CN114934065A - Oncolytic adenovirus construction method carrying immune checkpoint molecule TIM-3 antibody gene and application thereof - Google Patents

Oncolytic adenovirus construction method carrying immune checkpoint molecule TIM-3 antibody gene and application thereof Download PDF

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CN114934065A
CN114934065A CN202210235112.XA CN202210235112A CN114934065A CN 114934065 A CN114934065 A CN 114934065A CN 202210235112 A CN202210235112 A CN 202210235112A CN 114934065 A CN114934065 A CN 114934065A
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tim
alpha
gene
plasmid
carrying
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王毅刚
李强
黄飚
张蕾蕾
张慧梨
戴川景
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Zhejiang University Of Science And Technology Shaoxing Biomedical Research Institute Co ltd
Zhejiang Sci Tech University ZSTU
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Zhejiang Sci Tech University ZSTU
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Abstract

The invention discloses a construction method and application of oncolytic adenovirus carrying an immune checkpoint molecule TIM-3 antibody gene, which comprises the steps of firstly preparing pCA 13-alpha-TIM-3 plasmid carrying alpha-TIM-3 gene; digesting the pCA 13-alpha-TIM-3 plasmid with BglII to obtain an expression frame, and connecting the expression frame with the pGD55 plasmid to obtain pGD 55-alpha-TIM-3 plasmid; transforming the pGD 55-alpha-TIM-3 plasmid into an adenovirus skeleton plasmid Adeasy-1 escherichia coli BJ5183 with a deleted E3 region for recombination, transferring the recombinant adenovirus skeleton plasmid Adeasy-1 escherichia coli BJ5183 into competent cells to generate a GP73 promoter to replace an endogenous E1A promoter, and regulating and controlling E1A expression, deleting E1B 55kDa and E3 regions and carrying a recombinant adenovirus genome vector pAd-GD 55-alpha-TIM-3 of an alpha-TIM-3 gene; pAd-GD 55-alpha-TIM-3 is transfected into 293A cells, oncolytic adenovirus is obtained after the cells are diseased, the constructed oncolytic adenovirus can selectively kill tumor cells without basically affecting normal cells, and can effectively inhibit the growth of liver cancer transplantation tumor in vivo, thereby providing a basis of an immune check point antibody-oncolytic virus medicine for treating cancer.

Description

Oncolytic adenovirus construction method carrying immune checkpoint molecule TIM-3 antibody gene and application thereof
Technical Field
The invention belongs to the field of biotechnology and gene therapy, and relates to a construction method and application of an oncolytic adenovirus carrying an immune checkpoint molecule TIM-3 antibody gene, in particular to a construction method of an oncolytic adenovirus GD 55-alpha-TIM-3 carrying an immune checkpoint molecule TIM-3 antibody gene and regulated by a Golgi transmembrane glycoprotein 73(GP73) promoter, and application of the oncolytic adenovirus in liver cancer treatment.
Background
Primary liver cancers include hepatocellular carcinoma (HCC) (75% -85%) and intrahepatic bile duct cancer (10% -15%), among other rare types. Its occurrence, development and invasive metastasis are a long-term, complex, multi-factorial co-involved continuous process. Liver cancer is concerned because of its easy induction and high mortality, but the systematic treatment of liver cancer has been advanced only a limited amount in the last 10 years, and the current main means for treating liver cancer is surgical resection combined with radiotherapy and chemotherapy, but the response rate to liver cancer is still low and the prognosis is poor regardless of the drug Sorafenib approved by FDA for the management of advanced HCC, or Regorafenib and Lenvatinib approved in the last few years. Therefore, there is an urgent need to explore new therapies for liver cancer to improve the therapeutic effects of liver cancer.
In recent years, oncolytic viruses show good application prospects because they specifically infect and lyse tumor cells and have little effect on normal cells. The activity of an oncolytic virus depends on the biological relationship of its host-virus interaction. Oncolytic viruses are capable of directly or indirectly targeting tumor cells, replicating and expressing proteins thought to be cytotoxic to cell survival, or inducing an anti-tumor response upon expression of key tumor epitopes. By genetically engineering oncolytic viruses, gene promoters that are activated only in tumor cells can be used to control the replication machinery of the oncolytic virus. A cancer-targeted Gene-virus therapy Strategy (cancer Targeting Gene-viral therapy, CTGVT, a new Strategy for cancer therapy by combining the respective advantages of Gene therapy and virus therapy) is proposed for the first time internationally in 2001, namely, the cancer-targeted Gene-virus therapy Strategy can be specifically replicated in tumor cells, the expression quantity of carried genes in the tumor cells is increased by tens to hundreds of times, the anti-cancer effect is better than that of the Gene therapy or virus therapy, and the safety is improved.
Immune checkpoints are inhibitory pathways in the immune system that can modulate the magnitude and duration of immune responses. In some cases, the tumor will manipulate these immune checkpoint pathways, thereby developing resistance to the body's natural immune system, which can limit T cell activation when immune checkpoint molecules specifically bind to their corresponding ligands. In recent years, targeted antibody drugs based on blocking immune checkpoint molecules CTLA-4 and PD-1 have achieved relatively ideal effects in clinical treatment of tumors such as melanoma, renal cancer and lung cancer, so that development of antibody drugs for tumor treatment is rapidly developed, however, nearly half of patients with the tumors are insensitive to or resistant to treatment of the targeted CTLA-4 and PD-1; in addition, the antibody drug for treating solid tumors has the problems of difficult targeting inside the tumors, large antibody dosage, high cost and the like.
T-cell immunoglobulin domain and mucin domain molecule-3 (TIM-3) is a further tumor immunosuppressive receptor found following CTLA-4 and PD-1 and is considered a second generation tumor immunodetection point therapeutic target. TIM-3 was first identified by Monney, equal to 2002, on the surface of differentiated Th1 cells and was found to be involved in regulating macrophage (M.phi.) activation and exacerbating autoimmune encephalomyelitis disease. The interaction between TIM-3 and its ligands results in suppression of Th1 and Th17 responses and induces immune tolerance, supporting the inhibitory effect of TIM-3 in T cell-mediated immune responses. Preclinical studies of TIM-3 showed that it was expressed on tumor infiltrating lymphocytes along with PD-1, and later studies found that TIM-3 was widely expressed on a variety of immune cells such as NK, DC, M phi and tumor cells such as liver, ovarian and renal cancers. In recent years, researches show that TIM-3 is closely related to liver cancer, and the expression of TIM-3 in tumor-related macrophages (TAMs) and liver cancer tissues of HCC patients is obviously higher than that of tumor-related macrophages (TAMs) and normal liver tissues from mediating T cell dysfunction of hepatitis-related hepatocellular carcinoma (HCC) patients and predicting poor prognosis and the TIM-3 gene polymorphism influencing the development of HCC and liver cirrhosis of hepatitis virus patients. Therefore, TIM-3 may play an important role in liver cancer development and treatment resistance, and simultaneously provides a new idea and target for liver cancer immunotherapy.
The oncolytic virus and the immune checkpoint inhibitor are combined together, so that the local immune microenvironment of the tumor has higher immune activity by using the infection of the oncolytic virus, and the immune checkpoint inhibitor is used for reducing the immune inhibition condition in the tumor microenvironment, so that the obtained clinical effect is good. For example, the combined therapy of TEV, an oncolytic virus, and either the CTLA-4 antibody Iipilimumab or the PD-L1 antibody pembrolizumab has clinically achieved good results.
Based on the consideration, the gene expression frame of the TIM-3 antibody of the immune checkpoint molecule is inserted into the oncolytic adenovirus GD55 regulated and controlled by the GP73 promoter to obtain the recombinant virus GD 55-alpha-TIM-3, hopefully, the recombinant virus GD 55-alpha-TIM-3 can effectively inhibit the proliferation of liver cancer cells, the function of the alpha-TIM-3 in lymphocyte infiltration of the liver cancer cells is explored, and a new gene treatment scheme and a theoretical basis are provided for the treatment of liver cancer.
Disclosure of Invention
In order to solve the above problems in the prior art, the present invention provides a method for constructing an oncolytic adenovirus carrying an antibody gene of TIM-3, which specifically comprises: a method for constructing oncolytic adenovirus GD 55-alpha-TIM-3 which targets Golgi transmembrane glycoprotein 73(GP73) positive tumors and binds to an immune checkpoint molecule TIM-3 antibody.
The invention also aims to provide application of the oncolytic adenovirus GD 55-alpha-TIM-3 carrying the antibody gene of the immune checkpoint molecule TIM-3 in liver cancer drugs.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention relates to a construction method of oncolytic adenovirus carrying an immune checkpoint molecule TIM-3 antibody gene, which comprises the following steps:
1) preparing pCA 13-alpha-TIM-3 plasmid carrying alpha-TIM-3 gene;
2) carrying out enzyme digestion on the pCA 13-alpha-TIM-3 plasmid by Bgl II to obtain an expression frame carrying a CMV promoter, an alpha-TIM-3 gene and SV40 polyA, and connecting the expression frame with a pGD55 plasmid subjected to enzyme digestion by Bgl II to finally obtain a pGD 55-alpha-TIM-3 plasmid;
3) pGD 55-alpha-TIM-3 plasmid is digested and linearized by Pme I and then transformed into adenovirus skeleton plasmid Adeasy-1 escherichia coli BJ5183 with deletion of E3 region for homologous recombination, and then transformed into DH5 alpha competent cells to generate GP73 promoter to replace endogenous E1A promoter and regulate E1A expression and delete E1B 55kDa and E3 region, and recombinant adenovirus genome vector pAd-GD 55-alpha-TIM-3 carrying alpha-TIM-3 gene;
4) after the recombinant adenovirus genome vector pAd-GD 55-alpha-TIM-3 with correct recombinant identification and endotoxin removal is subjected to enzyme digestion linearization by PacI, the recombinant adenovirus genome vector pAd-GD 55-alpha-TIM-3 is transfected into HEK293A cells with good state to package adenovirus, and after the cells are diseased, the oncolytic adenovirus GD 55-alpha-TIM-3 is obtained.
Preferably, the procedure for preparing pCA13- α -TIM-3 plasmid carrying α -TIM-3 gene is as follows:
1) amplification of the α -TIM-3 gene: designing an alpha-TIM-3 gene primer, wherein Hind III and Xba I enzyme cutting sites are respectively arranged at the upstream and downstream, and amplifying an alpha-TIM-3 gene fragment by using KOD high fidelity PCR polymerase and a synthesized pCDNA3.1-alpha-TIM-3 as a template;
2) the alpha-TIM-3 gene fragment was digested with HindIII and XbaI, and ligated into pCA13 plasmid vector digested with HindIII and XbaI to obtain pCA 13-alpha-TIM-3.
Preferably, the α -TIM-3 gene fragment comprises a signal peptide, an α -TIM-3 heavy chain variable region, a human antibody heavy chain constant region fragment one, a human antibody hinge region, a human antibody heavy chain constant region fragment two, a human antibody heavy chain constant region fragment three, F2A linker, an α -TIM-3 light chain variable region, and a K chain constant region.
Preferably, the alpha-TIM-3 gene segment is a Signal peptide-VH-CH1-Hinge-CH2-CH3-F2A-Signal peptide-VL-CL-6 XHis fusion gene segment.
Preferably, the nucleotide sequence of the fusion gene fragment is shown as SEQ ID NO. 1.
Preferably, the plasmids pGD55, pGD55- α -TIM-3 and pAd-GD55- α -TIM-3 are all subjected to Fastap dephosphorylation.
Preferably, the recombinant identification method in the step 4) adopts MluI enzyme digestion identification.
The invention provides a recombinant vector, which is characterized in that: comprising an alpha-TIM-3 gene according to any one of claims 1 to 4.
The present invention is a pharmaceutical composition comprising the oncolytic adenovirus GD55- α -TIM-3 according to any one of claims 1-7.
The invention relates to application of oncolytic adenovirus GD 55-alpha-TIM-3 carrying an antibody gene of an immune checkpoint molecule TIM-3 in liver cancer drugs.
Has the advantages that: the dual-target oncolytic adenovirus GD 55-alpha-TIM-3 constructed by the invention can selectively kill tumor cells without influencing normal cells basically, has the effects of obviously weakening the tumor activity and inhibiting the tumor growth, can effectively inhibit the growth of liver cancer transplantable tumors in vivo, provides a basis of an immune check point antibody-oncolytic virus medicine for treating cancers, and particularly provides a new gene treatment basis for treating liver cancers.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a schematic structural diagram of a dual targeting oncolytic adenovirus GD 55-alpha-TIM-3.
FIG. 2 shows the Mlu I restriction map of the packaging plasmid pAd-GD 55-alpha-TIM-3 for oncolytic adenovirus.
FIG. 3 is a PCR amplification assay for the α -TIM-3 gene.
FIG. 4 is a specific expression diagram of an oncolytic adenovirus GD 55-alpha-TIM-3 mediated alpha-TIM-3 gene in hepatoma cell BEL-7404
FIG. 5 is a structural diagram of an antibody expressed by an oncolytic adenovirus GD 55-alpha-TIM-3 mediated alpha-TIM-3 gene in hepatoma cell BEL-7404.
FIG. 6 is a graph showing the dose-dependent effect of oncolytic adenovirus GD 55-alpha-TIM-3 on WRL-68 in normal liver cells in vitro.
FIG. 7 is a graph showing the dose-dependent effect of oncolytic adenovirus GD 55-alpha-TIM-3 on specific killing of hepatoma carcinoma cells MHCC-97H in vitro.
FIG. 8 is a graph showing the dose-dependent effect of oncolytic adenovirus GD 55-alpha-TIM-3 on specific killing of hepatoma carcinoma cell BEL-7404 in vitro experiments.
FIG. 9 is a graph of the specific killer dose-dependent effect of oncolytic adenovirus GD 55-alpha-TIM-3 on hepatoma cells Huh7 in vitro experiments.
FIG. 10 is a graph showing the time-dependent effect of oncolytic adenovirus GD 55-alpha-TIM-3 on specific killing of normal liver cells WRL-68 in vitro.
FIG. 11 is a graph showing the time-dependent effect of oncolytic adenovirus GD 55-alpha-TIM-3 on the specific killing of hepatoma cells MHCC-97H in vitro experiments.
FIG. 12 is a graph showing the time-dependent effect of oncolytic adenovirus GD 55-alpha-TIM-3 on the specific killing of hepatoma carcinoma cell BEL-7404 in vitro experiments.
FIG. 13 is a graph showing the time-dependent effect of oncolytic adenovirus GD 55-alpha-TIM-3 on the specific killing of hepatoma cells Huh7 in vitro.
FIG. 14 is a diagram of the pathological effect of oncolytic adenovirus GD 55-alpha-TIM-3 on liver normal cell WRL-68 in vitro experiments.
FIG. 15 is a diagram showing the pathological effect of oncolytic adenovirus GD 55-alpha-TIM-3 on hepatoma cells MHCC-97H in vitro experiments.
FIG. 16 is a diagram showing the pathological effect of oncolytic adenovirus GD 55-alpha-TIM-3 on hepatoma cell BEL-7404 in vitro experiment.
FIG. 17 is a diagram of the pathological effect of oncolytic adenovirus GD 55-alpha-TIM-3 on hepatoma cells Huh7 in vitro.
FIG. 18 is a schematic diagram of the tumor growth inhibition of oncolytic adenovirus GD 55-alpha-TIM-3 on hepatoma cell BEL-7404 and peripheral blood lymphocytes mixed tumor-bearing nude mice in vivo.
FIG. 19 is a schematic diagram of mouse body weight measurement.
Detailed Description
The invention will be further described with reference to the accompanying drawings, FIGS. 1-19, but the invention is not limited to the following examples.
The invention is characterized in that an antibody of a targeting immune checkpoint molecule TIM-3 is combined with oncolytic adenovirus of targeting GP73, and after the hepatoma cell is infected by virus, alpha-TIM-3 can be generated to be combined with the immune checkpoint molecule TIM-3 on the hepatoma cell or lymphocyte, so that the tumor inhibiting effect is enhanced.
The invention also provides the application of the oncolytic adenovirus GD 55-alpha-TIM-3 in the preparation of a medicament for treating liver cancer.
The animal tumor formation of the invention adopts a mode of mixing human liver cancer cells and human peripheral blood lymphocytes to form tumor to simulate immune microenvironment.
When the oncolytic adenovirus GD 55-alpha-TIM-3 is used for treating liver cancer, an intratumoral injection mode is adopted, and the dosage is 1 multiplied by 109 pfu/mouse counted by mice.
The invention is modified on the basis of ZD55, and comprises the steps of controlling an E1A gene and carrying an alpha-TIM-3 gene by utilizing a GP73 promoter, newly constructing GD 55-alpha-TIM-3, wherein the constructed GD 55-alpha-TIM-3 has obvious effects of weakening tumor activity and inhibiting tumor growth, and the anti-cancer effect is stronger than that of a control virus GD 55. Thus, the α -TIM-3 gene can be well used for immune checkpoint antibody-virus therapy of tumors.
Experiments prove that the constructed double-targeting oncolytic adenovirus GD 55-alpha-TIM-3 can selectively kill tumor cells without influencing normal cells basically, can effectively inhibit the growth of liver cancer transplantable tumor in vivo, and provides an immune checkpoint antibody-oncolytic virus medicine for treating cancer.
Example 1
A method for constructing oncolytic adenovirus GD 55-alpha-TIM-3 which targets GP73 positive tumors and is combined with an immune checkpoint molecule TIM-3 antibody.
1) Amplification of fusion gene fragment α -TIM-3.
The fusion gene fragment alpha-TIM-3 (Signal peptide-VH-CH1-Hinge-CH2-CH3-F2A-Signal peptide-VL-CL-6 XHis) module is schematically shown in FIG. 1 (complete nucleic acid sequence is shown in appendix SEQ ID NO. 1).
The respective module sequences of the fusion gene fragment α -TIM-3(Signal peptide-VH-CH1-Hinge-CH2-CH3-F2A-Signal peptide-VL-CL-6 XHis):
1. a signal peptide artificial sequence (signal peptide) shown as SEQ ID NO. 2;
2. the alpha-TIM-3 heavy chain variable region nucleic acid artificial sequence (alpha-TIM-3 heavy chain variable region) is shown as SEQ ID NO. 3;
3. a nucleic acid artificial sequence of the alpha-TIM-3 heavy chain constant region fragment I (human antibody heavy chain constant region fragment I) is shown as SEQ ID NO. 4;
4. a hinge region artificial sequence (human antibody hinge region) shown in SEQ ID NO. 5;
5. an alpha-TIM-3 heavy chain constant region fragment II nucleic acid artificial sequence (human antibody heavy chain constant region fragment II) shown as SEQ ID NO. 6;
6. an alpha-TIM-3 heavy chain constant region fragment trinucleotide artificial sequence (human antibody heavy chain constant region fragment III) shown as SEQ ID NO. 7;
7. an artificial sequence of F2A linker (F2A linker) as shown in SEQ ID NO. 8;
8. a signal peptide artificial sequence (signal peptide) shown as SEQ ID NO. 9;
9. the alpha-TIM-3 light chain variable region nucleic acid artificial sequence (alpha-TIM-3 light chain variable region) is shown as SEQ ID NO. 10;
10. an alpha-TIM-3K chain constant region nucleic acid artificial sequence (K chain constant region) shown as SEQ ID NO. 11;
11. 6 × His artificial sequence shown in SEQ ID NO. 12.
Designing alpha-TIM-3 gene primer, carrying HindIII and XbaI restriction enzyme cutting sites at the upstream and downstream, dissolving with sterile water, preparing to final concentration of 10 mu mol, and storing at-20 ℃.
TABLE 1 primers for the alpha-TIM-3 gene
Figure BDA0003539729140000091
The complete alpha-TIM-3 gene is obtained by KOD high fidelity PCR by using pCDNA3.1-alpha-TIM-3 plasmid synthesized by Shanghai strapdown bioengineering company Limited as a template, wherein a KOD high fidelity PCR system is as follows:
Figure BDA0003539729140000092
the KOD high fidelity PCR reaction program is:
Figure BDA0003539729140000101
preparing 1% agarose gel, running the PCR product for 35min by 135V electrophoresis, and taking a picture under ultraviolet light; after the target fragment was excised and placed in a 1.5mL EP tube, the α -TIM-3 gene was recovered according to the tapping recovery kit procedure.
2) Construction of pCA 13-alpha-TIM-3
(1) The alpha-TIM-3 gene obtained by PCR is subjected to double digestion by Hind III and Xba I, agarose gel electrophoresis and gel tapping recovery. The system is as follows:
Figure BDA0003539729140000102
(2) the pCA13 plasmid was digested simultaneously with HindIII and Xba I, electrophoresed on an agarose gel, and recovered by tapping. The system is as follows:
Figure BDA0003539729140000103
Figure BDA0003539729140000111
(3) and (2) connecting the alpha-TIM-3 gene fragment obtained by double enzyme digestion of Hind III and Xba I obtained in the step (1) with the Hind III and pCA13 vector obtained by double enzyme digestion of Xba I obtained in the step (2), calculating the adding volume ratio of the alpha-TIM-3 gene fragment X to the pCA13 vector fragment Y according to a formula V target fragment/V vector fragment (M target fragment/M vector fragment X10 xC vector fragment/C target fragment (V is adding volume; M is the number of fragment bases; C is DNA concentration after gel cutting recovery), adding each component according to a connecting system, and reacting for 4 hours at 16 ℃.
The linking system is as follows:
Figure BDA0003539729140000112
(4) transforming the reacted connection system by DH5 alpha, and transforming according to the conventional Escherichia coli transformation method;
then, 800 mu.L of LB liquid culture medium without Amp resistance is added into a 1.5-mL lep tube, after shaking culture is carried out for 1h at 37 ℃, centrifugation is carried out for 1min at 5000rpm, about 700 mu.L of supernatant is poured off, the bottom layer of deposited bacteria is evenly blown by a pipette gun, then the deposited bacteria are evenly mixed by a sterilized glass rod and spread in 100 mu.g/mL Amp resistance LB solid culture medium, and then the mixture is inversely placed into a 37 ℃ incubator for culture for 16 h.
(5) And (3) when macroscopic bacterial plaques grow out, selecting 6-8 monoclonals in an LB liquid culture medium containing 100 mu g/mL Amp resistance, shaking the bacteria by a shaking table until the bacteria liquid is turbid, taking 500 mu L of the bacteria liquid and 1:1 of glycerol for bacteria preservation, then extracting plasmids according to the instructions of the small plasmid extraction kit, taking 1 mu L of the plasmids and measuring the plasmid concentration in NanoDrop 2000.
(6) And (3) carrying out enzyme digestion identification on the pCA 13-alpha-TIM-3 plasmid extracted in the step (5) by using Hind III and Xba I, wherein an enzyme digestion system is as follows, carrying out sequencing verification on the plasmid which is correctly identified and can generate a band about 2300bp, and comparing an alpha-TIM-3 gene sequence obtained by sequencing with a fusion gene fragment alpha-TIM-3 (the complete nucleic acid sequence is shown in appendix SEQ ID NO.1) to determine whether mutation occurs.
Figure BDA0003539729140000121
3) Construction of pGD 55-alpha-TIM-3
(1) The constructed pCA 13-alpha-TIM-3 plasmid is subjected to single enzyme digestion by BglII, and the gel is cut and recovered to obtain an expression frame segment containing a CMV promoter, an alpha-TIM-3 gene and SV40 polyA;
Figure BDA0003539729140000122
(2) the pGD55 plasmid, which was stored in the laboratory, was cleaved with BglII and dephosphorylated with Fastap. The enzyme digestion system is as follows:
Figure BDA0003539729140000123
Figure BDA0003539729140000131
the FastAp dephosphorylation is because the pGD55 plasmid vector fragment after BglII single enzyme digestion can be self-connected, and in order to eliminate the influence, the phosphate group at the 5' end of the FastAp dephosphorylated plasmid vector fragment is required to be incapable of forming a phosphodiester bond with a group on the self vector and being incapable of being connected.
The dephosphorylation system was as follows:
Figure BDA0003539729140000132
(3) the expression cassette comprising the CMV promoter, the α -TIM-3 gene and the SV40 polyA obtained in step (1) of the FastAp dephosphorylated pGD55 vector obtained in step (2) was ligated according to the one-step cloning kit procedure.
(4) And (3) converting the reacted connection system obtained in the step (3), coating the transformed connection system in a 50 mu g/mL Kan-resistant LB solid culture medium, picking 6-8 monoclonals when colonies are visible by naked eyes after about 16 hours, shaking the monoclonals in an LB liquid culture medium containing 50 mu g/mL Kan resistance, taking 500 mu L of bacterial liquid and 1:1 glycerol when the bacterial liquid is turbid, preserving the bacterial liquid, extracting plasmids, and detecting the concentration by using NanoDrop 2000.
(5) Identifying the pGD 55-alpha-TIM-3 plasmid extracted in the step (4) by using Hind III single enzyme digestion, obtaining a plasmid with a band of about 400bp after enzyme digestion, wherein the enzyme digestion system of pGD 55-alpha-TIM-3 is as follows:
Figure BDA0003539729140000133
Figure BDA0003539729140000141
4) taking a pGD 55-alpha-TIM-3 with correct enzyme restriction identification, performing PmeI linearization and FastAp dephosphorylation, converting an enzyme restriction system after the FastAp dephosphorylation to a purchased BJ5183 competent cell containing Adeasy-1, coating the cell on LB solid culture medium with 50 mu g/mL Kana resistance, inverting the cell and putting the cell into a 37 ℃ incubator, culturing for 20h, then picking spots, shaking the cell for 18h, extracting a plasmid according to the specification of a plasmid extraction kit to obtain pAd-GD 55-alpha-TIM-3, namely generating a GP73 promoter to replace an E1A endogenous promoter, regulating and controlling E1A expression, deleting E1B 55kDa and E3 regions, and carrying a recombinant adenovirus genome vector pAd-GD 55-alpha-TIM-3 of an alpha-TIM-3 gene, performing enzyme restriction identification by using MluI, wherein the correct plasmid has a 5 band with the size of about 1200bp, 2000bp, 4800bp, 7000bp and 20000bp, and the results are shown in FIGS. 2-3.
The PmeI enzyme digestion system is as follows:
Figure BDA0003539729140000142
the MluI digestion system is as follows:
Figure BDA0003539729140000143
5) transforming the MluI restriction enzyme identification correct recombinant plasmid pAd-GD 55-alpha-TIM-3 into DH5 alpha competent cells, selecting plaque and shaking bacteria, preserving bacteria and extracting plasmids, performing restriction enzyme identification by using MluI (restriction enzyme system is the same as above), sequencing the plasmid with correct restriction enzyme band, comparing the sequencing result with fusion gene fragment alpha-TIM-3 (complete nucleic acid sequence is shown in appendix SEQ ID NO.1) to determine whether mutation occurs, and obtaining pAd-GD 55-alpha-TIM-3.
6) After the correctly sequenced pAd-GD 55-alpha-TIM-3 plasmid was linearized by Pac I and FastAp dephosphorylated, the plasmid pAd-GD 55-alpha-TIM-3 linearized DNA fragment was transfected into HEK293A cells according to lipo2000 Lipofectin instructions. Adding 5% CO 2 Culturing in a cell culture box at 37 deg.C for 7-10 days to obtain target oncolytic adenovirus GD 55-alpha-TIM-3 after the cells are diseased (i.e. HEK293A cells are found to be clustered into grape strain strings and part of cells are dropped to form plaques), collecting all cells and cell supernatant, and storing at-80 deg.C;
the PacI linearized digestion system is as follows:
Figure BDA0003539729140000151
example 2
Oncolytic adenovirus GD 55-alpha-TIM-3 in vitro experiment specifically kills liver normal cells WRL-68 and liver cancer cells MHCC-97H, BEL-7404 and Huh 7.
Laying the liver normal cells WRL-68 with good growth state, the liver cancer cells MHCC-97H, BEL-7404 and Huh7 on a 96-well plate according to 3000 cells/hole and a DMEM complete cell culture medium of 10% fetal bovine serum and 1% penicillin-streptomycin by 100 mu L/hole; after 12h, diluting oncolytic adenovirus GD 55-alpha-TIM-3 and oncolytic adenovirus GD55 constructed in the front of a laboratory according to different MOIs, wherein each group is provided with 5 multiple holes, and 10 mu L of virus solution is added into each hole; after the viruses are added for 24h, 48h, 72h and 96h respectively, 20 mu L of 5 mg/mL thiazole blue solution is added into each hole; standing at 37 deg.C for 5% CO 2 Treating the cells in a constant-temperature incubator for 4 hours, then removing the culture medium, and adding DMSO into the culture medium at 150 mu L/hole; after oscillating for 10min, measuring the light absorption value (OD490nm) by an enzyme-labeling instrument; and processing the data according to an MTT cell survival rate calculation formula to calculate the cell survival rate.
The above cell cultures were all at 37 ℃ and 5% CO 2 Culturing the cells in a constant-temperature incubator; the medium was 10% fetal bovine serum, 1% penicillin-streptomycin DMEM (DMEM from Gibco).
The results are shown in fig. 6-13, after the virus infects cells, along with the increase of MOI, the time is increased, the killing effect of the virus is stronger, the action effect of the target virus GD 55-alpha-TIM-3 and the control virus GD55 has strong dose dependence and time dependence, after 96h, 40MOI GD 55-alpha-TIM-3 can reduce the survival rate of BEL-7404 and Huh7 to below 30%, and the target virus GD 55-alpha-TIM-3 and the control virus GD55 have obvious difference.
Example 3
In vitro experiment of oncolytic adenovirus GD 55-alpha-TIM-3 on WRL-68 of liver normal cell and MHCC-97H, BEL-7404 and Huh7 of liver cancer cell
And (3) detecting the killing effect of the oncolytic adenovirus GD 55-alpha-TIM-3 on the CRC cell line by using a crystal violet experiment. Laying normal liver cells WRL-68 and liver cancer cells MHCC-97H, BEL-7404 and Huh7 with good growth conditions on a 24-well plate at 30000 cells/well; after 12h, target virus GD 55-alpha-TIM-3 and control virus GD55 were diluted according to different MOIs, and 100 μ L of virus solution was added to each well; after 48h, sucking out the cell culture solution, adding 500 mu L of crystal violet staining solution into each hole, and staining for 20min at room temperature; absorbing the crystal violet dye solution, washing with tap water, drying and taking a picture.
The above cell cultures were all at 37 ℃ and 5% CO 2 Culturing the cells in a constant-temperature incubator; the medium was 10% fetal bovine serum 1% cyan-streptomycin DMEM medium (DMEM purchased from Gibco).
The results are shown in fig. 14-17, the killing effect of the virus on the hepatoma cells is more and more obvious with the increase of the MOI of the virus, and the virus has a weaker effect on the WRL-68 of the normal liver cells, but the target virus GD 55-alpha-TIM-3 is not obviously different from the control virus GD 55. The experiments show that the target virus GD 55-alpha-TIM-3 can better inhibit the survival of hepatoma carcinoma cells compared with the control virus GD55, and basically does not influence the growth of normal hepatoma cells.
Example 4
Oncolytic adenovirus GD 55-alpha-TIM-3 in vivo experiment shows the effect of inhibiting the growth of subcutaneous tumors of tumor-bearing nude mice with hepatoma cells BEL-7404 mixed with human peripheral blood lymphocytes (PBMC) for tumor formation
The method comprises the steps of firstly obtaining PBMC, collecting approximately 30mL of venous blood aseptically through a heparin aseptic blood collection tube, diluting anticoagulated whole blood with aseptic PBS (phosphate buffer solution) 1:1, then evenly paving the diluted anticoagulated whole blood 1:1 on ficoll lymphocyte separation medium to form a clear interface, centrifuging by a horizontal rotor centrifuge at room temperature for 30min at 2000rpm, and setting the lifting acceleration to be the lowest. After centrifugation, the anticoagulated whole blood is divided into four layers in ficoll lymphocyte separation liquid, the middle white membrane layer is taken as PBMC, 10-15mL of PBMC is taken for washing and separating, the room temperature horizontal rotor centrifuge is centrifuged at 1500rpm for 10min, the lifting acceleration is set to be the highest, the washing step is repeated by using a PBMC cell complete culture medium (PRMI1640+ 10% fetal bovine serum + 1% cyan-streptomycin +3 mu g/mL of CD3 antibody +1 mu g/mL of CD28 antibody +100U/mL of IL-2+2mM glutamine) and the fine particles are resuspended by using a PBMC cell activation complete culture medium (PRMI1640+ 10% fetal bovine serum + 1% cyan-streptomycin +3 mu g/mL of CD3 antibody +1 mu g/mL of CD28 antibody +100U/mL of IL-2+2mM glutamine)Counting cells, and using six-hole plate according to 1 × 10 6 cells/mL, 2mL/well, were passaged once every three days (5 min at 1000 rpm) for two passages.
Activation of PBMC, BEL-7404 cells plated in six-well plates, 5X 10 5 cells/well, after the cells are attached, mitomycin of 2 mug/mL is added to treat the cells, after 2h the supernatant is removed and washed 2 times with PBS, and PBMC of 5 × 10 is added according to the ratio of tumor cells to PBMC of 1:10 6 cells/well coculture, PBMC were collected once two days and the activation procedure was repeated three times.
Animal experiments were conducted in compliance with regulations and standards established by the U.S. department of agriculture and the national institutes of health, and the requirements of the animal ethics committee of the university of chem. BALB/C female nude mice purchased from Shanghai Slek laboratory animal center at about 4 weeks of age were adaptively bred, and activated PBMC and hepatoma cell BEL-7404 cells were mixed at 5 weeks of age (activated PBMC: BEL-7404: 1:4 with 2X 10 in 100. mu.L PBS) 6 cells PBMC+8×10 6 cells BEL-7404) and subcutaneously inoculated into the right side of nude mice. The mouse needs 10 to 12 days for forming tumor, and when the transplanted tumor reaches 80 to 120mm 3 At this time, the mice were randomly divided into 3 groups (6 mice per group), and PBS (vehicle), GD55(1X 10) were injected separately 9 pfu/mouse), GD 55-alpha-TIM-3 (1X 10) 9 PFU/mouse). Oncolytic adenovirus was injected continuously for 2 days. Tumor size was measured with a vernier caliper every 5 days after virus injection.
The results are shown in FIGS. 18-19, compared with the PBS group, the objective virus GD 55-alpha-TIM-3 and the control virus GD55 can effectively inhibit the growth of tumor-bearing nude mice tumor with hepatoma cell BEL-7404 and human peripheral blood lymphocyte mixed tumor, and the objective virus GD 55-alpha-TIM-3 has stronger tumor growth inhibition effect than the control virus GD55, which indicates that the expression of alpha-TIM-3 plays an important anti-cancer growth effect.
Experiments prove that the constructed double-targeting oncolytic adenovirus GD 55-alpha-TIM-3 can selectively kill tumor cells without influencing normal cells basically, can effectively inhibit the growth of liver cancer transplantable tumor in vivo, and provides an immune checkpoint antibody-oncolytic virus medicine for treating cancer.
Finally, it should be noted that the present invention is not limited to the above embodiments, and many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
The invention name is as follows: oncolytic adenovirus construction method carrying immune checkpoint molecule TIM-3 antibody gene and application thereof
The first applicant: shaoxing biological medicine research institute, Inc., of Zhejiang university of science;
the second applicant: zhejiang university of science and engineering
1 α -TIM-3 complete nucleic acid sequence of SEQ ID NO:
ATGGATATGAGGGTTCCCGCCCAACTTCTGGGACTGTTGTTGCTTTGGCTTAGAGGGGCACGGTGCATGGAATTTGGTCTGTCCTGGGTCTTTCTGGTTGCTTTGCTGCGGGGCGTTCAGTGCCAGGTTCAGCTCGTGGAATCTGGGGGGGGTGTTGTGCAACCAGGCAGGTCACTGAGGCTTTCATGTGCGGCGTCCGGCTTTACTTTCAACTCCTACGGAATGCATTGGGTGAGGCAGGCCCCAGGAAAAGGCCTCGAGTGGGTGGCTGTGATCTGGTATGATGGGTCCAACAAGTATTATGGGGACAGCGTGAAAGGGAGATTTACCATCAGCAGAGACAACTCTAAGAATACACTGTACCTCCAGATGAACAGCCTGCGGGCAGAAGACACAGCTGTGTATTACTGCGCCATTTGGTTTGGCGAGATGTTCTCAGAGTATTTCCAACATTGGGGCCAGGGAACCCTTGTGACTGTAAGCAGCGCAAGCACTAAGGGGCCATCCGTCTTTCCCCTTGCTCCCAGTTCCAAGAGTACTTCAGGTGGAACTGCCGCTCTGGGTTGTCTTGTGAAGGATTATTTTCCCGAGCCCGTTACAGTCAGTTGGAATTCAGGTGCCCTGACTAGCGGTGTCCACACATTCCCTGCCGTGTTGCAGAGTAGCGGCTTGTACTCCCTTTCAAGCGTGGTCACAGTACCTAGCAGCAGCCTTGGGACCCAGACCTACATTTGTAATGTGAACCACAAGCCTTCAAATACCAAGGTTGATAAGCGGGTTGAGCCCAAGTCCTGCGACAAGACTCACACTTGCCCCCCCTGTCCTGCACCTGAACTGTTGGGGGGTCCTTCCGTGTTCTTGTTCCCACCGAAACCTAAGGATACTCTGATGATCTCCCGCACACCCGAGGTCACATGTGTTGTCGTGGATGTCTCTCACGAAGACCCCGAGGTTAAGTTCAACTGGTACGTGGATGGCGTGGAAGTGCACAACGCCAAGACAAAGCCCCGCGAAGAACAGTACAATTCAACCTACAGGGTGGTCAGTGTCCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAATGCAAGGTCTCCAACAAGGCTCTTCCTGCGCCCATCGAGAAGACGATTTCCAAGGCAAAAGGTCAGCCACGCGAGCCACAAGTCTACACCCTGCCCCCATCCAGGGAGGAGATGACAAAGAATCAGGTGTCCCTGACTTGTCTCGTCAAGGGGTTTTATCCATCTGATATCGCAGTCGAGTGGGAAAGTAACGGGCAGCCGGAAAATAACTATAAGACCACCCCCCCTGTCCTGGATTCTGACGGCAGTTTCTTCTTGTATTCCAAGCTGACAGTCGATAAAAGCCGATGGCAGCAAGGTAATGTGTTTAGCTGTAGCGTGATGCACGAGGCACTGCATAACCACTACACACAGAAGTCCCTCAGCCTTAGTCCCGGCAAAAGGGCCAAAAGAGCCCCTGTGAAACAGACTCTGAATTTCGATCTCCTGAAACTTGCAGGTGACGTCGAGTCAAACCCCGGTCCTATGGATATGAGGGTTCCCGCCCAACTGTTGGGTCTGCTGTTGCTGTGGTTGAGGGGGGCCAGATGCATGGACATGCGAGTGCCTGCTCAGCTGCTGGGACTGCTCTTGCTGTGGCTGCCCGGAGCTAGATGCGCTATCCAATTGACACAGTCACCCTCCTCATTGTCCGCCAGTGTGGGCGATAGGGTGACAATAACGTGTAGAGCCAGCCAGGGCATTTCCTCAGCCCTTGCTTGGTACCAGCAAAAGCCTGGAAAGGCACCAAAGTTGCTGATCTACGATGCCTCCAGCTTGGAGAGTGGTGTACCAAGTCGATTTTCCGGGTCCGGTTCAGGAACCGATTTTACTCTGACAATCTCTAGCCTTCAGCCAGAAGACTTTGCCACCTATTACTGTCAGCAGTTCAACTCTTACCCTCTGACGTTTGGGGGCGGAACGAAGGTAGAGATTAAACGGACTGTTGCCGCCCCTAGTGTTTTTATCTTTCCCCCTTCCGATGAGCAGCTGAAGAGCGGAACCGCTTCCGTGGTGTGCCTGCTTAACAATTTCTACCCAAGAGAAGCCAAGGTGCAGTGGAAAGTGGACAATGCTCTGCAGTCAGGTAATAGCCAAGAATCTGTGACAGAACAGGACTCTAAGGACAGTACCTACTCACTTAGTTCAACCCTCACACTGTCAAAGGCCGATTATGAGAAACATAAAGTTTATGCCTGTGAAGTCACTCATCAGGGGCTTTCCAGCCCTGTGACAAAAAGTTTTAATCGGGGCGAGTGCCATCACCATCACCACCATtag
SEQ ID NO.2 Signal peptide Artificial sequence:
ATGGATATGAGGGTTCCCGCCCAACTTCTGGGACTGTTGTTGCTTTGGCTTAGAGGGGCACGGTGC
the alpha-TIM-3 heavy chain variable region nucleic acid artificial sequence shown in SEQ ID NO. 3:
ATGGAATTTGGTCTGTCCTGGGTCTTTCTGGTTGCTTTGCTGCGGGGCGTTCAGTGCCAGGTTCAGCTCGTGGAATCTGGGGGGGGTGTTGTGCAACCAGGCAGGTCACTGAGGCTTTCATGTGCGGCGTCCGGCTTTACTTTCAACTCCTACGGAATGCATTGGGTGAGGCAGGCCCCAGGAAAAGGCCTCGAGTGGGTGGCTGTGATCTGGTATGATGGGTCCAACAAGTATTATGGGGACAGCGTGAAAGGGAGATTTACCATCAGCAGAGACAACTCTAAGAATACACTGTACCTCCAGATGAACAGCCTGCGGGCAGAAGACACAGCTGTGTATTACTGCGCCATTTGGTTTGGCGAGATGTTCTCAGAGTATTTCCAACATTGGGGCCAGGGAACCCTTGTGACTGTAAGCAGCGCAAGC
SEQ ID NO.4 alpha-TIM-3 heavy chain constant region fragment-nucleic acid artificial sequence:
ACTAAGGGGCCATCCGTCTTTCCCCTTGCTCCCAGTTCCAAGAGTACTTCAGGTGGAACTGCCGCTCTGGGTTGTCTTGTGAAGGATTATTTTCCCGAGCCCGTTACAGTCAGTTGGAATTCAGGTGCCCTGACTAGCGGTGTCCACACATTCCCTGCCGTGTTGCAGAGTAGCGGCTTGTACTCCCTTTCAAGCGTGGTCACAGTACCTAGCAGCAGCCTTGGGACCCAGACCTACATTTGTAATGTGAACCACAAGCCTTCAAATACCAAGGTTGATAAGCGGGTT
the artificial sequence of the hinge region of SEQ ID NO. 5:
GAGCCCAAGTCCTGCGACAAGACTCACACTTGCCCCCCCTGTCCT
SEQ ID NO.6 alpha-TIM-3 heavy chain constant region fragment two nucleic acid artificial sequence:
GCACCTGAACTGTTGGGGGGTCCTTCCGTGTTCTTGTTCCCACCGAAACCTAAGGATACTCTGATGATCTCCCGCACACCCGAGGTCACATGTGTTGTCGTGGATGTCTCTCACGAAGACCCCGAGGTTAAGTTCAACTGGTACGTGGATGGCGTGGAAGTGCACAACGCCAAGACAAAGCCCCGCGAAGAACAGTACAATTCAACCTACAGGGTGGTCAGTGTCCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAATGCAAGGTCTCCAACAAGGCTCTTCCTGCGCCCATCGAGAAGACGATTTCCAAGGCAAAAGGTCAGCCACGCGAGCCACAAGTCTACACCCTGCCCCCATCCAGG
three nucleic acid artificial sequences of SEQ ID NO.7 alpha-TIM-3 heavy chain constant region fragment:
GAGGAGATGACAAAGAATCAGGTGTCCCTGACTTGTCTCGTCAAGGGGTTTTATCCATCTGATATCGCAGTCGAGTGGGAAAGTAACGGGCAGCCGGAAAATAACTATAAGACCACCCCCCCTGTCCTGGATTCTGACGGCAGTTTCTTCTTGTATTCCAAGCTGACAGTCGATAAAAGCCGATGGCAGCAAGGTAATGTGTTTAGCTGTAGCGTGATGCACGAGGCACTGCATAACCACTACACACAGAAGTCCCTCAGCCTTAGTCCCGGCAAA
SEQ ID NO. 8F 2A linker artificial sequence:
GTGAAACAGACTCTGAATTTCGATCTCCTGAAACTTGCAGGTGACGTCGAGTCAAACCCCGGTCCT
SEQ ID NO.9 Signal peptide Artificial sequence:
ATGGATATGAGGGTTCCCGCCCAACTTCTGGGACTGTTGTTGCTTTGGCTTAGAGGGGCACGGTGC
SEQ ID NO.10 alpha-TIM-3 light chain variable region nucleic acid artificial sequence:
ATGGACATGCGAGTGCCTGCTCAGCTGCTGGGACTGCTCTTGCTGTGGCTGCCCGGAGCTAGATGCGCTATCCAATTGACACAGTCACCCTCCTCATTGTCCGCCAGTGTGGGCGATAGGGTGACAATAACGTGTAGAGCCAGCCAGGGCATTTCCTCAGCCCTTGCTTGGTACCAGCAAAAGCCTGGAAAGGCACCAAAGTTGCTGATCTACGATGCCTCCAGCTTGGAGAGTGGTGTACCAAGTCGATTTTCCGGGTCCGGTTCAGGAACCGATTTTACTCTGACAATCTCTAGCCTTCAGCCAGAAGACTTTGCCACCTATTACTGTCAGCAGTTCAACTCTTACCCTCTGACGTTTGGGGGCGGAACGAAGGTAGAGATTAAACGGACT
SEQ ID NO.11 alpha-TIM-3K chain constant region nucleic acid artificial sequence:
GTTGCCGCCCCTAGTGTTTTTATCTTTCCCCCTTCCGATGAGCAGCTGAAGAGCGGAACCGCTTCCGTGGTGTGCCTGCTTAACAATTTCTACCCAAGAGAAGCCAAGGTGCAGTGGAAAGTGGACAATGCTCTGCAGTCAGGTAATAGCCAAGAATCTGTGACAGAACAGGACTCTAAGGACAGTACCTACTCACTTAGTTCAACCCTCACACTGTCAAAGGCCGATTATGAGAAACATAAAGTTTATGCCTGTGAAGTCACTCATCAGGGGCTTTCCAGCCCTGTGACAAAAAGTTTTAATCGGGGCGAGTGC
SEQ ID NO 126 × His artificial sequence:
CATCACCATCACCACCAT

Claims (10)

1. the construction method of oncolytic adenovirus carrying immune checkpoint molecule TIM-3 antibody gene is characterized by comprising the following steps:
1) preparing pCA 13-alpha-TIM-3 plasmid carrying alpha-TIM-3 gene;
2) carrying out enzyme digestion on the pCA 13-alpha-TIM-3 plasmid by BglII to obtain an expression frame carrying a CMV promoter, an alpha-TIM-3 gene and SV40 polyA, and connecting the expression frame with a pGD55 plasmid subjected to enzyme digestion by BglII to finally obtain a pGD 55-alpha-TIM-3 plasmid;
3) pGD 55-alpha-TIM-3 plasmid is digested and linearized by Pme I and then transformed into adenovirus skeleton plasmid Adeasy-1 escherichia coli BJ5183 with deletion of E3 region for homologous recombination, and then transformed into DH5 alpha competent cells to generate GP73 promoter to replace endogenous E1A promoter and regulate E1A expression and delete E1B 55kDa and E3 region, and recombinant adenovirus genome vector pAd-GD 55-alpha-TIM-3 carrying alpha-TIM-3 gene;
4) after the recombinant adenovirus genome vector pAd-GD 55-alpha-TIM-3 with correct recombinant identification and endotoxin removal is subjected to enzyme digestion linearization by PacI, the recombinant adenovirus genome vector pAd-GD 55-alpha-TIM-3 is transfected into HEK293A cells with good state to package adenovirus, and after the cells are diseased, the oncolytic adenovirus GD 55-alpha-TIM-3 is obtained.
2. The method for constructing an oncolytic adenovirus carrying an immune checkpoint molecule TIM-3 antibody gene according to claim 1, wherein: the procedure for preparing pCA13- α -TIM-3 plasmid carrying α -TIM-3 gene was as follows:
1) amplification of the α -TIM-3 Gene: designing an alpha-TIM-3 gene primer, wherein Hind III and Xba I enzyme cutting sites are respectively arranged at the upstream and downstream, and amplifying an alpha-TIM-3 gene fragment by using KOD high fidelity PCR polymerase and a synthesized pCDNA3.1-alpha-TIM-3 as a template;
2) the α -TIM-3 gene fragment was digested simultaneously with HindIII and XbaI and ligated to pCA13 plasmid vector digested simultaneously with HindIII and XbaI to obtain pCA13- α -TIM-3.
3. The method for constructing an oncolytic adenovirus carrying an immune checkpoint molecule TIM-3 antibody gene according to claim 2, wherein: the alpha-TIM-3 gene fragment comprises a signal peptide, an alpha-TIM-3 heavy chain variable region, a human antibody heavy chain constant region fragment I, a human antibody hinge region, a human antibody heavy chain constant region fragment II, a human antibody heavy chain constant region fragment III, an F2A linker, an alpha-TIM-3 light chain variable region and a K chain constant region.
4. The method for constructing an oncolytic adenovirus carrying an immune checkpoint molecule TIM-3 antibody gene according to claim 2 or 3, characterized in that: the alpha-TIM-3 gene segment is a Signal peptide-VH-CH1-Hinge-CH2-CH3-F2A-Signal peptide-VL-CL-6 XHis fusion gene segment.
5. The method for constructing an oncolytic adenovirus carrying an immune checkpoint molecule TIM-3 antibody gene according to claim 2 or 3, characterized in that: the nucleotide sequence of the fusion gene fragment is shown as SEQ ID NO. 1.
6. The method for constructing an oncolytic adenovirus carrying an immune checkpoint molecule TIM-3 antibody gene according to claim 1, wherein: the pGD55 plasmid, pGD55- α -TIM-3 plasmid, pAd-GD55- α -TIM-3 plasmid were all subjected to Fastap dephosphorylation.
7. The method for constructing an oncolytic adenovirus carrying an immune checkpoint molecule TIM-3 antibody gene according to claim 1, wherein: the recombinant identification method in the step 4) adopts Mlu I enzyme digestion identification.
8. A recombinant vector characterized by: comprising an alpha-TIM-3 gene according to any one of claims 1 to 4.
9. A pharmaceutical composition comprising an oncolytic adenovirus GD55- α -TIM-3 according to any one of claims 1-7.
10. An application of oncolytic adenovirus GD 55-alpha-TIM-3 carrying immune checkpoint molecule TIM-3 antibody gene in liver cancer drugs.
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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101448941A (en) * 2006-03-23 2009-06-03 麒麟医药株式会社 Agonistic antibody directed against human thrombopoietin receptor
CN103079644A (en) * 2010-06-11 2013-05-01 协和发酵麒麟株式会社 Anti-TIM-3 antibody
CN103981155A (en) * 2014-04-14 2014-08-13 浙江理工大学 Construction method and application of targeting hepatoma oncolytic adenovirus
US20170157188A1 (en) * 2014-07-16 2017-06-08 Transgene S.A. Oncolytic virus for expression of immune checkpoint modulators
WO2018049261A1 (en) * 2016-09-09 2018-03-15 Icellhealth Consulting Llc Oncolytic virus expressing immune checkpoint modulators
US20180155412A1 (en) * 2015-05-13 2018-06-07 The Trustees Of The University Of Pennsylvania Aav-mediated expression of anti-influenza antibodies and methods of use thereof
US20180207273A1 (en) * 2015-07-29 2018-07-26 Novartis Ag Combination therapies comprising antibody molecules to tim-3
CN110295195A (en) * 2019-06-26 2019-10-01 浙江理工大学 The building and application of the oncolytic adenovirus GD55-GSDME of liver cancer targeting
CN111094341A (en) * 2017-07-31 2020-05-01 生物医学研究院 Antibodies having functional domains in the elbow region between the variable and constant domains
WO2021228106A1 (en) * 2020-05-12 2021-11-18 上海荣瑞医药科技有限公司 Oncolytic virus in combination with immune checkpoint inhibitor for treating tumors

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101448941A (en) * 2006-03-23 2009-06-03 麒麟医药株式会社 Agonistic antibody directed against human thrombopoietin receptor
CN103079644A (en) * 2010-06-11 2013-05-01 协和发酵麒麟株式会社 Anti-TIM-3 antibody
CN103981155A (en) * 2014-04-14 2014-08-13 浙江理工大学 Construction method and application of targeting hepatoma oncolytic adenovirus
US20170157188A1 (en) * 2014-07-16 2017-06-08 Transgene S.A. Oncolytic virus for expression of immune checkpoint modulators
US20180155412A1 (en) * 2015-05-13 2018-06-07 The Trustees Of The University Of Pennsylvania Aav-mediated expression of anti-influenza antibodies and methods of use thereof
US20180207273A1 (en) * 2015-07-29 2018-07-26 Novartis Ag Combination therapies comprising antibody molecules to tim-3
WO2018049261A1 (en) * 2016-09-09 2018-03-15 Icellhealth Consulting Llc Oncolytic virus expressing immune checkpoint modulators
CN111094341A (en) * 2017-07-31 2020-05-01 生物医学研究院 Antibodies having functional domains in the elbow region between the variable and constant domains
CN110295195A (en) * 2019-06-26 2019-10-01 浙江理工大学 The building and application of the oncolytic adenovirus GD55-GSDME of liver cancer targeting
WO2021228106A1 (en) * 2020-05-12 2021-11-18 上海荣瑞医药科技有限公司 Oncolytic virus in combination with immune checkpoint inhibitor for treating tumors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
汪琦等: "靶向人TIM3 单抗制备及对肝癌细胞增殖的影响", 免疫学技术与方法, vol. 35, no. 14, pages 1735 *

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