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Anti-tnf- alpha /cxcl10 double-targeting antibody and use thereof

A double-targeted antibody, antibody technology, applied in the direction of antibodies, antibody medical components, anti-animal/human immunoglobulin, etc.

Inactive Publication Date: 2016-01-20
METABOLIC ENG LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is not clearly known how the CXCL10 plays a role in the inflammatory response or immune response (besides chemotaxis) in these diseases, and how important the CXCL10 is as a therapeutic target

Method used

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  • Anti-tnf- alpha /cxcl10 double-targeting antibody and use thereof
  • Anti-tnf- alpha /cxcl10 double-targeting antibody and use thereof
  • Anti-tnf- alpha /cxcl10 double-targeting antibody and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0136] Embodiment 1: the production of human CXCL10 antigenic protein

[0137] To construct monoclonal antibodies against human CXCL10, human CXCL10 protein was expressed and purified. Specifically, to amplify human CXCL10, a mixture of spleen, placenta, liver, and kidney cDNA libraries was used as a template, which was inserted into pET22b vector. BL21(DE3) was used to transform pET22b-human CXCL10, and the obtained transformant was inoculated on an LB plate containing ampicillin, cultivated overnight, and then cultured with shaking at 37°C.

[0138] Thereafter, when the OD600 was 0.55, 0.5 mM IPTG was added to the cells, cultured overnight, and centrifuged at 6,500 rpm at 4° C. for 15 minutes to obtain a supernatant. The obtained supernatant was concentrated and reacted with Ni-NTA agarose, and then eluted by packing the column with agarose beads. After concentration, the resulting product was loaded onto a 15% SDS-PAGE gel and stained with Coomassie blue. To compare the ...

Embodiment 2

[0139] Embodiment 2: the construction of monoclonal antibody

[0140] 2-1. Panning process

[0141] Panning is the process of selecting only phage-displayed peptides on their surface that have the property of binding to target molecules (antibodies, enzymes, cell surface receptors, etc.) from a phage library displaying peptides on the phage capsid . To construct a phage antibody panel for the construction of monoclonal antibodies against CXCL10, phage panning was performed. 100 μg of the purified human CXCL10 antigen obtained in Example 1 was used in 2 ml of coating buffer (Na 2 CO 3 (Sigma, S7795) 1.59gNaHCO 3 (Sigma, S8875) 2.93gNaN 3 (Sigma, S2002), 0.2g) was coated at 4°C for about 16 hours, and diluted in PBS at room temperature for 2 hours, and then immunized with 4% skimmed milk ((BD, 232100)-4%, in 1XPBS) Reaction blocked in sorbent tube. 2 ml of phage library was added to the immunosorbent tube, allowed to react at room temperature for 2 hours, and then washed ...

Embodiment 3

[0156] Example 3: Classification and Research of Selected Monoclonal Phages

[0157] 3-1. Verification by fingerprint analysis

[0158] In order to verify the 10 monoclonal cells selected in Example 2 by fingerprint analysis, 1 μl of the selected monoclonal cells were mixed with 0.2 μl of TaqDNA polymerase (Gendocs, 5 U / μl), 50 p / μl of forward primer ( pelB5) (SEQ ID NO:41:5'-CTAGATAACGAGGGCAAATCATG-3') and reverse primer (cla3) (SEQ ID NO:42:5'-CGTCACCAATGAAACCATC-3') 0.2 μl each, 3 μl of 10X buffer and 0.6 μl of 10 mM dNTP mix and 24.8 μl of distilled water were mixed, and clone PCR (iCycleriQ, BIO-RAD) was performed under the following conditions for the PCR program shown in Table 3.

[0159] 【table 3】

[0160]

[0161] The clone PCR product thus obtained was identified on 1% agarose gel (SeakemLE, CAMERES50004), and 0.2 μl of BstNI (Roche 11288075001, 10 U / μl) was added to the PCR product, and it was heated at 37° C. React under the reaction conditions for 2 to 3 hou...

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Abstract

The present invention relates to a TNF-alpha (tumor necrosis factor-alpha) / CXCL-10 (C-X-C motif chemokine 10) double targeting antibody based on the IgG format. Specifically, it was verified that an antibody, in which scFv having a heavy chain variable region and a light chain variable region of the CXCL10 specific antibody binds to the C-terminus of the heavy chain variable region of the TNF-alpha specific antibody, is a bispecific antibody that effectively binds to both TNF- alpha and CXCL10, and thus the antibody can be useful as a double targeting antibody capable of identifying TNF- alpha / CXCL10. A composition of the present invention comprises a TNF- alpha / CXCL-10 double targeting antibody which effectively binds to both TNF- alpha and CXCL10. The double targeting antibody of the present invention has excellent TNF- alpha inhibitory activity and osteoclast differentiation inhibitory activity compared with the TNF- alpha or CXCL10 single targeting antibody. The composition of the present invention can be used in preventing or treating immunological disease.

Description

technical field [0001] The invention relates to a TNF-α / CXCL-10 dual-targeting antibody specifically combined with tumor necrosis factor alpha (TNF-α) and C-X-C motif chemokine 10 (CXCL10) and an application thereof. [0002] Related Patent Applications [0003] This application claims priority and benefit from Korean Patent Applications 10-2013-0057475 and 10-2013-0057762 filed on May 22, 2013, the disclosures of which are incorporated herein by reference in their entirety. Background technique [0004] In general, antibodies are formed by disulphide-bonding a heavy-chain polypeptide with a high molecular weight to a light-chain polypeptide with a low molecular weight to form a heterodimer, and again disulfide-bonding the two heterodimers. Polymers link to form tetramers. The polypeptide forming the heavy chain consists of four domains comprising, from the N-terminus, the variable domain, constant domain 1, constant domain 2, and constant domain 3, and the polypeptide for...

Claims

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Application Information

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IPC IPC(8): C07K16/46A61K39/395
CPCC07K16/46C07K16/24A61K2039/505A61K2039/545C07K2317/31C07K2317/622C07K2317/64C07K2317/76C07K2317/92C07K16/241A61P1/04A61P11/00A61P11/02A61P11/06A61P17/02A61P17/04A61P17/06A61P29/00A61P37/00A61P37/02A61P37/06A61P39/00C07K2317/565
Inventor 姜欣濉朴昭炫宋永旭申己澈李恩荣李恩奉朴荣祐朴范赞李东熙金东辰尹善河李基世李炫周金京珍金熙赞刘奭镐张明熙张世佚
Owner METABOLIC ENG LAB
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