WO2020116989A1 - Anti-cancer composition comprising in vivo cell injection chip - Google Patents

Abstract

The present invention relates to an anti-cancer pharmaceutical composition comprising a vivo cell injection chip as an active ingredient, the composition comprising: a porous three-dimensional cryogel scaffold comprising a structure in which first and second components of a hyaluronic acid derivative, having different chemical structures, are crosslinked; and cells cultured in chambers of the scaffold. The composition comprising the scaffold and the cells cultured in chambers of the scaffold exhibits a cell growth rate, a survival rate, a cancer cell killing ability, and a cancer metastasis reduction effect that is greater than those of a conventional two-dimensional culture, thereby being effectively usable in cancer treatment and cancer treatment methods or as a composition for cell cultures.

Description

Anticancer composition comprising in vivo cell input chip

The present invention is a porous three-dimensional cryogel scaffold comprising a structure in which a first component and a second component of a hyaluronic acid derivative having different chemical structures are crosslinked: and cells cultured in a chamber of the scaffold It relates to a method for treating a solid cancer and blood cancer comprising the step of administering to the subject an effective amount of an anticancer pharmaceutical composition or an anticancer pharmaceutical composition comprising an in vivo cell input chip as an active ingredient.

Currently, existing anti-cancer treatments mainly include surgical surgery, chemotherapy, and irradiation. However, due to serious side effects such as cytotoxicity and cancer metastasis and recurrence, the need for new treatments is increasing. Surgery, administration of anticancer drugs, and irradiation cause weakening of physical strength and reduced immunity. In addition, cancer cells remaining in the body after treatment are likely to cause cancer recurrence and metastasis, and the most common cause of death among cancer patients is cancer recurrence. Therefore, recently developed cancer treatments are focused on destroying only cancer cells with few side effects. The current research direction of anti-cancer therapy is currently being directed toward minimizing cancer as an existing treatment method and then completely removing residual cancer cells by cell immunotherapy. In addition, NK cells, cytotoxic T cells, and dendritic cells that selectively destroy cancer cells, which are cell immunotherapy targeting residual cancer cells, have recently attracted attention.

On the other hand, NK cells are a type of immune cells that show selective cytotoxicity to cancer cells. Unlike other immune cells, NK cells can immediately detect and remove cancer cells, and can differentiate cancer cells from normal cells through various immune receptors on the surface of NK cells. The number of NK cells in the body of cancer patients is lower than that of normal people, and the anticancer activity is also defective. In addition, dysfunction of NK cells is closely related to the development of cancer. In addition, NK cells are known to play a key role in regulating inflammation and immune responses by producing cytokines such as interferon gamma (IFN-γ) and thien alpha (TNF-α). NK cells play a key role in regulating the immune response through direct interaction with dendritic cells, macrophages, and T cells and indirect interactions through cytokines. It has been found that NK cells play an important role in the development of inflammatory diseases, autoimmune diseases and various intractable diseases (Front Immunol. 2017; 8:745).

On the other hand, cancer stem cells have strong resistance to anti-cancer agents and radiation therapy, and cancer stem cells that survive the attack of the anti-cancer agents are still in an incubated state after the cancer is cured, and then actively proliferate and differentiate again, and cancer metastases to other sites. cause. Therefore, if cancer stem cells are removed, it is highly possible to prevent cancer from recurring and effectively treat it. At this time, NK cells not only inhibit the development, proliferation, and metastasis of cancer cells, but also can effectively remove cancer stem cells, which are the most important for cancer recurrence. According to a recent study, when NK cells isolated from normal humans are injected into a patient, the immune rejection response is extremely low, unlike other immune cells, thereby increasing safety in the development of immune cell therapy products.

In order to overcome this, the NK cell transplantation did not last for a long time in the body of the cancer patient, and a method of periodically inserting a cytokine IL-2 capable of inducing NK cell proliferation after transplantation was attempted. However, IL-2 is a problem because it increases regulatory T cells (Tregs) that inhibit the anticancer immune response in addition to NK cells.

Green Cross Lab Cell announced the prospect of developing an anti-cancer immune cell treatment that can mass-produce using allogeneic NK cells. It has been announced that anti-cancer drugs using NK cells will be able to solve the safety and economic problems of existing immuno-cancer drugs. Even when transplanted to other people, NK cell therapy has no graft-versus-host disease (GVHD), and has already proven its anti-cancer function and safety through hematopoietic transplantation and 89 clinical trials worldwide are ongoing as of 2016. Eleven clinical trials are underway in Korea, and the effectiveness of anticancer drugs using NK cells is positive in clinical practice. According to the clinical phase 1 (MG4101), in which NK cells from healthy non-associated donors were expanded and administered, the NK cell anti-cancer immune cell therapy was safe even at the maximum dose and showed stable lesions in 47.1% of patients and progressive lesions in 52.9% without pretreatment. . Cultured allogeneic NK cells can be a useful and safe tumor therapy, but the handling of NK cells is very demanding, so pure culture is not easy and this problem must be solved.

NKMAX is an autologous NK cell treatment that uses patients' own cells and plans to submit a Phase 1/2 clinical trial application to the Food and Drug Administration this year. In addition, it aims to obtain drug approval in 2022 by demonstrating its stability and effectiveness, and has already begun production in March in Japan, has given it to patients, and is also preparing for clinical trials in the United States and Mexico.

In addition, Immunos Bio entered the Japanese market with the approval of the Japan Review Board for NK cell immunotherapy. It plans to start phase 1 clinical trials for liver cancer and brain tumors this year by conducting NK cell immunotherapy in cooperation with Nishihashi Clinic in Japan.

It has been reported that dendritic cells, NK cells, and T cells, which are therapeutic immune cells currently being tried in clinical trials, have a disadvantage in that a large amount of injected remains at the injection site or does not function in the circulation process in vivo and disappears. In order to overcome these drawbacks, in practice, attempts have been made to increase cancer treatment efficacy by injecting a large number of 109 therapeutic immune cells in vivo. However, it takes a lot of time and money to cultivate a large number of cells in vitro, and there is a problem that the treatment cost is very high.

In addition, a large number of cells cultured in vitro should be used in patients within a designated period, otherwise, there is a fatal disadvantage that a large number of therapeutic cells produced at a high cost must be disposed of (Nat Rev Immunol. 2018; 18(11):671-688). Therefore, in order to secure a large number of live NK cells and obtain NK cells having anticancer activity, it is necessary to cultivate a new three-dimensional culture type NK cell using a porous scaffold rather than a conventional cell culture method.

Porous scaffolds used in the bio and medical fields must be freely controlled in resolution or swelling ratio, depending on their purpose and purpose, but all porous scaffolds produced by the above-mentioned techniques have uniform resolution. Or, there is a disadvantage that the swelling ratio is very low. In particular, it is known that when using a low molecular weight component to control the degree of decomposition or lowering the crosslinking density, the mechanical properties are deteriorated.

On the other hand, cell therapies or cancer vaccines are mainly used for blood cancer-related diseases, and in solid cancers, most of them have a very low therapeutic effect. One of these reasons is a microenvironment factor that suppresses immune function around solid cancer. In fact, cells that lower the function of immune cells in the microenvironment (MDSC: myeoloid-derived stromal cells, Treg: regulatory T cells, TAM: tumor-assocaited macrophages) or immunosuppressive cytokines, metabolites, etc. , It is known to rapidly decrease the activity of immuno-activating substances and therapeutic immune cells (Nat Rev Immunol. 2016; 16(2):112-123).

In order to solve the above problems, the present invention is a porous three-dimensional cryogel scaffold comprising a structure in which a first component and a second component of a hyaluronic acid derivative having different chemical structures are crosslinked: and the scaffold It is an object of the present invention to provide a pharmaceutical composition for anti-cancer, an anti-cancer treatment method, and a method for manufacturing the scaffold comprising cells cultured in a chamber of a fold, the cell-injection chip in vivo as an active ingredient.

It is also an object of the present invention to provide a composition and scaffold that can be used for cancer treatment by culturing cells using a three-dimensional biodegradable scaffold and directly injecting them into a living body.

In order to achieve the above object, the present invention is a porous three-dimensional cryogel scaffold comprising a structure in which a first component and a second component of a hyaluronic acid derivative having different chemical structures are crosslinked: And it provides a pharmaceutical composition for anti-cancer comprising cells incubated in the chamber of the scaffold, an in vivo cell input chip as an active ingredient.

In addition, the present invention is a porous three-dimensional cryogel scaffold comprising a structure in which a first component and a second component of a hyaluronic acid derivative having different chemical structures are crosslinked: and cells cultured in a chamber of the scaffold It provides a cancer treatment method comprising the step of administering a pharmaceutical composition for anti-cancer to an individual in an effective amount, comprising, as an active ingredient, a cell-injection chip in vivo. More preferably, the cancer treatment method provides a method for treating solid cancer and blood cancer.

According to one embodiment, the first component is a hyaluronic acid methacrylate (HA-MA) derivative, and the second component is an oxidized hyaluronic acid methacrylate derivative (oxHA-MA) or hyaluronic acid Aldehyde methacrylate (HA-ald-MA).

According to a preferred embodiment, the first component is hyaluronic acid methacrylate, and the second component is hyaluronic acid aldehyde methacrylate (HA-ald-MA).

According to another preferred embodiment, the first component is hyaluronic acid methacrylate, and the second component is oxidized hyaluronic acid methacrylate (oxHA-MA).

The cells include Langerhans islet cells, Serto cells, dopaminergic neurons, stem cells, mesenchymal stem cells, umbilical cord blood cells, embryonic stem cells, neural stem cells, differentiated stem cells, T cells, natural killer (NK) cells, and B cells. One or more of the group consisting of.

In another specific example, the cell is a natural killer (NK) cell, and in one particular example, the natural killer (NK) cell is a chimeric antigen receptor-natural killer (CAR-NK) cell. The chimeric antigen receptor-natural killer (CAR-NK) cells are ecto-domain, hinge, transmembrane, and CD28 of cancer-specific anti-EGFR affibodies. , DAP10 and CD3ζ.

In addition, the present invention provides an anti-cancer pharmaceutical composition comprising the composition for in vivo cell injection.

In addition, the present invention provides a method of treating cancer by administering the composition for in vivo cell injection to an individual.

The cancer is solid cancer or blood cancer, and the solid cancer is characterized in that it is lung cancer, brain cancer, liver cancer, breast cancer, ovarian cancer, or colon cancer. In addition, the blood cancer is characterized by leukemia, multiple myeloma, aplastic anemia or malignant lymphoma.

In addition, the present invention provides a composition for cell culture comprising a porous three-dimensional cryogel scaffold comprising a structure in which hyaluronic acid methacrylate and oxidized hyaluronic acid methacrylate are crosslinked.

Porous three-dimensional cryogel scaffold comprising a structure in which a first component and a second component of a hyaluronic acid derivative having different chemical structures according to one aspect are crosslinked: and cells cultured in a chamber of the scaffold A pharmaceutical composition for anti-cancer containing an in vivo cell input chip as an active ingredient, wherein the scaffold exhibits a cell growth rate, survival rate, cancer cell killing ability, and cancer metastasis reduction effect compared to a conventional two-dimensional culture, It can be usefully used as a cancer treatment method or a cancer treatment composition.

1 is a porous three-dimensional cryogel scaffold comprising a structure in which a first component and a second component of a hyaluronic acid derivative having different chemical structures of the present invention are crosslinked, wherein the first component is hyaluronic acid meta An acrylate derivative, the second component is an oxidized hyaluronic acid methacrylate derivative (oxHA-MA), 3D engineered hyaluronic acid based niche for cell expansion, showing the manufacturing process and cell culture method of 3D ENHANCE scaffold.

Figure 2a shows an electron micrograph of the 3D ENHANCE scaffold prepared using hyaluronic acid and image images in the form. Figure 2b shows the results of spheroid formation in the NK92 cell line loaded (4 days after loading) on the 3D ENHANCE scaffold.

Figure 3 shows the results of comparing the pore structure of the 3D ENHANCE scaffold of the present invention with conventional gel. The 3D ENHANCE scaffold was a bio-degradable cryogel, and was produced based on hyaluronic acid in the same manner as the conventional gel.

Figure 4 shows the characteristics of the 3D ENHANCE scaffold and conventional gel of the bio-degradable cryogel of Figure 3b.

Figure 5a shows the decomposition process of the 3D ENHANCE scaffold of the present invention. In addition, Figure 5b shows the result of adjusting the ratio of the ratio of the hyaluronic acid and oxidized hyaluronic acid and the ratio of scaffold degradation by adjusting the ratio in the components of the 3D ENHANCE scaffold. In addition, Figure 5c is a living body according to various ratios of the 3D EVHANCE scaffold, wherein the first component of the present invention is hyaluronic acid methacrylate (HA-MA) and the second component is oxidized hyaluronic acid methacrylate (oxHA-MA). It shows the results of confirming that it does not affect the toxicity by measuring the weight after inserting the scaffold into the segmentation characteristics and the rat.

6 shows a comparison result with hyaluronic acid based-conventional gel and 2D culture, which are currently used as a 3D culture system. Figure 6a is a 48-well plate culture (2D culture) compared to the cell growth for conventional gel shows the results of measuring and comparing the number of cells recovered 5 days after cultivation, Figure 6b is using FACS, the 48 It shows the result of checking cell viability and dead cells for conventional gel compared to -well plate culture (2D culture).

Figure 7 shows the comparison results for the 3D ENHANCE scaffold and 2D cell culture (24 well plate culture or 48 well plate culture) of the bio-degradable cryogel of the present invention. FIG. 7A shows the results obtained by measuring the number of cells recovered 5 days after culturing cell growth, and FIG. 7B shows the 3D ENHANCE scaffold and 2D cell culture (24 well plate culture or 48 well) using FACS. plate culture). 7C and 7D show the results for cell killing or cell viability of natural killer cells, NK cells in the SK-SC1 scaffold.

Figure 8 is a HA-MA and oxHA-MA ratio of 3D-ENHANCE scaffolds different from each other and after loading the killer cells (Natural killer cell, NK cell), compared to 2D cell culture, proliferation and activity of the natural killer cells It shows the result of comparing. Figure 8a shows the results of comparing live cells with a fluorescence image through fluorescence microscopy. Figure 8b shows 2D culture and 3D ENHANCE scaffold NK cell proliferation, Figure 8c shows cell viability, cell viability, Figure 8d compares 2D culture and 3D ENHANCE scaffold for NK cell migration activity, Figure 8e shows K562 myelogeniys leukemia cell And, the effect on tumor lytic activity on Raji Burkitt's lymphoma cells is shown by comparing 2D culture and 3D ENHANCE scaffold. In addition, Figure 8f is an effect of increasing NK cell proliferation among NK cell activities in the SK-SC1 scaffold of the present invention, Figure 8g is an effect of increasing Nk cell migration, Figure 8h is a tumor for the K562 myelogeniys leukemia cell, and Raji Burkitt's lymphoma cell It shows the results confirmed by comparing the effect of increasing lytic activity or cell cytotoxicity with 2D cell culture. Figure 8i shows the results of confirming the genetic changes related to the NK cell cell proliferation, cytokine/chemokine, and NK-medicated cell cytotoxicity for the SK-SC1 scaffold. In addition, Figure 8j shows the effect of increasing gene expression related to NK cell proliferation, cytokine receptor interaction, and NK cell mediated cytotoxicity compared to 2D culture (2D) for the 3D ENHANCE scaffold.

9 is a porous three-dimensional cryogel scaffold comprising a structure in which the first and second components of the hyaluronic acid derivative are crosslinked, and FIG. 9A is a crosslinking process of Collagen/Hyaluronic acid and Hyaluronic acid derivative at low temperature ( cryogel), a scaffold having a pore size of 150-300 μm, and a scaffold containing collagen/Hyaluronic acid (collagen matrix scaffold). Figure 9b is a collagen / hyaluronic acid, Hyaluronic acid derivative at a low temperature cross-linking process (cryogel) produced by comparing the cell viability of the scaffold compared to 2D, Figure 9c includes the collagen / hyaluronic acid, The effect of cancer cell killing ability on hyaluronic acid scaffold containing natural killer cells is compared with 2D, and fluorescence image recording and cancer cell killing ability are graphed after observation with a fluorescence microscope. 9D and 9F show expression confirmation of 1,162 genes in the collagen matrix scaffold cultured NK-92, and FIGS. 9G and 9H show LTA (Lymphotoxin-) in the collagen matrix scaffold cultured NK-92. alpha), HSPE1 (Heat shock protein), HSPA5 (Glucose-regulated protein), CST7 (Cystatin-F, immune regulator), HYOU1 (Hypoxia up-regulated protein 1), etc. It is shown.

Figure 10 shows a comparison of the effect of cell therapy (adoptive cell transfer (ACT)) cells using two-dimensional cell culture (2D culture) and the 3D ENHANCE scaffold of the present invention. The cell therapy effect was confirmed by removing blood cancer cells by injecting blood cancer cells into mice and then injecting the two-dimensional cell culture (2D culture) and NK cells cultured in the 3D ENHANCE scaffold of the present invention. Figure 10A shows the results by using the in vivo imaging equipment for small animals, photographing fluorescent signals in vivo at high speed in real time. Fig. 10b shows the results at the survival rate, and Fig. 10c shows the results compared with the body weight.

Figure 11 shows the NK-CAR (natural killer cell-chimeric antigen receptor) containing EGFR affibody and NK 92 cells in the 3D ENHANCE scaffold of the present invention, and then through mice injected with MDA-MB-231 breast cancer cell line. It shows that cancer metastasis reduction effect was confirmed. Figure 11a shows the NK-CAR (natural killer cell-chimeric antigen receptor, zEGFR-CAR) information containing the EGFR affibody, Figure 11b is the breast cancer cell line MDA-MB-231 EGFR expression It was confirmed by FACS, Figure 11c shows the cancer cell lyctic activity of NK-92 and zEGFR-CAR, Figure 11d shows the outline of the experiment confirming the effect of reducing cancer metastasis through the mouse, Figure 11e is through FACS , MDA-MB-231 is a tumor marker for breast cancer cell line CK18 (cytokeratins 18) expression is measured and graphed, Figure 11f is the MDA-MB-231 breast cancer cell line in Figure 11d the mice injected with NK 92 and After injecting NK-CAR (natural killer cell-chimeric antigen receptor, chimeric antigen receptor-natural killer (CAR-NK)) containing EGFR affibody, the tumor marker CK18 expression was compared against breast cancer tissue and graphed. , Figure 11g shows that the tissue change, cancer cell metastasis reduction was confirmed by using immunohistochemistry staining. Figure 11h shows the results of confirming the lytic activity for cancer tissue of SK-SC1 scaffold containing NK cells.

The present invention is a porous three-dimensional cryogel scaffold comprising a structure in which a first component and a second component of a hyaluronic acid derivative having different chemical structures are crosslinked: and cells cultured in a chamber of the scaffold It provides an anti-cancer pharmaceutical composition comprising, as an active ingredient, a cell-injection chip in vivo.

The first component is hyaluronic acid methacrylate (HA-MA), and the second component is hyaluronic acid aldehyde methacrylate (HA-ald-MA) or oxidized hyaluronic acid meta Acrylate (oxHA-MA).

In addition, the present invention may be that the hyaluronic acid methacrylate (HA-MA) 1 to 2: oxidized hyaluronic acid methacrylate (oxHA-MA) is mixed in a weight ratio of 0 to 2.

The hyaluronic acid methacrylate (HA-MA) may be defined by the following Chemical Formula 1, and the hyaluronic acid aldehyde methacrylate (HA-ald-MA) may be defined by the following Chemical Formula 2, and the oxidized hyaluronic acid The methacrylate (oxHA-MA) may be defined by Formula 3 below.

In the present invention, the term “cryogel” refers to a porous hydrogel prepared at 0° C. or lower (subzero temperature), having an interconnected pore structure, and the diameter of the pores is two-step cooling ( cooling) technique can be controlled to 20 to 900 μm, but 200 μm is preferred.

In the present invention, the term "crosslinking" means that one polymer chain is covalently linked to the other polymer chain.

In the present invention, the term, "Scaffold (Scaffold)" refers to a substance that replaces a part of an damaged organ or tissue in vivo to secure or replace their functions, and in addition, to receive a desired site by accommodating 1 or 2 or more drugs It may be a material that can be transferred to, and may be a biodegradable material that can be completely decomposed and then disappeared after being maintained in vivo until it sufficiently performs its function and role, but is not limited thereto. In this connection, it also means a physical support and adhesive substrate made to enable in vitro culture of tissue cells and implantation in the body. In this regard, xenograft for treatment or autograft (Allograft) is also included.

Optionally, the scaffolds of the present invention can be used for cell delivery or for culturing cells. Cells can be stimulated to undergo differentiation or other physiological processes by the addition of appropriate growth factors. Culture media comprising one or more cytokines, growth factors, hormones, or mixtures thereof can be used to keep cells undifferentiated or to differentiate cells by a specific pathway. In this regard, the scaffold can be used to provide a biological environment for the settlement of cells in a bioreactor. It can also be used to study physiological and pathological processes, tumorigenic differentiation and angiogenesis. The cell transport can also be used as a cell transport for therapeutic use.

In the present invention, the term “drug” refers to small molecules, chemical substances, nucleic acids, nucleic acid derivatives, peptides, peptide derivatives, naturally occurring proteins, non-proteins, which are administered to a subject to treat a disease or dysfunction or otherwise affect the health of an individual. Naturally occurring proteins, glycoproteins and steroids. Non-limiting examples of drugs include polypeptides, such as enzymes, hormones, cytokines, antibodies or antibody fragments, antibody derivatives, drugs that affect metabolic functions, organic compounds, such as analgesics, antipyretics, anti-inflammatory agents, antibiotics, Cardiovascular drugs, drugs that affect kidney function, electrolyte metabolites, drugs acting on the central nervous system, chemotherapy compounds, receptor agonists and receptor antagonists. The drug may also include, but is not limited to, plasma proteins such as, for example, extracellular molecules, such as serum albumin, immunoglobulins, apolipoproteins, or transferrins, or proteins found on the surface of red blood cells or lymphocytes. Includes arguments. Thus, exemplary drugs include small molecules, chemicals, nucleic acids, nucleic acid derivatives, peptides, peptide derivatives, naturally occurring proteins, non-naturally occurring proteins, peptide-nucleic acids (PNA), staple peptides, phosphorodiamidate morpholino , Antisense drugs, RNA-based silencing drugs, aptamers, glycoproteins, enzymes, hormones, cytokines, interferons, growth factors, blood coagulation factors, antibodies, antibody fragments, antibody derivatives, toxin-conjugated antibodies, metabolic agonists, Analgesic, antipyretic, anti-inflammatory, antibiotic, antimicrobial, antiviral, antifungal, musculoskeletal, cardiovascular, renal, pulmonary, digestive disease, blood, urinary, metabolic, liver, neuro, anticancer, Drugs for treating stomach conditions, drugs for treating colon conditions, drugs for treating skin conditions, and drugs for treating lymph conditions. Any of the above drugs that can be mounted on the scaffold of the present invention can be applied, and not only a single drug but also a combination of drugs can be applied. Therefore, the scaffold of the present invention can be used without limitation as a therapeutic or tissue regeneration application for various diseases, and the selection and combination of these drugs can be appropriately selected by those skilled in the art.

The scaffold of the present invention is a porous scaffold, and the porous scaffold uses a main component of an extracellular matrix (ECM) such as alginate, gelatin, collagen, hyaluronic acid, and a physical/chemical crosslinking method. Can be manufactured.

In addition, the cancer of the pharmaceutical composition for anticancer of the present invention is solid cancer or blood cancer, characterized by natural killer (NK) cells, and the natural killer cell is a chimeric antigen receptor-natural killer (CA R-NK) cell. desirable. The chimeric antigen receptor (CAR) is characterized in that it comprises an ectodomain (ecto-domain) of cancer-specific anti-EGFR affibodies (tumor-specific zEGFR affibodies). In addition, the chimeric antigen receptor (CAR) includes a hinge, transmembrane, CD28, DAP10 and CD3ζ.

In the present invention, the term “affibody (affibody, affibo dies)” may mean an antibody mimetic capable of binding to a specific target protein (receptor). In general, the affibody consists of 20 to 150 amino acid residues, may be composed of 2 to 10 alpha helixes, and may include both affibody or affibody molecules capable of recognizing a specific receptor or target protein of a cell. Can be. In the present invention, in detail, an EGFR target may mean an aphibody capable of recognizing an EGFR protein. The EGFR is a growth factor receptor epidermal growth receptor, and has been reported to be overexpressed in many cancer cells such as solid cancer or blood cancer.

On the other hand, the pharmaceutical composition of the present invention includes a pharmaceutical or pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is commonly used in preparation, and the pharmaceutical composition of the present invention may further include a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

When the pharmaceutical composition of the present invention is used in a manner to be administered to an object, a unit dosage form suitable for intra-body administration of a patient according to a conventional method in the pharmaceutical field such as intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, etc. It can be formulated and administered as a formulation.

The preferred dosage of the pharmaceutical composition of the present invention depends on the patient's condition and body weight, the degree of disease, the drug form, the route of administration, and the duration, but can be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention can be used through an ampoule for injection. The ampoule for injection may be mixed with an injection solution immediately before use, and physiological saline, glucose, mannitol, Ringer's solution, etc. may be used as the injection solution. The composition or pharmaceutical preparation of the present invention thus prepared may be administered in the form of a mixture with cells used for transplantation and other uses, using administration methods conventionally used in the art. The actual dosage of the active ingredient should be determined in light of various relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and sex.

The composition of the present invention is a medium for suspending cells, a gene effective for solid cancer or blood cancer (e.g., anti-inflammatory cytokine gene, siRNA for inflammatory cytokine or anti-sense primer) (antisenseprim er)) or an expression vector containing the same, a cytokine that provides an autocrine or paracrine effect, a growth factor, and a combination thereof selected from the group consisting of One or more may be included. At this time, the medium may be the same type of medium as the culture medium, and does not contain serum, antibiotics, and antifungal agents at all.

The content of the composition for bio-injection included in the pharmaceutical composition of the present invention is not particularly limited, but may be included in an amount of 10 to 50% by weight, more specifically 20 to 40% by weight based on the total weight of the final composition.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, the term "pharmaceutically effective amount" of the present invention to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention By sufficient amount, the effective dose level is the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the invention used, the route of administration and the rate of discharge treatment The duration can be determined according to factors including drugs used in combination or coincidental with the composition of the present invention used and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects.

The dosage of the pharmaceutical composition of the present invention can be administered at one or multiple sites (for example, 2 to 50 sites) at a site where cancer is generated, and the dosage is specifically 1.0×105 to 1.0×108. The number of cells/kg (body weight), and more specifically, may be 1.0×10 6 to 1.0×10 7 cell number/kg (body weight).

Another aspect of the present invention provides a method of treating cancer, comprising administering the pharmaceutical composition to an individual in which solid cancer or blood cancer has occurred in organs, vascular tissues, and the like.

In addition, the treatment method may include the step of additionally administering another therapeutic drug or in parallel.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

<Example 1>> Preparation of porous three-dimensional cryogel scaffold comprising a structure in which the first component and the second component of the hyaluronic acid derivative are crosslinked

Substances with different degrees of decomposition under physiological and chemical conditions, after mixing the first component and the second component in an aqueous phase, float on a prepared mold, freeze at a low temperature, and gradually crosslink, and then crosslink the ice layer using a freeze-drying method. (ice crystal) was removed to prepare a cryogel scaffold having cross-linked pores. The first component is hyaluronic acid methacrylate (HA-MA), collagen (Collagen), and the corresponding second component is hyaluronic acid aldehyde methacrylate (HA-ald-MA), hyaluronic acid (Hyaluronic acid) or Oxidized hyaluronic acid methacrylate (oxHA-MA).

1-1. Preparation of a scaffold with the first component being hyaluronic acid methacrylate (HA-MA) and the second component being hyaluronic acid aldehyde methacrylate (HA-ald-MA) (manufactured by SK-SC1 scaffold)

Hyaluronic acid (Hyal uronic acid: HA, 500 kDa) is dissolved in 100 ml of distilled water at a concentration of 10 mg/ml, and 3.85 g of Methacrylic anhydride (MA) is added. HA-MA was prepared by adjusting the pH to 8 using 5N sodium hydroxide, stirring at room temperature and in the dark, and dialysis (12-14 KDa cutoff) in distilled water for 48 hours, followed by freeze-drying. Then, hyaluronic acid (HA, 500 kDa) was dissolved in 100 ml of distilled water at a concentration of 10 mg/ml, and 534 mg of sodium periodate (NaIO4) was added, followed by stirring for 24 hours in the dark and room temperature so that the oxidation reaction was sufficiently To get up. To terminate the oxidation reaction, 1 g of ethylene glycol was added and stirred at room temperature for 1 hour, then dialyzed in distilled water for 12 hours (12-14 KDa cutoff) and lyophilized to prepare HA-ald. . 1 g of the prepared HA-ald was dissolved in 100 ml of distilled water, and 3.85 g of methacryl anhydride (MA) was added. Subsequently, the pH was adjusted to 8 using 5N sodium hydroxide, and the mixture was stirred at dark room and room temperature, dialyzed in distilled water (12-14 KDa cutoff) for 48 hours, and lyophilized to prepare HA-ald-MA. The ratio of 100 mg of HA-MA and 100 mg of HA-ald-MA at a concentration of 10 mg/ml at 4° C. (HA-MA:HA-ald-MA=1:0, 2:1, 1:1) Dissolve in PBS. 30 mg of ammonium persulfate was added to the mixed solution and mixed. In order to induce the cross-linking reaction between HA-MA and HA-ald-MA, 60 µl of tetramethylethyldiamine (N,N,N',N'-tetramethylethylenediamine) was added to the mixed solution and thoroughly mixed. The mixture was quickly transferred to a PDMS mold and stored at -20°C for 24 hours to prepare a cryogel scaffold (SK-SC1 scaffold) containing HA-MA/HA-ald-MA. The scaffold was sterilized using 70% ethanol solution, and then washed 3 times using PBS.

1-2. Production of scaffolds with the first component being hyaluronic acid methacrylate (HA-MA) and the second component being oxidized hyaluronic acid methacrylate (oxHA-MA) (manufactured by 3D ENHANCE scaffold)

100 mg of 100 mg of Methacrylate Modified Hyaluronic Acid (HA-MA) and 100 mg of oxidized hyaluronic acid methacrylate-m odified oxidized HA (oxHA-MA) at a concentration of 10mg/ml at 4℃ Dissolve in PBS with (HA-MA:ox-MA=1:0, 2:1, 1:1) 30 mg of ammonium persulfate was added to the mixed solution and mixed with HA. To induce the crosslinking reaction of oxHA-MA, 60 µl of tetramethyl ethyldiamine (N,N,N',N'-tetramethylethylenediamine, TEMED) was added to the mixed solution, and the mixture was thoroughly mixed. mold) and stored at -20° C. for 24 hours to prepare a cryogel scaffold containing HA-MA/oxHA-MA.The scaffold was sterilized using a 70% ethanol solution, followed by PBS. Washed 3 times (FIG. 1) After recording the electrophotographic and morphological image photographs of the 3D ENHANCE scaffold prepared using hyaluronic acid as described above (FIG. 2A) through the method of dropping NK-92 cells, After loading hyaluronic acid in 3D-engineered hyaluronic acid-based niche for cell expansion (3D-ENHANCE) used for polymerization, the proliferation trend of cells was observed through a fluorescence microscope for 7 days (FIG. 2B).

<Example 2> Analysis of pore structure or decomposition process of porous three-dimensional polymerization scaffold

The confirmation of the pore structure of the 3D ENHANCE scaffold of the present invention was compared with the conventional gel (FIG. 3). The 3D ENHANCE scaffold is a bio-degradable cryogel and was produced based on hyaluronic acid in the same manner as the conventional gel. In the case of the 3D ENHANCE scaffold of the present invention, Bio-degradable cryogel, since it was made through cryogelation at -20°C for 24 hours, an interconnected pore structure of a certain size is generated therein. Cells grow in the form of spheroids in this constant size pore structure. The formation of spheroids in NK cells enhances the cell's proliferation, viability and killing power. In contrast, conventional gels do not have a uniform pore size and the pore structure itself is not formed properly. Therefore, it is difficult to load the cells and the cells do not properly settle within the pores. 4 shows the characteristics of the 3D ENHANCE scaffold and conventional gel of the 3D ENHANCE scaffold of the present invention bio-degradable cryogel.

Next, as shown in Figure 5a or 5b, the analysis of the degradation process of the 3D ENHANCE scaffold of the present invention. The biggest advantage of bio-degradable cryogel is that it can control the degree of scaffold degradation. Bio-degradable cryogel undergoes two major steps of decomposition. The first stage of decomposition occurs rapidly with the decomposition of oxidize d hyaluronic acid. The second stage of decomposition occurs relatively slowly due to the decomposition of hyaluronic acid (Fig. 5a). Therefore, by controlling the ratio of hyaluronic acid and oxidized hyaluronic acid, the degree of decomposition of scaffold can be controlled (FIG. 5B).

In addition, biodegradation to the SK-SC1 scaffold, the scaffold of which the first component of the present invention is hyaluronic acid methacrylate (HA-MA), and the second component is hyaluronic acid aldehyde methacrylate (HA-ald-MA) As a result of confirming the characteristics, it was confirmed that the decomposition was performed as shown in FIG. 5C, and the weight of the rat was measured after inserting the SK-SC1 scaffold into a rat or mouse, and the inserted scaffold did not cause toxicity to the rat. Was confirmed.

<Example 3> 3D scaffold and 2D cell culture comparison

As shown in FIG. 6, hyaluronic acid based- conventional gel, which is commonly used as a 3D culture system, was compared with 2D culture. After culturing the same amount of cells (1.25x10 ⁵ cells) in 48-well plate (2D) and conventional gel and measuring the number of cells recovered after 5 days, the cell growth rate was confirmed. Although it was confirmed that the cell growth rate was high, it was confirmed that it did not grow at all compared to the number of cells laid (Fig. 6A). In addition, the error range of the number of recovered cells was large and the cell viability was lower than that of the 48-well plate culture, as well as 80% or more of the dead cells (FIG. 6B ).

Then, the 3D ENHANCE scaffold of the present invention was compared with the 2D cell culture as shown in FIG. 7. As a result of comparison of the 3D ENHANCE scaffold and 2D culture (24 well plate culture or 48 well plate culture) of the bio-degradable cryogel of the present invention, in the case of bio-degradable cryogel culture (0.5x10 ⁵ cells) 5 days after cell culture 20 It was confirmed that embryonic abnormal cells grew. In comparison, cells decreased in 24-well plate culture (2.5x10 ⁵ cells) and increased approximately 5 times in 48-well plate culture (0.25x10 ⁵ cells) (FIG. 7A). In the case of measuring cell viability, dead cells were more than 70% in 24-well plate culture, and in 48-well plate culture, live cells and dead cells accounted for approximately half, but in bio-degradable cryogel, approximately 20% Only cells were dead (FIG. 7B ).

Next, as a result of comparing the cell viability of natural killer cells (NK) cells in the SK-SC1 scaffold as shown in Figure 7c or Figure 7d, cell culture in 2D cell culture (2D) cells after 5 days Although death increased rapidly, in the 3D and SK-SC1 scaffolds of the present invention, fewer cell deaths were observed than in 2D.

Then, 3D ENHANCE scaffold (3D) and 2D cell culture (2D) were compared using natural killer cells (NK cells). First, live cells were compared by fluorescence image through fluorescence microscopy (optical electron fluorescence microscopy) observation. Compared to 2D as shown in FIG. 8A, it was confirmed that the live cell increased, and as a result of comparing NK cell proliferation of 2D culture and 3D ENHANCE scaffold as shown in FIG. 8B, in 3D of the present invention than the 2D, cell It was confirmed that proliferation increased. In addition, Figure 8c is a result of confirming the cell viability, compared to 2D, it was found that the cell death in the 3D of the present invention is reduced (increased cell viability), 2D cell culture and 3D for NK cell migration activity as shown in Figure 8d As a result of comparing the ENHANCE scaffold, it was found that the NK-92 cell migration activity increased in 3D of the present invention compared to the 2D. In addition, as compared to 2D culture and 3D ENHANCE scaffold for the effect of tumor lytic activity on K562 myelogeniys leukemia cell and Raji Burkitt's lymphoma cell as shown in FIG. 8E, compared with 2D, cancer cell lysis in 3D ENHANCE scaffold of the present invention Tumor lytic activity increased. In addition, in relation to NK cell activity, as shown in FIG. 8j, as compared with 2D culture (2D), NK cell proliferation, cytokine receptor interaction, and NK cell mediated cytotoxicity-related gene expression were confirmed, compared to 2D, in the 3D ENHANCE scaffold of the present invention. It was confirmed that the expression of the gene is increased.

In addition, as a result of confirming NK cell activity in the SK-SC1 scaffold compared to 2D cell culture (2D), cell proliferation of NK cell line NK 92, as shown in FIG. 8F, and cell migration increased as shown in FIG. 8G. Confirmed. In addition, it was confirmed that the tumor lytic activity or cell cytotoxicity of the K562 m yelogeniys leukemia cell and Raji Burkitt's lymphoma cell increased as shown in FIG. 8h. In addition, as shown in Figure 8i, the NK cell cell proliferation, cytokine/chemokine, and NK-medicated cell cytotoxicity related gene changes for the SK-SC1 scaffold were confirmed.

<Example 4.> A porous three-dimensional cryogel scaffold comprising a structure in which the first and second components of a hyaluronic acid derivative are crosslinked, and a scaffold comprising collagen and hyaluron (collagen matrix scaffold) Fold) production and cell activity measurement

A porous three-dimensional cryogel scaffold comprising a structure in which the first component and the second component of the hyaluronic acid derivative are crosslinked, a scaffold containing collagen and hyaluronic acid, and a scaffold containing Collagen/H yaluronic acid Fold (collagen matrix scaffold) preparation and cell activity were measured (Figure 9). After the collagen/Hyaluronic acid and Hyaluronic acid derivatives were cross-linked at low temperature (cryogel), a scaffold with a pore size of 150-300 μm was produced (200 μm is preferred) (FIG. 9a), and the NK-92 cell line was injected. Through the method, the activity of NK cells was measured until 7 days after loading into Collagen/Hyaluronic acid porous scaffold (CS). It was confirmed that the NK cells cultured in the scaffold had increased cell viability (Viability, FIG. 9B) and cancer cell killing ability (Cytotoxicity) than 2D cell culture (2D) (FIG. 9C).

Next, NK-92 cells were cultured in a 2D cell culture (2D) or a scaffold (collagen matrix scaffold) containing Collagen/Hyaluronic acid to measure gene expression changes. After NK-92 cells were cultured in 2D or matrix, NGS (next generation sequencing) studies were conducted. On the 5th day, an increase or decrease in the expression of 1,162 genes was observed in the NK-92 cultured scaffold containing Collagen/Hyaluronic acid compared to 2D (FIGS. 9D and 9F ). In addition, as a result of confirming the gene expression level related to cancer cell killing ability, through the Volum plot analysis, the 5 genes that differed most from the 2D culture conditions were LTA (Lymphotoxin-alpha), HSPE1 (Heat shock protein), HSPA5 (Glucose-regulated) protein), CST7 (Cystatin-F, immune regulator), and HYOU1 (Hypoxia up-regulated protein 1) (FIG. 9g). In addition, it was confirmed that the mRNA expression of the gene related to the killing ability of NK cells was increased (FIG. 9h ).

<Example 5> Comparison of the effects of two-dimensional cell culture and Adoptive cell transfer (ACT) cell therapy using 3D-ENAHNCE

Adoptive cell transfer (ACT) is a typical anti-cancer immunotherapy method along with an immune checkpoint inhibitor. Therefore, in order to find out if NK cells grown on 3D-ENHANCE scaffolds are more effective than NK cells grown on two-dimensional cell culture in ACT therapy, blood cancer cells are injected into mice and then two-dimensional cell culture and NK cells grown on 3D-ENHANCE are respectively used. The injection was confirmed to remove the blood cancer cells. As a result, it was confirmed that blood cancer cells were more effectively removed from the mice injected with NK cells grown in 3D-ENHANCE, and the survival rate of the mice increased (FIG. 10).

<Example 6.> Validation of scaffold treatment effect on cancer tissue

6-1. Validation of the therapeutic effect of 3D-ENHANCE scaffold on cancer tissue

The animal experiment was conducted as shown in FIG. 11 to verify the therapeutic effect by implanting a 3D-ENHANCE scaffold containing NK cells in a region where cancer tissue was excised. After transplanting the breast cancer cell line MDA-MB-231 into mice, excised cancer cells were excised and 3D-ENAHNCE scaffolds were transplanted to confirm the degree of cancer cell metastasis. In the animal experiment, NK-CAR (natural killer cell-chimeric antigen receptor or chimeric antigen receptor-natural killer, CAR-NK, zEGFR-CAR) containing EGFR affibody, and NK-92 natural killer cells were used. After incubating the NK-92 and the zEGFR-CAR for 3 days, lyctic activity against the MDA-MB-231 breast cancer cell line was measured (FIG. 11C). After transplanting the breast cancer cell line MDA-MB-231 into the mouse as described above, the grown cancer cells are excised, and 3D-ENAHNCE (3D-ENAHNCE scaffold cultured after loading NK92 or zEGFR-CAR) is transplanted to the site, and cancer cells metastasize. The degree was confirmed. 3D ENHACE scaffold (zEGFR-CAR, zEGFR-CAR scaffold) test cultured after loading zEGFR-CAR of the present invention as shown in FIG. The group showed a cancer cell metastasis reduction effect of 40%. As described above, it was confirmed that the 3D-ENHANCE scaffold can be transplanted in vivo in a mouse and can effectively reduce the metastasis of cancer cells.

6-2. Validation of treatment effect on cancer tissues on SK-SC1 scaffold

After transplanting the breast cancer cell line MDA-MB-231 into the mouse by the method of Example 6-1, the grown cancer cells were excised and the SK-SC1 scaffold was transplanted to the site to confirm the degree of cancer cell metastasis. In the animal experiment, natural killer cells of zEGFR-CAR and NK-92 were used. After NK-92 and zEGFR-CAR were cultured for 3 days, the lyctic activity of the breast cancer cell line MDA-MB-231 cells was measured. As shown in Figure 11h, it was confirmed that EGFR was over-expressed in MDA-MB-231 cells, and that lyctic activity was active against the breast cancer cell line MDA-MB-231.

Claims (13)

1. A porous three-dimensional cryogel scaffold comprising a structure in which a first component and a second component of a hyaluronic acid derivative having different chemical structures are cross-linked, and comprising cells cultured in a chamber of the scaffold, An anti-cancer pharmaceutical composition comprising an in vivo cell input chip as an active ingredient.
2. The method of claim 1, wherein the first component is hyaluronic acid methacrylate (HA-MA) or a derivative thereof, and the second component is oxidized hyaluronic acid methacrylate (oxHA-MA), an oxHA-MA derivative (oxHA). -MA) or hyaluronic acid aldehyde methacrylate (HA-ald-MA) pharmaceutical composition for anticancer, characterized in that.
3. The pharmaceutical composition for anticancer according to claim 2, wherein the first component is hyaluronic acid methacrylate, and the second component is hyaluronic acid aldehyde methacrylate (HA-ald-MA).
4. The pharmaceutical composition for anticancer according to claim 2, wherein the first component is hyaluronic acid methacrylate and the second component is oxidized hyaluronic acid methacrylate (oxHA-MA).
5. The method of claim 1, wherein the cells are Langerhans islet cells, Serto cells, dopaminergic neurons, stem cells, mesenchymal stem cells, umbilical cord blood cells, embryonic stem cells, neural stem cells, differentiated stem cells, natural killer (NK) cells, And B cells comprising at least one pharmaceutical composition for anti-cancer.
6. The method of claim 1, wherein the cells are natural killer (NK) cells, characterized in that the pharmaceutical composition for cancer.
7. 7. The pharmaceutical composition for anticancer according to claim 6, wherein the natural killer (NK) cells are chimeric antigen receptor-natural killer (CAR-NK) cells.
8. The pharmaceutical composition for anticancer according to claim 7, wherein the chimeric antigen receptor (CAR) comprises an ecto-domain of a cancer-specific anti-EGFR affibodies.
9. The pharmaceutical composition for anti-cancer according to claim 8, wherein the CAR comprises a hinge, transmembrane, CD28, DAP10 and CD3ζ.
10. The pharmaceutical composition for anticancer according to claim 1, wherein the cancer is solid cancer or blood cancer.
11. The pharmaceutical composition for anticancer according to claim 10, wherein the solid cancer is lung cancer, brain cancer, liver cancer, breast cancer, ovarian cancer or colon cancer.
12. The pharmaceutical composition for anti-cancer according to claim 10, wherein the blood cancer is leukemia, multiple myeloma, aplastic anemia or malignant lymphoma.
13. A composition for cell culture comprising a porous three-dimensional cryogel scaffold comprising a cross-linked structure of hyaluronic acid methacrylate and oxidized hyaluronic acid methacrylate.

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