WO2022265092A1

WO2022265092A1 - Composition for cancer treatment

Info

Publication number: WO2022265092A1
Application number: PCT/JP2022/024277
Authority: WO
Inventors: 幸世野村
Original assignee: 国立大学法人東京大学
Priority date: 2021-06-19
Filing date: 2022-06-17
Publication date: 2022-12-22
Also published as: JPWO2022265092A1

Abstract

The present invention addresses the problem of providing a treatment method that is more practical than currently reported methods in cancer treatment using sodium iodide symporter (NIS)-MSCs, and a composition for cancer treatment that is to be used in the treatment method. More particularly, in the present invention, the means for solving the problem is a composition for cancer treatment that is characterized by comprising a combination of mesenchymal stem cells expressing NIS with an α-nuclide.

Description

Cancer therapeutic composition

The present invention relates to a composition for treating cancer using a combination of mesenchymal stem cells and a radionuclide, and a method for treating cancer using the composition for treatment.

Currently, cancer treatment is performed by combining various methods such as surgical treatment such as surgery, drug therapy using anticancer drugs, radiation therapy, and immune checkpoint inhibitors. survival rates have also improved. However, in addition to intractable cancers such as scirrhous gastric cancer, pancreatic cancer, and triple-negative breast cancer, gastrointestinal cancers such as gastric cancer, pancreatic cancer, and colorectal cancer, ovarian cancer, and ureteral cancer have metastasized. For peritoneal dissemination that occurs in the In particular, peritoneal dissemination is difficult to remove by surgical operation, and treatment with ordinary chemotherapeutic agents alone has little therapeutic effect and the prognosis is extremely poor.

In recent years, attempts have been made to develop new cancer treatment methods that focus on the tumor microenvironment (TME). So-called "cancer" consists of tumor tissue consisting of tumor cells, interstitial fibroblasts, cells forming blood vessels and lymphatics, infiltrating inflammatory cells, extracellular matrix such as collagen, and physiologically active substances. made up of quality. Cancer stroma grows by highly accumulating and proliferating stromal cells, and mesenchymal stem cells (MSCs) are incorporated during the growth process. MSCs are characterized by high proliferation and have the ability to differentiate into cells that constitute connective tissues such as bone, fat, cartilage and muscle, and play important roles in the maintenance and regeneration of various tissues. When MSCs are taken into the cancer stroma, they differentiate into various cancer stroma-associated cell types, including tumor vascular cells and cancer-associated fibroblasts (CAF) (for example, see Non-Patent Document 1). see). Due to the ability of MSCs to accumulate in the cancer stroma, MSCs are attracting attention as carriers for delivering cancer therapeutic genes and the like to the tumor microenvironment (see, for example, Non-Patent Document 2). ).

It has been reported that MSCs expressing sodium iodide symporter (NIS) can also be used to kill cancer cells by delivering radionuclides into the TME (Non-Patent Document 3, Non-Patent Document 4, etc.). NIS is a transmembrane glycoprotein with 13 transmembrane domains that can transport radionuclides such as the radionuclides ¹³¹ I, ¹²³ I, ¹²⁵ I, ¹²⁴ I, ^99m Tc, ¹⁸⁸ Re and ²¹¹ At. can. Non-Patent Document 3 reports that administration of NIS-expressing MSCs and ¹³¹ I to liver cancer model mice resulted in tumor regression. However, in the mouse experiment disclosed in Non-Patent Document 3, a very high dose of ¹³¹ I of 55.5 Mbq was used, and it is unrealistic to convert such a high dose to humans. is. Furthermore, multiple doses of ¹³¹ I may be necessary because the sensitivity of cells to the cytocidal β-rays emitted by ¹³¹ I varies depending on the cell cycle.

As described above, a treatment method that delivers radionuclides to cancer lesions using NIS-expressing MSCs (hereinafter also referred to as "NIS-MSCs") as a carrier is expected to have many excellent therapeutic effects. However, there are still many points to be improved and problems to be solved still exist.

In view of the above circumstances, the present invention provides a treatment method that is more practical than currently reported methods in the treatment of cancer using NIS-MSCs, and a therapeutic composition for cancer used in the treatment method Make the provision of goods a problem to be solved.

The inventors decided to use α nuclides instead of β-ray emitting nuclides as radionuclides to be combined with NIS-MSC. α-rays emitted by α-nuclides are a type of heavy ion beam, and compared to β-rays, they can impart high energy to a narrower range, so they are thought to exhibit high therapeutic effects locally. Also, unlike β rays, its cell-killing ability does not depend on the cell cycle. On the other hand, since the cell-killing effect of α-rays is localized, it was unclear whether α-nuclides incorporated into NIS-MSCs exert effective killing ability against cancer cells.
When the inventor administered ²¹¹ At, an α nuclide, to a mouse model of peritoneal dissemination, unexpectedly, the number of administrations (3 times) and the dose (4 MBq or 10 MBq) when using β nuclides were higher. It was found that a small dose (1 time) and dose (1.3MBq) exerted tumor contraction effect equal to or greater than that of β-ray nuclides.
The present invention has been completed based on the above findings.

That is, the present invention is the following (1) to (4).
(1) A cancer treatment composition comprising a combination of mesenchymal stem cells expressing a sodium iodide symporter (NIS) and an α nuclide. composition.
(2) The composition for cancer treatment according to (1) above, wherein the cancer is peritoneal dissemination.
(3) The composition for cancer treatment according to (1) above, wherein the mesenchymal stem cells are administered intraperitoneally, and the α nuclide is administered intraperitoneally or orally.
(4) The composition for cancer treatment according to any one of (1) to (3) above, wherein the α nuclide is ²¹¹ At.
In this specification, the sign "-" indicates a numerical range including the values on the left and right of it.

The present invention enables safer and more effective cancer treatment with less risk of radiation exposure than conventional methods. In particular, even intractable cancers such as peritoneal dissemination can be effectively treated.

Using a peritoneal dissemination mouse model, an experimental schedule for confirming the therapeutic effect of cancer by combined administration of NIS-MSCs and radionuclides is shown. FIG. 1 shows the results of counting radiation doses in various organs after administering NIS-MSCs intraperitoneally and ¹²⁵ I orally to peritoneal dissemination mouse models according to the experimental schedule shown in FIG. Figure 1 shows an image of peritoneal dissemination on the peritoneum after intraperitoneal administration of NIS-MSCs and oral administration of ¹³¹ I to a peritoneal dissemination mouse model. A is a control mouse, and B is an image of the peritoneum of a mouse orally administered with ^131I . The image shows the state of peritoneal dissemination on the peritoneum after intraperitoneal administration of NIS-MSC and oral administration of ²¹¹ At to a peritoneal dissemination mouse model. A is a control mouse, B is an orally administered ²¹¹ At, and C is an image of the peritoneum of a mouse intraperitoneally administered ²¹¹ At. The results of examining the survival rate of mice after intraperitoneal administration of NIS-MSCs and ²¹¹ At in peritoneal dissemination mouse models are shown.

A first embodiment for carrying out the present invention is a composition for treating cancer, comprising mesenchymal stem cells expressing sodium iodide symporter (NIS): MSC) and an α nuclide are combined to treat cancer (hereinafter also referred to as "the composition according to the present embodiment").
NIS is an integral membrane protein of approximately 650 amino acids, has 13 transmembrane domains and functions as an ion pump, pumping one iodide ion (I-) ^and two sodium ions (Na+) into the cell. It has the function of simultaneously transporting within So far, reports have been made on its utility as a new tool in disease treatment (see, for example, Non-Patent Document 4). The NIS used in the present embodiment may be derived from any animal species depending on the purpose of use. For example, human NIS consisting of the amino acid sequence represented by SEQ ID NO: 1 is preferable, but this It is not limited to arrays. In addition to naturally occurring NIS, its variants or homologues (e.g., consisting of an amino acid sequence having an identity of 90% or more with the amino acid sequence of naturally occurring NIS, simultaneously introducing iodide ions and sodium ions into cells) NIS variants or homologues having transport activity), for example, NIS variants disclosed in US8852576B2 may be used.

MSCs are a type of stem cell that differentiates into mesoderm-derived tissues (e.g., bone, cartilage, fat, blood vessels, cardiomyocytes, etc.), ectoderm-derived nerve cells and glia, and endoderm-derived stem cells. It is a cell with capacity. Human MSCs are characterized, for example, by being positive for cell surface markers such as CD73 and CD105 and negative for myeloid markers such as CD34, CD14, CD45 and MHC class II antigen. MSCs can be harvested from bone marrow, dental pulp, and fat. In addition, depending on the intended use of the collected MSCs, they may be modified, for example, immortalized using SV40 large T antigen or the like.

Expression of NIS in MSCs can be easily carried out based on common technical knowledge in the relevant technical field. For example, an expression vector containing NIS cDNA under the control of an appropriate promoter (e.g., cytomegalovirus-derived promoter (CMV IE promoter), etc.) in an expressible state is transfected into MSCs, and an appropriate selection marker is used. , MSCs containing the expression vector are selected, and NIS-expressing cells among the obtained cell clones can be used as NIS-expressing MSCs (NIS-MSCs).

The α-nuclide used in the present embodiment is preferably a nuclide that emits α-rays and is taken up into cells by NIS. Although not particularly limited, for example, ²¹¹ At is preferable.

The composition according to this embodiment is a cancer treatment composition characterized by administering NIS-MSCs and α nuclides separately. As mentioned above, NIS-MSCs accumulate in the cancer stroma and incorporate α nuclides into the cells, which enables efficient irradiation of α rays to tumor cells, resulting in the elimination of tumor cells. can be annihilated. That is, a composition administered in combination with NIS-MSCs and α nuclides can be used for cancer treatment.
Here, NIS-MSCs may be suspended in NIS-MSCs or in a solution capable of suspending NIS-MSCs, such as physiological saline or phosphate-buffered saline (PBS). Furthermore, the NIS-MSC suspension may contain pharmaceutically acceptable additives. Suspensions containing NIS-MSCs may be administered to tumor tissue (cancer foci) or tissue in which tumor cells reside, or may be administered intravenously. For example, when the cancer to be treated is peritoneal dissemination, the administration method of NIS-MSCs is preferably intraperitoneal administration.
In the composition according to this embodiment, alpha nuclides are administered after administration of NIS-MSCs. The administration interval between NIS-MSCs and α nuclides is not particularly limited, but is preferably about 1 to 7 days, for example. The α nuclide may be administered to a tumor tissue (cancer focus) or a tissue in which tumor cells are present, intravenous administration, or oral administration. In addition, the administration frequency of NIS-MSCs and α nuclides may be one administration or multiple administrations. For example, when the cancer to be treated is peritoneal dissemination, the administration method of the α nuclide (eg, ²¹¹ At) is preferably intraperitoneal administration or oral administration, more preferably intraperitoneal administration.
Treatment using the compositions of this embodiment can be performed alone or in combination with chemotherapy, radiation therapy, and the like.

Cancers (malignant tumors/malignant neoplasms) to be treated with the composition according to this embodiment are not particularly limited. Cell carcinoma, transitional cell carcinoma, adenocarcinoma, malignant gastrinoma, malignant melanoma, fibrosarcoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, malignant teratoma, angiosarcoma, Kaposi's sarcoma, osteosarcoma, chondrosarcoma , lymphangiosarcoma, malignant meningioma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, brain tumor, epithelial cell-derived neoplasm (epithelial carcinoma), basal cell carcinoma, adenocarcinoma, lip cancer, oral cancer, esophageal cancer, small bowel cancer and Gastrointestinal cancer such as gastric cancer, colon cancer, rectal cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, skin cancer such as squamous cell carcinoma and basal cell carcinoma, prostate cancer and kidney In addition to cell carcinoma, peritoneal dissemination resulting from the metastasis of these cancers, such as gastric cancer, pancreatic cancer, colorectal cancer, ovarian cancer, ureter cancer, etc. etc. can be mentioned.

The composition according to this embodiment may be provided in the form of a kit together with instructions such as an administration method. The composition contained in the kit effectively maintains the activity of the active ingredients (i.e., MSC or NIS-MSC and α nuclides) for a long period of time, and the ingredients do not adsorb to the inside of the container, and the ingredients are denatured. Each active ingredient is supplied separately in a container made of non-toxic material. In addition, alpha nuclides need to be supplied in a suitable form to prevent leakage of radiation.
In addition, the instructions for use of the kit may be printed on paper or the like, or may be stored and supplied on an electromagnetically readable medium such as a CD-ROM or DVD-ROM.

A second embodiment of the present invention is a cancer treatment method (hereinafter also referred to as "treatment method according to this embodiment") comprising administering a composition according to this embodiment to a patient.
The term “treatment” as used herein means to prevent or alleviate the progress and deterioration of the condition in a patient already suffering from cancer, and thereby to prevent or alleviate the progression and deterioration of cancer. It is the action to take.
In addition, the subject of treatment is not limited to humans, and may be mammals other than humans, such as mice, rats, dogs, cats, domestic animals such as cows, horses, and sheep, and primates such as monkeys, chimpanzees, and gorillas. Humans are particularly preferred.

Cancers (malignant tumors/malignant neoplasms) to be treated by the treatment method of the present embodiment are not particularly limited, but examples include hepatocellular carcinoma, cholangiocarcinoma, renal cell carcinoma, squamous cell carcinoma, and basal cell carcinoma. , transitional cell carcinoma, adenocarcinoma, malignant gastrinoma, malignant melanoma, fibrosarcoma, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, malignant teratoma, angiosarcoma, Kaposi's sarcoma, osteosarcoma, chondrosarcoma, lymphoid Vascular sarcoma, malignant meningioma, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, brain tumor, neoplasm of epithelial cell origin (epithelial carcinoma), basal cell carcinoma, adenocarcinoma, lip cancer, oral cancer, esophageal cancer, small bowel cancer and gastric cancer gastrointestinal cancer, colon cancer, rectal cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, skin cancer such as squamous cell carcinoma and basal cell carcinoma, prostate cancer and renal cell carcinoma In addition, cancers that these cancers have metastasized to, for example, gastrointestinal cancers such as gastric cancer, pancreatic cancer, colon cancer, ovarian cancer, and peritoneal dissemination caused by metastasis from ureteral cancer can be mentioned.

When this specification is translated into English and contains the words "a", "an" and "the" in the singular, the singular as well as the plural unless the context clearly indicates otherwise. shall also include those of
EXAMPLES The present invention will be further described below with reference to Examples, but these Examples are merely illustrations of embodiments of the present invention and do not limit the scope of the present invention.

1. Confirmation of radionuclide accumulation in the peritoneum C57BL/6 mice (Charles River Laboratories Japan, Inc.) were intraperitoneally injected with the transplantable mouse stomach cancer cell line YTN16 (1×10 ⁷ cells) to form peritoneal dissemination. . YTN16 is a cell line established by the inventors (Yamamoto et al., Cancer Res. 109:1480-1492 2018). Three weeks after administration of YTN16, macroscopically visible peritoneal dissemination had formed on the peritoneum. Mouse mesenchymal stem cells (MSC) (5×10 ⁶ cells/500 μL) in which human sodium iodide symporter (NIS) was forcibly expressed in peritoneally disseminated mice ) was administered intraperitoneally three times every other day (Fig. 1).
MSCs (NIS-MSCs) in which human NIS (SEQ ID NO: 1) was forcibly expressed were produced according to a previous report (Non-Patent Document 3). Specifically, the human NIS coding sequence (SEQ ID NO: 2) was inserted into the EcoRI site of pcDNA3.1plasmid (Thermo Fisher Scientific) having a CMV promoter, and C57BL/6 mouse bone marrow-derived MSCs (SV40 large T antigen-immortalized) were transfected using LipofectAMINE Plus (Thermo Fisher Scientific). Cells were then cultured (Mesenchymal Stem Cell Growth medium containing 10% FBS) and then selected in medium containing 1.0 mg/ml neomycin. Among the obtained cell clones, those with high NIS expression were used as NIS-MSCs for experiments.

Two days after the third administration of MSCs, 4 MBq of γ-emitting ¹²⁵ I was administered orally. The above administration of NIS-MSCs and ¹²⁵ I to mice was repeated on the same schedule. Thereafter, ¹²⁵ I and NIS-MSCs were administered once each to mice (Fig. 1). Two days after the last ¹²⁵ I dose, the mice were euthanized and the extent of ¹²⁵ I accumulation in the excised peritoneum was measured with a γ-counter.
FIG. 2 shows the measurement results of gamma-ray emission counts in several organs including the peritoneum. Black bars and white bars show the results of counting the amount of γ-rays emitted from NIS-MSC-administered mice and MSC-administered mice (without forced expression of NIS), respectively. In addition, gray bars indicate the results of counting the amount of γ-rays emitted from mice to which ¹²⁵ I was not administered. FIG. 2A shows that ¹²⁵ I accumulates in the peritoneum in the NIS-MSC-administered group. In other various organs, the counts indicated by white bars tended to be slightly higher than the ^counts indicated by black bars. It is considered that the accumulation in the peritoneum decreased the accumulation in other sites.
As described above, it was confirmed that the radionuclide ¹²⁵ I could be accumulated in the peritoneum where peritoneal dissemination was observed by using NIS-MSCs as a delivery carrier.

2. Investigation of the effect of β-emitting radionuclides on peritoneal dissemination In a model similar to the peritoneal dissemination mouse model described in 1 above, ¹³¹ I, which emits β-rays with a cell-killing effect, was used as a radionuclide. The experiment was conducted with the same administration schedule as the method. ¹³¹ I was administered orally at 4 MBq or 10 MBq per dose. Five weeks after the intraperitoneal administration of YTN16 (1×10 ⁷ cells), the mice were euthanized and the formation of peritoneal dissemination was observed. In addition, a group in which peritoneal seeding was formed in the same manner (group a), and a group in which peritoneal seeding was formed in the same manner, NIS-MSCs were administered in the same manner as the experimental group, and physiological saline was orally administered (group b), The above two groups were used as controls. There was no significant difference in peritoneal dissemination status between these two groups of controls.
Images of the peritoneum of ¹³¹ I-administered mice and control mice are shown in FIG. Control peritoneal surfaces showed nodules, or peritoneal seeding, composed of YTN16 cells (Fig. 3A, e.g., circled area), whereas in the peritoneum of mice treated with 10 MBq of ^131I , peritoneal A reduction and shrinkage of seeding nodules was observed (Fig. 3B).

3. Investigation of Effect of α-ray Emitting Radionuclide on Peritoneal Dissemination Next, the same experiment as in 2 above was performed using ²¹¹ At, which emits α ray, as the radionuclide. However, in 2 above, the procedure of administering NIS-MSCs three times and then administering ^131I once was repeated twice, and then NIS- ^MSCs were administered once and then ^131I was administered once. When using , a single dose of ²¹¹ At (orally or intraperitoneally) was performed after three doses of NIS-MSCs. The dose of ²¹¹ At was 1.3 MBq. As a result, oral administration of ²¹¹ At was found to reduce and reduce peritoneal dissemination (Fig. 4B), and intraperitoneal administration more effectively reduced and reduced dissemination (Fig. 4C).

As shown in 2 above, when the β nuclide ¹³¹ I was used, ¹³¹ I was administered multiple times (three times in 2 above). In contrast, ²¹¹ At was administered only once. Nevertheless, a single dose of ²¹¹ At exerts a reduction and reduction effect on dissemination equal to or greater than multiple doses of ¹³¹ I. In particular, when comparing FIG. 2 and FIG. 3, it can be seen that the use of α nuclides (FIG. 3) has a higher effect of reducing seeding. As described above, it was clarified that the use of ²¹¹ At, which is an α nuclide, exhibits a superior tumor regression effect. Alpha rays are heavy ion beams that break DNA double-strands and inflict great damage to cancer cells. Compared to beta rays, this effect is thought to be exerted regardless of the cell cycle. In addition, since alpha rays are heavy ion beams, they travel a short distance, cause little damage to surrounding normal cells other than cancer cells, and penetrate only a few cells. In this case, although it is excreted in urine and feces, there is no danger that the patient's body will be exposed to the surrounding people. Therefore, long-term isolation and hospitalization unlike ¹³¹ I is not required, and the therapeutic method using α nuclides disclosed by the present invention is considered to be remarkably superior to the conventional therapeutic methods using β nuclides.

4. Effect of ²¹¹ At on Prolonging Survival Period of Peritoneal Dissemination Model Mouse Further, in order to verify the therapeutic effect of peritoneal dissemination with ²¹¹ At, a mid-term experiment was conducted to examine the survival period of the peritoneal dissemination model mouse. Specifically, mice were ^peritoneally ^seeded in the same manner as in the above experiment. Intraperitoneal administration group, Group 3: ²¹¹ At combined with an antibody that attaches to cancer cells, and intraperitoneal administration group, Group 4: ²¹¹ At combined with an antibody that attaches to cancer cells, administered intravenously Administered group Group 5: A non-treated group was established, and the mice were bred until they died, and their survival conditions were observed.
The results are shown in the survival curve of FIG. In FIG. 5, the date on the horizontal axis is the number of days after ²¹¹ At administration. The state of peritoneal dissemination at the time of administration of ²¹¹ At was such that the mice died 10 days later in the case of no treatment. FIG. 5 shows that the ²¹¹ At-treated groups (Groups 1-4) had longer survival times than the untreated group (Group 5). Among them, the group (Group 1) in which ²¹¹ At was administered intraperitoneally after administration of NIS-MSC had the longest survival time.

These results demonstrate that the combined administration of NIS-MSCs and ²¹¹ At reduced the number and size of peritoneal dissemination. It was also confirmed that the therapeutic effect on cancer is significantly superior to the already reported effects of combination therapy with NIS-MSC and ^131I .

The therapeutic composition according to the present invention is effective in treating cancer, especially intractable cancers (for example, peritoneal dissemination). Therefore, the present invention is expected to be used in the medical field (particularly in the cancer treatment field).

Claims

A composition for treating cancer, characterized by combining mesenchymal stem cells expressing a sodium iodide symporter (NIS) and an α nuclide. .
The composition for cancer treatment according to claim 1, wherein the cancer is peritoneal dissemination.
The composition for cancer treatment according to claim 1, wherein the mesenchymal stem cells are administered intraperitoneally, and the α nuclide is administered intraperitoneally or orally.
4. The composition for cancer treatment according to any one of claims 1 to 3, wherein the alpha nuclide is 211 At.