CN1241632A - Gene engineering adenovirus and its application - Google Patents
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Abstract
The present invention construts two kinds of gene engineering adenovirus capable of being replicated selectively in human tumor cells with P53 function deficiency for ultimately killing tumor cells. Its tumor resisting curative effect is determined through the human tumor model transplanted to mouse body and test data shows that the gene engineering adenovirus can be used in treating specific tumor.
Description
The present invention relates to gene engineering adenovirus, they are massive duplication and final kill tumor cell in the tumour cell of p53 afunction optionally, but they can not duplicate in normal cell.In addition, the invention still further relates to the pharmaceutical composition that contains this gene engineering adenovirus and this gene engineering adenovirus and be used for the treatment of purposes in the pharmaceutical composition of cancer in preparation.
Cancer is a kind of general name of heterology disease, it is characterized by the indeterminate growth and the propagation of improper cell.It has been generally acknowledged that Normocellular propagation is subjected to the adjusting of the gene of two kinds of mutual antagonisms, i.e. proto-oncogene and tumor suppressor gene.Proto-oncogene can promote the growth of cell, and tumor suppressor gene then suppresses the growth of cell.When gene is undergone mutation, promptly proto-oncogene is activated or the tumor suppressor gene inactivation, all can quicken the growth of tumour cell.Usually the inactivation of tumor suppressor gene (comprising p53, the RB gene) and the activation of proto-oncogene take place simultaneously, consequently produce the tumour cell of infinite multiplication.
All might cause the function of cell cycle regulation gene to change although dissimilar genes changes, and produce improper cell proliferation.It has been generally acknowledged that needs multiple factor to act on simultaneously and normal cell could be transformed into tumour cell.Its accurate molecular pathways and consequential cellular change cause the mechanism of cell malignant proliferation not understood as yet.But existing many reports show, more intracellular albumen play an important role in the regulation and control of cell generation cycle, when their function changes, tend to cause the vicious transformation of cell, for example when p53 gene product inactivation, can make the growth of cell out of hand.
Although many discoveries have been arranged, the treatment for cancer overwhelming majority is still used conventional methods of treatment at present, as radiation and chemotherapy.The therapeutic index of these methods is very low, and side effect is very big, often brings a lot of miseries to patient, even life is on the hazard, as inhibition of marrow etc.
In recent years, there has been the method for scientist's applying gene treatment to revise or the allelic defective of additional people, be used for the treatment of some heredopathias, but success ratio has been less so far.Its method that mainly adopts is that one section oligonucleotide sequence is transduceed in people's cell, in the hope of expressing normal functional protein, corrects certain function of allelic defective or compensation cell.Application carrier is a duplicate deficit type recombinant adenovirus.As carrying out oncotherapy in tumour necrosis factor (TNF) and interleukin I I (IL-2) transfered cell.In United States Patent (USP) 7769623, WO9514101 etc., put down in writing with replication-defective adenoviral specific gene has been delivered to cancer patients's focus, expressed protein by the specific inhibition tumour of described genes encoding to reach the purpose of treatment cancer in described focus.In external test, this method is successful, but in vivo, efficient reduction greatly.
This gene therapy methods up to the present, the proto-oncogene of rectification of defects or tumor suppressor gene effectively.The characteristics of cell biology of tumour self makes does not also have a kind of effective means can treat cancer now, and all gene therapy methods are all too expensive, and this has also limited its application.
Adenovirus can infect respiratory tract, eye, gi tract and bladder, has identified 47 serotypes.According to its hemagglutinative function characteristic, tumorigenicity, to the conversion and the G+C per-cent of culturing cell, adenovirus hominis is divided into 7 hypotypes.
The replication cycle of adenovirus is divided into early stage and two stages of late period, and late period is from viral dna replication.After the infection, adenovirus is closed DNA and proteinic synthetic in the cell rapidly.Its shutdown mechanism it be unclear that, but the protein synthesis in the cell is suppressed rapidly, and cell DNA is synthetic then a little later.In a virus circulation of 32-36 hour, every cell produces 10
4Individual virion.
11 genes are arranged in the adenoviral gene group, wherein the E1A district in early days gene be very important in synthetic.Studies show that E1A protein can be in conjunction with tumor suppressor gene RB105 (retinoblastoma 105kd), and E1B albumen can be in conjunction with the P53 tumor suppressor protein.E1B itself can not transformant, but with E1A transformant stably.E3 contains in the district adjusting and with the host relevant gene is reacted in infection.
Under the normal circumstances, adenovirus major effectively duplicates in cell, the E1B district genes encoding of its nucleotide sequence synthesizes P55 albumen, this albumen can be with the P53 protein binding in the host cell, thereby and make its inactivation stop apoptotic generation, enable the virus to reproduce themselves, the cell that has lacked the P53 protein function like this can be appointed viral massive duplication.In this way, wild-type adenovirus can be in having the proteic normal cell of functional P53 massive duplication.
Method with recombinant adenovirus treatment tumour is disclosed in the United States Patent (USP) 5677178, method is employed to be the reorganization embedded virus dl1520 that is made up by adenovirus II type (Ad2) and adenovirus V-type (Ad5), because this recombinant virus is not the natural adenovirus that is present in the Mammals, therefore, this virus in human body survival condition and potential may suddenly change and caused harm still be difficult to determine.In addition, the used virus of this method has also kept 1/9 E3 district, and there is the effect that suppresses the human immune system in the E3 district, and the part reservation in this district has proved and can make this virus produce resistance, thereby might exist in the human body midium or long term.Like this because the atavism of virus, or because the influence of other factors, this recombinant adenovirus can revert back to the wild-type virus that can express in the normal human cell, thereby human body is had a negative impact.
Because the propagation of replication-defective adenoviral is with specific host, be that tumour cell is a target, therefore after specified target tissue disappears, the described replication-defective adenoviral that requires to destroy the particular target tissue also should disappear in acceptor thereupon, in order to avoid make described replication-defective adenoviral produce unwanted sudden change in vivo and keep in vivo, and the host is had a negative impact.Therefore, this has high as far as possible target-specific with regard to the replication-defective adenoviral that requires to be used to destroy the particular target tissue, thereby it can only be lived in specified target tissue.
For this reason, the inventor has made up a kind of novel recombinant adenovirus after research and testing, and it derives from Ad5.Because Ad5 type adenovirus is natural being present in the Mammals, its is with after deliberation very clear and definite of human mutual relationship, and E3 district gene is all removed.Like this, after this virus was brought into play drug effect in vivo, the immunity system of human body self can be with it removing, thereby had avoided retaining in issuable harm in the body.Therefore, the security of this novel recombinant adenovirus is better than the improved recombinant replication-defective adenoviral of prior art.
The purpose of this invention is to provide a kind of improved recombinant replication-defective adenoviral, and contain this viral pharmaceutical composition, in addition, the present invention also provides the method for selectivity kill tumor cell.
Another object of the present invention provides the method for selectivity kill tumor cell, this method comprises, under the condition that infects, the defective recombinant adenovirus of the E1B district p53 defective of kill tumor cell significant quantity is contacted with the cell population that contains tumour cell, produce the cell population that infects thus, described virus is the virus of the two sudden changes of E1B/E1a, and can further include a negative selection gene that links to each other with viral promotors.
On the other hand, the invention provides the pharmaceutical composition that contains recombinant replication-defective adenoviral and pharmaceutically acceptable carrier.
In addition, the present invention also provides the defective recombinant adenovirus to be used for the treatment of purposes in the pharmaceutical composition of tumour in preparation.
Fig. 1 .S98 gene engineering adenovirus collection of illustrative plates.Fig. 2. in normal cell, the duplicating of S98 gene engineering adenovirus.The pharmacodynamics test of Fig. 3 .S98-001.
Replication-defective adenoviral of the present invention is the Ad5 through transforming. The genome of Ad5 contains 35935 nucleotides. Ad5 is fully aware of with human relation, and obvious self-healing property is arranged. In the clinical trial that the Ad5 complete sequence is used for the people, the characteristic of normal cell never occured to transform.
Recombinant replication-defective adenoviral S98-001 and the S98-002 as shown in fig. 1 that the present invention makes up. In S98-001, added the TGA terminator codon at the 2025 nucleotides places in E1B district, lacked simultaneously nucleotides 2501-3328, lacked simultaneously the whole E3 district that comprises nucleotides 27865-30995. Because E1B coding P55 albumen, thus above-mentioned disappearance so that replication-defective adenoviral can only be produced deficiency P55 protein, and described deficiency P55 albumen reduces greatly with the binding ability of p53 gene outcome or with it can not in conjunction with. Therefore, described deficiency Ad5 can not copy in the normal cell that contains normal p53 gene outcome, and optionally massive duplication in the tumour cell of p53 functional defect, finally the kill tumor cell by the massive duplication of described adenovirus. Because this adenovirus is the p53 deficiency, therefore, after interior tumor cell was all killed basically, these adenovirus finally also can be dead because not copying in normal cell. Therefore, these replication-defective viruses are safe for normal cell. The E3 district is the zone relevant with host immune system, and having of this district may make adenovirus resist host immune system, therefore in the present invention, has lacked the E3 district fully. In addition, because recombined adhenovirus of the present invention has lacked the E3 district fully, thereby can bring the possibility of harm to human body after further having reduced this cell entry human body. The specificity of replication-defective adenoviral of the present invention just can be killed a normal cell for whenever killing 100-1000 tumour cell.
Except the sudden change in above 55KD district, described sudden change adenovirus also may have the disappearance of P19 protein function, and it can strengthen the pathology effect of cell, and its corresponding gene is the p19cyt mutator. But, also there is a lot of sudden changes to keep functional p19 gene, this is in order to keep the integrality of viral DNA in the process of infection cell.
Another used recombinant replication-defective adenoviral of the present invention is S98-002, and this virus has lacked the nucleotides 2501-3328 in the 55kD and the whole E3 district that comprises nucleotides 27865-30995. Its purpose also is for the specificity that improves described replication-defective adenoviral and lethality, make described virus by conventional method of administration and form of medication, arrive specific target area, kill tumor cell optionally, after tumour cell was all killed, described virus was also because losing the thereupon death of environment of relying and copying.
Recombinant replication-defective adenoviral of the present invention can be by conventional route of administration, as intravenous administration.Dosage is 10
9PFU-10
12PFU/ patient, preferred 10
9PFU-10
10The PFU/ individuality.
Embodiment
The structure of embodiment 1. plasmids
PXC-1 and pBHG11 purchase the Biosystems in Microbix, and PXC-1 contains adenovirus hominis 5 types (Ad5) gene order, from bp22 to 5790.PBHG11 contains the Ad5 gene order, on two places disappearances arranged: the disappearance in E1 district, from bp188 to 1339, promptly be used to form the packaging signal of viral DNA glutelin and the disappearance in E3 district, from bp27,865 to 30,995.The DNA of pBHG11 does not have infectivity, but the cotransfection of pXC-1 and pBHG11 can have infective virus by the homologous recombination generation.
Go up Ad5 pulsating subclone from bp1338 to 2501DNA in order to make up pXC-1, use following two synthetic oligonucleotide segments, amplify the PCR product from pXC-1, primer is as follows: HZ1 (5 '-CTATCCTGAGACGCCCGAC-3 ') and HZ2 (5 '-GATCGGATCCAGGTCTCCAGTAAGTGGTAGCTGC-3 ') (following graticule place is the BglII site) also are cloned into (Promega) on the pGEM-T carrier with it, produce HZ102.In order to make up HZ103, with Xbal and BglII digestion HZ102, the segment that obtains is connected with the pXC-1 that cuts with same method.
In order to make up a termination site at pXC-1 bp2025 place, two oligonucleotide chain HZ3 (5 '-AAAGGATAAATGGAGTAAAGAAACC-3 ') HZ4 (5 '-CAGATGGGTTTCTTCACTCCATTTATCC-3 ') is used to the process of transgenation, adopt QuickChange (Strategene), and produce plasmid HZ104, detect the sequence of change with the method for order-checking.In order to make up HZ105, with BglII and Xbal digestion HZ104, the segment of release connects with the pXC-1 with identical method cutting.
The generation of S98 virus is by two eclipsed plasmid DNA, obtains with the method for homologous recombination.Select the plaque that obtains, and in the HYH cell, increase.Viral DNA obtains from Qiagen blood test kit, analyzes with PCR and Southern-Blot method.
The generation of S98-001 is that HZ105 DNA obtains with pBHG11 DNA cotransfection.Equally, S98-002 and S98-100 obtain with pBHG11 and HZ103 DNA or pXC-1 DNA cotransfection.
S98-100 is the S98 wild-type adenovirus, and it contains the disappearance in E3 district, from map distance 77.5mu to the 86.2mu (see figure 1).It can be used as positive control, detects the specificity and the lethality of S98 virus.
S98-001 is that one sudden change takes place in the E1B district adenovirus, this sudden change is the nucleotide sequence that has lacked one section synthetic 495R albumen (55KD), and with the relevant synthetic mRNA-13s of 495R, the sequence of 14s and 14.5s, but do not influence the proteic structure of its 175R (19KD).S98-002 is another Ad mutant strain, and is similar with S98-001, except it still has synthetic mRNA-13s, the sequence of 14s and 14.5s.No matter be S98-001 or S98-002, can both produce and have functional E1B 19KD albumen, apoptosis promptly still capable of inhibiting cell.
The structure of S98 virus is by PCR, and order-checking and Southern method are proved conclusively.Our hypothesis: genetically engineered S98 adenovirus, do not express E1B 55KD albumen when it, it should can't duplicate in normal cell, but can be in the p53 deficient cell massive duplication.
Embodiment 2. usefulness plaque ethodses carry out vitro detection
At first adopt plaque ethods to detect the energy for growth of this viroid in the p53 deficient cell, promptly the detection by quantitative specific virus infects the ability of a certain cell strain.Plaque ethods is used following cell strain: ovarian cancer cell strain (OVCAR-3, the p53 mutant strain), hepatoma cell strain (Hep3B, the p53 mutant strain) becomes to bite cell plastid knurl JEG-3 (U373, p53 mutant strain), colon cancer cell line (SW620, p53 mutant strain and RKO, the normal strain of p53), mammary gland normal cell (HBL-100, the normal strain of p53).Because HYH cell expressing E1A and E1B albumen, S98 virus can effectively form plaque to these cell strains.
Table 1 has shown the average percent of the reproduction copies of tested virus.For all clone, the titre of a specific virus is carried out stdn with it to the titre of HYH cell, have comparability between virus and the corresponding clone like this.When the titre of a certain clone was stdn with the titre of HYH clone, the quantity of recombinant virus can same S98-100, and promptly wild-type adenovirus has comparability.The S98 genetically engineered virus can be used to assess the validity of virus replication with the ratio of wild-type adenovirus, and to the specificity of tumour cell.For example, when ratio less than 100, show that tested virus forms the ability of plaque less than wild-type adenovirus.On the contrary, when ratio greater than 100 the time, it is stronger to show that then this virus forms the ability of energy force rate wild-type adenovirus formation plaque of plaque.
From then on during plaque detected, can be observed: at first, S98-001 and S98-002 had the advantage of significantly duplicating in the cell of p53 defective.For example: S98-001 compares with S98-100 with S98-002, can more effective p53 deficient cells generation pathology, Hep3B, ovarian cancer cell, the colon cancer cell of inducing.Secondly, S98-001 and S98-002 in the normally functioning cancer cells of p53 and normal people's cell, the number that virus replication takes place and cause lysis seldom, its quantity that forms plaque is the minimizing of the order of magnitude.For example, in the normally functioning colon cancer cell RPO of p53, the plaque quantity that S98-001 and S98-002 produce than S98-100 is lacked 470 times and 250 times respectively.In the normal breast cancer cell of people, also obtained similar result.And forming the energy force rate S98-001 and the S98-002 of plaque in the HBL-100 cell, S98-100 exceeds 3000 times and 1000 times respectively.Therefore, the lysis of gene engineering adenovirus mediation, wild-type adenovirus is the minimizing of the order of magnitude in the normally functioning cell of p53 relatively.
This experiment shows, S98-001 and the S98-002 plaque number that (being HBL-100 and RPO) forms in the normally functioning cell of p53 obviously be less than in the cancer cells of p53 functional defect (be OVCAR-3, Hep3B, U373, SW620) the plaque number of Xing Chenging.Table 1 plaque ethods is represented the copy choice of S98 gene engineering adenovirus in the human tumor cells of p53 defective
Annotate:
1.OVCAR-3 be the ovarian cancer cell line of p53 defective type
2.Hep3B be the hepatoma cell line of p53 defective type
3.U373 be the glioma cell line of p53 defective type
4.SW620 be the colon carcinoma cell line of p53 defective type
5.RKO be the normally functioning colon carcinoma cell line of p53
6.HBL-100 be the normally functioning normal breast cell of p53
The test of embodiment 3. cell in vitro
The 2nd experiment is to observe the pathology that characteristics that different virus duplicates and cell produce.Its experimentation is as described below.Come cells infected with the virus infection amount that increases progressively gradually, and monitor cytogenetic pathology.When observing MOI and be 0.01, wild-type adenovirus can make the complete cracking of the monolayer cell of cultivation, and this is the terminal point of experiment.Select people's microtubule endotheliocyte (hMVEC derives from people's lung), a kind of former generation non-renewable cell, vitro detection it to the susceptibility of S98 genetically engineered virus and wild-type adenovirus.
The result shows, when S98-100 when MOI is 0.01, can make the complete cracking of monolayer cell of cultivation in 10 days.On the contrary, the normal monolayer cell that S98-002 infects, at identical time point, MOI is respectively 10,1.0, and 0.1 and 0.01 o'clock, do not present tangible cytopathy.Have only as MOI during, just can observe corresponding cytopathic phenomenon than wild-type adenovirus big 100 times and 1000 times.Therefore, the S98 gene engineering adenovirus is compared with wild-type adenovirus, and the cytopathy that human normal cell line is caused significantly reduces.
The experiment of embodiment 4. cytogamy
In order to determine whether the duplicating with the S98 gene engineering adenovirus in the cell and normal cell of p53 defective of virus, the cytopathy that produces is relevant, we experimentize with different virus titers in the cell of p53 defective and normal cell, and selected cell is hMVEC.When cell grows to the 70%-90% fusion, infected 90 minutes with wild-type adenovirus S98-001 and S98-002 respectively, MOI is 10.Infect after 48 hours,, discharge virus three freeze thawing of cell.Supernatant HYH cell titration.The viral number that S98-001 and S98-002 were produced behind the cells infected at 48 hours, the viral number that produces in same cell strain and same cell cycle with wild-type adenovirus carries out standardized calculation.The results are shown in Fig. 3.
Test shows that to normal cell, the S98 genetically engineered virus is compared with wild-type, and the titre of virus is less than 100 times.And to OVCAR-3 clone, S98-001 and S98-002 be S98-100 virus titer 30%.This proves that again the duplicate amount of S98 gene engineering adenovirus in human normal cell line seldom.
Embodiment 5. experimentation on animalies
At first with C33A cell (human cervical carcinoma, the p53 defective type), A549 cell (people's cancer of the stomach, p53 normal type) be subcutaneously injected in the female athymic nude mouse, when gross tumor volume during greater than 200 μ l, directly S98-001/S98-002 is expelled in the tumor tissues, with the positive contrast of S98-100, the PBS solution of 10% glycerine is as negative control.Every kind of virus is inoculated each 10 C33A and A549 tumour respectively.The injected dose of virus is respectively 10
8PFU/mm
3, 10
7PFU/mm
3, 10
6PFU/mm
3, 10
5PFU/mm
3, injection was injected 6 days altogether continuously.Write down the growth of tumor situation weekly one time, in 6 weeks of continuous recording, the results are shown in table 2.
Table 2 S98-001 animal pharmacodynamics testing data (n=10)
| Fate | The 1st group | The 2nd group | The 3rd group | The 4th | Control group | |
| 0 | 100 * | 100 | 100 | 100 | 100 | |
| 7 | 85.87 | 142.55 | 160.35 | 242.85 | 245.19 | |
| 14 | 84.78 | 139.08 | 189.80 | 235.94 | 354.16 | |
| 21 | 75.02 | 161.66 | 244.28 | 274.31 | 424.55 | |
| 28 | 63.51 | 216.88 | 276.37 | 300.03 | 545.92 |
Annotate:
1. the injected dose of the used virus of 1-4 group is respectively 10 in the table
9Granule number/mm
3, 10
8Granule number/mm
3, 10
7Granule number/mm
3, 10
6Granule number/mm
3
2
*Knurl volume with the same day after the administration is 100.
The result shows, S98-001 be inoculated into the C33A nude mice on one's body after, promptly observe gross tumor volume after 1 week and reduce, this trend is maintained always, and is obvious further after 5 weeks, after the 6th week, gross tumor volume to be to taper to 1/3 of original volume, promptly dwindled closely 70%, and a tumour completely dissolve is wherein arranged.After stopping administration, gross tumor volume is still constantly dwindling.And in negative control group, after administration, gross tumor volume continues to increase, and after 6 weeks, is nearly 4 times of former gross tumor volume.Therefore, compare with negative control group, the gross tumor volume of S98-001 administration group has dwindled more than 10 times.For the A549 nude mice, after 6 weeks, S98-001 administration group is very little with the gross tumor volume difference of negative control group, all continues to increase, at last until diabrosis.For S98-100, under same dosage, no matter which kind of tumor model all can make gross tumor volume obviously dwindle, and its degree is greater than S98-001 (94%).Thereby confirmed that S98-001 optionally kills p53 defective type tumour cell.
Gene engineering adenovirus of the present invention can carry out purifying through the ordinary skill in the art, and the gene engineering adenovirus behind the purifying can be made conventional dosage form such as freeze-dried preparation, suspension etc. with this area pharmaceutically acceptable carrier commonly used (as damping fluid etc.) and use.
Method of the present invention also is applicable to other recombinant viruses of preparation, as human papillomavirus, and SV40, they all lack and can combine and make it the corresponding viral protein of inactivation with P53 albumen.As the human papillomavirus mutant strain normal E6 albumen of can not encoding, this albumen can be with the P53 protein binding, thus it can be in the tumour cell of p53 defective massive duplication, and final kill tumor cell.Accordingly, in normal cell, it can not duplicate.
The present invention also comprises the series of steps to the tumour patient medication, promptly gives the tumour patient medication of the p53 defective of particular type with this recombinant virus.
Although above describe the present invention in detail with embodiment, this area those skilled in the art will appreciate that, can carry out any change and not depart from scope of the present invention the present invention.
Claims (9)
1. the replication-defective adenoviral of a reorganization is characterized in that this virus can not be expressed can and lack the E3 district with endocellular function p53 tumor suppressor gene product bonded albumen.
2. the described recombinant replication-defective adenoviral of claim 1, the wherein said virus E1B P55 polypeptide of can not encoding.
3. claim 1 or 2 described recombinant replication-defective adenovirals, described virus are S98-001 (preserving number: CCTCC-V98003) and S98-002.
4. the described recombinant replication-defective adenoviral of claim 1, wherein said virus can be in the tumour cell of p53 defective massive duplication, form infective virion.
5. pharmaceutical composition, said composition contain any described recombinant replication-defective adenoviral and pharmaceutically acceptable carrier among the claim 1-4 of kill tumor cell significant quantity.
6. the method for selectivity kill tumor cell, this method comprises, under the condition that infects, any described recombinant replication-defective adenoviral contacts with the cell population that contains tumour cell among the claim 1-4 with kill tumor cell significant quantity, produces the cell population that infects thus.
7. according to the method for claim 6, wherein said tumour cell is the p53 gene defection type.
8. the method for selectivity kill tumor cell, this method comprises, under the condition that infects, the defective recombinant adenovirus of the E1B district p53 defective of kill tumor cell significant quantity is contacted with the cell population that contains tumour cell, produce the cell population that infects thus, described virus is the virus of the two sudden changes of E1B/E1a, and can further include a negative selection gene that links to each other with viral promotors.
9. any described defective recombinant adenovirus is used for the treatment of purposes in the pharmaceutical composition of tumour in preparation among the claim 1-4.
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