WO2004007703A1 - Replicability virus which can express anti-tumour antibody with high efficiency and the use of it - Google Patents

Replicability virus which can express anti-tumour antibody with high efficiency and the use of it Download PDF

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Publication number
WO2004007703A1
WO2004007703A1 PCT/CN2003/000565 CN0300565W WO2004007703A1 WO 2004007703 A1 WO2004007703 A1 WO 2004007703A1 CN 0300565 W CN0300565 W CN 0300565W WO 2004007703 A1 WO2004007703 A1 WO 2004007703A1
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antibody
promoter
gene
tumor
virus
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PCT/CN2003/000565
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French (fr)
Chinese (zh)
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Qijun Qian
Qin Yang
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Sino-Gene Biotechnology Ltd.
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Priority to AU2003257770A priority Critical patent/AU2003257770A1/en
Publication of WO2004007703A1 publication Critical patent/WO2004007703A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention relates to the field of life sciences, and in particular, to a virus that can proliferate in tumor cells capable of efficiently expressing antibodies or antibody fragments for treating tumors and uses thereof.
  • the monoclonal antibody preparation technology founded by Kohler and Milstein in 1975 provides a new method for tumor treatment-tumor-directed therapy.
  • great enthusiasm and unrealistic expectations were given, but the early clinical studies were very unsatisfactory.
  • the main reasons are: 1.
  • mouse-derived antibodies were used.
  • Anti-murine antibodies (HAMA) will be produced in the human body, which will neutralize therapeutic mouse-derived antibodies. It is quickly cleared, so the mouse-derived antibody has a short half-life in the human body, so the effect is not exact. At the same time, the mouse-derived antibody can cause an allergic reaction in the human body, resulting in toxic side effects; 2.
  • the affinity and specificity of the antibody is not high, Most antibodies, especially some genetically engineered small molecule antibodies or humanized antibodies, have low affinity and low specificity, so they cannot effectively target tumor cells, so their clinical antitumor efficacy is not clear; 3. Differences and modulation of antigens are also factors that affect the success of antibody therapy. With the development of modern biotechnology in the past 5 years, two key technologies of antibody technology have made breakthroughs. 1. The maturation of humanized antibody technology and human antibody preparation technology can basically overcome the use of mouse-derived antibodies to produce anti-antibodies for human use.
  • tumor-specific proliferation viruses are to take advantage of the fact that such viruses can only specifically replicate and proliferate in tumor cells. As the virus replicates and proliferates in tumor cells, tumor cells are lysed and virus particles are released. Infect other tumor cells again, and proliferate and lyse again. This produces a magnifying effect, allowing the virus to spread to various tissues and organs throughout the body, infecting all tissues and organs, and thus infecting all tumor cells, thereby killing local and metastatic tumors. Since tumor proliferative virus is basically unable to proliferate in normal cells, it does not affect normal cells surrounding tumor cells.
  • the application of tumor-specific proliferative virus alone is still limited.
  • the tumor The mechanism of tumor formation is very complicated and the heterogeneity is very strong. There are obvious differences between patients and patients, between tumors and tumors, and between different tumor cells in the same tumor.
  • the P53 gene mutation As far as the P53 gene mutation is concerned, the tumor of a tumor patient Cell P53 gene mutation, which does not mean that the patient's tumor cell P53 gene is mutated, so the virus proliferated by a certain tumor mechanism is not enough to kill all tumor cells.
  • physical factors such as fibrosis, incorporation into normal cells, and necrotic areas in tumor tissues may also limit virus spread.
  • the inventors creatively combined tumor antibody treatment with tumor-specific proliferation virus treatment methods, and first proposed an antibody-virus treatment method for tumors, that is, using tumor-specific proliferation viruses to carry antibodies or antibody fragment genes for treating tumors, Successfully achieved virus-specific replication and proliferation in tumor cells, forming high-concentration viruses, directly destroying tumor cells, and at the same time in tumor cells, the virus was replicated to achieve a large number of antibodies or antibody fragment genes for tumor treatment, generating synergy effect.
  • the tumor proliferating virus carrying the antibody or antibody fragment gene for tumor treatment specifically replicates and proliferates in tumor tissue, the antibody or antibody fragment that highly expresses the tumor is produced inside the tumor tissue, Therefore, it overcomes the difficulty that antibodies or antibody fragments that treat tumors are difficult to enter into solid tumor tissues; tumor proliferative viruses that carry the antibodies or antibody fragment genes that treat tumors are cheap to produce, thereby solving the high cost of mass production of antibodies or antibody fragments that treat tumors
  • the problem is therefore better than tumor antibody therapy in terms of efficacy and economy.
  • it is more effective than treating tumor-specific proliferation virus alone.
  • the virus genome includes a nucleotide sequence encoding an antibody or an antibody fragment for treating a tumor.
  • Another object of the present invention is to provide a method for treating tumors using the virus of the present invention, which comprises the steps of: 1) infecting the virus with tumor cells in vitro or in vivo, and 2) restricting the virus to selective replication in tumor cells. And proliferation, resulting in an increase in the number of copies of a nucleotide sequence encoding an antibody or antibody fragment for treating a tumor and an increase in expression of the antibody or antibody fragment for treating a tumor in a tumor cell; thereby inhibiting tumor formation, growth, and metastasis.
  • the method of the invention further comprises administering a chemical anti-tumor drug before, at the same time and / or after the virus of the invention infects tumor cells.
  • Another object of the present invention is to provide the use of the virus of the present invention for inhibiting the growth of tumor cells.
  • the invention finally provides a pharmaceutical composition comprising the recombinant virus of the invention and a pharmaceutically acceptable carrier.
  • Figure 1 shows a proliferating adenovirus SG500 that carries the humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2), the telomerase catalytic subunit gene promoter, and the hypoxic promoter that control the expression of E1A and E1B, respectively.
  • -HER SG502 expresses humanized antibodies in mice.
  • Figure 2 shows a proliferating adenovirus SG500 that carries the humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2), the telomerase catalytic subunit gene promoter, and the hypoxic promoter that control the expression of E1A and E1B, respectively.
  • -HER SG502 in the treatment of transplanted human ovarian cancer SKOV3 in mice.
  • the present invention provides a class of recombinant viruses in which a nucleotide sequence encoding an antibody or antibody fragment for treating a tumor is inserted into a virus genome capable of specifically proliferating in tumor cells.
  • the virus selectively proliferates in tumor cells, but does not substantially proliferate in normal cells.
  • the virus replicates and proliferates inside tumor cells, As a result, the copy number of the nucleotide sequence encoding the antibody or antibody fragment for treating a tumor is increased, thereby increasing the gene expression amount of the antibody or antibody fragment for treating a tumor.
  • the transcription of at least one essential gene for proliferation in the recombinant virus of the present invention is effectively controlled by a cis-acting element that is specifically activated by tumor cells.
  • the recombinant virus of the present invention contains at least one protein having a function of a region necessary for proliferation, and thus cannot substantially proliferate in normal cells, but can specifically proliferate in tumor cells.
  • viruses can be used in the present invention, as long as they can specifically proliferate in one or more tumor cells.
  • the virus is an adenovirus or herpes simplex virus.
  • the necessary regions for virus proliferation according to the present invention vary depending on the virus used, but are easily known to those skilled in the art.
  • "the transcription of genes necessary for virus proliferation is effectively controlled by cis-acting elements specifically activated by tumor cells” refers to The relative position can be varied depending on the virus used, as long as the transcription of the gene necessary for proliferation can be effectively controlled by cis-acting elements that are specifically activated by tumor cells.
  • the cis-acting element of tumor cell-specific activation contained in the virus of the present invention can be placed in an appropriate position before the transcription start site of the essential gene for virus proliferation.
  • a cis-acting element that is specifically activated by tumor cells can replace the virus's own promoter.
  • the virus of the present invention may further comprise at least one cis-acting element that is specifically activated by tumor cells in the region between the transcription initiation site and the encoding initiation site of the virus essential genes Recombinant virus.
  • the cis-acting element may be selected from: a telomerase catalytic subunit gene promoter, telomerase
  • RNA component gene promoter hypoxia response element, cell S-phase specific promoter (E2F promoter), alpha-fetoprotein enhancer and promoter, carcinoembryonic antigen enhancer and promoter Activators, tyrosinase enhancers and promoters, urokinase fibrin activator enhancers and promoters, ErbB2 enhancers and promoters, ErbB3 enhancers and promoters, ErbB4 enhancers and promoters, DF3 breast cancer related Antigen enhancers, prostaglandin-specific antigen enhancers and promoters, adrenostatin enhancers and promoters, Orip in EB virus, FR enhancer in EB virus Orip, and EB virus BamHI C-promoter.
  • E2F promoter cell S-phase specific promoter
  • alpha-fetoprotein enhancer and promoter carcinoembryonic antigen enhancer and promoter Activators
  • the gene necessary for proliferation may be an early virus gene, including but not limited to one or more of the following: E1A, E1B, E2, or E4.
  • the virus according to the present invention may also be a recombinant virus comprising at least one protein in a region necessary for proliferation and having a specific function in tumor cells.
  • the loss of protein function may be a point mutation, deletion, and / or insertion mutation of a coding gene, so that the function of the protein is lost.
  • the virus is a recombinant adenovirus, wherein the adenovirus ElB55Kda gene has a point mutation, a deletion mutation, and / or an insertion mutation, resulting in abnormal ElB55Kda protein function.
  • the virus is a recombinant adenovirus, wherein the adenovirus ElB 19kDa gene has a point mutation, a deletion mutation, and / or an insertion mutation, resulting in abnormal E1B 19kDa protein function.
  • the virus is a recombinant adenovirus, wherein the adenovirus E1A gene undergoes point mutation, deletion mutation and / or insertion mutation, resulting in abnormal E1A protein function.
  • the virus is a recombinant herpes simplex virus, wherein the herpes simplex virus ICP6 gene causes abnormal function of the ICP6 protein through point mutation, deletion mutation, and insertion mutation.
  • the virus is a recombinant herpes simplex virus, wherein the herpes simplex virus copies the ICP34.5 gene through a point mutation, a deletion mutation, or an insertion mutation, resulting in an abnormal function of the double copy ICP34.5 protein.
  • the nucleotide sequence encoding the antibody or antibody fragment for treating tumor includes, but is not limited to, the antibody or antibody selected from anti-neoangiogenesis.
  • the nucleotide sequence of the fragment, the nucleotide sequence of the antibody or antibody fragment encoding the anti-tumor cell growth factor receptor, the nucleotide sequence of the antibody or antibody fragment encoding the anti-tumor cell membrane antigen, the idiotype of the anti-tumor antigen The nucleotide sequence of a cloned antibody or antibody fragment.
  • any antibody or antibody fragment protein capable of treating tumors can be used in the present invention.
  • the antibody may be selected from the following table:
  • the nucleotide sequence encoding the antibody or antibody fragment for treating tumor is preferably operable with a promoter. connection.
  • the promoter includes, but is not limited to, the SV40 promoter, the RSV LTR promoter, the human cytomegalovirus IE promoter, and / or cis-acting elements that are specifically activated by tumor cells, wherein the cis-acting elements can be selected from: Telomerase catalytic subunit gene promoter, telomerase RNA component gene promoter, hypoxia response element, cell S-phase specific promoter (E2F promoter), alpha-fetoprotein enhancer and promoter, carcinoembryonic antigen enhancer and promoter, tyrosinase enhancer and promoter, urokinase fibrin activator enhancer and promoter, ErbB2 enhancer and Promoter, ErbB3 enhancer and promoter, ErbB4 enhancer and promoter, DF3 breast cancer-associated antigen enhancer, prostaglandin-specific antigen enhancer and promoter, adrenal relaxin enhancer and promoter, EB virus Orip, FR enhancer in EB virus O
  • any promoter whether homologous or heterologous, that can effectively direct the expression of antibodies or antibody fragments for treating tumors according to the present invention can be used in the present invention.
  • operably linked refers to the manner in which several elements juxtaposed are connected in such a way that each element can perform its intended function.
  • a promoter can transcribe the nucleotide sequence that follows Signal peptides can guide subsequent peptide chains into the cell's secretory pathway, and so on.
  • a tumor-specific virus is constructed so that the virus specifically proliferates and replicates in tumor cells, but does not proliferate and replicate in normal cells, mainly through the following methods:
  • the regulation of gene transcription activation is affected by the interaction of trans-acting factors (such as transcription factors) and cis-acting elements. When a certain transcription factor is missing or present, it can affect the transcription level of the gene.
  • the invention uses tumor tissue to specifically activate cis-acting elements (including promoters or enhancers) to control the target gene, so that the target gene is specifically expressed in tumor cells, but not expressed or expressed at low levels in normal cells.
  • the tumor tissue-specific promoter or enhancer is used to control the essential genes for virus proliferation and replication, so that the essential genes for viral proliferation can only be expressed in tumor cells, resulting in the virus can only proliferate in tumor cells and in normal cells Basically no proliferation.
  • the tumor cell-specific activation cis-acting element of the present invention may be any of the following:
  • TERT Telomerase catalytic subunit
  • telomere activity About 90% of tumor cells have telomerase activity, and most normal cells are negative for telomerase, which indicates that telomerase can be used as a characteristic marker of tumor cells. So far, human telomerase consists of three It consists of 1. RNA component (hTERC :), which serves as an endogenous template for repeated telomere synthesis; 2. Telomerase binding protein (TEP1); 3. Telomerase catalytic subunit, TERT), also known as telomerase reverse transcriptase gene. RNA components and telomerase catalytic subunits are necessary components for telomerase activity. Recent studies have shown that the telomerase catalytic subunit (hTERT) plays a decisive role in telomere activity.
  • TERT Telomerase binding protein
  • telomerase catalytic subunits are expressed at high levels in most tumor cells or tumor cell lines, but not in normal cells. Or low-level expression. Therefore, the telomerase catalytic subunit promoter and the telomerase RNA component gene promoter are activated in a large number of tumor cells.
  • the cis-acting elements of human and mouse TERT genes have been recently cloned. Their sequences have high GC content, lack TATA and CAAT boxes, but contain multiple binding sites for transcription factors, including transcription activation factors Myc, SP1, and transcription. Inhibitors Madl> p53, MZF2 and other binding sites.
  • hTERT human TERT gene
  • the core region of hTERT gene promoter contains E-box (CACGTG) and SP1 binding sites.
  • E-box CACGTG
  • SP1 binding sites the expression of SP1 transcription factor is up-regulated in telomerase-negative somatic cells, and its binding site promotes the initiation of hTERT gene transcription, in contrast, the main determinant of regulating hTERT gene expression is E-box binding factors, such as Myc, Madl, etc.
  • Myc protein can significantly increase the activity of the hTERT gene promoter, thereby promoting gene transcription and enhancing telomerase activity.
  • E-box upstream of the transcription start site (- 187bp--182b)
  • E-box (+ 22bp ⁇ + 27bp) downstream of the hTERT gene transcription start site and before the translation start site (- 187bp--182b)
  • This downstream E-box can be combined by Myc and Madl. They play a role in activating and inhibiting the transcriptional activity of hTERT genes.
  • Myc and Madl They play a role in activating and inhibiting the transcriptional activity of hTERT genes.
  • the latest research believes that the E-box sequence downstream of the transcription start site of the hTERT gene promoter is the target site of the negative regulation mechanism of telomerase negative cells.
  • the E-box sequence downstream of the transcription start site Copy number can significantly inhibit hTERT gene promoter activity in telomerase-negative cells, and in telomerase-positive cells, the E-box sequence may be transformed into an activation factor such as the Myc family member USF (Upstream stimulatory factor ) Binds and activates positive regulatory sites for hTERT gene transcription.
  • an activation factor such as the Myc family member USF (Upstream stimulatory factor ) Binds and activates positive regulatory sites for hTERT gene transcription.
  • a silencer Silencer
  • approximately 400bp 776bp ⁇ 378bp
  • MZF2 Myeloid-specific zinc finger protein 2 binding units
  • HRE Hypoxia response element
  • hypoxia is common in solid tumors.
  • Most solid tumor cells express the transcription factor-hypoxia-inducible factor la (HIF-1 ⁇ ) efficiently.
  • the transcription factor can be combined with a hypoxia-response element (HRE) to promote its transcription, so the hypoxia-response element (HRE) can activate its transcription level in solid tumors.
  • HRE hypoxia-response element
  • HRE can be derived from the promoter of the angiogenesis factor gene, the promoter of the erythropoietin gene, the promoter of the glucose carrier protein 1 gene, the promoter of the heme oxygenase gene, and the promoter of the inducible nitric oxide synthase gene. Child and so on.
  • E2F-1 gene is related to the cell cycle, it is a widely expressed growth-regulating gene, and it exhibits peak transcriptional activity in the S phase of cells.
  • the RB protein and other members of the RB family can form specific complexes with E2F-1 to curb its ability to activate transcription.
  • E2F-1 is down-regulated by RB.
  • E2F-1 cis-acting element is activated in most tumor cells.
  • AFP Alpha-fetoprotein
  • Alpha-fetoprotein is a carcinoembryonic protein, which is mainly limited to differentiated tissues of origin of endoderm (such as vitelline, fetal liver, viscera, etc.). Its expression level varies greatly with tissues and developmental stages. The reason why AFP is of clinical concern is that the plasma concentration of AFP increases in most patients with liver cancer, and the plasma concentration of AFP increases in some patients with advanced disease. The increase of AFP in the patient's plasma is caused by the expression of AFP in hepatocellular carcinoma, and AFP expression does not appear in normal tissues around liver cancer. Therefore, the expression of AFP genes is characteristic of liver cancer cells.
  • AFP transcriptional cis-acting elements are sensitive to cell proteins (such as transcription factors and common factors) associated with AFP-producing cells, such as AFP-binding proteins. At least one AFP promoter and one AF enhancer must be included in the transcriptional cis-acting element. Cell-specific transcriptional cis-acting elements derived from the A ⁇ gene have been identified. At 5, the sequence immediately upstream of the start of transcription contains AFP regulatory regions (including promoters, putative silencers and enhancers). In the AFP regulatory region, the human AFP enhancer is located between -3954 and -3335, and the human AFP promoter is located between -174 and +29 (both relative to the transcription start point).
  • Juxtaposing these two elements produces a fully functional AFP transcriptional cis-acting element.
  • Ido et al. Described a 259 bp promoter fragment that is specifically expressed in liver cancer cells. (Nt-230 to +29).
  • the AFP enhancer is located between -3954 and -3335, and contains two regions named and 8 respectively.
  • the promoter region contains the typical TATA box and CAAT box.
  • AFP transcriptional regulatory elements preferably include an enhancer region. Of course, it is even better that the transcriptional cis-acting element of AFP contains two enhancers.
  • the transcriptional cis-acting element on AFP may include any number of structural elements, and is not limited to one promoter, one enhancer; it may be one AFP enhancer and AFP promoter; or one AFP promoter and one heterogeneous enhancer; Or heterologous promoter and AFP enhancer; or multiple combinations of the aforementioned elements.
  • AFP transcription The promoter and enhancer in the cis-acting element and the sequence encoding the gene of interest can exist in any direction and distance, as long as the AFP cell-specific transcriptional activity exists within the element.
  • the adenoviral vector can also contain an AFP transcriptional regulatory element endogenous silencer, which can also be deleted.
  • CEA Carcinoembryonic Antigen Enhancer
  • CEA is a tumor-associated glycoprotein antigen with a size of 18,000 Daltons. It mainly occurs in the tumors of the gastrointestinal endoderm, such as colorectal cancer, gastric cancer, and pancreatic cancer. It is also found in some other adenocarcinomas such as breast and lung cancer. There are performances. Because CEA can be detected in the circulation of patients with most CEA-positive tumors, CEA has received clinical attention. In lung cancer, CEA can be detected in the circulation in about 50% of cases, CEA concentrations in adenocarcinoma exceed 20ng / L, and nearly 50% of patients with gastric cancer have positive CEA.
  • CEA showed cell-specific activity in the 5'-end sequence.
  • the CEA promoter region located at the first 424 nucleotides of the gene 5 upstream of the start of transcription, demonstrates its cells by showing higher promoter activity in CEA-producing cells than in non-CEA-producing Hela cell lines. Specificity.
  • the cell-specific enhancer region is also 3 000565
  • CEA promoter The CEA promoter, putative silencer, and enhancer elements appear to be contained within a region of 14.5 Kb from the starting transcription point. Further studies on the CEA gene 5 terminal region revealed that two regions (-13.6 to -10.7Kb, -6.1KbA to -4.0Kb) upstream of the transcription initiation point, when they are linked to a multimerized promoter, The expression of the reporter gene in Lovo and SW1463 cell lines producing CEA was increased and specific. Richard et al. Also positioned the promoter in a region between -90bp and + 69bp from the start of transcription, with sequences between -41bp and -18bp necessary for expression.
  • PCT / GB / 02546 describes a series of five, cell-specific fragments that include the following sequences: nt-299 to nt + 69; nt-90 to nt + 69; nt-13600 to -ntl0600, nt-6100 to nt-3800 (all relative to the start of transcription).
  • the -402 to +69 fragment in the CEA gene can also provide tissue-specific transcriptional activity to the linked gene.
  • CEA cis-acting elements mentioned here are derived from mammalian cells, including but not limited to human cells. Therefore, any cis-acting element of CEA can be used as long as the necessary functional regions are present in the carrier.
  • a cis-acting element that specifically expresses human tyrosinase gene in melanocytes.
  • This element contains a 20-bp long sequence called the tyrosinase distal element (TDE), which contains the -CATGTG sequence, located between -1874 and -1835 from the start of transcription.
  • TDE tyrosinase distal element
  • a promoter containing a human tyrosinase gene sequence from -209 to +61 was found to direct specific expression of the gene in melanocytes.
  • the mouse tyrosinase 5 has a similar sequence in the gene bank. The smallest promoter of the mouse TRP-1 gene has been identified, including sequences between -44 and +107 for the transcription start point.
  • a melanocyte-specific transcriptional regulatory element derived from the human tyrosinase gene 5 terminal sequence comprises a sequence from 231 to +65, or containing a sequence between -1956 and -1716.
  • the control element may also include the above two juxtaposed sequences. It has been reported that sequences from -956 to -1716 from the transcriptional start point of human tyrosinase can cooperate with the same or heterologous promoter to activate the reporter gene specific expression in melanocytes. Thus, in some examples, melanocyte-specific transcriptional regulatory elements include heterologous promoters and sequences from-1956 to-1716 operatively linked to them.
  • Urokinase plasminogen activator (uPA) enhancer and promoter uPA protein and its receptor uPAR which are expressed in most common tumors, and play an important role in tumor metastasis. They involve breast cancer, rectal cancer, prostate cancer, liver cancer, lung cancer, and uterine cancer. The sequence of cis-acting elements related to the transcription of uPA and uPAR has been extensively studied.
  • the C-erbB2 / neu gene is a transforming gene that encodes a 185KD epithelial growth factor receptor-related transmembrane protein.
  • the C-erbB2 / neu protein is expressed during fetal development; in adults, this protein is difficult to detect in many normal tissue epitheliums by immunohistochemistry.
  • C-erbB2 / neu gene amplification and overexpression are related to many human tumors, such as breast cancer, ovarian cancer, uterine cancer, prostate cancer, gastric cancer and lung cancer. In breast and ovarian cancer, the consequences of C-erbB2 / neu protein overexpression have been fully studied.
  • C-erbB2 / neu protein is overexpressed in 20% of 40% of breast cancer and 30% of ovarian cancer, which is related to the poor prognosis of these two types of diseases.
  • ErbB3 enhancer and promoter which is a breast cancer cell-specific activation enhancer and promoter.
  • ErbB4 enhancer and promoter which is a specific activation enhancer and promoter of breast cancer and gastric cancer cells.
  • MUC1 gene protein products (known as mucin, MUC1 protein, epidermal sialulin, polymorphic epidermal mucin or PEM; EMA; DF3 antigen; NPGP; PAS-O or CA15.3 antigen) usually in the stomach, pancreas, Lung, trachea, kidney, uterus, salivary glands, mammary glands, and apical expression of ductal epithelial cells.
  • mucin MUC1 protein, epidermal sialulin, polymorphic epidermal mucin or PEM
  • EMA DF3 antigen
  • NPGP apical expression of ductal epithelial cells.
  • Overexpression of mucin can be found in approximately 75 ⁇ 90% of human breast cancers.
  • the regulation of mucin expression is related to the degree of differentiation of breast cancer.
  • MUCI gene Overexpression of the MUCI gene in human breast cancer cell lines MCF-7 and 2R-75- appears to occur at the transcription level.
  • the regulatory sequence of the MUCI gene including a 0.9 Kb sequence upstream of the transcription start point, has been cloned, and a cis-acting element contained in it appears to be related to cell-specific transcription.
  • the transcriptional cis-acting element of MUC1- can be obtained from mammalian cells, including but not limited to human cells, and of course, it is preferentially expressed in human cells.
  • the transcriptional cis-acting element of MUC1 contains the entire 0.9 Kb sequence of the MUCI gene 5 terminal.
  • the transcriptional cis-acting element of MUCI can also contain the following sequences linked to the promoter (all relative to the MUCI transcription start point): -725 to +31, nt-743 to nt + 33, nt-750 to nt + 33 And nt-598 to +485.
  • the prostaglandin-specific antigen enhancer is located at nt-5322 ⁇ nt-3739 of the prostaglandin-specific antigen start transcription site, and the promoter is located at nt-540 ⁇ nt + 12 of the prostaglandin-specific antigen start transcription site
  • the enhancer and promoter are specifically activated in prostate cells and prostate cancer cells.
  • Adrenal relaxin enhancer and promoter which can be specifically activated in prostate cells and prostate cancer cells.
  • the FR in the Epstein-Barr virus Orip in combination with the herpes simplex virus thymidine kinase basic promoter or the SV40 basic promoter is a cis-acting element that is specifically activated in EB virus infected or latently infected cells.
  • the present invention provides a type of proliferative recombinant virus capable of specifically killing tumor cells.
  • the transcription of at least one gene necessary for virus proliferation is effectively controlled by cis-acting elements that are specifically activated by tumor cells.
  • the cis-acting element is inserted in the region between the transcription start site and the coding start site.
  • the cis-acting element is specifically activated in tumor cells to produce transcriptional activity, but is not activated in normal cells to produce transcriptional activity.
  • the cis-acting element may be one of the following sequences: a telomerase catalytic subunit gene promoter, a telomerase RNA component gene promoter, a hypoxia response element, a cell S-phase specific promoter (E2F promoter) , Alpha-fetoprotein enhancer and promoter, Carcinoembryonic antigen enhancer and promoter, Tyrosinase enhancer and promoter, Urokinase fibrin activator enhancer and promoter, ErbB2 enhancer and promoter, ErbB3 enhancer Promoter and promoter, ErbB4 enhancer and promoter, DF3 breast cancer-associated antigen enhancer, prostaglandin-specific antigen enhancer and promoter, adrenostatin enhancer and promoter, Orip in Epstein-Barr virus, Orip in EB virus FR enhancer, EB virus BamHI C-promoter.
  • E2F promoter cell S-phase specific promoter
  • virus early expressing gene Necessary for the above virus to multiply is a virus early expressing gene.
  • herpes simplex virus is used, and the gene necessary for virus proliferation contains the early and early expression gene ICP4.
  • the virus may be an adenovirus.
  • the virus proliferation essential gene contains at least one of the following early expressed genes of adenovirus: E1A, E1B, E2, E4.
  • the loss of viral protein function can be any of the following: point mutation, deletion mutation, and / or insertion mutation in the gene of adenovirus E1B 55kDa, resulting in abnormal function of E1B 55kDa protein.
  • the point mutation, deletion mutation and / or insertion mutation of the adenovirus ElB 19kDa gene caused the ElB 19kDa protein to malfunction.
  • Adenovirus E1A genes have point mutations, deletion mutations, and / or insertion mutations that cause the E1A protein to malfunction.
  • Point mutations, deletion mutations and / or insertion mutations in the ICP6 gene of herpes simplex virus have caused ICP6 protein to malfunction.
  • Point mutations, deletion mutations and / or insertion mutations in the herpes simplex virus ICP34.5 gene cause abnormal protein function of ICP34.5.
  • RNA-dependent protein kinase PLR
  • Ras double-stranded RNA-dependent protein kinase
  • the Ras gene is an oncogene, and when it is activated abnormally, it can become cancerous.
  • Respiratory intestinal filter virus replication and proliferation depend on the signal pathway of abnormal activation of Ras, that is, replication and proliferation in tumor cells with high Ras expression.
  • the transformation in this area mainly focuses on the adenovirus coat protein Fiber, Penton and Hexon, especially in the fibrin, which is most common in the HI loop or C-terminus of the head, including insertion
  • Some ligands, short peptides, and Fab regions of antibodies or antibody fragments for treating tumors have high affinity on the surface of tumor membranes.
  • a nucleus encoding an antibody or an antibody fragment for treating a tumor includes, but is not limited to, a nucleotide sequence selected from the group consisting of an antibody or antibody fragment encoding anti-angiogenesis, a nucleotide sequence encoding an antibody or antibody fragment encoding an anti-tumor cell growth factor receptor, and encoding an anti-tumor cell membrane antigen.
  • the nucleotide sequence of the antibody or antibody fragment, the nucleotide sequence encoding the antitumor antigen idiotype monoclonal antibody or antibody fragment are examples of any antibody or antibody fragment protein capable of treating tumors.
  • An antibody is a glycoprotein specific for the binding of a specific antigen.
  • a natural antibody of about 150,000 Daltons is a heterotetraglycan protein consisting of two identical light chains (L) and two identical heavy chains (H). The light and heavy chains are linked by a covalent disulfide bond.
  • Each heavy chain is composed of a variable region (VH) and multiple constant regions.
  • Each light chain is composed of a variable region (VL) and a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain.
  • variable regions The more conserved regions in the variable regions of antibodies are called framework regions (FR).
  • the variable regions of the natural heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4, respectively). Three hypervariable regions are sandwiched between the four framework regions, and they are roughly in a folded configuration. It is connected by three connection areas. The hypervariable regions in each chain are closely held together by the FR region and together with the hypervariable regions of the other chain form the antigen-binding portion of the antibody.
  • the hypervariable region (CDR) of an antibody refers to the amino acid residues in the antibody that are responsible for antigen binding.
  • the hypervariable region includes amino acid residues from the complementarity determining region "i.e. tCDR".
  • the constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cytotoxicity involved in antibodies.
  • Papain digestion of antibodies produces two identical antigen-binding fragments (called “F ab” fragments, each fragment having a single antigen-binding site) and a residual “Fc” fragment (the name reflects its ability to crystallize easily) ).
  • F ab antigen-binding fragments
  • Fc residual fragment
  • pepsin produced an F (ab,) 2 fragment that had two antigen-binding sites and was still able to cross-link with the antigen.
  • " ⁇ " is the smallest antibody fragment that contains all antigen recognition and binding sites. This region consists of a dimer of a heavy chain and a light chain variable region that are non-covalently tightly bound. In this configuration, The three hypervariable regions in each variable region interact.
  • the VH-VL dimer defines an antigen binding site on the surface. Six hypervariable regions together constitute the specificity of antibody-antigen binding. However, even a single variable region (or half of the Fv, which contains only three variable regions against the original specificity), can recognize and bind the antigen, except that its affinity is lower than the complete binding site.
  • the Fab fragment also contains the constant region of the light chain and the first constant region (CH1) of the heavy chain.
  • Fab, fragments differ from Fab fragments in that there are several more residues at the carboxy terminus of the heavy chain CH1 region (including one or more cysteine residues from the hinge region).
  • Fab, -SH herein means that the cysteine residue of the constant region carries a free sulfhydryl group.
  • F (ab,) 2 antibody fragments were originally produced as Fab, fragment pairs with hinge cysteine in between.
  • antibody fragment includes a part of an intact antibody, usually an antigen binding region or a variable region of the intact antibody.
  • antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments; diabody; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • the antibody or antibody fragment for treating tumor is an antibody or antibody fragment protein against neoangiogenesis.
  • an antibody or antibody fragment protein capable of anti-angiogenesis can be used in the virus of the present invention.
  • the nucleotide sequence encoding the anti-angiogenesis antibody or antibody fragment may be selected from one of the following: a nucleotide sequence of an anti-vascular endothelial growth factor antibody or an antibody fragment, and an anti-vascular endothelial growth factor receptor 2
  • the nucleotide sequence of the antibody or antibody fragment, the nucleotide sequence of the anti-integrin ⁇ ⁇ P 3 antibody or antibody fragment, and the nucleotide sequence of the antibody or antibody fragment that inhibits neovascular endothelial production may be selected from one of the following: a nucleotide sequence of an anti-vascular endothelial growth factor antibody or an antibody fragment, and an anti-vascular endothelial growth factor receptor 2
  • Vascular endothelial growth factor plays a key role in angiogenesis. Promote angiogenesis and play an important role in tumor formation and metastasis.
  • Anti-endovascular growth factor antibodies and antibody fragments can block the binding of vascular endothelial growth factor and its receptors (especially vascular endothelial growth factor receptor 2), inhibit tumor neovascularization, and thereby inhibit tumor growth and metastasis.
  • American Genentech's anti-vascular endothelial growth factor chimeric antibody (Avastin, another name is Bevacizumab) is already in the phase III clinical trial stage of advanced solid tumors.
  • KDR vascular endothelial growth factor receptor 2
  • the antibody or antibody fragment for treating a tumor is a nucleotide sequence encoding an antibody or antibody fragment against an anti-tumor cell growth factor receptor.
  • an antibody or antibody fragment protein having an anti-tumor cell growth factor receptor can be used in the present invention.
  • the nucleotide sequence of the antibody or antibody fragment encoding the anti-tumor cell growth factor receptor may be selected from one of the following: the nucleotide sequence of the antibody or antibody fragment of the anti-epidermal growth factor receptor 1 The nucleotide sequence of an antibody or antibody fragment of growth factor receptor 2.
  • Human epidermal growth factor receptor plays a key role in the transformation of many malignant tumor cells, and it is overexpressed in many tumor cells.
  • the human epidermal growth factor receptor is also known as the ErbB receptor.
  • HER1 also known as ErbBl or EGFR
  • HER2 also known as ErbBl or Neu
  • epidermal growth factor receptor 31 HER3, also called ErbB3
  • epidermal growth factor receptor 4 HER4, also called ErbB4
  • ImClone Systems' IMC-C225 chimeric antibody against epidermal growth factor receptor 1 has obvious effects on a variety of advanced tumors
  • the antibody has been clinically tested in m and iv stages.
  • Abgenix's human antibody ABX-EGF against epidermal growth factor receptor 1 in the United States has obvious therapeutic effects on a variety of advanced tumors.
  • the antibody has been in phase II clinical trials.
  • Herceptin also known as Trastuzumab
  • a humanized antibody against epidermal growth factor receptor 2 from Genentech of the United States, was approved for clinical application by the US FDA in 1998. It combined with chemotherapy produced significant clinical effects.
  • the company's other humanized antibody against epidermal growth factor receptor 1, 2C4 has also entered a phase I clinical trial.
  • the antibody or antibody fragment for treating tumors is a nucleotide sequence encoding an antibody or antibody fragment against an anti-tumor cell membrane antigen.
  • an antibody or antibody fragment protein capable of having an anti-tumor cell membrane antigen can be used in the present invention.
  • the nucleotide sequence of the antibody or antibody fragment encoding the anti-tumor cell membrane antigen can be selected from one of the following: the nucleotide sequence of the antibody or antibody fragment of anti-CD20, the nucleoside of the antibody or antibody fragment of anti-MUC1 Acid sequence.
  • the anti-CD20 chimeric antibody Rituxan also known as Rituximab
  • Rituximab The anti-CD20 chimeric antibody
  • MUC1 is widely present in a variety of tumors.
  • Antisoma's anti-MUC1 humanized antibody has entered clinical trials.
  • the antibody or antibody fragment for treating a tumor is a nucleotide sequence encoding an idiotypic monoclonal antibody or antibody fragment of an anti-tumor antigen.
  • an idiotypic monoclonal antibody or antibody fragment of an anti-tumor antigen Those skilled in the art know that any protein that can have an idiotype monoclonal antibody or antibody fragment of an anti-tumor antigen can be used in the present invention.
  • the nucleotide sequence of the idiotype monoclonal antibody or antibody fragment encoding the anti-tumor antigen may be selected from one of the following: the nucleotide sequence of the idiotype monoclonal antibody or antibody fragment of anti- ⁇ 7-1A, Nucleotide sequence of carcinoembryonic antigen idiotypic monoclonal antibody or antibody fragment, nucleotide sequence of anti-GD3 idiotype monoclonal antibody or antibody fragment Column, the nucleotide sequence of a unique monoclonal antibody or antibody fragment against MUC1.
  • Anti-idiotypic mAb (also known as Ab2) is also called anti-idiotypic antibody, which mimics the antigen on the surface of tumor cells, which can cause cytotoxic T cells and helper T cells to respond, thereby achieving tumor treatment. the goal of.
  • 17-1A is highly expressed in epithelial-derived tumor cells.
  • Germany approved the anti-17-1A unique monoclonal antibody (Panorex) produced by Glaxo Wellcome / Centocor to treat colon cancer.
  • ImClone Systems Inc. 's anti-GD3 idiotypic monoclonal antibody BEC2 antibody IMC-1C11 has entered phase II clinical trials of tumors.
  • the chain of spinal antibody (immunoglobulin) can be classified into two distinctly different types (called kappa ( ⁇ ) and lambda ( ⁇ )) based on the amino acid sequence of its constant region.
  • immunoglobulins can be divided into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgGT and IgM, some of which can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, IgG4, IgA-1 And IgA-2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called ot, P, ⁇ , ⁇ , and 4.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • ⁇ member 4 ' may be understood as IgA, IgD, IgE, IgGT or IgM, including natural antibodies, humanized antibodies or chimeric antibodies.
  • antibody fragment is removed intact antibody A portion of the antibody portion that still retains the antitumor effect of the antibody, including, for example, single-chain antibodies, Fv, Fab ', Fab, single-chain antibodies or antigen-binding regions in F (ab,) 2 antibodies, dimer antibodies, linear Antibodies, etc.
  • the Fab fragment also includes the constant region of the light chain and the first constant region (CH1) of the heavy chain.
  • Fab differs from Fab fragments in that the carboxy terminus of the heavy chain CHI region has a few more amino acid residues, including one or several cysteine residues from the hinge region. Sour.
  • the F (ab,) 2 antibody fragment was originally derived from a pair of Fab 'fragments linked by hinge region cysteine.
  • An Fv fragment is the smallest antibody fragment that contains a complete antigen recognition and binding site. It is a dimer formed by a heavy chain variable region and a light chain variable region that are tightly and covalently combined.
  • the three hypervariable regions of each variable region in its conformation interact to determine the antigen-binding site on the surface of the VH-VL dimer. Therefore, the six hypervariable regions together determine their specificity of binding to the antigen.
  • a single variable region or half of an Fv fragment containing three hypervariable regions specific for an antigen
  • a “single-chain Fv” or 3 ⁇ 4Fv "antibody fragment contains the VH and VL regions of an antibody, which are combined into the form of a single polypeptide chain.
  • the Fv polypeptide chain also contains a polypeptide linker between VH and VL so that sFv forms the ideal configuration for binding to antigens.
  • Diabodies are small fragments of antibodies that include two antigen-binding sites.
  • the heavy chain variable region (VH) and the light chain variable region (VL) are linked into the same polypeptide chain.
  • the linker used is very short, so that two variable regions on the same chain cannot be paired with each other. In this way, the variable region on one chain only has to pair with the complementary region on the other chain to form two antigen-binding sites.
  • Linear antibodies can be bispecific or monospecific, and consist of a pair of Fd pieces VH-CH1-VH-CH1) connected end-to-end to form a pair of antigen-binding site regions. Linear antibodies can be bispecific or monospecific.
  • the nucleotide sequence encoding the antibody or antibody fragment for treating a tumor may be linked in unison with the nucleotide sequence of the anti-oncogene to produce a fusion gene.
  • the anti-oncogene can be a vascular inhibitory gene, a cytokine gene, a prodrug converting enzyme gene, a cytotoxic gene, or the like.
  • the anti-oncogene may be a vascular suppressor gene, which suppresses tumors.
  • the formation of new blood vessels can block the nutritional supply of tumor cells, leading to the death of tumor cells due to inadequate nutrition, and the tumors obviously shrink or even completely disappear.
  • the formation of tumor neovascularization is inhibited, and the purpose of blocking the pathway of tumor metastasis is also achieved.
  • the angiostatin genes can be: endostatin gene, angiostatin gene, Kringlel-4 structure in plasma plasminogen, Kringlel-5 structure, Kringlel-3 structure, Kringlel-3 plus Kringle5 structure , Thrombospondin gene, platelet factor 4 gene, plasminogen activating factor inhibitor (PAI) gene and fibronectin gene.
  • the anti-oncogene may also be a cytokine gene.
  • Cytokine genes can activate immune cells and increase hematopoietic function.
  • the cytokine gene can be selected from the group consisting of: interleukin 2, interleukin 12, granulose-single colony stimulating factor, tumor necrosis factor, interferon-c, interferon-P, interferon-Y, Light, and Flt3 ligand.
  • Its anticancer gene may be a prodrug converting enzyme gene.
  • Prodrug converting enzyme genes can transform non-toxic drugs into toxic drugs, thereby enhancing the killing of tumor cells.
  • the prodrug conversion gene may be: herpes simplex virus thymidine kinase, bacterial P-lactamase and E. coli cytosine deaminase;
  • anti-oncogene can be a cytotoxic gene, such as a Pseudomonas aeruginosa exotoxic fragment.
  • the nucleotide sequence encoding the antibody or antibody fragment for treating a tumor may be controlled by a promoter.
  • the promoter may be, for example, one of the following promoters: Simian vis 40 (SV40, conventionally abbreviated as conventional) promoter, Rous Sarcoma Virus (RSV, conventionally referred to as RSV) LTR promoter, Human cytomegalovirus (conventionally called HCMV) IE promoter, murine cytomegalovirus (MCMV) IE promoter and human adenovirus major late expression promoter (conventionally referred to as MLP), can also be It is the natural promoter of this antibody gene.
  • the nucleotide sequence may be under the control of a viral endogenous promoter.
  • an intron is included between the transcription initiation site of the promoter expressed by the antibody or antibody fragment and the translation initiation site of the antibody. Insertion of introns can greatly increase the expression of antibodies. Those skilled in the art know that any intron that can increase the expression of an antibody can be used in the present invention.
  • the intron may be a heterozygous intron.
  • the heterozygous intron may be, for example, one of the following: a third guide sequence containing the major late-stage mRNA of the adenovirus 5, a splice site and an immunoglobulin 3, a heterozygous intron of the splice site, containing The first leading sequence of the adenovirus's major late mRNA is 5, a splice site and an immunoglobulin 3, a heterozygous intron for the splice site.
  • the recombinant virus of the present invention may further include a nucleotide sequence encoding a secretory signal peptide in a nucleotide sequence encoding an antibody or antibody fragment for treating a tumor.
  • a signal peptide capable of causing the secretion of antibodies or antibody fragments can be used in the present invention.
  • the signal peptide may be, for example, one of the following: an antibody or an antibody fragment self-signal peptide, an M-oncoprotein signal peptide, and a tumor necrosis factor ⁇ signal peptide.
  • the nucleotide sequence encoding the antibody or antibody fragment for treating a tumor may be inserted into a non-proliferation-required region in the viral genome.
  • the nucleotide sequence encoding the antibody or antibody fragment for treating tumors of the present invention may be a nucleotide sequence encoding an antibody or antibody fragment for anti-angiogenesis, and a nucleoside encoding an anti-tumor cell growth factor receptor or antibody fragment. Acid sequence, or a nucleotide sequence encoding an antibody or antibody fragment against a tumor cell membrane antigen.
  • this modified virus vector can be used to kill a particular target cell in some cell mixtures.
  • this modified virus can selectively proliferate in the target cell, so that this kind of Target cells are selectively killed by the proliferating virus; in vitro culture or in animals by mixing the modified virus with the cell complex, the virus can only proliferate on the target cell, also This means that other than the target cells, other cells cannot be killed by this virus. Because the virus multiplies and expands in the target cell, the target cell in the mixed cell is killed. Once the target cell is destroyed, the virus cannot reproduce again.
  • a method for treating tumors using the virus of the present invention includes the following steps: 1) infecting the virus with tumor cells in vitro or in vivo, and 2) limiting the virus to be limited to tumor cells.
  • Replication and proliferation lead to an increase in the number of copies of the nucleotide sequence encoding the antibody or antibody fragment that treats the tumor in the tumor cells and an increase in the expression of the antibody or antibody fragment that treats the tumor; thereby inhibiting the formation of tumor blood vessels and specifically killing it directly Tumor cells inhibit tumor formation, growth and metastasis.
  • the mammals described in the present invention include, but are not limited to, humans, monkeys, cattle, sheep, pigs, dogs, cats, and the like.
  • a method for treating mammals, especially human tumors, using the virus of the present invention which further comprises administering a chemical before, simultaneously and / or after the virus of the present invention infects tumor cells.
  • Antitumor drugs are also provided.
  • the virus proposed by the present invention can be used with conventional chemotherapy drugs (such as cisplatin, 5-fluorouracil mitomycin C, etc.), biotoxins (such as snake toxin), monoclonal antibodies or antibodies for tumor treatment Fragments are used together to treat tumors, which are more effective as antitumor drugs.
  • conventional chemotherapy drugs such as cisplatin, 5-fluorouracil mitomycin C, etc.
  • biotoxins such as snake toxin
  • monoclonal antibodies or antibodies for tumor treatment Fragments are used together to treat tumors, which are more effective as antitumor drugs.
  • the virus of the present invention is used in combination with X-rays to produce a more effective antitumor effect.
  • the present invention provides a class of recombinant viruses, which can specifically inhibit the growth of tumor cells by replicating and proliferating in tumor cells.
  • the present invention provides a class of recombinant viruses used to infect tumor cells in vitro.
  • the virus replicates and proliferates in tumor cells, resulting in an increase in the number of nucleotide sequences that encode antibodies or antibody fragments that treat tumors and the expression of antibodies or antibody fragments that treat tumors. The amount increases.
  • the present invention provides a class of recombinant viruses that can be used to selectively replicate and proliferate in tumor cells in vivo, resulting in nucleotide sequences encoding antibodies or antibody fragments that treat tumors. Increasing the number of copies in the column and increasing its expression inhibits tumor formation, growth, and metastasis. At the same time, the virus can proliferate in and be limited to tumor cells, and can specifically and directly kill tumor cells.
  • the use of the virus of the present invention for inhibiting the growth of tumor cells is provided.
  • recombinant virus there are many ways to deliver the recombinant virus to target cells, including but not limited to liposomes, conventional transfection methods (such as calcium phosphate precipitation or electroporation) well known in the art, direct injection, and intravenous infusion.
  • the delivery method mainly depends on the specific recombinant virus (including its morphology) and the type and location of the target cell (ie, whether the cell is in vitro or in vivo).
  • a packaged recombinant virus it can be in a suitable physiologically acceptable carrier It is administered at a dose of about 10 4 to about 10 14.
  • the multiplicity of infection usually ranges from about 0.001 to 100.
  • a polynucleotide is administered (ie, unpackaged as a virus)
  • the dose may be about 0.01 to about 1000 g.
  • the dosage can be determined based on the common sense of the virus (which can be conveniently obtained from published literature) or can be determined empirically.
  • the administration of the recombinant virus can be given once or multiple times, depending on its purpose
  • the use and the host's immune response ability can also be administered by multiple simultaneous injections. If an immune response is not desired, the immune response can be reduced with various immunosuppressants so that repeated administrations can be made without generating a strong immune response.
  • compositions such as pharmaceutical compositions, containing a recombinant virus described herein.
  • Such compositions can be administered in vivo.
  • these compositions further comprise a pharmaceutically acceptable excipient.
  • These compositions which may contain an effective amount of the recombinant virus of the present invention in a pharmaceutically acceptable excipient are suitable in unit dosage form, sterile parenteral solution or suspension, sterile parenteral solution Or an oral solution or suspension, an oil-in-water or water-in-oil emulsion and the like are administered systemically to an individual.
  • Parenteral and parenteral drug delivery formulations are known in the art, see Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing (1990).
  • compositions also include lyophilized and / or reconstituted forms of the recombinant viruses of the invention (including those packaged as viruses).
  • the invention also provides methods of treatment, wherein an effective amount of a recombinant virus described herein is administered to an individual.
  • Treatment with recombinant viruses can be used in patients with tumors (such as hepatocellular carcinoma). It can also be used in people at high risk for tumors, such as those with a family history of the disease and / or individuals who have had a disease that has been removed or otherwise treated (such as chemotherapy).
  • the determination of suitability for administration of the recombinant virus of the invention will be based in particular on assessable clinical parameters such as serological indicators and histological examination of tissue biopsy samples.
  • Pharmaceutical compositions containing recombinant viruses are usually administered. The pharmaceutical composition is as described above.
  • the amount of recombinant virus used depends on many factors, such as the specific type of recombinant virus, the route of administration, the physical condition of the individual, the degree of disease development, and the specific antibody or antibody fragment gene used.
  • the amount is about 10 4 to about 10 14 , preferably about 10 4 to about 10 12 , and more preferably about 10 4 to about 10 1 ⁇ .
  • the amount is from about 0.01 g to about 100 g, preferably from 0.01 g to about 500 g, more preferably from about 0.5 g to about 200 g.
  • More than one recombinant virus can be administered simultaneously or sequentially. It is usually given periodically while monitoring any response. It can be administered intratumorally, intravenously or intraperitoneally.
  • the present invention has the following beneficial effects: Animal experiments prove that the recombinant virus of the present invention can be used to treat tumors.
  • the virus of the present invention can specifically proliferate in tumor cells, but does not substantially proliferate in normal cells, so that antibodies can be targeted to be expressed in tumor cells to achieve specific inhibition or killing of tumor cells, thereby being highly efficient and low.
  • Human adenovirus has six different subgenus, divided into A, B, C, D, E and F. They have different philophilic, tumorigenic, and diseased histories of host cells.
  • the present invention is further exemplified by taking type 5 (Ad5) in the subgenus Adenovirus C as an example.
  • Ad5 type 5
  • the construction means of the present invention can be implemented by those skilled in the art.
  • Example 1 Construction of a cloned vector carrying a telomerase catalytic subunit gene promoter. Genomic DNA was extracted from fresh normal human liver tissue, and the telomerase catalytic subunit gene promoter was amplified using nested polymerase chain reaction (PCR) technology (for methods, see PCR Protools Current Methods and Applications, editor of White BA, Humana Press Inc. 1993 published a document 1), at the promoter 5, the three restriction sites of EcoR I, Not I and Bgl ⁇ were inserted, and at the promoter 3, the two sites of Xho I and BamH I were added .
  • PCR polymerase chain reaction
  • Primer 2 5 'AAC GTG GCC AGC GGC AGC ACC TC 3, (SEQ ID NO: 2);
  • Primer 4 5
  • Primer 1 and primer 2 were amplified by PCR for the first time, and a 370 bp fragment was recovered. Then, a second PCR amplification of 370 fragments was performed with primer 3 and primer 4. A 270 bp fragment was recovered, and digested with EcoR I + BamH I. This gene was inserted into the pUC19 vector (purchased from ATCC, USA), and was named pUC-hTERTp.
  • Primer 5 5 TCG AGG ACG CAC GTG GGC GCA CGT GGG CGC ACG TGG GAT TTA AAT A 3 (SEQ ID NO: 5);
  • Primer 6 5 AGC TTA TTT AAA TCC CAC GTG CGC CCA CGT GCG CCC ACG TGC GCC T 3, (SEQ ID NO: 6).
  • Primer 5 and primer 6 were denatured, renatured, phosphorylated using phosphokinase, and inserted into the Xho I and Hind III digestion sites in pUC-hTERTp (for the method, see the second edition of the Molecular Cloning Experiment Guide, J. Sam Edited by Brooke et al., Science Press. 1996. 1).
  • the telomerase catalytic subunit gene promoter contained nt-213 to nt in the human telomerase catalytic subunit gene promoter. +47 and 3 E-box sequences downstream, and EcoR I, Not I, and Bgl II restriction sites in the upstream sequence of the entire promoter, and Swa in the downstream sequence of the entire promoter I restriction sites.
  • Example 2 Construction of an attenuated proliferative adenovirus vector carrying an anti-oncogene telomerase catalytic subunit gene promoter to control E1A expression.
  • the pXC.l vector was purchased from Microbix Biosystem Inc. (Toronto), Canada.
  • pXC.l contains a type 5 adenovirus sequence bp22-5790. Seven new and unique restriction sites were created at 552bp in this vector, namely Age l, Bst BI, Not I. Spe I, Sal L Xho I ⁇ Swa I, and this site is located before the E1A start code 12bp, the method uses positional mutation double PCR technology (see the previous fan literature 1).
  • the primers are
  • Primer 7 (5, -primer, containing EcoR I digestion site): 5, TTC AAG AAT TCT CAT GTT TG 3, (SEQ ID NO: 8);
  • Primer 8 (3, -primer, add ATT TAA ATC TCG AGT CGA CAC TAG TGC GGC CGC TTC GAA CCG GT) at the 5 end: 5, ATT TAA ATC TCG AGT CGA CAC TAG TGC GGC CGC TTC GAA CCG GTG TCG GAG CGG CTC GGA 3, (SEQ ID NO: 9); Primer 9 (5,-primer, add ACC GGT TCG AAG CGG to the 5, end
  • Primer 10 (3, -primer, containing Xba I digestion site): 5, TTC TCT AGA CAC AGG TGA TG 3, (SEQ ID NO: 11).
  • the PCR product fragment was inserted into the pGEM-T-easy vector (for the method, refer to the operating instructions of Promega) and named as pGEM-T-Ela.
  • This fragment was subjected to DNA sequencing, and the sequencing results showed that: TTC GAA GCG GCC GCA CTA GTG TCG ACT CGA GAT TTA AAT CCG GT was inserted into the pXC.l plasmid bp552 site, thereby generating 7 new Age I, Bst BI, Not I, Spe I, Sal I, Xho I and Swa I restriction sites, and other sequences are the same as pXC.l.
  • PGEM-T-ElA and pXC.l plasmids were digested with EcoR I and Xba I, and the fragments cut out from pGEM-T-ElA were inserted into the EcoR I and Xba I sites in pXC.l plasmid to make pXC.l 552 Inserts a TTC GAA GCG GCC GCA CTA GTG TCG ACT CGA GAT TTA AAT CCG GT to generate 7 Age I, Bst BI, Not I, Spell, Sal I, Xho I, and Swa I restriction sites. The dot was located 12bp before the E1A start code, and the plasmid was named pQW.
  • pUC-hTERT-Ep was digested with Not I + Swa I, and the fragments were inserted into the Not l + Swa l digestion site in pQW plasmid, and primers 4 and 7 were used for PCR amplification, respectively. This indicates that the promoter of the telomerase catalytic subunit gene has been inserted into the Not I + Swa I site of the pQW plasmid, that is, 12 bp upstream of the start codon of the adenovirus type 5 E1A, and named pQW-hTERT-Ep.
  • Example 3 Construction of an attenuated proliferative adenovirus vector carrying an anti-oncogene telomerase catalytic subunit gene promoter and an hypoxic promoter to control the expression of E1A and E1B, respectively.
  • Hypoxia-responsive element (HRE) combined with basic human cytomegalovirus promoter
  • HRE hypoxia-acting element
  • mini-CMVp The sequence of the basic human giant cell promoter (mini-CMVp) is as follows:
  • hypoxia-acting element combined with the basic human giant cell promoter (mini-CMVp) contains the above two sequences, and the Spel restriction sites are added at the 5, -terminus and 3, -terminus, respectively.
  • HRE combined with the basic human giant cell promoter (mini-CMVp) was used for full-length gene synthesis by Shanghai Shenneng Gaming Biotechnology Co., Ltd. After sequencing, it was named pMD-HRE, and its nucleotide sequence was as follows:
  • the pQW-hTER-Ep plasmid was deleted from the E1B promoter, which is located at the ntl926-nt2043 position in the pQW-hTER-Ep plasmid (that is, ntl595-ntl713 position), delete this position and create a new, unique Spe I digestion site, this site is located 4bp before the E1B start code, and its method uses position mutation double PCR technology (see the method above Reference 1).
  • the primers are:
  • Primer 13 5 ACT AGT ATG GAG GCT TGG GAG TGT TTG 3, (SEQ ID NO: 17);
  • Primer 14 5 GGC CAG AAA ATC CAG CAG GTA 3, (SEQ ID NO: 18).
  • the PCR product fragment was inserted into the pGEM-T-easy vector (for the method, refer to the operating instructions of Promega) and named as pGEM-T-ElB.
  • the DNA was sequenced from this fragment.
  • the sequencing results showed that the pQW-hTER-Ep plasmid ntl926-nt2043 was deleted and inserted to generate a new Spe I restriction site.
  • the other sequences were the same as pQW-hTER-E.
  • the pGEM-T-ElB and pQW-hTER-Ep plasmids were double digested with Kpn I and Xba I, and the fragments cut out from pGEM-T-ElB were inserted into the Kpn I and Xba I sites in pQW-hTER-E plasmid so that The pQW-hTER-E plasmid, ntl926-nt2043, was deleted and inserted to generate a new Spl restriction site, which was located 4bp before the E1B start code. This plasmid was named pQW2-Spe.
  • the pMD-HRE was digested with Spe I, and the fragment containing the hypoxia-reactive element (HRE) combined with the basic human giant cell promoter (mini-CMVp) was cut and inserted into the Spe l digestion site in the pQW2-Spe plasmid.
  • the PCR technology was used to verify the positive and negative directions of insertion.
  • Primer 15 5, AGG TCT ATA TAA GCA GAG CTC 3 '(SEQ ID NO: 19). PCR was performed with primers 14 and 15. The length of the PCR product was 518 bp. This indicates that the hypoxia-acting element (HRE) combined with the basic human giant cell promoter (mini-CMVp) was inserted into the upstream region of E1B and named pQW-hTERT-Ep. --HRE.
  • HRE hypoxia-acting element
  • mini-CMVp basic human giant cell promoter
  • Example 4 Human antibody SG-EGFR gene carrying anti-human epidermal growth factor receptor 1 (EGFR), humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2), and anti-human CD20 Construction of human and mouse chimeric antibody SG-CD20 gene expression vector
  • pShuttle-CMV was purchased from Qbiogene in the United States and contains a human cytomegalovirus promoter (CMV IE) and SV40 poly A tailed signal.
  • PCR technology was used to clone the human cytomegalovirus promoter (CMV IE) and SV40 poly A tailed signal
  • CMV IE human cytomegalovirus promoter
  • SV40 poly A tailed signal Add PAC I digestion sites in the upstream and downstream positions, and insert multiple cloning sites between its human cytomegalovirus promoter (CMV IE) and SV40 poly A tailing signals, respectively EcoR I, Sal L Hind III , Xho L BamH I and Xba I digestion sites, the method uses site-directed mutation double PCR technology (for methods see PCR Protools Current Methods and Applications, editor of White BA, Humana Press Inc. 1993-Literature 1).
  • the primers were used to perform PCR on pShuttle-CM
  • Primer 16 5;
  • Primer 17 5 GAG AAG CTT GTC GAC GAA TTC CTA GCG GAT CTG ACG GTT CAC 3, (SEQ ID NO: 21).
  • the primers 18 and 19 were used to perform PCR on pShuttle-CMV, and the product length was 293bp.
  • Primer 18 5 'GAA TTC GTC GAC AAG CTT CTC GAG GGA TCC ATC TAG ATA ACT GAT CAT A 3, (SEQ ID NO: 22); Primer 19: 5 'ATA GTT TAG CGG CCG CTT AAT TAA GAT AC A TTG ATG AGT TTG 3' (SEQ ID NO: 23).
  • the primer 16 and primer 19 were used to perform PCR on the upper fragment, and the product length was 893bp
  • the polycistronic (IRES) of encephalomyocarditis virus (EMCV) was derived from pIRES-EYFP, and the plasmid was purchased from Clontech. Primer 5 and primer 6 were used for PCR, and the amplification length was 624bp.
  • Primer 20 5, CCG GAA TTC ATC GAT TCT GTC GAC CTG CAG GAA TTG CCC CTC TCC CTC 3, (SEQ ID NO: 24); Primer 21: 5, TGC TCT AGA CCC GGG CTC GAG GGA TCC TTA ATC ATC GTG TTT TTC AAA G 3, (SEQ ID NO: 25).
  • EcoR I + Xba I was double digested and inserted into the EcoR I + Xba I digestion site of pUC19, followed by sequencing and named pUC19-IRES.
  • the EcoR I + Xba I double digestion was inserted into the EcoR I + Xba I digestion site of pClonelO and named pClonel2.
  • Plasmid pClonel2 is a dual gene expression vector containing encephalomyocarditis virus (EMCV) polycistronic (IRES). Its promoter is the human cytomegalovirus promoter (CMV IE). It contains two polyclonal sites. The restriction sites of the cloning site were EcoR I, Cla L Sai l, and Pst l, and the restriction sites of the second multi-cloning site were BamH I, Xho I, Xma l, and Xba l.
  • EMCV encephalomyocarditis virus
  • IVS human cytomegalovirus promoter
  • the human antibody SG-EGFR against human epidermal growth factor receptor 1 was synthesized by Shanghai JBES Gene Technology Co., Ltd., and the variable regions of its light chain and heavy chain genes were targeted by the American Abgenix company.
  • the light chain gene of the human antibody ABX-EGF of the epidermal growth factor receptor 1 is the same as the variable region of the heavy chain gene (see Jakobovits, et al. PCT WO98 / 50433).
  • the pUC19 plasmid containing the SG-EGFR heavy chain gene and introducing the BamH I restriction site in the upstream region of the heavy chain and the Xba 1 restriction site in the downstream region of the heavy chain was named pUC-SG-EGFRH.
  • the pUC19 plasmid containing the SG-EGFR light chain gene and introducing EcoR I in the upstream region of the light chain and the Sal I restriction site in the downstream region of the light chain was pUC-SG-EGFRL.
  • the human antibody SG-EGFR against human epidermal growth factor receptor 1 was synthesized by Shanghai JBES Gene Technology Co., Ltd., and the variable regions of its light chain and heavy chain genes were targeted by the American Abgenix company.
  • the light chain gene of the human antibody ABX-EGF of the epidermal growth factor receptor 1 is the same as the variable region of the heavy chain gene (see Jakobovits, et al. PCT WO98 / 50433).
  • the pUC19 plasmid containing the SG-EGFR heavy chain gene and introducing the BamH I restriction site in the upstream region of the heavy chain and the Xba I restriction site in the downstream region of the heavy chain was named pUC-SG-EGFRH.
  • the pUC19 plasmid containing the SG-EGFR light chain gene and introducing EcoR I in the upstream of the light chain and Sal I in the downstream of the light chain was pUC-SG "EGFRL.
  • PUC-SG-EGFRH was excised using BamH I + Xba I, and inserted into the P Clonel2 vector BamH I + Xba I, named paonel2-SG-EGFRH.
  • EcoR I + Sal I was used to excise pUC-SG-EGFRL and insert it into the pClonel2-SG-EGFRH vector EcoR I + Sal I. It was named pClonel2-SG-EGFR.
  • the humanized antibody SG-HER gene of human epidermal growth factor receptor 2 (Her2) was synthesized by Shanghai JBES Gene Technology Co., Ltd.
  • the variable region of the light chain gene and the heavy chain gene is from Genentech in the United States.
  • the light chain gene of the company's humanized antibody Herceptin against epidermal growth factor receptor 2 is the same as the variable region of the heavy chain gene (see Carter et al., United States Patent No: 5,821,337) 0 contains the SG-HER heavy chain gene
  • the pUC19 plasmid that introduced the BamH I restriction site in the upstream region of the heavy chain and the Xho I restriction site in the downstream region of the heavy chain was named pUC-SG-HERH.
  • the pUC19 plasmid containing the SG-HER light chain gene and introducing EcoR I in the upstream region of the light chain and the Sal I restriction site in the downstream region of the light chain was pUC-SG-HERL.
  • PUC-SG-HERH was excised using BamH I + Xho I, and inserted into the pClonel2 vector BamH I + Xho I, named pCkmel2-SG-HERH.
  • BamH I + Xho I BamH I + Xho I
  • pCkmel2-SG-HERH Use EcoR I + Sal I to cut pUC-SG-HERL, insert it into the P Clonel2-SG-HERH vector EcoR I + Sal I, and name it pClonel2-SG-HERo
  • the human-mouse chimeric antibody SG-CD20 gene of anti-human CD20 was synthesized by Shanghai JBES Gene Technology Co., Ltd.
  • the variable region of the light chain gene and the heavy chain gene was compared with the human and mouse against CD20 of the United States IDEC Corporation.
  • the chimeric antibody IDEC-C2B8 (Rituximab) has the same light chain gene as the variable region of the heavy chain gene (see Anderson et al. United States Patent No: 6,399,061)
  • the pUC19 plasmid that introduced the Xba I digestion site into the BamH I digestion site and the heavy chain downstream region was named pUC-SG-CD20H. It contained the SG-CD20 light chain gene and introduced EcoR I and the light chain downstream region in the upstream region
  • the pUC19 plasmid introduced into the Sal I restriction site is pUC-SG-CD20L.
  • the end of the constant region Sai l pUC-SG-CD20H was excised using BamH I + Xba I, and inserted into the pClonel2 vector BamH I + Xba I, named pClonel2-SG-CD20H.
  • EcoR I + Sal I was used to cut pUC-SG-CD20L and insert it into the pClonel2-SG- CD20H vector EcoR I + Sal I. It was named pCkmel2-SG-CD20.
  • an intron is inserted between the transcription start site of the promoter and the translation start site of the antibody, and the intron is the third, 5 splicing sequence of the main advanced mRNA of the adenovirus Site and an immunoglobulin 3, a heterozygous intron for the splice site.
  • the heterozygous intron nucleotide sequence was synthesized by Shanghai Jibeisi Gene Technology Co., Ltd.
  • the Spe I restriction site was introduced into the upstream region of the intron and the EcoR I restriction site was introduced into the downstream region of the intron.
  • the pUC19 plasmid was named pUC-Intron.
  • Primer 22 5 'AAT TAC TAG TCA GGA ATT CA 3' (SEQ ID NO: 33).
  • Primer 23 5 'AGC TTG AAT TCC TGA CTA GT3' (SEQ ID NO: 34).
  • the two DNA oligonucleotide fragments were each mixed with 0.1 g, denatured at 100 t ⁇ for 5 minutes, and then slowly cooled to renaturation. After renaturation, T4 phage polynucleotide phosphoric acid was used for phosphorylation.
  • the phosphorylated linker was inserted into the EcoR I and Hind III sites of the pClonelO vector and named pClonelO-Linker. The multiple cloning sites of this vector were Spe I, EcoR I, Sal L Hind III, Xho I, and BamH I. And Xba l.
  • PUC-Intron was digested with Spe I + EcoR I, and the fragment was inserted into the Spe I + EcoR I digestion site of PDC315-linker, named pYQ20.
  • the multiple cloning sites of this vector were EcoR I, Sal L Hind III, Xho I, BamH I and Xba I.
  • PClonel2-SG-HER was cut out with EcoR I + Xho I, and inserted into the pYQ20 vector EcoR I + Xho I. It was named pYQ20-SG-HER.
  • Example 5 Human antibody SG-EGFR gene against human epidermal growth factor receptor 1 (EGFR), humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2), and anti-human CD20 Construction of human and mouse chimeric antibody SG-CD20 gene adenovirus vector
  • pBHGlO was purchased from Microbix Biosystem Inc. (Toronto), Canada. will pClonel2-SG-EGFR, pClonel2-SG-HER, pClonel2-SG-CD20 and pYQ20-SG-HER were digested with PAC I, inserted into the PAC I digestion sites of pBGHIO, and named pBGH10-SG-EGFR, pBGH10-SG-HE, pBGH10-SG-CD20 and pBGH10-YQSG-HER.
  • Example 6 Humans carrying anti-human epidermal growth factor receptor 1 (EGFR) antibody SG-EGFR gene, humanized antibodies against human epidermal growth factor receptor 2 (Her2) SG-HER gene and human anti-CD20
  • EGFR epidermal growth factor receptor 1
  • Her2 human epidermal growth factor receptor 2
  • HER human epidermal growth factor receptor 2
  • human anti-CD20 The telomerase catalytic subunit gene promoter and hypoxic promoter of murine chimeric antibody SG-CD20 gene control the recombination of proliferating adenoviruses expressing E1A and E1B, respectively.
  • the 293 cell line was purchased from Microbix Biosystem Inc. (Toronto), Canada. It was transformed from human embryonic kidney cells by shearing type 5 adenovirus DNA. It contains and expresses type 5 adenovirus E1 region. Transfection rate. A plasmid containing an adenovirus type 5 left arm and a plasmid containing an adenovirus type 5 right arm were co-transfected into 293 cells, and infectious adenovirus could be produced by homologous recombination.
  • telomerase catalytic subunit gene promoter was used to control the proliferation of adenoviruses expressing E1A and E1B, respectively.
  • Epidermal growth factor receptor 1 human antibody SG-EGFR gene proliferative adenovirus SG500-EGFR, telomerase catalytic subunit gene promoter and hypoxic promoter respectively control E1A and E1B expression proliferative adenovirus Anti-human epidermal growth factor receptor 2 (Her2) humanized antibody SG-HER gene proliferation adenovirus SG500-HER, telomerase catalytic subunit gene promoter and hypoxic promoter control E1A and E1B expression proliferation, respectively Adenovirus anti-human CD20 human mouse chimeric antibody SG-CD20 gene proliferation Adenovirus SG500-CD20 and telomerase catalytic subunit gene promoter and hypoxic promoter control the proliferation of adenovirus expression of E1A and E1B and Proliferating adenovirus SG500-YQ-HER, a humanized antibody against the human epidermal growth factor receptor 2 (Her2) gene and inserting a hybrid intron downstream of the human C
  • SG500-HER is an adenovirus type 5, which inserts the telomerase catalytic subunit gene promoter upstream of the El A coding start site and the hypoxia gene promoter upstream of the E1B coding start site, accompanied by 28133 -30818bp (partial sequence of E3 region) was deleted and a light chain containing a human cytomegalovirus (HCMV) IE promoter and a humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2) was inserted in E3 , Heavy chain genes and encephalomyocarditis virus polycistronic, SV40 poly A tail signal gene sequences, the other DNA sequences of the virus are the same as type 5 adenovirus.
  • HCMV human cytomegalovirus
  • SG500-CD20 (SG503) is an adenovirus type 5 in E1A
  • the telomerase catalytic subunit gene promoter is inserted upstream of the coding start site and the hypoxic gene promoter is inserted upstream of the E1B coding start site, with a deletion of 28133-30818bp (partial sequence of the E3 region), and the insertion at E3 contains Human cytomegalovirus (HCMV) IE promoter, light chain, heavy chain gene of human mouse-chimeric antibody SG-CD20 gene containing anti-human CD20, polycistronic of encephalomyocarditis virus, SV40 poly A tail signal gene sequence, The other DNA sequences of the virus are the same as those of adenovirus type 5.
  • HCMV Human cytomegalovirus
  • SG500-YQ-HER (SG-YQ-502) is a type 5 adenovirus.
  • the telomerase catalytic subunit gene promoter is inserted upstream of the E1A coding start site and the hypoxia gene promoter is inserted upstream of the E1B coding start site.
  • E3 containing the human cytomegalovirus (HCMV) IE promoter
  • the third guide sequence containing the major late-stage mRNA of adenovirus 5, the splice site and An immunoglobulin 3, a heterologous intron at the splice site, a light chain, a heavy chain gene, and an encephalomyocarditis virus containing the humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2)
  • the cistron, SV40 poly A tail signal gene sequence, and other viral DNA sequences are the same as type 5 adenovirus.
  • Example 7 A proliferating adenovirus carrying the humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2) and having a telomerase catalytic subunit gene promoter and a hypoxic promoter controlling the expression of E1A and E1B, respectively SG500-HER (SG502) specifically proliferates and replicates tumor cells with telomerase activity in vitro.
  • Human ovarian cell cancer cell line SKOV3, human breast cancer cell line BT-474 and normal fibroblast cell line BJ were purchased from ATCC in the United States.
  • SG502 was infected with human ovarian cancer cell line SKOV3 with telomerase activity.
  • Example 8 The telomerase catalytic subunit gene promoter and hypoxia promoter of the humanized antibody SG-HER gene carrying human epidermal growth factor receptor 2 (Her2) control E1A and Humanized antibody expressed in E1B-proliferated adenovirus SG500-HER (SG502) mice
  • the control adenovirus Ad5-Lac Z or recombinant adenovirus (SG502) was administered to Balb / c mice at 1 x 10 9 pfu and 5 x 10 9 pf «in the tail vein, and sandwich enzyme-linked immunosorbent assay was applied 1 week later.
  • ELISA sandwich enzyme-linked immunosorbent assay
  • Example 10 A telomerase catalytic subunit gene promoter and a hypoxia promoter carrying the humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2), a proliferating adenovirus SG500 that controls the expression of E1A and E1B, respectively -HER (SG502) in transplanted tumors in mice
  • a telomerase catalytic subunit gene promoter and a hypoxia promoter carrying the humanized antibody SG-HER gene of anti-human epidermal growth factor receptor 2 (Her2) proliferating adenovirus SG500-HER (E500) SG502) is used to treat transplanted tumors in mice. Studies have shown that it can efficiently express humanized antibodies against human epidermal growth factor receptor 2 (Her2) and inhibit human epidermal growth factor receptor 2 (Her2) tumor formation, growth and metastasis. . At the same time, the virus can proliferate in and be limited to tumor cells and can specifically kill tumor cells directly.
  • mice 4-5 week old mice were subcutaneously inoculated with human ovarian cell line SKOV3 cells 1 X 107, two weeks after the administration of l xl 0 9 pfu recombinant adenovirus SG502 proliferation or treatment with a control adenovirus Ad5-Lac Z of the same dose, which is not the treatment group and control adenovirus treated group after 4 weeks 3-fold increase in tumor
  • the SG502 in the treatment group did not increase the tumor significantly. See Figure 2 for the results.
  • Cell-specific adenovirus vectors includes an internal ribosome entry site

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Abstract

The invention provided a replicability virus which can express anti-tumour antibody or its segment with high efficiency and the use of it. The propagate of this virus was only in tumour cell. The nucleotide sequence which code anti-tumour antibody or its segment was inserted into the replicability virus genome, and the copies of this nucleotide sequence increased with the propagate of this replicability virus in tumour cell, so the virus can express anti-tumour antibody or its segment with high efficiency in tunour cell and restrained the upgrowth and tranfer of tumours.

Description

高效表达治疗肿瘤的  Highly expressive for treating tumors
抗体的复制型病毒及其用途 技术领域  Antibody-replicating virus and use thereof
本发明涉及生命科学领域， 具体涉及一种能高效表达治疗肿 瘤的抗体或抗体片段的肿瘤细胞内增殖的病毒及其用途。  The present invention relates to the field of life sciences, and in particular, to a virus that can proliferate in tumor cells capable of efficiently expressing antibodies or antibody fragments for treating tumors and uses thereof.
背景技术 Background technique
Kohler和 Milstein在 1975年创立的单克隆抗体制备技术， 为肿瘤的治疗提供一种新的方法一一肿瘤的导向治疗法。 在肿瘤 导向治疗研究初期， 人们给予极大的热情和不切合实际的期望， 但早期的临床研究的疗效十分不理想。 其主要原因为： 1. 在早期 的临床试验中应用的是鼠源性抗体， 在人体内会产生针对鼠源性 抗体的抗抗体（HAMA ) ， 它会中和治疗性的鼠源性抗体， 使其 被快速清除， 因此鼠源性抗体在人体内半衰期很短， 故疗效不确 切， 同时鼠源性抗体会导致人体过敏反应， 从而产生毒副作用； 2. 抗体的亲和力和特异性不高， 多数抗体特别是一些基因工程小 分子抗体或人源化抗体自身亲和力较低， 特异性不高， 从而不能 有效靶向肿瘤细胞， 因此其临床上抗肿瘤疗效不确切； 3. 肿瘤自 身抗原表达的差异及抗原的调变也是影响抗体治疗成功的因素。 近 5年来随着现代生物技术的发展， 抗体技术两个关键性技术获 得突破， 1. 人源化抗体技术和人抗体制备技术的成熟， 基本上可 以克服鼠源性抗体人用产生抗抗体的问题； 同时由于在人源化抗 体技术和人抗体中采用人的 Fc片段 (可结晶的片段， 是指用木瓜 酶消化免疫球蛋白所得的片段， 其分子量大约为 50 000)， 在人体 内的半衰期从鼠源性抗体小于 20 小时到人源化抗体和人抗体的 半衰期为数天、 甚至到 21天之久； 另外在人的 Fc片段的改造可 进一步提高对肿瘤的杀伤。 2. 抗体库的建立和筛选以及多价重组 抗体制备技术的发展使人们能够直接获得特异性强和亲和力高的 单克隆抗体， 如 SLAM 法 （ Selected Lymphocyte Antibody Method )制备的抗体的亲和力比杂交瘤技术制备的提高 1000倍。 抗体技术的发展终于使导向治疗研究走出低谷， 并在近几年取得 了突破性进展， 应用抗体治疗肿瘤初显曙光。 The monoclonal antibody preparation technology founded by Kohler and Milstein in 1975 provides a new method for tumor treatment-tumor-directed therapy. In the early days of tumor-directed treatment research, great enthusiasm and unrealistic expectations were given, but the early clinical studies were very unsatisfactory. The main reasons are: 1. In the early clinical trials, mouse-derived antibodies were used. Anti-murine antibodies (HAMA) will be produced in the human body, which will neutralize therapeutic mouse-derived antibodies. It is quickly cleared, so the mouse-derived antibody has a short half-life in the human body, so the effect is not exact. At the same time, the mouse-derived antibody can cause an allergic reaction in the human body, resulting in toxic side effects; 2. The affinity and specificity of the antibody is not high, Most antibodies, especially some genetically engineered small molecule antibodies or humanized antibodies, have low affinity and low specificity, so they cannot effectively target tumor cells, so their clinical antitumor efficacy is not clear; 3. Differences and modulation of antigens are also factors that affect the success of antibody therapy. With the development of modern biotechnology in the past 5 years, two key technologies of antibody technology have made breakthroughs. 1. The maturation of humanized antibody technology and human antibody preparation technology can basically overcome the use of mouse-derived antibodies to produce anti-antibodies for human use. Problems; At the same time, due to the use of human Fc fragments in humanized antibody technology and human antibodies (crystallizable fragments refer to fragments obtained by digesting immunoglobulins with papain, its molecular weight is about 50,000). The half-life is from mouse-derived antibodies to less than 20 hours to half-lives of humanized antibodies and human antibodies of several days, or even 21 days. In addition, the modification of human Fc fragments can further improve tumor killing. 2. Establishment and screening of antibody libraries and multivalent recombination The development of antibody preparation technology enables people to directly obtain monoclonal antibodies with high specificity and affinity. For example, the affinity of antibodies prepared by the SLAM method (Selected Lymphocyte Antibody Method) is 1000 times higher than that of hybridoma technology. The development of antibody technology has finally brought the research on guided therapy out of the trough, and has made breakthrough progress in recent years. The application of antibodies to treat tumors has dawned.
但目前对于抗体治疗实体瘤仍存在着两个难题： 1. 由于实体 肿瘤的细胞被致密的基质包裹， 抗体难以穿透这一屏障而到达肿 瘤细胞； 大多数实体肿瘤有淋巴回流障碍， 导致间质内压力升高， 因而阻止了抗体由血液中进入肿瘤实质； 小部分进入肿瘤内部的 抗体， 首先遇到血管周围的肿瘤细胞而被结合， 使得抗体无法到 达距离血管较远的肿瘤细胞。 2. 由于治疗肿瘤的抗体需要量极 多， 目前的生物工程生产比较困难； 同时由于需要量极多， 要求 其产品纯度极高， 因此价格非常昂贵。 故目前应用抗体治疗大体 积的实体肿瘤的疗效仍不理想， 很多研究建议对于实体肿瘤的抗 体治疗主要应用于肿瘤的微小残留或微转移灶中， 但这种疾病的 治疗需要长时间及大规模的多中心临床试验才能评估其疗效， 这 也使抗体在实体瘤的临床应用及推广产生一定的限制作用。  However, there are still two problems for antibody treatment of solid tumors: 1. Because the cells of solid tumors are wrapped by a dense matrix, it is difficult for antibodies to penetrate this barrier to reach tumor cells; most solid tumors have lymphatic reflux disorders, which leads to The increased intraplasmic pressure prevents the antibody from entering the tumor parenchyma from the blood; a small portion of the antibody that enters the interior of the tumor first encounters tumor cells around the blood vessel and is bound, making it impossible for the antibody to reach tumor cells that are far from the blood vessel. 2. Due to the extremely large amount of antibodies required to treat tumors, current bioengineering production is difficult; at the same time, because of the large amount of antibodies required, and the purity of their products is extremely high, the price is very expensive. Therefore, the curative effect of antibody treatment of large-volume solid tumors is still not satisfactory. Many studies suggest that antibody treatment of solid tumors is mainly used in small residual tumors or micrometastases. However, the treatment of this disease requires long-term and large-scale Only multi-center clinical trials can evaluate its efficacy, which also makes the clinical application and promotion of antibodies in solid tumors have a certain limiting effect.
众所周知， 长期以来， 肿瘤基因治疗的方法存在着基因的转 染有效率和抗癌基因的表达量均较低的缺陷。 而肿瘤特异性增殖 病毒的治疗方法是利用该类病毒只能特异性地在肿瘤细胞内复制 和增殖的性质， 随着病毒在肿瘤细胞内的复制和增殖导致肿瘤细 胞裂解， 释放出病毒颗粒， 再次感染其它肿瘤细胞， 再次增殖、 裂解， 如此产生放大效应， 使得病毒弥散到全身各个組织及脏器， 感染所有组织及脏器， 因而能感染所有肿瘤细胞， 从而杀灭局部 及转移的肿瘤。 由于肿瘤增殖病毒基本上不能在正常细胞中增殖， 所以不影响肿瘤细胞周围的正常细胞。  As we all know, for a long time, the methods of tumor gene therapy have the defects of efficient gene transfection and low expression of anti-cancer genes. The treatment method for tumor-specific proliferation viruses is to take advantage of the fact that such viruses can only specifically replicate and proliferate in tumor cells. As the virus replicates and proliferates in tumor cells, tumor cells are lysed and virus particles are released. Infect other tumor cells again, and proliferate and lyse again. This produces a magnifying effect, allowing the virus to spread to various tissues and organs throughout the body, infecting all tissues and organs, and thus infecting all tumor cells, thereby killing local and metastatic tumors. Since tumor proliferative virus is basically unable to proliferate in normal cells, it does not affect normal cells surrounding tumor cells.
但单独应用肿瘤特异性增殖病毒治疗仍有局限性， 首先， 肿 瘤形成的机制非常复杂， 异质性非常强， 病人与病人之间， 肿瘤 与肿瘤之间， 同一肿瘤不同肿瘤细胞之间均存在明显差异，就 P53 基因突变而言， 某一肿瘤病人的肿瘤细胞 P53基因突变， 这并不 意味着该病人的所有肿瘤细胞 P53基因都发生突变， 因此利用某 一肿瘤机制而增殖的病毒不足以杀灭所有肿瘤细胞。 其次， 肿瘤 组织中的物理因素如纤维化、 混入正常细胞以及坏死区域也可能 限制病毒扩散。 再次， 某此肿瘤的病毒受体（如柯萨奇病毒受体） 表达不充分限制病毒对其感染。 第四， 病人的免疫反应限制病毒 增殖和播散。目前，美国 ONYX药物公司尝试单独应用 Elb 55kDa 蛋白缺失型病毒（ONYX-015 ) 治疗肿瘤， 其临床有效率只有 15 - 20 % ( Nemunaitis, J. 等人， 癌症研究 ( Cancer Res. )， 2000， 60(22):6359； US 5677178; US 5801029 ) 。 However, the application of tumor-specific proliferative virus alone is still limited. First, the tumor The mechanism of tumor formation is very complicated and the heterogeneity is very strong. There are obvious differences between patients and patients, between tumors and tumors, and between different tumor cells in the same tumor. As far as the P53 gene mutation is concerned, the tumor of a tumor patient Cell P53 gene mutation, which does not mean that the patient's tumor cell P53 gene is mutated, so the virus proliferated by a certain tumor mechanism is not enough to kill all tumor cells. Second, physical factors such as fibrosis, incorporation into normal cells, and necrotic areas in tumor tissues may also limit virus spread. Third, the expression of viral receptors (such as Coxsackie virus receptor) in a certain tumor is insufficient to limit the infection of the virus. Fourth, the patient's immune response limits virus proliferation and spread. At present, ONYX Pharmaceutical Company of the United States has tried to apply Elb 55kDa protein-deficient virus (ONYX-015) alone to treat tumors, and its clinical effectiveness is only 15-20% (Nemunaitis, J. et al., Cancer Res., 2000, 60 (22): 6359; US 5677178; US 5801029).
本发明人创造性地将肿瘤的抗体治疗与肿瘤特异性增殖病毒 治疗方法相结合， 首次提出了肿瘤的抗体-病毒治疗方法， 即： 采用肿瘤特异性增殖病毒携带治疗肿瘤的抗体或抗体片段基因， 成功实现了在肿瘤细胞内病毒特异性复制和增殖， 形成高浓度病 毒， 直接破坏肿瘤细胞， 同时在肿瘤细胞内随病毒复制而实现所 携带的治疗肿瘤的抗体或抗体片段基因大量表达，产生协同作用。 本发明所述抗体-病毒治疗方法， 由于携带治疗肿瘤的抗体或抗 体片段基因的肿瘤增殖病毒在肿瘤组织中特异性复制及增殖， 其 高效表达肿瘤的抗体或抗体片段是在肿瘤組织内部产生， 因此克 服了治疗肿瘤的抗体或抗体片段难以进入实体瘤组织的困难； 同 时携带治疗肿瘤的抗体或抗体片段基因的肿瘤增殖病毒生产成本 低廉， 从而解决了治疗肿瘤的抗体或抗体片段大量生产成本高的 难题， 因而从疗效及经济上均优于肿瘤的抗体治疗法。 同时也比 单纯采用肿瘤特异性增殖病毒方法进行治疗更为有效。  The inventors creatively combined tumor antibody treatment with tumor-specific proliferation virus treatment methods, and first proposed an antibody-virus treatment method for tumors, that is, using tumor-specific proliferation viruses to carry antibodies or antibody fragment genes for treating tumors, Successfully achieved virus-specific replication and proliferation in tumor cells, forming high-concentration viruses, directly destroying tumor cells, and at the same time in tumor cells, the virus was replicated to achieve a large number of antibodies or antibody fragment genes for tumor treatment, generating synergy effect. According to the antibody-virus treatment method of the present invention, since the tumor proliferating virus carrying the antibody or antibody fragment gene for tumor treatment specifically replicates and proliferates in tumor tissue, the antibody or antibody fragment that highly expresses the tumor is produced inside the tumor tissue, Therefore, it overcomes the difficulty that antibodies or antibody fragments that treat tumors are difficult to enter into solid tumor tissues; tumor proliferative viruses that carry the antibodies or antibody fragment genes that treat tumors are cheap to produce, thereby solving the high cost of mass production of antibodies or antibody fragments that treat tumors The problem is therefore better than tumor antibody therapy in terms of efficacy and economy. At the same time, it is more effective than treating tumor-specific proliferation virus alone.
发明内容 本发明的一个目的在于提供一种重组病毒， 该病毒能特异性 地在肿瘤细胞内复制， 同时在该病毒基因组中包含一种编码治疗 肿瘤的抗体或抗体片段的核苷酸序列。 Summary of the invention It is an object of the present invention to provide a recombinant virus that can specifically replicate in tumor cells, and at the same time, the virus genome includes a nucleotide sequence encoding an antibody or an antibody fragment for treating a tumor.
本发明的另一个目的在于提供一种利用本发明的病毒治疗肿 瘤的方法， 其包括如下步骤： 1 )将该病毒体外或体内感染肿瘤细 胞， 2 )使病毒基本上限于肿瘤细胞内选择性复制及增殖， 导致在 肿瘤细胞内编码治疗肿瘤的抗体或抗体片段的核苷酸序列拷贝数 增加及治疗肿瘤的抗体或抗体片段表达量增加； 由此抑制肿瘤形 成、 生长及转移。 适当地， 本发明所述方法还包括在本发明所述 病毒感染肿瘤细胞之前、 同时和 /或之后施用化学抗肿瘤药物。  Another object of the present invention is to provide a method for treating tumors using the virus of the present invention, which comprises the steps of: 1) infecting the virus with tumor cells in vitro or in vivo, and 2) restricting the virus to selective replication in tumor cells. And proliferation, resulting in an increase in the number of copies of a nucleotide sequence encoding an antibody or antibody fragment for treating a tumor and an increase in expression of the antibody or antibody fragment for treating a tumor in a tumor cell; thereby inhibiting tumor formation, growth, and metastasis. Suitably, the method of the invention further comprises administering a chemical anti-tumor drug before, at the same time and / or after the virus of the invention infects tumor cells.
本发明的又一目的在于提供了一种本发明所述的病毒用于抑 制肿瘤细胞的生长的用途。 本发明最后提供了包含本发明重组病 毒及药学可接受载体的药物組合物。  Another object of the present invention is to provide the use of the virus of the present invention for inhibiting the growth of tumor cells. The invention finally provides a pharmaceutical composition comprising the recombinant virus of the invention and a pharmaceutically acceptable carrier.
附图说明 BRIEF DESCRIPTION OF THE DRAWINGS
图 1 显示携带抗人表皮生长因子受体 2(Her2)的人源化抗体 SG-HER基因以及端粒酶催化亚基基因启动子和缺氧启动子分别 控制 E1A及 E1B表达的增殖腺病毒 SG500-HER(SG502)在小鼠 体内表达人源化抗体。  Figure 1 shows a proliferating adenovirus SG500 that carries the humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2), the telomerase catalytic subunit gene promoter, and the hypoxic promoter that control the expression of E1A and E1B, respectively. -HER (SG502) expresses humanized antibodies in mice.
图 2 显示携带抗人表皮生长因子受体 2(Her2)的人源化抗体 SG-HER基因以及端粒酶催化亚基基因启动子及缺氧启动子分别 控制 E1A及 E1B表达的增殖腺病毒 SG500-HER(SG502)在棵鼠 体内治疗移植人卵巢癌 SKOV3的效果。  Figure 2 shows a proliferating adenovirus SG500 that carries the humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2), the telomerase catalytic subunit gene promoter, and the hypoxic promoter that control the expression of E1A and E1B, respectively. -HER (SG502) in the treatment of transplanted human ovarian cancer SKOV3 in mice.
具体实施方式 detailed description
在一个实施方案中， 本发明提供一类重组病毒， 其中编码治 疗肿瘤的抗体或抗体片段的核苷酸序列插入到能够在肿瘤细胞内 特异性增殖的病毒基因組中。该病毒选择性地在肿瘤细胞内增殖， 而在正常细胞内基本不增殖。 通过病毒在肿瘤细胞内复制增殖， 导致编码治疗肿瘤的抗体或抗体片段的核苷酸序列拷贝数增加， 从而治疗肿瘤的抗体或抗体片段基因表达量增加。 In one embodiment, the present invention provides a class of recombinant viruses in which a nucleotide sequence encoding an antibody or antibody fragment for treating a tumor is inserted into a virus genome capable of specifically proliferating in tumor cells. The virus selectively proliferates in tumor cells, but does not substantially proliferate in normal cells. The virus replicates and proliferates inside tumor cells, As a result, the copy number of the nucleotide sequence encoding the antibody or antibody fragment for treating a tumor is increased, thereby increasing the gene expression amount of the antibody or antibody fragment for treating a tumor.
在一个优选的实施方案中， 本发明的重組病毒中有至少一种 增殖必需基因的转录受肿瘤细胞特异性激活的顺式作用元件有效 控制。  In a preferred embodiment, the transcription of at least one essential gene for proliferation in the recombinant virus of the present invention is effectively controlled by a cis-acting element that is specifically activated by tumor cells.
在另一个优选的实施方案中， 本发明的重组病毒中包含至少 一种增殖必需区蛋白功能缺失， 从而基本上不能在正常细胞内增 殖， 但能在肿瘤细胞内特异性增殖。  In another preferred embodiment, the recombinant virus of the present invention contains at least one protein having a function of a region necessary for proliferation, and thus cannot substantially proliferate in normal cells, but can specifically proliferate in tumor cells.
本发明中可以选用多种野生型病毒， 只要它们在一种或多种 肿瘤细胞中可特异性增殖。 优选地， 所述病毒是腺病毒或单纯疱 奢病毒。  A variety of wild-type viruses can be used in the present invention, as long as they can specifically proliferate in one or more tumor cells. Preferably, the virus is an adenovirus or herpes simplex virus.
本发明所述病毒增殖必需区依所采用的病毒不同而不同， 但 容易为本领域技术人员知晓。 本发明所述所述 "病毒增殖必需基 因的转录受肿瘤细胞特异性激活的顺式作用元件的有效控制 "是 指病毒增殖必需基因与肿瘤细胞特异性激活的顺式作用元件在病 毒基因组中的相对位置依所用病毒的不同可以是多种多样的， 只 要该增殖必需基因的转录能够受到肿瘤细胞特异性激活的顺式作 用元件有效控制即可。 在一个实施方案中， 本发明所述病毒中包 含的肿瘤细胞特异性激活的顺式作用元件可以在病毒增殖必需基 因的转录起始位点之前适当的位置。 在本发明的另一个实施方案 中， 肿瘤细胞特异性激活的顺式作用元件可以替代病毒自身的启 动子。 在本发明的另一个实施方案中， 本发明所述病毒还可以是 在病毒增殖必需基因的转录起始点与编码起始位点之间区域包含 至少一种肿瘤细胞特异性激活的顺式作用元件的重组病毒。 其中 所述顺式作用元件可选自： 端粒酶催化亚基基因启动子， 端粒酶 The necessary regions for virus proliferation according to the present invention vary depending on the virus used, but are easily known to those skilled in the art. According to the present invention, "the transcription of genes necessary for virus proliferation is effectively controlled by cis-acting elements specifically activated by tumor cells" refers to The relative position can be varied depending on the virus used, as long as the transcription of the gene necessary for proliferation can be effectively controlled by cis-acting elements that are specifically activated by tumor cells. In one embodiment, the cis-acting element of tumor cell-specific activation contained in the virus of the present invention can be placed in an appropriate position before the transcription start site of the essential gene for virus proliferation. In another embodiment of the invention, a cis-acting element that is specifically activated by tumor cells can replace the virus's own promoter. In another embodiment of the present invention, the virus of the present invention may further comprise at least one cis-acting element that is specifically activated by tumor cells in the region between the transcription initiation site and the encoding initiation site of the virus essential genes Recombinant virus. The cis-acting element may be selected from: a telomerase catalytic subunit gene promoter, telomerase
RNA 组份基因启动子， 缺氧反应元件， 细胞 S 期特异性启动子 (E2F 启动子)， 甲胎蛋白增强子及启动子， 癌胚抗原增强子及启 动子， 酪氨酸酶增强子及启动子， 尿激酶纤维蛋白激活剂增强子 及启动子， ErbB2 增强子及启动子， ErbB3 增强子及启动子， ErbB4增强子及启动子， DF3乳腺癌相关抗原增强子， 前列腺素 特异性抗原增强子及启动子， 腺血管舒緩素增强子及启动子， EB 病毒中的 Orip, EB病毒 Orip中的 FR增强子， EB病毒 BamHI C-启动子。 RNA component gene promoter, hypoxia response element, cell S-phase specific promoter (E2F promoter), alpha-fetoprotein enhancer and promoter, carcinoembryonic antigen enhancer and promoter Activators, tyrosinase enhancers and promoters, urokinase fibrin activator enhancers and promoters, ErbB2 enhancers and promoters, ErbB3 enhancers and promoters, ErbB4 enhancers and promoters, DF3 breast cancer related Antigen enhancers, prostaglandin-specific antigen enhancers and promoters, adrenostatin enhancers and promoters, Orip in EB virus, FR enhancer in EB virus Orip, and EB virus BamHI C-promoter.
当所述病毒为腺病毒时， 所述增殖必需基因可以是病毒早期 基因， 包括但不限于下述中的一种或多种: E1A、 E1B、 E2或 E4。  When the virus is an adenovirus, the gene necessary for proliferation may be an early virus gene, including but not limited to one or more of the following: E1A, E1B, E2, or E4.
本发明所述的病毒还可以是在增殖必需区包含至少一种蛋白 功能缺失且能在肿瘤细胞内特异性增殖的重組病毒。 其中所述蛋 白功能缺失可以是对编码基因的点突变、 删除和 /或插入突变， 从 而使所述蛋白的功能缺失。 在本发明的一个实施方案中， 所述病 毒是一种重组腺病毒，其中该腺病毒 ElB55Kda基因发生点突变、 缺失突变和 /或插入突变， 导致 ElB55Kda蛋白质功能异常。 在本 发明的一个实施方案中， 所述病毒是一种重组腺病毒， 其中该腺 病毒 ElB 19kDa基因发生点突变、 缺失突变和 /或插入突变， 导致 E1B 19kDa蛋白质功能异常。 在本发明的又一个实施方案中， 所 述病毒是一种重组腺病毒， 其中该腺病毒 E1A基因发生点突变、 缺失突变和 /或插入突变， 导致 E1A蛋白质功能异常。  The virus according to the present invention may also be a recombinant virus comprising at least one protein in a region necessary for proliferation and having a specific function in tumor cells. The loss of protein function may be a point mutation, deletion, and / or insertion mutation of a coding gene, so that the function of the protein is lost. In one embodiment of the present invention, the virus is a recombinant adenovirus, wherein the adenovirus ElB55Kda gene has a point mutation, a deletion mutation, and / or an insertion mutation, resulting in abnormal ElB55Kda protein function. In one embodiment of the present invention, the virus is a recombinant adenovirus, wherein the adenovirus ElB 19kDa gene has a point mutation, a deletion mutation, and / or an insertion mutation, resulting in abnormal E1B 19kDa protein function. In still another embodiment of the present invention, the virus is a recombinant adenovirus, wherein the adenovirus E1A gene undergoes point mutation, deletion mutation and / or insertion mutation, resulting in abnormal E1A protein function.
在本发明的一个实施方案中，所述病毒是重組单纯疱疹病毒， 其中该单纯疱疹病毒 ICP6 基因通过点突变、 缺失突变、 插入突 变， 导致 ICP6 蛋白质功能异常。 在本发明的一个实施方案中， 所述病毒是重組单纯疱疹病毒， 其中该单纯疱疹病毒 拷贝 ICP34.5 基因通过点突变、 缺失突变、 插入突变， 导致双拷贝 ICP34.5蛋白质功能异常。  In one embodiment of the present invention, the virus is a recombinant herpes simplex virus, wherein the herpes simplex virus ICP6 gene causes abnormal function of the ICP6 protein through point mutation, deletion mutation, and insertion mutation. In one embodiment of the present invention, the virus is a recombinant herpes simplex virus, wherein the herpes simplex virus copies the ICP34.5 gene through a point mutation, a deletion mutation, or an insertion mutation, resulting in an abnormal function of the double copy ICP34.5 protein.
本发明所述的病毒中， 编码治疗肿瘤的抗体或抗体片段的核 苷酸序列包括， 但不限于选自编码抗新生血管生成的抗体或抗体 片段的核苷酸序列， 编码抗肿瘤细胞生长因子受体的抗体或抗体 片段的核苷酸序列， 编码抗肿瘤细胞膜抗原的抗体或抗体片段的 核苷酸序列， 编码抗肿瘤抗原的独特型单克隆抗体或抗体片段的 核苷酸序列。 本领域技术人员知晓， 只要能够具有治疗肿瘤作用 的抗体或抗体片段蛋白， 均可被用于本发明。 In the virus of the present invention, the nucleotide sequence encoding the antibody or antibody fragment for treating tumor includes, but is not limited to, the antibody or antibody selected from anti-neoangiogenesis. The nucleotide sequence of the fragment, the nucleotide sequence of the antibody or antibody fragment encoding the anti-tumor cell growth factor receptor, the nucleotide sequence of the antibody or antibody fragment encoding the anti-tumor cell membrane antigen, the idiotype of the anti-tumor antigen The nucleotide sequence of a cloned antibody or antibody fragment. Those skilled in the art know that any antibody or antibody fragment protein capable of treating tumors can be used in the present invention.
在本发明中， 所述的抗体可以选自下表中：  In the present invention, the antibody may be selected from the following table:
治疗性抗体 批准曰期 抗体名称 靶向抗原 抗体种类 公司名称 适应证 或临床试 验Therapeutic antibody Approval date Antibody name Targeted antigen Antibody type Company name Indication or clinical test
Panorex EpCam GlaxoSmithKline, Dukes' Panorex EpCam GlaxoSmithKline, Dukes'
鼠源性单抗 1995(德国） (Edrecolomab) (17-IA) Centocor 结肠癌  Murine mAb 1995 (Germany) (Edrecolomab) (17-IA) Centocor Colon Cancer
Rituxan  Rituxan
CD20 人-鼠嵌合抗体 Genentech 淋巴瘤 1997 (rituximab)  CD20 human-mouse chimeric antibody Genentech lymphoma 1997 (rituximab)
Herceptin  Herceptin
HER-2 人源化抗体 Genentech 乳腺癌 1998 (Trastuzumab)  HER-2 Humanized Antibody Genentech Breast Cancer 1998 (Trastuzumab)
人源化抗体 (偶 急性髓性  Humanized antibody (even acute myeloid
Mylotarg CD33 Wyeth Laboratories 2000  Mylotarg CD33 Wyeth Laboratories 2000
联毒素） 白血病  (Toxin) Leukemia
慢性淋巴  Chronic lymphatic
Millennium  Millennium
Campath CD52 人源化抗体 细胞性白 2001  Campath CD52 Humanized Antibody Cellular White 2001
Pharmaceuticals  Pharmaceuticals
血病  Blood disease
头颈部肿  Head and neck swelling
IMC-C225 EGFR 人 -鼠嵌合抗体 ImCIone System ΠΙ  IMC-C225 EGFR human-mouse chimeric antibody ImCIone System II
瘤  Tumor
抗 Titan  Anti-Titan
CeaVac 鼠源性单抗 大肠癌 nr id(CEA)* Pharmaceuticals  CeaVac mouse monoclonal antibody colorectal cancer nr id (CEA) * Pharmaceuticals
抗 ImCIone System  Anti-ImCIone System
BEC2 鼠源性单抗 肺癌 ΠΙ id(GD3)* Merck KGaA  BEC2 mouse monoclonal antibody lung cancer Π id (GD3) * Merck KGaA
Avastin  Avastin
VEGF 人源化鼠单抗 Genentech 肿瘤 m (Bevacizumab)  VEGF Humanized Mouse mAb Genentech Tumor m (Bevacizumab)
Epratuzumab  Epratuzumab
CD22 人源化单抗 Immunomedics 淋巴瘤 ηι (LymphoCide)  CD22 Humanized mAb Immunomedics Lymphoma ηι (LymphoCide)
Smart M195 急性髓性  Smart M195 Acute Myeloid
CD33 人源化鼠单抗 Protein Design Labs m (Zamyl) 白血病 HER-2/C CD33 Humanized Mouse MAb Protein Design Labs m (Zamyl) Leukemia HER-2 / C
Medarex, Immuno  Medarex, Immuno
MDX-210 D64(Fc Y 双功能抗体 m  MDX-210 D64 (Fc Y bifunctional antibody m
Designed Molecules  Designed Molecules
RI)  RI)
OvaRex CA125 鼠源性单抗 Altarex 卵巢癌 m OvaRex CA125 mouse monoclonal antibody Altarex ovarian cancer m
TheraCIMh-R TheraCIMh-R
EGFR 人源化鼠单抗 不详 头颈部癌 π 3  EGFR humanized mouse monoclonal antibody unknown head and neck cancer π 3
鼠源性单抗-化  Mouse monoclonal antibody
SGN-15 不详 Seattle Genetics 前列腺癌 π  SGN-15 Unknown Seattle Genetics Prostate Cancer π
疗药  Cure
抗 Titan  Anti-Titan
TriGem 鼠源性单抗 黑色素瘤 π id(GD2)* Pharmaceuticals  TriGem mouse monoclonal antibody melanoma π id (GD2) * Pharmaceuticals
Smart ID10 HLA DR 人源化抗体 Protein Design Labs 淋巴瘤 π  Smart ID10 HLA DR Humanized Antibody Protein Design Labs Lymphoma π
C242  C242
人源化鼠单抗- Humanized mouse mAb-
SB-408075 CanAg ImmunoGen 实体瘤 Ι /Π SB-408075 CanAg ImmunoGen solid tumor Ι / Π
化疗药交联物  Chemotherapeutic cross-links
antigen  antigen
a V P 3 Applied Molecular 大肠癌，  a V P 3 Applied Molecular colorectal cancer,
Vitaxin 人源化鼠单抗 ι /π integrin Evolution 肉瘤  Vitaxin humanized mouse mAb / π integrin Evolution sarcoma
4B5 人源抗体 Viventia Biotech 黑色素瘤 ΐ /π mesotheli  4B5 human antibody Viventia Biotech melanoma ΐ / π mesotheli
SSl(dsFv)PE38 dsFv-PE38 NeoPharm 实体瘤 ι /π n  SSl (dsFv) PE38 dsFv-PE38 NeoPharm solid tumor ι / π n
ABX-EGF EGFR 人的抗体 Abgenix 实体瘤 ι /π  ABX-EGF EGFR human antibody Abgenix solid tumor ι / π
墚  Liang
CEA、 放  CEA, put
iPentacea 双特异抗体 IBC Pharmaceuticals 肿瘤 Ι /Π  iPentacea Bispecific Antibody IBC Pharmaceuticals Tumor Ι / Π
射性核素  Radionuclide
2C4  2C4
(Omnitarg™ ,P HER2 人源化抗体 Genentech 肿瘤 π ertuzumab)  (Omnitarg ™, P HER2 humanized antibody Genentech tumor π ertuzumab)
BrevaRex MUC1 鼠源性单抗 AltaRex 肿瘤 I  BrevaRex MUC1 Murine mAb AltaRex Tumor I
• 抗独特型单抗， 扩号中为其所模拟的抗原 此外， 在本发明所述的病毒中， 所述编码治疗肿瘤的抗体或 抗体片段的核苷酸序列还优选地与启动子可操作连接。 所述启动 子包括， 但不限于 SV40启动子、 RSV LTR启动子、 人巨细胞病 毒 IE启动子和 /或肿瘤细胞特异性激活的顺式作用元件， 其中所 迷顺式作用元件可选自： 端粒酶催化亚基基因启动子， 端粒酶 RNA 組份基因启动子， 缺氧反应元件， 细胞 S 期特异性启动子 (E2F 启动子)， 甲胎蛋白增强子及启动子， 癌胚抗原增强子及启 动子， 酪氨酸酶增强子及启动子， 尿激酶纤维蛋白激活剂增强子 及启动子， ErbB2 增强子及启动子， ErbB3 增强子及启动子， ErbB4增强子及启动子， DF3乳腺癌相关抗原增强子， 前列腺素 特异性抗原增强子及启动子， 腺血管舒緩素增强子及启动子， EB 病毒中的 Orip, EB病毒 Orip中的 FR增强子， EB病毒 BamHI C-启动子。 任何能有效指导本发明所述治疗肿瘤的抗体或抗体片 段表达的启动子， 无论同源还是异源， 均能用于本发明。 在本发 明的上下文中， 术语 操作连接"是指并置的几个元件之间的 连接方式能够使各元件以各自的预定功能发挥作用。 例如启动子 能够使其后的核苷酸序列发生转录， 信号肽能够引导其后的肽链 进入细胞的分泌途径， 等等。 • Anti-idiotype monoclonal antibody, which is the antigen it mimics in the extension. In addition, in the virus of the present invention, the nucleotide sequence encoding the antibody or antibody fragment for treating tumor is preferably operable with a promoter. connection. The promoter includes, but is not limited to, the SV40 promoter, the RSV LTR promoter, the human cytomegalovirus IE promoter, and / or cis-acting elements that are specifically activated by tumor cells, wherein the cis-acting elements can be selected from: Telomerase catalytic subunit gene promoter, telomerase RNA component gene promoter, hypoxia response element, cell S-phase specific promoter (E2F promoter), alpha-fetoprotein enhancer and promoter, carcinoembryonic antigen enhancer and promoter, tyrosinase enhancer and promoter, urokinase fibrin activator enhancer and promoter, ErbB2 enhancer and Promoter, ErbB3 enhancer and promoter, ErbB4 enhancer and promoter, DF3 breast cancer-associated antigen enhancer, prostaglandin-specific antigen enhancer and promoter, adrenal relaxin enhancer and promoter, EB virus Orip, FR enhancer in EB virus Orip, EB virus BamHI C-promoter. Any promoter, whether homologous or heterologous, that can effectively direct the expression of antibodies or antibody fragments for treating tumors according to the present invention can be used in the present invention. In the context of the present invention, the term "operably linked" refers to the manner in which several elements juxtaposed are connected in such a way that each element can perform its intended function. For example, a promoter can transcribe the nucleotide sequence that follows Signal peptides can guide subsequent peptide chains into the cell's secretory pathway, and so on.
本发明中， 构建肿瘤特异性病毒， 使得病毒在肿瘤细胞内特 异性增殖及复制， 而在正常细胞不增殖及复制， 主要通过以下方 法实现：  In the present invention, a tumor-specific virus is constructed so that the virus specifically proliferates and replicates in tumor cells, but does not proliferate and replicate in normal cells, mainly through the following methods:
(1) 对病毒增殖必需基因的选择性控制： 基因转录激活的调 节受反式作用因子（如转录因子）与顺式作用元件相互作用的影 响。 当缺乏或出现某些转录因子会影响基因的转录水平。 本发明 应用肿瘤组织特异性激活顺式作用元件（包括启动子或增强子） 控制目的基因， 使该目的基因特异性在肿瘤细胞内表达， 而在正 常的细胞内不表达或低水平表达。 应用肿瘤組织特异性启动子或 增强子控制病毒增殖及复制的必需基因， 从而使病毒增殖必需基 因只能在肿瘤的细胞中表达，导致病毒只能在肿瘤的细胞内增殖， 而在正常细胞内基本不增殖。  (1) Selective control of genes necessary for virus proliferation: The regulation of gene transcription activation is affected by the interaction of trans-acting factors (such as transcription factors) and cis-acting elements. When a certain transcription factor is missing or present, it can affect the transcription level of the gene. The invention uses tumor tissue to specifically activate cis-acting elements (including promoters or enhancers) to control the target gene, so that the target gene is specifically expressed in tumor cells, but not expressed or expressed at low levels in normal cells. The tumor tissue-specific promoter or enhancer is used to control the essential genes for virus proliferation and replication, so that the essential genes for viral proliferation can only be expressed in tumor cells, resulting in the virus can only proliferate in tumor cells and in normal cells Basically no proliferation.
本发明所迷肿瘤细胞特异性激活顺式作用元件可以是下述任 何一种：  The tumor cell-specific activation cis-acting element of the present invention may be any of the following:
A. 端粒酶催化亚基 (TERT)启动子及端粒酶 RNA组份基因 启动子 A. Telomerase catalytic subunit (TERT) promoter and telomerase RNA component genes Promoter
大约 90%肿瘤细胞具有端粒酶活性， 而极大多数正常细胞端 粒酶均为阴性， 这表明端粒酶可作为肿瘤细胞一种特征性标记， 到目前为止， 人的端粒酶由三个部分组成，1.RNA 组份 (hTERC：)， 它作为端粒重复合成的内源性模板 ;2. 端粒酶结合蛋白 (TEP1);3. 端粒酶催化亚基 (telomerase catalytic subunit, 简写 TERT)， 也有 人称为端粒綠逆转录醇基因 (telomerase reverse transcriptase gene)。 RNA組份和端粒酶催化亚基是端粒酶活性所必需的成份。 最近研究表明端粒酶催化亚基 (hTERT) 在端粒晦活性中起决定 作用， 在极大多数肿瘤细胞或肿瘤细胞株中端粒酶催化亚基呈高 水平表达， 而正常细胞中不表达或低水平表达。 因此端粒酶催化 亚基启动子及端粒酶 RNA 组份基因启动子在极大多肿瘤细胞内 呈激活状态。  About 90% of tumor cells have telomerase activity, and most normal cells are negative for telomerase, which indicates that telomerase can be used as a characteristic marker of tumor cells. So far, human telomerase consists of three It consists of 1. RNA component (hTERC :), which serves as an endogenous template for repeated telomere synthesis; 2. Telomerase binding protein (TEP1); 3. Telomerase catalytic subunit, TERT), also known as telomerase reverse transcriptase gene. RNA components and telomerase catalytic subunits are necessary components for telomerase activity. Recent studies have shown that the telomerase catalytic subunit (hTERT) plays a decisive role in telomere activity. Telomerase catalytic subunits are expressed at high levels in most tumor cells or tumor cell lines, but not in normal cells. Or low-level expression. Therefore, the telomerase catalytic subunit promoter and the telomerase RNA component gene promoter are activated in a large number of tumor cells.
人与鼠的 TERT基因的顺式作用元件最近均已被克隆出来， 其序列 GC含量高， 缺少 TATA和 CAAT盒， 但含有多个转录因 子的结合位点， 包括转录激活因子 Myc、 SP1 和转录抑制因子 Madl> p53、 MZF2等结合位点。通过对人的 TERT基因（hTERT) 启动子 5，端进行截短， 构建不同长度的启动子缺失突变体， 来 研究该启动子的活性， 结果发现， 在转录起始位点上游存在一个 至少 181bp的启动子核心区域， 这个区域对保持该启动子的活性 很重要。 hTERT基因启动子核心区域内含有 E-box ( CACGTG ) 和 SP1结合位点。 虽然 SP1转录因子在端粒酶阴性的体细胞中表 达上调， 它与其结合位点的结合对 hTERT基因转录的起始有促 进作用， 但相比之下， 调节 hTERT基因转录表达的主要决定因 素是与 E-盒 (E-box)结合的因子， 如 Myc、 Madl等。 Myc蛋白的 高表达， 可以明显提高 hTERT基因启动子的活性， 从而促进基 因转录和增强端粒酶活性。 除转录起始位点上游的 E-box ( - 187bp - - 182b ) 以外， 在 hTERT基因转录起始位点下游、 翻 译起始位点之前还存在一个 E-box ( + 22bp ~ + 27bp ) , 这个下 游的 E-box 可以被 Myc 和 Madl 结合， 分别发挥激活和抑制 hTERT基因转录活性的作用。 最新的研究认为， hTERT基因启 动子转录起始位点下游的 E-box序列， 是端粒酶阴性细胞负向调 节机制的靶向位点， 我们在转录起始位点下游添加 E-box序列拷 贝数， 可以明显抑制端粒酶阴性细胞的 hTERT基因启动子活性， 而在端粒酶阳性的细胞中， E-box序列即有可能转变为能被激活 因子如 Myc家族成员 USF ( Upstream stimulatory factor ) 结合、 并激活 hTERT基因转录的正向调节位点。 同样通过启动子 5，端 序列截短构建不同的缺失突变体的实验还发现:在 hTERT基因启 动子核心序列上游还存在一个沉默子（Silencer ) , 约 400bp ( - 776bp ~― 378bp ) , 其间含有多个 MZF2 ( Myeloid-specific zinc finger protein 2 ) 结合单元（Motif) 。 对这些位点引入突变， 结 果引起 hTERT基因转录的激活， 而 MZF2的过度表达及与其结 合单元的相互作用则可以引起 hTERT基因转录的下调和端粒酶 活性的降低。 实验证明 hTERT基因启动子核心序列上游存在的 沉默子是 hTERT基因的负向调节区， MZF2是 hTERT基因的负 向调节因子。 MZF2 不仅在骨髓细胞中表达， 在不同組织来源的 细胞系中也有表达， 表示 MZF2对 hTERT基因的负向调节功能 具有普遍性。 这样看来， hTERT基因的表达和端粒酶的活性被多 种因素包括激活因子和抑制因子所牢牢控制， 其调控的主要部位 位于 hTERT基因转录的启动子结构区。 The cis-acting elements of human and mouse TERT genes have been recently cloned. Their sequences have high GC content, lack TATA and CAAT boxes, but contain multiple binding sites for transcription factors, including transcription activation factors Myc, SP1, and transcription. Inhibitors Madl> p53, MZF2 and other binding sites. By truncating the 5 end of the human TERT gene (hTERT) promoter and constructing promoter deletion mutants of different lengths, the activity of the promoter was studied. As a result, at least 181 bp upstream of the transcription start site was found. The promoter core region, this region is important to maintain the activity of the promoter. The core region of hTERT gene promoter contains E-box (CACGTG) and SP1 binding sites. Although the expression of SP1 transcription factor is up-regulated in telomerase-negative somatic cells, and its binding site promotes the initiation of hTERT gene transcription, in contrast, the main determinant of regulating hTERT gene expression is E-box binding factors, such as Myc, Madl, etc. The high expression of Myc protein can significantly increase the activity of the hTERT gene promoter, thereby promoting gene transcription and enhancing telomerase activity. Except the E-box upstream of the transcription start site (- 187bp--182b), there is an E-box (+ 22bp ~ + 27bp) downstream of the hTERT gene transcription start site and before the translation start site. This downstream E-box can be combined by Myc and Madl. They play a role in activating and inhibiting the transcriptional activity of hTERT genes. The latest research believes that the E-box sequence downstream of the transcription start site of the hTERT gene promoter is the target site of the negative regulation mechanism of telomerase negative cells. We added the E-box sequence downstream of the transcription start site Copy number can significantly inhibit hTERT gene promoter activity in telomerase-negative cells, and in telomerase-positive cells, the E-box sequence may be transformed into an activation factor such as the Myc family member USF (Upstream stimulatory factor ) Binds and activates positive regulatory sites for hTERT gene transcription. Similarly, the experiment of constructing different deletion mutants by promoter 5 truncation of the terminal sequence also found that: a silencer (Silencer), approximately 400bp (-776bp ~ 378bp), is present upstream of the core sequence of the hTERT gene promoter. Multiple MZF2 (Myeloid-specific zinc finger protein 2) binding units (Motif). Mutations were introduced into these sites, resulting in activation of hTERT gene transcription, and overexpression of MZF2 and interaction with its binding unit can cause down regulation of hTERT gene transcription and decrease in telomerase activity. The experiment proved that the silencer upstream of the core sequence of hTERT gene promoter is the negative regulatory region of hTERT gene, and MZF2 is the negative regulatory factor of hTERT gene. MZF2 is not only expressed in bone marrow cells, but also in cell lines of different tissues, indicating that MZF2 has universal negative regulation of hTERT gene. In this way, the expression of hTERT gene and telomerase activity are tightly controlled by a variety of factors including activators and inhibitors. The main site of its regulation is located in the promoter structural region of hTERT gene transcription.
B. 缺氧反应元件 (HRE)  B. Hypoxia response element (HRE)
肿瘤细胞不断增殖， 其不断增多的肿瘤细胞数导致细胞耗氧 量不断增加， 因此在实体瘤中普遍存在着缺氧状态。 极大多数实 体瘤细胞均高效表达转录因子一缺氧诱导因子 l a ( HIF-1 α ) ， 该转录因子可与缺氧反应元件 (hypoxia-response element, HRE) 结合， 促进其转录， 因此在实体瘤中缺氧反应元件 (HRE)可激活 其转录水平。 HRE可来源于血管生长因子基因的启动子、 促红细 胞生成素基因的启动子、 葡萄糖载体蛋白 1基因的启动子、 血红 素加氧酶基因的启动子和诱导型一氧化氧合酶基因的启动子等。 Tumor cells continue to proliferate, and the increasing number of tumor cells leads to an increase in the oxygen consumption of the cells. Therefore, hypoxia is common in solid tumors. Most solid tumor cells express the transcription factor-hypoxia-inducible factor la (HIF-1α) efficiently. The transcription factor can be combined with a hypoxia-response element (HRE) to promote its transcription, so the hypoxia-response element (HRE) can activate its transcription level in solid tumors. HRE can be derived from the promoter of the angiogenesis factor gene, the promoter of the erythropoietin gene, the promoter of the glucose carrier protein 1 gene, the promoter of the heme oxygenase gene, and the promoter of the inducible nitric oxide synthase gene. Child and so on.
C. 细胞 S期特异性启动子 (E2F-1启动子）  C. Cell S-phase specific promoter (E2F-1 promoter)
E2F - 1基因的表达同细胞周期相关，它是广泛表达的生长调 控基因， 在细胞 S期呈现出峰值转录活性。 RB蛋白及 RB家族的 其它成员， 可与 E2F - 1 組成特异性复合体， 遏制其激活转录的 能力。 因而， E2F - 1受到 RB的下调控。 在多种肿瘤细胞中， 由 于 Rb/P16INK4 o /Cydin D信号转导途径异常， 从而导致 E2F-1 基因高表达。 因此 E2F-1顺式作用元件在极大多数肿瘤细胞处于 激活状态。 The expression of E2F-1 gene is related to the cell cycle, it is a widely expressed growth-regulating gene, and it exhibits peak transcriptional activity in the S phase of cells. The RB protein and other members of the RB family can form specific complexes with E2F-1 to curb its ability to activate transcription. Thus, E2F-1 is down-regulated by RB. In various tumor cells, since the Rb / P16INK4 o / C ydin D abnormal signal transduction pathway, resulting in high expression of E2F-1 gene. Therefore, E2F-1 cis-acting element is activated in most tumor cells.
D. 甲胎蛋白（AFP) 增强子及启动子  D. Alpha-fetoprotein (AFP) enhancer and promoter
甲胎蛋白是一癌胚蛋白， 它主要限于内胚层起源的分化組织 (如卵黄嚢、 胎肝、 内脏等） 。 它的表达水平随组织及发育阶段 而有较大的变化。 AFP在临床受到关注的原因是因为在大多数肝 癌病人 AFP血浆浓度增高， 一些疾病晚期的病人 AFP血浆浓度 也升高。 病人血浆中 AFP增高是由肝细胞癌的 AFP表达引起的， 在肝癌周围的正常组织未出现 AFP的表达。 因此， 表达 AFP基 因是肝癌细胞的特性。  Alpha-fetoprotein is a carcinoembryonic protein, which is mainly limited to differentiated tissues of origin of endoderm (such as vitelline, fetal liver, viscera, etc.). Its expression level varies greatly with tissues and developmental stages. The reason why AFP is of clinical concern is that the plasma concentration of AFP increases in most patients with liver cancer, and the plasma concentration of AFP increases in some patients with advanced disease. The increase of AFP in the patient's plasma is caused by the expression of AFP in hepatocellular carcinoma, and AFP expression does not appear in normal tissues around liver cancer. Therefore, the expression of AFP genes is characteristic of liver cancer cells.
据已出版的报道， AFP转录顺式作用元件对产生 AFP的细胞 相关的细胞蛋白（如转录因子及共同因子）敏感， 例如 AFP结合 蛋白。 转录顺式作用元件中至少需包含一个 AFP 启动子和一个 AF 增强子。源自 ΑΓ 基因的细胞特异性转录顺式作用元件已被 鉴定。在 5，端距转录起始点上游近的序列中含有 AFP调控区（包 括启动子、 假定沉默子及增强子） 。 在 AFP调控区中， 人 AFP增强子位于 -3954到 -3335之间， 人 AFP启动子位于 -174到 +29 (均相对于转录起始点） 。 并置这 两种元件可产生一有全面功能的 AFP转录顺式作用元件。 Ido等 文献出处描述了在肝癌细胞中特异表达的一长约 259bp启动子片 段。 （nt-230到 +29 ) 。 AFP增强子位于 -3954到 -3335之间， 包 含两个区， 分别命名为 和8。 启动子区包含了典型的 TATA盒 和 CAAT盒。 AFP转录调控元件最好要包含一个增强子区。当然， AFP的转录顺式作用元件含有两个增强子就更好。 According to published reports, AFP transcriptional cis-acting elements are sensitive to cell proteins (such as transcription factors and common factors) associated with AFP-producing cells, such as AFP-binding proteins. At least one AFP promoter and one AF enhancer must be included in the transcriptional cis-acting element. Cell-specific transcriptional cis-acting elements derived from the AΓ gene have been identified. At 5, the sequence immediately upstream of the start of transcription contains AFP regulatory regions (including promoters, putative silencers and enhancers). In the AFP regulatory region, the human AFP enhancer is located between -3954 and -3335, and the human AFP promoter is located between -174 and +29 (both relative to the transcription start point). Juxtaposing these two elements produces a fully functional AFP transcriptional cis-acting element. Ido et al. Described a 259 bp promoter fragment that is specifically expressed in liver cancer cells. (Nt-230 to +29). The AFP enhancer is located between -3954 and -3335, and contains two regions named and 8 respectively. The promoter region contains the typical TATA box and CAAT box. AFP transcriptional regulatory elements preferably include an enhancer region. Of course, it is even better that the transcriptional cis-acting element of AFP contains two enhancers.
关于 AFP 的转录顺式作用元件可以包含任何数目的结构元 件， 而不是限于一个启动子， 一个增强子； 它可以是一个 AFP增 强子同 AFP启动子； 或一个 AFP启动子同一个异种增强子； 或 异种启动子同 AFP增强子； 或前述元件的多重组合。 AFP的转录 顺式作用元件中的启动子和增强子同编码感兴趣基因的序列可以 以任何方向及距离存在， 只要元件内 AFP细胞特异性转录活性存 在。腺病毒载体中还可包含一个 AFP的转录调控元件内源性沉默 子， 该元件亦可删除。  The transcriptional cis-acting element on AFP may include any number of structural elements, and is not limited to one promoter, one enhancer; it may be one AFP enhancer and AFP promoter; or one AFP promoter and one heterogeneous enhancer; Or heterologous promoter and AFP enhancer; or multiple combinations of the aforementioned elements. AFP transcription The promoter and enhancer in the cis-acting element and the sequence encoding the gene of interest can exist in any direction and distance, as long as the AFP cell-specific transcriptional activity exists within the element. The adenoviral vector can also contain an AFP transcriptional regulatory element endogenous silencer, which can also be deleted.
E. 癌胚抗原 (CEA) 增强子及启动子  E. Carcinoembryonic Antigen (CEA) Enhancer and Promoter
CEA是一大小为 18000道尔顿、 与肿瘤相关的糖蛋白抗原， 主要出现在胃肠道内胚层生发的肿瘤， 如结直肠癌、 胃癌、 胰腺 癌，在一些其他腺癌如乳腺癌、肺癌亦有表现。由于在大多数 CEA 阳性肿瘤的病人循环中可以检测到 CEA, 因而 CEA在临床上受 到关注。 在肺癌， 约 50%的病例可在循环中检测到 CEA, 在腺癌 CEA的浓度超过 20ng/L, 近 50%的胃癌患者血清 CEA阳性。  CEA is a tumor-associated glycoprotein antigen with a size of 18,000 Daltons. It mainly occurs in the tumors of the gastrointestinal endoderm, such as colorectal cancer, gastric cancer, and pancreatic cancer. It is also found in some other adenocarcinomas such as breast and lung cancer. There are performances. Because CEA can be detected in the circulation of patients with most CEA-positive tumors, CEA has received clinical attention. In lung cancer, CEA can be detected in the circulation in about 50% of cases, CEA concentrations in adenocarcinoma exceed 20ng / L, and nearly 50% of patients with gastric cancer have positive CEA.
CEA的 5，端序列中呈现出细胞特异性活性。 CEA启动子区， 位于该基因 5，端距转录起点上游的头 424个核苷酸， 通过在产 生 CEA细胞比非产生 CEA的 Hela细胞株表现出更高的启动子 活性而展示出它的细胞特异性。 另外， 细胞特异性增强子区也被 3 000565 CEA showed cell-specific activity in the 5'-end sequence. The CEA promoter region, located at the first 424 nucleotides of the gene 5 upstream of the start of transcription, demonstrates its cells by showing higher promoter activity in CEA-producing cells than in non-CEA-producing Hela cell lines. Specificity. In addition, the cell-specific enhancer region is also 3 000565
发现， 可参阅 PCT/GB/02456。 CEA启动子、 假定沉默子及增强 子元件似乎包含在距起始转录点 14.5Kb的区域中。 对 CEA基因 5，端区域进一步研究发现， 距转录起始点上游的两个区 (-13.6 到 -10.7Kb,-6.1KbA到 -4.0Kb)， 当它们同多聚体化启动子相连时，可 使报告基因在产生 CEA的 Lovo及 SW1463细胞株的表达增高且 具特异性。 Richard 等也将启动子定位于距转录起始点 -90bp 到 +69bp之间的区域， 其中 -41bp到 -18bp之间的序列对表达来说是 必需的。 PCT/GB/02546描述了一系列 5， 端呈现细胞特异性的片 段， 包括如下序列： nt-299到 nt+69; nt-90到 nt+69; nt-13600到 -ntl0600, nt-6100 到 nt-3800 (均相对于转录起始点而言） 。 另 外，通过 CEA基因中的 -402到 +69的片段也可使相连基因具有组 织特异性转录活性。 Findings can be found in PCT / GB / 02456. The CEA promoter, putative silencer, and enhancer elements appear to be contained within a region of 14.5 Kb from the starting transcription point. Further studies on the CEA gene 5 terminal region revealed that two regions (-13.6 to -10.7Kb, -6.1KbA to -4.0Kb) upstream of the transcription initiation point, when they are linked to a multimerized promoter, The expression of the reporter gene in Lovo and SW1463 cell lines producing CEA was increased and specific. Richard et al. Also positioned the promoter in a region between -90bp and + 69bp from the start of transcription, with sequences between -41bp and -18bp necessary for expression. PCT / GB / 02546 describes a series of five, cell-specific fragments that include the following sequences: nt-299 to nt + 69; nt-90 to nt + 69; nt-13600 to -ntl0600, nt-6100 to nt-3800 (all relative to the start of transcription). In addition, the -402 to +69 fragment in the CEA gene can also provide tissue-specific transcriptional activity to the linked gene.
这里所提及的 CEA顺式作用元件来自哺乳动物细胞，包括但 不局限于人类细胞。 因此， 只要载体中必需功能区存在， 任何一 种 CEA的顺式作用元件皆可被利用。  The CEA cis-acting elements mentioned here are derived from mammalian cells, including but not limited to human cells. Therefore, any cis-acting element of CEA can be used as long as the necessary functional regions are present in the carrier.
F. 酪氨酸酶增强子及启动子  F. Tyrosinase enhancer and promoter
人酪氨酸酶基因在黑色素细胞特异性表达的顺式作用元件。 该元件含有一长约 20bp名为酪氨酸酶远端元件（TDE ) 的序列， 包含- CATGTG序列， 位于距转录起始点的 - 1874到 - 1835之 间。 一包含人酪氨酸酶基因从- 209到 + 61 间序列的启动子， 被 发现可以引导基因在黑色素细胞的特异性表达。 与此相似， 小鼠 酪氨酸酶 5，端也有类似的一段序列登录基因库。 小鼠的 TRP - 1 基因的最小启动子已被识别， 包括对于转录起始点 - 44 到 + 107 之间的序列。  A cis-acting element that specifically expresses human tyrosinase gene in melanocytes. This element contains a 20-bp long sequence called the tyrosinase distal element (TDE), which contains the -CATGTG sequence, located between -1874 and -1835 from the start of transcription. A promoter containing a human tyrosinase gene sequence from -209 to +61 was found to direct specific expression of the gene in melanocytes. Similarly, the mouse tyrosinase 5 has a similar sequence in the gene bank. The smallest promoter of the mouse TRP-1 gene has been identified, including sequences between -44 and +107 for the transcription start point.
在一些实例中， 来自人酪氨酸酶基因 5，端序列的黑色素细 胞特异性的转录调控元件包含了相对于转录起始点一 231到 + 65, 或含有 - 1956到 - 1716之间的序列。黑色素细胞特异性的转录调 N2003/000565 In some examples, a melanocyte-specific transcriptional regulatory element derived from the human tyrosinase gene 5, terminal sequence comprises a sequence from 231 to +65, or containing a sequence between -1956 and -1716. Melanocyte-specific transcription regulation N2003 / 000565
控元件还可以包括以上两个并置的序列。 据报道， 距人酪氨酸酶 转录起始点 - 1956 到 - 1716 间的序列能协同同种或异种启动子 所启动的可搡作连接报告基因在黑色素细胞特异性表达。 因而， 在一些实例中， 黑色素细胞特异性的转录调控元件包含了异种启 动子及与之可操作相连的从 - 1956到 - 1716间的序列。 The control element may also include the above two juxtaposed sequences. It has been reported that sequences from -956 to -1716 from the transcriptional start point of human tyrosinase can cooperate with the same or heterologous promoter to activate the reporter gene specific expression in melanocytes. Thus, in some examples, melanocyte-specific transcriptional regulatory elements include heterologous promoters and sequences from-1956 to-1716 operatively linked to them.
G. 尿激酶纤维蛋白溶酶原激活剂 （uPA )增强子及启动子 uPA蛋白及其受体 uPAR, 在大多数常见肿瘤中有表达， 且 在肿瘤的转移过程中发挥着重要的作用。 它们涉及乳腺癌、 直肠 癌、 前列腺癌、 肝癌、 肺癌、 子宫癌。 有关 uPA及 uPAR的转录 顺式作用元件的序列已得到广泛的研究。  G. Urokinase plasminogen activator (uPA) enhancer and promoter uPA protein and its receptor uPAR, which are expressed in most common tumors, and play an important role in tumor metastasis. They involve breast cancer, rectal cancer, prostate cancer, liver cancer, lung cancer, and uterine cancer. The sequence of cis-acting elements related to the transcription of uPA and uPAR has been extensively studied.
H. ErbB2增强子及启动子  H. ErbB2 enhancer and promoter
其为乳腺癌细胞特异性激活增强子及启动子。 C-erbB2/neu 基因为一转化基因， 它编码一 185KD的上皮生长因子受体相关性 跨膜蛋白。 在人类， C-erbB2/neu 蛋白在胎儿发育过程中表达； 在成人， 这种蛋白用免疫组化的方法在许多正常組织上皮难以检 测到。 C-erbB2/neu 基因的扩增及过度表达， 同人类的许多肿瘤 相关， 如乳腺癌、 卵巢癌、 子宫癌、 前列腺癌、 胃癌及肺癌。 在 乳腺癌及卵巢癌， C-erbB2/neu 蛋白过度表达的后果已得到充分 的研究。 C-erbB2/neu蛋白在 20 w 40%的乳腺癌及 30%的卵巢癌 中过度表达， 它同这两种类型疾病较差的预后相关。  It is a breast cancer cell-specific activation enhancer and promoter. The C-erbB2 / neu gene is a transforming gene that encodes a 185KD epithelial growth factor receptor-related transmembrane protein. In humans, the C-erbB2 / neu protein is expressed during fetal development; in adults, this protein is difficult to detect in many normal tissue epitheliums by immunohistochemistry. C-erbB2 / neu gene amplification and overexpression are related to many human tumors, such as breast cancer, ovarian cancer, uterine cancer, prostate cancer, gastric cancer and lung cancer. In breast and ovarian cancer, the consequences of C-erbB2 / neu protein overexpression have been fully studied. C-erbB2 / neu protein is overexpressed in 20% of 40% of breast cancer and 30% of ovarian cancer, which is related to the poor prognosis of these two types of diseases.
人、 大鼠、 小鼠的 C-erbB2/neuTREs已被识别且表现出针对 表达 C-erbB2/neu细胞的特异性转录活性。  Human, rat, and mouse C-erbB2 / neuTREs have been identified and have shown specific transcriptional activity against C-erbB2 / neu expressing cells.
ErbB3增强子及启动子， 其为乳腺癌细胞特异性激活增强子 及启动子。  ErbB3 enhancer and promoter, which is a breast cancer cell-specific activation enhancer and promoter.
ErbB4增强子及启动子， 其为乳腺癌及胃癌细胞特异性激活 增强子及启动子。  ErbB4 enhancer and promoter, which is a specific activation enhancer and promoter of breast cancer and gastric cancer cells.
I. DF3乳腺癌相关抗原 (MUC1)增强子 其为乳腺癌细胞特异性激活增强子及启动子。 MUC1基因的 蛋白产物 （已知的有粘蛋白、 MUC1蛋白、 表皮涎素、 多形表皮 粘蛋白或 PEM; EMA; DF3抗原； NPGP; PAS-O或 CA15.3抗 原） 通常在胃、 胰腺、 肺、 气管、 肾脏、 子宫、 唾液腺、 乳腺的 腺体及管道的上皮细胞的顶端表达。 在人类约 75^ 90%的乳腺癌 中可发现粘蛋白的过度表达。 粘蛋白的表达调控同乳腺癌的分化 程度有一定关系。 I. DF3 breast cancer-associated antigen (MUC1) enhancer It is a breast cancer cell-specific activation enhancer and promoter. MUC1 gene protein products (known as mucin, MUC1 protein, epidermal sialulin, polymorphic epidermal mucin or PEM; EMA; DF3 antigen; NPGP; PAS-O or CA15.3 antigen) usually in the stomach, pancreas, Lung, trachea, kidney, uterus, salivary glands, mammary glands, and apical expression of ductal epithelial cells. Overexpression of mucin can be found in approximately 75 ^ 90% of human breast cancers. The regulation of mucin expression is related to the degree of differentiation of breast cancer.
MUCI基因在人乳腺癌细胞株 MCF-7和 2R-75-的过度表达 似乎发生在转录水平上。 MUCI基因的调控序列， 包括距离转录 起始点上游 0.9Kb的序列已被克隆， 其中包含的一顺式作用元件 似乎同细胞特异性转录相关。  Overexpression of the MUCI gene in human breast cancer cell lines MCF-7 and 2R-75- appears to occur at the transcription level. The regulatory sequence of the MUCI gene, including a 0.9 Kb sequence upstream of the transcription start point, has been cloned, and a cis-acting element contained in it appears to be related to cell-specific transcription.
MUC1-的转录顺式作用元件可以从哺乳动物细胞取得， 包括 但不限于人类细胞， 当然优先在人类细胞表达。 MUC1的转录顺 式作用元件包含 MUCI基因 5， 端的全部 0.9Kb的序列。 MUCI 的转录顺式作用元件也可包含同启动子相连的如下序列 （均相对 于 MUCI转录起始点而言）： -725到 +31, nt-743到 nt+33， nt-750 到 nt+33及 nt-598到 +485等。  The transcriptional cis-acting element of MUC1- can be obtained from mammalian cells, including but not limited to human cells, and of course, it is preferentially expressed in human cells. The transcriptional cis-acting element of MUC1 contains the entire 0.9 Kb sequence of the MUCI gene 5 terminal. The transcriptional cis-acting element of MUCI can also contain the following sequences linked to the promoter (all relative to the MUCI transcription start point): -725 to +31, nt-743 to nt + 33, nt-750 to nt + 33 And nt-598 to +485.
J. 前列腺素特异性抗原增强子及启动子  J. Prostaglandin-specific antigen enhancers and promoters
该前列腺素特异性抗原增强子位于前列腺素特异性抗原起始 转录位点的 nt-5322 ~ nt-3739, 启动子位于前列腺素特异性抗原 起始转录位点的 nt-540 ~ nt+12,该增强子与启动子特异性激活于 前列腺细胞及前列腺癌细胞。  The prostaglandin-specific antigen enhancer is located at nt-5322 ~ nt-3739 of the prostaglandin-specific antigen start transcription site, and the promoter is located at nt-540 ~ nt + 12 of the prostaglandin-specific antigen start transcription site The enhancer and promoter are specifically activated in prostate cells and prostate cancer cells.
K. 腺血管舒緩素增强子及启动子，其可被特异性激活于前列 腺细胞及前列腺癌细胞。  K. Adrenal relaxin enhancer and promoter, which can be specifically activated in prostate cells and prostate cancer cells.
L. 爱泼斯坦氏 -巴尔氏病毒 (Epstein-Barr virus, 按常规 称为 EB病毒）中的 Orip、EB病毒 Orip中的 FR、EB病毒 BamHI C-启动子、 EB病毒中的 Orip联合 EB病毒 BamHI C-启动子、 03 000565 L. Orip in Epstein-Barr virus (conventionally called EB virus), FR in EB virus Orip, EB virus BamHI C-promoter, Orip in EB virus combined with EB virus BamHI C-promoter, 03 000565
EB病毒 Orip中的 FR联合单纯疱疹病毒胸腺嘧啶核苷激酶基本 启动子或 SV40基本启动子、在 EB病毒感染或潜伏感染细胞中特 异性激活的顺式作用元件。 The FR in the Epstein-Barr virus Orip in combination with the herpes simplex virus thymidine kinase basic promoter or the SV40 basic promoter is a cis-acting element that is specifically activated in EB virus infected or latently infected cells.
本发明提供了一类能特异性杀灭肿瘤细胞的增殖型重组病 毒， 它至少有一个病毒增殖必需基因的转录受肿瘤细胞特异性激 活的顺式作用元件有效控制， 其中在病毒增殖必需基因的转录起 始位点与编码起始位点之间区域插入所述顺式作用元件。 该顺式 作用元件特异性在肿瘤细胞内激活， 产生转录活性， 而在正常细 胞内不激活， 不能产生转录活性。 该顺式作用元件可以是下述顺 序之一： 端粒酶催化亚基基因启动子， 端粒酶 RNA组份基因启动 子， 缺氧反应元件， 细胞 S期特异性启动子 (E2F启动子)， 甲胎 蛋白增强子及启动子， 癌胚抗原增强子及启动子， 酪氨酸酶增强 子及启动子， 尿激酶纤维蛋白激活剂增强子及启动子， ErbB2增 强子及启动子， ErbB3增强子及启动子， ErbB4增强子及启动子， DF3乳腺癌相关抗原增强子， 前列腺素特异性抗原增强子及启动 子， 腺血管舒緩素增强子及启动子， EB病毒中的 Orip, EB病 毒 Orip中的 FR增强子， EB病毒 BamHI C-启动子。 上述病毒增 殖所必需的为病毒早期表达基因。 在本发明的一个实施方案中， 使用单纯疱疹病毒， 其病毒增殖必需基因含有早早期表达基因 ICP4。 在本发明又一实施方案中， 上述病毒可以采用腺病毒。 所 述病毒增殖必需基因至少含有以下一个腺病毒的早期表达基因： E1A、 E1B、 E2、 E4。  The present invention provides a type of proliferative recombinant virus capable of specifically killing tumor cells. The transcription of at least one gene necessary for virus proliferation is effectively controlled by cis-acting elements that are specifically activated by tumor cells. The cis-acting element is inserted in the region between the transcription start site and the coding start site. The cis-acting element is specifically activated in tumor cells to produce transcriptional activity, but is not activated in normal cells to produce transcriptional activity. The cis-acting element may be one of the following sequences: a telomerase catalytic subunit gene promoter, a telomerase RNA component gene promoter, a hypoxia response element, a cell S-phase specific promoter (E2F promoter) , Alpha-fetoprotein enhancer and promoter, Carcinoembryonic antigen enhancer and promoter, Tyrosinase enhancer and promoter, Urokinase fibrin activator enhancer and promoter, ErbB2 enhancer and promoter, ErbB3 enhancer Promoter and promoter, ErbB4 enhancer and promoter, DF3 breast cancer-associated antigen enhancer, prostaglandin-specific antigen enhancer and promoter, adrenostatin enhancer and promoter, Orip in Epstein-Barr virus, Orip in EB virus FR enhancer, EB virus BamHI C-promoter. Necessary for the above virus to multiply is a virus early expressing gene. In one embodiment of the present invention, herpes simplex virus is used, and the gene necessary for virus proliferation contains the early and early expression gene ICP4. In yet another embodiment of the present invention, the virus may be an adenovirus. The virus proliferation essential gene contains at least one of the following early expressed genes of adenovirus: E1A, E1B, E2, E4.
(2). 病毒在正常细胞复制必需而在肿瘤细胞中非必需的基因 编码的蛋白功能选择性地缺失：  (2). The function of the protein encoded by genes necessary for virus replication in normal cells but not in tumor cells is selectively deleted:
正常细胞与肿瘤存在着某些基因表达上的差异。 某些病毒在 正常细胞内复制所必需的基因， 在肿瘤细胞内并不需要。 因此， 去除这些基因编码的蛋白功能有望使病毒在肿瘤细胞内特异性复 制， 而不能在正常细胞中复制。 There are differences in the expression of certain genes between normal cells and tumors. Certain viruses require genes that replicate in normal cells and are not needed in tumor cells. Therefore, removing the function of the proteins encoded by these genes is expected to allow the virus to specifically revert in tumor cells. Control and cannot replicate in normal cells.
病毒蛋白功能缺失可以是以下任何一种:腺病毒 E1B 55kDa 的基因发生点突变、 缺失突变和 /或插入突变， 导致 E1B 55kDa 蛋白质功能异常。 腺病毒 ElB 19kDa的基因发生点突变、 缺失突 变和 /或插入突变， 导致 ElB 19kDa蛋白质功能异常。腺病毒 E1A 的基因发生点突变、缺失突变和 /或插入突变， 导致 E1A蛋白质功 能异常。 单纯疱疹病毒 ICP6基因发生点突变、 缺失突变和 /或插 入突变， 导致 ICP6 的蛋白质功能异常。 单纯疱疹病毒 ICP34.5 基因发生点突变、 缺失突变和 /或插入突变， 导致 ICP34.5的蛋白 质功能异常。  The loss of viral protein function can be any of the following: point mutation, deletion mutation, and / or insertion mutation in the gene of adenovirus E1B 55kDa, resulting in abnormal function of E1B 55kDa protein. The point mutation, deletion mutation and / or insertion mutation of the adenovirus ElB 19kDa gene caused the ElB 19kDa protein to malfunction. Adenovirus E1A genes have point mutations, deletion mutations, and / or insertion mutations that cause the E1A protein to malfunction. Point mutations, deletion mutations and / or insertion mutations in the ICP6 gene of herpes simplex virus have caused ICP6 protein to malfunction. Point mutations, deletion mutations and / or insertion mutations in the herpes simplex virus ICP34.5 gene cause abnormal protein function of ICP34.5.
(3) . 依赖于肿瘤细胞某个信号转导途径异常而产生的病毒复 制：  (3). Viral replication that depends on abnormal signal transduction pathways of tumor cells:
呼吸道肠道过滤性病毒 （Reovirus)感染后其早期病毒基因转 录可激活双链 RNA依赖的蛋白激酶 （PKR), 而该激酶可抑制该 病毒的其它基因的转录，从而使病毒不能有效复制。 当细胞中 Ras 处于激活状态则可抑制该激酶， 从而使该病毒活跃复制。 Ras 基 因是一个癌基因， 当它被不正常激活时， 即可能发生癌变。 呼吸 道肠道过滤性病毒复制及增殖依赖于 Ras异常激活的信号途径， 即在 Ras高表达的肿瘤细胞内复制及增殖。  Reovirus infection of the respiratory tract with early viral gene transcription can activate double-stranded RNA-dependent protein kinase (PKR), which inhibits the transcription of other genes of the virus, making the virus unable to replicate efficiently. When Ras is activated in the cell, the kinase can be inhibited and the virus can actively replicate. The Ras gene is an oncogene, and when it is activated abnormally, it can become cancerous. Respiratory intestinal filter virus replication and proliferation depend on the signal pathway of abnormal activation of Ras, that is, replication and proliferation in tumor cells with high Ras expression.
(4) . 选择性进入肿瘤细胞：  (4). Selective access to tumor cells:
改变它们表面的结合蛋白可使其与某些特定的肿瘤组织结 合， 从而使病毒只能感染特定的肿瘤组织。 目前这方面改造主要 集中于腺病毒外壳蛋白 纤维蛋白（Fiber)、 五邻体（ penton )及六 邻体蛋白（hexon), 尤其在纤维蛋白中头部 HI环中或 C末端最常 见， 包括插入肿瘤膜表面具有高亲和力的某种配体、 短肽及治疗 肿瘤的抗体或抗体片段 Fab区域。  Changing the binding proteins on their surface allows them to bind to certain tumor tissues, so that the virus can only infect specific tumor tissues. At present, the transformation in this area mainly focuses on the adenovirus coat protein Fiber, Penton and Hexon, especially in the fibrin, which is most common in the HI loop or C-terminus of the head, including insertion Some ligands, short peptides, and Fab regions of antibodies or antibody fragments for treating tumors have high affinity on the surface of tumor membranes.
本发明的上述病毒中， 编码治疗肿瘤的抗体或抗体片段的核 苷酸序列包括， 但不限于选自编码抗新生血管生成的抗体或抗体 片段的核苷酸序列， 编码抗肿瘤细胞生长因子受体的抗体或抗体 片段的核苷酸序列， 编码抗肿瘤细胞膜抗原的抗体或抗体片段的 核苷酸序列， 编码抗肿瘤抗原的独特型单克隆抗体或抗体片段的 核苷酸序列。 本领域技术人员知晓， 只要能够具有治疗肿瘤作用 的抗体或抗体片段蛋白， 均可被用于本发明。 In the above virus of the present invention, a nucleus encoding an antibody or an antibody fragment for treating a tumor The nucleotide sequence includes, but is not limited to, a nucleotide sequence selected from the group consisting of an antibody or antibody fragment encoding anti-angiogenesis, a nucleotide sequence encoding an antibody or antibody fragment encoding an anti-tumor cell growth factor receptor, and encoding an anti-tumor cell membrane antigen. The nucleotide sequence of the antibody or antibody fragment, the nucleotide sequence encoding the antitumor antigen idiotype monoclonal antibody or antibody fragment. Those skilled in the art know that any antibody or antibody fragment protein capable of treating tumors can be used in the present invention.
抗体（Ab )是一种对特异抗原的结合特异性的糖蛋白。 天然 抗体约 150000道尔顿的异四聚糖蛋白， 其由两个相同的轻链（L ) 和两个相同的重链（H ) 组成。 轻链与重链通过一个共价二硫键 相连。 每条重链是由一个可变区（VH )和多个恒定区组成。 每条 轻链是由一个可变区（VL )和一个恒定区组成； 轻链的恒定区与 重链的第一个恒定区相对， 轻链的可变区与重链的可变区相对。  An antibody (Ab) is a glycoprotein specific for the binding of a specific antigen. A natural antibody of about 150,000 Daltons is a heterotetraglycan protein consisting of two identical light chains (L) and two identical heavy chains (H). The light and heavy chains are linked by a covalent disulfide bond. Each heavy chain is composed of a variable region (VH) and multiple constant regions. Each light chain is composed of a variable region (VL) and a constant region; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain.
抗体的可变区中较为保守的区域称为框架区（FR )。 天然重 链和轻链的可变区中各自包含四个 FR区（分别是 FR1、 FR2、 FR3 和 FR4)， 在 4个框架区中间夹了 3个高变区， 它们大致上成 折叠构型， 由三个连接区相连。 每条链中的高变区通过 FR 区紧 密的靠在一起并与另一链的高变区一起形成了抗体的抗原结合部 位。  The more conserved regions in the variable regions of antibodies are called framework regions (FR). The variable regions of the natural heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4, respectively). Three hypervariable regions are sandwiched between the four framework regions, and they are roughly in a folded configuration. It is connected by three connection areas. The hypervariable regions in each chain are closely held together by the FR region and together with the hypervariable regions of the other chain form the antigen-binding portion of the antibody.
抗体的高变区（CDR)指抗体中负责抗原结合的氨基酸残基。 高变区包括来自 ^补决定区"即 tCDR"的氨基酸残基。  The hypervariable region (CDR) of an antibody refers to the amino acid residues in the antibody that are responsible for antigen binding. The hypervariable region includes amino acid residues from the complementarity determining region "i.e. tCDR".
恒定区不直接参与抗体与抗原的结合， 但是它们表现出不同 的效应子功能， 例如参与抗体的依赖于抗体的细胞毒性。  The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cytotoxicity involved in antibodies.
木瓜蛋白酶消化抗体产生了两个相同的抗原结合片段（称为 "F ab "片段， 每个片段有单个抗原结合位点） 以及一个残余的 "Fc "片段（该名称反映了其容易结晶的能力） 。 用胃蛋白酶处 理产生了一个 F ( ab，） 2片段， 该片段有两个抗原结合位点， 并 仍能与抗原交联。 Ψν "是最小的抗体片段， 它含有全部抗原识别和结合位点。 该区域由非共价地紧密结合的一个重链和一个轻链可变区的二聚 物組成。 在该构型中， 各可变区中的三个高变区相互作用， 在Papain digestion of antibodies produces two identical antigen-binding fragments (called "F ab" fragments, each fragment having a single antigen-binding site) and a residual "Fc" fragment (the name reflects its ability to crystallize easily) ). Treatment with pepsin produced an F (ab,) 2 fragment that had two antigen-binding sites and was still able to cross-link with the antigen. "Ψν" is the smallest antibody fragment that contains all antigen recognition and binding sites. This region consists of a dimer of a heavy chain and a light chain variable region that are non-covalently tightly bound. In this configuration, The three hypervariable regions in each variable region interact.
VH-VL二聚物表面上界定了抗原结合位点。 6个高变区共同组成 抗体抗原结合特异性。 然而， 即使是单个可变区 （或 Fv的一半， 它只包含对抗原有特异性三个可变区） ， 也能识别并结合抗原， 只是其亲和力比完整的结合位点低。 The VH-VL dimer defines an antigen binding site on the surface. Six hypervariable regions together constitute the specificity of antibody-antigen binding. However, even a single variable region (or half of the Fv, which contains only three variable regions against the original specificity), can recognize and bind the antigen, except that its affinity is lower than the complete binding site.
Fab 片段还含有轻链的恒定区和重链的第一个恒定区 ( CH1 ) 。 Fab，片段与 Fab片段的不同之处在于， 在重链 CH1 区的羧基端多几个残基（包括来自铰链区的一个或多个半胱氨 酸）。 Fab，- SH在本文表示恒定区的半胱氨酸残基携带了游离巯 基的 Fab，。 ： F ( ab，） 2抗体片段最初是以 Fab，片段对的形式 产生的， 在其间有铰链半胱氨酸。 The Fab fragment also contains the constant region of the light chain and the first constant region (CH1) of the heavy chain. Fab, fragments differ from Fab fragments in that there are several more residues at the carboxy terminus of the heavy chain CH1 region (including one or more cysteine residues from the hinge region). Fab, -SH herein means that the cysteine residue of the constant region carries a free sulfhydryl group. : F (ab,) 2 antibody fragments were originally produced as Fab, fragment pairs with hinge cysteine in between.
在本发明中， 术语 "抗体片段" 包括完整抗体的一部分， 通 常是完整抗体的抗原结合区或可变区。抗体片段的例子包括 Fab、 Fab ' , F ( ab ' ) 2和 Fv片段； 二体（ diabody ) ； 线性抗体； 单链抗体分子； 和由抗体片段形成的多特异性抗体。  In the present invention, the term "antibody fragment" includes a part of an intact antibody, usually an antigen binding region or a variable region of the intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments; diabody; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
在一个实施方案中， 本发明的重組病毒中， 所述治疗肿瘤的 抗体或抗体片段为抗新生血管生成的抗体或抗体片段蛋白。 本领 域技术人员知晓， 只要能够具有抗新生血管生成的抗体或抗体片 段蛋白， 均可被用于本发明的病毒中。 所述编码抗新生血管生成 的抗体或抗体片段的核香酸序列可选自下述之一种： 抗血管内皮 生长因子的抗体或抗体片段的核苷酸序列， 抗血管内皮生长因子 受体 2的抗体或抗体片段的核苷酸序列， 抗整合素的 α ν Ρ 3的抗 体或抗体片段的核苷酸序列， 抑制新生血管内皮生成的抗体或抗 体片段的核苷酸序列。  In one embodiment, in the recombinant virus of the present invention, the antibody or antibody fragment for treating tumor is an antibody or antibody fragment protein against neoangiogenesis. Those skilled in the art know that any antibody or antibody fragment protein capable of anti-angiogenesis can be used in the virus of the present invention. The nucleotide sequence encoding the anti-angiogenesis antibody or antibody fragment may be selected from one of the following: a nucleotide sequence of an anti-vascular endothelial growth factor antibody or an antibody fragment, and an anti-vascular endothelial growth factor receptor 2 The nucleotide sequence of the antibody or antibody fragment, the nucleotide sequence of the anti-integrin α ν P 3 antibody or antibody fragment, and the nucleotide sequence of the antibody or antibody fragment that inhibits neovascular endothelial production.
血管内皮生长因子 (VEGF)在血管生成中起关键作用， 它通过 促进血管生成从而在肿瘤生成及转移中发挥重要作用。 抗血管内 管生长因子的抗体及抗体片段可阻断血管内皮生长因子与其受体 (特別血管内皮生长因子受体 2)结合， 抑制肿瘤新生血管的生成， 从而抑制肿瘤的生长及转移。 美国 Genentech公司的抗血管内皮 生长因子的嵌合性抗体 (Avastin, 另一个名称为 Bevacizumab)已 处于晚期实体瘤的 ΠΙ期临床试验阶段。 美国 ImClone Systems公 司的针对抗血管内皮生长因子受体 2(KDR)的抗体 IMC-1C11 已 进入肿瘤的 I期临床试验。 整合素的 α ν ρ 3通过细胞间基质与内 皮细胞之间信号传递从而参予新生血管的生成， 抗整合素的 α ν Ρ 3的抗体可抑制新生血管生成， 从而抑制肿瘤的形成。 Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis. Promote angiogenesis and play an important role in tumor formation and metastasis. Anti-endovascular growth factor antibodies and antibody fragments can block the binding of vascular endothelial growth factor and its receptors (especially vascular endothelial growth factor receptor 2), inhibit tumor neovascularization, and thereby inhibit tumor growth and metastasis. American Genentech's anti-vascular endothelial growth factor chimeric antibody (Avastin, another name is Bevacizumab) is already in the phase III clinical trial stage of advanced solid tumors. ImClone Systems' IMC-1C11 antibody against vascular endothelial growth factor receptor 2 (KDR) has entered phase I clinical trials of tumors. Integrin α ν ρ 3 so that the signal transmission by the participating neovascularization between the matrix and the intercellular endothelial cells, an anti [alpha] v integrin antibody Ρ 3 can inhibit angiogenesis, thereby inhibiting tumor formation.
在一个实施方案中， 本发明的重組病毒中， 所述治疗肿瘤的 抗体或抗体片段为编码抗肿瘤细胞生长因子受体的抗体或抗体片 段的核苷酸序列。 本领域技术人员知晓， 只要能够具有抗肿瘤细 胞生长因子受体的抗体或抗体片段蛋白， 均可被用于本发明。 其 中所述编码抗肿瘤细胞生长因子受体的抗体或抗体片段的核苷酸 序列可选自下述之一种： 抗表皮生长因子受体 1 的抗体或抗体片 段的核苷酸序列， 抗表皮生长因子受体 2的抗体或抗体片段的核 苷酸序列。  In one embodiment, in the recombinant virus of the present invention, the antibody or antibody fragment for treating a tumor is a nucleotide sequence encoding an antibody or antibody fragment against an anti-tumor cell growth factor receptor. Those skilled in the art know that any antibody or antibody fragment protein having an anti-tumor cell growth factor receptor can be used in the present invention. The nucleotide sequence of the antibody or antibody fragment encoding the anti-tumor cell growth factor receptor may be selected from one of the following: the nucleotide sequence of the antibody or antibody fragment of the anti-epidermal growth factor receptor 1 The nucleotide sequence of an antibody or antibody fragment of growth factor receptor 2.
人表皮生长因子受体 (human epidermal growth factor receptor)在多种恶性肿瘤细胞转化上起关键作用， 它在多种肿瘤 细胞中过表达。 人表皮生长因子受体也被称为 ErbB 受体， 目前 已有四个成员被识別， 分别表皮生长因子受体 1(HER1, 又称为 ErbBl或 EGFR；)、 表皮生长因子受体 2(HER2， 又称为 ErbBl或 Neu)、 表皮生长因子受体 31(HER3, 又称为 ErbB3)及表皮生长 因子受体 4(HER4， 又称为 ErbB4)。 阻断表皮生长因子受体信号 传导可抑制肿瘤细胞生长。 美国 ImClone Systems公司的针对表 皮生长因子受体 1嵌合抗体 IMC-C225对多种晚期肿瘤有明显的 治疗作用， 目前该抗体已在 m及 iv期临床试验。 美国另一家生物 公司 Abgenix 公司的针对表皮生长因子受体 1 的人的抗体 ABX-EGF 对多种晚期肿瘤有明显的治疗作用， 目前该抗体已在 Π期临床试验。 美国 Genentech公司的针对表皮生长因子受体 2 的人源化抗体 Herceptin (又称为 Trastuzumab) 己于 1998年美国 FDA批准进行临床应用， 它与化疗联合产生明显的临床疗效。 该 公司的另一个针对表皮生长因子受体 1的人源化抗体 2C4亦己进 入 I期临床试验。 Human epidermal growth factor receptor plays a key role in the transformation of many malignant tumor cells, and it is overexpressed in many tumor cells. The human epidermal growth factor receptor is also known as the ErbB receptor. Currently four members have been identified, namely epidermal growth factor receptor 1 (HER1, also known as ErbBl or EGFR;) and epidermal growth factor receptor 2 ( HER2, also known as ErbBl or Neu), epidermal growth factor receptor 31 (HER3, also called ErbB3) and epidermal growth factor receptor 4 (HER4, also called ErbB4). Blocking epidermal growth factor receptor signaling can inhibit tumor cell growth. ImClone Systems' IMC-C225 chimeric antibody against epidermal growth factor receptor 1 has obvious effects on a variety of advanced tumors For the therapeutic effect, the antibody has been clinically tested in m and iv stages. Abgenix's human antibody ABX-EGF against epidermal growth factor receptor 1 in the United States has obvious therapeutic effects on a variety of advanced tumors. Currently, the antibody has been in phase II clinical trials. Herceptin (also known as Trastuzumab), a humanized antibody against epidermal growth factor receptor 2 from Genentech of the United States, was approved for clinical application by the US FDA in 1998. It combined with chemotherapy produced significant clinical effects. The company's other humanized antibody against epidermal growth factor receptor 1, 2C4, has also entered a phase I clinical trial.
在一个实施方案中， 本发明的重组病毒中， 所述治疗肿瘤的 抗体或抗体片段为编码抗肿瘤细胞膜抗原的抗体或抗体片段的核 苷酸序列。 本领域技术人员知晓， 只要能够具有抗肿瘤细胞膜抗 原的抗体或抗体片段蛋白， 均可被用于本发明。 其所述编码抗肿 瘤细胞膜抗原的抗体或抗体片段的核苷酸序列可选自下迷之一种: 抗 CD20的抗体或抗体片段的核苷酸序列， 抗 MUC1的抗体或抗 体片段的核苷酸序列。  In one embodiment, in the recombinant virus of the present invention, the antibody or antibody fragment for treating tumors is a nucleotide sequence encoding an antibody or antibody fragment against an anti-tumor cell membrane antigen. Those skilled in the art know that any antibody or antibody fragment protein capable of having an anti-tumor cell membrane antigen can be used in the present invention. The nucleotide sequence of the antibody or antibody fragment encoding the anti-tumor cell membrane antigen can be selected from one of the following: the nucleotide sequence of the antibody or antibody fragment of anti-CD20, the nucleoside of the antibody or antibody fragment of anti-MUC1 Acid sequence.
美国 Genentech公司的抗 CD20的嵌合抗体 Rituxan(又称为 Rituximab)用于治疗 B-细胞淋巴瘤，已于 1997年批准临床正式应 用。 MUC1广泛存于多种肿瘤中， 美国 Antisoma公司抗 MUC1 人源化抗体己进入临床试验。  The anti-CD20 chimeric antibody Rituxan (also known as Rituximab) from Genentech of the United States is used for the treatment of B-cell lymphoma, and has been officially approved for clinical application in 1997. MUC1 is widely present in a variety of tumors. Antisoma's anti-MUC1 humanized antibody has entered clinical trials.
在一个实施方案中， 本发明的重组病毒中， 所述治疗肿瘤的 抗体或抗体片段为编码抗肿瘤抗原的独特型单克隆抗体或抗体片 段的核苷酸序列。 本领域技术人员知晓， 只要能够具有抗肿瘤抗 原的独特型单克隆抗体或抗体片段的蛋白， 均可被用于本发明。 其所述编码抗肿瘤抗原的独特型单克隆抗体或抗体片段的核苷酸 序列可选自下述之一种:抗 Γ7-1Α的独特型单克隆抗体或抗体片段 的核苷酸序列， 抗癌胚抗原的独特型单克隆抗体或抗体片段的核 苷酸序列， 抗 GD3 的独特型单克隆抗体或抗体片段的核苷酸序 列， 抗 MUC1的独特 单克隆抗体或抗体片段的核苷酸序列。 抗独特型单克隆抗体 (anti-idiotypic mAb, 也可用 Ab2表示) 又称之抗个体基因型抗体， 它模拟肿瘤细胞表面的抗原， 可引起 细胞毒 T 细胞和辅助 T 细胞反应， 从而达到治疗肿瘤的目的。 In one embodiment, in the recombinant virus of the present invention, the antibody or antibody fragment for treating a tumor is a nucleotide sequence encoding an idiotypic monoclonal antibody or antibody fragment of an anti-tumor antigen. Those skilled in the art know that any protein that can have an idiotype monoclonal antibody or antibody fragment of an anti-tumor antigen can be used in the present invention. The nucleotide sequence of the idiotype monoclonal antibody or antibody fragment encoding the anti-tumor antigen may be selected from one of the following: the nucleotide sequence of the idiotype monoclonal antibody or antibody fragment of anti-Γ7-1A, Nucleotide sequence of carcinoembryonic antigen idiotypic monoclonal antibody or antibody fragment, nucleotide sequence of anti-GD3 idiotype monoclonal antibody or antibody fragment Column, the nucleotide sequence of a unique monoclonal antibody or antibody fragment against MUC1. Anti-idiotypic mAb (also known as Ab2) is also called anti-idiotypic antibody, which mimics the antigen on the surface of tumor cells, which can cause cytotoxic T cells and helper T cells to respond, thereby achieving tumor treatment. the goal of.
17-1A在上皮来源的肿瘤细胞内高表达， 德国于 1997年已批准由 Glaxo Wellcome/Centocor公司生产的抗 17-1A独特型单克隆抗 体 (Panorex)用于治疗结肠癌。 美国 ImClone Systems公司的针对 抗的 GD3的独特型单克隆抗体 BEC2抗体 IMC-1C11已进入肿瘤 的 ΠΙ期临床试验。 17-1A is highly expressed in epithelial-derived tumor cells. In 1997, Germany approved the anti-17-1A unique monoclonal antibody (Panorex) produced by Glaxo Wellcome / Centocor to treat colon cancer. ImClone Systems Inc.'s anti-GD3 idiotypic monoclonal antibody BEC2 antibody IMC-1C11 has entered phase II clinical trials of tumors.
脊推动物抗体（免疫球蛋白） 的 链 "， 可根据其恒定区 的氨基酸序列归为明显不同两类（称为 kappa( κ )和 lambda( λ ) 中一类。  The chain of spinal antibody (immunoglobulin) can be classified into two distinctly different types (called kappa (κ) and lambda (λ)) based on the amino acid sequence of its constant region.
根据其重链恒定区的氨基酸序列， 免疫球蛋白可以分为不同 的种类。 主要有 5类免疫球蛋白： IgA、 IgD, IgE、 IgGT和 IgM, 其中一些还可进一步分成亚类（同种型），如 IgG-l、IgG-2、IgG-3、 IgG4、 IgA-1和 IgA-2。 对应于不同类免疫球蛋白的重链恒定区称 为 ot、 Ρ、 ε、 丫和4。 不同类免疫球蛋白的亚基结构和三维构 型是众所周知的。  Depending on the amino acid sequence of the constant region of their heavy chains, immunoglobulins can be divided into different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgGT and IgM, some of which can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, IgG4, IgA-1 And IgA-2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called ot, P, ε, γ, and 4. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
在本发明的上下文中， 术语 4 ^体"应理解为可以是 IgA、 IgD、 IgE、 IgGT 或 IgM, 包括天然抗体、 人源化抗体或嵌合抗 体。 术语 "抗体片段"除去了完整抗体的一部分、 但仍然保留了 抗体的抗肿瘤效应的抗体部分， 包括例如单链抗体、 Fv、 Fab' 、 Fab, 单链抗体或 F(ab，)2抗体中的抗原结合区、 二体抗体、 线性 抗体等等。 In the present context, the term ^ member 4 'may be understood as IgA, IgD, IgE, IgGT or IgM, including natural antibodies, humanized antibodies or chimeric antibodies. The term "antibody fragment" is removed intact antibody A portion of the antibody portion that still retains the antitumor effect of the antibody, including, for example, single-chain antibodies, Fv, Fab ', Fab, single-chain antibodies or antigen-binding regions in F (ab,) 2 antibodies, dimer antibodies, linear Antibodies, etc.
Fab 片段亦包括轻链的恒定区和重链的第一恒定区域 ( CH1 )。 Fab，和 Fab片段的不同在于重链 CHI区的羧基末端多 了一些氨基酸残基， 这其中包括来自铰链区的一个或几个半胱氨 酸。 F(ab，)2抗体片段最初来自于由铰链区半胱氨酸相连的成对 Fab'片段。 The Fab fragment also includes the constant region of the light chain and the first constant region (CH1) of the heavy chain. Fab differs from Fab fragments in that the carboxy terminus of the heavy chain CHI region has a few more amino acid residues, including one or several cysteine residues from the hinge region. Sour. The F (ab,) 2 antibody fragment was originally derived from a pair of Fab 'fragments linked by hinge region cysteine.
Fv 片段是包含有完整的抗原识别和结合位点的最小抗体片 段， 它是由一个重链可变区和一个轻链可变区通过紧密、 共价结 合形成的二聚体。 在它构象中每一个可变区的三个超变区相互作 用从而决定位于 VH-VL 二聚体表面的抗原结合位点。 因此， 六 个超变区共同决定其与抗原结合的特异性。 然而， 即使是单个可 变区（或者包含有与抗原特异的三个超变区的 Fv片段的一半）也 有识别和结合抗原的能力， 尽管它的亲和力比整个结合位点来得 低。  An Fv fragment is the smallest antibody fragment that contains a complete antigen recognition and binding site. It is a dimer formed by a heavy chain variable region and a light chain variable region that are tightly and covalently combined. The three hypervariable regions of each variable region in its conformation interact to determine the antigen-binding site on the surface of the VH-VL dimer. Therefore, the six hypervariable regions together determine their specificity of binding to the antigen. However, even a single variable region (or half of an Fv fragment containing three hypervariable regions specific for an antigen) has the ability to recognize and bind antigens, although it has a lower affinity than the entire binding site.
"单链 Fv "或 ¾ Fv"抗体片段包含抗体的 VH和 VL区， 它 们结合成单个多肽链的形式。 一般来说， 该 Fv多肽链还包含 VH 和 VL之间的多肽连接物， 以使 sFv形成能与抗原结合的理想构 形。  A "single-chain Fv" or ¾Fv "antibody fragment contains the VH and VL regions of an antibody, which are combined into the form of a single polypeptide chain. Generally, the Fv polypeptide chain also contains a polypeptide linker between VH and VL so that sFv forms the ideal configuration for binding to antigens.
二体 (Diabodies)是包括两个抗原结合位点的抗体小片段。 重 链可变区 （VH ) 与轻链可变区 （VL ) 连接成同一条多肽链， 所 用的连接物非常短，使得同一条链上的两个可变区不能相互配对。 这样， 一条链上的可变区只得与另一条链上的互补区配对结合， 从而形成两个抗原结合位点。  Diabodies are small fragments of antibodies that include two antigen-binding sites. The heavy chain variable region (VH) and the light chain variable region (VL) are linked into the same polypeptide chain. The linker used is very short, so that two variable regions on the same chain cannot be paired with each other. In this way, the variable region on one chain only has to pair with the complementary region on the other chain to form two antigen-binding sites.
线性抗体 (Linear antibody)可以是双特异性或单特异性， 是 由首尾相连的一对 Fd 片 VH-CH1-VH-CH1 ) 构成， 从而形成一 对抗原结合位区。 线性抗体可以是双特异性或单特异性。  Linear antibodies can be bispecific or monospecific, and consist of a pair of Fd pieces VH-CH1-VH-CH1) connected end-to-end to form a pair of antigen-binding site regions. Linear antibodies can be bispecific or monospecific.
在本发明的重組病毒中， 所迷编码治疗肿瘤的抗体或抗体片 段的核苷酸序列可与抗癌基因的核苷酸序列读框一致地连接在一 起， 产生融合基因。 该抗癌基因可以是血管抑制基因、 细胞因子 基因、 前药转换酶基因或细胞毒性基因等。  In the recombinant virus of the present invention, the nucleotide sequence encoding the antibody or antibody fragment for treating a tumor may be linked in unison with the nucleotide sequence of the anti-oncogene to produce a fusion gene. The anti-oncogene can be a vascular inhibitory gene, a cytokine gene, a prodrug converting enzyme gene, a cytotoxic gene, or the like.
所述抗癌基因可以是血管抑制基因， 血管抑制基因抑制肿瘤 新生血管形成， 可阻断肿瘤细胞的营养供应， 从而导致肿瘤细胞 因营养不足而死亡， 肿瘤明显萎缩乃至完全消褪。 同时抑制肿瘤 新生血管的形成， 也达到阻断肿瘤转移的通路的目的。 其中所迷 的血管抑制基因可以是： 内皮抑素基因， 血管生成抑素基因， 血 浆纤维蛋白溶酶原中 Kringlel-4 结构、 Kringlel-5 结构、 Kringlel-3结构、 Kringlel-3加上 Kringle5结构、 血小板反应蛋 白基因、 血小板因子 4基因、 纤溶酶原激活因子抑制剂 (PAI)基因 及纤维结合素基因。 The anti-oncogene may be a vascular suppressor gene, which suppresses tumors The formation of new blood vessels can block the nutritional supply of tumor cells, leading to the death of tumor cells due to inadequate nutrition, and the tumors obviously shrink or even completely disappear. At the same time, the formation of tumor neovascularization is inhibited, and the purpose of blocking the pathway of tumor metastasis is also achieved. The angiostatin genes can be: endostatin gene, angiostatin gene, Kringlel-4 structure in plasma plasminogen, Kringlel-5 structure, Kringlel-3 structure, Kringlel-3 plus Kringle5 structure , Thrombospondin gene, platelet factor 4 gene, plasminogen activating factor inhibitor (PAI) gene and fibronectin gene.
所述抗癌基因还可以是细胞因子基因。 细胞因子基因具有激 活免疫细胞， 增加造血功能等。 细胞因子基因可选自： 白细胞介 素 2、 白细胞介素 12、 粒 -单集落刺激因子、 肿瘤坏死因子、 干扰 素 - c、 干扰素 - P、 干扰素 - Y、 Light及 Flt3配体。  The anti-oncogene may also be a cytokine gene. Cytokine genes can activate immune cells and increase hematopoietic function. The cytokine gene can be selected from the group consisting of: interleukin 2, interleukin 12, granulose-single colony stimulating factor, tumor necrosis factor, interferon-c, interferon-P, interferon-Y, Light, and Flt3 ligand.
其抗癌基因可以是前药转换酶基因。 前药转换酶基因可使无 毒性药物转变成毒性药物， 从而增强对肿瘤细胞的杀伤。 所述的 前药转换晦基因可以是： 单纯疱疹病毒胸腺嘧啶激酶、 细菌 P -内 酰胺酶及大肠杆菌胞嘧啶脱氨酶；  Its anticancer gene may be a prodrug converting enzyme gene. Prodrug converting enzyme genes can transform non-toxic drugs into toxic drugs, thereby enhancing the killing of tumor cells. The prodrug conversion gene may be: herpes simplex virus thymidine kinase, bacterial P-lactamase and E. coli cytosine deaminase;
其抗癌基因可以是细胞毒性基因， 例如： 绿脓杆菌外毒片段。 在本发明的重组病毒中， 编码治疗肿瘤的抗体或抗体片段的 核苷酸序列可以受启动子控制。 该启动子可以为例如以下启动子 之一： 猴病毒 40 ( Simian vi s40， 按常规简称为 SV40 )启动子、 劳斯氏肉瘤病毒 (Rous Sarcoma Virus, 按常规筒称为 RSV) LTR 启动子、 人巨细胞病毒 (按常规筒称为 HCMV)IE启动子， 鼠巨细 胞重組病毒 (按常规简称为 MCMV)IE 启动子及人腺病毒主要晚 期表达启动子 (按常规简称为 MLP), 也可以是该抗体基因的天然 启动子。 或者， 所述核苷酸序列也可以处于病毒内源性的启动子 的控制之下。  Its anti-oncogene can be a cytotoxic gene, such as a Pseudomonas aeruginosa exotoxic fragment. In the recombinant virus of the present invention, the nucleotide sequence encoding the antibody or antibody fragment for treating a tumor may be controlled by a promoter. The promoter may be, for example, one of the following promoters: Simian vis 40 (SV40, conventionally abbreviated as conventional) promoter, Rous Sarcoma Virus (RSV, conventionally referred to as RSV) LTR promoter, Human cytomegalovirus (conventionally called HCMV) IE promoter, murine cytomegalovirus (MCMV) IE promoter and human adenovirus major late expression promoter (conventionally referred to as MLP), can also be It is the natural promoter of this antibody gene. Alternatively, the nucleotide sequence may be under the control of a viral endogenous promoter.
在本发明的重組病毒中， 任选地还可以在控制编码治疗肿瘤 的抗体或抗体片段表达的启动子的转录起始位点与抗体的翻译起 始位点之间包含内含子。插入内含子后可使抗体表达量大大增加。 本领域技术人员知晓， 只要能够具有增加抗体的表达的内含子， 均可被用于本发明。 其所述的内含子可以为杂合内含子。 杂合内 含子可以是例如下述之一:含有腺病毒主要晚期 mRNA的第三个 引导顺序的 5，剪接位点和一个免疫球蛋白的 3， 剪接位点的杂合 内含子，含有腺病毒主要晚期 mRNA的第一个引导顺序的 5，剪接 位点和一个免疫球蛋白的 3， 剪接位点的杂合内含子。 In the recombinant virus of the present invention, optionally, it can also be used in the control coding to treat tumors. An intron is included between the transcription initiation site of the promoter expressed by the antibody or antibody fragment and the translation initiation site of the antibody. Insertion of introns can greatly increase the expression of antibodies. Those skilled in the art know that any intron that can increase the expression of an antibody can be used in the present invention. The intron may be a heterozygous intron. The heterozygous intron may be, for example, one of the following: a third guide sequence containing the major late-stage mRNA of the adenovirus 5, a splice site and an immunoglobulin 3, a heterozygous intron of the splice site, containing The first leading sequence of the adenovirus's major late mRNA is 5, a splice site and an immunoglobulin 3, a heterozygous intron for the splice site.
任选地， 本发明的重组病毒还可以在编码治疗肿瘤的抗体或 抗体片段的核苷酸序列中含有编码分泌型信号肽核苷酸序列。 本 领域技术人员知晓， 只要能够具有导致抗体或抗体片段分泌的信 号肽， 均可被用于本发明。 所迷的信号肽可以是例如下述中的一 种： 抗体或抗体片段自身信号肽， M-成瘤蛋白信号肽， 肿瘤坏死 因子 α的信号肽。  Optionally, the recombinant virus of the present invention may further include a nucleotide sequence encoding a secretory signal peptide in a nucleotide sequence encoding an antibody or antibody fragment for treating a tumor. Those skilled in the art know that any signal peptide capable of causing the secretion of antibodies or antibody fragments can be used in the present invention. The signal peptide may be, for example, one of the following: an antibody or an antibody fragment self-signal peptide, an M-oncoprotein signal peptide, and a tumor necrosis factor α signal peptide.
本发明中， 所述编码治疗肿瘤的抗体或抗体片段的核苷酸序 列可以插入在病毒基因組中的非增殖必需区域。 本发明所述的编 码治疗肿瘤的抗体或抗体片段的核苷酸序列可以是编码抗新生血 管生成的抗体或抗体片段的核苷酸序列， 编码抗肿瘤细胞生长因 子受体或抗体片段的核苷酸序列， 或者编码抗肿瘤细胞膜抗原的 抗体或抗体片段的核苷酸序列。 随着病毒在肿瘤细胞内复制， 使 编码治疗肿瘤的抗体或抗体片段的核苷酸序列拷贝数增加， 使肿 瘤细胞高效表达治疗肿瘤的抗体或抗体片段， 从而抑制肿瘤血管 形成， 抑制肿瘤形成、 生长及转移。 同时， 这种修饰后的病毒载 体可被用于在某些细胞混合物中杀灭某种特殊靶细胞， 通过给予 修飾后的病毒， 能选择性地在该种靶细胞中增殖， 从而使这种靶 细胞被增殖的病毒选择性的杀灭； 在体外培养或在动物体内通过 将修饰后的病毒与细胞复合物混合， 病毒只能在靶细胞增殖， 也 就是说除了靶细胞， 其它细胞不能被这种病毒杀死。 由于病毒在 靶细胞内增殖及扩增， 从而使混合细胞中的该靶细胞被杀灭， 一 旦靶细胞被破坏， 病毒不能够再增殖。 In the present invention, the nucleotide sequence encoding the antibody or antibody fragment for treating a tumor may be inserted into a non-proliferation-required region in the viral genome. The nucleotide sequence encoding the antibody or antibody fragment for treating tumors of the present invention may be a nucleotide sequence encoding an antibody or antibody fragment for anti-angiogenesis, and a nucleoside encoding an anti-tumor cell growth factor receptor or antibody fragment. Acid sequence, or a nucleotide sequence encoding an antibody or antibody fragment against a tumor cell membrane antigen. As the virus replicates in tumor cells, the copy number of the nucleotide sequence encoding the antibody or antibody fragment that treats the tumor increases, so that tumor cells efficiently express the antibody or antibody fragment that treats the tumor, thereby inhibiting tumor blood vessel formation, inhibiting tumor formation, Growth and transfer. At the same time, this modified virus vector can be used to kill a particular target cell in some cell mixtures. By giving the modified virus, it can selectively proliferate in the target cell, so that this kind of Target cells are selectively killed by the proliferating virus; in vitro culture or in animals by mixing the modified virus with the cell complex, the virus can only proliferate on the target cell, also This means that other than the target cells, other cells cannot be killed by this virus. Because the virus multiplies and expands in the target cell, the target cell in the mixed cell is killed. Once the target cell is destroyed, the virus cannot reproduce again.
本发明的又一方面， 提供了一种利用本发明的病毒治疗肿瘤 的方法，其包括如下步骤： 1 )将该病毒体外或体内感染肿瘤细胞， 2 )使病毒基本上限于肿瘤细胞内选择性复制及增殖， 导致在肿瘤 细胞内编码治疗肿瘤的抗体或抗体片段的核苷酸序列拷贝数增加 及治疗肿瘤的抗体或抗体片段表达量增加； 由此抑制肿瘤血管的 形成， 特异性直接杀灭肿瘤细胞， 抑制肿瘤形成、 生长及转移。 本发明中所述的哺乳动物包括但不限于人， 猴， 牛， 羊， 猪， 犬， 猫等等。  According to another aspect of the present invention, a method for treating tumors using the virus of the present invention is provided, which includes the following steps: 1) infecting the virus with tumor cells in vitro or in vivo, and 2) limiting the virus to be limited to tumor cells. Replication and proliferation lead to an increase in the number of copies of the nucleotide sequence encoding the antibody or antibody fragment that treats the tumor in the tumor cells and an increase in the expression of the antibody or antibody fragment that treats the tumor; thereby inhibiting the formation of tumor blood vessels and specifically killing it directly Tumor cells inhibit tumor formation, growth and metastasis. The mammals described in the present invention include, but are not limited to, humans, monkeys, cattle, sheep, pigs, dogs, cats, and the like.
在本发明的又一方面， 还提供了一种利用本发明的病毒治疗 哺乳动物、 尤其是人类肿瘤的方法， 其中还包括在本发明所迷病 毒感染肿瘤细胞之前、 同时和 /或之后施用化学抗肿瘤药物。  In yet another aspect of the present invention, there is also provided a method for treating mammals, especially human tumors, using the virus of the present invention, which further comprises administering a chemical before, simultaneously and / or after the virus of the present invention infects tumor cells. Antitumor drugs.
为了进一步提高治疗效果， 本发明提出的病毒可以与常规化 疗药物（如顺铂、 5-氟脲嘧啶丝裂霉素 C等） 、 生物毒素（如蛇 毒素） 、 单克隆治疗肿瘤的抗体或抗体片段一起用于治疗肿瘤， 其作为抗肿瘤药物效果更好。 在本发明的又一实施方案中， 本发 明的病毒与 X-线联合应用， 可产生更为有效的抗肿瘤效应。  In order to further improve the therapeutic effect, the virus proposed by the present invention can be used with conventional chemotherapy drugs (such as cisplatin, 5-fluorouracil mitomycin C, etc.), biotoxins (such as snake toxin), monoclonal antibodies or antibodies for tumor treatment Fragments are used together to treat tumors, which are more effective as antitumor drugs. In still another embodiment of the present invention, the virus of the present invention is used in combination with X-rays to produce a more effective antitumor effect.
本发明提供一类重组病毒， 所述病毒本身通过在肿瘤细胞中 的复制和增殖， 可以特异性抑制肿瘤细胞的生长。  The present invention provides a class of recombinant viruses, which can specifically inhibit the growth of tumor cells by replicating and proliferating in tumor cells.
本发明提供一类重組病毒， 用于体外感染肿瘤细胞， 病毒在 肿瘤细胞内复制及增殖， 导致编码治疗肿瘤的抗体或抗体片段的 核苷酸序列拷贝数增加及治疗肿瘤的抗体或抗体片段表达量增 加。  The present invention provides a class of recombinant viruses used to infect tumor cells in vitro. The virus replicates and proliferates in tumor cells, resulting in an increase in the number of nucleotide sequences that encode antibodies or antibody fragments that treat tumors and the expression of antibodies or antibody fragments that treat tumors. The amount increases.
本发明提供一类重組病毒， 可用于体内选择性地在肿瘤细胞 内复制及增殖， 导致编码治疗肿瘤的抗体或抗体片段的核苷酸序 列拷贝数增加及其表达量增加， 抑制肿瘤形成、 生长及转移。 同 时该病毒能在而且仅限于肿瘤细胞内增殖， 也能特异性直接杀灭 肿瘤细胞。 The present invention provides a class of recombinant viruses that can be used to selectively replicate and proliferate in tumor cells in vivo, resulting in nucleotide sequences encoding antibodies or antibody fragments that treat tumors. Increasing the number of copies in the column and increasing its expression inhibits tumor formation, growth, and metastasis. At the same time, the virus can proliferate in and be limited to tumor cells, and can specifically and directly kill tumor cells.
本发明再一方面， 提供了本发明所迷的病毒用于抑制肿瘤细 胞的生长的用途。  In another aspect of the present invention, the use of the virus of the present invention for inhibiting the growth of tumor cells is provided.
重组病毒向靶细胞的送递有多种方式， 包括但不限于脂质体， 本领域熟知的常规转染方法（如磷酸钙沉淀或电穿孔） ， 直接注 射， 及静脉内灌注。 送递方法主要取决于特定重组病毒（包括其 形态） 以及靶细胞的类型和位置（即细胞在体外还是在体内） „ 若使用包装的重组病毒， 则其可在适当的生理学上可接受载 体中以剂量约 104至约 1014给予。感染复数通常范围是约 0.001至 100。 若所给予的是多核苷酸（即未包装成病毒）， 则其剂量可以 是约 0.01 至约 1000 g。具体的给药量可根据该病毒的有关常 识 （可方便地得自如已公布的文献） 而定， 亦可凭经验确定。 重 組病毒的给予可以是一次， 也可以是多次， 这取决于其目的用途 和宿主的免疫应答能力， 还可多重地同时注射给药。 若不期望产 生免疫应答， 则可用各种免疫抑制剂减少免疫应答， 以便能重复 给药而不产生强免疫应答。 There are many ways to deliver the recombinant virus to target cells, including but not limited to liposomes, conventional transfection methods (such as calcium phosphate precipitation or electroporation) well known in the art, direct injection, and intravenous infusion. The delivery method mainly depends on the specific recombinant virus (including its morphology) and the type and location of the target cell (ie, whether the cell is in vitro or in vivo). „If a packaged recombinant virus is used, it can be in a suitable physiologically acceptable carrier It is administered at a dose of about 10 4 to about 10 14. The multiplicity of infection usually ranges from about 0.001 to 100. If a polynucleotide is administered (ie, unpackaged as a virus), the dose may be about 0.01 to about 1000 g. Specifically The dosage can be determined based on the common sense of the virus (which can be conveniently obtained from published literature) or can be determined empirically. The administration of the recombinant virus can be given once or multiple times, depending on its purpose The use and the host's immune response ability can also be administered by multiple simultaneous injections. If an immune response is not desired, the immune response can be reduced with various immunosuppressants so that repeated administrations can be made without generating a strong immune response.
本发明还包括内含本文所述重組病毒的组合物， 如药物组合 物。 这类组合物可体内给药。 优选地， 这些组合物更进一步包含 药物学上可接受的赋形剂。 这些可包含有效量的本发明重组病毒 于药物学上可接受的赋形剂中的組合物适于以单位剂量形式、 无 菌的胃肠道外溶液或悬浮液、 无菌的非胃肠道外溶液或口服液或 悬浮液， 水包油或油包水乳液等全身性给药于个体。 非胃肠道外 和胃肠道外药物送递配方为本领域已知， 可参见 Remington's药 物科学， 18版， Mack Publishing (1990)。 药物组合物还包括本发 明重组病毒的冻干形式和 /或重建形式（包括包装成病毒的那些）。 本发明还提供治疗方法， 其中将有效量的本文所述重組病毒 施用于个体。 应用重组病毒的治疗方法可用于肿瘤（如肝细胞癌） 患者。 还可用于肿瘤高危人群， 如那些有此类病的家族史和 /或有 已切除或以其它形式 （如化疗） 治疗过的疾病的个体。 决定是否 适于施用本发明的重组病毒将特别依据可评估的临床参数， 如血 清学指标及组织活检样品的组织学检查。 通常施用含重组病毒的 药物组合物。 药物组合物如上迷。 The invention also includes compositions, such as pharmaceutical compositions, containing a recombinant virus described herein. Such compositions can be administered in vivo. Preferably, these compositions further comprise a pharmaceutically acceptable excipient. These compositions which may contain an effective amount of the recombinant virus of the present invention in a pharmaceutically acceptable excipient are suitable in unit dosage form, sterile parenteral solution or suspension, sterile parenteral solution Or an oral solution or suspension, an oil-in-water or water-in-oil emulsion and the like are administered systemically to an individual. Parenteral and parenteral drug delivery formulations are known in the art, see Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing (1990). Pharmaceutical compositions also include lyophilized and / or reconstituted forms of the recombinant viruses of the invention (including those packaged as viruses). The invention also provides methods of treatment, wherein an effective amount of a recombinant virus described herein is administered to an individual. Treatment with recombinant viruses can be used in patients with tumors (such as hepatocellular carcinoma). It can also be used in people at high risk for tumors, such as those with a family history of the disease and / or individuals who have had a disease that has been removed or otherwise treated (such as chemotherapy). The determination of suitability for administration of the recombinant virus of the invention will be based in particular on assessable clinical parameters such as serological indicators and histological examination of tissue biopsy samples. Pharmaceutical compositions containing recombinant viruses are usually administered. The pharmaceutical composition is as described above.
重组病毒的用量取决于多种因素， 如具体的重組病毒类型， 给药途径， 个体的身体状况， 疾病发展程度， 所用的具体抗体或 抗体片段基因。  The amount of recombinant virus used depends on many factors, such as the specific type of recombinant virus, the route of administration, the physical condition of the individual, the degree of disease development, and the specific antibody or antibody fragment gene used.
若施用包装的重組病毒， 则用量为约 104至约 1014, 优选约 104至约 1012， 更优选约 104至约 101β。 若施用多核苷酸， 则用量 为约 O.Ol g至约 lOO g, 优选 O.l g至约 500 g, 更优选约 0.5 g至约 200 g。 可同时或先后施用不止一种重組病毒。 通常 是周期性给药， 同时监控任何反应。 可经如肿瘤内、 静脉内或腹 膜内给药。 If a packaged recombinant virus is administered, the amount is about 10 4 to about 10 14 , preferably about 10 4 to about 10 12 , and more preferably about 10 4 to about 10 1β . If a polynucleotide is applied, the amount is from about 0.01 g to about 100 g, preferably from 0.01 g to about 500 g, more preferably from about 0.5 g to about 200 g. More than one recombinant virus can be administered simultaneously or sequentially. It is usually given periodically while monitoring any response. It can be administered intratumorally, intravenously or intraperitoneally.
与现有的肿瘤治疗方法相比， 本发明具有如下有益效果： 动物实验证明， 本发明的重組病毒可用于治疗肿瘤。  Compared with the existing tumor treatment methods, the present invention has the following beneficial effects: Animal experiments prove that the recombinant virus of the present invention can be used to treat tumors.
本发明病毒能够在肿瘤细胞内特异性增殖， 而在正常细胞内 基本上不增殖， 从而能够靶向性地使抗体在肿瘤细胞内表达， 达 到特异性地抑制或杀死肿瘤细胞、从而高效低毒治疗肿瘤的目的。  The virus of the present invention can specifically proliferate in tumor cells, but does not substantially proliferate in normal cells, so that antibodies can be targeted to be expressed in tumor cells to achieve specific inhibition or killing of tumor cells, thereby being highly efficient and low. The purpose of toxic treatment of tumors.
人类腺病毒有 6个不同亚属， 分为 A、 B、 C, D、 E及F。 它们对宿主细胞的亲嗜性、 致瘤性及疾病性史并不相同。 本发明 以腺病毒 C亚属中的 5型 (Ad5)作为例证，对本发明加以进一步具 体说明。 本发明构建手段均是现有技术人员能够实现。  Human adenovirus has six different subgenus, divided into A, B, C, D, E and F. They have different philophilic, tumorigenic, and diseased histories of host cells. The present invention is further exemplified by taking type 5 (Ad5) in the subgenus Adenovirus C as an example. The construction means of the present invention can be implemented by those skilled in the art.
实施例  Examples
例 1: 携带端粒酶催化亚基基因启动子的克隆栽体的构建。 自新鲜的正常人肝組织中抽提基因组 DNA， 应用巢式多聚酶 链反应（PCR)技术扩增端粒酶催化亚基基因启动子（方法参见 PCR Protools Current Methods and Applications, White BA主 编， Humana Press Inc. 1993年出版一文献 1)，在启动子 5， 端入 EcoR I、Not I及Bgl Π三个酶切位点， 在启动子 3， 端加入 Xho I、 BamH I两个酶切位点。 Example 1: Construction of a cloned vector carrying a telomerase catalytic subunit gene promoter. Genomic DNA was extracted from fresh normal human liver tissue, and the telomerase catalytic subunit gene promoter was amplified using nested polymerase chain reaction (PCR) technology (for methods, see PCR Protools Current Methods and Applications, editor of White BA, Humana Press Inc. 1993 published a document 1), at the promoter 5, the three restriction sites of EcoR I, Not I and Bgl Π were inserted, and at the promoter 3, the two sites of Xho I and BamH I were added .
引物 1: 5， CGG GCT CCC AGT GGA TTC 3' ( SEQ ID NO: Primer 1: 5, CGG GCT CCC AGT GGA TTC 3 '(SEQ ID NO:
1 ) ； 1 ) ;
引物 2: 5' AAC GTG GCC AGC GGC AGC ACC TC 3， ( SEQ ID NO: 2 ) ；  Primer 2: 5 'AAC GTG GCC AGC GGC AGC ACC TC 3, (SEQ ID NO: 2);
引物 3: 5， GGA ATT CGC GGC CGC AGA TCT CAC AGA CGC CCA GGA ACC 3， ( SEQ ID NO: 3 ) ；  Primer 3: 5, GGA ATT CGC GGC CGC AGA TCT CAC AGA CGC CCA GGA ACC 3, (SEQ ID NO: 3);
引物 4: 5， CGG GAT CCTG CT CGA GTG GCC GGG GCC AGG GCT TC 3， ( SEQ ID NO: 4 ) 。  Primer 4: 5, CGG GAT CCTG CT CGA GTG GCC GGG GCC AGG GCT TC 3, (SEQ ID NO: 4).
引物 1与引物 2进行第一次 PCR扩增， 回收 370bp片段， 再 用引物 3与引物 4对 370片段进行第二次 PCR扩增， 回收 270bp 片段，应用 EcoR I+BamH I对酶切， 将该基因插入 pUC19载体 (购 于美国 ATCC公司）， 将其命名为 pUC-hTERTp。  Primer 1 and primer 2 were amplified by PCR for the first time, and a 370 bp fragment was recovered. Then, a second PCR amplification of 370 fragments was performed with primer 3 and primer 4. A 270 bp fragment was recovered, and digested with EcoR I + BamH I. This gene was inserted into the pUC19 vector (purchased from ATCC, USA), and was named pUC-hTERTp.
合成含有 3 xE-box的连接头 Composite connector with 3 x E-box
引物 5: 5， TCG AGG ACG CAC GTG GGC GCA CGT GGG CGC ACG TGG GAT TTA AAT A 3，（ SEQ ID NO: 5 ) ；  Primer 5: 5, TCG AGG ACG CAC GTG GGC GCA CGT GGG CGC ACG TGG GAT TTA AAT A 3 (SEQ ID NO: 5);
引物 6: 5， AGC TTA TTT AAA TCC CAC GTG CGC CCA CGT GCG CCC ACG TGC GCC T 3， ( SEQ ID NO: 6 ) 。  Primer 6: 5, AGC TTA TTT AAA TCC CAC GTG CGC CCA CGT GCG CCC ACG TGC GCC T 3, (SEQ ID NO: 6).
将引物 5与引物 6进行变性， 复性， 应用磷酸激酶进行磷酸 化,将其插入 pUC-hTERTp中 Xho I与 Hind III酶切位点（方法参 见分子克隆实验指南第二版， J.萨姆布鲁克等主编， 科学出版社. 1996年出版一文献 2)。 将该质粒进行测序， 其测序结果见下： 5， gaattcgcgg ccgcagatct cacagacgcc caggaccgcg cttcccacgt ggcggaggga ctggggaccc gggcacccgt cctgcccctt caccttccag ctccgcctcc tccgcgcgga ccccgccccg tcccgacccc tcccgggtcc ccggcccagc cccctccggg ccctcccagc ccctcccctt cctttccgcg gccccgccct ctcctcgcgg cgcgagtttc aggcagcgct gcgtcctgct gcgcacgtgg gaagccctgg ccccggccac tcgacgcacg tgggcgcacg tgggcgcacg tgggatttaa ataagct 3， ( SEQ ID NO: 7 ) 。 Primer 5 and primer 6 were denatured, renatured, phosphorylated using phosphokinase, and inserted into the Xho I and Hind III digestion sites in pUC-hTERTp (for the method, see the second edition of the Molecular Cloning Experiment Guide, J. Sam Edited by Brooke et al., Science Press. 1996. 1). The plasmid was sequenced, and the sequencing results are as follows: 5, gaattcgcgg ccgcagatct cacagacgcc caggaccgcg cttcccacgt ggcggaggga ctggggaccc gggcacccgt cctgcccctt caccttccag ctccgcctcc tccgcgcgga ccccgccccg tcccgacccc tcccgggtcc ccggcccagc cccctccggg ccctcccagc ccctcccctt cctttccgcg gccccgccct ctcctcgcgg cgcgagtttc aggcagcgct gcgtcctgct gcgcacgtgg gaagccctgg ccccggccac tcgacgcacg tgggcgcacg tgggcgcacg tgggatttaa ataagct 3, (SEQ ID NO: 7).
结果表明端粒酶催化亚基基因启动子正确， 将其命名为 pUC-hTERT-Ep, 该端粒酶催化亚基基因启动子含有人端粒酶催 化亚基基因启动子中 nt-213 至 nt+47 以及在其下游加入了 3 个 E-box序列，并在整个启动子的上游序列中加入了 EcoR I、 Not I 与 Bgl II酶切位点， 在整个启动子的下游序列中加入了 Swa I酶 切位点。  The results showed that the telomerase catalytic subunit gene promoter was correct. It was named pUC-hTERT-Ep. The telomerase catalytic subunit gene promoter contained nt-213 to nt in the human telomerase catalytic subunit gene promoter. +47 and 3 E-box sequences downstream, and EcoR I, Not I, and Bgl II restriction sites in the upstream sequence of the entire promoter, and Swa in the downstream sequence of the entire promoter I restriction sites.
例 2:可携带抗癌基因的端粒酶催化亚基基因启动子控制 E1A 表达的減毒增殖腺病毒载体的构建。  Example 2: Construction of an attenuated proliferative adenovirus vector carrying an anti-oncogene telomerase catalytic subunit gene promoter to control E1A expression.
pXC.l载体购于加拿大 Microbix Biosystem Inc.(Toronto), pXC.l含有 5型腺病毒序列 bp22-5790。 在该载体中 552bp处创 立 7个新的、 唯一的酶切位点， 分别为 Age l、 Bst BI、 Not I. Spe I、 Sal L Xho I ^ Swa I, 该位点位于 E1A起始密码前 12bp， 其 方法采用定位突变双次 PCR技术 (方法参见前迷文献 1)。 其引物 分别为  The pXC.l vector was purchased from Microbix Biosystem Inc. (Toronto), Canada. pXC.l contains a type 5 adenovirus sequence bp22-5790. Seven new and unique restriction sites were created at 552bp in this vector, namely Age l, Bst BI, Not I. Spe I, Sal L Xho I ^ Swa I, and this site is located before the E1A start code 12bp, the method uses positional mutation double PCR technology (see the previous fan literature 1). The primers are
引物 7(5，-引物， 含有 EcoR I酶切位点)： 5， TTC AAG AAT TCT CAT GTT TG 3，（ SEQ ID NO: 8 ) ；  Primer 7 (5, -primer, containing EcoR I digestion site): 5, TTC AAG AAT TCT CAT GTT TG 3, (SEQ ID NO: 8);
引物 8(3，-引物, 在 5， 端加入 ATT TAA ATC TCG AGT CGA CAC TAG TGC GGC CGC TTC GAA CCG GT): 5， ATT TAA ATC TCG AGT CGA CAC TAG TGC GGC CGC TTC GAA CCG GTG TCG GAG CGG CTC GGA 3，（ SEQ ID NO: 9 ); 引物 9( 5，-引， 在 5， 端加入 ACC GGT TCG AAG CGG Primer 8 (3, -primer, add ATT TAA ATC TCG AGT CGA CAC TAG TGC GGC CGC TTC GAA CCG GT) at the 5 end: 5, ATT TAA ATC TCG AGT CGA CAC TAG TGC GGC CGC TTC GAA CCG GTG TCG GAG CGG CTC GGA 3, (SEQ ID NO: 9); Primer 9 (5,-primer, add ACC GGT TCG AAG CGG to the 5, end
ACC GGT TCG AAG CGG CCG CAC TAG TGT CGA CTC GAG ATT TAA ATC CGG TGA CTG AAA ATG AGA CAT ATT A 3' ( SEQ ID NO: 10 ) ; ACC GGT TCG AAG CGG CCG CAC TAG TGT CGA CTC GAG ATT TAA ATC CGG TGA CTG AAA ATG AGA CAT ATT A 3 '(SEQ ID NO: 10);
引物 10(3，-引物， 含有 Xba I酶切位点)： 5， TTC TCT AGA CAC AGG TGA TG 3， ( SEQ ID NO: 11 ) 。  Primer 10 (3, -primer, containing Xba I digestion site): 5, TTC TCT AGA CAC AGG TGA TG 3, (SEQ ID NO: 11).
采用定位突变双次 PCR 技术， PCR 产物片段插入 pGEM-T-easy载体中（方法参见 Promega公司操作说明书）， 命名 为 pGEM-T-Ela。 将该片段进行 DNA测序， 其测序结果显示： 在 pXC.l质粒 bp552位点中插入 TTC GAA GCG GCC GCA CTA GTG TCG ACT CGA GAT TTA AAT CCG GT， 从而产生 7个新的 Age I、 Bst BI、 Not I、 Spe I、 Sal I、 Xho I和 Swa I酶 切位点， 其它序列与 pXC.l相同。 应用 EcoR I与 Xba I双酶切 pGEM-T-ElA及 pXC.l质粒， 将 pGEM-T-ElA中切出片段插入 pXC.l质粒中 EcoR I及 Xba I位点中，使 pXC.l中 552插入一个 TTC GAA GCG GCC GCA CTA GTG TCG ACT CGA GAT TTA AAT CCG GT, 从而产生 7个 Age I、 Bst BI、 Not I, Spe l、 Sal I、 Xho I和 Swa I酶切位点，该位点位于 E1A起始密码前 12bp, 将该质粒命名为 pQW。  Using localized mutation double PCR technology, the PCR product fragment was inserted into the pGEM-T-easy vector (for the method, refer to the operating instructions of Promega) and named as pGEM-T-Ela. This fragment was subjected to DNA sequencing, and the sequencing results showed that: TTC GAA GCG GCC GCA CTA GTG TCG ACT CGA GAT TTA AAT CCG GT was inserted into the pXC.l plasmid bp552 site, thereby generating 7 new Age I, Bst BI, Not I, Spe I, Sal I, Xho I and Swa I restriction sites, and other sequences are the same as pXC.l. PGEM-T-ElA and pXC.l plasmids were digested with EcoR I and Xba I, and the fragments cut out from pGEM-T-ElA were inserted into the EcoR I and Xba I sites in pXC.l plasmid to make pXC.l 552 Inserts a TTC GAA GCG GCC GCA CTA GTG TCG ACT CGA GAT TTA AAT CCG GT to generate 7 Age I, Bst BI, Not I, Spell, Sal I, Xho I, and Swa I restriction sites. The dot was located 12bp before the E1A start code, and the plasmid was named pQW.
pUC-hTERT-Ep分别用 Not I+Swa I酶切， 其酶切片段分别 插入 pQW质粒中 Not l+Swa l酶切位点中， 分别应用引物 4及 7 进行 PCR扩增， 扩出 1310bp, 这表明端粒酶催化亚基基因启动 子已正向插入 pQW质粒 Not I+Swa I位点， 即腺病毒 5型 E1A 起始密码子上游区 12bp处， 命名为 pQW-hTERT-Ep。  pUC-hTERT-Ep was digested with Not I + Swa I, and the fragments were inserted into the Not l + Swa l digestion site in pQW plasmid, and primers 4 and 7 were used for PCR amplification, respectively. This indicates that the promoter of the telomerase catalytic subunit gene has been inserted into the Not I + Swa I site of the pQW plasmid, that is, 12 bp upstream of the start codon of the adenovirus type 5 E1A, and named pQW-hTERT-Ep.
例 3. 可携带抗癌基因的端粒酶催化亚基基因启动子及缺氧 启动子分别控制 E1A及 E1B表达的減毒增殖腺病毒载体的构建。 缺氧作用元件（HRE)联合基础人巨细胞病毒启动子Example 3. Construction of an attenuated proliferative adenovirus vector carrying an anti-oncogene telomerase catalytic subunit gene promoter and an hypoxic promoter to control the expression of E1A and E1B, respectively. Hypoxia-responsive element (HRE) combined with basic human cytomegalovirus promoter
(mini-CMVp)由 5个拷贝的 HRE及基础人巨细胞启动子组成。其 缺氧作用元件 (HRE)来源于血管内皮生长因子的启动子序列，其 5 个拷贝的 HRE的核苷酸序列如下: 5， CCA CAG TGC ATA CGT GGG CTC CAA CAG GTC CTC TT CCA CAG TGC ATA CGT GGG CTC CAA CAG GTC CTC TT CCA CAG TGC ATA CGT GGG CTC CAA CAG GTC CTC TT CCA CAG TGC ATA CGT GGG CTC CAA CAG GTC CTC TT CCA CAG TGC ATA CGT GGG CTC CAA CAG GTC CTC TT 3， (SEQ ID NO:12)„ (mini-CMVp) consists of 5 copies of HRE and a basic human giant cell promoter. Its hypoxia-acting element (HRE) is derived from the promoter sequence of vascular endothelial growth factor. The nucleotide sequence of its five copies of HRE is as follows: 5. CCA CAG TGC ATA CGT GGG CTC CAA CAG GTC CTC TT CCA CAG TGC ATA CGT GGG CTC CAA CAG GTC CTC TT CCA CAG TGC ATA CGT GGG CTC CAA CAG GTC CTC TT CCA CAG TGC ATA CGT GGG CTC CAA CAG GTC CTC TT CCA CAG TGC ATA CGT GGG CTC CAA CAG GTC CTC TT 3, (SEQ ID NO : 12) „
基础人巨细胞启动子 (mini-CMVp)的序列如下：  The sequence of the basic human giant cell promoter (mini-CMVp) is as follows:
5， GGT AGG CGT GTA CGG TGG GAG GTC TAT ATA AGC AGA GCT CGT TTA GTG AAC CGT CAG ATC 3， (SEQ ID NO:13)。 5. GGT AGG CGT GTA CGG TGG GAG GTC TAT ATA AGC AGA GCT CGT TTA GTG AAC CGT CAG ATC 3, (SEQ ID NO: 13).
缺氧作用元件 (HRE)联合基础人巨细胞启动子 (mini-CMVp) 含有以上两个序列， 并在 5，-末端与 3， -末端分别加入 Spe l酶切 位点， 缺氧作用元件 (HRE)联合基础人巨细胞启动子 (mini-CMVp) 由上海申能博彩生物科技有限公司进行全长基因合成。 经测序正 确， 命名为 pMD-HRE， 其核苷酸序列如下：  The hypoxia-acting element (HRE) combined with the basic human giant cell promoter (mini-CMVp) contains the above two sequences, and the Spel restriction sites are added at the 5, -terminus and 3, -terminus, respectively. HRE) combined with the basic human giant cell promoter (mini-CMVp) was used for full-length gene synthesis by Shanghai Shenneng Gaming Biotechnology Co., Ltd. After sequencing, it was named pMD-HRE, and its nucleotide sequence was as follows:
5'ACTAGTCCACAGTGCATACGTGGGCTCCAACAGGTCCT CTTCCACAGTGCATACGTGGGCTCCAACAGGTCCTCTTC CACAGTGCATACGTGGGCTCCAACAGGTCCTCTTCCACA GTGCATACGTGGGCTCCAACAGGTCCTCTTCCACAGTGC ATACGTGGGCTCCAACAGGTCCTCTTGGTAGGCGTGTAC GGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACC GTCAGATCACTAGT 3' ( SEQ ID NO: 14 ) 。 5'ACTAGTCCACAGTGCATACGTGGGCTCCAACAGGTCCT CTTCCACAGTGCATACGTGGGCTCCAACAGGTCCTCTTCCACAGTGCATACGTGGGCTCCAACAGGTCCTCTTCCACA GTGCATACGTGGGCTCCAACAGGTCCTCTTCCACAGTGC ATACGTGGGCTCCAACAGGTCCTCTTGGTAGGCGTGTGCACGTGTAC
在 pQW-hTER-Ep质粒中使其缺失 E1B启动子，该启动子位 于 pQW-hTER-Ep质粒中 ntl926-nt2043位置（即在 5型腺病毒的 ntl595-ntl713位置)， 将该位置缺失并创立 1个新的、 唯一的 Spe I酶切位点， 该位点位于 E1B起始密码前 4bp, 其方法采用定位 突变双次 PCR技术 (方法参见前述文献 1)。 其引物分别为： The pQW-hTER-Ep plasmid was deleted from the E1B promoter, which is located at the ntl926-nt2043 position in the pQW-hTER-Ep plasmid (that is, ntl595-ntl713 position), delete this position and create a new, unique Spe I digestion site, this site is located 4bp before the E1B start code, and its method uses position mutation double PCR technology (see the method above Reference 1). The primers are:
引物 11: 5， TCA CCT GTG TCT AGA GAA TGC 3， (SEQ ID NO:15);  Primer 11: 5, TCA CCT GTG TCT AGA GAA TGC 3, (SEQ ID NO: 15);
引物 12: 5， CTC CCA AGC CTC CAT ACT AGT TTA AAC ATT ATC TCA CCC TTT A 3' (SEQ ID NO: 16)；  Primer 12: 5, CTC CCA AGC CTC CAT ACT AGT TTA AAC ATT ATC TCA CCC TTT A 3 '(SEQ ID NO: 16);
引物 13: 5， ACT AGT ATG GAG GCT TGG GAG TGT TTG 3， (SEQ ID NO:17);  Primer 13: 5, ACT AGT ATG GAG GCT TGG GAG TGT TTG 3, (SEQ ID NO: 17);
引物 14: 5， GGC CAG AAA ATC CAG CAG GTA 3， (SEQ ID NO:18)。  Primer 14: 5, GGC CAG AAA ATC CAG CAG GTA 3, (SEQ ID NO: 18).
PCR产物片段插入 pGEM-T-easy载体中（方法参见 Promega 公司操作说明书）， 命名为 pGEM-T-ElB。 将该片段进行 DNA测 序， 其测序结果显示： 在 pQW-hTER-Ep质粒 ntl926-nt2043位 点缺失并插入产生 1 个新的 Spe I 酶切位点， 其它序列与 pQW-hTER-E 相同。 应用 Kpn I与 Xba I双酶切 pGEM-T-ElB 及 pQW-hTER-Ep 质粒， 将 pGEM-T-ElB 中切出片段插入 pQW-hTER-E 质粒中 Kpn I 及 Xba I 位点中， 使在 pQW-hTER-E 质粒 ntl926-nt2043位点缺失并插入产生 1个新的 Spe l酶切位点， 该位点位于 E1B起始密码前 4bp,将该质粒命名 为 pQW2-Spe。  The PCR product fragment was inserted into the pGEM-T-easy vector (for the method, refer to the operating instructions of Promega) and named as pGEM-T-ElB. The DNA was sequenced from this fragment. The sequencing results showed that the pQW-hTER-Ep plasmid ntl926-nt2043 was deleted and inserted to generate a new Spe I restriction site. The other sequences were the same as pQW-hTER-E. The pGEM-T-ElB and pQW-hTER-Ep plasmids were double digested with Kpn I and Xba I, and the fragments cut out from pGEM-T-ElB were inserted into the Kpn I and Xba I sites in pQW-hTER-E plasmid so that The pQW-hTER-E plasmid, ntl926-nt2043, was deleted and inserted to generate a new Spl restriction site, which was located 4bp before the E1B start code. This plasmid was named pQW2-Spe.
将 pMD-HRE用 Spe I酶切， 回收含有缺氧作用元件 (HRE) 联合基础人巨细胞启动子（mini-CMVp)片 断， 将其插入 pQW2-Spe质粒中的 Spe l酶切位点。 应用 PCR技术分别验证其 插入的正反方向。  The pMD-HRE was digested with Spe I, and the fragment containing the hypoxia-reactive element (HRE) combined with the basic human giant cell promoter (mini-CMVp) was cut and inserted into the Spe l digestion site in the pQW2-Spe plasmid. The PCR technology was used to verify the positive and negative directions of insertion.
引物 15: 5， AGG TCT ATA TAA GCA GAG CTC 3 '(SEQ ID NO:19)。 用引物 14及引物 15进行 PCR，其 PCR产物长度为 518bp， 这表明缺氧作用元件（HRE)联合基础人巨细胞启动子 (mini-CMVp) 正 向 插 入 E1B 上 游 区 ， 命 名 pQW-hTERT-Ep--HRE。 Primer 15: 5, AGG TCT ATA TAA GCA GAG CTC 3 '(SEQ ID NO: 19). PCR was performed with primers 14 and 15. The length of the PCR product was 518 bp. This indicates that the hypoxia-acting element (HRE) combined with the basic human giant cell promoter (mini-CMVp) was inserted into the upstream region of E1B and named pQW-hTERT-Ep. --HRE.
例 4.分别携带抗人表皮生长因子受体 l(EGFR)的人的抗体 SG-EGFR 基因、 抗人表皮生长因子受体 2(Her2)的人源化抗体 SG-HER基因及抗人 CD20的人鼠嵌合抗体 SG-CD20基因的表达 载体的构建  Example 4. Human antibody SG-EGFR gene carrying anti-human epidermal growth factor receptor 1 (EGFR), humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2), and anti-human CD20 Construction of human and mouse chimeric antibody SG-CD20 gene expression vector
pShuttle-CMV 购于美国 Qbiogene, 含有人巨细胞病毒启动 子 (CMV IE)及 SV40 poly A加尾信号， 应用 PCR技术克隆人巨 细胞病毒启动子 (CMV IE)及 SV40 poly A加尾信号， 并在上下游 位置中分别加入 PAC I 酶切位点， 在其人巨细胞病毒启动子 (CMV IE)及 SV40 poly A加尾信号之间插入多克隆位点， 分别为 EcoR I、 Sal L Hind III、 Xho L BamH I及 Xba I酶切位点， 其 方法采用定位突变双次 PCR 技术（方法参见 PCR Protools Current Methods and Applications, White BA 主编， Humana Press Inc. 1993年出版 -文献 1)。其引物分别为应用引物 16与引物 17对 pShuttle-CMV进行 PCR,其产物长度为 621bp。  pShuttle-CMV was purchased from Qbiogene in the United States and contains a human cytomegalovirus promoter (CMV IE) and SV40 poly A tailed signal. PCR technology was used to clone the human cytomegalovirus promoter (CMV IE) and SV40 poly A tailed signal Add PAC I digestion sites in the upstream and downstream positions, and insert multiple cloning sites between its human cytomegalovirus promoter (CMV IE) and SV40 poly A tailing signals, respectively EcoR I, Sal L Hind III , Xho L BamH I and Xba I digestion sites, the method uses site-directed mutation double PCR technology (for methods see PCR Protools Current Methods and Applications, editor of White BA, Humana Press Inc. 1993-Literature 1). The primers were used to perform PCR on pShuttle-CMV using primer 16 and primer 17, respectively, and the product length was 621 bp.
引物 16: 5， GGG GTA CCT TAA TTA AGA TCT TAG TAA TCA ATT ACG GGG TCA V ( SEQ ID NO: 20 )；  Primer 16: 5, GGG GTA CCT TAA TTA AGA TCT TAG TAA TCA ATT ACG GGG TCA V (SEQ ID NO: 20);
引物 17:5， GAG AAG CTT GTC GAC GAA TTC CTA GCG GAT CTG ACG GTT CAC 3， ( SEQ ID NO: 21 ) 。  Primer 17: 5, GAG AAG CTT GTC GAC GAA TTC CTA GCG GAT CTG ACG GTT CAC 3, (SEQ ID NO: 21).
应用引物 18与引物 19对 pShuttle-CMV进行 PCR，其产物长 度为 293bp。  The primers 18 and 19 were used to perform PCR on pShuttle-CMV, and the product length was 293bp.
引物 18: 5' GAA TTC GTC GAC AAG CTT CTC GAG GGA TCC ATC TAG ATA ACT GAT CAT A 3， ( SEQ ID NO: 22 ) ； 引物 19: 5' ATA GTT TAG CGG CCG CTT AAT TAA GAT AC A TTG ATG AGT TTG 3' ( SEQ ID NO: 23 ) 。 Primer 18: 5 'GAA TTC GTC GAC AAG CTT CTC GAG GGA TCC ATC TAG ATA ACT GAT CAT A 3, (SEQ ID NO: 22); Primer 19: 5 'ATA GTT TAG CGG CCG CTT AAT TAA GAT AC A TTG ATG AGT TTG 3' (SEQ ID NO: 23).
应用引物 16与引物 19对上迷片断进行 PCR,其产物长度为 893bp  The primer 16 and primer 19 were used to perform PCR on the upper fragment, and the product length was 893bp
应用 Kpn I与 Not I双酶切后，插入 pBluescript中.进行测序, 正确者命名为 pClonelO，其 PAC I酶切后长度为 861bp。  After double digestion with Kpn I and Not I, insert it into pBluescript. Sequencing, the correct one was named pClonelO, and the length after PAC I digestion was 861bp.
脑心肌 炎病毒（EMCV)的 多 顺反子（IRES)来源 于 pIRES-EYFP, 该质粒购于美国 Clontech公司。 应用引物 5与引 物 6对其进行 PCR， 其扩增长度为 624bp。  The polycistronic (IRES) of encephalomyocarditis virus (EMCV) was derived from pIRES-EYFP, and the plasmid was purchased from Clontech. Primer 5 and primer 6 were used for PCR, and the amplification length was 624bp.
引物 20: 5， CCG GAA TTC ATC GAT TCT GTC GAC CTG CAG GAA TTG CCC CTC TCC CTC 3， ( SEQ ID NO: 24 ) ； 引物 21: 5， TGC TCT AGA CCC GGG CTC GAG GGA TCC TTA ATC ATC GTG TTT TTC AAA G 3， （ SEQ ID NO: 25 ) 。  Primer 20: 5, CCG GAA TTC ATC GAT TCT GTC GAC CTG CAG GAA TTG CCC CTC TCC CTC 3, (SEQ ID NO: 24); Primer 21: 5, TGC TCT AGA CCC GGG CTC GAG GGA TCC TTA ATC ATC GTG TTT TTC AAA G 3, (SEQ ID NO: 25).
用 EcoR I+Xba I双酶切插入 pUC19的 EcoR I+Xba I酶切位 点中,进行测序， 命名为 pUC19-IRES。  EcoR I + Xba I was double digested and inserted into the EcoR I + Xba I digestion site of pUC19, followed by sequencing and named pUC19-IRES.
用 EcoR I+Xba I双酶切分别插入 pClonelO的 EcoR I+Xba I 酶切位点中，命名为 pClonel2。  The EcoR I + Xba I double digestion was inserted into the EcoR I + Xba I digestion site of pClonelO and named pClonel2.
质粒 pClonel2 为含有脑心肌炎病毒 (EMCV)的多顺反子 (IRES)的双基因表达载体， 其启动子为人巨细胞病毒启动子 (CMV IE), 含有两个多克隆位点， 第一个多克隆位点的酶切位点 依次为 EcoR I、 Cla L Sai l及 Pst l， 第二个多克隆位点的酶切位 点依次为 BamH I、 Xho I、 Xma l及 Xba l。  Plasmid pClonel2 is a dual gene expression vector containing encephalomyocarditis virus (EMCV) polycistronic (IRES). Its promoter is the human cytomegalovirus promoter (CMV IE). It contains two polyclonal sites. The restriction sites of the cloning site were EcoR I, Cla L Sai l, and Pst l, and the restriction sites of the second multi-cloning site were BamH I, Xho I, Xma l, and Xba l.
抗人表皮生长因子受体 l(EGFR)的人抗体 SG-EGFR由上海 捷倍思基因技术有限公司进行全长基因合成， 其轻链基因与重链 基因的可变区与美国 Abgenix公司的针对表皮生长因子受体 1的 人的抗体 ABX-EGF 的轻链基因与重链基因的可变区相同（参阅 Jakobovits, et al. PCT WO98/50433)。含有 SG-EGFR重链基因并 在重链上游区引入 BamH I酶切位点和重链下游区引入 Xba 1酶 切位点的 pUC19质粒命名为 pUC-SG-EGFRH。 含有 SG-EGFR 轻链基因并在轻链上游区引入 EcoR I和轻链下游区引入 Sal I 酶切位点的 pUC19质粒为 pUC-SG-EGFRL。 The human antibody SG-EGFR against human epidermal growth factor receptor 1 (EGFR) was synthesized by Shanghai JBES Gene Technology Co., Ltd., and the variable regions of its light chain and heavy chain genes were targeted by the American Abgenix company. The light chain gene of the human antibody ABX-EGF of the epidermal growth factor receptor 1 is the same as the variable region of the heavy chain gene (see Jakobovits, et al. PCT WO98 / 50433). The pUC19 plasmid containing the SG-EGFR heavy chain gene and introducing the BamH I restriction site in the upstream region of the heavy chain and the Xba 1 restriction site in the downstream region of the heavy chain was named pUC-SG-EGFRH. The pUC19 plasmid containing the SG-EGFR light chain gene and introducing EcoR I in the upstream region of the light chain and the Sal I restriction site in the downstream region of the light chain was pUC-SG-EGFRL.
SG-EGFR的重链基因的核苷酸序列：  Nucleotide sequence of SG-EGFR heavy chain gene:
抗人表皮生长因子受体 l(EGFR)的人抗体 SG-EGFR由上海 捷倍思基因技术有限公司进行全长基因合成， 其轻链基因与重链 基因的可变区与美国 Abgenix公司的针对表皮生长因子受体 1的 人的抗体 ABX-EGF 的轻链基因与重链基因的可变区相同（参阅 Jakobovits, et al. PCT WO98/50433)。含有 SG-EGFR重链基因并 在重链上游区引入 BamH I酶切位点和重链下游区引入 Xba I酶 切位点的 pUC19质粒命名为 pUC-SG-EGFRH。 含有 SG-EGFR 轻链基因并在轻链上游区引入 EcoR I和轻链下游区引入 Sal I 酶切位点的 pUC19质粒为 pUC-SG"EGFRL。  The human antibody SG-EGFR against human epidermal growth factor receptor 1 (EGFR) was synthesized by Shanghai JBES Gene Technology Co., Ltd., and the variable regions of its light chain and heavy chain genes were targeted by the American Abgenix company. The light chain gene of the human antibody ABX-EGF of the epidermal growth factor receptor 1 is the same as the variable region of the heavy chain gene (see Jakobovits, et al. PCT WO98 / 50433). The pUC19 plasmid containing the SG-EGFR heavy chain gene and introducing the BamH I restriction site in the upstream region of the heavy chain and the Xba I restriction site in the downstream region of the heavy chain was named pUC-SG-EGFRH. The pUC19 plasmid containing the SG-EGFR light chain gene and introducing EcoR I in the upstream of the light chain and Sal I in the downstream of the light chain was pUC-SG "EGFRL.
SG-EGFR的重链基因的核苷酸序列: Nucleotide sequence of SG-EGFR heavy chain gene:
5'CGCGGATCCACC^TGGAGTTTTGGCTGAGCTGGGTT  5'CGCGGATCCACC ^ TGGAGTTTTGGCTGAGCTGGGTT
BamHI 重链信号肽  BamHI heavy chain signal peptide
TTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGTCTCTG  TTCCTTGTTGCTATTTTAAAAGGTGTCCAGTGTGTCTCTG
可变区开始 Variable region start
GTGGCTCCGTCAGCAGTGGTGATTACTACTGGACCTGGAGTGGCTCCGTCAGCAGTGGTGATTACTACTGGACCTGGA
TTCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGATTCGGCAGTCCCCAGGGAAGGGACTGGAGTGGATTGGA
CACATCTATTACAGTGGGAACACCAATTATAACCCCTCCCCACATCTATTACAGTGGGAACACCAATTATAACCCCTCCC
TCAAGAGTCGACTCACCATATCAATTGACACGTCCAAGACTCAAGAGTCGACTCACCATATCAATTGACACGTCCAAGAC
TCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCTGCGGATCAGTTCTCCCTGAAGCTGAGTTCTGTGACCGCTGCGGA
CACGGCCATTTATTACTGTGTGCGAGATCGAGTGACTGG TGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGT CTCTTC^GCCTCCACCAAGGGCCCATCGGTCTTCCCCCT 可变区结束， 恒定区开始 CACGGCCATTTATTACTGTGTGCGAGATCGAGTGACTGG TGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGT CTCTTC ^ GCCTCCACCAAGGGCCCATCGGTCTTCCCCCT End of the variable region and start of the constant region
GGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGG GGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGG
CCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG
TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC
GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGC
TTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAG
CCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAACCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAA
TCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCA
CCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC
CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT
GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGAGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGA
CCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGA
GGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT
GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA
GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACGGTCTCCAACAAAGCCCTCCCCCAGCCCCCATCGAGAAAAC
CATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT
GTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAAGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAA
CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCC
CAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC
CGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACT
CCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCT
CCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA AGAGCCTCTCCCTGTCTCCGGGTAA4TAGTAATCTAG AA 恒定区末尾 Xba lCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGA AGAGCCTCTCCCTGTCTCCGGGTAA4TAGTAATCTAG AA End of constant region Xba l
GCTTGGG 3' ( SEQ ID NO: 26 )。 GCTTGGG 3 '(SEQ ID NO: 26).
SG-EGFR的轻链基因的核苷酸序列: Nucleotide sequence of the light chain gene of SG-EGFR:
5'CCGGAATTCACC TGGAAGCCCCAGCTCAGCTTCTC  5'CCGGAATTCACC TGGAAGCCCCAGCTCAGCTTCTC
EcoR I 轻链信号肽  EcoR I light chain signal peptide
TTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA^CC  TTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA ^ CC
可变区开始 Variable region start
ATCACTTGCCAGGCGAGTCAGGACATCAGCAACTATTTAAATCACTTGCCAGGCGAGTCAGGACATCAGCAACTATTTAA
ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCC
TGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATC
AAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTT
CACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATAT
TTCTGTCAACACTTTGATCATCTCCCGCTCGCTTTCGGCG TTCTGTCAACACTTTGATCATCTCCCGCTCGCTTTCGGCG
■ 可变区结束， 恒定区开始 ■ End of variable region, start of constant region
CTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATC TGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTAT CCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCC CTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG GACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTG ACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTC ACAAAGAGCTTCAACAGGGGAGAGTGTGATAAGTCGAC3' CTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATC TGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTAT CCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCC CTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG GACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTG ACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTC ACAAAGAGCTTCAACAGGGGAGAGTGTGATAAGTCGAC3 '
恒定区结束  End of constant region
( SEQ ID NO: 27 )。 应用 BamH I+Xba I 将 pUC-SG-EGFRH切下， 定向插入 PClonel2载体 BamH I+Xba I中， 命名为 paonel2-SG-EGFRH。 应用 EcoR I+Sal I 将 pUC-SG-EGFRL 切下， 定向插入 pClonel2-SG- EGFRH 载体 EcoR I+Sal I 中， 命名 为 pClonel2-SG-EGFR。 (SEQ ID NO: 27). PUC-SG-EGFRH was excised using BamH I + Xba I, and inserted into the P Clonel2 vector BamH I + Xba I, named paonel2-SG-EGFRH. EcoR I + Sal I was used to excise pUC-SG-EGFRL and insert it into the pClonel2-SG-EGFRH vector EcoR I + Sal I. It was named pClonel2-SG-EGFR.
抗人表皮生长因子受体 2(Her2)的人源化抗体 SG-HER基因 由上海捷倍思基因技术有限公司进行全长基因合成， 其轻链基因 与重链基因的可变区与美国 Genentech公司的针对表皮生长因子 受体 2的人源化抗体 Herceptin的轻链基因与重链基因的可变区 相同（参阅 Carter et al. , United States Patent No: 5,821,337)0 含 有 SG-HER重链基因并在重链上游区引入 BamH I酶切位点和重 链下游区引入 Xho I 酶切位点的 pUC19 质粒命名为 pUC-SG-HERH。 含有 SG-HER 轻链基因并在轻链上游区引入 EcoR I 和轻链下游区引入 Sal I 酶切位点的 pUC19 质粒为 pUC-SG-HERL。 The humanized antibody SG-HER gene of human epidermal growth factor receptor 2 (Her2) was synthesized by Shanghai JBES Gene Technology Co., Ltd. The variable region of the light chain gene and the heavy chain gene is from Genentech in the United States. The light chain gene of the company's humanized antibody Herceptin against epidermal growth factor receptor 2 is the same as the variable region of the heavy chain gene (see Carter et al., United States Patent No: 5,821,337) 0 contains the SG-HER heavy chain gene The pUC19 plasmid that introduced the BamH I restriction site in the upstream region of the heavy chain and the Xho I restriction site in the downstream region of the heavy chain was named pUC-SG-HERH. The pUC19 plasmid containing the SG-HER light chain gene and introducing EcoR I in the upstream region of the light chain and the Sal I restriction site in the downstream region of the light chain was pUC-SG-HERL.
SG-HER的重链基因的核苷酸序列： Nucleotide sequence of SG-HER heavy chain gene:
5'GGATCCACC^TGAGCACTGAAAGCATGATCCGGGAC  5'GGATCCACC ^ TGAGCACTGAAAGCATGATCCGGGAC
BamH I TNF信号肽  BamH I TNF signal peptide
GTGGAGCTGGCCGAGGAGGCGCTCCCCAAGAAGACAGG GTGGAGCTGGCCGAGGAGGCGCTCCCCAAGAAGACAGG
GGGGCCCCAGGGCTCCAGGCGGTGCTTGTTCCTCAGCCGGGGCCCCAGGGCTCCAGGCGGTGCTTGTTCCTCAGCC
TCTTCTCCTTCCTGATCGTGGCAGGCGCCACCACGCTCTTCTTCTCCTTCCTGATCGTGGCAGGCGCCACCACGCTCT
TCTGCCTGCTGCACTTTGGAGTGATCGGCCCCCAGAGGG TCTGCCTGCTGCACTTTGGAGTGATCGGCCCCCAGAGGG
kAGAGTTCCCCAGGGACCTCTCTCTAATCAGCCCTCTGGkAGAGTTCCCCAGGGACCTCTCTCTAATCAGCCCTCTGG
[CCCAGGCAIGAGGTTCAGCTGGTGGAGTCTGGCGGTGGC [CCCAGGCAIGAGGTTCAGCTGGTGGAGTCTGGCGGTGGC
可变区开始  Variable region start
CTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCA GCTTCTGGCTTCAACATTAAAGACACCTATATACACTGGG TGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCA AGGATTTATCCTACGAATGGTTATACTAGATATGCCGATAG CGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAA AAACACAGCCTACCTGCAGATGAACAGCCTGCGTGCTGA GGACACTGCCGTCTATTATTGTTCTAGATGGGGAGGGGA CGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCCT GGTCACCGTCTCCTCGGCCTCCACCAAGGGCCCATCGGT CTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCA GCTTCTGGCTTCAACATTAAAGACACCTATATACACTGGG TGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCA AGGATTTATCCTACGAATGGTTATACTAGATATGCCGATAG CGTCAAGGGCCGTTTCACTATAAGCGCAGACACATCCAA AAACACAGCCTACCTGCAGATGAACAGCCTGCGTGCTGA GGACACTGCCGTCTATTATTGTTCTAGATGGGGAGGGGA CGGCTTCTATGCTATGGACTACTGGGGTCAAGGAACCCT GGTCACCGTCTCCTCGGCCTCCACCAAGGGCCCATCGGT
可变区结束， 恒定区开始  End of variable region, start of constant region
CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGG CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGG
CACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCC
CGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA
CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCT
CAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCT
CCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGACCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGA
ATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTG
AGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCC
TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGA
GGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCT
CACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTA
CAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCAT
CGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGACGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA
ACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCT GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA TGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGT GCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCT TCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA CACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAGTA ACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCT GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCACA TGGGCAGCCGGAGAACAACTACAAGACCACGCCCACCCCCC GCTGGACTCCGACGGCTCCTTCTTCCTCGCAGGCCATCAGTCAC
恒定区结束  End of constant region
ACTCGAG 3' ( SEQ ID NO: 28 )。  ACTCGAG 3 '(SEQ ID NO: 28).
Xho l  Xho l
5'CCGGAATTCACC^TGGAAGCCCCAGCTCAGCTTCTC 5'CCGGAATTCACC ^ TGGAAGCCCCAGCTCAGCTTCTC
EcoR I 轻链信号肽
Figure imgf000043_0001
EcoR I light chain signal peptide
Figure imgf000043_0001
可变区开始 Variable region start
TCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTG
TGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGG
ATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCAGATGTGAATACTGCTGTAGCCTGGTATCAACAGAAACCAG
GAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCCTGAAAAGCTCCGAAACTACTGATTTACTCGGCATCCTTCCT
CTACTCTGGAGTCCCTTCTCGCTTCTCTGGATCCAGATCTCTACTCTGGAGTCCCTTCTCGCTTCTCTGGATCCAGATCT
GGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCG
GAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTAGAAGACTTCGCAACTTATTACTGTCAGCAACATTATACTA
CTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCA 可变区结束， 恒定区开始 CTCCTCCCACGTTCGGACAGGGTACCAAGGTGGAGATCA End of the variable region and start of the constant region
TGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTG CCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACA GTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCA GGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCT ACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACT ACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGG GGAGAGTGTTGATAAGTCGAC 3' (SEQ ID NO: 29)。 TGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTG CCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACA GTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCA GGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCT ACAGCCTCAGCAGCACCCTCGCGCTGAGCAAAGCAGACT ACGAGAAACACGTGTACGCCTGCGCGAAGTCTCAGAGACCCCCCAGAG
恒定区结束 Sai l  End of constant zone Sai l
应用 BamH I+Xho I 将 pUC-SG-HERH 切下， 定向插入 pClonel2载体 BamH I+Xho I中， 命名为 pCkmel2-SG-HERH。 应用 EcoR I+Sal I 将 pUC-SG-HERL 切下， 定向插入 PClonel2-SG-HERH 载 体 EcoR I+Sal I 中 ， 命 名 为 pClonel2-SG-HERo PUC-SG-HERH was excised using BamH I + Xho I, and inserted into the pClonel2 vector BamH I + Xho I, named pCkmel2-SG-HERH. Use EcoR I + Sal I to cut pUC-SG-HERL, insert it into the P Clonel2-SG-HERH vector EcoR I + Sal I, and name it pClonel2-SG-HERo
抗人 CD20的人鼠嵌合抗体 SG-CD20基因由上海捷倍思基因 技术有限公司进行全长基因合成， 其轻链基因与重链基因的可变 区与 美国 IDEC 公司 的针对 CD20 的人鼠嵌合抗体 IDEC-C2B8(Rituximab)的轻链基因与重链基因的可变区相同（参 阅 Anderson et al. United States Patent No: 6,399,061)» 含有 SG-CD20重链基因并在重链上游区引入 BamH I酶切位点和重链 下游区引入 Xba I 酶切位点的 pUC19 质粒命名为 pUC- SG-CD20H.含有 SG-CD20轻链基因并在轻链上游区引入 EcoR I 和轻链下游区引入 Sal I 酶切位点的 pUC19 质粒为 pUC-SG-CD20L„  The human-mouse chimeric antibody SG-CD20 gene of anti-human CD20 was synthesized by Shanghai JBES Gene Technology Co., Ltd. The variable region of the light chain gene and the heavy chain gene was compared with the human and mouse against CD20 of the United States IDEC Corporation. The chimeric antibody IDEC-C2B8 (Rituximab) has the same light chain gene as the variable region of the heavy chain gene (see Anderson et al. United States Patent No: 6,399,061) The pUC19 plasmid that introduced the Xba I digestion site into the BamH I digestion site and the heavy chain downstream region was named pUC-SG-CD20H. It contained the SG-CD20 light chain gene and introduced EcoR I and the light chain downstream region in the upstream region The pUC19 plasmid introduced into the Sal I restriction site is pUC-SG-CD20L.
SG-CD20的重链基因的核苷酸序列： Nucleotide sequence of the heavy chain gene of SG-CD20:
5'CGCGGATCCACCkTGGAGTTTTGGCTGAGCTGGGTT  5'CGCGGATCCACCkTGGAGTTTTGGCTGAGCTGGGTT
BamH I 重链信号肽
Figure imgf000045_0001
BamH I heavy chain signal peptide
Figure imgf000045_0001
可变区开始 可变区结束， 恒定区开始 Start of variable region End of variable region, start of constant region
CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTC TCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAG GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGC GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGA GAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA CGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAA GAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAG CCTCTCCCTGTCTCCGGGTAAATAGTAATCTAGA 3' CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTC TCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAG GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGC GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGA GAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA CGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAA GAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAG CCTCTCCCTGTCTCCGGGTAAATAGTAATCTAGA 3 '
恒定区结束 Xba l  End of constant region Xba l
( SEQ ID NO: 30 )。  (SEQ ID NO: 30).
SG-CD20的轻链基因的核苷酸序列: Nucleotide sequence of the light chain gene of SG-CD20:
5'CCGGAATTCACC^TGGAAGCCCCAGCTCAGCTTCTC  5'CCGGAATTCACC ^ TGGAAGCCCCAGCTCAGCTTCTC
EcoR I Kappa轻链信号肽  EcoR I Kappa light chain signal peptide
TTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGACAA  TTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGACAA
可变区开始 Variable region start
ATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTC
CAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCACAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCA
AGTGTAAGTTACATCCACTGGTTCCAGCAGAAGCCAGGAAGTGTAAGTTACATCCACTGGTTCCAGCAGAAGCCAGGA
TCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGTCCTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGG
CTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTG
GGACTTCTTACTCTCTCACCATCAGCAGAGTGGAGGCTGGGACTTCTTACTCTCTCACCATCAGCAGAGTGGAGGCTG
AAGATGCTGCCACTTATTACTGCCAGCAGTGGACTAGTAAAAGATGCTGCCACTTATTACTGCCAGCAGTGGACTAGTAA
CCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATCA AACGTACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCC 可变区结束， 恒定区开始 CCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATCA AACGTACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCC The variable region ends and the constant region begins
ATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGT GTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGT ACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTC CCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCA CCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAG ACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCC ATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA GGGGAGAGTGTTGATAAGTCGAC 3' ( SEQ ID NO: 31 )。  ATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGT GTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGT ACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTC CCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCA CCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAG ACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCC ATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA GGGGAGAGTGTTGATAAGTCGAC 3 '(SEQ ID NO: 31).
恒定区结束 Sai l 应用 BamH I+Xba I将 pUC-SG-CD20H切下， 定向插入 pClonel2载体 BamH I+Xba I中， 命名为 pClonel2-SG-CD20H。 应用 EcoR I+Sal I 将 pUC-SG-CD20L 切下， 定向插入 pClonel2-SG- CD20H 载体 EcoR I+Sal I 中， 命名 为 pCkmel2-SG-CD20。  The end of the constant region Sai l pUC-SG-CD20H was excised using BamH I + Xba I, and inserted into the pClonel2 vector BamH I + Xba I, named pClonel2-SG-CD20H. EcoR I + Sal I was used to cut pUC-SG-CD20L and insert it into the pClonel2-SG- CD20H vector EcoR I + Sal I. It was named pCkmel2-SG-CD20.
为进一步提高抗体表达量， 在启动子的转录起始位点与抗体 的翻译起始位点中插入内含子， 该内含子为腺病毒主要晚期 mRNA 的第三个引导顺序的 5，剪接位点和一个免疫球蛋白的 3， 剪接位点的杂合内含子。 杂合内含子核苷酸序列由上海捷倍思基 因技术有限公司进行全长合成， 并在内含子上游区引入 Spe I酶 切位点和内含子下游区引入 EcoR I酶切位点的 pUC19质粒命名 为 pUC - Intron。  In order to further increase the expression level of the antibody, an intron is inserted between the transcription start site of the promoter and the translation start site of the antibody, and the intron is the third, 5 splicing sequence of the main advanced mRNA of the adenovirus Site and an immunoglobulin 3, a heterozygous intron for the splice site. The heterozygous intron nucleotide sequence was synthesized by Shanghai Jibeisi Gene Technology Co., Ltd. The Spe I restriction site was introduced into the upstream region of the intron and the EcoR I restriction site was introduced into the downstream region of the intron. The pUC19 plasmid was named pUC-Intron.
杂合内含子的核苷酸序列：  Nucleotide sequence of heterozygous intron:
ACTAGTTAACCAGTCACAGTCGCAAGGTAGGCTGAGCAC CGTGGCGGGCGGCAGCGGGTGGCGGTCGGGGTTGTTTCACTAGTTAACCAGTCACAGTCGCAAGGTAGGCTGAGCAC CGTGGCGGGCGGCAGCGGGTGGCGGTCGGGGTTGTTTC
TGGCGGAGGTGCTGCTGATGATGTAATTAAAGTAGGCGGTGGCGGAGGTGCTGCTGATGATGTAATTAAAGTAGGCGG
TCTTGAGACGGCGGATGGTCGAGGTGAGGTGTGGCAGGTCTTGAGACGGCGGATGGTCGAGGTGAGGTGTGGCAGG
CTTGAGATCGATCTGGCCATACACTTGAGTGACAATGACACTTGAGATCGATCTGGCCATACACTTGAGTGACAATGACA
TCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGGTCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGG
TCCAACCGAATTC 3' ( SEQ ID NO: 32 )。 TCCAACCGAATTC 3 '(SEQ ID NO: 32).
合成两个 DNA寡核苷酸片段做一个连接头， 其 DNA寡核苷 酸序列为：  Synthesize two DNA oligonucleotide fragments as a linker, and the DNA oligonucleotide sequence is:
引物 22: 5' AAT TAC TAG TCA GGA ATT CA 3' ( SEQ ID NO: 33 )。  Primer 22: 5 'AAT TAC TAG TCA GGA ATT CA 3' (SEQ ID NO: 33).
引物 23: 5' AGC TTG AAT TCC TGA CTA GT3' ( SEQ ID NO: 34 )。  Primer 23: 5 'AGC TTG AAT TCC TGA CTA GT3' (SEQ ID NO: 34).
将两个 DNA寡核苷酸片段各 O.l g混合， lOOt^变性 5分钟， 然后緩慢降温复性，复性后应用 T4噬菌体多核苷酸激酸进行磷酸 化。将该磷酸化后的连接头插入 pClonelO载体的 EcoR I和 Hind III位点，命名为 pClonelO-Linker,该载体的多克隆位点分别 Spe I、 EcoR I、 Sal L Hind III、 Xho I、 BamH I及 Xba l。  The two DNA oligonucleotide fragments were each mixed with 0.1 g, denatured at 100 t ^ for 5 minutes, and then slowly cooled to renaturation. After renaturation, T4 phage polynucleotide phosphoric acid was used for phosphorylation. The phosphorylated linker was inserted into the EcoR I and Hind III sites of the pClonelO vector and named pClonelO-Linker. The multiple cloning sites of this vector were Spe I, EcoR I, Sal L Hind III, Xho I, and BamH I. And Xba l.
应用 Spe I+EcoR I 酶切 pUC-Intron， 其片段插入 PDC315-linker的 Spe I+EcoR I酶切位点中， 命名为 pYQ20, 该 载体的多克隆位点分别 EcoR I、 Sal L Hind III、 Xho I, BamH I 及 Xba I。将 pClonel2-SG-HER用 EcoR I+Xho I切下， 定向插入 pYQ20载体 EcoR I+Xho I中， 命名为 pYQ20-SG-HER。  PUC-Intron was digested with Spe I + EcoR I, and the fragment was inserted into the Spe I + EcoR I digestion site of PDC315-linker, named pYQ20. The multiple cloning sites of this vector were EcoR I, Sal L Hind III, Xho I, BamH I and Xba I. PClonel2-SG-HER was cut out with EcoR I + Xho I, and inserted into the pYQ20 vector EcoR I + Xho I. It was named pYQ20-SG-HER.
例 5. 分别携带抗人表皮生长因子受体 l(EGFR)的人的抗体 SG-EGFR 基因、 抗人表皮生长因子受体 2(Her2)的人源化抗体 SG-HER基因及抗人 CD20的人鼠嵌合抗体 SG-CD20基因的腺病 毒载体的构建  Example 5. Human antibody SG-EGFR gene against human epidermal growth factor receptor 1 (EGFR), humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2), and anti-human CD20 Construction of human and mouse chimeric antibody SG-CD20 gene adenovirus vector
pBHGlO购于加拿大 Microbix Biosystem Inc.(Toronto)。 将 pClonel2-SG-EGFR 、 pClonel2-SG-HER, pClonel2-SG-CD20 和 pYQ20-SG-HER分别用 PAC I酶切，分别插入 pBGHIO的 PAC I酶切位点，分别命名为 pBGH10-SG-EGFR、 pBGH10-SG-HE , pBGH10-SG-CD20及 pBGH10-YQSG-HER。 pBHGlO was purchased from Microbix Biosystem Inc. (Toronto), Canada. will pClonel2-SG-EGFR, pClonel2-SG-HER, pClonel2-SG-CD20 and pYQ20-SG-HER were digested with PAC I, inserted into the PAC I digestion sites of pBGHIO, and named pBGH10-SG-EGFR, pBGH10-SG-HE, pBGH10-SG-CD20 and pBGH10-YQSG-HER.
例 6 分别携带抗人表皮生长因子受体 l(EGFR)的人的抗体 SG-EGFR 基因、 抗人表皮生长因子受体 2(Her2)的人源化抗体 SG-HER基因及抗人 CD20的人鼠嵌合抗体 SG-CD20基因的端粒 酶催化亚基基因启动子及缺氧启动子分别控制 E1A及 E1B表达 的增殖腺病毒的重组。  Example 6 Humans carrying anti-human epidermal growth factor receptor 1 (EGFR) antibody SG-EGFR gene, humanized antibodies against human epidermal growth factor receptor 2 (Her2) SG-HER gene and human anti-CD20 The telomerase catalytic subunit gene promoter and hypoxic promoter of murine chimeric antibody SG-CD20 gene control the recombination of proliferating adenoviruses expressing E1A and E1B, respectively.
293细胞株购于加拿大 Microbix Biosystem Inc.(Toronto),是 由剪切的 5型腺病毒 DNA转化人胚胎肾细胞而成，它含有及表达 5型腺病毒 E1区， 腺病毒 DNA对其具有高转染率。 将含有 5型 腺病毒左臂的质粒联合含有 5型腺病毒右臂的质粒共转染 293细 胞， 通过同源重组可产生具有感染力的腺病毒。 我们将 pQW-hTERT-Ep-HRE 分 别 与 pBGH10-SG-EGFR 、 pBGH10-SG-HER, pBGH10-SG-CD20及 pBGH10-YQSG-HER 通过 Lipofectamine共转染至 293细株。 共转染后 9-14天出现病 毒空斑， 经过三次病毒空斑纯化， 分别得到端粒酶催化亚基基因 启动子及缺氧启动子分别控制 E1A及 E1B表达的增殖腺病毒并 携带抗人表皮生长因子受体 l(EGFR)的人的抗体 SG-EGFR基因 的增殖腺病毒 SG500-EGFR、 端粒酶催化亚基基因启动子及缺氧 启动子分别控制 E1A及 E1B表达的增殖腺病毒并抗人表皮生长 因子受体 2(Her2)的人源化抗体 SG-HER 基因的增殖腺病毒 SG500-HER, 端粒酶催化亚基基因启动子及缺氧启动子分別控制 E1A 及 E1B 表达的增殖腺病毒并抗人 CD20 的人鼠嵌合抗体 SG-CD20基因的增殖腺病毒 SG500-CD20及端粒酶催化亚基基因 启动子及缺氧启动子分别控制 E1A及 E1B表达的增殖腺病毒并 抗人表皮生长因子受体 2(Her2)的人源化抗体 SG-HER基因并在 人 CMV 启动子下游插入杂合内含子的增殖腺病毒 SG500-YQ-HER,分别 记为 SG501 、 SG502 、 SG503 及 SG-YQ-502。 The 293 cell line was purchased from Microbix Biosystem Inc. (Toronto), Canada. It was transformed from human embryonic kidney cells by shearing type 5 adenovirus DNA. It contains and expresses type 5 adenovirus E1 region. Transfection rate. A plasmid containing an adenovirus type 5 left arm and a plasmid containing an adenovirus type 5 right arm were co-transfected into 293 cells, and infectious adenovirus could be produced by homologous recombination. We co-transfected pQW-hTERT-Ep-HRE with pBGH10-SG-EGFR, pBGH10-SG-HER, pBGH10-SG-CD20, and pBGH10-YQSG-HER into 293 strains via Lipofectamine. Viral plaques appeared 9 to 14 days after co-transfection. After three viral plaque purifications, a telomerase catalytic subunit gene promoter and a hypoxic promoter were used to control the proliferation of adenoviruses expressing E1A and E1B, respectively. Epidermal growth factor receptor 1 (EGFR) human antibody SG-EGFR gene proliferative adenovirus SG500-EGFR, telomerase catalytic subunit gene promoter and hypoxic promoter respectively control E1A and E1B expression proliferative adenovirus Anti-human epidermal growth factor receptor 2 (Her2) humanized antibody SG-HER gene proliferation adenovirus SG500-HER, telomerase catalytic subunit gene promoter and hypoxic promoter control E1A and E1B expression proliferation, respectively Adenovirus anti-human CD20 human mouse chimeric antibody SG-CD20 gene proliferation Adenovirus SG500-CD20 and telomerase catalytic subunit gene promoter and hypoxic promoter control the proliferation of adenovirus expression of E1A and E1B and Proliferating adenovirus SG500-YQ-HER, a humanized antibody against the human epidermal growth factor receptor 2 (Her2) gene and inserting a hybrid intron downstream of the human CMV promoter, and is denoted as SG501, SG502, SG503 and SG-YQ-502.
由上述方法构建的重組病毒的列表如下:  The list of recombinant viruses constructed by the above method is as follows:
重组病毒 ^ 含 Ad5左臂质粒 含 Ad5右臂质粒 Recombinant virus ^ Ad5 left arm plasmid included Ad5 right arm plasmid included
SG500-EGFR SG501 pQW-hTERT-Ep--HRE PBHG10-SG-EGFR SG500-EGFR SG501 pQW-hTERT-Ep--HRE PBHG10-SG-EGFR
SG500-HER SG502 pQ -hTERT-Ep-HRE PBHG10-SG-HERSG500-HER SG502 pQ -hTERT-Ep-HRE PBHG10-SG-HER
SG500-CD20 SG503 pQ -hTERT-Ep--HRE PBHG10-SG-CD20SG500-CD20 SG503 pQ -hTERT-Ep--HRE PBHG10-SG-CD20
SG500-YQ-HER SG-YQ-502 pQW-hTERT-Ep-HRE PBHG10-YQSG-HER 腺病毒在 293细胞中大量繁殖， 应用氯化铯梯度离心的方法 大量纯化腺病毒 (具体方法参见前述文献 2 出版)。 SG500-EGFR ( SG501 ) 为 5型腺病毒， 在 E1A编码起始位点上游插入端粒酶 催化亚基基因启动子以及 E1B编码起始位点上游插入缺氧基因启 动子,同时伴有 28133-30818bp(E3区部分序列) 缺失， 并在 E3插 入含有人巨细胞病毒 (HCMV) IE启动子、 含有抗人表皮生长因子 受体 l(EGFR)的人的抗体 SG-EGFR基因的轻链、 重链基因及脑 心肌炎病毒多顺反子、 SV40 poly A加尾信号基因序列， 病毒其他 DNA序列与 5型腺病毒相同。 SG500-HER ( SG502 )为 5型腺病 毒， 在 El A编码起始位点上游插入端粒酶催化亚基基因启动子以 及 E1B 编码起始位点上游插入缺氧基因启动子，同时伴有 28133-30818bp(E3 区部分序列） 缺失， 并在 E3插入含有人巨细 胞病毒 (HCMV) IE启动子、 含有抗人表皮生长因子受体 2(Her2) 的人源化抗体 SG-HER基因的轻链、 重链基因及脑心肌炎病毒多 顺反子、 SV40 poly A加尾信号基因序列， 病毒其他 DNA序列与 5型腺病毒相同。 SG500-CD20 ( SG503 ) 为 5型腺病毒， 在 E1A 编码起始位点上游插入端粒酶催化亚基基因启动子以及 E1B编码 起始位点上游插入缺氧基因启动子，同时伴有 28133-30818bp(E3 区部分序列） 缺失, 并在 E3插入含有人巨细胞病毒 (HCMV) IE 启动子、 含有抗人 CD20的人鼠嵌合抗体 SG-CD20基因的轻链、 重链基因及脑心肌炎病毒多顺反子、 SV40 poly A加尾信号基因序 列， 病毒其他 DNA序列与 5 型腺病毒相同。 SG500-YQ-HER ( SG-YQ-502 )为 5型腺病毒， 在 E1A编码起始位点上游插入端 粒酶催化亚基基因启动子以及 E1B编码起始位点上游插入缺氧基 因启动子,同时伴有 28133-30818bp(E3 区部分序列） 缺失， 并在 E3插入含有人巨细胞病毒 (HCMV) IE启动子、 含有腺病毒主要 晚期 mRNA 的第三个引导顺序的 5，剪接位点和一个免疫球蛋白 的 3， 剪接位点的杂合内含子、含有抗人表皮生长因子受体 2(Her2) 的人源化抗体 SG-HER基因的轻链、 重链基因及脑心肌炎病毒多 顺反子、 SV40 poly A加尾信号基因序列， 病毒其他 DNA序列与 5型腺病毒相同。 SG500-YQ-HER SG-YQ-502 pQW-hTERT-Ep-HRE PBHG10-YQSG-HER Adenoviruses multiply in 293 cells. Adenoviruses are purified by cesium chloride gradient centrifugation method (for details, please refer to the aforementioned reference 2) publishing). SG500-EGFR (SG501) is a type 5 adenovirus. The telomerase catalytic subunit gene promoter is inserted upstream of the E1A coding start site and the hypoxia gene promoter is inserted upstream of the E1B coding start site. 30818bp (partial sequence of E3 region) was deleted, and the light chain and heavy chain of human antibody SG-EGFR gene containing human cytomegalovirus (HCMV) IE promoter and human anti-human epidermal growth factor receptor 1 (EGFR) were inserted in E3 Chain gene and encephalomyocarditis virus polycistronic, SV40 poly A tail signal gene sequence, the other DNA sequences of the virus are the same as type 5 adenovirus. SG500-HER (SG502) is an adenovirus type 5, which inserts the telomerase catalytic subunit gene promoter upstream of the El A coding start site and the hypoxia gene promoter upstream of the E1B coding start site, accompanied by 28133 -30818bp (partial sequence of E3 region) was deleted and a light chain containing a human cytomegalovirus (HCMV) IE promoter and a humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2) was inserted in E3 , Heavy chain genes and encephalomyocarditis virus polycistronic, SV40 poly A tail signal gene sequences, the other DNA sequences of the virus are the same as type 5 adenovirus. SG500-CD20 (SG503) is an adenovirus type 5 in E1A The telomerase catalytic subunit gene promoter is inserted upstream of the coding start site and the hypoxic gene promoter is inserted upstream of the E1B coding start site, with a deletion of 28133-30818bp (partial sequence of the E3 region), and the insertion at E3 contains Human cytomegalovirus (HCMV) IE promoter, light chain, heavy chain gene of human mouse-chimeric antibody SG-CD20 gene containing anti-human CD20, polycistronic of encephalomyocarditis virus, SV40 poly A tail signal gene sequence, The other DNA sequences of the virus are the same as those of adenovirus type 5. SG500-YQ-HER (SG-YQ-502) is a type 5 adenovirus. The telomerase catalytic subunit gene promoter is inserted upstream of the E1A coding start site and the hypoxia gene promoter is inserted upstream of the E1B coding start site. Accompanied by a deletion of 28133-30818bp (partial sequence of the E3 region), and inserted in E3 containing the human cytomegalovirus (HCMV) IE promoter, the third guide sequence containing the major late-stage mRNA of adenovirus 5, the splice site and An immunoglobulin 3, a heterologous intron at the splice site, a light chain, a heavy chain gene, and an encephalomyocarditis virus containing the humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2) The cistron, SV40 poly A tail signal gene sequence, and other viral DNA sequences are the same as type 5 adenovirus.
例 7: 携带抗人表皮生长因子受体 2(Her2)的人源化抗体 SG-HER基因并且具有端粒酶催化亚基基因启动子及缺氧启动子 分别控制 E1A及 E1B表达的增殖腺病毒 SG500-HER(SG502)体 外在具有端粒酶活性的肿瘤细胞特异性增殖、 复制。  Example 7: A proliferating adenovirus carrying the humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2) and having a telomerase catalytic subunit gene promoter and a hypoxic promoter controlling the expression of E1A and E1B, respectively SG500-HER (SG502) specifically proliferates and replicates tumor cells with telomerase activity in vitro.
人卵巢细胞癌细胞株 SKOV3、 人乳腺癌细胞株 BT-474及正 常成纤维细胞株 BJ均购于美国 ATCC公司。  Human ovarian cell cancer cell line SKOV3, human breast cancer cell line BT-474 and normal fibroblast cell line BJ were purchased from ATCC in the United States.
将 SG502 分别感染具有端粒酶活性的人卵巢癌细胞株 SKOV3. 人乳腺癌细胞株 BT-474及正常成纤维细胞株 BJ, 细 胞按 5 xlO5/孔接种 6孔板， 分別感染重组腺病毒 SG502及野生 型 5型腺病毒 1 xl 06pfu， 48小时后应用 293细胞株测定其病毒 滴度， 具体方法参见前述文献 2, 48小时增殖倍数结果为： SKOV3 BT-474 BJ SG502 was infected with human ovarian cancer cell line SKOV3 with telomerase activity. Human breast cancer cell line BT-474 and normal fibroblast cell line BJ. Cells were inoculated into 6-well plates at 5 x 10 / well and infected with recombinant adenovirus respectively. SG502 and wild type adenovirus type 5 1 xl 0 6 pfu, 48 hours after the application of 293 cells was measured virus titer, refer to the specific method of Document 2, a multiple of 48 hours proliferation results: SKOV3 BT-474 BJ
SG502 230,000 180,000 50  SG502 230,000 180,000 50
野生型腺病毒 260,000 150,000 156,000 例 8: 携带抗人表皮生长因子受体 2(Her2)的人源化抗体 SG-HER基因的端粒酶催化亚基基因启动子及缺氧启动子分别控 制 E1A及 E1B表达的增殖腺病毒 SG500-HER(SG502)小鼠体内 表达人源化抗体 Wild-type adenovirus 260,000 150,000 156,000 Example 8: The telomerase catalytic subunit gene promoter and hypoxia promoter of the humanized antibody SG-HER gene carrying human epidermal growth factor receptor 2 (Her2) control E1A and Humanized antibody expressed in E1B-proliferated adenovirus SG500-HER (SG502) mice
将对照腺病毒 Ad5-Lac Z或重组腺病毒 (SG502)分别以 1 χ 109pfu及 5 xl09pf«在尾静脉给予 Balb/c小鼠治疗， 1周后应用 夹心法酶联免疫吸附试验（ELISA ) 定量检测其小鼠血清的表达 量， 结果可见 SG502体内感染后， 其小鼠血清有大量人源化抗体 表达， 其表达水平与给予的病毒剂量成正比， 其动物体内人源化 抗体的血清浓度高于文献中 Herceptin 的治疗浓度， 而对照腺病 毒 Ad5-Lac Z则阴性。 结果见图 1。 The control adenovirus Ad5-Lac Z or recombinant adenovirus (SG502) was administered to Balb / c mice at 1 x 10 9 pfu and 5 x 10 9 pf «in the tail vein, and sandwich enzyme-linked immunosorbent assay was applied 1 week later. (ELISA) Quantitative detection of the expression of mouse serum, the results showed that after SG502 infection in vivo, the mouse serum has a large amount of humanized antibody expression, the expression level is directly proportional to the dose of the virus, the humanized antibody in its animal body The serum concentration was higher than the therapeutic concentration of Herceptin in the literature, while the control adenovirus Ad5-Lac Z was negative. The results are shown in Figure 1.
例 10: 携带抗人表皮生长因子受体 2(Her2)的人源化抗体 SG-HER基因的端粒酶催化亚基基因启动子及缺氧启动子分别控 制 E1A及 E1B表达的增殖腺病毒 SG500-HER(SG502)在棵鼠体 内治疗移植肿瘤  Example 10: A telomerase catalytic subunit gene promoter and a hypoxia promoter carrying the humanized antibody SG-HER gene against human epidermal growth factor receptor 2 (Her2), a proliferating adenovirus SG500 that controls the expression of E1A and E1B, respectively -HER (SG502) in transplanted tumors in mice
携带抗人表皮生长因子受体 2(Her2)的人源化抗体 SG-HER 基因的端粒酶催化亚基基因启动子及缺氧启动子分别控制 E1A及 E1B表达的增殖腺病毒 SG500-HER(SG502)在棵鼠体内治疗移植 肿瘤，研究表明其可高效表达抗人表皮生长因子受体 2(Her2)的人 源化抗体， 抑制人表皮生长因子受体 2(Her2)肿瘤形成、 生长及转 移。 同时该病毒能在而且仅限于肿瘤细胞内增殖， 也能特异性直 接杀灭肿瘤细胞。  A telomerase catalytic subunit gene promoter and a hypoxia promoter carrying the humanized antibody SG-HER gene of anti-human epidermal growth factor receptor 2 (Her2) proliferating adenovirus SG500-HER (E500) SG502) is used to treat transplanted tumors in mice. Studies have shown that it can efficiently express humanized antibodies against human epidermal growth factor receptor 2 (Her2) and inhibit human epidermal growth factor receptor 2 (Her2) tumor formation, growth and metastasis. . At the same time, the virus can proliferate in and be limited to tumor cells and can specifically kill tumor cells directly.
将 4-5周龄的棵鼠皮下接种人卵巢细胞株 SKOV3 细胞 1 X 107, 两周后给予 l xl 09pfu增殖型重組腺病毒 SG502治疗或用相 同剂量的对照腺病毒 Ad5-Lac Z, 其未治疗组及对照腺病毒治疗 組 4周后肿瘤体增加 3倍以上， 而治疗組 SG502则肿瘤增加不明 显， 结果参见图 2。 4-5 week old mice were subcutaneously inoculated with human ovarian cell line SKOV3 cells 1 X 107, two weeks after the administration of l xl 0 9 pfu recombinant adenovirus SG502 proliferation or treatment with a control adenovirus Ad5-Lac Z of the same dose, which is not the treatment group and control adenovirus treated group after 4 weeks 3-fold increase in tumor Above, the SG502 in the treatment group did not increase the tumor significantly. See Figure 2 for the results.
本发明通过上述实施例进行了详细说明， 但本发明并不限于 此。 本领域普通技术人员完全清楚， 可以对本发明作各种变更和 修饰， 而仍然落入本发明的精神和范围。  The present invention has been described in detail through the above embodiments, but the present invention is not limited thereto. Those skilled in the art are fully aware that various changes and modifications can be made to the present invention, and still fall within the spirit and scope of the present invention.
本发明引用了下述参考文献。 它们均单独地全文引入本文作为 参考- 参考文献  The following references are cited in the present invention. They are individually incorporated in their entirety as references-References
有关病毒中至少一个增殖必需基因的转录受肿瘤细胞特异性 激活的顺式作用元件有效控制导致病毒肿瘤细胞内特异性增殖的 相关专利及文献：  Related patents and literatures related to the transcription of at least one gene necessary for proliferation in the virus are effectively controlled by tumor cell-specific activated cis-acting elements that lead to specific proliferation in viral tumor cells:
1. Henderson, et al. United States Patent No. US 6,432,700 Bl Date of patent: Aug.13,2002, Adenovirus vectors containing heterologous transcription regulatory elements and methods of using same  1. Henderson, et al. United States Patent No. US 6,432,700 Bl Date of patent: Aug.13,2002, Adenovirus vectors containing heterologous transcription regulatory elements and methods of using same
2. Henderson, et al. PCT WO 01/73093 Cell-specific adenovirus vectors comprising an internal ribosome entry site  2. Henderson, et al. PCT WO 01/73093 Cell-specific adenovirus vectors includes an internal ribosome entry site
3. Hallenbeck, et al. United States Patent No. US 5,998,205 Vectors for tissue-specific replication  3. Hallenbeck, et al. United States Patent No. US 5,998,205 Vectors for tissue-specific replication
4. Molnar-kim-ber, et al. PCT WO 01/23004 Replicating selective adenoviruses for use in cancer therapy.  4. Molnar-kim-ber, et al. PCT WO 01/23004 Replicating selective adenoviruses for use in cancer therapy.
5. Martuza, et al. United States Patent No. US 5,728,379 Tumor - or cell-specific herpes simplex virus replication  5. Martuza, et al. United States Patent No. US 5,728,379 Tumor-or cell-specific herpes simplex virus replication
6.钱其军等. 中国发明专利申请号 99124108 —种能特异性杀灭 E B病毒相关肿瘤的重组病毒及其构建方法 6. Qian Qijun, et al. Chinese Invention Patent Application No. 99124108 — Species that can be specifically killed Recombinant virus of EB virus related tumor and its construction method
7. Qian Qijun PCT WO0244347 A VIRUS WHICH CAN EXPRESS TUMOR ANGIOSTATIN FACTOR WITH HIGH EFFICIENCY IN SPECIFIC TUMOR CELLS AND THE USE OF IT  7. Qian Qijun PCT WO0244347 A VIRUS WHICH CAN EXPRESS TUMOR ANGIOSTATIN FACTOR WITH HIGH EFFICIENCY IN SPECIFIC TUMOR CELLS AND THE USE OF IT
8. Qian Qijun PCT WO03006640 A SPECIFIC PROLIFERATION IN TUMOUR CELL WHICH CAN EXPRESS ANTIONCOGENE WITH HIGH EFFICIENCY AND THE USE OF IT 有关病毒包含至少一种增殖必需区蛋白功能缺失， 从而不能 在正常细胞内增殖， 但能在肿瘤细胞内特异性增殖的相关专利及 文献：  8. Qian Qijun PCT WO03006640 A SPECIFIC PROLIFERATION IN TUMOUR CELL WHICH CAN EXPRESS ANTIONCOGENE WITH HIGH EFFICIENCY AND THE USE OF IT Relevant patents and literatures on specific proliferation:
1. Molnar-Kimber, et al. United States Patent 6,428,968 Combined therapy with a chemotherapeutic agent and an oncolytic virus for killing tumor cells in a subject  1. Molnar-Kimber, et al. United States Patent 6,428,968 Combined therapy with a chemotherapeutic agent and an oncolytic virus for killing tumor cells in a subject
2. McCormick, et al. United States Patent 5,677,178 Cytopathic viruses for therapy and prophylaxis of neoplasia  2. McCormick, et al. United States Patent 5,677,178 Cytopathic viruses for therapy and prophylaxis of neoplasia
3. McCormick, et al. United States Patent 5,846,945 Cytopathic viruses for therapy and prophylaxis of neoplasia  3. McCormick, et al. United States Patent 5,846,945 Cytopathic viruses for therapy and prophylaxis of neoplasia
4. Qian Qijun PCT WO0244347 A VIRUS WHICH CAN EXPRESS TUMOR ANGIOSTATIN FACTOR WITH HIGH EFFICIENCY IN SPECIFIC TUMOR CELLS AND THE USE OF IT  4. Qian Qijun PCT WO0244347 A VIRUS WHICH CAN EXPRESS TUMOR ANGIOSTATIN FACTOR WITH HIGH EFFICIENCY IN SPECIFIC TUMOR CELLS AND THE USE OF IT
5. Coffey, et al. PCT WOOl/35970 Viruses for the treatment of cellular proliferative disorders  5. Coffey, et al. PCT WOOl / 35970 Viruses for the treatment of cellular proliferative disorders

Claims

1. 一种重组病毒， 其特征在于该病毒能特异性地在肿瘤细胞 内复制， 同时在该病毒基因组中包含一种编码治疗肿瘤的抗体或 抗体片段的核苷酸序列。 A recombinant virus, characterized in that the virus can specifically replicate in tumor cells, and at the same time, the virus genome contains a nucleotide sequence encoding an antibody or an antibody fragment for treating a tumor.
2. 根据权利要求 1 所述的重組病毒， 其特征在于所迷病毒中 至少一个增殖必需基因的转录受至少一种肿瘤细胞特异性激活的 顺式作用元件有效控制。  The recombinant virus according to claim 1, characterized in that the transcription of at least one essential gene for proliferation in the virus is effectively controlled by at least one cis-acting element specifically activated by tumor cells.
3. 根据权利要求 1 所述的重组病毒， 其特征在于所迷病毒包 含至少一种增殖必需区蛋白功能缺失， 从而不能在正常细胞内增 殖， 但能在肿瘤细胞内特异性增殖。  3. The recombinant virus according to claim 1, characterized in that the virus contains at least one protein that is essential for the function of the proliferative region, so that it cannot proliferate in normal cells, but can specifically proliferate in tumor cells.
4. 根据权利要求 2所迷的重组病毒， 其特征在于所述顺式作 用元件选自下述中的至少一种： 端粒酶催化亚基基因启动子， 端 粒酶 RNA组份基因启动子， 缺氧反应元件， 细胞 S期特异性启 动子， 甲胎蛋白增强子及启动子， 癌胚抗原增强子及启动子， 酪 氨酸酶增强子及启动子，尿激酶纤维蛋白激活剂增强子及启动子， ErbB2增强子及启动子， ErbB3增强子及启动子， ErbB4增强 子及启动子， DF3乳腺癌相关抗原增强子， 前列腺素特异性抗原 增强子及启动子， 腺血管舒緩素增强子及启动子， EB 病毒中的 Orip, EB病毒 Orip中的 FR增强子， EB病毒 BamHI C-启动 子。  4. The recombinant virus according to claim 2, characterized in that the cis-acting element is selected from at least one of the following: a telomerase catalytic subunit gene promoter, a telomerase RNA component gene promoter Hypoxia response element, S-phase specific promoter, alpha-fetoprotein enhancer and promoter, carcinoembryonic antigen enhancer and promoter, tyrosinase enhancer and promoter, urokinase fibrin activator enhancer And promoter, ErbB2 enhancer and promoter, ErbB3 enhancer and promoter, ErbB4 enhancer and promoter, DF3 breast cancer-related antigen enhancer, prostaglandin-specific antigen enhancer and promoter, adrenal relaxin enhancer And promoter, Orip in EB virus, FR enhancer in EB virus Orip, EB virus BamHI C-promoter.
5. 根据权利要求 4所述的重组病毒， 其特征在于所述病毒为 腺病毒， 所述病毒增殖必需基因为以下腺病毒早期表达基因中的 一种或多种：E1A、 E1B、 E2或 E4。  5. The recombinant virus according to claim 4, characterized in that the virus is an adenovirus, and the virus essential gene for proliferation is one or more of the following early adenovirus-expressed genes: E1A, E1B, E2, or E4 .
6. 根据权利要求 4所述的重组病毒， 其特征在于所述病毒为 腺病毒， 所述病毒增殖必需基因为腺病毒晚期表达基因。  6. The recombinant virus according to claim 4, wherein the virus is an adenovirus, and the gene necessary for virus proliferation is an adenovirus late-expressing gene.
7. 根据权利要求 3 所迷的重组病毒， 其特征在于所述病毒是 重组腺病毒， 其中该腺病毒 ElB55Kda、 ElB19Kda和 /或 E1A基 因编码的相应蛋白功能缺失。 7. The recombinant virus according to claim 3, characterized in that said virus is Recombinant adenovirus, wherein the corresponding protein encoded by the adenovirus ElB55Kda, ElB19Kda and / or E1A function is deleted.
8. 根据权利要求 3所述的重组病毒， 其特征在于所述病毒是 重组单纯疱疹病毒， 该单纯疱疹病中 ICP6和 /或双拷贝 ICP34.5 基因编码的相应蛋白功能缺失。  8. The recombinant virus according to claim 3, wherein the virus is a recombinant herpes simplex virus, and the corresponding protein encoded by the ICP6 and / or double-copy ICP34.5 gene in the herpes simplex disease has a loss of function.
9. 根据权利要求 1所迷的重组病毒， 其特征在于所述编码治 疗肿瘤的抗体或抗体片段的核苷酸序列可选自下迷： 编码抗新生 血管生成的抗体或抗体片段的核苷酸序列， 编码抗肿瘤细胞生长 因子受体的抗体或抗体片段的核苷酸序列， 编码抗肿瘤细胞膜抗 原的抗体或抗体片段的核苷酸序列， 编码抗肿瘤抗原的独特型单 克隆抗体或抗体片段的核苷酸序列。  9. The recombinant virus according to claim 1, characterized in that the nucleotide sequence encoding the antibody or antibody fragment for treating tumors can be selected from the following: a nucleotide encoding an antibody or antibody fragment for anti-angiogenesis Sequence, the nucleotide sequence of an antibody or antibody fragment encoding an anti-tumor cell growth factor receptor, the nucleotide sequence of an antibody or antibody fragment encoding an anti-tumor cell membrane antigen, an idiotypic monoclonal antibody or antibody fragment encoding an anti-tumor antigen Nucleotide sequence.
10. 根据权利要求 9所述的重組病毒， 其特征在于所述编码抗 新生血管生成的抗体或抗体片段的核苷酸序列可选自下述： 抗血 管内皮生长因子的抗体或抗体片段的核苷酸序列， 抗血管内皮生 因子受体 2的抗体或抗体片段的核苷酸序列， 抗整合素的 a v p 3 的抗体或抗体片段的核苷酸序列， 抑制新生血管内皮生成的抗体 或抗体片段的核苷酸序列。 10. The recombinant virus according to claim 9, characterized in that the nucleotide sequence encoding the anti-angiogenesis antibody or antibody fragment can be selected from the following: a nucleus of an anti-vascular endothelial growth factor antibody or antibody fragment Nucleotide sequences, nucleotide sequences of antibodies or antibody fragments against vascular endothelial factor receptor 2, nucleotide sequences of antibodies or antibody fragments against av p 3 of integrin, antibodies that inhibit neovascular endothelial production or Nucleotide sequence of an antibody fragment.
11. 根据权利要求 9所述的重组病毒， 其特征在于所述编码抗 肿瘤细胞生长因子受体的抗体或抗体片段的核苷酸序列可选自下 述： 抗表皮生长因子受体 1 的抗体或抗体片段的核苷酸序列， 抗 表皮生长因子受体 2的抗体或抗体片段的核苷酸序列。  The recombinant virus according to claim 9, wherein the nucleotide sequence of the antibody or antibody fragment encoding the anti-tumor cell growth factor receptor is selected from the following: an antibody against epidermal growth factor receptor 1 Or the nucleotide sequence of an antibody fragment, the nucleotide sequence of an antibody or antibody fragment against epidermal growth factor receptor 2.
12. 根据权利要求 9所迷的重組病毒， 其特征在于所迷编码抗 肿瘤细胞膜抗原的抗体或抗体片段的核苷酸序列可选自下述:抗 17-1A的抗体或抗体片段的核苷酸序列， 抗 CD20的抗体或抗体 片段的核苷酸序列， 抗 MUC1的抗体或抗体片段的核苷酸序列。  12. The recombinant virus according to claim 9, characterized in that the nucleotide sequence of the antibody or antibody fragment encoding the anti-tumor cell membrane antigen can be selected from the following: anti-17-1A antibody or nucleoside of the antibody fragment Acid sequence, the nucleotide sequence of an anti-CD20 antibody or antibody fragment, the nucleotide sequence of an anti-MUC1 antibody or antibody fragment.
13. 根据权利要求 9所述的重組病毒， 其特征在于所述编码抗 肿瘤抗原的独特型单克隆抗体或抗体片段的核苷酸序列可选自下 述:抗 17-1A的独特型单克隆抗体或抗体片段的核苷酸序列， 抗癌 胚抗原的独特型单克隆抗体或抗体片段的核苷酸序列，抗 GD3的 独特型单克隆抗体或抗体片段的核苷酸序列， 抗 MUC1的独特型 单克隆抗体或抗体片段的核苷酸序列。 13. The recombinant virus according to claim 9, characterized in that the nucleotide sequence of the idiotype monoclonal antibody or antibody fragment encoding the anti-tumor antigen can be selected from the following Description: Nucleotide sequence of anti-17-1A idiotype monoclonal antibody or antibody fragment, Nucleotide sequence of idiotype monoclonal antibody or antibody fragment of anti-carcinoembryonic antigen, anti-GD3 idiotype monoclonal antibody or antibody The nucleotide sequence of the fragment, the nucleotide sequence of the anti-MUC1 idiotype monoclonal antibody or antibody fragment.
14. 根据权利要求 1所述的重組病毒， 其特征在于所述抗体或 抗体片段可以是 IgG或 IgM或者其片段。  14. The recombinant virus according to claim 1, wherein the antibody or antibody fragment is IgG or IgM or a fragment thereof.
15. 根据权利要求 1所迷的重組病毒， 其特征在于所迷抗体或 抗体片段可以是： 单链抗体， Fv， Fab', Fab, 单链抗体的核苷酸 序列或 F(ab，)2抗体中的抗原结合区， 二体抗体， 或线性抗体。  15. The recombinant virus according to claim 1, characterized in that the antibody or antibody fragment can be: a single chain antibody, Fv, Fab ', Fab, the nucleotide sequence of a single chain antibody or F (ab,) 2 Antigen-binding regions in antibodies, dimers, or linear antibodies.
16. 根据权利要求 1所述的重组病毒， 其特征在于所述抗体或 抗体片段是人抗体、 人源化抗体或嵌合抗体或者其片段。  16. The recombinant virus according to claim 1, wherein the antibody or antibody fragment is a human antibody, a humanized antibody or a chimeric antibody, or a fragment thereof.
17. 根据权利要求 1所述的重組病毒， 其特征在于所述编码治 疗肿瘤的抗体或抗体片段的核苷酸序列可与抗癌基因的核苷酸序 列相融合， 产生融合基因， 该抗癌基因为血管抑制基因、 细胞因 子基因、 前药转换酶基因及细胞毒性基因之一种;其中所述的血管 抑制基因可以是:内皮抑素基因， 血管生成抑素基因， 血浆纤维蛋 白溶酶原中 Kringlel-4结构、 Kringlel-5结构、 KringIel-3结构、 Kringlel-3加上 Kringle5结构、 血小板反应蛋白基因、 血小板因 子 4基因、纤溶酶原激活因子抑制剂 (PAI)基因及纤维结合素基因； 所述的细胞因子基因可以是： 白细胞介素 2、 白细胞介素 12、 粒- 单集落刺激因子、 肿瘤坏死因子、 干扰素 - α、 干扰素 - Ρ、 干扰素 - Y、 Light及 Flt3配体； 所述的前药转换酶基因可以是： 单纯疱 疹病毒胸腺嘧啶激酶、 细菌 内酰胺酶及大肠杆菌胞嘧啶脱氨 酶； 所述的细胞毒性基因可以是： 绿脓杆菌外毒片段。  17. The recombinant virus according to claim 1, characterized in that the nucleotide sequence encoding the antibody or antibody fragment for treating a tumor can be fused with the nucleotide sequence of an anti-cancer gene to generate a fusion gene, and the anti-cancer The gene is one of angiostatin gene, cytokine gene, prodrug converting enzyme gene and cytotoxic gene; the vasostatin gene may be: endostatin gene, angiostatin gene, plasma plasminogen Kringlel-4 structure, Kringlel-5 structure, KringIel-3 structure, Kringlel-3 plus Kringle5 structure, thrombospondin gene, platelet factor 4 gene, plasminogen activating factor inhibitor (PAI) gene and fibronectin Gene; The cytokine gene may be: interleukin 2, interleukin 12, granulocyte-single colony stimulating factor, tumor necrosis factor, interferon-α, interferon-P, interferon-Y, Light and Flt3 Ligand; The prodrug converting enzyme gene may be: herpes simplex virus thymidine kinase, bacterial lactamase and E. coli Pyrimidine deaminase; the cytotoxic gene may be: Pseudomonas aeruginosa outer toxic fragment.
18. 根据权利要求 1所述的重組病毒， 其特征在于编码治疗肿 瘤的抗体或抗体片段的核苷酸序列的表达还受启动子控制， 所述 启动子可选自下迷之一种： SV40启动子， RSV LTR启动子， 人巨 细胞病毒 IE启动子， 肿瘤细胞特异性激活的顺式作用元件， 鼠巨 细胞重組病毒 IE启动子， 人腺病毒主要晚期表达启动子。 18. The recombinant virus according to claim 1, characterized in that the expression of the nucleotide sequence encoding an antibody or antibody fragment for treating a tumor is further controlled by a promoter, and the promoter can be selected from one of the following: SV40 Promoter, RSV LTR promoter, human giant Cytovirus IE promoter, cis-acting element specifically activated by tumor cells, mouse cytomegalovirus recombinant IE promoter, human adenovirus major late expression promoter.
19. 根据权利要求 1所述的重組病毒， 其特征在于所迷病毒为 腺病毒， 其中在控制所述抗体或抗体片段表达的启动子的转录起 始位点与抗体的翻译起始位点之间可操作连接了内含子， 所迷启 动子可选自下述之一种： 含有腺病毒主要晚期 mR A的第三个引 导顺序的 5，剪接位点和一个免疫球蛋白的 3， 剪接位点的杂合内 含子，含有腺病毒主要晚期 mRNA的第一个引导顺序的 5，剪接位 点和一个免疫球蛋白的 3， 剪接位点的杂合内含子。  19. The recombinant virus according to claim 1, wherein the virus is an adenovirus, wherein the transcription initiation site of the promoter controlling the expression of the antibody or antibody fragment and the translation initiation site of the antibody are between Introns are operably linked, and the promoter may be selected from one of the following: a third guide sequence containing the major late mR A of the adenovirus 5, a splice site and an immunoglobulin 3, a splice The heterozygous intron at the site contains 5 of the first leading sequence of the adenovirus's major late mRNA, the splice site and an immunoglobulin of 3, and the heterozygous intron at the splice site.
20. 根据权利要求 18所迷的重組病毒， 其中所述肿瘤细胞特 异性激活的顺式作用元件可选自： 端粒酶催化亚基基因启动子, 端粒酶 RNA组份基因启动子， 缺氧反应元件， 细胞 S期特异性 启动子，甲胎蛋白增强子及启动子，癌胚抗原增强子及启动子， 酪 氨酸酶增强子及启动子，尿激酶纤维蛋白激活剂增强子及启动子， ErbB2增强子及启动子， ErbB3增强子及启动子， ErbB4增强 子及启动子， DF3乳腺癌相关抗原增强子， 前列腺素特异性抗原 增强子及启动子， 腺血管舒緩素增强子及启动子， EB 病毒中的 Orip, EB病毒 Orip中的 FR增强子， EB病毒 BamHI C-启动 子。  20. The recombinant virus according to claim 18, wherein the cis-acting element that is specifically activated by the tumor cell is selected from the group consisting of a telomerase catalytic subunit gene promoter, a telomerase RNA component gene promoter, Oxygen response element, S-phase specific promoter, alpha-fetoprotein enhancer and promoter, carcinoembryonic antigen enhancer and promoter, tyrosinase enhancer and promoter, urokinase fibrin activator enhancer and promoter Promoter, ErbB2 enhancer and promoter, ErbB3 enhancer and promoter, ErbB4 enhancer and promoter, DF3 breast cancer-related antigen enhancer, prostaglandin-specific antigen enhancer and promoter, adrenal relaxin enhancer and promoter Promoter, Orip in EB virus, FR enhancer in EB virus Orip, EB virus BamHI C-promoter.
21. 根据权利要求 1所述的重组病毒， 其特征在于编码治疗肿 瘤的抗体或抗体片段的核苷酸序列可操作连接了编码分泌型信号 肽的核苷酸序列。  The recombinant virus according to claim 1, wherein the nucleotide sequence encoding the antibody or antibody fragment for treating tumor is operably linked to the nucleotide sequence encoding the secretory signal peptide.
22. 根据权利要求 21 所述的重组病毒， 其特征在于该分泌型 信号肽可以是下述中的一种： 抗体或抗体片段自身信号肽， M-成 瘤蛋白信号肽， 肿瘤坏死因子 α的信号肽等等。  22. The recombinant virus according to claim 21, wherein the secreted signal peptide is one of the following: an antibody or antibody fragment self-signal peptide, an M-oncogene protein signal peptide, a tumor necrosis factor alpha Signal peptides and more.
23. 权利要求 1 - 22任一项所迷的重組病毒的应用， 其特征在 于将有效量的该重组病毒用于体外感染肿瘤细胞， 从而杀死或抑 制肿瘤细胞。 23. The use of a recombinant virus according to any one of claims 1 to 22, characterized in that an effective amount of the recombinant virus is used to infect tumor cells in vitro, thereby killing or inhibiting Control tumor cells.
24. 根据权利要求 1-22 任一项所述的重组病毒用于治疗哺乳 动物、 尤其是人类肿瘤的方法， 其包括如下步骤： 1 )将该病毒体 外或体内感染肿瘤细胞， 2 )使病毒基本上限于肿瘤细胞内选择性 复制及增殖， 导致在肿瘤细胞内编码治疗肿瘤的抗体或抗体片段 的核苷酸序列拷贝数增加及治疗肿瘤的抗体或抗体片段的表达量 增加； 由此特异性直接杀灭肿瘤细胞， 抑制肿瘤形成、 生长及转 移。  24. A method for treating a mammal, especially a human tumor using a recombinant virus according to any one of claims 1-22, comprising the steps of: 1) infecting the virus with tumor cells in vitro or in vivo, 2) infecting the virus It is basically limited to selective replication and proliferation in tumor cells, resulting in an increase in the number of copies of a nucleotide sequence encoding an antibody or antibody fragment for treating a tumor and an increase in expression of the antibody or antibody fragment for treating a tumor in the tumor cell; Directly kill tumor cells and inhibit tumor formation, growth and metastasis.
25. 根据权利要求 24所迷的方法， 其中还包括在权利要求 1 - 11任一项所述重組病毒感染肿瘤细胞之前、同时和 /或之后施用 化学抗肿瘤药物的步驟。  25. The method according to claim 24, further comprising the step of administering a chemical anti-tumor drug before, simultaneously and / or after the recombinant virus of any one of claims 1 to 11 infects tumor cells.
26. 根据权利要求 1-22 所述的重组病毒用于抑制肿瘤细胞的 生长的用途。  26. Use of a recombinant virus according to claims 1-22 for inhibiting the growth of tumor cells.
27. 根据权利要求 1 - 22所述的重组病毒在制备可用于治疗肿 瘤的药物中的用途。  27. Use of a recombinant virus according to claims 1 to 22 in the manufacture of a medicament useful for treating tumors.
28. 一种药物组合物， 其中包含权利要求 1-22 任一项所述的 重组病毒以及药学上可接受的载体.  28. A pharmaceutical composition comprising the recombinant virus according to any one of claims 1-22 and a pharmaceutically acceptable carrier.
29. 根据权利要求 28 的药物组合物， 其中还包含其它肿瘤抑 制和 /或治疗剂。  29. The pharmaceutical composition according to claim 28, further comprising other tumor suppressing and / or therapeutic agents.
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