEQ ID NO: 2 and homologous sequences thereof.

The composition according to any one of claims 3 to 7, wherein the anti-oncogene is selected from the hTERT C27 gene having the nucleic acid sequence of SEQ ID NO: 3 and homologous sequences thereof.

The composition according to any one of claims 3-8, wherein the cytokine gene is selected from the IL-2 gene having the nucleic acid sequence of SEQ ID NO: 4 and homologous sequences thereof.

10. Use of a composition according to any of claims 3-9 for the preparation of a medicament for the prevention or treatment of cancer, said cancer being a solid tumor.

The use according to claim 10, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, rectal colon cancer, and glial parenchyma.

The use according to claim 10 or 11, wherein the cancer is a primary cancer or a metastatic cancer.

13. Use of the construct Adv-HGFK1 of claim 1 for the manufacture of a medicament for the treatment of cancer and for the prevention of cancer recurrence and metastasis.

14. Use of the construct AAV-HGFK1 of claim 2 for the manufacture of a medicament for the treatment of cancer and for the prevention of cancer recurrence and metastasis.

The use according to claim 13 or 14, wherein the cancer is selected from the group consisting of liver cancer, colorectal cancer, and glioma cancer.

An antibody against the anti-angiogenic factor HGFK1 produced by a polypeptide selected from the group consisting of an anti-angiogenic factor HGFK1 and an immunogenic fragment thereof.

17. A method of preventing or treating cancer in a subject, comprising preventing or treating the individual An effective amount of the composition of claim 1 or 2 or the composition of any of claims 3-10, wherein the subject requires the administration.

18. The method of claim 17, wherein the individual is a mammal.

19. The method of claim 17, wherein the individual is a human.

20. The method of claim 17, wherein the administering is selected from the group consisting of intravenous, intratumoral, oral, intraperitoneal, intrathecal administration.

21. The method of claim 17, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, rectal colon cancer, and glial parenchyma.

22. A method of inhibiting the growth of a tumor cell comprising contacting an effective amount of the construct of claim 1 or 2 or the composition of any of claims 3-10 with a tumor cell.

23. The method of claim 22, wherein the contacting is transfection or transformation of the tumor cells.

24. The method of claim 22 or 23, wherein the tumor cells are selected from the group consisting of hepatocellular carcinoma, colorectal cancer, and glial cancer cells.