WO2020166727A1 - Oncolytic virus using human adenovirus type 35 as base - Google Patents

Abstract

The present invention provides a recombinant oncolytic adenovirus type 35 in which an hTERT promotor or a Survivin promotor and the E1 gene are incorporated into the genome of adenovirus type 35.

Description

Oncolytic virus based on human adenovirus type 35

The present invention relates to an oncolytic virus based on human adenovirus type 35, and a pharmaceutical composition containing the virus.

Oncolytic virus is expected to be a novel anti-cancer agent because it infects and proliferates specifically in cancer cells and kills cancer cells. Up to now, an oncolytic virus consisting of 10 or more kinds of various viruses has been developed, and the oncolytic adenovirus (Ad) is one of the most clinically developed oncolytic viruses. On the other hand, there are 67 serotypes of Ad alone derived from humans, and Ad derived from various animal species other than human has also been identified, but most of them are being developed as oncolytic Ad. Is derived from human type 5 Ad. In the case of the Ad vector lacking the proliferative ability (mainly used as a vaccine vector for infectious diseases), vectorization other than human type 5 Ad is in progress, but it is almost used as an oncolytic virus. Limited to human type 5 Ad.

With the progress of gene therapy, the characteristics and functions of various gene therapy vectors have become clearer as well as improved. In recent years, the proper use of each vector has advanced depending on the disease, administration method, and administration site (target cell). Ad, by reversely utilizing the fact that it is accompanied by an immune reaction, is used as a vaccine vector against emerging/re-emerging infectious diseases such as HIV (Human Immunodeficiency Virus), Ebola hemorrhagic fever, highly pathogenic influenza, or cancer cell-specific virus amplification. By virtue of this, non-clinical and clinical trials are actively progressing as an oncolytic virus that kills cancer cells.

Regarding the oncolytic virus, the herpesvirus IMLYGICTM (Talimogene Laherparepvec) carrying the GM-CSF gene was approved by the US FDA as a therapeutic agent for melanoma in 2015, but the Ad-based oncolytic virus is also active. Development has been advanced. Initially, the virus ONYX-015 (dl1520) deficient in the E1B-encoded 55 kDa protein was noticed so as to specifically replicate and kill cells p53-deficient (many cancer cells). However, it was not approved in the United States. By the way, in 2006, Oncorine H101, a virus preparation almost equivalent to ONYX-015, was approved as a drug in China. At present, a tumor-specific promoter that expresses the E1A (and E1B) gene essential for Ad replication to cause virus replication in a tumor-specific manner and kill cells is often used. The anti-tumor effect of oncolytic Ad is not only the direct cell killing effect associated with virus replication, but also the stimulation of DAMPs (damage-associated molecular patterns) associated with cancer cell death and the induction of immune system such as anti-virus immunity. It is also considered to make a large contribution, and from the viewpoint of efficiently activating antitumor immunity, it has been attempted to incorporate various cytokines or immune-related genes that activate the immune system into oncolytic Ad. ..

However, most of the oncolytic Ad that have been developed so far have human type 5 Ad as the basic skeleton. In order to change the infected area and improve the ability to infect cancer cells, oncolytic Ad in which a foreign peptide is inserted into the fiber region that plays an important role in cell infection, and Ad derived from other serotypes Oncolytic Ad substituted with fiber has also been developed, but the basic skeleton is derived from human type 5 Ad. There are very few examples of producing oncolytic Ad with a basic structure other than human type 5 Ad. For example, PsiOxus Therapeutics has a clinical study of a chimeric virus (Enadenotucirev; ColoAd1) consisting of human type 3 and type 11 Ad. Testing is in progress.

On the other hand, particularly in the case of Ad vaccine vectors (non-proliferative Ad vectors) against infectious diseases, studies using Ad vectors derived from species other than human type 5 Ad and serotypes have been actively conducted. Among them, human type 35 Ad belonging to subgroup B group, which is different from human type 5 Ad belonging to subgroup C group, has a very low existing antibody retention rate in humans, and as an infectious receptor, all except human erythrocytes are excluded. It has the characteristic of using CD46, which is expressed in cells of E. coli, in particular, highly expressed in cancer cells, as a receptor, and was considered to be promising as a vaccine vector against infectious diseases. However, it has been revealed that the human type 35 Ad vector has higher innate immunity-inducing ability than the conventional human type 5 Ad vector (Non-patent document 1: J. Virol., 88, 10354, 2014), and for this reason. It was revealed that the expression period of the antigen gene was shortened and the vaccine effect was diminished.
Prior Art Document Non-Patent Document 1: J. Virol. , 88, 10354, 2014

In the present invention, an oncolytic virus for treating cancer by human type 35 Ad has been desired.

Therefore, the present inventors have succeeded in developing an oncolytic virus preparation having human 35 type Ad as a basic skeleton and completed the present invention.
That is, the present invention is as follows.
(1) A recombinant oncolytic type 35 adenovirus in which the hTERT promoter or Survivin promoter and the E1 gene are integrated into the type 35 adenovirus genome.
(2) A recombinant oncolytic type 35 adenovirus in which the hTERT promoter or Survivin promoter and the E1A gene are integrated into the type 35 adenovirus genome.
(3) A recombinant oncolytic type 35 adenovirus in which the hTERT promoter or Survivin promoter, E1A gene, E1B promoter and E1B gene are integrated into the type 35 adenovirus genome.
(4) The recombinant oncolytic type 35 adenovirus according to (2) or (3), wherein the E1A gene is a mutant type.
(5) The recombinant oncolytic type 35 adenovirus according to any one of (1) to (4), which further comprises a gene encoding a cell death induction-related protein or an immunostimulation-related protein.
(6) A pharmaceutical composition comprising the recombinant oncolytic type 35 adenovirus according to any one of (1) to (5). Here, examples of the pharmaceutical composition include those for treating cancer, and examples of the cancer include breast cancer.
(7) The recombinant oncolytic type 35 adenovirus according to any one of (1) to (5) or the pharmaceutical composition according to (6) 6 is administered to a cancer patient. How to treat cancer.
(8) The treatment method according to (7), wherein the cancer patient is a breast cancer patient.
Effect of the invention

The high innate immunity-inducing ability of human type 35 Ad, on the contrary, leads to the enhancement of anti-tumor immunity in the case of an oncolytic virus for the purpose of treating cancer, and is expected to be a great advantage.

The present inventors have started the development of an oncolytic virus preparation having human type 35 Ad as a basic skeleton and succeeded in the development (FIG. 1). In the present invention, the modification of each viral gene suitable for the oncolytic virus revealed in the studies so far was carried out to prepare an optimal oncolytic Ad basic virus for cancer treatment. In FIG. 1, (3) shDicer cassette attachment and (4) mutant E1a use can be selected as necessary and are not necessarily essential configurations. In the present invention, for example, a shDicer cassette that enhances the antitumor effect can be inserted (Fig. 2). However, the gene to be inserted is not limited to the shDicer cassette, and a gene encoding a cell death induction-related protein or an immunostimulation-related protein can be inserted. The shDicer cassette, a gene encoding a cell death induction-related protein, and a gene encoding an immunostimulatory-related protein may be inserted individually or in appropriate combination.
Examples of the gene encoding the cell death induction-related protein include, but are not limited to, a gene encoding p53, a gene encoding p38, a gene encoding SAPK, and the like.
Examples of the gene encoding the immunostimulation-related protein include a gene encoding PD-1 and a gene encoding LAG-3.

Further, in one embodiment of the present invention, an immune checkpoint inhibitor or radiation therapy can be used in combination. So far, the Ad-derived E1B 55 kDa protein inhibits DNA repair by degrading the Mre11/Rad50/NBS1 (MRN) protein complex induced by double-stranded DNA damage in cancer cells caused by radiation It has been demonstrated to dramatically increase sensitivity (Fig. 3). In addition, the present inventors have a track record of developing the 35-type Ad vector for the first time in the world (Patent No. 4237449; Gene Ther., 10, 1041, 2003), and have the best technology and knowledge on Ad modification in Japan and overseas. Have

In the present invention, an oncolytic virus having a human 35 type Ad as a basic skeleton is prepared, and a viral gene (promoter, use of mutant E1a gene, etc.) that influences viral replication and antitumor effect is modified to produce a foreign gene. Optimize by inserting. A variety of modified human type 35 Ad was examined for virus replication ability, antitumor effect, innate immunity and antitumor immune activation ability under both in vitro and in vivo conditions, and a new oncolytic alternative to human type 5 Ad To clarify the usefulness of Ad.

It is a schematic diagram of an example of the oncolytic type 35 adenovirus (Ad) of the present invention. It is a figure which shows the mechanism of Ad replication ability improvement by knockdown of Dicer expression. It is a figure which shows the mechanism of the synergistic effect of oncolytic Ad and radiation. A: When normal cancer cells not infected with oncolytic Ad are irradiated, a repair mechanism of DNA damage works. B: When normal cancer cells infected with oncolytic Ad were irradiated, Ad-derived E1B 55 kDa protein induces Mre11/Rad50 induced by double-strand DNA damage in cancer cells due to radiation. Degrading the /NBS1(MRN) protein complex inhibits DNA repair and dramatically enhances radiation sensitivity. It is a figure which shows the strategy for oncolytic type 35 Ad (OAd35) construction. It is a figure which shows the result of the flow cytometric analysis of CAR and CD46 expression in a human cancer cell. Cells were incubated with anti-CAR (RmcB) antibody or anti-CD46 (M177) antibody. After washing the cells, the cells were incubated with a PE-conjugated goat anti-mouse Ig secondary antibody prior to FCM analysis. Gray histograms show isotype control antibodies and red histograms show anti-CAR (RmcB) or anti-CD46 (M177) antibodies. It is a figure which shows the cancer cell lytic activity of OAd5 and OAd35. OAd5 and OAd35 were infected with a human cancer cell line with VP (vector particle)/cell shown in the figure. After incubation for 5 days, cells were stained with crystal violet reagent. It is a figure which shows the result of infecting a human cancer cell line with 300 VP/cell of oncolytic type 5 Ad (OAd5) and oncolytic type 35 Ad (OAd35). Cell viability was determined by WST-8 assay at each time point shown in the figure. The data was normalized by the data of the mock infection group. It is a figure which shows the result of infecting a normal human cell with VP/cell shown in the figure by OAd5 and OAd35. After incubation for 5 days, cells were stained with crystal violet reagent. It is a figure which shows the cancer cell lytic activity of OAd5 and OAd35. It is a figure which shows the cancer cell lytic activity of OAd5 and OAd35. It is a figure which shows the result of adhesion of OAd to the cancer cell surface. Human cancer cell lines were infected with OAd5 and OAd35 at 100 VP/cell and incubated at 4°C. After a 1.5 hour incubation, the copy number of the viral genome of OAd was determined by real-time PCR analysis. It is a figure which shows Ad genome copy number in the cancer cell after oncolytic Ad(OAd) infection. Human cancer cell lines were infected with OAd5 and OAd35 at 100 VP/cell. At each time point shown in the figure, the copy number of the viral genome of OAd was determined by real-time PCR analysis. It is a figure which shows the cancer cell lytic activity of OAd in the presence of anti-Ad5 serum. HepG2 cells (A) and T24 cells (B) were infected with OAd in the presence of mouse anti-Ad5 serum. After incubation for 5 days, cell viability was determined by WST-8 assay. Data were normalized by data from mock infection group. It is a figure which shows the measurement result of the tumor volume after intratumor administration of OAd35. OAd was injected intratumorally at a dose of 2.4×10 ⁹ VP/mouse. Arrows indicate the days after virus injection (day 0 and day 3). Tumor volume is the mean tumor volume ± S.M. E. Expressed as ^* P<0.05 (vs. PBS). (N=5)

The present invention will be described in detail below. The scope of the present invention is not limited to these descriptions, and other than the following examples, the present invention can be appropriately modified and implemented within a range not impairing the gist of the present invention. The present specification includes the entire US provisional application No. 62/804,334 (filed on February 12, 2019), which is the basis for claiming priority of the present application. Further, all the publications cited in the present specification, for example, prior art documents, and publications, patent publications and other patent documents are incorporated herein by reference.

In the present invention, oncolytic human type 35 Ad was developed as a new oncolytic virus replacing conventional oncolytic human type 5 Ad, and its usefulness was evaluated. Oncolytic human type 35 Ad has an extremely low antibody retention rate in humans as compared with conventional oncolytic human type 5 Ad, so that the influence of existing antibodies is eliminated, and it is expressed in a wide range of human cells as an infection receptor. It was shown that infection is enhanced by using CD46 whose expression is increased in cancer cells. Further, the high innate immunity-inducing ability of human type 35 Ad leads to enhancement of anti-tumor immunity in the case of oncolytic virus for the purpose of treating cancer, and is expected to be an extremely great advantage (Table 1). ..

In the present invention, first, a plasmid having the entire genome of human type 35 Ad was prepared, and then a system capable of producing various modified oncolytic human type 35 Ad genomes by simple recombination of the plasmid was developed. This plasmid is linearized by cutting at unique restriction enzyme sites present at both ends of the viral genome, and the desired virus can be produced by transfecting any cell (for example, HEK293 cell).

Furthermore, the present inventors have revealed that knocking down Dicer enhances Ad replication (WO2015/25940; Mol. Cancer Ther., 16, 251, 2017, etc.). Therefore, in the present invention, the shDicer cassette can be added, which makes it possible to enhance the cell killing effect (FIG. 2). Further, in the present invention, it is also possible to add a mutation to a sequence that avoids activation of innate immunity of the E1a gene, which is essential for viral growth, and thereby optimizes a tumor-specific promoter (use of Survivin or hTERT promoter). , ADP (adenovirus death protein) gene retention function and the like can be imparted, and more excellent oncolytic human type 35 Ad can be obtained.

Further, in the present invention, various oncolytic human type 35 Ad viruses are analyzed for viral characteristics such as virus replication ability and cytotoxic activity. Further, the virus of the present invention is expected to have an innate immune activating effect and an antitumor immunity inducing effect, and a tumor growth suppressing effect in combination with an immune checkpoint inhibitor.
On the other hand, the Ad-derived E1B 55 kDa protein inhibits DNA repair by degrading the Mre11/Rad50/NBS1 (MRN) protein complex induced by double-strand DNA damage in cancer cells caused by radiation, resulting in radiation sensitivity. It has been proved to dramatically increase (Cancer Res., 70, 9339, 2010). Therefore, in the present invention, oncolytic Ad and radiation therapy can be combined, and thereby a synergistic antitumor effect can be expected (FIG. 3).

Oncolytic virus is expected to be a novel anti-cancer agent because it infects and proliferates specifically in cancer cells and kills cancer cells. Up to now, oncolytic viruses based on 10 or more viruses have been developed, and oncolytic adenovirus (Ad) is one of the most clinically developed oncolytic viruses. Most of the oncolytic Ad that have been developed so far are based on type 5 Ad (Ad5) belonging to the subgroup C group, but in recent years, (i) Ad5 is an Ad5 infection receptor, Coxsackievirus. Problems such as difficulty in infecting and adenovirus receptor (CAR)-negative cells and (ii) that many adults carry a neutralizing antibody against Ad5 have become apparent. On the other hand, type 35 Ad (Ad35) belonging to subgroup B is characterized in that it uses CD46 expressed in all human cells except erythrocytes as an infection receptor and has a low antibody retention rate in adults. .. From this, it is expected that the Ad35-based oncolytic Ad can infect a wide range of cancer cells including CAR-negative cells and can avoid the influence of anti-Ad5 antibody.

In one aspect of the present invention, E1 gene (divided into E1A gene and E1B gene), which is essential for Ad self-proliferation, is mounted downstream of a tumor-specific promoter so that tumor cell-specific growth can be achieved. Design Ad. As a tumor-specific promoter, a human telomerase Reverse Transcriptase (hTERT) promoter, which is highly active in many human cancer cells, is used. As the promoter, the Survivin promoter can also be used. An E1 gene expression cassette designed to insert the E1A gene downstream of the promoter and to express the E1B gene from the virus original promoter is used for the type 5 and 35 type Ad genomes. It was inserted into the E1 deletion region. The E1A gene and E1B gene of OAd5 are used in oncolytic Ad5 (OAd5), and the E1A gene and E1B gene of OAd35 are used in oncolytic Ad35 (OAd35), respectively. A plasmid encoding the Ad genome into which the E1 gene expression cassette had been inserted was treated with a restriction enzyme to linearize it, and then each OAd was recovered by introducing the gene into a packaging cell. As packaging cells, H1299 cells were used for OAd5, and HEK293 cells were used for OAd35. These OAds were allowed to act on various cultured cells, and the cell killing effect and Ad genome amount were measured.

In order to examine the cell-killing effect of each OAd produced on cancer cells, production was performed on CAR-positive human cancer cells (HepG2 cells, A549 cells, H1299 cells) and CAR-negative human cancer cells (T24 cells, MCF-7 cells). The oncolytic Ad5 (OAd5) and the oncolytic Ad35 (OAd35) were allowed to act, and the cell viability 5 days after the viral action was evaluated. As a result, both OAds showed a high cell killing effect on CAR-positive cells. On the other hand, while OAd5 did not show a significant cell killing effect on CAR-negative cells at all virus doses, OAd35 showed 300 VP/cell or more in T24 cells and 10 VP/cell in MCF-7 cells. Almost all cells were killed by the above.

Also, each OAd did not show a remarkable cell killing effect on various normal human cells. Next, each OAd was allowed to act on the above cancer cell lines, and the amount of virus particles bound to the cell surface was examined. As a result, each OAd efficiently binds to CAR-positive cells while it binds to CAR-negative cells. On the other hand, only OAd35 showed high binding ability. Furthermore, when each anti-Ad5 serum was incubated with each OAd for 30 minutes and then allowed to act on various cancer cells, the cell killing effect of OAd5 was remarkably attenuated by the anti-Ad5 antibody, whereas that of OAd35 was not inhibited by the anti-Ad5 serum. It did not receive and showed a high cell killing effect.

The OAd35 developed in the present invention showed a higher cell killing effect on human cancer cells regardless of the expression of CAR, as compared with the conventional OAd5. It also showed a high cell killing effect even in the presence of anti-Ad5 antibody. From the above, it was shown that OAd35 can overcome the problems of OAd5.

<Treatment method and pharmaceutical composition>
The present invention provides a pharmaceutical composition containing the recombinant oncolytic type 35 adenovirus. The present invention also provides a method for treating cancer, which comprises administering the recombinant oncolytic type 35 adenovirus or the pharmaceutical composition to a cancer patient.
The type of tumor (cancer) to be treated is not limited and includes, for example, brain tumor, head and neck cancer, gastric cancer, colon cancer, lung cancer, liver cancer, prostate cancer, pancreatic cancer, esophageal cancer, bladder cancer, gallbladder cancer. , Bile duct cancer, breast cancer, uterine cancer, thyroid cancer, ovarian cancer, leukemia, lymphoma, sarcoma, mesenchymal tumor, and the like, with breast cancer being preferred.

The pharmaceutical composition of the present invention can be applied to the affected area as it is, or any known method, for example, injection in a tumor, vein, muscle, intraperitoneal or subcutaneous, or inhalation from nasal cavity, oral cavity or lung, It can also be introduced into a living body (target cell or organ) by oral administration, intravascular administration using a catheter or the like.

Further, the recombinant oncolytic type 35 adenovirus of the present invention may be used as it is, or a known pharmaceutically acceptable carrier such as an excipient, a bulking agent, a binder, a lubricant, or a known addition. Agents (buffering agents, isotonicity agents, chelating agents, coloring agents, preservatives, perfumes, flavoring agents, sweetening agents, etc.) can be mixed.

The pharmaceutical composition of the present invention includes oral administration agents such as tablets, capsules, powders, granules, pills, solutions, syrups, parenteral administration agents such as injections, external preparations, suppositories, and eye drops. It can be administered orally or parenterally depending on the form. Preferably, local injection into tumor, muscle, abdominal cavity and the like, injection into vein and the like are exemplified.

The dose of recombinant oncolytic type 35 adenovirus is appropriately selected according to the type of active ingredient, administration route, administration subject, age, weight, sex of patient, symptoms and other conditions. The dose is preferably about 10 ⁶ to 10 ¹¹ PFU, preferably about 10 ⁹ to 10 ¹¹ PFU, and can be administered once a day or divided into several times.
It should be noted that the recombinant oncolytic type 35 adenovirus of the present invention does not prevent the combined use with known anticancer agents, radiation and the like.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

1. Experimental material
Cells HEK293 cells are DULBECCO'S MODIFIED EAGLE'S MEDIUM (DMEM) (10% Fetal Bovine Serum (FBS), 100 U/mL penicillin, 1 mM L-glutamine-containing), A549 cells, HepGNH cells, F24C cells, T24 cells, F24 cells, and T cells. −5 cells contained DMEM (containing 10% FBS, 100 μg/mL streptomycin, and 100 U/mL penicillin), and H1299 and MCF-7 cells contained RPMI1640 MEDIUM (containing 10% FBS, 100 μg/mL streptomycin, and 100 U/mL penicillin). It was used for cultivation in the presence of 37° C., saturated vapor pressure and 5% CO ₂ . All the FBSs used in this example were used after being inactivated at 56° C. for 30 minutes.

Oncolytic Ad based on the plasmid Ad5, pAdHM3-hmE1 which is a plasmid for producing OAd5 was prepared as follows.

First, the region containing BGH poly (A) sequence fragment and hTERT promoter (SEQ ID NO: 11) was amplified by PCR using pHMTERT-L (plasmid with luciferase gene inserted downstream of hTERT promoter) as template DNA. By doing so, a BGH poly(A) sequence fragment and an hTERT promoter fragment were obtained. Next, pHM15 (shuttle plasmid; Hum Gene Ther. 2003 Sep1; 14(13): 1265-77.) was ligated with the EcoRI/ApaI-treated fragment and the BGH poly(A) sequence fragment described above to obtain pHM15-BGH. Obtained. Next, the pHM15-BGH fragment treated with BamHI/PstI and the hTERT promoter fragment that could be amplified by PCR were ligated to obtain pHM15-hTERT carrying the BGH poly(A) sequence downstream of the hTERT promoter.

Next, a fragment obtained by treating pHM15-hTERT with EcoRI/BamHI and a synthetic oligo DNA encoding the EcoRI-EcoRV-BamHI recognition sequence were ligated to obtain pHM15-hTERT-EEB. Next, the E1 gene was amplified by the PCR method using pTG3602 having the full length of the Ad genome as a template DNA to obtain an E1 gene fragment. Next, p3xFLAG-CMV-10 (a cloning plasmid having a multi-cloning site; reference URL:
https://www. sigmaaldrich. com/catalog/product/sigma/e7658? lig=ja&region=JP) was ligated to the EcoRV-treated fragment and the E1 gene fragment to obtain p3xFLAG-CMV-E1. Next, the fragments obtained by treating each of pHM15-hTERT-EEB and p3xFLAG-CMV-E1 with EcoRI/BamHI were ligated to obtain pHM15-hTERT-E1 plasmid having the E1 gene downstream of the hTERT promoter.

In order to delete the XbaI site on pHM15-hTERT-E1, pHM15-hTERT-E1-ΔXbaI was obtained using the Quick Change Lighting Mutagenesis kit and the primers. In addition, a fragment obtained by treating p3xFLAG-CMV-E1 with SacI was self-ligated to obtain p3xFLAG-CMV-E1Δss. Then, p3xFLAG-CMV-E1ΔssΔXbaI was obtained by subjecting p3xFLAG-CMV-E1Δss to XbaI treatment, smoothing with Klenow treatment, and self-ligation. Next, pHM15-hTERT-E1-ΔXbaI and p3xFLAG-CMV-E1ΔssΔXbaI were ligated with BamHI/SacI-treated fragments to obtain pHM15-hTERT-E1-2ΔXbaI. Next, pE1-BS (FASMAC) and pHM15-hTERT-E1-2ΔXbaI, which are plasmids purchased by artificially synthesizing a sequence in which the LTCHE motif of Ad E1 gene was replaced with the VTSHD motif, were treated with SacI/BspEI, respectively. Ligation was performed to obtain pHM15-hTERT-mE1-2ΔXbaI.

Then, the fragment treated with SpeI of pHM15-hTERT-mE1-2ΔXbaI and the fragment treated with XbaI of pHM3 (shuttle plasmid; Hum Gene Ther. 1998 Nov 20;9(17):2577-83) were ligated to obtain pHM3-hmE1. Obtained. Next, the fragments obtained by treating pHM3-hmE1 and pHM5 with I-CeuI/PI-SceI respectively were ligated to obtain pHM5-hmE1 which is a plasmid carrying a cassette expressing the Ad1 E1 gene under the control of hTERT promoter. Finally, pAdHM3-hmE1 was obtained by ligating the fragments of pAdHM3 and pHM5-hmE1, which are plasmids carrying the Ad5 genome lacking the E1 gene, with I-CeuI/PI-SceI.

The plasmid AdAd2-based oncolytic Ad for OAd35 production, pAdMS2-hTERT-mE1, was constructed as follows. First, pAd35-mE1-1, which is a plasmid in which the 5′ITR of Ad35 and the middle of the E1 gene (Ad35 2804 MfeI) are installed downstream of the survivin promoter, and the middle of the E1 gene of Ad35 to the middle of the pIX gene (Ad35 2804). The plasmid pAd35-mE1-2, which has a sequence of about 4603 bp), was artificially synthesized (outsourced to Genewiz).

Fragmet obtained by treating these plasmids with MfeI/SpeI was ligated to obtain pAd35-surv-mE1. Next, a fragment of pAdMS2, which is a plasmid carrying the Ad35 genome lacking the E1 gene, treated with SwaI and a fragment of pAd35-surv-mE1 treated with SalI were transformed into E. coli. E. coli BJ5183 (recBC and sbcBC) was transformed at the same time to induce homologous recombination to obtain pAdMS2-surv-mE1 in which the cassette expressing the Ad1 E1 gene under the control of survivin promoter was mounted in the E1-deficient region. ..

Finally, using pHMTERT-L as a template DNA and using the following primers, hTERT promoter fragment 2 obtained by amplifying the gene by the PCR method and pAdMS2-surv-mE1 treated with PmeI are subjected to In-Fusion reaction. Thus, pAdMS2-hTERT-mE1 was prepared.

Oncolytic Ad (OAd)
By linearizing the plasmid DNAs obtained by treating pAdHM3-hmE1 and pAdMS2-hTERT-mE1 with PacI and SbfI, respectively, and transfecting them into HEK293 cells using Lipofectamine 2000 (Invitrogen), OAd-tANB (OAd5) and OAd35, respectively. -TANB(OAd35) was obtained. Then, OAd-tANB was subjected to a third-fourth infection with H1299 cells and OAd35 with HEK293 cells in a large-scale preparation. Each obtained Ad was purified by density gradient centrifugation of cesium chloride, and dialyzed against a solution consisting of 10 mM Tris (pH 7.5), 1 mM MgCl ₂ , and 10% glycerol. OAd35-sANB was obtained by transfecting pAdMS2-surv-mE1 with SbfI and then transfecting HEK293 cells. The shDicer expression cassette was inserted downstream of the E1B expression cassette of pAdMS2-hTERT-mE1 and pAdMS2-surv-mE1 to obtain pAdMS2-hTERT-mE1-shDicer and pAdMS2-surv-mE1-shDicer. After treating these plasmids with SbfI, they were transfected into HEK293 cells to obtain OAd35-tANB-shDicer and OAd35-sANB-shDicer.
The physical titer (VP) of each Ad was measured by the method of Maizel et al.

2. Evaluation method
Flow cytometric analysis 5×10 ⁵ cells of each cell were suspended in 100 μL of PBS containing 3% FBS, and Mouse anti-CAR monoclonal antibody (RmcB), Mouse anti-CD46 monoclonal anti-body (M177) or Pur anti-MoI anti-molar (M177) was added. The reaction was allowed to proceed on the ice for 1 hour while protected from light. After washing with 4 mL of PBS containing 2% FBS, the mixture was centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed by suction.

The cells were suspended again in 100 μL of PBS containing 2% FBS, 1 μL of PE-labeled Goat Anti-Mouse Ig was added, and the reaction was carried out on ice for 30 minutes in the dark. After washing with 4 mL of PBS containing 2% FBS, centrifugation was carried out at 1500 rpm for 5 minutes, the supernatant was removed by suction, suspended in 500 μL of PBS containing 2% FBS, and the expression was analyzed using a flow cytometer (MACS Quant Analyzer; Miltenyi Biotec). Data were analyzed by FCS multicolor data analysis software (Flojo).

Determination of Ad Genome Copy Number After collecting each cell, Total DNA was extracted using DNeasy Blood & Tissue Kit (QIAGEN), and Ad genome copy number was quantitatively determined using THUDERBIRD SYBR qPCR Mix (TOYOBO) and the following primers. PCR was performed. For the measurement, StepOnePlus real-time PCR systems (Applied Biosystems) were used.

Crystal Violet Assay Oncolytic Ad was allowed to act on each cell at 100 to 1000 VP/cell, and after 5 days, live cells were stained with crystal violet.

Cell viability assay Oncolytic Ad was allowed to act on each cell at 300 VP/cell, and cell viability was measured using Cell Counting Kit-8 (Dojindo Laboratories).

Immunization with Ad5 vector Anti-Ad5 immunity was induced in C57BL/6 mice by tail vein administration of E1-deficient Ad5 vector at 1×10 ¹⁰ VP/mouse. Blood was collected by orbital blood sampling 19 days after Ad5 vector administration, and allowed to stand at 37° C. for 1 hour for complete blood coagulation, and then left at 4° C. overnight. The next day, centrifugation (3,000 rpm, 5 minutes) was performed, and the supernatant was collected as anti-Ad5 serum.

Oncolytic activity of each OAd in the presence of anti-Ad5 serum Antiserum collected from a mouse immunized with E1-deficient Ad5 vector and each oncolytic Ad were diluted and mixed in a medium containing no FBS. The mouse antiserum was serially diluted every two-fold dilution so that the final dilution ratio was 1/400 to 1/1600, and the oncolytic Ad was adjusted to 300 VP/cell (total 50 mL). The prepared solution was incubated for 30 minutes and then allowed to act on the cells. After 1.5 hours, an equal amount of a medium containing 20% FBS was added, and after 5 days, cell viability was measured using Cell Counting Kit-8 (Dojindo Laboratories).

Intratumoral administration to Mouse xenograph model H1299 cells (3×10 ⁶ cells per mouse) containing 50% Matrigel (Corning, Corning, NY) at the age of 5 weeks in female BALB/c nu/nu mice (Japan SLC, Hamamatsu, Japan) ) Was subcutaneously injected into the right flank. After tumors grew to approximately 5-6 mm in diameter, mice were randomly assigned to three groups. PBS, OAd5, and OAd35 were injected intratumorally into each group at a dose of 2.4×10 ⁹ VP/mouse and reinjected 3 days later. Tumor size was measured every 3 days. The tumor volume was calculated by the following formula.
Tumor volume (mm ³ )=1/2×a×b ² (a indicates the longest dimension (major axis), b indicates the shortest dimension (minor axis)).

3. result
Method for Producing Oncolytic Ad35 (OAd35) In this example, by mounting the E1 gene (divided into E1A gene and E1B gene), which is essential for Ad self-proliferation, downstream of a tumor-specific promoter, The oncolytic Ad was designed to grow in. As a tumor-specific promoter, a human Telomerase Reverse Transcriptase (hTERT) promoter (SEQ ID NO: 11), which has high activity in many human cancer cells, or a Survivin promoter was used. First, in the production of oncolytic type 35 Ad (OAd35), the E1A gene (569 to 1441 bp (SEQ ID NO: 6)) was used as a cancer cell-specific promoter with reference to the gene sequence information of Ad35 (SEQ ID NO: 5). An E1 gene expression cassette was constructed so that it was inserted downstream of a certain hTERT promoter or the Survivin promoter, and the Ad35 E1B gene was expressed from the virus original promoter (FIG. 4). Here, as the E1A gene, a mutant E1A gene (SEQ ID NO: 7) was used instead of the wild-type E1A gene (SEQ ID NO: 6). The mutation is a mutation in the nucleotide sequence that avoids the activation of innate immunity of the E1A gene, which is essential for virus growth, and is specifically the 343rd T(th) in the nucleotide sequence of SEQ ID NO:6. Thymine) is replaced with G (glycine), the 350th G is replaced with C (cytosine), and the 357th A (adenine) is replaced with T (in short, in the base sequence of SEQ ID NO: 6). The 343rd to 357th bases "TTGCACTGCTATGAA" are replaced with "GTGCACTCCTATGAT". The mutation can be appropriately performed using a well-known gene recombination technique.

As the E1B gene promoter region and E1B gene region of Ad35, 1442 to 3400 bp (SEQ ID NO: 8) were inserted downstream of the E1A expression cassette with reference to the gene sequence information of Ad35 (SEQ ID NO: 5). Here, the promoter region of the E1B gene is presumed to be 1442-1610 bp (SEQ ID NO: 9), and the E1B gene region is 1611-3400 bp (SEQ ID NO: 10).
Thus, as the E1 gene expression cassette, "wild type E1A gene + E1B gene promoter region + E1B gene" (SEQ ID NO: 12) and "mutant E1A gene + E1B gene promoter region + E1B gene" (SEQ ID NO: 13) were used. ..
Further, a donor plasmid was prepared in which about 450 bp (1 to 455 bp) upstream of the E1 deletion region of Ad35 and about 1200 bp (3401 to 4634 bp) downstream were added as homology arms to both sides of the E1 gene expression cassette.

The donor plasmid and the plasmid encoding the Ad35 genome were linearized by restriction enzyme treatment, and E. E. coli BJ5183 (recBC and sbcBC) was transformed at the same time to induce homologous recombination to insert the E1 gene expression cassette into the E1 deletion region of the Ad35 genome (FIG. 4). Similarly, for oncolytic type 5 Ad (OAd5), an E1 gene expression cassette designed to insert the E1A gene of Ad5 downstream of the hTERT promoter and to express the E1B gene of Ad5 from the virus original promoter was prepared. It was inserted into the E1 deletion region of the type 5 Ad genome.

A plasmid encoding the Ad genome into which the E1 gene expression cassette had been inserted was treated with a restriction enzyme to linearize it, and then the gene was introduced into HEK293 cells, which are packaging cells, to recover OAd5 and OAd35. After that, H1299 cells for OAd5 and HEK293 cells for OAd35 were quaternized to prepare a large amount. Since HEK293 cells have the E1 gene of Ad5 in their genome, amplification of OAd5 in HEK293 cells causes homologous recombination between the E1 gene expression cassette in the OAd5 genome and the E1 gene region in the cells. Since the wild-type Ad5 may be contaminated, H1299 cells not having the E1 gene were used for amplification of OAd5.

After each OAd was prepared in a large amount, it was purified by cesium chloride density gradient centrifugation. The physical titer of each OAd, Virus Particle (VP)/cell, was measured. As a result, about OAd35, a titer about 10 times lower than that of OAd5 was obtained. The physical titer of OAd35 was about 10 times lower than that of OAd5. The ratio of physical and biological titers of OAd35 was 1:8.5, which was comparable to that of OAd5 (Table 3).

Expression of Ad Infectious Receptor in Human Cancer Cell Lines Ad5 and Ad35, which are the basis of OAd, prepared this time have CAR and CD46 as infectious receptors, respectively. Therefore, each of 5 types of human cancer cell lines (HepG2 cells; human liver cancer-derived, A549 cells; human lung cancer-derived, H1299 cells; human lung cancer-derived, T24 cells; human bladder cancer-derived, MCF-7 cells; human breast cancer-derived) The expression level of the receptor was measured by flow cytometry (Fig. 5).

As a result, CAR and CD46 were both highly expressed in HepG2 cells, A549 cells, and H1299 cells. On the other hand, in MCF-7 cells, CD46 was highly expressed, but CAR was only slightly expressed. Moreover, CAR was not expressed at all in T24 cells, but CD46 was highly expressed. From these results, hereinafter, HepG2 cells, A549 cells, and H1299 cells were used as CAR-positive cells, and T24 cells and MCF-7 cells were used as CAR-negative cells.

Cell-killing effect of each OAd in human cultured cells Next, in order to examine the cell-killing effect of each OAd produced in this time in the human cancer cell line, each OAd was allowed to act on various human cancer cell lines, and after 5 days of virus action, crystal was added. Violet staining was performed to measure cell viability (Fig. 6A). With crystal violet staining, only live cells are stained blue-violet.

As a result, CAR-positive HepG2 cells showed high cell killing effect in both OAd acting groups, and almost all cells were killed at all virus doses. Further, although CAR was sufficiently expressed in A549 cells, no sufficient cell killing effect was observed in the OAd5 acting group, and a high cell killing effect was shown in the OAd35 acting group. Although H1299 cells expressed CAR and CD46 similarly to HepG2 cells and A549 cells, the cell killing effect was higher in the OAd5 acting group than in the OAd35 acting group.

On the other hand, CAR-negative T24 cells did not show a cell-killing effect at all viral doses in the OAd5 action group, whereas almost all cells in the OAd35 action group died at 300 VP/cell. In MCF-7 cells, almost all cells were dead at 300 VP/cell in the OAd5 acting group and 10 VP/cell in the OAd35 acting group. This is presumably because OAD5 could be infected because CAR expression was slightly observed in MCF-7 cells. Among all the cell lines, Ad35 had the highest killing effect on MCF-7, which is a breast cancer cell. This phenomenon cannot be explained only by the expression of receptors such as CAR and CD45, and the unknown life cycle differences (infection pathway, uncoating, nuclear translocation, transcription translation, viral particle formation, etc.) Is considered to be the cause. Therefore, the virus of the present invention is considered to be particularly useful for treating breast cancer.

Furthermore, in order to quantitatively measure the cell killing effect of each OAd, the cell viability was measured over time using the WST-8 reagent (Fig. 6B). As a result, results correlated with the results of crystal violet staining were obtained. Specifically, in HepG2 cells, both OAd action groups showed a high cell killing effect, but in A549 cells, T24 cells, H1299 cells and MCF-7 cells, a significantly higher cell killing effect in the OAd35 action group than in the OAd5 action group. Showed the effect.

Next, in order to examine the safety of each OAd against human normal cells, various human normal cells (MRC-5 cells; human fetal lung fibroblasts, NHLF; adult lung fibroblasts, HUVEC; human umbilical vein endothelium) Each cell was treated with each OAd and subjected to crystal violet staining in the same manner as above to measure cell viability (FIG. 6C). As a result, no significant cell killing effect was shown in each OAd acting group. In all human normal cells, cell death was observed at high virus doses (300, 1000 VP/cell) in the OAd35 acting group. It is considered that this is because the virus was infected at a dose extremely higher than the dose assumed in the in vivo administration, which caused damage to the cells and cell death was observed.
Furthermore, the cell-killing effect on various cancer cells was examined using each OAd (FIG. 6D, FIG. 6E).
As a result of the above, it was revealed that the newly produced OAd35 exhibits a cell killing effect specifically in cancer cells.

Cell-Binding Ability of Each OAd Next, in order to examine the binding ability of each OAd to each human cancer cell line, each OAd was allowed to act for 1.5 hours at 4° C., and the genome of OAd bound to the cell surface was examined. The amount was measured by the quantitative PCR method (Fig. 7). As a result, both OAd5 and OAd35 showed similar Ad genome amounts to CAR-positive cells, suggesting that both OAds can efficiently bind to CAR-positive cells. On the other hand, CAR-negative cells showed a higher Ad genome amount in the OAd35 acting group than in OAd5. From these results, it was suggested that although OAd5 is difficult to bind to CAR-negative cells, OAd35 has high binding ability regardless of CAR expression in target cells.

Infective proliferation ability of each OAd in various human cancer cells Next, in order to examine whether cell death in human cancer cells due to each OAd action shown in FIGS. 6A, 6B, 6D, and 6E is due to Ad proliferation, Each OAd was made to act on various human cancer cells, and the Ad genome amount was measured 24 hours and 72 hours after the virus action (FIG. 8). As a result, in various human cancer cells, the amount of Ad genome increased from 24 hours to 72 hours after both OAds. Particularly, in CAR-negative cells, the Ad genome greatly increased in the OAd35 acting group as compared with the OAd5 acting group. From the above results, it was shown that OAd35 can efficiently infect and propagate not only CAR-positive cells but also CAR-negative cells.

Cell-killing effect of each OAd in the presence of anti-Ad5 serum Lastly, in order to examine whether Oad35 produced this time infects target cells even in the presence of an antibody against Ad5 and shows a cell-killing effect, 1/400 dilution to 1 After incubating each OAd with anti-Ad5 serum that had been serially diluted 2-fold to a dilution of 1600 for 30 minutes, the anti-Ad5 serum acted on various human cancer cells, and the cell-killing effect after 5 days of virus action was measured.

The anti-Ad5 serum was obtained by injecting the E1-deficient Ad5 vector into the C57BL/6 mouse by tail vein administration at 1×10 ¹⁰ VP/mouse to induce anti-Ad5 immunity. As a result, in HepG2 cells, in the presence of anti-Ad5 serum, cell death was suppressed in the OAd5 acting group, whereas it was not suppressed in the OAd35 acting group (FIG. 9A). In T24 cells, cell death by OAd35 was not suppressed even in the presence of anti-Ad5 serum (Fig. 9B). From the above results, it was suggested that OAd35 can infect target cells even in the presence of anti-Ad5 antibody.

In order to investigate the anti-tumor effect of OAd35 in vivo, OAd was intratumorally administered to xenograft model in which a tumor was subcutaneously formed in mice with H1299 cells (Fig. 10). After intratumoral administration of OAd5 and OAd35, tumor growth was significantly suppressed. The in vitro oncolytic activity against H1299 cells was higher in OAd5 than in OAd35 (Fig. 6A), but there was no significant difference between OAd5 and OAd35 in the tumor growth inhibitory effect in vivo. From these results, it was shown that OAd35 has a sufficient antitumor effect in vivo.

4. Discussion In this example, the development of a novel oncolytic Ad based on Ad35 was investigated, and the properties of OAd35 such as the cell-killing effect in a human cultured cell line were examined. The OAd35 developed this time was designed so that the E1A gene was inserted downstream of the hTERT promoter or the Survivin promoter and the E1B gene was expressed from the original promoter of the E1B gene (FIG. 4).

The E1 gene expression cassette is an important factor that controls the growth of Ad and various designs have been made. In this example, the hTERT promoter was used. The hTERT promoter is reported to exhibit high activity in 80% or more of cancer cells, and is considered to be useful because it can be applied to many cancer cells. Since the plasmid encoding the OAd35 genome prepared in this Example has restriction enzyme sites inserted on both sides of the hTERT promoter region of the E1 gene expression cassette, it does not lose tumor specificity and is superior in activity to hTERT. When a tumor-specific promoter is developed or when a cancer cell of a specific tissue is targeted, the promoter can be easily exchanged.

When the cell killing effect of OAd35 in human cancer cells was measured using crystal violet staining and WST-8 reagent, OAd35 efficiently infected both CAR-positive cells and CAR-negative cells and showed a high cell-killing effect ( 6A and 6B). Although the fiber of Ad35 does not bind to the Ad5 infection receptor CAR, but binds to the Ad35 infection receptor CD46, it is considered that OAd35 was able to efficiently infect both CAR positive and negative cells.

Since there is a report that the expression of CAR decreases as the malignancy of cancer increases, OAd35 has high malignancy and can efficiently infect even cancer cells whose CAR expression is decreased or disappeared. Be expected. Further, although A549 cells expressed 90% or more of both CAR and CD46, the cell killing effect by OAd5 was low, and the high cell killing effect by OAd35 was observed.

On the other hand, OAd5 showed a higher cell-killing effect on H1299 cells, which are other human lung cancer cell lines, and therefore OAd5 showed a lower cell-killing effect on A549 cells. It is considered that the cause is not a specific phenomenon, but a difference in the life cycle of Ad5 and Ad35 (infection pathway, uncoating, nuclear translocation, transcription translation, virus particle formation, etc.).

The binding of OAd35 to the surface of various human cancer cells was examined. CAR-positive cells showed similar binding ability to both OAds, whereas CAR-negative cells exhibited OAD35 having a significantly higher binding ability. (Fig. 7).
The infection and proliferation ability of OAd5 and OAd35 in various human cancer cells was examined, and both of the OAds showed high infection and proliferation ability after 24 to 72 hours in various human cancer cells (FIG. 8). Interestingly, genomic amplification of OAd5 was also found in CAR-negative cells, but Ad5 is not only a CAR-dependent infection pathway, but also αV integrin-dependent infection on the cell surface and blood coagulation factor X (FX)-dependent. It is considered that OAd5 is infected via these pathways because it is possible to infect the cells even in the general infection.

It has been reported that more than 80% of adults have anti-Ad5 antibody by natural exposure, which is a big problem in cancer treatment using oncolytic Ad. This time, the cell-killing effect of OAd35 in the presence of anti-Ad5 antibody was examined, but the cell-killing effect of OAd5 was suppressed in the presence of anti-Ad5 antibody, whereas it was not suppressed by OAd35 (FIG. 9). Therefore, OAd35 is expected to exert an antitumor effect even on experimental animals and humans having anti-Ad5 antibody.

Currently, the intratumoral administration protocol is most used in cancer treatment using OAd. In the case of intratumoral administration, when anti-Ad5 antibody is present, oncolytic Ad leaked into the blood is immediately trapped by the antibody, and it is difficult to exert a therapeutic effect on metastatic lesions. On the other hand, Ad35 has a low antibody retention rate and does not accumulate in a specific tissue. Since it was shown in this example that OAd35 was not affected by anti-Ad5 antibody, not only the antitumor effect of intratumoral administration of OAd35 but also the metastasis after intravenous administration were observed during in vivo and clinical administration. Can also be expected to have therapeutic effects on cancer

The high innate immunity-inducing ability of human type 35 Ad in the present invention is extremely useful in the case of an oncolytic virus for the purpose of treating cancer, on the contrary that it can lead to enhancement of antitumor immunity.

SEQ ID NOs: 1-4: Synthetic DNA
SEQ ID NO: 7: Synthetic DNA
SEQ ID NO: 13: Synthetic DNA

Claims (10)

1. Recombinant oncolytic type 35 adenovirus in which the hTERT promoter or Survivin promoter and E1 gene are integrated into the type 35 adenovirus genome.
2. A recombinant oncolytic type 35 adenovirus in which the hTERT promoter or Survivin promoter and the E1A gene are integrated into the type 35 adenovirus genome.
3. A recombinant oncolytic type 35 adenovirus in which the hTERT promoter or Survivin promoter, E1A gene, E1B promoter and E1B gene are integrated into the type 35 adenovirus genome.
4. The recombinant oncolytic type 35 adenovirus according to claim 2 or 3, wherein the E1A gene is of a mutant type.
5. The recombinant oncolytic type 35 adenovirus according to any one of claims 1 to 4, further comprising a gene encoding a cell death induction-related protein or an immunostimulation-related protein.
6. A pharmaceutical composition comprising the recombinant oncolytic type 35 adenovirus according to any one of claims 1 to 5.
7. The pharmaceutical composition according to claim 6, which is for treating cancer.
8. The pharmaceutical composition according to claim 7, wherein the cancer is breast cancer.
9. A recombinant oncolytic type 35 adenovirus according to any one of claims 1 to 5 or a pharmaceutical composition according to any one of claims 6 to 8 is administered to a cancer patient. How to treat cancer.
10. The treatment method according to claim 9, wherein the cancer patient is a breast cancer patient.

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