PREPARATION METHOD OF NK CELLS AND USE THEREOF IN THE TREATMENT OF CANCER

Abstract

The present disclosure relates to a method for preparing NK cells and an application thereof in treatment of a cancer. The present disclosure provides a pharmaceutical composition prepared from NK cells and a monoclonal antibody of CD105, which can be used for the treatment of colorectal cancer and has a good application prospect.

Claims

1-6. (canceled)

7. A pharmaceutical composition comprising: (a) a monoclonal antibody of CD105, (b) NK cells, or (c) a combination of (a) and (b); and a pharmaceutically acceptable carrier; wherein the monoclonal antibody of CD105 is a monoclonal antibody named 1A4, which comprises a light chain variable region having an amino acid sequence of SEQ ID NO:1, and a heavy chain variable region having an amino acid sequence of SEQ ID NO:2; and wherein the NK cells are prepared by the following procedure: isolating umbilical cord blood mononuclear cells by using Ficoll-Hypaque density gradient centrifugation method, washing the isolated mononuclear cells with PBS for 3 times, and then suspending them in EX vivo-15 serum-free culture medium; adjusting the concentration of the mononuclear cells to 3×10.sup.6 cells/ml, adding 5ml of cell suspension, and cytokines IL-2 (1000u/ml), IL-15 (50ng/ml), and IL-21 (30ng/ml) to a culture flask coated with anti-CD3 antibody for continuous culture in 5% CO.sub.2 at 37° C. for 18 days, during which, depending on the color of the culture medium and the size of NK cell colonies, supplementing the culture medium in half amount and supplementing the cytokines in full amount; and for supplementing the cytokines for the first time, adding 1000u/ml of IL-2, 50ng/m1 of IL-15, and 30ng/m1 of IL-21, and from the second time, adding 1000u/ml of the cytokine IL-2 and 50ng/m1 of IL-15.

8. A method for treating colorectal cancer in a subject in need thereof, comprising the step of administrating to the subject a therapeutically effective amount of: (a) NK cells, (b) a monoclonal antibody of CD105, or (c) a combination of (a) and (b); wherein the NK cells are prepared by the following procedure: isolating umbilical cord blood mononuclear cells by using Ficoll-Hypaque density gradient centrifugation method, washing the isolated mononuclear cells for 3 times with PBS, and then, suspending them in EX vivo-15 serum-free culture medium; adjusting the concentration of the mononuclear cells to 3 x10.sup.6 cells/ml, adding 5m1 of cell suspension, and cytokines IL-2 (1000 u/ml), IL-15 (50 ng/ml), and IL-21 (30 ng/ml) to a culture flask coated with anti-CD3 antibody for continuous culture in 5% CO.sub.2 at 37° C. for 18 days; during which, depending on the color of the culture medium and the size of NK cell colonies, supplementing the culture medium in half amount and supplementing the cytokines in full amount; wherein for supplementing cytokines for the first time, adding 1000u/ml of IL-2, 50ng/m1 of IL-15, and 30ng/m1 of IL-21, and from the second time, adding 1000u/ml of the cytokine IL-2 and 5Ong/m1 of IL-15; and wherein the monoclonal antibody of CD105 is a monoclonal antibody named 1A4, which comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region having an amino acid sequence of SEQ ID NO: 2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 shows the antibody subtype identification results.

[0023] FIG. 2 shows the inhibitory effect of the monoclonal antibody on LOVO cells.

[0024] FIG. 3 shows the inhibitory effect of drugs on transplanted tumors.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present disclosure is further described below by way of examples, but is not limited to the scope of the described examples. The experimental methods that do not specify specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product description.

EXAMPLE 1

[0026] Preparation of NK Cells

[0027] Umbilical cord blood mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation method, washed with PBS three times, and suspended in EX vivo-15 serum-free culture medium. The mononuclear cell concentration was adjusted to 3×10.sup.6 cells/ml. To a culture flask coated with anti-CD3 antibody, 5m1 of cell suspension and cytokines IL-2 (1000u/m1), IL-15 (50ng/ml), and IL-21 (30ng/m1) were added. The culture flask was then placed in an incubator with 5% CO.sub.2 at 37° C., and cultured for 18 days. During the period, depending on the color of the culture medium and the size of NK cell colonies, the culture medium was supplemented in half amount, and the cytokines were supplemented in full amount. For supplementing the cytokines for the first time, 1000 u/ml of IL-2, 50 ng/ml of IL-15, and 30 ng/ml of IL-21 were added. From the second time, 1000 u/ml of the cytokine IL-2 and 50 ng/ml ml of IL-15 were added. After 18 days, cell phenotypes before and after cell expansion were detected by flow cytometry, and the CD56+CD3-cells reached (81.53±2.53)%.

Example 2

[0028] Preparation of Anti-CD105 Monoclonal Antibody

[0029] 1. Preparation of Antigen

[0030] According to the amino acid sequence of human CD105, a highly immunogenic epitope peptide, also called antigenic peptide FVLRSAYSSCGMQVSASMISNEAVVNILSSSSPQRK, was obtained by screening, and was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0031] 2. Preparation of Hybridoma Cells

[0032] BALB/c mice were immunized. First immunization: 100 μL, of CFA was added to 100 μL, of 0.5 mg/mL CD105 antigenic peptide for emulsification, and the obtained emulsion was used to immunize 8-week-old mice. Second immunization: 3 weeks after completion of the first immunization, 100 μL, of IFA was added to 100 μL, of 0.5 mg/mL CD105 antigenic peptide for emulsification, and the obtained emulsion was used to immunize the mice for the second time. Third immunization: Third immunization was performed 3 weeks after completion of the second immunization. The method was the same as that for the second immunization. On the 10th day after completion of the third immunization, tail vein blood was collected to measure the antibody titer. A 96-well ELISA plate was coated with 1 ug/mL CD105 antigenic peptide solution. 1:100 mouse serum was diluted at multiple times in the 96-well ELISA plate to measure the antibody titer in mouse serum. Three mice with an antibody titer of over 1:10000 were selected for cell fusion. The spleens were taken from the three immunized mice, and placed into a sterile tissue culture dish with 4 mL of cell culture medium (containing 2% RPMI1640, 0.2% NaHCO.sub.3, 1% Penicillin-streptomycin, and 10% inactivated fetal bovine serum), and smashed. NS-1 myeloma cells and mouse spleen cells were mixed in a 50mL centrifuge tube at a ratio of 1:10, and centrifuged at 1500 r/min for 5 min. After removal of the supernatant, 1mL 50% PEG3000 was slowly added. After washing the PEG3000 off with RPMI1640 medium, hybridoma cells were screened by cloning method in hemiglial medium, which comprises: culturing hybridoma cells in a 96-well cell culture plate (37° C., 5% CO2); and 3 days later, using indirect ELISA method to screen the positive grown cell clone wells, with an antibody positive rate of hybridoma cells being 9.53%. The four most significantly positive monoclonal hybridoma cells were cloned and subcloned by limiting dilution method.

[0033] Finally, two hybridoma cell lines which can secret anti-CD105 monoclonal antibody were obtained, named 1A4 and 4C6 respectively.

[0034] Mice were immunized with the two hybridoma cell lines, respectively, to collect ascites fluid. The monoclonal antibodies in the ascites fluid were routinely purified by using protein A column, followed by protein concentrating for use. As determined by Lowry method, the antibody 1A4 had a concentration of 2.1 mg/mL and the 4C6 had a concentration of 2.3 mg/mL.

EXAMPLE 3

[0035] Subtype identification and titer evaluation of the monoclonal antibody 1A4 The mouse monoclonal antibody typing kit was used for identification the subtype, and the operation steps were carried out in strict accordance with the instructions. The results were shown in FIG. 1.

[0036] The monoclonal antibody 1A4 was identified as immunoglobulin G1 class (FIG. 1).

[0037] Determination of titer of the monoclonal antibody: the concentrated antibody was serially diluted at 1: 1000, 1: 2000, 1: 4000, 1: 8000, 1: 16 000, 1: 32 000, 1: 64 000, 1: 128 000, 1: 256 000, and 1: 512 000, and then the dilutions were added to the microtiter plate coated with the antigenic peptide, to measure the A450 nm values by indirect ELISA method, which were then plotted versus the dilution degree values. The dilution degree value when A450 nm was 0.1 was defined as its titer. As a result, the titer of the monoclonal antibody 1A4 was about 1:512000.

EXAMPLE 4

[0038] Affinity identification and sequence identification of the monoclonal antibody 1A4

[0039] The binding ability of the monoclonal antibody 1A4 to the antigenic peptide was identified by SPR method. Specifically, the binding kinetics of the monoclonal antibody 1A4 to the antigenic peptide was measured by the surface plasmon resonance (SRP) method with the BIAcoreX100 instrument, the antigenic peptide was directly coated on the CMS biosensor chip. For kinetic measurements, the monoclonal antibody 1A4 was serially diluted three-fold with HBS-EP+1X buffer, injected at 25° C. for 120 s, dissociated for 30 min, and regenerated by adding 10 mM glycine-HC1 (pH 2.0) for 120 s. The equilibrium dissociation constant (kD) of the monoclonal antibody 1A4 and the antigenic peptide was calculated using a simple one-to-one Languir binding model. The calculation results were shown in Table 1.

TABLE-US-00001 TABLE 1 Dissociation constants of the monoclonal antibody Monoclonal antibodies Equilibrium dissociation constant the monoclonal antibody 1A4 2.43E−11

[0040] It can be seen from Table 1 that the dissociation constant of the monoclonal antibody 1A4 was 2.43E-11, which indicates that the monoclonal antibody 1A4 of the present disclosure has a better effect of binding antigenic peptides.

[0041] The sequences of the light chain variable region and heavy chain variable region of the monoclonal antibody were identified by PCR, as follows:

TABLE-US-00002 Light chain variable region (SEQ ID NO: 1) DIVITQSPALAAASPGEKVTITCAVSGGISDIYLHWYQQKSGIS PKPWIYSTSWIAGGVPARFSGSGSGTSYSLTITSMEAEDAATYY CDDWSCIPLCFGAGTKLELK Heavy chain variable region (SEQ ID NO: 2) EVQLEESGTELRRPGASVKLSCKASGYIFSSYLMSWIKQRPGQG LEWIGGIYVGSSDTRYTSGFAGKATLTADKSSSTAYMQLSSLAS EDSAVYYCAGSNMMEDCWGLGTTLAVSS

EXAMPLE 5

[0042] Experiment of the monoclonal antibody 1A4 inhibiting cells

[0043] Human colon cancer LOVO cells (Cat. No.: CL-0144, Wuhan Procell Life Science & Technology Co., Ltd.)

[0044] The cytotoxic effect of the monoclonal antibody 1A4 on LOVO cells was detected by MTT method. LOVO cells in the logarithmic growth phase were digested with 0.25% trypsin, and made into a single cell suspension, which was then added to a 96-well culture plate, with 100 μl per well, and at a final cell density of 3×10′ cells/ml. Several experimental groups and a control group were set up with four replicates in each group. The cells were cultured at 37° C., 5% CO2 for 24h. For the experimental groups, 200 μl of the monoclonal antibody solution with a final mass concentration of 0.1, 0.2, 0.3, 0.4, or 0.5 mg/ml was added respectively. For the control group, mouse IgG was added for 24 h of incubation. 20 μl of 0.5 mg/ml MTT solution was added to each well, and incubated in the dark for another 4 h. The supernatant was discarded, and 200 μl of DMSO was added to each well for shaking, and the absorbance at 490 nm was measured with a microplate reader. The measurement was repeated for three times. The cell inhibition rate (%) was calculated as follows: the inhibition rate =(the value for the control group—the value for the experimental group)/the value for the control group x 100%.

[0045] It can be seen from FIG. 2 that the monoclonal antibody 1A4 has an inhibitory effect on the growth of LOVO cells, and with the increasing of the antibody concentration, the inhibition rate of the growth of LOVO cells also increases, indicating a significant dose correlation, and the inhibition rate reached up to 85% at a concentration of 0.5 mg/ml.

EXAMPLE 6

[0046] Western blotting to detect the experimental groups for the effect on the expression of apoptotic proteins

[0047] LOVO cells in the logarithmic growth phase were digested with 0.25% trypsin, and made into a single cell suspension, which was then added to a 6-well culture plate, at a final cell density of 3 x10.sup.5 cells/ml. The cells were cultured at 37° C., 5% CO.sub.2 for 24 h. The culture medium was poured. For the experimental group 1, the monoclonal antibody was added at a final concentration of 0.5mg/ml; for the experimental group 2, the monoclonal antibody was added at a final concentration of 0.5mg/m1 and concurrently NK cells were added at a final concentration of 3×10.sup.5 cells/ml; and for the experimental group 3, NK cells were added at a final concentration of 3×10.sup.5 cells/ml; and for the control group, DMEM medium was added, for 24 hours of treatment. The isolated LOVO cells were lysed with RAPI lysis buffer on ice to extract the total proteins, which were then separated by 12% SDS-PAGE. Specifically, the gel was placed in the membrane transfer buffer at 90 V for 45 min, during which the proteins were transferred onto the PVDF membrane. To the PVDF membrane, the primary antibody was added and incubated for 1 h, followed by washing 3 times with the washing buffer. Then the secondary antibody labeled with human HRP was added and incubated for 1 h, followed by washing and ECL color development. The expressions of caspase-3 and cleaved caspase-3 were detected by Western blotting. The results were shown as the relative expression level over the expression level of the control group, which were shown in Table 2.

TABLE-US-00003 TABLE 2 Results of the relative expression levels of apoptotic proteins Relative expression Relative expression level Group level of caspase-3 of cleaved caspase-3 Experimental group 1 0.71 ± 0.05 1.33 ± 0.11 Experimental group 2 0.59 ± 0.03 1.63 ± 0.09 Experimental group 3 0.83 ± 0.06 1.21 ± 0.07

[0048] As can be seen from the results in Table 2, for all the three experimental groups the signal of the cleaved caspase-3 was enhanced and the signal of caspase-3 was weakened, where the combination of the monoclonal antibody and NK cells can induce apoptosis of LOVO tumor cells most effectively, with the relative expression of the cleaved caspase-3 reaching 1.63±0.09.

Example 7

[0049] In vivo anti-tumor experiment to observe the effect of NK cells and the monoclonal antibody of the present disclosure alone or in combination on the growth of LOVO cell transplanted tumor

[0050] The LOVO cells in the logarithmic growth phase were digested and washed twice with PBS. The cell density was adjusted to 1×10.sup.7 cells/ml, and 0.2 ml was subcutaneously inoculated into each BALB/nu male nude mouse. When the tumor mass grew to 100mm.sup.3, nude mice were randomly divided into normal saline control group, NK combined with the monoclonal antibody group, NK group, and the monoclonal antibody treatment group, with 6 mice per group. Mice were injected intravenously with drugs, where 100 μl of NK cells at a concentration of 11×10.sup.7/ml, and the monoclonal antibody at 5mg/kg were injected once every 4 days for a total of 4 weeks. The maximum longitudinal diameter (a) and the maximum transverse diameter (b) of the tumor in nude mice were measured with a vernier caliper every 4 days after starting the treatment, and the growth of the tumor was observed. The tumor volume =0.5×a×b.sup.2. On the 28th day, the nude mice were sacrificed, to isolate and weigh the tumor tissue, and calculate the tumor inhibition rate. Tumor inhibition rate (%)=(1 -the average tumor mass for the experimental group/the average tumor mass for the control group) x100%.

[0051] It can be seen from FIG. 3 that the inhibitory effect of the NK cells combined with the monoclonal antibody administration group on the transplanted tumor was significantly stronger than the effect of the two single drugs (FIG. 3). The NK cells combined with the monoclonal antibody group had the most significant inhibition on the increasing of tumor weight, and the tumor's volume was only about 130 mm.sup.3, indicating a good inhibitory effect.