Ficoll

EXAMPLE 36

Peripheral blood mononuclear cells were prepared from blood samples obtained from llama No. 45 and No. 46 using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA extracted was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored after filter sterilization at 4° C. for further use.

EXAMPLE 12

Peripheral blood mononuclear cells were prepared from blood samples obtained from llama No. 146 and No. 147 using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA extracted was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored after filter sterilization at 4° C. for further use.

Example 25

This experiment was to evaluate the effect of killing cancer cells by treating MDA-MB-231 cells (human breast cancer cells) with the test substance GI-101 alone or in combination with the TGF-beta signal inhibitor Vactosertib substance in an in vitro environment.

MDA-MB-231 cells were purchased from the Korea cell line bank and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). For use in cancer cell killing test, the cells were harvested using trypsin (Gibco), and then suspended in RPMI1640 medium, and then dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution. The cells suspended in RPMI1640 medium were carefully layered on ficoll solution. The cell layer with a low specific gravity formed by centrifuging at room temperature at 350×g for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350×g for 5 minutes. The separated cell layer was made into a suspension of 2×10⁵ cells/mL with FBS-free RPMI1640 medium. The cancer cell suspension was stained at 37° C. for 1 hour using CELLTRACKER™ Deep Red Dye (Thermo) in order to track proliferation or inhibition of the proliferation of cancer cells. After staining, it was centrifuged at 1300 rpm for 5 minutes, and then it was washed with FBS-free RPMI1640 medium, and then suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 2×10⁵ cells/mL. The cancer cell suspension was added to each well of a 96-well microplate (Corning) by 50 μl (1×10⁴ cells), and then stabilized in an incubator (37° C., 5% CO₂) for 1 hour.

Human peripheral blood mononuclear cells (PBMCs) were used in order to identify the effect of killing cancer cells by GI-101. The human PBMCs were purchased from Zen-Bio, and the PBMCs stored frozen were placed in a 37° C. water bath, and thawed as quickly as possible, and then transferred to RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco), and centrifuged at 1300 rpm for 5 minutes. The separated cell layer was suspended in RPMI1640 medium, and then dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution in the same manner as the cancer cell line. The cells suspended in RPMI1640 medium were carefully layered on ficoll solution. The cell layer with a low specific gravity formed by centrifuging at room temperature at 350×g for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350×g for 5 minutes. The separated cell layer was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 5×10⁵ cells/mL. The PBMC suspension was dispensed 50 μl into each well of a 96-well microplate (Corning) in which cancer cell line has been dispensed, depending on the conditions.

In order to identify the effect of killing the cells, a CytoTox Green reagent (INCUCYTE™ CytoTox Green, Satorius) that binds to the DNA of cells to be killed was prepared in 1 μl per 1 mL of RPMI1640 medium containing 5% human AB serum (Sigma). The prepared medium was used for dilution of the test substance, and the effect of killing the cells could be quantitatively identified by staining the cells to be killed when the test substance was co-cultured with cancer cell lines and PBMCs.

Vactosertib power was dissolved in DMSO (Sigma) to a concentration of 48.4 mM, and diluted using RPMI1640 medium containing a CytoTox Green reagent, and then used in the experiment at a final concentration of 12.1 nM (50 μL) per well of a 96-well microplate.

GI-101 was diluted by ⅓ using RPMI1640 medium containing a CytoTox Green reagent, and then used in the experiment at final concentrations of 0.4 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM by 50 μl per well of a 96-well microplate.

The prepared test substance was placed in each well of a 96-well microplate in which cancer cell lines and PBMCs were dispensed depending on the conditions, and cultured in an incubator (37° C., 5% CO₂) for 24 hours, and the proliferation or death of cancer cells was observed through the real-time cell imaging analysis equipment IncuCyte S3 (Satorious). The death of cancer cells was quantified by the integrated intensity of the cells stained in green with a CytoTox Green reagent.

As a result, it was identified that the group having received a combination of GI-101 and Vactosertib exhibited the excellent effect of killing cancer cells as compared with the group having received each drug alone.