CN113969266B

CN113969266B - Recombinant oncolytic adenovirus and application thereof

Info

Publication number: CN113969266B
Application number: CN202111249158.9A
Authority: CN
Inventors: 孔北华; 王秋曼; 苑存忠; 吴焕; 宋坤; 张青
Original assignee: Qilu Hospital of Shandong University
Current assignee: Qilu Hospital of Shandong University
Priority date: 2021-10-26
Filing date: 2021-10-26
Publication date: 2023-10-13
Anticipated expiration: 2041-10-26
Also published as: CN113969266A

Abstract

The invention provides a recombinant oncolytic adenovirus and application thereof, wherein the recombinant oncolytic adenovirus is constructed based on a 5-type adenovirus, a genome of the recombinant oncolytic adenovirus comprises a nucleotide sequence for encoding a BiTE antibody or an antibody fragment, and the nucleotide sequence for encoding the BiTE antibody or the antibody fragment is positioned between an element mCMV and an MCS-3Flag of the virus. The recombinant oncolytic adenovirus improves the targeting of T cells to tumor cells, avoids adverse reaction caused by systemic administration of BiTE, and increases the killing effect of oncolytic adenovirus on solid tumors through the activation of BiTE on immune microenvironment.

Description

Recombinant oncolytic adenovirus and application thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to a recombinant oncolytic adenovirus and application thereof.

Background

The information disclosed in the background of the invention is intended to enhance an understanding of the general background of the invention, and this disclosure should not necessarily be taken as an acknowledgement or any form of suggestion that this information has become known to a person of ordinary skill in the art.

Ovarian cancer is the most lethal tumor of the female reproductive system. Due to the advances in therapeutic methods such as screening, diagnosis, surgery, etc., the survival rate of many cancers has been greatly improved, but the survival rate of ovarian cancer has not changed much for decades. The standard treatment regimen for ovarian cancer is surgery and platinum-paclitaxel combination chemotherapy, although some progress has been made, most patients still relapse, metastasize, and resist drugs very quickly, and once this occurs, most patients face no effective method available. Thus, there is an urgent need to improve therapies for ovarian cancer patients.

Tumor immunotherapy has become one of the most promising therapeutic approaches in oncology, and it exerts an anti-tumor effect mainly by activating the immune system to induce tumor immune monitoring or reverse tumor immune escape. Research has shown that ovarian cancer is an immune tumor, and the survival rate of ovarian cancer can be greatly improved due to the existence 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 poor survival. Based on the above, epithelial ovarian cancer patients may benefit from immunotherapy.

Oncolytic adenoviruses (Oncolytic adenovirus, OAd) are purposefully engineered for adenovirus gene structure to enable the virus to selectively replicate in specific tumor cells without disrupting tumor cells and while having no killing power to surrounding normal cells. OAds can promote anti-tumor immune responses through a variety of mechanisms. OAds induce a pro-inflammatory environment that recruits T cells and breaks the physical barrier of T cell infiltration, increasing T cell infiltration of tumors. OAds can stimulate the production of pro-inflammatory cytokines and the consumption of immunosuppressive cells in the tumor microenvironment. The proinflammatory environment created by OAds infection can also convert immunosuppressive cell types (e.g., M2 macrophages) to a more antitumor phenotype. In addition, OAdss can up-regulate pathways involved in antigen processing and presentation, including increasing Tumor Associated Antigen (TAA) release, increasing expression of Major Histocompatibility Complex (MHC) class I and MHC class II on APC and tumor cells, and thereby increasing the recognition of tumor cells by the immune system.

Bispecific T cell engagers (bites) are Bispecific single chain antibodies consisting of two single chain variable fragments (ScFv), one of which binds to a tumor-associated antigen on a cancer cell and the other of which binds to the T cell surface marker CD3, which can simultaneously target cancer cells and T cells, physically bridge the T cells to form a T cell-BiTE-tumor cell complex, induce immune synapse formation, stimulate T cell activation, and kill tumor cells. BiTE binds to T cell antigen positive target cells, presents independently of HLA, and can activate any T cell to bind to and destroy adjacent target cells. In addition, biTE-mediated T cell activation can overcome the limiting factors of tumor-associated immunosuppression, resulting in the activation and proliferation of depleted tumor-specific T cells. The Blina ememab (Blina) was a CD19 BiTE that was rapidly approved by the FDA in 2014 for the treatment of recurrent or refractory B-ALL, significantly better than standard chemotherapy regimens. However, biTE serum has a short 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 permeation problems of tumor microenvironments, dose limiting toxicity due to off-target effects, and the like.

MUC16 is one of the members of the mucin family (MUC) and belongs to the type I transmembrane mucin. MUC16 is the largest glycoprotein in the MUC family, whose gene is located on human chromosome 19p13.2, encoded by 179kb genomic DNA, and encoded 22152 amino acids in total. The protein consists of two domains: a release domain (CA-125) and a retention domain (MUC-CD). Wherein the release domain is a large cleavage and release domain consisting of multiple repeats released into the blood as a serum marker CA-125 for ovarian cancer; the retention domains, including residual non-repetitive extracellular fragments, transmembrane regions and cytoplasmic tails (18), remain on the cell surface and are only expressed at low levels in normal tissues, including the corneal surface of the uterus, fallopian tubes, ovaries and eyes, and thus can be an attractive target. There are currently methods of treating ovarian cancer using antibodies directed against MUC16, such as the ago Fu Shan anti (oregavisab) is an anti-MUC 16 antibody that has now entered phase III clinical settings.

Disclosure of Invention

In order to increase the efficacy of the BiTE and reduce adverse side effects caused by continuous systemic administration, improve the targeting of the oncolytic adenovirus and promote the killing effect of the oncolytic adenovirus on solid tumors, the invention provides a recombinant oncolytic adenovirus capable of expressing the BiTE (especially expressing MUC 16-BiTE), the recombinant adenovirus can simultaneously target a T cell CD3 antigen and a tumor cell antigen (MUC 16) to realize bridging of tumor cells and T cells, the BiTE coded by the recombinant adenovirus has obviously enhanced cytotoxicity, can bypass or reverse the immunosuppressive microenvironment of the tumor, is shown to obviously promote the killing effect on the solid tumors, especially ovarian cancer, and greatly reduces the action time. The results of in vitro cell experiments show that even for ovarian cancer cells with resistance, the survival rate of cancer cells can be as low as 10% or less after the killing experiment is carried out for 24 hours.

Specifically, the present invention provides the following technical features, and one or more of the following technical features are combined to form the technical scheme of the present invention.

In a first aspect of the invention, the invention provides a recombinant oncolytic adenovirus constructed on the basis of adenovirus type 5 (adenoviruses 5, ad 5) comprising in its genome a nucleotide sequence encoding a BiTE antibody or antibody fragment located between the elements mCMV and MCS-3Flag of the virus. For example, in some embodiments of the invention, the structure of the recombinant oncolytic adenoviruses of the invention is shown in FIG. 1.

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

In some embodiments of the invention, the tumor antigen or tumor-associated antigen comprises one or more of Mesothelin (Mesothelin), folate receptor alpha (FR alpha), MUC-16, EGFR, HER2, CD133, NKG2D, MUC1, WTI, NY-ES0-1, survivin.

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

The MUC16-BiTE comprises a human MUC16 sequence fragment and two single-chain antibody fragment (scFvs) sequences of CD3 epsilon.

In some embodiments of the invention, the two single chain antibody fragments of MUC16 sequence and CD3 epsilon are linked by a DNA sequence encoding a G4S linker.

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

signal peptide-VL (MUC 16) -Linker1-VH (MUC 16) -Linker2-VH (CD 3) -Linker3-VL (CD 3).

In some embodiments of the invention, the end of the MUC16-BiTE structure may contain an affinity tag, preferably a 6 XHis tag.

In some embodiments of the invention, the end of the MUC16-BiTE structure may contain an affinity tag, preferably a 6 XHis tag. In embodiments of the invention, a 6 XHis tag affinity tag may be used to purify the virus. It is noted that he is optional and can be removed, for example, in the final product, although the skilled person can choose other affinity tags and can also be removed in the final product.

In the MUC16-BiTE structure, the nucleotide sequence of VL (MUC 16) is shown in SEQ ID NO:4, the nucleotide sequence of VH (MUC 16) is shown in SEQ ID NO:5, the nucleotide sequence of VH (CD 3) is set forth in SEQ ID NO:6, the nucleotide sequence of VL (CD 3) is shown in SEQ ID NO: shown in fig. 7.

In some embodiments of the invention, linker1, linker2 or Linker3 is a series of one or more groups of G4S linkers; preferably, the nucleotide sequence of Linker1 is set forth in SEQ ID NO:8, the nucleotide sequence of Linker2 is shown as SEQ ID NO:9, the nucleotide sequence of Linker3 is shown as SEQ ID NO: shown at 10.

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

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

1) Adenovirus type 5 in which the deletion gene of E1B 55KD gene is silenced or the mutation of E1B 55KD gene is not expressed; for example, on the self genome of adenovirus, the E1B 55KD gene is deleted by a molecular biological method or the initiation codon point mutation is carried out, so that the E1B 55KD gene is not expressed, and the like, and the oncolytic adenovirus constructed by the method has the capacity of copying and amplifying in P53 defective tumor cells, thereby playing the oncolytic role of the virus.

2) Comprising one or more expression control sequences that regulate initiation of the E1 region; for example, the expression control sequence comprises at least one specific promoter, the E1 region of adenovirus is started by using the specific promoter, the replicative property of the virus is relatively controlled, the safety of oncolytic virus is improved, and the promoter comprises one or more of tumor specific antigen, telomerase and survivin promoter; and under the control of these promoters, the virus remains replication competent and is capable of expressing BiTE and/or other proteins.

3) The capsid protein is modified by the hydrophilicity, and the adenovirus capsid protein is modified by the transformation based on the 2), thereby changing the hydrophilicity of the virus and obtaining the specific targeted tumor cells.

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

In some embodiments of the invention, the pharmaceutical composition or pharmaceutical formulation of the invention comprises at least one recombinant oncolytic adenovirus of the invention, and, on that basis, the pharmaceutical composition or pharmaceutical formulation may further comprise at least one pharmaceutical carrier or pharmaceutically acceptable adjuvant, or other therapeutically effective agent. Suitable pharmaceutical excipients may be of the type known in the art, such as solvents, buffers, diluents, etc., and may be described, for example, in the pharmaceutical excipients handbook (Handbook of Parmaceutical Excipients) by the author Paul J Shrekey et al. Those skilled in the art can make selections as desired. The pharmaceutical composition or pharmaceutical formulation 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 invention, the present invention provides the use of a recombinant oncolytic adenovirus as described in the first aspect above or a pharmaceutical composition as described in the second aspect above for the manufacture of an anti-tumour medicament. Wherein the tumor is in particular an ovarian cancer, including ovarian cancer that develops resistance.

In the embodiment of the invention, the recombinant oncolytic adenovirus OAd-MUC16-BiTE has good killing effect on ovarian cancer cells, has good oncolytic efficacy on various ovarian cancer cells including OVCAR3, CAOV3, A2780, HEY and SKOV3, and can remarkably improve the killing capacity under the action of T cells, wherein the improvement on the killing effect is particularly remarkable on ovarian cancer SKOV3 cells which show resistance to viruses, and the target cell survival rate can be as low as 10% or less after 24 hours of cell killing experiments. In vitro, based on ovarian cancer PDX model, better anti-tumor effect is also shown. Compared with the simple administration of MUC16-BiTE or the combined use of MUC16-BiTE and an immunosuppression point inspection agent (such as PD-1), the recombinant oncolytic adenovirus OAd-MUC16-BiTE of the invention shows more excellent effect, because the therapy also comprises the direct cracking effect of the virus on tumor cells and the anti-tumor immunity effect of indirectly causing hosts, and the better killing effect can be achieved by the synergistic effect of various treatment methods.

In a fourth aspect of the invention, the invention provides a method of treating a solid tumor comprising administering to a subject a therapeutically effective amount of a recombinant oncolytic adenovirus as described in the first aspect above or a composition or pharmaceutical formulation comprising a recombinant oncolytic adenovirus of the invention. The solid tumor is especially ovarian cancer.

Wherein the "subject" refers to an animal, preferably a mammal, most preferably a human, who has been the subject of treatment, observation or experiment. By "therapeutically effective amount" is meant that amount of active compound or pharmaceutical agent, including a compound of the present invention, which causes a biological or medical response in a tissue system, animal or human that is sought by a researcher, veterinarian, medical doctor or other medical personnel, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated.

The compounds and pharmaceutical compositions of the invention may be used clinically in mammals, including humans and animals, with optimal dosages depending on the particular treatment. Typically starting from a small dose, the dose is gradually increased until the most suitable dose is found.

In one embodiment of the invention, the recombinant oncolytic adenoviruses described in the first aspect of the invention above may be administered in combination with a checkpoint inhibitor such as a PD-1 or PDL1 inhibitor or CAR-T or a chemotherapeutic or anti-vascular inhibitor.

Compared with the prior art, the invention has the advantages that: the present invention provides a recombinant oncolytic adenovirus that can express and secrete a bispecific antibody, particularly MUC16-BiTE, that can bridge tumor cells and T cells, induce immune synapse formation, and stimulate T cell activation; the recombinant oncolytic adenovirus can dissolve tumor cells, increase T cell infiltration, generate a pro-inflammatory environment, promote the immune activation of solid tumors and overcome the immune suppression of the tumors. In particular, the OAd-MUC16-BiTE obtained after recombination can target the T cell CD3 antigen and the tumor cell MUC16 antigen simultaneously, so that the targeting of the oncolytic adenovirus is enhanced, the adverse reaction caused by the whole body administration of the BiTE is avoided, the killing effect of the oncolytic adenovirus on solid tumors is enhanced through the activation of the BiTE on the immune microenvironment, the action time is greatly shortened, for example, the enhanced killing effect is shown on various ovarian cancer cells and tumor cells with resistance, and the action time is remarkably shortened.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:

fig. 1: OAd-MUC16-BiTE structure.

Fig. 2: virus titer detection picture, A is 100 times bright field view, B is 100 times fluorescent view, and C is virus titer detection data.

Fig. 3: OAd-MUC16-BiTE expression MUC16-BiTE.

Fig. 4: results of killing of individual ovarian cancer cells by OAd-MUC16-BiTE in example 4.

Fig. 5: results of OAd-MUC16-BiTE mediated killing of ovarian cancer cells SKOV3 by T cells in example 4.

Fig. 6: results of in vivo oncology experiments in example 5.

Detailed Description

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or materials used in the present invention may be purchased in conventional manners, and unless otherwise indicated, they may be used in conventional manners in the art or according to the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

Example 1Construction of MUC16-BiTE

DNA sequences encoding two single chain antibody fragments (scFvs) recognizing human MUC16 and CD3 epsilon with a DNA sequence encoding G ₄ The DNA sequences of the S-junctions are joined to produce a MUC 16-targeted BiTE (MUC 16-BiTE), wherein the anti-MUC 16 scFV is directed against the human MUC16 cytoplasmic retention domain (MUC-CD). An immunoglobulin signal peptide human IgK signal peptide for secretion by mammals was added at the N-terminus and a 6 XHis tag for detection was added at the C-terminus.

The structural general formula of the MUC16-BiTE is as follows:

signal peptide-VL (MUC 16) -Linker1-VH (MUC 16) -Linker2-VH (CD 3) -Linker3-VL (CD 3) -His.

The CDS region sequence and amino acid sequence of the MUC16-BiTE gene are shown below, wherein, of the amino acid or nucleotide sequences described below, the signal peptide sequence is underlined in dotted lines, the VL (MUC 16) sequence is underlined in single line, the VH (MUC 16) sequence is underlined in double line, the VH (CD 3) sequence is underlined in single wave, and the VL (CD 3) sequence is underlined in double wave.

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

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

example 2Construction of recombinant adenovirus OAd-MUC16-BiTE

1. Adenovirus package

1.1 construction of plasmid

Synthesis the DNA sequence encoding MUC16-BiTE described above was inserted into the shuttle plasmid using Gibson assembly techniques. Plasmid construction was confirmed to be correct by DNA sequencing.

1.2 plasmid transfection procedure

1) 24h before transfection, HEK293 cells in logarithmic growth phase are digested by 0.25% trypsin, the cell density is adjusted to 30% -40% by DMEM medium containing 10% FBS, and the cells are re-inoculated in a cell culture bottle at 37 ℃ and 5% CO ₂ Culturing in an incubator. And the cell can be used for transfection after the cell density reaches 50 to 60 percent after about 24 hours. Cell status is critical for viral packaging and therefore good cell status and fewer passages need to be ensured.

2) The cell culture medium was replaced with serum-free DMEM medium 2h before transfection.

3) The prepared DNA solution (5. Mu.g of the over-expressed shuttle plasmid pHBAd shuttle plasmid carrying the foreign gene and 5. Mu.g of helper plasmid pBHG loxDeltaE 1,3 Cre) was added to a sterile centrifuge tube, mixed well with DMEM, adjusted to a total volume of 50. Mu.l, and incubated at room temperature for 5 minutes.

4) Lipofectamine 2000 reagent was gently shaken well, 10. Mu.l Lipofectamine 2000 reagent was mixed with 50. Mu.l DMEM in another tube and incubated at room temperature for 5 minutes.

5) The diluted DNA was mixed with diluted Lipofectamine 2000, and the mixture was gently inverted and mixed without shaking.

6) After mixing, incubation was performed for 20 minutes at room temperature to form transfected complexes of DNA with Lipofectamine 2000 dilutions.

7) Transferring the mixed solution of DNA and Lipofectamine 2000 into culture solution of HEK293 cells, mixing, and adding 5% CO at 37deg.C ₂ Culturing in a cell culture incubator.

8) After culturing for 6 to 8 hours, the medium containing the transfection mixture was poured out, 2ml of PBS was added to each bottle of cells, and the flask was gently shaken left and right to wash the residual transfection mixture, followed by pouring out.

9) 5ml of cell culture medium containing 10% serum is added into each bottle of cells, and the mixture is heated to 37 ℃ and 5% CO ₂ Culturing in the incubator.

10 The growth of the transfected cells is observed every day, and if the cell culture medium turns yellow obviously, a proper amount of fresh whole culture solution is added as appropriate.

1.2 microscopic observations after transfection

About 10-15 days after transfection, HEK293 cells began to fall, and some cells appeared cytopathic (cytopathic effect, CPE).

1.3 harvesting of recombinant adenoviruses

After the majority of cells appeared to be typical of CPE and had 50% of the cells had been de-walled, the cells were collected by low-speed centrifugation and resuspended in 2ml DMEM, repeatedly frozen and thawed 3 times at-70℃at 37℃and centrifuged at 7000g for 5min at 4℃and the viral supernatant was collected and stored at-70 ℃.

The structure of the OAd-MUC16-BiTE obtained by construction is shown in figure 1.

2. Amplification of adenoviruses

2.1 round 1 amplification

HEK293 cells in good growth in 1T 25 cell culture flask were 4-fold diluted and transferred to another 1T 25 cell culture flask, and cultured in DMEM medium containing 10% FBS at 37℃under 5% CO2 (this condition was used for cell culture in the following expansion). When the cells reach 60% confluence, discarding the old culture solution, adding 2mL of crude extract obtained after the replication defective adenovirus is successfully recombined into a culture flask, placing the culture flask into a cell culture box for incubation for 90min, and finally adding 3mL of complete culture solution into the culture flask and continuing to culture. When most cells were subjected to typical CPE and 50% of the cells had been subjected to cell wall removal, the cells were collected by low-speed centrifugation and resuspended in 2ml DMEM, repeatedly frozen and thawed 3 times at-70℃at 37℃and centrifuged at 7000g for 5min at 4℃and the virus supernatant was collected and stored at-70 ℃.

2.2 round 2 amplification

HEK293 cells with good growth state in 1T 25 cell culture flask are all transferred into 1T 75 cell culture flask, and the culture is continued with complete culture medium. When the cells reach 90% confluence, discarding the old culture solution, adding 2mL of virus solution obtained by amplification in round 1 into a culture flask, placing the culture flask into a cell culture box for incubation for 90min, and finally adding 10mL of complete culture solution into the culture flask and continuing to culture. When most cells were subjected to typical CPE and 50% of the cells had been subjected to cell wall removal, the cells were collected by low-speed centrifugation and resuspended in 10ml DMEM, repeatedly frozen and thawed 3 times at-70℃at 37℃and centrifuged at 7000g for 5min at 4℃and the virus supernatant was collected and stored at-70 ℃.

3. Adenovirus titer detection

3.1 adenovirus titre assay-endpoint dilution method

1) 24 hours prior to the experiment, 100. Mu.l HEK293 cell suspension, approximately 1X10, was added to each well of a 96-well plate ³ A cell;

2) 12 sterile Ep tubes were prepared, 990. Mu.l of complete broth was added to the first Ep tube, and 900. Mu.l of complete broth was added to each of the remaining 11 tubes.

3) Dilution of the virus liquid to be tested: 10 μl of adenovirus stock solution was added to 990 μl Ep tube for 1:100 dilutionRelease (10) ^-2 ) The method comprises the steps of carrying out a first treatment on the surface of the Then, starting from this, 100. Mu.l of the diluent was added to a 900. Mu.l Ep tube and diluted 1:10 (10 ^-3 ) Until diluted to 10 ^-13 。

4) The 96-well plates were removed from the incubator and cells from each well were confirmed to grow well under a microscope. The old culture broth was pipetted off and then 10 were sequentially added ^-13 To 10 ^-6 The diluted virus solutions were added to 96-well plates, one row was occupied by each dilution, 90. Mu.l of virus dilution was added to each of wells 1-10 of each row, and 90. Mu.l of complete medium without virus was added to each of wells 11-12 of each row as controls.

5) The 96-well plate was placed at 37℃in 5% CO ₂ The cell culture was continued in the incubator.

6) Cytopathic effect was observed after 10d and CPE wells were counted, the positive rate for each row was calculated, and virus titer (Spearman-Karber Method) was calculated.

Note that: positive results were obtained as soon as there was a small spot or some cells appeared CPE.

4.2 method for calculating virus titer

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

x＝10 ^-1 To 10 ^-13 Sum of CPE positivity at sequential dilutions

* The formula uses the condition:

a. the negative control had no CPE and growth inhibition;

b. the wells to which the virus crude extract with minimum dilution concentration were added all had CPE.

Detection of viral titres as shown in figure 2, viral titres were calculated, x=9.2, and were 1.0e+10 (PFU/ml).

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

Human ovarian cancer cells HEY were infected with moi=100 OAd (MOI is multiplicity of infection), OAd-MUC16-BiTE, respectively, HEY empty cells and virus-infected HEY cell proteins were extracted after 24 hours, respectively, and western blot electrophoresis detection was performed with anti-His antibodies:

an antibody: purified anti-His Tag anti-body (biolegend # 362601) dilution ratio 1:1000; the molecular weight of the protein of interest is about 58kDa.

An antibody: the dilution ratio of the Anti-Beta-action Anti-ibody (protein 66009-1-Ig) is 1:2000; beta-actin has a molecular weight of 42kDa.

And (2) secondary antibody: dilution ratio of the Goat Anti-Rabbit IgG H & L (HRP) (abcam ab 6721) 1:20000.

analysis of results: as shown in FIG. 3, it was found that adenovirus OAd-MUC16-BiTE infected HEY cells had a distinct target band at the 58kDa locus, whereas control HEY null cells and OAd infected HEY cells did not have a band at the corresponding loci, indicating that adenovirus OAd-MUC16-BiTE could infect cells and highly express MUC16-BiTE within cells.

Example 4In vitro tumor killing experiment

1. Detection of killing ability of ovarian cancer cells Using CCK8 method

1) Ovarian cancer cell lines (OVCAR 3, CAOV3, A2780, HEY, SKOV 3) 5×10 ³ Spread in 96-well plates.

2) A specified amount of OAd or OAd-MUC16-BiTE was added, and the control group was not virus added.

3) At 24h, 48h, 72h, 96h after incubation, 10 μl CCK-8 (MCE) was added to each well and absorbance was measured at 450nm using a microplate reader (BIO-RAD) after 2h at 37deg.C.

4) Cell viability was calculated as follows:

cell viability= (experimental OD value-blank OD value)/(negative control OD value-blank OD value).

Killing of each ovarian cancer cell line is shown in figure 5.

2. Detection of killing Capacity against target cells (ovarian cancer cells SKOV 3) Using the Luciferase luminescence intensity method

Grouping condition:

experimental group: OAd +Tcell group, OAd-MUC 16-BiTE+Tcell group;

blank control group: a virus-free, effector-free T cell group;

negative control group: no virus group;

1) Target cells SKOV3-LUC or SKOV3-MUC stably expressing luciferases16 by 1X10 ⁴ Spreading in 96-well plate;

2) Adding effector cell T cells into target cells according to the ratio of the effective target ratio of 5:1;

3) Viruses were added to the wells at moi=10, and PBS was added to the corresponding volumes for the single T cell group or negative control group;

4) After 24 hours, carrying out luminescence intensity detection of luciferases on target cells;

specific procedure according to Bright-Lumi ^TM Firefly luciferase reporter assay kit (Biyun Tian, RG 051M) instructions.

5) Tumor suppression ratio=1- (experimental group lumen value-blank group lumen value)/(negative control group lumen value-blank group lumen value) ×100%.

The experimental results are shown in FIG. 5.

The results of the in vitro killing experiments are shown in fig. 4 and 5.

As can be seen from the experimental results of FIG. 4, the oncolytic adenovirus OAd-MUC16-BiTE carrying the bispecific antibody MUC16-BiTE of the present invention has no significant difference in the lytic activity against ovarian cancer cells from the parental virus in most cases, and exhibits time dependence. Thus, the insertion of MUC16-BiTE has little effect on viral replication or oncolytic activity. It is worth noting that OAd-MUC16-BiTE has oncolytic efficacy in all cell lines tested, but ovarian cancer SKOV3 cells are partially resistant, kill slower, and increasing the virus dose does not counteract this resistance.

From the results of FIG. 5, it can be seen that the killing ability against target cells, especially SKOV3 cells resistant to viruses, can be significantly enhanced in the presence of T cells. And the effect of OAd-MUC16-BiTE on T cell killing is antigen specific. Cell killing experiments were performed for 24 hours, and target cell viability was already as low as below 10%.

In combination with the above, OAd-MUC16-BiTE kills target cells with antigen specificity, can remarkably improve killing ability to ovarian cancer cells in the presence of T cells, and greatly reduce action time.

Example 5In vivo tumor killing experiment

1) An ovarian cancer PDX model was constructed.

2) PDX P2 generation tumors were homogenized and inoculated subcutaneously into mice.

3) After 1 week, mice were randomly divided into 5 groups (5 animals per group) and 5×10 intratumorally injected on day 0 and day 3 ⁹ PFU OAd or OAd-MUC16-BiTE or PBS, 1X10 mice were injected via the tail vein 1 day after each round of virus injection ⁷ T cells or an equal volume of PBS.

4) At the indicated analysis time, all mice were euthanized with intraperitoneal injection of sodium pentobarbital (200 mg/kg) and the tumors were resected.

In vivo tumor killing results as shown in figure 6, PBS group showed the fastest tumor growth, OAd-MUC16-BiTE showed similar anti-tumor efficacy to the parental virus without T cells. The administration of T cells significantly enhanced the anti-tumor efficacy of OAd-MUC16-BiTE and was significantly more potent than the OAd-treated group administered T cells.

The above examples show that the recombinant oncolytic adenovirus OAd-MUC16-BiTE can target tumor cells to express and secrete bispecific antibody MUC16-BiTE, and the tumor site stimulates tumor infiltrating T cells without systemic toxicity by activating an immune microenvironment through BiTE, so that the killing effect of oncolytic adenovirus on ovarian cancer can be remarkably improved.

The foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Qilu Hospital at Shandong university

<120> a recombinant oncolytic adenovirus and uses thereof

<130> 202124120

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 1572

<212> DNA

<213> artificial sequence

<400> 1

atggacatgc gggtgctggc acagctgctg ggcctgctgc tgctgtgctt cccaggagcc 60

agatgtgaca ttgagctcac ccagtctcca tcctccctgg ctgtgtcagc aggagagaag 120

gtcactatga gctgcaaatc cagtcagagt ctgctcaaca gtagaacccg aaagaaccag 180

ttggcttggt accagcaaaa accaggacag tctcctgaac tgctgatcta ctgggcatcc 240

actaggcaat ctggagtccc tgatcgcttc acaggcagtg gatctgggac agatttcact 300

ctcaccatca gcagtgtgca ggctgaagac ctggcagttt attactgcca gcaatcttat 360

aatctactca cgttcggtcc tgggaccaag ctggaggtca aacggggtgg aggcggcagt 420

ggcggaggtg ggagcggagg gggcggttcc gtgaagctgc aggagtcagg gggaggcttc 480

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 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 cacctttact 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 aggtgggagc ggagggggcg gttcc 45

<210> 9

<211> 15

<212> DNA

<213> artificial sequence

<400> 9

ggtggcgggg gatct 15

<210> 10

<211> 45

<212> DNA

<213> artificial sequence

<400> 10

ggtggcgggg gaagcggcgg cggtggatcc ggtggagggg gcagt 45

Claims

1. A recombinant oncolytic adenovirus, characterized in that the virus is constructed based on adenovirus type 5, comprising in its genome a nucleotide sequence encoding a BiTE antibody, said nucleotide sequence encoding a BiTE antibody being located between the elements mCMV and MCS-3Flag of the virus;

the BiTE is MUC16-BiTE;

the nucleotide sequence of MUC16-BiTE is shown as SEQ ID NO: 1.

2. The recombinant oncolytic adenovirus according to claim 1, wherein the BiTE targets both T cell CD3 antigen and tumor antigen or tumor-associated antigen.

3. The recombinant oncolytic adenovirus of claim 2, wherein said tumor antigen or tumor-associated antigen comprises one or more of mesothelin, folate receptor alpha, MUC-16, EGFR, HER2, CD133, NKG2D, MUC1, WTI, NY-ES0-1, survivin.

4. The recombinant oncolytic adenovirus according to claim 1, wherein the amino acid sequence of MUC16-BiTE is as set forth in SEQ ID NO: 2.

5. The recombinant oncolytic adenovirus of any one of claims 1-4, wherein said adenovirus type 5 is selected from the group consisting of:

1) Adenovirus type 5 in which the deletion gene of E1B 55KD gene is silenced or the mutation of E1B 55KD gene is not expressed;

2) Comprising one or more expression control sequences that regulate initiation of the E1 region;

3) The capsid protein is modified with affinity.

6. The recombinant oncolytic adenovirus according to claim 5, wherein the expression control sequence comprises at least one specific promoter comprising one or more of a tumor specific antigen, telomerase, and survivin promoter.

7. A pharmaceutical composition or pharmaceutical formulation comprising the recombinant oncolytic adenovirus of any one of claims 1-6.

8. Use of a recombinant oncolytic adenovirus according to any one of claims 1-6 or a pharmaceutical composition according to claim 7 for the preparation of an anti-tumor medicament.

9. The use according to claim 8, wherein the tumour is ovarian cancer.