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Application of monoclonal antibody NJ001-1 to preparation of drug for inhibiting invasion and metastasis of NSCLC (non-small-cell lung cancer)

A monoclonal antibody, drug technology

Active Publication Date: 2015-12-09
NANJING MARKERLINE BIOMEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Taking clinically used platinum-based antineoplastic drugs-cisplatin and carboplatin as examples, although platinum-based drugs can significantly inhibit tumor proliferation and have a wide anticancer spectrum, they cannot inhibit tumor invasion and metastasis

Method used

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  • Application of monoclonal antibody NJ001-1 to preparation of drug for inhibiting invasion and metastasis of NSCLC (non-small-cell lung cancer)
  • Application of monoclonal antibody NJ001-1 to preparation of drug for inhibiting invasion and metastasis of NSCLC (non-small-cell lung cancer)
  • Application of monoclonal antibody NJ001-1 to preparation of drug for inhibiting invasion and metastasis of NSCLC (non-small-cell lung cancer)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Cell migration detection

[0026] Collect lung adenocarcinoma cells SPC-A-1 in the logarithmic growth phase to make a single cell suspension, dilute 2×10 5 The cells / well were added to a 6-well cell culture plate, cultured overnight, the culture supernatant was discarded, and washed once with 500 μl / well of Hank's solution. Antibody groups (50 μg / mL, 100 μg / mL and 150 μg / mL three groups): monoclonal antibody NJ001-1 solution was added to each well, and the final concentrations were 50 μg / mL, 100 μg / mL and 150 μg / mL per well; the control group (0 μg / mL / mL): Add 20ml of culture medium to each well. Cultured for 24h and 48h after intervention, for cell migration detection. Three parallel wells were set up in each group, and the experiment was repeated 3 times.

[0027] 2. Detection of cell migration ability (scratch test)

[0028] Principle: When the cells grow to become a monolayer state, artificially create a blank area on the fused monolayer cells, called "scrat...

Embodiment 2

[0039] (1) Cell invasion detection

[0040] Collect SPC-A-1 and A549 in the logarithmic growth phase to make a single cell suspension, 8×10 4 Cells / well were added to 24-well cell culture plates. Antibody groups (50 μg / mL, 100 μg / mL and 150 μg / mL three groups): add monoclonal antibody NJ001-1 solution to the outer chamber of each well, and the final concentrations were 50 μg / mL, 100 μg / mL and 150 μg / mL per well; (0 μg / mL): Add 500 μl of culture solution to each well. After the intervention, cultured for 24 hours, used for cell invasion detection. Three parallel wells were set up in each group, and the experiment was repeated 3 times.

[0041] (2) Detection of cell invasion ability (Transwell assay)

[0042] Principle: Put the transwell chamber into the culture plate, the chamber is called the upper chamber, and the inside of the culture plate is called the lower chamber. The upper chamber is added with the upper layer culture solution, and the lower chamber is added with t...

Embodiment 3T

[0055] Example 3 Detection of TIMP-3 expression

[0056] RT-PCR:

[0057] 1) Sample preparation

[0058] The culture medium in the culture plate was discarded, and 700 μl Trizol reagent was added to each well, and the cells were repeatedly blown and blown with Trizol for digestion and lysis. After the cells were completely lysed, the solution was collected in an enzyme-free EP tube and stored at -70°C.

[0059] 2) RNA extraction

[0060] (1) Adjust the desktop refrigerated high-speed centrifuge to 4°C;

[0061] (2) Take out the Trizol-added cell mass from the -70°C refrigerator;

[0062] (3) After the cell mass is dissolved, use a pipette tip to gently blow or vortex to mix, and vortex for 1 min;

[0063] (4) Place at room temperature (15-25°C) for 5 minutes;

[0064] (5) Add 140 μl of chloroform, gently cover the tube tightly, and shake vigorously for 15 seconds;

[0065] (6) Stand at room temperature for 2 to 3 minutes;

[0066] (7) 12000g×15min at 4°C, adjust the ce...

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Abstract

The invention discloses application of a monoclonal antibody NJ001-1 to preparation of a drug for inhibiting invasion and metastasis of NSCLC (non-small-cell lung cancer). The monoclonal antibody NJ001-1 is secreted by a hybridoma cell strain NM001-1 with the preservation number CCTCC NO: C201172. The monoclonal antibody NJ001-1 is capable of inhibiting migration and invasion of lung adenocarcinoma cells effectively and increasing TIMP-3mRNA and protein expression remarkably. Under the action of the monoclonal antibody NJ001-1, specific antigen of the monoclonal antibody NJ001-1 can be combined with transcription factors FOXP1 (forkhead box protein P1 ) in lung adenocarcinoma cell nucleuses to inhibit transcription inhibition effect on a TIMP-3 gene promoter region.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals and relates to the application of monoclonal antibody NJ001-1 in the preparation of inhibiting NSCLC invasion and metastasis. Background technique [0002] The invasion and metastasis of lung cancer are the hallmarks and characteristics of its malignancy, and they are also the main reasons that affect the treatment effect and cause death of lung cancer patients. Lung cancer can be divided into small cell lung cancer and non-small cell lung cancer (Non-small-cell Lung Cancer, NSCLC), of which NSCLC accounts for about 85%, and among NSCLC, lung adenocarcinoma is the most common. NSCLC has an insidious onset, rapid development, and poor prognosis. The median survival period of untreated patients is only 4 to 5 months, the 1-year survival rate is about 10%, and the 5-year survival rate is less than 5%. Most NSCLC patients have developed distant metastasis (stage IV) at the time of initial clinical d...

Claims

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Application Information

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IPC IPC(8): A61K39/395A61P35/00
CPCA61K39/39558A61K2300/00
Inventor 潘世扬徐建王芳黄珮珺
Owner NANJING MARKERLINE BIOMEDICAL TECH CO LTD
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