CN113969266A

CN113969266A - Recombinant oncolytic adenovirus and application thereof

Info

Publication number: CN113969266A
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: 2022-01-25
Anticipated expiration: 2041-10-26
Also published as: CN113969266B

Abstract

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

Description

Recombinant oncolytic adenovirus and application thereof

Technical Field

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

Background

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

Ovarian cancer is the most lethal tumor of the female reproductive system. The survival rate of many cancers has improved greatly due to the progress of leading-edge research, screening, diagnosis, surgery and other treatment methods, but the survival rate of ovarian cancer has not changed greatly for decades. The standard treatment for ovarian cancer is surgery and platinum-paclitaxel combination chemotherapy, and despite some advances, most patients still relapse, metastasize, and are resistant quickly, and once this occurs, most patients face no effective treatment available. Thus, there is an urgent need for improved therapies for ovarian cancer patients.

Tumor immunotherapy, one of the most promising therapies in oncology, has been shown to exert antitumor effects, primarily by activating the immune system to induce tumor immune surveillance or to reverse tumor immune escape. The research proves that the ovarian cancer is an immune tumor, and the survival rate of the ovarian cancer can be greatly improved due to the existence of tumor infiltrating lymphocytes (especially CD3+ T cells). Meanwhile, immune evasion mechanism mediated by suppressor cells such as T regulatory cells is associated with poor survival. Based on the above, epithelial ovarian cancer patients may benefit from immunotherapy.

Oncolytic adenovirus (oncolytical adenovirus, OAd) is a purposeful modification of adenovirus gene structure, which enables the virus to selectively replicate in specific tumor cells, continuously lyses the tumor cells, and has no lethality to surrounding normal cells. OAds can promote anti-tumor immune responses by a variety of mechanisms. OAds induce a pro-inflammatory environment that recruits T cells and disrupts the physical barrier to T cell infiltration, increasing T cell infiltration of tumors. OAds can stimulate the production of proinflammatory cytokines and the depletion of immunosuppressive cells in the tumor microenvironment. The pro-inflammatory environment created by OAds infection may also transform immunosuppressive cell types (e.g., M2 macrophages) into a more antitumor phenotype. In addition, OAdss may also upregulate pathways involved in antigen processing and presentation, including increased release of Tumor Associated Antigens (TAAs), increased expression of Major Histocompatibility Complex (MHC) class I and MHC class II on APCs and tumor cells, thereby enhancing the ability of the immune system to recognize tumor cells.

Bispecific T-cell engagers (BiTE) Bispecific single chain antibodies consisting of two single chain variable fragments (ScFv), one of which binds to a tumor associated antigen on cancer cells and the other binds to the T-cell surface marker CD3, can target cancer cells and T-cells simultaneously, physically bridge the T-cells to form a T-cell-BiTE-tumor cell complex, induce immune synapse formation, stimulate T-cell activation, kill tumor cells. BiTE binds to T cell antigen positive target cells, presents independently of HLA, and can activate the binding and destruction of any T cell to adjacent target cells. In addition, BiTE-mediated T cell activation can overcome the limiting factor of tumor-associated immunosuppression, resulting in the activation and proliferation of depleted tumor-specific T cells. Bornatuzumab (Blinatumomab) is CD19 BiTE that was rapidly approved by the FDA in 2014 for the treatment of relapsed or refractory B-ALL, significantly better than standard chemotherapy regimens. However, BiTE has a short serum half-life and requires continuous intravenous administration for a long period of time, which is highly toxic. In addition, the use of BiTE in solid tumors is limited in efficacy due to osmotic problems in the tumor microenvironment and dose-limiting toxicity due to off-target effects.

MUC16 is one of the members of the mucin family (MUC) and belongs to the type I transmembrane mucin. MUC16, the largest glycoprotein in the MUC family, is located on human chromosome 19p13.2 and is encoded by 179kb genomic DNA, which encodes 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 repetitive sequences, which is released into the blood as the serum marker CA-125 of ovarian cancer; the retention domain, which includes the residual non-repetitive extracellular segment, transmembrane region and cytoplasmic tail (18), remains on the cell surface and is expressed only at low levels in normal tissues, including the uterus, fallopian tube, ovary and corneal surface of the eye, and thus can serve as an attractive target. There are currently methods of treating ovarian cancer using antibodies to MUC16, such as agovacizumab (oregozomab), an anti-MUC 16 antibody that has currently entered phase III clinics.

Disclosure of Invention

In order to increase the efficacy of BiTE, reduce adverse side effects caused by continuous systemic administration, improve the targeting property of oncolytic adenovirus and improve the killing effect of oncolytic adenovirus on solid tumors, the invention provides the recombinant oncolytic adenovirus capable of expressing BiTE (especially expressing MUC16-BiTE), wherein the recombinant virus can simultaneously target a T cell CD3 antigen and a tumor cell antigen (MUC16) to realize bridging of tumor cells and T cells, the BiTE coded by the recombinant virus has obviously enhanced cytotoxicity and can bypass or reverse the immunosuppressive microenvironment of tumors, the killing effect on solid tumors, especially ovarian cancers is obviously improved, and the action time is greatly reduced. The results of in vitro cell experiments show that the cancer cell survival rate can be as low as 10% or less even when the killing experiment is carried out for 24 hours for the ovarian cancer cells with resistance.

Specifically, the present invention provides the following technical features, and one or a combination 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 constructed on the basis of adenovirus type 5 (adenvirus 5, Ad5), 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 recombinant oncolytic adenovirus of the invention has a structure as shown in FIG. 1.

In some embodiments of the invention, the BiTE targets both the T cell CD3 antigen and a 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 α), MUC-16, EGFR, HER2, CD133, NKG2D, MUC1, WTI, NY-ES0-1, Survivin.

In a more preferred embodiment of the present invention, the BiTE is MUC 16-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 MUC16 sequence and the two single chain antibody fragments of CD3 epsilon are linked by a DNA sequence encoding a G4S linker.

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

signal peptide-VL (MUC16) -Linker1-VH (MUC16) -Linker2-VH (CD3) -Linker3-VL (CD 3).

In some embodiments of the invention, the MUC16-BiTE structure may comprise an affinity tag at the end, preferably 6 × His tag.

In some embodiments of the invention, the MUC16-BiTE structure may comprise an affinity tag at the end, preferably 6 × His tag. In embodiments of the invention, a 6 × His tag affinity tag may be used to purify the virus. It is noted that this is optional and can be removed, for example, in the final product, although the skilled person can also select other affinity tags and also be removed in 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 as SEQ ID NO: 5, the nucleotide sequence of VH (CD3) is shown in SEQ ID NO: 6, the nucleotide sequence of VL (CD3) is shown in SEQ ID NO: shown in fig. 7.

In some embodiments of the invention, Linker1, Linker2, or Linker3 is a group of G4S linkers or a series of groups of G4S linkers; preferably, the nucleotide sequence of Linker1 is shown 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 invention, the nucleotide sequence of MUC16-BiTE is as set forth in SEQ ID NO: 1, the amino acid sequence of MUC16-BiTE is shown in SEQ ID NO: 2, respectively.

In some embodiments of the invention, the adenovirus type 5 of the invention is selected from the group consisting of:

1) adenovirus type 5 which is not expressed by deletion gene silencing of E1B 55KD gene or mutation of E1B 55KD gene; for example, in the self genome of adenovirus, the gene E1B 55KD is deleted by a molecular biological method, or the start codon point mutation is carried out, so that the gene E1B 55KD is not expressed, and the oncolytic adenovirus constructed by the method has the capability of replicating and amplifying a P53 defective tumor cell, thereby playing the role of virus oncolytic.

2) Contains one or more expression control sequences which regulate the initiation of the E1 region; for example, the expression control sequence comprises at least one specific promoter, and the specific promoter is used for promoting the E1 region of the adenovirus, so that the replication of the virus is relatively controlled, and the safety of the oncolytic virus is improved, wherein 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 of the adenovirus is modified by tropism, and the virus modifies the capsid protein of the adenovirus on the basis of 2) to change the tropism of the virus, so that specific targeting tumor cells can be obtained.

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 present invention, the pharmaceutical composition or pharmaceutical preparation of the present invention comprises at least one recombinant oncolytic adenovirus of the present invention, and, based thereon, the pharmaceutical composition or pharmaceutical preparation may further comprise at least one pharmaceutical carrier or pharmaceutically acceptable adjuvant, or other therapeutically effective agent. Suitable pharmaceutical Excipients may be of a kind known in the art, such as solvents, buffers, diluents and the like, and may be, for example, those described in the Handbook of pharmaceutical Excipients (Handbook of pharmaceutical Excipients) by the authors Paul J Sheskey et al. The skilled person can select 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 present invention, the present invention provides the use of the recombinant oncolytic adenovirus of the first aspect or the pharmaceutical composition of the second aspect in the preparation of an anti-tumor medicament. Wherein the tumor is especially ovarian cancer, and the ovarian cancer comprises resistant ovarian cancer.

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 obviously improve killing capability under the action of T cells, wherein the improvement on the killing effect is particularly obvious for ovarian cancer SKOV3 cells showing resistance to viruses, and the target cell survival rate can be reduced to below 10 percent after the cell killing experiment is carried out for 24 hours. In vitro, based on the ovarian cancer PDX model, also showed better antitumor effect. Compared with the mode of singly applying MUC16-BiTE or combining MUC16-BiTE and an immune suppression point check agent (such as PD-1), the recombinant oncolytic adenovirus OAd-MUC16-BiTE shows more excellent effect, because the treatment also comprises the direct lysis of the virus to tumor cells and the indirect anti-tumor immunity effect of a host, and a plurality of treatment methods are synergistic to achieve better killing effect.

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 according to 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 object of treatment, observation or experiment. By "therapeutically effective amount" is meant an amount of active compound or pharmaceutical agent, including a compound of the present invention, that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other medical professional, 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 present invention may be administered clinically to mammals, and the optimum dosage for both human and animal subjects will depend upon the particular treatment. Usually starting with a small dose and gradually increasing the dose until the most suitable dose is found.

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

Compared with the prior art, the invention has the advantages that: the invention provides a recombinant oncolytic adenovirus, which can express and secrete a bispecific antibody, especially MUC16-BiTE, which can bridge tumor cells and T cells, induce immune synaptogenesis and stimulate the activation of the T cells; the recombinant oncolytic adenovirus can dissolve tumor cells, increase T cell infiltration, generate proinflammatory environment, promote solid tumor immune activation and overcome tumor immunosuppression. Particularly, OAd-MUC16-BiTE obtained after recombination can simultaneously target a T cell CD3 antigen and a tumor cell MUC16 antigen, not only enhances the targeting property of the oncolytic adenovirus, but also avoids adverse reactions caused by systemic administration of the BiTE, increases the killing effect of the oncolytic adenovirus on solid tumors through the activation effect of the BiTE on immune microenvironment, greatly shortens the action time, shows enhanced killing effect on various ovarian cancer cells and resistant tumor cells, and obviously shortens the action time.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1: OAd-MUC16-BITE structural schematic diagram.

FIG. 2: the virus titer detection pictures show that A is a 100-fold bright field, B is a 100-fold fluorescence field, and C is virus titer detection data.

FIG. 3: OAd-MUC16-BITE expresses MUC 16-BITE.

FIG. 4: results of the killing effect of OAd-MUC16-BITE on individual ovarian cancer cells in example 4.

FIG. 5: results of the killing effect of OAd-MUC 16-BiTE-mediated T cells on ovarian cancer cells SKOV3 in example 4.

FIG. 6: results of the in vivo tumoricidal experiment in example 5.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

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 starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with 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 embodiments and materials described herein are intended to be exemplary only.

Example 1Construction of MUC16-BITE

DNA sequences encoding two single chain antibody fragments (scFvs) recognizing human MUC16 and CD3 epsilon are ligated with a DNA sequence encoding G₄The DNA sequences of the S-linker were ligated to generate a BITE targeting MUC16 (MUC16-BITE), in which the anti-MUC 16 scFV was directed against humanMUC16 cytoplasmic Retention Domain (MUC-CD). And an immunoglobulin signal peptide human IgK signal peptide for mammal secretion is added at the N end, and 6 XHis tag for detection is added at the C end.

The structural general formula of MUC16-BITE is as follows:

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

The CDS region sequence and amino acid sequence of MUC16-BiTE gene are shown below, wherein, in the following amino acid or nucleotide sequence, the signal peptide sequence is underlined with dotted lines, the VL (MUC16) sequence is underlined with single lines, the VH (MUC16) sequence is underlined with double lines, the VH (CD3) sequence is underlined with single waves, and the VL (CD3) sequence is underlined with double waves.

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

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

example 2Construction of recombinant adenovirus OAd-MUC16-BiTE

1. Adenovirus packaging

1.1 construction of plasmids

Synthesis the DNA sequence encoding MUC16-BiTE described above was inserted into a shuttle plasmid using Gibson assembly technology. The plasmid construction was confirmed by DNA sequencing.

1.2 plasmid transfection procedure

1) 24h before transfection, HEK293 cells in the logarithmic growth phase were digested with 0.25% trypsin and cultured in DMEM containing 10% FBSThe cell density is adjusted to 30-40% by nutrient medium, and the cell is re-inoculated in a cell culture bottle at 37 ℃ and 5% CO₂Culturing in an incubator. The cell can be used for transfection after the cell density reaches 50% -60% after about 24 h. The cell state is critical for virus packaging and therefore it is desirable to ensure good cell state and a low number of passages.

2) The cell culture medium was changed to serum-free DMEM medium 2h before transfection.

3) The prepared DNA solution (5. mu.g of over-expressed shuttle plasmid pHBAd shuttle plasmid carrying foreign gene and 5. mu.g of helper plasmid pBHGlox. DELTA.E 1,3Cre) was added to a sterilized centrifuge tube and mixed with DMEM uniformly, adjusted to a total volume of 50. mu.l, and incubated at room temperature for 5 minutes.

4) Lipofectamine 2000 was gently shaken, and 10. mu.l Lipofectamine 2000 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 the diluted Lipofectamine 2000, and the mixture was gently inverted and mixed without shaking.

6) After mixing, incubation was performed at room temperature for 20 minutes to form a transfection complex of DNA with Lipofectamine 2000 dilution.

7) Transferring the mixture of DNA and Lipofectamine 2000 to HEK293 cell culture medium, mixing, and culturing at 37 deg.C with 5% CO₂Culturing in a cell culture box.

8) After culturing for 6-8 h, pouring out the culture medium containing the transfection mixture, adding 2ml of PBS (phosphate buffer solution) into each bottle of cells, slightly shaking the culture bottle left and right to wash the residual transfection mixture, and then pouring out.

9) 5ml of cell culture medium containing 10% serum was added to each flask of cells, and the mixture was incubated at 37 ℃ with 5% CO₂And continuously 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 examination after transfection

Approximately 10-15 days after transfection, HEK293 cells began to shed, and some cells developed cytopathic effect (CPE).

1.3 harvesting of recombinant adenovirus

After most of the cells showed typical CPE and 50% of the cells were detached, the cells were collected by centrifugation at low speed and resuspended in 2ml DMEM, frozen and thawed repeatedly at-70 ℃/37 ℃ and shaken 3 times, centrifuged at 4 ℃ and 7000g for 5min, and the virus supernatant was collected and stored at-70 ℃.

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

2. Amplification of adenovirus

2.1 round 1 amplification

HEK293 cells in good growth state in 1T 25 cell culture flask were diluted 4 times and transferred to another 1T 25 cell culture flask, and cultured in 10% FBS-containing DMEM at 37 ℃ under 5% CO2 (cells were cultured under these conditions in the following expansion). And 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 the culture bottle, placing the culture bottle into a cell incubator for incubation for 90min, and finally supplementing 3mL of complete culture solution into the culture bottle and continuing to culture. When most cells show typical CPE and 50% of the cells are detached from the wall, the cells are collected by low-speed centrifugation and are resuspended in 2ml DMEM, the cells are repeatedly frozen and thawed at-70 ℃/37 ℃ and shaken for 3 times, and are centrifuged for 5min at 4 ℃ and 7000g, and virus supernatant is collected and stored at-70 ℃.

2.2 round 2 amplification

All the HEK293 cells in 1T 25 flask with good growth status were transferred into 1T 75 flask and the culture was continued with complete medium. And when the cells reach 90% confluence, discarding the old culture solution, adding 2mL of virus solution obtained by the 1 st round of amplification into the culture bottle, placing the culture bottle in a cell incubator for incubation for 90min, and finally supplementing 10mL of complete culture solution into the culture bottle and continuing culture. When most cells show typical CPE and 50% of the cells are detached from the wall, the cells are collected by low-speed centrifugation and are resuspended in 10ml DMEM, the cells are repeatedly frozen and thawed at-70 ℃/37 ℃ and shaken for 3 times, and are centrifuged for 5min at 4 ℃ and 7000g, and virus supernatant is collected and stored at-70 ℃.

3. Adenovirus titer detection

3.1 adenovirus titer determination-end point dilution method

1) 24 hours before the experiment, 100. mu.l HEK293 cell suspension containing approximately 1X10 was added to each well of a 96-well plate³(ii) individual cells;

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

3) Diluting virus liquid to be tested: mu.l adenovirus stock solution was added to 990. mu.l Ep tube for 1:100 dilution (10)^-2) (ii) a Starting from this, 100. mu.l of the dilution were then added to 900. mu.l of Ep tube for a 1:10 dilution (10)^-3) Until it is diluted to 10^-13。

4) The 96-well plate was removed from the incubator and the cells in each well were determined to grow well under the microscope. The old culture medium is discarded, and then 10 are sequentially added^-13To 10^-6Diluted virus was added to 96-well plates, one row for each dilution, 90. mu.l of virus dilution was added to each well from 1-10 of each row, and 90. mu.l of complete medium without virus was added to each well from 11-12 of each row as a control.

5) Placing 96-well plate at 37 deg.C and 5% CO₂And continuing culturing in the cell culture box.

6) Cytopathic effects were observed after 10d and CPE wells were counted, the positivity per row was calculated, and the virus titer was calculated (Spearman-Karber Method).

Note: positive results were obtained if only a small spot or some cells showed CPE.

4.2 Virus titer calculation method

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

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

Formula usage conditions:

a. negative controls had no CPE and growth inhibition;

b. the wells to which the minimal dilution of the viral crude extract was added all had CPE.

The virus titer was measured as shown in fig. 2, and calculated to be 9.2, 1.0E +10 (PFU/ml).

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

Human ovarian cancer cell HEY is infected by MOI 100OAd (MOI is multiplicity of infection) and OAd-MUC16-BITE respectively, HEY empty cells and HEY cell proteins infected by virus are extracted respectively after 24 hours, and western blot electrophoresis detection is carried out by using an anti-His antibody:

a first antibody: purified anti-His Tag Antibody (biolegend #362601) dilution ratio 1: 1000; the molecular weight of the target protein is about 58 kDa.

A first antibody: Anti-Beta-Actin antibody (Proteintetech 66009-1-Ig) dilution ratio 1: 2000; the molecular weight of beta-actin is 42 kDa.

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

and (4) analyzing results: the results are shown in FIG. 3, and it can be seen from the results that adenovirus OAd-MUC 16-BiTE-infected HEY cells have a distinct target band at the 58kDa position, while control HEY empty cells and OAd-infected HEY cells have no band at the corresponding position, indicating that adenovirus OAd-MUC16-BiTE can infect cells and highly express MUC16-BiTE in the cells.

Example 4In vitro tumor killing experiment

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

1) Ovarian cancer cell lines (OVCAR3, CAOV3, A2780, HEY, SKOV3) 5X 10³Spread on 96-well plates.

2) The negative control group was prepared by adding OAd or OAd-MUC16-BITE in the prescribed amount and no virus.

3) At 24h, 48h, 72h, 96h after incubation, 10. mu.l of CCK-8(MCE) was added to each well, and after standing at 37 ℃ for 2h, the absorbance was measured at 450nm using a microplate reader (BIO-RAD).

4) Calculating the cell survival rate according to the following formula:

cell survival rate ═ (experimental OD value-blank OD value)/(negative control OD value-blank OD value).

The killing effect on each ovarian cancer cell line is shown in fig. 5.

2. Killing ability to target cells (ovarian cancer cell SKOV3) was examined by Luciferase luminescence intensity method

Grouping condition:

experimental groups: OAd + T cell group, OAd-MUC16-BITE + T cell group;

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

negative control group: a virus-free group;

1) SKOV3-LUC or SKOV3-MUC16 of target cells stably expressing Luciferase are mixed according to the proportion of 1x10⁴Spreading in 96-well plate;

2) adding effector cell T cells into target cells according to the ratio of an effector target to the target being 5: 1;

3) the virus was added to the wells at MOI 10, and the single T cell group or negative control group was added to the corresponding volume of PBS;

4) detecting the luminous intensity of Luciferase on the target cells after 24 h;

the specific operation is according to Bright-Lumi^TMFirefly luciferase reporter gene assay kit (Biyunyan, RG051M) instructions.

5) Tumor inhibition rate is 1- (experimental group lumen value-blank group lumen value)/(negative control group lumen value-blank group lumen value) × 100%.

The results of the experiment 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 invention has no significant difference in the lytic activity to ovarian cancer cells and the parental virus in most cases, and shows time dependence. Thus, the insertion of MUC16-BiTE had little effect on viral replication or oncolytic activity. It is noteworthy that OAd-MUC16-BITE all had oncolytic potency in the cell lines tested, but ovarian cancer SKOV3 cells were partially resistant and killed more slowly, and increasing the virus dose did not counteract this resistance.

As can be seen from the results in fig. 5, the killing ability against target cells, especially SKOV3 cells resistant to viruses, was significantly improved in the presence of T cells. And the effect of OAd-MUC16-BITE on T cell killing was antigen-specific. After 24 hours of cell killing experiment, the survival rate of target cells is reduced to below 10%.

In conclusion, the killing of OAd-MUC16-BITE to target cells is antigen specificity, the killing capability to ovarian cancer cells can be obviously improved in the presence of T cells, and the action time is greatly shortened.

Example 5In vivo tumor killing experiment

1) The ovarian cancer PDX model is constructed.

2) PDX P2 tumor generation was homogenized and inoculated subcutaneously into mice.

3) After 1 week, mice were randomized into 5 groups (5 mice per group) and intratumorally injected with 5 × 10 on

days

0 and 3⁹PFU OAd or OAd-MUC16-BITE or PBS, 1 day after each virus injection, mice were injected 1X10 by tail vein⁷T cells or an equal volume of PBS.

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

In vivo tumoricidal results are shown in FIG. 6, with the PBS group showing the fastest tumor growth and OAd-MUC16-BITE showing similar anti-tumor efficacy to the parental virus in the absence of T cells. The administration of T cells significantly enhanced the antitumor efficacy of OAd-MUC16-BiTE and was significantly more effective than the OAd treatment 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, activate immune microenvironment through BiTE, stimulate tumor infiltration T cells at tumor sites without systemic toxicity, and can significantly increase the killing effect of the oncolytic adenovirus on ovarian cancer.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement 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 of Shandong university

<120> recombinant oncolytic adenovirus and application thereof

<130> 202124120

<160> 10

<170> PatentIn version 3.5

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<211> 1572

<212> DNA

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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 constructed on the basis of adenovirus type 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.

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

3. The recombinant oncolytic adenovirus of claim 2, wherein the 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 of claim 1 or 2, wherein the BiTE is MUC 16-BiTE.

5. The recombinant oncolytic adenovirus of claim 4, wherein the MUC16-BITE comprises two single chain antibody fragment sequences of a human MUC16 sequence fragment and CD3 epsilon;

preferably, the MUC16 sequence and two single chain antibody fragments of CD3 epsilon encode G₄The DNA sequences of the S-linker are ligated.

Preferably, the structural formula of MUC16-BiTE is as follows: signal peptide-VL (MUC16) -Linker1-VH (MUC16) -Linker2-VH (CD3) -Linker3-VL (CD 3);

preferably, the end of the MUC16-BiTE structure may contain an affinity tag, preferably 6 × His tag;

preferably, the signal peptide is an immunoglobulin signal peptide, preferably a human IgK signal peptide.

6. The recombinant oncolytic adenovirus of claim 5, wherein the nucleotide sequence of VL (MUC16) is set forth in SEQ ID NO: 4 is shown in the specification;

preferably, the nucleotide sequence of VH (MUC16) is as set forth in SEQ ID NO: 5 is shown in the figure;

preferably, the nucleotide sequence of VH (CD3) is as set forth in SEQ ID NO: 6;

preferably, the nucleotide sequence of VL (CD3) is as set forth in SEQ ID NO: 7;

preferably, Linker1, Linker2 or Linker3 is a group G₄S linker or groups G₄S linker is connected in series;

preferably, Linker1 has the amino acid sequence as shown in SEQ ID NO: 8, Linker2 has an amino acid sequence as shown in SEQ ID NO: 9, Linker3 has an amino acid sequence shown in SEQ ID NO: 10, or a pharmaceutically acceptable salt thereof.

7. The recombinant oncolytic adenovirus of claim 5, wherein the nucleotide sequence of MUC16-BiTE is as set forth in SEQ ID NO: 1;

preferably, the amino acid sequence of MUC16-BiTE is as shown in SEQ ID NO: 2, respectively.

8. The recombinant oncolytic adenovirus of any one of claims 1-7, wherein the adenovirus type 5 is selected from the group consisting of:

1) adenovirus type 5 which is not expressed by deletion gene silencing of E1B 55KD gene or mutation of E1B 55KD gene;

2) contains one or more expression control sequences which regulate the initiation of the E1 region; preferably, the expression control sequence comprises at least one specific promoter, wherein the specific promoter comprises one or more of tumor specific antigen, telomerase and survivin promoters;

3) the capsid protein is modified with tropism.

9. A pharmaceutical composition or pharmaceutical formulation comprising the recombinant oncolytic adenovirus of any one of claims 1-8.

10. Use of the recombinant oncolytic adenovirus of any one of claims 1-8 or the pharmaceutical composition of claim 9 for the preparation of an anti-tumor medicament;

preferably, the tumor is ovarian cancer.