KR101616705B1 - The specific binding molecules-nanofibers complex and method for activating the same - Google Patents

Abstract

The present invention relates to a specific binding molecule-nanofiber complex and an immune cell activation method using the same. The nanofibers to which the specific binding molecules of the present invention are bound can be easily and conveniently used because the binding and separation of antibodies and target cells therefrom are quick and easy to use, and the target cells can be separated specifically and efficiently, Particularly, about 95% or more of cells can be obtained without loss of cell number. Therefore, the nanofiber having the specific binding molecule of the present invention can provide a disadvantage of the cell separation method using existing beads (about 50% Can be usefully used for simple, rapid and effective cell sorting, in addition to a high content of antibody or aptamer-conjugated nanofibers, which can compensate for the cell loss of the cells. In addition, by directly activating the cells thus isolated, it is very easy to use for cell activity experiments.

Description

Specific binding molecule-nanofiber complex and method for activating the same using the same.

The present invention relates to a specific binding molecule-nanofiber complex and an immune cell activation method using the same.

Isolation of cells is one of the most important technologies in various fields such as biologic or biologic application in the diagnosis or treatment of diseases, and its use is increasing in order to isolate a desired specific cell from a mixture of various cells [McCloskey KE , et al., Anal Chem, 2003, Dec 15: 75 (24): 6868-6874; McCloskey KE, et al., Biotechnol Prog, 2003, May-Jun; 19 (3): 899-907]. Therefore, due to this importance, many studies on cell separation technology are under way in many research groups.

When cells with different specific gravities are separated, they can be separated by velocity sedimentation. On the other hand, when there is little difference between cells, such as discriminating between sensitized cells and unsensitized cells, it is necessary to separate cells from information stained with a fluorescent antibody or information through direct microscopic observation. For example, a cell sorter (Kamarck, M.E., Methods Enzymol., Et al., &Quot; Cell Enzyme Immunoassay ") is used to label cells with fluorescence and to fractionate cells according to the intensity of the label. Vol. 151, p. 150-165, 1987). In recent years, a microfluidic device (Micro Total Analysis, 98) has been used for separating microparticles flowing in a laminar flow , pp. 77-80, Kluwer Academic Publishers, 1998; Analytical Chemistry, 70, pp. 1909-1915, 1998).

Fluorescence activated cell sorter (FACS), which is one of the most widely used cell sorting methods, is a device for analyzing surface antigens of glass cells such as lymphocytes or separating cells by surface antigen or the like using a laser beam When particles or cells in a liquid state pass through a certain detection area, each particle or cell is rapidly measured, and the intensity of fluorescence of each cell is measured. Using the intensity of the fluorescence, And can be selectively separated. Fluorescent dyes or monoclonal antibodies can be used to identify the immune status of the cell's surface and interior to separate cells. However, there is a disadvantage that complex manipulation techniques of the device, highly skilled technicians, time consuming and expensive machines are required [Lancioni CL, et al., J Immunol Methods, 2009, May 15; 344 (1) 15-25].

In addition, the magnetic activated cell sorter, which is one of the currently used methods, separates a desired specific cell with the magnetic field by using an antibody or its corresponding substance having a magnetic particle attached thereto At this time, a bead necessary for cell separation enters the cell by binding to the cell, thereby causing a change in the state of the cell.

On the other hand, antibodies are widely used for the detection, isolation and measurement of biological substances using antibodies specific for cell surface antigens because of their specificity to the antigen. It is necessary to establish a method for isolating highly pure cells that are simpler and easier to stabilize, which have the same cell characteristics, in order to isolate specific cells for cell therapy or to detect specific cells. In addition, the technology to develop an in vitro nano platform that can be applied to patients with cancer and autoimmune diseases by in vitro activation of the separated cells without any separate process is currently a technology at the global stage.

It is an object of the present invention to provide a specific binding molecule-nanofiber complex and a method of activating an immune cell using the same.

Since electrospun nanofibers have a large surface area in a small space and have a good characteristic that they are durable, very easy to handle, can be manufactured in various forms, and are easy to chemically bond various substances It provides a very good directional approach in the application of cell separation.

Thus, the present inventors devised cell separation and cell activation in a single platform, thereby establishing a more convenient and effective one-step cell separation and activation platform.

Furthermore, the present inventors have found that activation and sustained release of immune cells bound to nanofibers can be controlled depending on the type of antibody. For example, when cells are separated by binding anti-CD4 antibody to normal nanofibers, the binding of nanofibers to isolated CD4 + T cells is so strong that the activated cells mainly remain in the nanofiber matrix and very small amounts of cells Was gradually released with time. However, when CD3 + T cells were isolated by binding anti-CD3 antibody to nanofibers and cultured, the affinity between anti-CD3 antibody and immune cells was low, indicating that a large amount of activated immune cells were separated from nanofibers more rapidly Thereby completing the present invention.

In order to achieve the above object,

1) preparing a nanofiber by electrospinning a polymer solution; And

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2) A method for producing a specific binding molecule-nanofiber composite for cell separation, which comprises mixing and binding specific binding molecules to the nanofibers of step 1).

The present invention also provides a specific binding molecule-nanofiber composite prepared according to the above method.

In addition,

1) mixing and culturing cells with the specific binding molecule-nanofiber composite; And

2) separating the cells bound to the complex from the mixture of step 1). The present invention also provides a method for separating cells using nanofibers to which specific binding molecules are bound.

3) a method of activating cells using a specific binding molecule-nanofiber complex in which the cells of step 2) are bound.

The nanofibers to which the specific binding molecules of the present invention are bound can be easily and conveniently used because the binding and separation of antibodies and target cells therefrom are quick and easy to use, and the target cells can be separated specifically and efficiently, Particularly, about 95% or more of cells can be obtained without loss of cell number. Therefore, the nanofiber having the specific binding molecule of the present invention can provide a disadvantage of the cell separation method using existing beads (about 50% Can be usefully used for simple, rapid and effective cell sorting, in addition to a high content of antibody or aptamer-conjugated nanofibers, which can compensate for the cell loss of the cells. In addition, by directly activating the cells thus isolated, it is very easy to use for cell activity experiments.

FIG. 1 schematically shows the manufacturing process of nanofibers.
Figure 2 shows the results of Example 3, which is an optical microscope image of cells released from nanofiber complexes conjugated with anti-CD4 or anti-CD3 monoclonal antibodies.
Figure 3 shows the activation and increase of T cells isolated from the anti-CD3 monoclonal antibody-nanofiber complex as a result of Example 4 over time.

The following examples are provided to further understand the present invention, and the scope and contents of the present invention can not be construed to be reduced or limited.

[Example 1] Preparation of materials

4-nitrophenyl butyrate (4-NPB) and N, N-dimethylformamide (DMF) were purchased from Sigma (ST Louis, MO, USA) and polystyrene , PS, MW = 950, 400), polystyrene-co-maleic anhydride (PSMA, MW = 224,000) and tetrahydrofuran (THF) were obtained from Aldrich (Milwaukee, ), And ethanol was purchased from Deajung (Siheung, Korea). Seven - week - old C57BL / 6 mice were purchased from Narabiotech (Seoul, Korea) and reared in Korea University animal room. All mice used in the experiment were 7-10 weeks of age. All procedures were conducted with the approval of Korea University Experimental Animal Management Committee (KUIACUC-2009-105). Mouse CD3 (145-2C11), CD4 (GK1.5, RM4-4), CD8 (53-6.7), NK1.1 (SEQ ID NO: (PK136) and CD25 (PC61.5) monoclonal antibodies (mAbs), phycoerythrin (PE), or allophycocyanin (APC) were purchased from eBioscience (San Diego, Calif.). An anti-mouse CD19 (6D5) mAb conjugated with Peridinin Chlorophyll Protein Complex (PerCP) was purchased from BioLegend (San Diego, Calif.). All other reagents were purchased from Sigma and Aldrich at the highest commercially available grade.

[Example 2] Production of nanofiber

1, the polymer solution was prepared by mixing a 2: 1 weight ratio mixture of polystyrene (PS) and styrene-co-maleic anhydride (PSMA) in tetrahydrofuran (THF ). First, the mixed solution was completely dissolved in the PS and PSMA mixture for 3 hours using a magnetic stirrer. Then, an acetone solution was added to the polymer solution to reduce the viscosity of the polymer solution. The polymer solution was filled into a 5 mL plastic syringe equipped with a 30 gauge stainless steel injection needle. The operating conditions of the voltage using the high voltage supply were 7 kV and the solution was supplied at 0.1 mL / hr using a syringe pump (PHD-2000 Infusion, Harvard Apparatus, Holliston, Mass., USA). The electrospun nanofibers were collected on a clean aluminum foil (connected to the bottom) at a suitable distance (7-10 cm) from the needle tip.

[Example 3] Optical microscope image of cells released from anti-CD3 monoclonal antibody-nanofiber composite and anti-CD4 monoclonal antibody-nanofiber composite

As shown in FIG. 2, the cells bound to the specific binding molecule-nanofiber composite, that is, the anti-CD3 monoclonal antibody-nanofiber composite (CD3 NF) and the anti-CD4 monoclonal antibody-nanofiber composite (CD4 NF) CD3 monoclonal antibody 1 mg / ml, anti-CD28 monoclonal antibody 1 mg / ml for 7 days to take a photograph under a microscope.
Next, experiments were conducted to determine whether cells attached to the anti-CD3 monoclonal antibody-nanofiber complex (CD3 NF) and anti-CD4 monoclonal antibody-nanofiber complex (CD4 NF) could be activated or released from the scaffold .
T cells attached to anti-CD3 monoclonal antibody-nanofiber complex (CD3 NF) and anti-CD4 monoclonal antibody-nanofiber complex (CD4 NF), namely CD3 + T cells and CD4 + T cells, CD3 < / RTI > monoclonal antibody, 1 mg / ml anti-CD28 monoclonal antibody, which leads to activation of T cells via stimulation of T cell receptor / CD3 complex and coadministering CD28.
However, when the anti-CD4 monoclonal antibody-nanofiber complex conjugated with CD4 + T cells in the presence of a soluble anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody, was cultured for 7 days, most of the CD4 + CD4 + T cells were collected from the culture medium adhered to the CD4 monoclonal antibody-nanofiber complex.
In contrast, when the anti-CD3 monoclonal antibody-nanofiber complexes were cultured for 7 days, CD3 + T cells were isolated from the anti-CD3 monoclonal antibody-nanofiber complex, and showed a more active form on the microscope.

[Example 4] Time-dependent activation and increase of CD3 + T cells isolated from anti-CD3 monoclonal antibody-nanofiber complexes

In order to confirm the degree of activation of CD3 + T cells released from the anti-CD3 monoclonal antibody-nanofiber complex, CD25 level, which is an activation marker of the cell, was confirmed after incubation for 7 days. As a result, as shown in FIG. 3, it was confirmed that the level of CD25, which is an activation marker of CD3 + T cells isolated from the anti-CD3 monoclonal antibody-nanofiber complex, is greatly elevated.

Claims (2)

delete 1) preparing polystyrene and polystyrene maleic anhydride copolymers in a weight ratio of 2: 1 to prepare nanofibers by electrospinning a polymer solution dissolved in tetrahydrofuran;
2) preparing a specific binding molecule-nanofiber composite by mixing an anti-CD3 monoclonal antibody, which is a specific binding molecule for CD3 + T cells, with the nanofibers of step 1);
3) mixing the cells containing the specific binding molecule-nanofiber composite and the CD3 + T cells of step 2) and culturing them to obtain a specific binding molecule-nanofiber complex in which CD3 + T cells are bound; And
4) adding the anti-CD3 monoclonal antibody and the anti-CD28 monoclonal antibody to the specific binding molecule-magnetic nanofiber complex to which the CD3 + T cells are bound in step 3), and culturing the CD3 + T cell, A method of in vitro activation by separating CD3 + T cells from specific binding molecule-nanofiber complexes.

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