KR102351107B1 - Hydrogel composition for pH control having antitumor effect and uses thereof - Google Patents

Abstract

It relates to a hydrogel composition for pH adjustment having an anti-tumor effect and a use thereof, and according to a hydrogel composition for pH adjustment according to an aspect and a pharmaceutical composition comprising the same, by buffering the pH of the tumor microenvironment (TME) , has an effect that can be usefully used as a cancer treatment.

Description

A hydrogel composition for pH control having an antitumor effect and uses thereof

It relates to a hydrogel composition for pH adjustment having an anti-tumor effect and a use thereof.

Immuno-oncology drugs, called third-generation anticancer drugs, are one of the most recent treatments, and they bind to the PD-1 receptor of immune cells and suppress the 'immune checkpoint', thereby activating the immune system and attacking cancer cells. These immune checkpoint inhibitory anticancer drugs have already been approved for use in melanoma and lung cancer by the US Food and Drug Administration (FDA), and are steadily expanding the pipeline for other indications. However, despite the success of immune checkpoint inhibitors, there are only a few patients who show therapeutic effects. Accordingly, researchers' efforts to increase the effectiveness of the drugs are continuing.

Immune checkpoint inhibitors are receiving active attention as new tumor immunotherapy with FDA approval in 2010. Immune checkpoint inhibitors are a concept that includes immunosuppressive regulatory molecules that prevent immune cells from attacking their own cells, and the expression of PD-L1 on the surface of tumor cells creates an immune-suppressive environment within the tumor. The currently developed immune checkpoint inhibitors have limited therapeutic effects in non-immune tumors, have side effects due to non-selective immune responses, and have high cost limitations. Accordingly, the necessity and importance of the study of immune checkpoint inhibitor antibody co-administration and mechanism (tumor microenvironment control) has emerged.

Immune checkpoint inhibitors currently in clinical trials are in clinical trials for Tremelimumab and Ipilimumab, which target CTLA-4, and Medimmune and Pfizer, and Nivolumab, which targets PD-1 ), CT-011, Lamvrolizumab, and AMP-224 are undergoing clinical trials at BMS, Curetech, Merck, and Ampleimmune, and MEDI4376 targeting PD-L1 is clinically conducted at Medimmune and AstraZeneca, etc. is in progress

Clinical application of single antibodies targeting PD-1 or PD-L1 to block the immune checkpoint has shown long-term cancer reduction in renal, melanoma and lung cancer patients. However, in the case of refractory solid cancer, even when the immune system is activated, due to the ability to escape from the activated anti-tumor immune response, it has been reported that there is a limitation in tumor recurrence in a small number of patients when an immune checkpoint inhibitor antibody is used. In order to improve these limitations, the need to develop a technology for maximizing the anticancer effect has emerged through the control study of the relevant tumor microenvironment.

Republic of Korea Patent Registration No. 10-1666792

One aspect is a biodegradable polymer; And to provide a hydrogel composition for pH adjustment comprising a basic material.

Another aspect is to provide a pharmaceutical composition for preventing or treating cancer comprising the hydrogel composition.

One aspect is a biodegradable polymer; And it provides a hydrogel composition for pH adjustment comprising a basic material.

In one embodiment, the biodegradable polymer may have temperature sensitivity.

The temperature-sensitive polymer exists in a solution state at room temperature and exists in a viscous gel state near body temperature, and may mean a polymer capable of sol-to-gel phase transition.

The sol-gel phase change may mean that the sol and the gel move from one state to another by a change in external conditions such as temperature, pressure, or pH. For example, it may mean changing from a sol state to a gel state or from a gel state to a sol state. The sol-gel transition may be reversible or irreversible.

In the above aspect, the sol-gel transition from the sol state to the gel state of the composition may occur at any temperature of 30 to 40°C. Any temperature from 30 to 40°C, any temperature from 31 to 40°C, any temperature from 32 to 40°C, any temperature from 33 to 40°C, any temperature from 34 to 40°C, any temperature from 35 to 40°C, from 32 to Any temperature of 39°C, any temperature of 33-38°C, any temperature of 34-38°C, any temperature of 35-38°C, for example, 31°C, 32°C, 33°C, 34°C, 35°C, It may occur at 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, but is not limited thereto. For example, the composition in a sol state may be converted to a gel state at a temperature close to body temperature, for example, about 37°C. In this way, if it gels rapidly near body temperature, it can be more usefully used for tissue engineering applications. Since the composition according to the present invention exists as an aqueous solution at room temperature and gels due to an immediate phase transition near body temperature, the structure can be maintained even in a wet body environment without a separate cross-linking agent treatment. In addition, it does not cause inflammation at the injection site, so it has excellent biocompatibility, is applied topically in vivo, and can be adhered to, for example, a desired soft tissue defect site and maintained for a long time. It can be easily injected into the body using a phosphorus syringe.

The term “hydrogel” may refer to a three-dimensional structure composed of a hydrophilic polymer network, and may exhibit properties such as high water content, porous structure, soft physical properties and biocompatibility. The polymer of the present invention can be immediately changed into a hydrogel form through a phase transition at 30 to 40 °C. Therefore, in the present invention, an instantaneous phase transition from the body to the hydrogel may be possible by the temperature-sensitive polymer.

The term “biodegradable” can mean that a polymer can be chemically degraded in the body to form non-toxic compounds.

In one embodiment, the biodegradable polymer may be an amphiphilic polymer.

The amphiphilic polymer is polyethylene glycol (polyethyleneglycol; PEG), polyisopropylacrylamide (poly N-isopropylacrylamide; PNIPAM), polymethylacrylate (PMA), polymethylmethacrylate (polymethylmethacrylate; PMMA), polyacryl amide, polyacrylic acid (PAA), polymethacrylic acid (PMAA), polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA), methyl cellulose , Polyethylene oxide (PEO), collagen (Collagen), alginate (Alginate), chitosan (Chitosan), and linear, block, graft or dendrimer hydrophilic polymer block including those selected from the group consisting of combinations thereof; And, polypropylene oxide (PPO), polylactic acid (polylactic acid; PLA, polylactide glycolide copolymer (poly lactide-coglycolide; PLGA), polycaprolactone (polycaprolactone), polyvinyl acetate (polyvinylacetate; PVAc), It may be made of a linear, block, graft, or dendrimer hydrophobic polymer block including those selected from the group consisting of a polypeptide (Polypeptide) and combinations thereof.

The amphiphilic polymer may include an amphiphilic triblock copolymer. For example, (PPO)x-(PEO)y-(PPO)z (each of x, y, and z is independently 3 to 10000) may include a polymer. Here, the PPO represents polypropylene oxide and PEO represents polyethylene oxide, respectively.

In the present invention, Pluronic® F-127 was used as an example of the amphipathic triblock copolymer, but the present invention is not limited thereto, and all amphiphilic triblock copolymers of the following pullulonic model names may be used: P85, P84, P123, P104 , P103, N3, L92, L81, L64, L62, L61, L43, L35, L31, L121, L101, L10, FT L61, F88, F87, F77, F68, F38, F127, F108, 31R, 25R, 17R, 10R, etc.

In addition, since other commercially available polymers such as Lutrol, Poloxamer, and Epan have a structure similar to the above-described pluronic-based amphiphilic triblock copolymer, these can also be used.

In one embodiment, the basic compound _{may be selected from the group consisting of NaHCO 3} , Na ₂ CO ₃ , NaOH, basic amino acids and amines, but is not limited thereto.

The basic amino acid includes, for example, arginine, lysine, or histidine.

In one embodiment, the hydrogel may have pores having a diameter of 10 to 200 nm.

The hydrogel may be porous including a plurality of pores on at least one portion of the surface and the inside, and in the present invention, a basic material may be supported in the pores.

In one embodiment, the hydrogel may activate T cells in a tumor.

The T cells may be CD4+ T cells, CD8+ T cells or NK T cells.

Another aspect provides a pharmaceutical composition for preventing or treating cancer comprising the hydrogel composition.

In one embodiment, the formulation of the pharmaceutical composition may be an injection.

The term “pharmaceutical composition” may refer to a molecule or compound that confers some beneficial effect upon administration to a subject. Advantageous effects include enabling diagnostic decisions; amelioration of a disease, symptom, disorder or condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and responding to a disease, symptom, disorder or condition in general.

The cancer refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body, wherein the cancer or cancerous tissue may include a tumor. Exemplary conditions include cancers such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovial sarcoma, mesothelioma, Ewing tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, brain cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous adenocarcinoma, papillary carcinoma, papillary adenocarcinoma , cystic adenocarcinoma, medullary cancer, bronchogenic cancer, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic cancer, Wilms tumor, cervical cancer, Hodgkin lymphoma, non-Hodgkin's lymphoma, testicular cancer , lung cancer, small cell lung cancer, brain cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngoma, ependymocytoma, pineal tumor, hemangioblastoma, auditory neuroma, oligodendroglioma, meningioma, melanoma, multiple myeloma, neuroblastoma, retina blastoma and leukemia. In some specific embodiments, the cancer to be treated is a solid cancer such as breast cancer, ovarian cancer, colon cancer, prostate cancer, bone cancer, colorectal cancer, gastric cancer, lymphoma, malignant melanoma, liver cancer, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, thyroid cancer , kidney cancer, cancer of the bile duct, brain cancer, cervical cancer, sinus cancer, bladder cancer, esophageal cancer, Hodgkin's disease and adrenal cortical cancer.

The pharmaceutical composition may be administered parenterally during clinical administration and may be used in the form of general pharmaceutical formulations. Parenteral administration may refer to administration via a route other than oral administration, such as rectal, intravenous, peritoneal, muscle, arterial, transdermal, nasal, inhalation, ocular and subcutaneous. When the pharmaceutical composition of the present invention is used as a pharmaceutical, it may further contain one or more active ingredients exhibiting the same or similar function.

When formulating the pharmaceutical composition, it is usually prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base of the suppository, Witepsol, macrogol, Tween 61, cacao butter, liulinji, glycerogelatin, etc. may be used.

In addition, the pharmaceutical composition can be used by mixing with various pharmaceutically acceptable carriers such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, ascorbic acid to increase stability or absorption Antioxidants such as (Ascorbic acid) or Glutathione, Chelating agents, low molecular weight proteins or other Stabilizers may be used as pharmaceuticals.

In addition, the present invention can be provided as an injectable cancer treatment agent by having the pharmaceutical composition as described above have a mechanical strength suitable for injection. For example, the hydrogel composition according to the present invention can be applied as an injection for injection into a tumor in the body.

Since the composition of the present invention is bonded to a temperature-sensitive polymer and can change to an aqueous solution state at room temperature and a gel state in the body, when applied as an injection, it can be easily injected into a local area of the body through an injection needle without blockage, and when injected into the body It can be gelled so that the desired effect can be sustained for a long time. When the composition of the present invention is injected through an injection needle, it may be injected into the body using, for example, 15 to 26 G, 15 to 21 G needles.

The composition of the present invention for parenteral administration may include sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders prepared immediately before use as sterile solutions or suspensions. Examples of suitable sterile aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, physiological saline, ethanol, polyols (eg, glycerol, propylene glycol, polyethylene glycol, etc.) and mixtures thereof, vegetable oils (eg, olive oil), and injectable organic esters (eg, ethyl oleate). For example, in the case of a dispersing agent and suspending agent, a coating material such as lecithin may be used to maintain an appropriate specific size, and a surfactant may be used to maintain appropriate fluidity. In addition, parenteral compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Sterilization of the injectable formulation can be used, for example, by filtration through a sterile filter, or by pre-sterilizing the components of the mixture before mixing, during preparation, or immediately before administration.

Any of the terms or elements mentioned in the pharmaceutical composition as mentioned in the description of the claimed hydrogel composition is understood as being referred to in the description of the claimed hydrogel composition.

According to the hydrogel composition for pH adjustment and the pharmaceutical composition comprising the same according to an aspect, by buffering the pH of the tumor microenvironment (TME), there is an effect that can be usefully used as a cancer treatment agent.

1 is a diagram _{showing the antitumor effect of pH e-} MIG by increasing the pH within the tumor.
2 is a graph showing the results of flow cytometry analysis of CD8 + T cell proliferation according to pH.
3 is a graph showing the results of measuring IFNγ production according to pH by ELISA.
Figure 4a is a _{photograph showing the change of pH e-} MIG to the sol-gel phase with temperature; Figure 4b is a photograph showing that the pH within the _e -MIG in the tumor when administered in the pH _e -MIG tumor.
5 is a graph of pH _e -MIG containing NaHCO ₃ shows that the pH increased by discharging the NaHCO _3.
6 is a cryo-transmission electron microscope (cryo-TEM) observation photograph showing the pore structure _{of pH e-MIG.}
7 is a graph measuring the pH before and after injection _{of pH e-MIG into the tumor.}
8 is a diagram showing the injection cycle _{of pH e-} MIG into the tumor of the mouse.
9 is a graph showing the results of flow cytometry analysis of tumor-infiltrating T cells when _{pH e-MIG is injected into a mouse tumor.}
10 is a graph showing the results of flow cytometric analysis comparing T cells when _{pH e-MIG is injected and when not.}
11 is a graph of flow cytometry analysis comparing the CD8 + T cell / T _{reg ratio between the pH e-MIG injection and the non-injection.}
12 is a graph showing the results of flow cytometry analysis of bone marrow cells in tumors when _{pH e-MIG is injected.}
13 is a graph showing the results of flow cytometry analysis of the bone marrow cell frequency in the case of _{pH e-MIG injection and the case where it is not.}
14 is a graph showing the results of flow cytometry analysis of the proliferation of CEF (CMV-EBV-Flu) antigen-specific human CD8 + T cells upon blocking of PD-1 and / or TIGIT according to pH.
15 is a graph showing the results of flow cytometry analysis of IFNγ secretion upon blocking of PD-1 and / or TIGIT according to pH.
16 is a graph showing the injection cycle of pHe-MIG and anti-TIGIT / anti-PD-1 antibody, and a graph showing the tumor volume in each case.
17 is a graph showing the results of flow cytometry analysis of TNF-α and IFN-γ secretion in the case where pHe-MIG and anti-TIGIT / anti-PD-1 antibody were injected together and when not.
18 is a graph showing the results of flow cytometry analysis of 4-1BB secretion in the case where pHe-MIG and anti-TIGIT / anti-PD-1 antibody were injected together and when not.
Figure 19 shows the immune checkpoint receptor expression of CD8 + T cells in the case where pHe-MIG and anti-TIGIT / anti-PD-1 antibody were co-injected using t-stochastic neighbor embedding (t-SNE). This is the graph shown.
20 is a graph showing the results of flow cytometry analysis of immune checkpoint receptor expression in CD8 + T cells when pHe-MIG and anti-TIGIT / anti-PD-1 antibody are co-injected and when not.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. NaHCO ₃ pH containing _e -Manufacture of MIG

In the present invention, it was intended to prepare a pH-modulating injectable gel (pH _{e-MIG), which is a pH-adjusted injection gel.}

Unless otherwise noted, reagents were purchased from Sigma-Aldrich and used as received: Pluronic® F-127, sodium bicarbonate (NaHCO ₃ ), lactic acid, phosphate buffered saline (PBS). ) (pH 7.4, 1X, Gibco®).

Specifically, all hydrogel samples were prepared by adding Pluronic® F-127 powder to a PBS solution at a concentration of 20% (w / v). NaHCO ₃ powder was dissolved in the hydrogel solution at concentrations of 50, 100 and 200 mM, respectively. The hydrogel mixture was then placed on a rotor at 4° C. overnight to obtain pH _{e- MIG containing NaHCO 3 . All pH e-} MIG samples were placed on ice before use.

Experimental Example 1. NaHCO on human CD8 + T cells ₃ analysis of the effect of

Since tumor progression is promoted in acidic conditions, an experiment was performed to analyze the effect of _{NaHCO 3 on human CD8 + T cells in acidic conditions.}

Specifically, peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors through density gradient centrifugation. CD8 + T cells were isolated by negative selection using a magnetic isolation kit (STEMCELL Technologies). To achieve a lower pH (acidic), 15 mM L-lactic acid (Sigma-Aldrich) was added to the culture medium. For activation, CD8 + T cells were stimulated with anti-CD3 (clone UCHT1, 5 μg/mL) and anti-CD28 (clone CD28.2, 2 μg/ml) antibodies in plate-bound form. Neutralizing monoclonal antibodies or isotype controls against human PD-1 (MK-3475, Pembrolizumab) and TIGIT (MBSA43) were used. It was then incubated for 3 days under normal, acidic and buffered conditions with _{40 mM NaHCO 3 .} Proliferation of CellTrace Violet (CTV)-labeled CD8 + T cells was assessed by quantification of CTV dilution. To measure antigen-specific memory T cell responses, PBMCs were stimulated for 5-6 days with CEF pooled peptides (JPT Peptide Technologies) under different pH conditions. Cell supernatants were collected and IFNγ production was measured by ELISA.

As a result, in the NaHCO ₃ buffered condition, the proliferation of CD8 + T cells and IFNγ production occurred similar to that of the normal condition compared to the acidic condition ( FIGS. 2 and 3 ).

Through this, it _{was confirmed that NaHCO 3} is helpful for the anti-tumor effect by increasing the pH and proliferating CD8 + T cells.

2 is a graph showing the results of flow cytometry analysis of CD8 + T cell proliferation according to pH.

3 is a graph showing the results of measuring IFNγ production according to pH by ELISA.

Experimental Example 2. Hydrogel Characterization of pHe-MIG

The pH _e -MIG was made with Pluronic® F-127, an amphiphilic triblock copolymer, and was characterized.

Specifically, Pluronic® F-127 reversibly changes the sol-gel phase according to temperature, and thus pHe-MIG containing _{100 mM NaHCO 3} It showed a gel phase when placed at room temperature, and a sol phase at 4°C (FIG. 4a).

In addition, 6-week-old male C57BL/6 mice were purchased from Oriental Bio (Korea), and 1x10 ⁶ MC38 cells in 1xHBSS were subcutaneously transplanted into the mice. After forming a solid tumor with a volume of about 100 mm ³ (± 25 mm ³ _{), FITC-loaded pH e-} MIG was locally injected into the tumor. Then, the tumor was excised to excite the FITC dye by UV light with a wavelength of 365 nm. As a result, it was confirmed that the pH _e- MIG in a gel state in the tumor was locally located at the injection site (FIG. 4b).

The property of reversibly changing the sol-gel phase of _{pH e- MIG means that NaHCO 3} can be easily introduced into the gel matrix and can impart curing ability to the tumor microenvironment (TME).

Figure 4a is a _{photograph showing the change of pH e-} MIG to the sol-gel phase with temperature; Figure 4b is a photograph showing that the pH within the _e -MIG in the tumor when administered in the pH _e -MIG tumor.

Experimental Example 3. NaHCO in pHe-MIG ₃ Emission analysis

An experiment was performed to analyze the release of _{pH e} -MIG NaHCO _{3 .}

Specifically, a simulated TME fluid solution was prepared at a pH of 6.6 by slowly adding lactic acid to the PBS solution while monitoring the pH change at 37°C. 100 μl of pHe-MIG sample was injected into 3 ml of simulated TME solution at concentrations of 50, 100 and 200 mM, respectively. Changes in pH of the solutions were measured at 0, 0.5, 1, 2, 4, 8 and 24 h time points. As a result, there was a sharp increase in pH at the beginning, and the pH was gradually increased until completely saturated (FIG. 5).

In addition, in _{order to confirm the morphology of pHe-MIG containing 100 mM NaHCO 3} , it was observed with a cryo-transmission electron microscope (cryo-TEM) in a Tecnai F20 having a 200 kV field emission gun. An aliquot of pHe-MIG sample was placed on a TEM grid, followed by a Vitrobot® with liquid ethane. Samples were prepared using a plunge freezer. As a result, a nanopore structure with a size of ~50-100 nm due to the Pluronic® F-127 gel of pHe-MIG was observed (FIG. 6). This means that when pHe-MIG was injected into the TME solution, the initial pH was rapidly increased as described in the above experiment, due to the burst release of _{NaHCO 3 due to the nanopore structure.}

In order to confirm the pH buffering effect of pHe-MIG in vivo, pHe-MIG _{having a concentration of 100 mM NaHCO 3} was injected into the tumor and the pH was measured at 0.5 and 72 hours. After calibration by connecting the pH electrode (InLab Nano, Mettler Toledo) to the Orion star A211 pH meter (Thermo fisher), the pH electrode was inserted into the center of the tumor. Triplicate measurements were taken at each time point and averaged. As a result, the pH of the tumor before injection was acidic at 6.69 ± 0.01, and the pH after injection of pHe-MIG rapidly increased to 7.10 ± 0.02 ( FIG. 7 ). However, the pH at 72 h after injection was slightly reduced to 7.00 ± 0.01, which is due to the combination _{of biodegradation of pHe-MIG and NaHCO 3 saturation.} This result means that NaHCO ₃ of pHe-MIG is released not only in vitro but also in vivo.

5 is a graph of pH _e -MIG containing NaHCO ₃ shows that the pH increased by discharging the NaHCO _3.

6 is a cryo-transmission electron microscope (cryo-TEM) observation photograph showing the pore structure _{of pH e-MIG.}

7 is a graph measuring the pH before and after injection _{of pH e-MIG into the tumor.}

Experimental Example 4. Analysis of the immune-friendly TME production effect of pHe-MIG

Flow cytometry was performed to determine the effect of pHe-MIG on TME.

Specifically, 6-week-old male C57BL/6 mice were purchased from Oriental Bio (Korea), and 1x10 ⁶ MC38 cells in 1xHBSS were subcutaneously transplanted. After solid tumors with a volume of about 100 mm ³ (± 25 mm ³ _{) were formed, pHe-MIG containing 100 mM NaHCO 3} and Pluronic® F-127 (IG) without _{NaHCO 3} as a control were each administered. Insulin syringes (28.5 gauge, 0.5 inch needle; BD) were injected into the tumor centers of mice 3 times every 72 hours ( FIG. 8 ). Then, the tumor tissue was mechanically minced and treated with collagenase IV (250 units/ml, Worthington) and DNAse I (100 μg/ml, Roche) at 37°C for 30 min. Tumor-infiltrating leukocytes were obtained by density separation in ^{Lymphopure™ (Biolegend).} Prepared single cells were blocked with anti-mouse CD16/32 (BioLegend) or human BD Fc Block™ (BD), followed by FACS buffer (2% FBS and 0.1% sodium azide) at 4°C for 20 min. Surface staining was carried out in PBS containing Dead cells were excluded using the Zombie NIR Fixable viability kit (Biolegend). Cytokine intracellular staining was performed after restimulation with PMA/ionomycin in the presence of Golgi-Plug for 5 hours. Flow cytometry was performed using a Cytoflex flow cytometer (Beckman Coulter) and FlowJo software (v.10.5.3, TreeStar). The following fluorescent staining-conjugated anti-human antibodies were used: anti-CD8 (SK1), anti-CD45RA (HI100), anti-CD45RO (UCHL1) were purchased from Biolegend; Antibodies to mouse proteins are: anti-CD45 (30-F11), anti-CD3 (17A2), anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD11b (M1/ 70), anti-Ly6C (HK1.4), anti-MHC-II (AF6-120.1), anti-F4/80 (BM8), anti-CD24 (M1/69), anti-CD11c (N418) from Biolegend was purchased, and anti-Foxp3 (FJK-16s) was purchased from eBioscience.

As a result, it was confirmed that injection of pHe-MIG caused an increase in CD45 + immune cells and infiltration of CD3 + and CD8 + T cells into the tumor ( FIGS. 9 and 10 ). In addition, the CD8 + T cell/Treg ratio was much higher in the pHe-MIG injected tumors compared to the control group (Fig. 11), indicating the induction of an effective anti-tumor immune response. In addition, there was an increase in TAM1 in pHe-MIG treated tumors compared to controls, although the proportion of immunosuppressive myeloid populations including MDSCs and TAM2 was decreased . (Fig. 12, 13). These results suggest that pHe-MIG generates immune-friendly TMEs for T cell activation by accurately regulating local extracellular pH.

8 is a diagram showing the injection cycle _{of pH e-} MIG into the tumor of the mouse.

9 is a graph showing the results of flow cytometry analysis of tumor-infiltrating T cells when _{pH e-MIG is injected into a mouse tumor.}

10 is a graph showing the results of flow cytometric analysis comparing T cells when _{pH e-MIG is injected and when not.}

11 is a graph of flow cytometry analysis comparing the CD8 + T cell / T _{reg ratio between the pH e-MIG injection and the non-injection.}

12 is a graph showing the results of flow cytometry analysis of bone marrow cells in tumors when _{pH e-MIG is injected.}

13 is a graph showing the results of flow cytometry analysis of the bone marrow cell frequency in the case of _{pH e-MIG injection and the case where it is not.}

Experimental Example 5. Analysis of the effect of pHe-MIG as an immune checkpoint inhibitor

Flow cytometry was performed to determine the efficacy of pHe-MIG containing NaHCO _{3 as an immune checkpoint inhibitor.}

Specifically, peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors through density gradient centrifugation. CD8 + T cells were isolated by negative selection using a magnetic isolation kit (STEMCELL Technologies). To achieve a lower pH (acidic), 15 mM L-lactic acid (Sigma-Aldrich) was added to the culture medium. For activation, CD8 + T cells were stimulated with anti-CD3 (clone UCHT1, 5 μg/mL) and anti-CD28 (clone CD28.2, 2 μg/ml) antibodies in plate-bound form. Neutralizing monoclonal antibodies or isotype controls against human PD-1 (MK-3475, Pembrolizumab) and TIGIT (MBSA43) were used. As a control, an IgG antibody (BioX cell) was used. It was then incubated for 3 days under normal, acidic and buffered conditions with _{40 mM NaHCO 3 .} Proliferation of CellTrace Violet (CTV)-labeled CD8 + T cells was assessed by quantification of CTV dilution. To measure antigen-specific memory T cell responses, PBMCs were stimulated for 5-6 days with CEF pooled peptides (JPT Peptide Technologies) under different pH conditions. Cell supernatants were collected and IFNγ production was measured by ELISA.

As a result, the proliferation and IFNγ secretion of CEF (CMV-EBV-Flu) antigen-specific human CD8 + T cells upon PD-1 and/or TIGIT blockade at normal pH were impaired by extracellular acidity, whereas NaHCO ₃ mediated culture was recovered by the medium ( FIGS. 14 and 15 ).

These results indicate _{that pHe-MIG containing NaHCO 3} can act as an immune checkpoint inhibitor and normally regulate CD8 + T cell proliferation and IFNγ secretion by raising the pH.

14 is a graph showing the results of flow cytometry analysis of the proliferation of CEF (CMV-EBV-Flu) antigen-specific human CD8 + T cells upon blocking of PD-1 and / or TIGIT according to pH.

15 is a graph showing the results of flow cytometry analysis of IFNγ secretion upon blocking of PD-1 and / or TIGIT according to pH.

Experimental Example 6. Analysis of the antitumor effect of pHe-MIG

Specifically, 6-week-old male C57BL/6 mice were purchased from Oriental Bio (Korea), and 1x10 ⁶ MC38 cells in 1xHBSS were subcutaneously transplanted. After forming solid tumors with a volume of about 100 mm ³ (± 25 mm ³ ), tumor-bearing mice were divided into 4 groups: (1) IG; (2) pHe-MIG; (3) IG and anti-TIGIT/anti-PD-1 antibodies; (4) pHe-MIG and anti-TIGIT/anti-PD-1 antibodies. Then, pHe-MIG containing 100 mM NaHCO _{3 and Pluronic® F-127 (IG) without NaHCO 3} as a control were each administered with an insulin syringe (28.5 gauge, 0.5 inch needle; BD) 3 every 72 hours. Injected into the tumor center of the mouse mice ( FIG. 16 ). To test the therapeutic effect of pHe-MIG, 25 μg of anti-TIGIT (cloning 1G9, BioXcell) and 25 μg of anti-PD-1 (cloning J43, BioXcell) antibodies were intraperitoneally administered on days 7, 10 and 13. Administered via intravenous injection. Measurements were made twice weekly with calipers and the tumor volume ^{was calculated as (length Х width 2)} /2. When the tumor exceeds 2,000 mm ³ , mice were euthanized and tumor growth inhibition (TGI%) was calculated. As a result, the combination treatment of anti-PD-1 and TIGIT antibody after pHe-MIG injection showed 75% tumor growth inhibition (TGI), whereas the co-treated IG showed only ~47% TGI ( FIG. 16 ). These results suggest that pHe-MIG has an antitumor effect.

In addition, the tumor tissue was mechanically minced and treated with collagenase IV (250 units/ml, Worthington) and DNAse I (100 μg/ml, Roche) at 37° C. for 30 min. Tumor-infiltrating leukocytes were obtained by density separation in ^{Lymphopure™ (Biolegend).} Prepared single cells were blocked with anti-mouse CD16/32 (BioLegend) or human BD Fc Block™ (BD), followed by FACS buffer (2% FBS and 0.1% sodium azide) at 4°C for 20 min. Surface staining was carried out in PBS containing Dead cells were excluded using the Zombie NIR Fixable viability kit (Biolegend). Cytokine intracellular staining was performed after restimulation with PMA/ionomycin in the presence of Golgi-Plug for 5 hours. Flow cytometry was performed using a Cytoflex flow cytometer (Beckman Coulter) and FlowJo software (v.10.5.3, TreeStar). The following fluorescent staining-conjugated anti-human antibodies were used: anti-CD8 (SK1), anti-PD-1 (EH12.2H7), anti-LAG3 (11C3C65), anti-TIM3 (F38-2E2), anti -IFNγ (4S.B3), anti-TNF (MAb11) were purchased from Biolegend; Antibodies to mouse proteins are: anti-CD8 (53-6.7), anti-PD-1 (29F.1A12), anti-LAG3 (C9B7W), anti-TIM3 (B8.2C12), anti-IFN- γ (XMG1.2), anti-TNF (C92-605) and 4-1BB antibodies were purchased from Biolegend. As a result, when pHe-MIG and anti-PD-1 / TIGIT antibody were co-treated, higher levels of TNF-α, IFN-γ and 4-1BB were expressed ( FIGS. 17 and 18 ). This means that pHe-MIG significantly enhanced the cytotoxic CD8 + T cell response. In addition, when tumor-infiltrating CD8 + T cells were observed using the co-inhibitory immune checkpoint receptors PD-1, Lag-3 and Tim-3, indicating the exhaustion of CD8 + T cells, pHe-MIG and anti- Low expression of immune checkpoint receptors on CD8 + T cells was observed when PD-1 / TIGIT antibody was co-treated. Visualized through the t-SNE method. (Fig. 19, 20). This implies a less depleted T cell phenotype, and thus the results suggest that pHe-MIG synergistically potentiates the antitumor effect of PD-1 and TIGIT inhibitor therapy by preventing T cell depletion.

16 is a graph showing the injection cycle of pHe-MIG and anti-TIGIT / anti-PD-1 antibody, and a graph showing the tumor volume in each case.

17 is a graph showing the results of flow cytometry analysis of TNF-α and IFN-γ secretion in the case where pHe-MIG and anti-TIGIT / anti-PD-1 antibody were injected together and when not.

18 is a graph showing the result of flow cytometry analysis of 4-1BB secretion in the case where pHe-MIG and anti-TIGIT/anti-PD-1 antibody were co-injected and when not.

Figure 19 shows the immune checkpoint receptor expression of CD8 + T cells when pHe-MIG and anti-TIGIT / anti-PD-1 antibody are co-injected using t-SNE (t-stochastic neighbor embedding) and when not. This is the graph shown.

20 is a graph showing the results of flow cytometry analysis of immune checkpoint receptor expression in CD8 + T cells when pHe-MIG and anti-TIGIT / anti-PD-1 antibody are co-injected and when not.

Claims (10)

Pluronic F-127 polymer; And comprising a basic material, as a hydrogel composition for adjusting the pH of the tumor microenvironment,
A hydrogel composition for adjusting the pH of a tumor microenvironment, wherein the basic material is released from the hydrogel. The method according to claim 1, wherein the hydrogel is a sol-gel phase transition occurs depending on the temperature, the hydrogel composition for adjusting the pH of the tumor microenvironment. delete delete The method according to claim 1, wherein the basic material is NaHCO ₃ , Na ₂ CO ₃ , NaOH, basic amino acids and amines selected from the group consisting of, the hydrogel composition for adjusting the pH of the tumor microenvironment. delete The method according to claim 1, The hydrogel will have a pore of a diameter of 10 to 200nm, the hydrogel composition for adjusting the pH of the tumor microenvironment. The method according to claim 1, wherein the hydrogel is to activate the T cells in the tumor, the hydrogel composition for adjusting the pH of the tumor microenvironment. An anticancer adjuvant for preventing or treating cancer, comprising the hydrogel composition according to any one of claims 1, 2, 5, 7 and 8, and administered in combination with an immune checkpoint inhibitor. The anticancer adjuvant according to claim 9, wherein the formulation of the anticancer adjuvant is an injection.

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