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Gremlin-1 antibody

A technology of antibodies and growth factor receptors, applied in the direction of antibodies, anti-inflammatory agents, anti-tumor drugs, etc., can solve the problems that the effects have not been studied in detail, and achieve the effect of preventing or treating cancer or immune diseases

Active Publication Date: 2015-01-28
SEOUL NAT UNIV R&DB FOUND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gremlin-1 is overexpressed in different human tumors including cancers of the cervix, endometrium, lung, ovary, kidney, breast, colon and pancreas (Namkoong H et al., (2006) BMC Cancer, 6:74; Sha G et al. (2009) Fertil Steril.,91:350-358), but its role in carcinogenesis has not been studied in detail

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Expression and purification of Gremlin-1

[0067] 1-1: Cell culture

[0068] A549, HeLa, A172 and A431 cells were obtained from the Korean Cell Line Bank (Seoul, Korea), and human umbilical vein endothelial cells (HUVEC) were obtained from Invitrogen (Carlsbad, CA). Meanwhile, A549, A172 and A431 cells were cultured in RPMI-1640 medium (Welgene) supplemented with 10% FBS, and HeLa cells were cultured in MEM medium (Welgene) supplemented with 10% FBS, while HUVEC endothelial cells Cultured in growth medium-2 (EGM-2, Lonza, Walkersville, MD).

[0069] 1-2: Preparation of Gremlin-1 expression vector and cell transfection

[0070] Gremlin cDNA was amplified from a human cervical tissue cDNA library as described in Namkoong H et al. (2006) BMC Cancer 6:74. Next, HindIII and XhoI restriction enzyme sites were introduced into the 5' and 3' ends of the gremlin cDNA by PCR using the following PCR primers:

[0071] 5'-CCC AAG CTT ATG AGC CGC ACA GCC TAC AC-3'...

Embodiment 2

[0086] Example 2: Generation of Gremlin-1 Antibody

[0087] 2-1: Immunization

[0088] 5 μg of gremlin-1-Fc was mixed with 2 mL of phosphate-buffered saline (PBS) and incubated at 37° C. for 30 minutes, and the mixture was dissolved in 2% squalene containing detoxified endotoxin MPL (monophosphorylated lipid A species) and mycobacterial cell wall components (TDW and CWS) in an oil-in-water emulsion adjuvant (Sigma, St. Louis, Mo) and injected into New Zealand white rabbits . Immunization was performed three times at 3-week intervals. Antibody titers of immunized rabbits were determined by enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase (HRP)-conjugated mouse anti-rabbit IgG polyclonal antibody (Pierce Chemical Co., Rockford, IL). ) as the secondary antibody.

[0089] Total RNA was obtained from spleen and bone marrow of immunized rabbits using TRI reagent (Invitrogen). Extracted spleen and bone marrow were homogenized in TRI reagent at 50% outpu...

Embodiment 3

[0114] Example 3: Analysis of the interaction between Gremlin-1 and cancer cells

[0115] To examine whether gremlin-1 directly interacts with cancer cells, gremlin-1 was incubated with four cancer cells (A549, HeLa, A172 and A431) and then analyzed by flow cytometry.

[0116] Specifically, adherent cells were trypsinized and washed with 1% (w / v) BSA in phosphate buffered saline (PBS). Suspension cells were harvested by centrifugation at 500 xg for 2 minutes and washed with 1% (w / v) BSA in PBS. All cells were incubated with His-tagged gremlin-1 (R&D Systems, Minneapolis, MN) at a final concentration of 100 nM in 1% (w / v) BSA in PBS. Cells were then washed twice with 1% (w / v) BSA in PBS and incubated with FITC-conjugated His antibody (Abcam, Cambridge, UK) at a final concentration of 5 mg / ml at 37°C for 30 minutes in the dark. Cells were then washed twice with 1% (w / v) BSA in PBS and resuspended in 500 μL of PBS before analysis on a FACSCanto II flow cytometer (BD Bioscienc...

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Abstract

The present invention relates to a Gremlin-1 antibody that inhibits Gremlin-1 in a manner independent of bone morphogenetic protein (BMP) or vascular endothelial growth factor receptor-2 (VEGFR-2), thus providing the effect of treating cancer. The antibody of the present invention inhibits cell migration, cell invasion and cell proliferation which are dependent on Gremlin-1, and therefore, can be effectively used in treating cancer or immune disease.

Description

technical field [0001] The present invention relates to gremlin-1 antibodies, and more particularly, to gremlin-1 antibodies that act independently of bone morphogenetic protein (BMP) or vascular endothelial growth factor receptor-2 (VEGFR2) Inhibition of gremlin-1 in a way to treat cancer. Background technique [0002] Gremlin-1 (20.7kDa), an antagonist of bone morphogenetic protein (BMP), is a protein that shares structures with cysteine-rich regions, cysteine-knot motifs, and members of the TGF-β superfamily A protein consisting of 184 amino acids. This protein is evolutionarily conserved and the human gremlin gene (GREM1) has been mapped to chromosome 15q13-q15 (Topol LZ et al., (1997) Mol. Cell Biol., 17:4801-4810; Topol LZ et al., Cytogenet Cell Genet., 89:79-84). Gremlin-1 is a secreted protein and three isoforms have been reported (Topol LZ et al., J. Biol. Chem., 275:8785-8793). Isoform 1 is the most common isoform, while isoforms 2 and 3 have deletions of amino...

Claims

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Application Information

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IPC IPC(8): C07K16/18A61K39/395G01N33/53A61P35/00
CPCC07K16/18C07K2317/73A61P1/04A61P1/16A61P7/06A61P9/00A61P11/00A61P11/06A61P13/12A61P17/00A61P17/06A61P17/14A61P19/02A61P21/00A61P25/00A61P29/00A61P35/00A61P37/02A61P37/06A61P37/08C07K16/22C07K16/2863C07K2317/24C07K2317/70C07K2317/76A61K39/395C07K2317/21C07K2317/565C07K2317/622
Inventor 金弦起郑埈昊金珉秀尹秀珉
Owner SEOUL NAT UNIV R&DB FOUND
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