Novel oncolytic adenovirus vector carrying membrane type CD40L as well as preparation method and application of novel oncolytic adenovirus vector

Abstract

The invention relates to the technical field of biological medicines, in particular to a novel oncolytic adenovirus vector carrying membrane type CD40L as well as a preparation method and application thereof. A plasmid skeleton of the recombinant plasmid is type-5 adenovirus, the recombinant plasmid has an E3 region nucleotide sequence with double deletion of a Gp19k fragment and an ADP fragment, and the E3 region nucleotide sequence comprises a nucleotide sequence of an mCD40L coding region. The oncolytic adenovirus vector can effectively prevent wild type CD40L from being sheared into soluble CD40L after expressing the cell surface, reduce the immunosuppression microenvironment state and reduce the systemic side effects; the compound has a good tumor inhibition effect, and has a wide application prospect in preparation of various preparations for treating tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a novel oncolytic adenovirus vector carrying membrane-type CD40L, a preparation method and an application thereof. Background Art

[0002] Adenovirus is a non-integrated, non-enveloped, double-stranded DNA virus with a genome size of about 30-38kb. It is a virus particle with a diameter of 80-110nm. The capsid is icosahedral and consists of 240 hexons and 12 pentons. Adenovirus vector is one of the most widely used vectors in preclinical research and clinical trials of oncolytic viruses. Adenovirus vectors have attracted much attention from researchers because of their good biosafety, easy access to high-titer viruses, and large capacity for foreign genes. According to statistics, adenovirus vectors account for 41.9% of oncolytic virus products that have been clinically tested. Oncolytic virus is a virus that exists naturally or is genetically engineered to replicate and lyse only in tumor cells. As a new type of anti-tumor drug, it has shown good development prospects.

[0003] With the rapid development of tumor immunotherapy, oncolytic viruses are gradually attracting attention because of their strong immunogenicity and the ability to carry and express exogenous immunostimulatory genes. Literature reports that adenovirus vectors carrying multiple immune targets such as GM-CSF, IFN-β, and IL12 can enhance anti-tumor immunity. CD40L is the natural trimeric ligand of CD40 and is a 39-kDa type II membrane glycoprotein that is expressed on a variety of cell types, including activated CD4+T cells, activated B cells, memory CD8 T cells, activated natural killer cells, granulocytes, endothelial cells, smooth muscle cells, macrophages, and activated platelets. A large number of studies have shown that CD40-CD40L plays a vital role in the function of CD8+ cytotoxic T lymphocytes (CTLs) in immune responses and is required for adaptive immune responses. In the past 20 years, CD40 has been a star target for the development of new anti-tumor immune drugs. CD40 agonists not only activate DCs, but also reverse TAM polarization and promote the formation of tertiary lymphoid structures. Therefore, CD40L has become the most commonly carried APC activation immune target by oncolytic viruses.

[0004] However, wild-type CD40L (wtCD40L) can be cleaved into soluble CD40L (sCD40L) after being expressed on the cell surface. Many reports have shown that sCD40L can increase the immunosuppressive microenvironment and increase systemic side effects.

[0005] In addition, the E3 gene expression product of the adenovirus vector has nothing to do with the replication of the viral genome, and its main function is to destroy the host's immune defense mechanism. The expression products of the E3 gene include gp 19K protein, RIDα&β and 14.7Kd protein, and adenovirus death protein; among them, the expression product of the E3 gene, gp 19K protein, can bind to the heavy chain of the MHC class I molecule on the endoplasmic reticulum to prevent it from being transported to the cell surface, thereby delaying the expression of MHC I; the expression products of the E3 gene, RIDα&β and 14.7Kd protein, can inhibit TNF-induced cell apoptosis, promote Fas degradation, and downregulate TNF receptor levels; the expression product of the E3 gene, adenovirus death protein (ADP), also known as 11.6Kd, can lyse cells and release virus particles in the late stage of viral infection. The genes in the E3 region include Gp19k, ADP, E3B, etc. These genes all share an expression frame, and they share the same promoter and polyA tail.

[0006] After deleting a gene in the E3 region, inserting an exogenous gene can be used for the expression of the exogenous gene, thereby achieving the goal of embedding the exogenous gene into an oncolytic adenovirus product. However, it is currently unknown which genes in the E3 region will not affect the anti-tumor effect of the oncolytic adenovirus and may even enhance anti-tumor immunity. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a novel oncolytic adenovirus vector carrying membrane-type CD40L, a preparation method and an application thereof.

[0008] The technical solution of the present invention to solve the above technical problems is as follows:

[0009] The invention provides a recombinant plasmid, the plasmid skeleton of the recombinant plasmid is type 5 adenovirus, the recombinant plasmid has an E3 region nucleotide sequence with double deletion of a Gp19k fragment and an ADP fragment, and the E3 region nucleotide sequence includes a nucleotide sequence of an mCD40L coding region.

[0010] Furthermore, the nucleotide sequence of the mCD40L coding region is shown as SEQ ID No.1.

[0011] Furthermore, the nucleotide sequence of the E3 region is shown in SEQ ID No.2.

[0012] Furthermore, the nucleotide sequence of the recombinant plasmid is shown in SEQ ID No.3.

[0013] The present invention provides a novel oncolytic adenoviral vector carrying membrane-type CD40L, which is prepared using the recombinant plasmid as described above. The oncolytic adenoviral vector is a conditionally replicative adenoviral vector.

[0014] Furthermore, its nucleotide sequence is shown in SEQ ID No.4.

[0015] The present invention also provides a method for preparing the novel oncolytic adenoviral vector carrying membrane-type CD40L as described above, comprising the following steps:

[0016] S1, synthesizing a first nucleotide sequence, and preparing a first plasmid by homologous recombination of the first nucleotide sequence and an adenovirus 5 backbone plasmid; the first nucleotide sequence is an E3 region nucleotide sequence having a double deletion of a Gp19k fragment and an ADP fragment;

[0017] S2, inserting the coding region nucleotide sequence of mCD40L into the first plasmid to obtain the recombinant plasmid; the insertion position of the coding region nucleotide sequence of mCD40L is the position where the Gp19k fragment is deleted, and the fibrin coding region of the recombinant plasmid has a deletion;

[0018] S3, using the Ad5 / 35 chimeric backbone plasmid and the second plasmid to prepare the novel oncolytic adenovirus vector carrying membrane-type CD40L by homologous recombination;

[0019] S4. Perform enzyme digestion, transfection and purification on the novel oncolytic adenoviral vector carrying membrane-type CD40L.

[0020] Furthermore, in step S1, the position where the Gp19k fragment is deleted has specific restriction sites BstBI and ClaI.

[0021] The present invention also provides the use of the novel oncolytic adenovirus vector carrying membrane-type CD40L as described above in the preparation of a cancer treatment preparation, wherein the cancer treatment preparation is a drug, a pharmaceutical raw material, a vaccine or a targeted inhibitor.

[0022] The present invention also provides a preparation for treating cancer, wherein the preparation comprises the above-mentioned recombinant plasmid and / or the above-mentioned novel Ad5 / 35 chimeric oncolytic adenovirus vector.

[0023] The beneficial effects of the present invention are:

[0024] (1) The recombinant plasmid of the present invention carries the target gene mCD40L in its gene, which can avoid the immunosuppressive effect of sCD40L and enhance the anti-tumor immune activation effect of mCD40L;

[0025] (2) The deletion of Gp19k and ADP in the recombinant plasmid of the present invention can both increase the anti-tumor immune effect and increase the viral load of tumor cell progeny;

[0026] (3) The novel oncolytic adenovirus vector carrying membrane-type CD40L of the present invention can effectively prevent wild-type CD40L from being cleaved into soluble CD40L (sCD40L) after being expressed on the cell surface, thereby reducing the immunosuppressive microenvironment and systemic side effects;

[0027] (4) The novel oncolytic adenovirus vector carrying membrane-type CD40L of the present invention has a good tumor-suppressing effect and has broad application prospects in the preparation of various preparations for treating tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of the structure of the novel oncolytic adenoviral vector carrying membrane-type CD40L of the present invention, and each plasmid vector prepared in Example 1;

[0029] Figure 2 This is a novel oncolytic adenoviral vector carrying membrane-type CD40L of the present invention, and in Example 1, the plasmid map of M20-5;

[0030] Figure 3 This is a novel oncolytic adenoviral vector carrying membrane-type CD40L of the present invention, and in Example 1, the plasmid map of Ad5 / 35-mCD40L;

[0031] Figure 4 This is a novel oncolytic adenovirus vector carrying membrane-type CD40L of the present invention, in Example 1, the map of M20-5 oncolytic adenovirus;

[0032] Figure 5 This is a novel oncolytic adenovirus vector carrying membrane-type CD40L of the present invention. In Example 1, the Ad5 / 35-mCD40L oncolytic adenovirus map;

[0033] Figure 6 This is a schematic diagram of the structure of the novel oncolytic adenovirus vector carrying membrane-type CD40L of the present invention, and different conditional replicative adenovirus E3 deletion fragments in Example 2;

[0034] Figure 7 This is a novel oncolytic adenovirus vector carrying membrane-type CD40L of the present invention. In Example 2, the tumor volume change diagram of the golden hamster HapT1 tumor model is shown. Figure 7 A in the middle is the Hamster group, Figure 7 B is the PBS group, Figure 7 C in the middle is the Ad-A group, Figure 7 D in the middle is the M20-1 group, Figure 7 Middle E is the M22-1 group;

[0035] Figure 8This is a novel oncolytic adenovirus vector carrying membrane-type CD40L of the present invention. Example 3 is a comparison chart of the properties of oncolytic adenovirus recombinants carrying different CD40L nucleotide sequences. Figure 8 A in the middle is a schematic diagram of sCD40L shearing. Figure 8 B in the middle is a schematic diagram of the structure of each virus. Figure 8 C in the middle is the WB test result. Figure 8 D in the middle is the result of flow cytometry antibody detection. Figure 8 E in the middle is the positive rate test result, Figure 8 The mean fluorescence intensity of F is detected in Figure 8 Middle G is a comparison chart of sCD40L content in secretion supernatant;

[0036] Fig. 9 This is a flow chart of the cell preparation method used in Example 4 for the novel oncolytic adenoviral vector carrying membrane-type CD40L of the present invention;

[0037] Fig.10 The novel oncolytic adenoviral vector carrying membrane-type CD40L of the present invention, in Example 4, the flow cytometry detection results, Fig.10 A in the middle is the number of MHCII+ cells, Fig.10 B in the middle is the number of CD80+ cells, Fig.10 Middle C is the number of CD86+ cells; Fig.10 D in the middle is the percentage of CD80+ cells to CD11b in the RAW264.7 cell line;

[0038] Fig.11 This is a novel oncolytic adenoviral vector carrying membrane-type CD40L of the present invention. In Example 4, a comparison chart of the content of components secreted by each virus is shown. Fig.11 A in the middle is IL-12P70, Fig.11 B in the middle is IL-12P40, Fig.11 C in the middle is IFN-r, Fig.11 D in the middle is CXCL10, Fig.11 E in the middle stands for MIG. Fig.11 F in the middle is sCD40L;

[0039] Fig.12 The novel oncolytic adenoviral vector carrying membrane-type CD40L of the present invention, in Example 5, the inhibitory effect of each plasmid vector on subcutaneous tumors of ovarian cancer; Fig.12 A in the middle is a flow chart for establishing the ID8-VEGF ovarian cancer subcutaneous tumor model; Fig.12 Middle B is a comparison of the volume changes of the subcutaneous tumor of ovarian cancer in mice in each experimental group at different times; Fig.12 Middle C is a comparison chart of the survival curves of each experimental group; Fig.12 Middle D is the graph showing the volume changes of subcutaneous tumors of ovarian cancer in mice in the PBS group; Fig.12Middle E is the graph showing the volume changes of subcutaneous tumors of ovarian cancer in mice in the M20 group;

[0040] Fig.12 Middle F is the graph showing the volume changes of subcutaneous tumors of ovarian cancer in mice in the M20-3 group; Fig.12 Middle G is the graph showing the volume changes of subcutaneous tumors of ovarian cancer in mice in the M20-5 group;

[0041] Fig.13 The novel oncolytic adenoviral vector carrying membrane-type CD40L of the present invention, in Example 5, the inhibitory effect of each plasmid vector on Hepa1-6 liver cancer tumors; Fig.13 A in the middle is a flowchart for establishing the Hepa1-6 liver cancer model; Fig.13 Middle B is a comparison of the volume changes of Hepa1-6 liver cancer tumors in mice of each experimental group at different times; Fig.13 Middle C is the graph showing the changes in the volume of Hepa1-6 liver cancer tumors in mice in the PBS group; Fig.13 D in the middle is the change of Hepa1-6 liver cancer tumor volume in mice in the M20 group; Fig.13 Middle E is the graph showing the changes in tumor volume of Hepa1-6 liver cancer in mice in the M20-3 group; Fig.12 Middle F is the graph showing the changes in the volume of Hepa1-6 liver cancer tumors in mice in the M20-5 group;

[0042] Fig.14 This is a novel oncolytic adenovirus vector carrying membrane-type CD40L of the present invention. In Example 6, the effect of each plasmid vector on immune cells in subcutaneous tumor tissue of ovarian cancer is shown in the figure. Fig.14 A in the middle is CD45+ cells. Fig.14 B in the middle is a dendritic cell. Fig.14 C in the middle is a mature DC cell. Fig.14 D in the middle is CD3+ cells, Fig.14 Middle E is CD4+, Fig.14 F in the middle is CD8+ cells, Fig.14 G in the middle is for activated CD4+ cells. Fig.14 Middle H is activated CD8+ cells;

[0043] Fig.15 This is a novel oncolytic adenoviral vector carrying membrane-type CD40L of the present invention. In Example 6, the changes in the cytokine content in the subcutaneous tumor tissue of ovarian cancer by each plasmid vector are shown in FIG. Fig.15 A in the middle is CD40L cells, Fig.15 B in the middle is CXCL9, Fig.15 C in the middle is CXCL10, Fig.15 D in the middle is IL12, Fig.15 E in the middle is IL10, Fig.15 F in the expression is TNFa. Fig.15 In the middle, G stands for IL2. Fig.15Middle H is IFN-γ;

[0044] Fig.16 This is a novel oncolytic adenoviral vector carrying membrane-type CD40L of the present invention. In Example 7, the oncolytic effect results of each plasmid vector on different cancer cells are compared. Fig.16 A in the middle is RKN, Fig.16 B in the middle is OVCAR8, Fig.16 The C in the middle is SKOV3, Fig.16 D in the middle is CaoV3;

[0045] Fig.17 This is a novel oncolytic adenoviral vector carrying membrane-type CD40L of the present invention. In Example 7, the expression comparison diagram of each plasmid vector is as follows: Fig.17 A is a comparison chart of the expression levels of each plasmid vector in SKOV3. Fig.17 Middle B is a comparison chart of the CD40L content expressed by each plasmid vector;

[0046] Fig.18 This is a novel oncolytic adenovirus vector carrying membrane-type CD40L of the present invention, and a comparison chart of oncolytic adenovirus replication characteristics of each plasmid vector in Example 7;

[0047] Fig.19 The novel oncolytic adenoviral vector carrying membrane-type CD40L of the present invention, in Example 7, the apoptosis detection results of each vector plasmid, Fig.19 A in the middle is the PBS group, Fig.19 Middle B is the M20 group, Fig.19 Middle C is the M20-F35 group, Fig.19 D in the middle is the oAd5 / 35-mCD40L group. DETAILED DESCRIPTION

[0048] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0049] The recombinant plasmid of the invention has a plasmid skeleton of type 5 adenovirus, and the recombinant plasmid has an E3 region nucleotide sequence with double deletion of Gp19k fragment and ADP fragment, and the E3 region nucleotide sequence includes the nucleotide sequence of the mCD40L coding region.

[0050] The recombinant plasmid of the present invention inserts the nucleotide sequence of the mCD40L coding region into the E3 region, which can avoid the immunosuppressive effect of sCD40L and enhance the anti-tumor immune activation effect of mCD40L. The deletion of Gp19k and ADP in the E3 region can increase the anti-tumor immune effect and the tumor cell progeny virus carrying capacity.

[0051] Preferably, the nucleotide sequence of the mCD40L coding region is shown as SEQ ID No.1.

[0052] Preferably, the nucleotide sequence of the E3 region is as shown in SEQ ID No.2.

[0053] Preferably, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID No.3.

[0054] The novel oncolytic adenoviral vector carrying membrane-type CD40L of the present invention is prepared using the recombinant plasmid as described above, and the oncolytic adenoviral vector is a conditionally replicative adenoviral vector.

[0055] Preferably, the nucleotide sequence of the novel oncolytic adenoviral vector carrying membrane-bound CD40L is shown in SEQ ID No.4.

[0056] In this novel oncolytic adenovirus vector carrying membrane-type CD40L, in addition to the aforementioned double deletion of Gp19k and ADP in the E3 region and the introduction of the target gene mCD40L, its E1A region also has a 27bp deletion, the specific deletion position is the 920-946th position of the CR2 region, and the sequence deletion of the E1A can make the Ad 5 adenovirus plasmid backbone meet the requirements of conditional replication. At the same time, the HVR1 and HVR5 in the Hexon region of the disease vector are replaced with the HVR1 and HVR5 of Ad 35, and the spike protein gene sequence is partially replaced with the fiber protein gene sequence of Ad 35 to change the virus's invasion mechanism, so that it enters the cell through the membrane auxiliary protein (CD46) instead of the Coxsackie virus and adenovirus receptor (CAR) receptor.

[0057] The preparation method of the novel oncolytic adenovirus vector carrying membrane-type CD40L of the present invention comprises the following steps:

[0058] S1. The first nucleotide sequence is synthesized by gene synthesis technology, which can be named as E3 delgp19k / delADP fragment. The first nucleotide sequence and the adenovirus 5 backbone plasmid Pad-E1A / Δ27bp are co-transformed into the competent BJ5183 by homologous recombination to obtain the conditional replicative adenovirus 5 backbone plasmid with double deletion of gp19k and ADP in the E3 region, which is the first plasmid and can be named as m20; the position where the Gp19k fragment is deleted has specific restriction sites BstBI and ClaI, and the two specific restriction sites are added after deleting the Gp19k fragment.

[0059] S2. Using specific enzyme cutting sites BstBI and ClaI, the target gene mCD40L is inserted into the first plasmid m20 by adopting in vitro homologous recombination technology to obtain a recombinant plasmid, which can be named m20-5; the insertion position of the nucleotide sequence of the coding region of mCD40L is the position where the Gp19k fragment is deleted, and the fibrin coding region of the recombinant plasmid has a deletion.

[0060] S3. A novel oncolytic adenovirus vector carrying membrane-type CD40L was prepared by homologous recombination of Ad5 / 35 chimeric backbone plasmid and recombinant plasmid.

[0061] S4. Perform enzyme digestion, transfection and purification on the novel oncolytic adenoviral vector carrying membrane-type CD40L.

[0062] Preferably, the backbone plasmid is digested with PacI and then transfected into HEK293 cells to obtain the final oncolytic adenovirus carrying the immune target.

[0063] The novel oncolytic adenovirus vector carrying membrane-type CD40L of the present invention can be used in the preparation of cancer treatment preparations, which are drugs, pharmaceutical raw materials, vaccines or targeted inhibitors.

[0064] The preparation for treating cancer of the present invention comprises the above-mentioned recombinant plasmid and / or the above-mentioned novel Ad5 / 35 chimeric oncolytic adenovirus vector.

[0065] The present invention is described below by means of specific examples.

[0066] Example 1 Preparation of oncolytic adenovirus

[0067] In this example, BJ5183 homologous recombination technology, gene synthesis technology and in vitro ligation technology were used to construct conditional replicative adenovirus vectors m20-5 and oAd5 / 35-mCD40L, and vector m20-3 containing the wild-type CD40L (wtCD40L) nucleotide sequence was constructed as a comparison for subsequent examples.

[0068] The plasmid vector structures of this example are as follows Figure 1 The specific preparation process of this embodiment is as follows:

[0069] 1. Preparation of m20 plasmid: (1) The E3 delGp19k / delADP fragment (puc57-E3delGp19k / delADP) was obtained by gene synthesis. Using puc57-E3 delGp19k / delADP as a template and PUC57-F and PUC57-R as primers, the E3 delGp19k / delADP product was obtained by PCR amplification. The specific sequences of PUC57-F and PUC57-R are shown in Table 1.

[0070] Table 1

[0071] Primer name sequence PUC57-F GTAAAACGACGGCCAGTG PUC57-R GGAAACAGCTATGACCATG

[0072] (2) Pad-E1A / Δ27bp was used as the adenovirus backbone plasmid and linearized by SpeI restriction enzyme digestion.

[0073] (3) The products of (1) and (2) were simultaneously transformed into the competent cells of Bj5183, and the plasmids after homologous recombination were further identified by plasmid size, PCR and sequencing.

[0074] (4) The plasmid with the correct sequence was transformed into competent T1, and the bacteria were picked, shaken, plasmid extracted, and PCR sequencing was performed to obtain Pad-E1 / Δ27--E3 / DelGp19k / delADP, which is the m20 plasmid. Its nucleotide sequence is shown in SEQ ID NO.5.

[0075] 2. Preparation of m20-3 plasmid and m20-5 plasmid: (1) In vitro gene synthesis of wtCD40L gene and mCD40L gene. The sequence of mCD40L gene is shown in SEQ ID No.1.

[0076] (2) The gene fragment carrying the homologous recombination arm outside the vector was obtained by PCR. The primers are shown in Table 2.

[0077] Table 2

[0078] Primer name sequence Gp19k-mCD40Lmut1-F gtattaggccaaaggcgcaTTATGATCGAGACCTACAGTCAGCCGT Gp19k-mCD40Lmut1-R gcatatcccacatagagtTTCGAAtcaCAATTTAAGTAGCCCAAAGCTGC Gp19k-mCD40L-F gtattaggccaaaggcgcaTTatgatagaaacatacagccaacct Gp19k-mCD40L-R gcatatcccacatagagtTTCGAAtcagagtttgagtaagccaa

[0079] (3) The m20 plasmid was digested with BSTBI and the digested product was purified.

[0080] (4) The products of (1) and (2) were connected in vitro using the NEBuider HiFi DNA Assembly Master Mix Kit.

[0081] (5) The m20 plasmid linearized fragment and the wtCD40L or mCD40L product were ligated in vitro using the NEBuider HiFi DNA Assembly Master Mix Kit. The reaction conditions were shown in Table 3. After the reaction, the m20-3 plasmid and the m20-5 plasmid were obtained. The ligation products were transformed into T1 competent cells, and clones were selected for sequencing and identification. Among them, the E3 region sequence in the m20-5 plasmid is shown in SEQ ID No. 2.

[0082] The nucleotide sequence of the m20-5 plasmid is shown in SEQ ID No. 3, and the maps of the m20-3 plasmid and the m20-5 plasmid are shown in Figure 2and Figure 3 shown.

[0083] Table 3

[0084] m20 (BstBI) digestion product 50ng wtCD40L or mCD40L PCR product 50ng NEBuider HiFi DNA Assembly master Mix 10ul DdH20 To 20ul Reaction conditions and time 50℃15min

[0085] (6) The m20, m20-3, and m20-5 plasmids were digested with PacI and purified, and then co-transfected with the Ad5 / 35 chimeric backbone plasmid into HEK293A cells to finally obtain the adenovirus oncolytic product.

[0086] Among them, the m20-5 oncolytic adenovirus map is as follows Figure 4 As shown, the Ad5 / 35-mCD40L oncolytic adenovirus map is as follows Figure 5 As shown, the gene sequence of Ad5 / 35-mCD40L oncolytic adenovirus is shown in SEQ ID No.4.

[0087] Example 2 Effects of different E3 region deletions on the anti-tumor effects of oncolytic adenovirus recombinants

[0088] like Figure 6 As shown, conditional replicative adenoviruses with single or combined deletions of Gp19k, ADP and E3B in the E3 region were constructed. Specifically, Ad-A was Gp19k deleted in the E3 region, M20-1 was Gp19k and ADP deleted, and M22-1 was Gp19k and E3B deleted.

[0089] The HapT1 tumor model of golden hamsters was established. Ad-A, M20-1 and M22-1 were intratumorally administered, and PBS was used as the control group. The changes in tumor volume were as follows: Figure 7 As shown. Figure 7 It can be seen that the conditional replication oncolytic adenovirus with combined deletion of Gp19k and ADP is not weaker than the oncolytic virus with single Gp19k deletion, and E3B deletion weakens the anti-tumor effect. Therefore, m20 was selected as the backbone vector.

[0090] Example 3 Comparison of properties of oncolytic adenovirus recombinants carrying different CD40L nucleotide sequences

[0091] After wtCD40L is expressed, it is easily cleaved by a variety of enzymes to produce sCD40L. The present invention uses GSG liner to replace the amino acid region that is susceptible to cleavage, while retaining the intracellular region, transmembrane region and extracellular active region, as shown in the schematic diagram Figure 8 As shown in A, the amino acid sequence is shown in SEQ ID No.6.

[0092] In this example, control oncolytic viruses m20 (empty virus with Gp19k and ADP regions deleted from the E3 region), m20-3 (oncolytic adenovirus vector carrying wtCD40L), and m20-5 (oncolytic adenovirus vector carrying mCD40L) were constructed based on the original CD40L design. The schematic diagrams of the virus structures are shown in Figure 8 As shown in B.

[0093] After transfecting ID8-VEGF cells with m20, m20-3, and m20-5, the cells were collected and their supernatants were taken for WB detection of CD40L expression, as shown in Figure 8 As shown in C, ELISA results showed that m20-3 and m20-5 were overexpressed in the transfected cells.

[0094] CD40L flow cytometry antibody was used to detect the expression of m20-3 and m20-5 on the cell surface. Figure 8 As shown in D, the expression level of m20-5 was significantly higher than that of m20-3.

[0095] The positive rate, mean fluorescence intensity, and sCD40L content in the secretion supernatant were statistically analyzed. Figure 8 As shown in E and F, the positive rate and average fluorescence intensity of m20-5 were significantly higher than those of other plasmids, while the sCD40L content in the secretion supernatant was significantly lower than those of other plasmids.

[0096] Example 4 Detection of functional activity of mCD40L expressed by M20-5 carrying mCD40L

[0097] In this example, the functional activity of mCD40L expressed by M20-5 cells carrying mCD40L was detected. The cell preparation method used in this example is as follows: Fig. 9 As shown, mouse monocytes were isolated and induced into BMDCs, and then matured by adding IL-4 and GM-CSF cytokines.

[0098] The above cells were co-cultured with ID8-VEGF cells transfected with m20, m20-3 and m20-5 for 24 hours. After culture, it was found that M20-5 could significantly enhance the activation ability of DC. The number of MHCII+, CD80+ and CD86+ cells was detected by flow cytometry. The test results are as follows: Fig.10 As shown, through Fig.10 It can be seen that the number of the above cells of m20-5 increased significantly, and the results of each experiment showed that m20-5 activated BMDC with the strongest effect. Fig.11 As shown, the secretion of IL-12, IFN-r and CXCL10 by m20-5 also increased, while sCD40L was still lower than that of m20-3.

[0099] Example 5 Pharmacodynamics test of oncolytic adenovirus carrying mCD40L

[0100] like Fig.12 As shown, in this example, an ID8-VEGF ovarian cancer subcutaneous tumor model was established, and PBS, m20 (control virus), m20-3 and m20-5 were administered respectively to observe their tumor inhibition effect and survival. Fig.12 It can be seen that m20-5 showed enhanced tumor inhibition effect on ID8-VEGF ovarian cancer subcutaneous tumors compared with m20-3 (wtCD40L) and m20, and at the same time, m20-5 prolonged its survival.

[0101] like Fig.13 As shown in the figure, a Hepa1-6 liver cancer model was established, and the same administration method as above was used to observe its tumor inhibition effect. Fig.13 The experimental results show that m20-5 has a stronger tumor inhibitory effect on Hepa1-6 liver cancer tumors.

[0102] Example 6 Effect of oncolytic adenovirus carrying mCD40L on the ovarian cancer microenvironment

[0103] The ID8-VEGF subcutaneous tumor model established in Example 5 was used, and PBS, m20, m20-3 and m20-5 were injected intratumorally for 4 times (once every other day). The tumor tissue was removed 7 days later, and the changes of immune cells in the tumor tissue were analyzed by multicolor flow cytometry.

[0104] The analysis results are as follows Fig.14 and Fig.15 The results showed that the M1 / M2 ratio in the m20-5 group was significantly increased, and the DC maturity increased (CD80 + / CD86 + ), further activating CD8+T cells, and at the same time, the inflammatory cytokine m20-5 group also increased synchronously, indicating that the oncolytic adenovirus m20-5 carrying mCD40L can reshape the ovarian cancer microenvironment.

[0105] Example 7 Verification of the oncolytic effect and CD40L expression of Ad5 / 35-mCD40L chimeric oncolytic adenovirus

[0106] In this example, the oncolytic effect and CD40L expression of Ad5 / 35-mCD40L chimeric oncolytic adenovirus (m20-5-F35) were verified. Fig.16 As shown, the results showed that Ad5 / 35-mCD40L had a stronger oncolytic effect on RKN and OVCAR8, as Fig.17 As shown, Ad5 / 35-mCD40L can successfully express membrane-type CD40L, such as Fig.18As shown in Figure 2, Ad5 / 35-mCD40L possesses oncolytic adenovirus replication characteristics. Fig.19 As shown, apoptosis detection revealed that Ad5 / 35-mCD40L induced a stronger apoptotic effect in OVCAR8.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A recombinant plasmid, characterized in that: The plasmid backbone of the recombinant plasmid is adenovirus type 5, the recombinant plasmid has an E3 region nucleotide sequence with double deletion of the Gp19k fragment and the ADP fragment, and the E3 region nucleotide sequence includes the nucleotide sequence of the mCD40L coding region.

2. The recombinant plasmid according to claim 1, characterized in that The nucleotide sequence of the mCD40L coding region is shown in SEQ ID No.

1.

3. The recombinant plasmid according to claim 2, characterized in that The nucleotide sequence of the E3 region is shown in SEQ ID No.

2.

4. The recombinant plasmid according to claim 3, characterized in that The nucleotide sequence of the recombinant plasmid is shown in SEQ ID No.

3.

5. A novel oncolytic adenoviral vector carrying membrane-bound CD40L, characterized in that: The oncolytic adenovirus vector is prepared using the recombinant plasmid according to any one of claims 1 to 4, and is a conditionally replicative adenovirus vector.

6. The novel oncolytic adenoviral vector carrying membrane-bound CD40L according to claim 5, characterized in that: Its nucleotide sequence is shown in SEQ ID No.

4.

7. A method for preparing a novel oncolytic adenoviral vector carrying membrane-bound CD40L as claimed in claim 5 or 6, characterized in that: The following steps are involved: S1, synthesizing a first nucleotide sequence, and preparing a first plasmid by homologous recombination of the first nucleotide sequence and an adenovirus 5 backbone plasmid; the first nucleotide sequence is an E3 region nucleotide sequence having a double deletion of a Gp19k fragment and an ADP fragment; S2, inserting the coding region nucleotide sequence of mCD40L into the first plasmid to obtain the recombinant plasmid; the insertion position of the coding region nucleotide sequence of mCD40L is the position where the Gp19k fragment is deleted, and the fibrin coding region of the recombinant plasmid has a deletion; S3, using the Ad5 / 35 chimeric backbone plasmid and the recombinant plasmid to prepare the novel oncolytic adenovirus vector carrying membrane-type CD40L through homologous recombination; S4. Perform enzyme digestion, transfection and purification on the novel oncolytic adenoviral vector carrying membrane-type CD40L.

8. The method for preparing the novel Ad5 / 35 chimeric oncolytic adenovirus vector according to claim 7, characterized in that: In the step S1, the position where the Gp19k fragment is missing and the position where the ADP is missing have specific restriction sites BstBI and ClaI, respectively.

9. Use of the novel oncolytic adenovirus vector carrying membrane-type CD40L as claimed in claim 5 or 6 in the preparation of a preparation for treating cancer, characterized in that: The cancer treatment preparation is a drug, a pharmaceutical raw material, a tumor therapeutic vaccine or a targeted inhibitor.

10. A preparation for treating cancer, characterized in that: The preparation comprises the recombinant plasmid according to any one of claims 1 to 3 and / or the novel Ad5 / 35 chimeric oncolytic adenovirus vector according to claim 5 or 6.

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