CN113969266B - Recombinant oncolytic adenovirus and application thereof - Google Patents

Abstract

The present invention provides a recombinant oncolytic adenovirus and its application. The recombinant oncolytic adenovirus is constructed based on type 5 adenovirus, and its genome contains a nucleotide sequence encoding a BiTE antibody or antibody fragment. The encoding BiTE antibody or antibody The nucleotide sequence of the fragment is located between the viral elements mCMV and MCS-3Flag. The recombinant oncolytic adenovirus of the present invention improves the targeting of T cells to tumor cells and avoids the adverse reactions caused by systemic administration of BiTE. Moreover, through the activation effect of BiTE on the immune microenvironment, the oncolytic adenovirus increases the effect on entities. tumor killing effect.

Description

A recombinant oncolytic adenovirus and its application

Technical field

The present invention relates to the field of biomedicine technology, and specifically relates to a recombinant oncolytic adenovirus and its application.

Background technique

The information disclosed in the Background of the Invention is intended to enhance an understanding of the general background of the invention, and the disclosure should not necessarily be considered an admission or in any way imply that the information is already known as the prior art to a person of ordinary skill in the art.

Ovarian cancer is the most lethal female reproductive system tumor. Survival rates for many cancers have improved greatly due to cutting-edge research and advances in screening, diagnosis, surgery and other treatments, but ovarian cancer survival rates have changed little in decades. The standard treatment plan for ovarian cancer is surgery and platinum-paclitaxel combined chemotherapy. Although certain progress has been made, the vast majority of patients still relapse, metastasize, and become drug-resistant quickly. Once this happens, most patients will face ineffective treatment. methods are available. Therefore, there is an urgent need to improve therapies for patients with ovarian cancer.

Tumor immunotherapy has become one of the most promising treatments in oncology. It mainly exerts anti-tumor effects by activating the immune system to induce tumor immune surveillance or reverse tumor immune escape. Studies have proven that ovarian cancer is an immune tumor, and the survival rate of ovarian cancer can be greatly improved by the presence of tumor-infiltrating lymphocytes (especially CD3+ T cells). At the same time, immune evasion mechanisms mediated by suppressive cells such as T regulatory cells are associated with poorer survival. Based on the above, patients with epithelial ovarian cancer may benefit from immunotherapy.

Oncolytic adenovirus (OAd) is a purposeful modification of the adenovirus gene structure, so that the virus can selectively replicate in specific tumor cells without breaking down the tumor cells, while at the same time having no lethality to surrounding normal cells. OAds can promote anti-tumor immune responses through multiple mechanisms. OAds induce a pro-inflammatory environment, which can recruit T cells and destroy the physical barriers to T cell infiltration, increasing T cell infiltration in tumors. OAds can stimulate the production of pro-inflammatory cytokines and the depletion of immunosuppressive cells in the tumor microenvironment. The pro-inflammatory environment generated by OAds infection can also convert immunosuppressive cell types, such as M2 macrophages, into a more anti-tumor phenotype. In addition, OAdss can also upregulate pathways involved in antigen processing and presentation, including increasing tumor-associated antigen (TAA) release and increasing the expression of major histocompatibility complex (MHC) class I and MHC class II on APC and tumor cells, thereby improving The ability of the immune system to recognize tumor cells.

Bispecific T-cell engagers (BiTE) are bispecific single-chain antibodies composed of two single-chain variable fragments (ScFv), one of which binds to tumor-associated antigens on cancer cells, and the other T cell surface marker CD3 can target cancer cells and T cells at the same time, physically bridging T cells to tumor cells to form a T cell-BiTE-tumor cell complex, inducing the formation of immune synapses, stimulating T cell activation, and killing tumors. cell. BiTE binds to T cell antigen-positive target cells, independent of HLA presentation, and can activate any T cell to bind to and destroy adjacent target cells. Furthermore, BiTE-mediated T cell activation can overcome limiting factors of tumor-associated immunosuppression, leading to activation and proliferation of exhausted tumor-specific T cells. Blinatumomab is a CD19BiTE that received rapid approval from the FDA in 2014. It is used to treat relapsed or refractory B-ALL and is significantly better than standard chemotherapy regimens. However, BiTE has a short serum half-life, requires long-term continuous intravenous administration, and is highly toxic. In addition, the application of BiTE in solid tumors has limited effects due to penetration problems in the tumor microenvironment and dose-limiting toxicity due to off-target effects.

MUC16 is a member of the mucin family (MUC) family and belongs to type I transmembrane mucins. MUC16 is the largest glycoprotein in the MUC family. Its gene is located on human chromosome 19p13.2 and is encoded by 179 kb of genomic DNA, encoding a total of 22,152 amino acids. The protein consists of two domains: a release domain (CA-125) and a retention domain (MUC-CD). The release domain is a large cleavage and release domain composed of multiple repeated sequences, which is released into the blood and becomes the serum marker of ovarian cancer CA-125; the retention domain includes residual non-repeated extracellular fragments, trans- The membrane region and cytoplasmic tail (18), are retained on the cell surface and are expressed only at low levels in normal tissues, including the uterus, fallopian tubes, ovaries, and the corneal surface of the eye, and thus serve as an attractive target. There are already methods to treat ovarian cancer using antibodies against MUC16. For example, Oregovomab is an anti-MUC16 antibody that has entered Phase III clinical trials.

Contents of the invention

In order to increase the efficacy of BiTE and reduce the adverse side effects caused by continuous systemic administration, while improving the targeting of oncolytic adenovirus and enhancing the killing effect of oncolytic adenovirus on solid tumors, the present invention provides a method that can express BiTE (especially It is a recombinant oncolytic adenovirus expressing MUC16-BiTE). The recombinant virus can simultaneously target T cell CD3 antigen and tumor cell antigen (MUC16) to achieve bridging between tumor cells and T cells. The BiTE encoded by the recombinant virus has Significantly enhanced cytotoxicity, and the ability to bypass or reverse the immunosuppressive microenvironment of tumors, showing a significant increase in the killing effect on solid tumors, especially ovarian cancer, and a greatly reduced action time. In vitro cell experiment results show that even for resistant ovarian cancer cells, the survival rate of cancer cells can be as low as less than 10% after the killing experiment is carried out for 24 hours.

Specifically, the present invention provides the following technical features, and the combination of one or more of the following technical features constitutes the technical solution of the present invention.

In a first aspect of the invention, the invention provides a recombinant oncolytic adenovirus, which is constructed based on adenovirus type 5 (Ad5) and whose genome contains a nucleotide sequence encoding a BiTE antibody or antibody fragment. , the nucleotide sequence encoding a BiTE antibody or antibody fragment is located between the viral elements mCMV and MCS-3Flag. For example, in some embodiments of the present invention, the structure of the recombinant oncolytic adenovirus of the present invention is shown in Figure 1.

In some embodiments of the invention, the BiTE targets T cell CD3 antigen and tumor antigen or tumor-associated antigen simultaneously.

In some embodiments of the invention, the tumor antigen or tumor-associated antigen includes mesothelin, folate receptor α (FRα), MUC-16, EGFR, HER2, CD133, NKG2D, MUCl, WTI, One or more of NY-ES0-1 and Survivin.

In a preferred embodiment of the present invention, the BiTE is MUC16-BiTE.

The MUC16-BiTE contains a human MUC16 sequence fragment and two single-chain antibody fragments (scFvs) sequences of CD3ε.

In some embodiments of the invention, the MUC16 sequence and two single-chain antibody fragments of CD3ε are linked with a DNA sequence encoding a G4S linker.

Specifically, the present invention provides a MUC16-BiTE with the following structure:

Signal peptide-VL(MUC16)-Linker1-VH(MUC16)-Linker2-VH(CD3)-Linker3-VL(CD3).

In some embodiments of the present invention, the end of the above-mentioned MUC16-BiTE structure may contain an affinity tag, preferably 6×His tag.

In some embodiments of the present invention, the end of the above-mentioned MUC16-BiTE structure may contain an affinity tag, preferably 6×His tag. In embodiments of the invention, 6×His tag affinity tags can be used to purify viruses. It should be noted that it is optional and can be removed, for example, from the final product. Of course, technicians can also choose other affinity tags and can also be removed from the final product.

In the above MUC16-BiTE structure, the nucleotide sequence of VL (MUC16) is shown in SEQ ID NO: 4, the nucleotide sequence of VH (MUC16) is shown in SEQ ID NO: 5, and the nucleotide sequence of VH (CD3) The nucleotide sequence of is shown in SEQ ID NO: 6, and the nucleotide sequence of VL (CD3) is shown in SEQ ID NO: 7.

In some embodiments of the invention, Linker1, Linker2 or Linker3 is a group of G4S linkers or a series of multiple groups of G4S linkers; preferably, the nucleotide sequence of Linker1 is as shown in SEQ ID NO: 8, and the nucleotide sequence of Linker2 is as shown in SEQ ID NO: 8. The sequence is shown in SEQ ID NO: 9, and the nucleotide sequence of Linker3 is shown in SEQ ID NO: 10.

In some preferred embodiments of the present invention, the nucleotide sequence of MUC16-BiTE is shown in SEQ ID NO: 1, and the amino acid sequence of MUC16-BiTE is shown in SEQ ID NO: 2.

In some embodiments of the invention, the type 5 adenovirus of the invention is selected from the following viruses:

1) Type 5 adenovirus with E1B 55KD gene deletion and gene silencing or E1B 55KD gene mutation not expressing; for example, in the adenovirus' own genome, the E1B 55KD gene is deleted through molecular biology methods, or the start codon is mutated, By preventing the expression of the E1B 55KD gene, the oncolytic adenovirus constructed through this method has the ability to replicate and amplify in P53-deficient tumor cells, thereby exerting the oncolytic effect of the virus.

2) Containing one or more expression control sequences that regulate the initiation of the E1 region; for example, the expression control sequence includes at least one specific promoter, and the specific promoter is used to activate the E1 region of the adenovirus to relatively control the replication of the virus. , improve the safety of oncolytic viruses, the promoters include one or more of tumor-specific antigen, telomerase and survivin promoters; and under the control of these promoters, the virus maintains the ability to replicate and can Express BiTE and/or other proteins.

3) The capsid protein has been modified for tropism. Based on 2), this type of virus can modify the adenovirus capsid protein to change the tropism of the virus, so that it can specifically target tumor cells.

In a second aspect of the present invention, the present invention provides a pharmaceutical composition or pharmaceutical preparation comprising the recombinant oncolytic adenovirus described in the above first aspect.

In some embodiments of the present invention, the pharmaceutical composition or pharmaceutical preparation of the present invention contains at least one recombinant oncolytic adenovirus of the present invention, and, based on this, the pharmaceutical composition or pharmaceutical preparation It may also contain at least one pharmaceutical carrier or pharmaceutically acceptable excipient, or other therapeutically effective agent. Among them, suitable pharmaceutical excipients can be of the types known in the art, such as solvents, buffers, diluents, etc., for example, they can be found in the "Handbook of Parmaceutical Excipients" compiled by the author Paul JSheskey et al. recorded. Those skilled in the art can make selections as needed. The pharmaceutical composition or pharmaceutical preparation may be in solid, semi-solid or liquid form, which may be prepared according to any conventional method known in the art.

In a third aspect of the present invention, the present invention provides the use of the recombinant oncolytic adenovirus described in the first aspect or the pharmaceutical composition described in the second aspect in the preparation of anti-tumor drugs. Wherein, the tumor is particularly ovarian cancer, and the ovarian cancer includes resistant ovarian cancer.

In the embodiment of the present invention, the recombinant oncolytic adenovirus OAd-MUC16-BiTE of the present invention has a good killing effect on ovarian cancer cells, and is effective against a variety of ovarian cancer cells including OVCAR3, CAOV3, A2780, HEY, and SKOV3. It has good oncolytic efficacy, and can significantly improve the killing ability under the action of T cells. Among them, the killing effect is particularly significant for ovarian cancer SKOV3 cells that are resistant to viruses, as shown in the cell killing experiment for 24 hours. The target cell survival rate can be as low as less than 10%. In vitro, it also showed better anti-tumor effects based on the ovarian cancer PDX model. Compared with simply administering MUC16-BiTE, or combining MUC16-BiTE with an immunosuppressive checkpoint agent (such as PD-1), the recombinant oncolytic adenovirus OAd-MUC16-BiTE of the present invention shows better Excellent effect, because this therapy also includes the direct lysis of tumor cells by the virus and the indirect anti-tumor immune effect of the host. Multiple treatment methods work synergistically to achieve better killing effects.

In a fourth aspect of the present invention, the present invention provides a method for treating solid tumors, which includes administering to a subject a therapeutically effective amount of the recombinant oncolytic adenovirus described in the first aspect or comprising the present invention. Compositions or pharmaceutical preparations of recombinant oncolytic adenoviruses. The solid tumor refers in particular to ovarian cancer.

Wherein, the "subject" refers to an animal that has been the subject of treatment, observation or experiment, preferably a mammal, and most preferably a human. The "therapeutically effective amount" refers to the amount of active compounds or agents, including the compounds of the present invention, that can cause the biological or biological effects of tissue systems, animals or humans pursued by researchers, veterinarians, doctors or other medical personnel. Medical response, which includes alleviation or partial alleviation of symptoms of the disease, syndrome, condition or disorder being treated.

The compounds and pharmaceutical compositions of the present invention can be used clinically in mammals, including humans and animals. The optimal dosage will depend on the specific treatment. Usually, you start with a small dose and gradually increase the dose until you find the most suitable dose.

In one embodiment of the present invention, the recombinant oncolytic adenovirus described in the first aspect of the present invention can be combined with checkpoint inhibitors such as PD-1 or PDL1 inhibitors or CAR-T or chemotherapy drugs or anti-vascular inhibitors administered in combination.

Compared with the prior art, the advantage of the present invention is that the present invention provides a recombinant oncolytic adenovirus that can express and secrete a bispecific antibody, especially MUC16-BiTE, which can bridge tumor cells. and T cells, inducing immune synapse formation and stimulating T cell activation; allowing the recombinant oncolytic adenovirus to dissolve tumor cells, increase T cell infiltration, generate a pro-inflammatory environment, promote immune activation of solid tumors and overcome tumor immunosuppression . In particular, the OAd-MUC16-BiTE obtained after recombination can simultaneously target T cell CD3 antigen and tumor cell MUC16 antigen, which not only enhances the targeting of oncolytic adenovirus, but also avoids the adverse reactions caused by systemic administration of BiTE. , and through the activation of the immune microenvironment by BiTE, the killing effect of oncolytic adenovirus on solid tumors is increased, and the action time is greatly shortened, such as for a variety of ovarian cancer cells and resistant tumor cells. It produces enhanced killing effect and significantly shortens the action time.

Description of the drawings

The description and drawings that constitute a part of the present invention are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. Below, the embodiments of the present invention are described in detail with reference to the accompanying drawings, wherein:

Figure 1: Schematic structural diagram of OAd-MUC16-BiTE.

Figure 2: Pictures of virus titer detection, A is the 100x bright field field of view, B is the 100x fluorescence field of view, and C is the virus titer detection data.

Figure 3: Detection results of OAd-MUC16-BiTE expressing MUC16-BiTE.

Figure 4: Results of the killing effect of OAd-MUC16-BiTE on various ovarian cancer cells in Example 4.

Figure 5: Results of OAd-MUC16-BiTE-mediated T cell killing of ovarian cancer cells SKOV3 in Example 4.

Figure 6: Results of the in vivo tumor killing experiment in Example 5.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as familiar to one skilled in the art. The reagents or raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in accordance with conventional methods in the art or in accordance with product instructions. In addition, any methods and materials similar or equivalent to those described can be used in the method of the present invention. The preferred implementation methods and materials described in this article are for demonstration purposes only.

Example 1 Construction of MUC16-BiTE

A BiTE targeting MUC16 (MUC16-BiTE) was generated by ligating DNA sequences encoding two single-chain antibody fragments (scFvs) that recognize human MUC16 and CD3ε with a DNA sequence encoding a G ₄ S linker, in which anti-MUC16 scFV Targeting the human MUC16 cytoplasmic retention domain (MUC-CD). A human IgK signal peptide, an immunoglobulin signal peptide used for mammalian secretion, is added to the N-terminal and a 6×His tag is added to the C-terminal for detection.

The general structural formula of MUC16-BiTE is:

Signal peptide-VL(MUC16)-Linker1-VH(MUC16)-Linker2-VH(CD3)-Linker3-VL(CD3)-His.

The gene CDS region sequence and amino acid sequence of MUC16-BiTE are as follows. Among the following amino acid or nucleotide sequences, the signal peptide sequence is represented by a dotted line underline, the VL (MUC16) sequence is represented by a single straight underline, and the VH (MUC16) sequence is represented by a dotted line underline. ) sequence is represented by double straight underline, VH(CD3) sequence is represented by single wavy underline, and VL(CD3) sequence is represented by double wavy underline.

The sequence of the CDS region of the MUC16-BiTE gene (SEQ ID NO.1) is:

The amino acid sequence information of MUC16-BiTE is (SEQ ID NO.2):

Example 2 Construction of recombinant adenovirus OAd-MUC16-BiTE

1. Adenovirus packaging

1.1 Construction of plasmid

The above DNA sequence encoding MUC16-BiTE was synthesized and inserted into the shuttle plasmid using Gibson assembly technology. Confirm plasmid construction was correct by DNA sequencing.

1.2 Plasmid transfection process

1) 24 hours before transfection, digest HEK293 cells in the logarithmic growth phase with 0.25% trypsin, adjust the cell density to 30% to 40% with DMEM medium containing 10% FBS, and re-inoculate into cell culture bottles. 37 ℃, 5% _CO2 incubator. It can be used for transfection in about 24 hours when the cell density reaches 50% to 60%. Cell status is critical for virus packaging, so good cell status and a low number of passages are needed.

2) Change the cell culture medium to serum-free DMEM medium 2 hours before transfection.

3) Add the prepared DNA solution (5 μg of the overexpression shuttle plasmid pHBAd shuttle plasmid carrying foreign genes and 5 μg of the helper plasmid pBHG loxΔE1,3Cre) into a sterilized centrifuge tube and mix evenly with DMEM, adjust the total volume to 50 μl, and add Incubate at room temperature for 5 minutes.

4) Gently shake Lipofectamine 2000 reagent, mix 10 μl Lipofectamine 2000 reagent with 50 μl DMEM in another tube, and incubate at room temperature for 5 minutes.

5) Mix the diluted DNA with the diluted Lipofectamine 2000, and mix gently without shaking.

6) After mixing, incubate at room temperature for 20 minutes to form a transfection complex of DNA and Lipofectamine 2000 dilution.

7) Transfer the mixture of DNA and Lipofectamine 2000 to the culture medium of HEK293 cells, mix well, and culture in a 37°C, 5% CO ₂ cell culture incubator.

8) After culturing for 6 to 8 hours, pour out the culture medium containing the transfection mixture. Add 2 ml of PBS solution to each bottle of cells. Gently shake the culture bottle left and right to wash the remaining transfection mixture, and then pour it out.

9) Add 5 ml of cell culture medium containing 10% serum to each bottle of cells, and continue culturing in a 37°C, 5% _CO2 incubator.

10) Observe the cell growth status after transfection every day. If the cell culture medium turns yellow obviously, add an appropriate amount of fresh whole culture medium as appropriate.

1.2 Microscopic observation after transfection

About 10 to 15 days after transfection, HEK 293 cells began to fall off, and some cells developed cytopathic effect (CPE).

1.3 Harvest of recombinant adenovirus

When most of the cells show typical CPE and 50% of the cells have detached, collect the cells by low-speed centrifugation and resuspend in 2 ml of DMEM. Repeat freeze-thaw and shake at -70℃/37℃ for 3 times, and centrifuge at 7000g for 5 minutes at 4℃. , collect the virus supernatant and store it at -70°C.

The structure of the constructed OAd-MUC16-BiTE is shown in Figure 1.

2. Amplification of Adenovirus

2.1 First round of amplification

HEK293 cells that were growing well in one T25 cell culture flask were diluted 4-fold and transferred to another T25 cell culture flask. They were cultured in DMEM culture medium containing 10% FBS at 37°C and 5% CO2 (the following expansion This condition was used to culture cells in Zengzhong). When the cells reach 60% confluence, discard the old culture medium, add 2 mL of crude extract harvested after successful recombination of replication-deficient adenovirus to the culture flask, place the culture flask in a cell culture incubator and incubate for 90 minutes, and finally add 2 mL of crude extract to the culture flask. Add 3 mL of complete culture solution to the bottle and continue culturing. When most cells show typical CPE and 50% of the cells are detached, collect the cells by low-speed centrifugation and resuspend them in 2 ml DMEM. Repeat freezing and thawing at -70°C/37°C and shaking three times, and centrifuge at 4°C and 7000g. 5 min, collect the virus supernatant and store it at -70°C.

2.2 Second round of amplification

Transfer all the HEK293 cells that are growing well in a T25 cell culture flask into a T75 cell culture flask and continue culturing with complete medium. When the cells reach 90% confluence, discard the old culture medium, add 2 ml of the virus liquid obtained in the first round of amplification to the culture bottle, place the culture bottle in the cell culture incubator and incubate for 90 minutes, and finally add additional virus to the culture bottle. Add 10 mL of complete culture medium and continue culturing. When most cells show typical CPE and 50% of the cells are detached, collect the cells by low-speed centrifugation and resuspend them in 10 ml DMEM. Repeat freezing and thawing at -70°C/37°C and shaking three times, and centrifuge at 4°C and 7000g. 5 min, collect the virus supernatant and store it at -70°C.

3. Adenovirus titer detection

3.1 Adenovirus titer determination-end point dilution method

1) 24 hours before the experiment, add 100 μl of HEK293 cell suspension to each well of the 96-well plate, containing approximately 1x10 ³ cells;

2) Prepare 12 sterile Ep tubes, add 990 μl of complete culture medium to the first Ep tube, and add 900 μl of complete culture medium to each of the remaining 11 tubes.

3) Dilution of the virus liquid to be tested: Add 10 μl of adenovirus stock solution into a 990 μl Ep tube to make a 1:100 dilution (10 ^-2 ); then use this as a starting point, and add 100 μl of the dilution solution into a 900 μl Ep tube. Dilute 1:10 (10 ^-3 ) until diluted to 10 ^-13 .

4) Take out the 96-well plate from the incubator and confirm under a microscope that the cells in each well are growing well. Aspirate away the old culture medium, and then add 10 ^-13 to 10 ^-6 diluted virus solutions into the 96-well plate in sequence. Each dilution occupies one row. Add 90 μl of virus diluent to each well in the 1-10 wells of each row. Add 90 μl of virus-free complete medium to wells 11-12 of each row as a control.

5) Place the 96-well plate in a 37°C, 5% _CO2 cell culture incubator to continue culturing.

6) Observe the cytopathic phenomenon after 10 days, count the CPE wells, calculate the positive rate of each row, and calculate the virus titer (Spearman-Karber Method).

Note: As long as there is a small spot or some cells showing CPE, it is considered positive.

4.2 Calculation method of virus titer

Virus titer=10(x+0.8)(PFU/ml)

x=Sum of CPE positive rate under sequential dilutions from 10 ^-1 to 10 ^-13

*Conditions for using the formula:

a. The negative control has no CPE and growth inhibition;

b. CPE is present in all wells where minimum dilution concentration of virus crude extract is added.

The detection of virus titer is shown in Figure 2. The virus titer is calculated, X=9.2, and the virus titer is 1.0E+10 (PFU/ml).

Example 3 Detection of OAd-MUC16-BiTE Expression MUC16-BiTE

Use MOI = 100OAd (MOI is the multiplicity of infection) and OAd-MUC16-BiTE to infect human ovarian cancer cells HEY respectively. After 24 hours, extract the proteins of HEY empty cells and HEY cells infected with the virus, and use anti-His antibodies for western blot electrophoresis detection. :

Primary antibody: Purified anti-His Tag Antibody (biolegend#362601) dilution ratio 1:1000; the molecular weight of the target protein is approximately 58kDa.

Primary antibody: Anti-Beta-Actin antibody (Proteintech 66009-1-Ig) dilution ratio 1:2000; β-actin molecular weight is 42kDa.

Secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (abcam ab6721) dilution ratio 1:20000.

Result analysis: The results are shown in Figure 3. From the results, it can be seen that HEY cells infected with adenovirus OAd-MUC16-BiTE have an obvious target band at the 58kDa position, while control HEY empty cells and OAd-infected HEY cells have no bands at the corresponding positions. , indicating that adenovirus OAd-MUC16-BiTE can infect cells and highly express MUC16-BiTE in cells.

Example 4 In vitro tumor killing experiment

1. Use CCK8 method to detect the killing ability of ovarian cancer cells

1) 5×10 ³ ovarian cancer cell lines (OVCAR3, CAOV3, A2780, HEY, SKOV3) were spread on a 96-well plate.

2) Add the specified amount of OAd or OAd-MUC16-BiTE, and the one without adding virus serves as the negative control group.

3) At 24h, 48h, 72h, and 96h after incubation, add 10 μl CCK-8 (MCE) to each well, place it at 37°C for 2 hours, and then use a microplate reader (BIO-RAD) to measure the absorbance at 450 nm.

4) Calculate cell survival rate, the formula is as follows:

Cell survival rate = (OD value of experimental group - OD value of blank control group) / (OD value of negative control group - OD value of blank control group).

The killing effect on various ovarian cancer cell lines is shown in Figure 5.

2. Use Luciferase luminescence intensity method to detect the killing ability of target cells (ovarian cancer cell SKOV3)

Grouping situation:

Experimental groups: OAd+T cell group, OAd-MUC16-BiTE+T cell group;

Blank control group: virus-free and effector T cell group;

Negative control group: virus-free group;

1) Plate the target cells SKOV3-LUC or SKOV3-MUC16 stably expressing Luciferase (luciferase) at 1×10 ⁴ in a 96-well plate;

2) Add effector T cells to target cells at a ratio of 5:1;

3) Add the virus into the well according to MOI=10, and add the corresponding volume of PBS to the single T cell group or negative control group;

4) After 24 hours, detect the luminescence intensity of Luciferase on the target cells;

The specific operation was carried out according to the instructions of Bright-Lumi ^TM Firefly Luciferase Reporter Gene Detection Kit (Beyotime, RG051M).

5) Tumor inhibition rate = 1-(Lumen value of the experimental group-Lumen value of the blank control group)/(Lumen value of the negative control group-Lumen value of the blank control group)×100%.

The experimental results are shown in Figure 5.

The results of the in vitro killing experiment are shown in Figures 4 and 5.

It can be seen from the experimental results in Figure 4 that in most cases, the oncolytic adenovirus OAd-MUC16-BiTE carrying the bispecific antibody MUC16-BiTE of the present invention has no significant difference in lytic activity against ovarian cancer cells compared with the parent virus, and both Exhibits time dependence. Therefore, insertion of MUC16-BiTE has little effect on viral replication or oncolytic activity. It is worth noting that OAd-MUC16-BiTE had oncolytic efficacy in all cell lines tested, but ovarian cancer SKOV3 cells were partially resistant and killed slower, and increasing the dose of virus did not offset this resistance. sex.

It can be seen from the results in Figure 5 that the killing ability of target cells can be significantly improved in the presence of T cells, especially SKOV3 cells that are resistant to viruses. And the effect of OAd-MUC16-BiTE on T cell killing is antigen-specific. The cell killing experiment was carried out for 24 hours, and the target cell survival rate had dropped to less than 10%.

Based on the above, OAd-MUC16-BiTE is antigen-specific in killing target cells, can significantly improve the killing ability of ovarian cancer cells in the presence of T cells, and greatly reduce the action time.

Example 5 In vivo tumor killing experiment

1) An ovarian cancer PDX model was constructed.

2) Homogenize the PDX P2 generation tumors and inoculate them subcutaneously into mice.

3) One week later, the mice were randomly divided into 5 groups (5 mice in each group), and 5 × 10 ⁹ PFUOAd or OAd-MUC16-BiTE or PBS were injected intratumorally on days 0 and 3, and each round of virus injection One day later, mice were injected with 1×10 ⁷ Tcells or an equal volume of PBS through the tail vein.

4) At the designated analysis time, all mice were euthanized by intraperitoneal injection of sodium pentobarbital (200 mg/kg), and then the tumors were excised.

The in vivo tumor killing results are shown in Figure 6. The PBS group showed the fastest tumor growth, and OAd-MUC16-BiTE showed similar anti-tumor efficacy to the parent virus in the absence of T cells. The administration of T cells significantly enhanced the anti-tumor efficacy of OAd-MUC16-BiTE, and the effect was obvious compared with the OAd treatment group administered T cells.

The above examples show that the recombinant oncolytic adenovirus OAd-MUC16-BiTE of the present invention can target tumor cells to express and secrete the bispecific antibody MUC16-BiTE, activate the immune microenvironment through BiTE, and stimulate tumor-infiltrating T cells at the tumor site without Systemic toxicity can significantly increase the killing effect of oncolytic adenovirus on ovarian cancer.

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the foregoing embodiments. Modify the recorded technical solutions, or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Shandong University Qilu Hospital

<120> A recombinant oncolytic adenovirus and its application

<130> 202124120

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 1572

<212> DNA

<213> Artificial sequence

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gtgaagcctg gagggtccct caaagtctcc tgtgcagcct ctggattcac tttcagtagc 540

tatgccatgt cctgggttcg cctgagtccg gagatgaggc tggagtgggt cgcaaccatt 600

agcagtgctg gtggttacat cttctattct gacagtgtgc agggacgatt caccatttcc 660

agagacaatg ccaagaacac cctgcacctg caaatgggca gtctgaggtc tggggacacg 720

gccatgtatt actgtgcaag gcagggattt ggtaactacg gtgattacta tgctatggac 780

tactggggcc aagggaccac ggtcaccgtc tcctcaggtg gcgggggatc tgatatcaaa 840

ctgcagcagt caggggctga actggcaaga cctggggcct cagtgaagat gtcctgcaag 900

acttctggct acacctttac taggtacacg atgcactggg taaaacagag gcctggacag 960

ggtctggaat ggattggata cattaatcct agccgtggtt atactaatta caatcagaag 1020

ttcaaggaca aggccacatt gactacagac aaatcctcca gcacagccta catgcaactg 1080

agcagcctga catctgagga ctctgcagtc tattactgtg caagatatta tgatgatcat 1140

tactgccttg actactgggg ccaaggcacc actctcacag tctcctcagg tggcggggga 1200

agcggcggcg gtggatccgg tggagggggc agtgacattc agctgaccca gtctccagca 1260

atcatgtctg catctccagg ggagaaggtc accatgacct gcagagccag ttcaagtgta 1320

agttacatga actggtacca gcagaagtca ggcacctccc ccaaaagatg gatttatgac 1380

acatccaaag tggcttctgg agtcccttat cgcttcagtg gcagtgggtc tgggacctca 1440

tactctctca caatcagcag catggaggct gaagatgctg ccacttatta ctgccaacag 1500

tggagtagta acccgctcac gttcggtgct gggaccaagc tggagctgaa acatcatcac 1560

catcaccatt ga 1572

<210> 2

<211> 523

<212> PRT

<213> Artificial sequence

<400> 2

Met Asp Met Arg Val Leu Ala Gln Leu Leu Gly Leu Leu Leu Leu Cys

1 5 10 15

Phe Pro Gly Ala Arg Cys Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ala Val Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser

35 40 45

Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Gln Leu Ala Trp Tyr

50 55 60

Gln Gln Lys Pro Gly Gln Ser Pro Glu Leu Leu Ile Tyr Trp Ala Ser

65 70 75 80

Thr Arg Gln Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly

85 90 95

Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala

100 105 110

Val Tyr Tyr Cys Gln Gln Ser Tyr Asn Leu Leu Thr Phe Gly Pro Gly

115 120 125

Thr Lys Leu Glu Val Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Val Lys Leu Gln Glu Ser Gly Gly Gly Phe

145 150 155 160

Val Lys Pro Gly Gly Ser Leu Lys Val Ser Cys Ala Ala Ser Gly Phe

165 170 175

Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Leu Ser Pro Glu Met

180 185 190

Arg Leu Glu Trp Val Ala Thr Ile Ser Ser Ala Gly Gly Tyr Ile Phe

195 200 205

Tyr Ser Asp Ser Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

210 215 220

Lys Asn Thr Leu His Leu Gln Met Gly Ser Leu Arg Ser Gly Asp Thr

225 230 235 240

Ala Met Tyr Tyr Cys Ala Arg Gln Gly Phe Gly Asn Tyr Gly Asp Tyr

245 250 255

Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

260 265 270

Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu

275 280 285

Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr

290 295 300

Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln

305 310 315 320

Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn

325 330 335

Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser

340 345 350

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

355 360 365

Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp

370 375 380

Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly

385 390 395 400

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr

405 410 415

Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met

420 425 430

Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln

435 440 445

Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val

450 455 460

Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser

465 470 475 480

Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr

485 490 495

Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr

500 505 510

Lys Leu Glu Leu Lys His His His His His

515 520

<210> 3

<211> 66

<212> DNA

<213> Artificial sequence

<400> 3

atggacatgc gggtgctggc acagctgctg ggcctgctgc tgctgtgctt cccaggagcc 60

agatgt 66

<210> 4

<211> 339

<212> DNA

<213> Artificial sequence

<400> 4

gacattgagc tcacccagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 60

atgagctgca aatccagtca gagtctgctc aacagtagaa cccgaaagaa ccagttggct 120

tggtaccagc aaaaaccagg acagtctcct gaactgctga tctactgggc atccactagg 180

caatctggag tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240

atcagcagtg tgcaggctga agacctggca gtttattact gccagcaatc ttataatcta 300

ctcacgttcg gtcctgggac caagctggag gtcaaacgg 339

<210> 5

<211> 366

<212> DNA

<213> Artificial sequence

<400> 5

gtgaagctgc aggagtcagg gggaggcttc gtgaagcctg gagggtccct caaagtctcc 60

tgtgcagcct ctggattcac tttcagtagc tatgccatgt cctgggttcg cctgagtccg 120

gagatgaggc tggagtgggt cgcaaccatt agcagtgctg gtggttacat cttctattct 180

gacagtgtgc agggacgatt caccatttcc agagacaatg ccaagaacac cctgcacctg 240

caaatgggca gtctgaggtc tggggacacg gccatgtatt actgtgcaag gcagggattt 300

ggtaactacg gtgattacta tgctatggac tactggggcc aagggaccac ggtcaccgtc 360

tcctca 366

<210> 6

<211> 357

<212> DNA

<213> Artificial sequence

<400> 6

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttat aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt ctcctca 357

<210> 7

<211> 318

<212> DNA

<213> Artificial sequence

<400> 7

gacattcagc tgacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60

atgacctgca gagccagttc aagtgtaagt tacatgaact ggtaccagca gaagtcaggc 120

acctccccca aaagatggat ttatgacaca tccaaagtgg cttctggagt cccttatcgc 180

ttcagtggca gtgggtctgg gacctcatac tctctcacaa tcagcagcat ggaggctgaa 240

gatgctgcca cttattactg ccaacagtgg agtagtaacc cgctcacgtt cggtgctggg 300

accaagctgg agctgaaa 318

<210> 8

<211> 45

<212> DNA

<213> Artificial sequence

<400> 8

ggtggaggcg gcagtggcgg aggtggggagc ggagggggcg gttcc 45

<210> 9

<211> 15

<212> DNA

<213> Artificial sequence

<400> 9

ggtggcgggggatct 15

<210> 10

<211> 45

<212> DNA

<213> Artificial sequence

<400> 10

ggtggcgggg gaagcggcgg cggtggatcc ggtggagggg gcagt 45

Claims (9)

1. A recombinant oncolytic adenovirus, characterized in that the virus is constructed based on type 5 adenovirus, and its genome contains a nucleotide sequence encoding a BiTE antibody, and the nucleotide sequence encoding a BiTE antibody is located in an element of the virus between mCMV and MCS-3Flag; The BiTE is MUC16-BiTE; The nucleotide sequence of MUC16-BiTE is shown in SEQ ID NO: 1. 2. The recombinant oncolytic adenovirus according to claim 1, wherein the BiTE targets T cell CD3 antigen and tumor antigen or tumor-associated antigen simultaneously. 3. The recombinant oncolytic adenovirus according to claim 2, wherein the tumor antigen or tumor-associated antigen includes mesothelin, folate receptor alpha, MUC-16, EGFR, HER2, CD133, NKG2D, One or more of MUC1, WTI, NY-ES0-1, and Survivin. 4. The recombinant oncolytic adenovirus according to claim 1, wherein the amino acid sequence of MUC16-BiTE is shown in SEQ ID NO: 2. 5. The recombinant oncolytic adenovirus according to any one of claims 1 to 4, characterized in that the type 5 adenovirus is selected from the following viruses: 1) Type 5 adenovirus with E1B 55KD gene deletion, gene silencing or E1B 55KD gene mutation with no expression; 2) Contains one or more expression control sequences that regulate the initiation of the E1 region; 3) Its capsid protein has been modified with tropism. 6. The recombinant oncolytic adenovirus according to claim 5, wherein the expression control sequence includes at least one specific promoter, and the specific promoter includes tumor-specific antigen, telomerase and survival rate. one or more of the promoters. 7. A pharmaceutical composition or pharmaceutical preparation comprising the recombinant oncolytic adenovirus according to any one of claims 1 to 6. 8. Use of the recombinant oncolytic adenovirus according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7 in the preparation of anti-tumor drugs. 9. The application according to claim 8, characterized in that the tumor is ovarian cancer.