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IgG antigen epitope and application thereof as target

An antigenic epitope and site technology, applied in the field of tumor diagnosis and treatment in immunology, can solve the problem of inability to prepare diagnostic or therapeutic drugs.

Active Publication Date: 2018-10-02
BEIJING SIG BIOPHARMACEUTICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the specific structure and properties of the unique epitope recognized by RP215 and the detailed mechanism of tumorigenesis and development driven by RP215-IgG are still unknown. diagnostic or therapeutic drug

Method used

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  • IgG antigen epitope and application thereof as target
  • IgG antigen epitope and application thereof as target
  • IgG antigen epitope and application thereof as target

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Determination of the functional structure of RP215-IgG

[0050] 1. Purify IgG from cancer tissue

[0051] (1) Use protein G-Sepharose 4Fast Flow (GE healthcare, USA) to purify tumor IgG in advance from non-small cell lung cancer tissues, breast cancer or ovarian cancer tissues, or PDX tumor models established by lung squamous cell carcinoma (LSCC). The purpose of using the PDX tumor model is to exclude the influence of IgG derived from peripheral blood.

[0052] (2) Purify RP215-IgG with RP215 affinity column.

[0053] RP215 affinity column preparation method: monoclonal antibody RP215 is coupled with Sepharose 4FastFlow (GE Healthcare, USA) activated by CNBr, reference Picture 1-1 A. According to the instructions, proceed as follows: ① After 330 mg CNBr-sepharese 4B is activated by 1 mM hydrochloric acid, use coupling buffer (0.1M NaHCO 3 , 0.5M NaCl, pH8.3) balance. ② Dissolve 5 mg of RP215 antibody in coupling buffer, add it to activated CNBr agarose gel packing a...

Embodiment 2

[0079] Example 2 IgG epitope is used as a target to identify non-B cell-derived IgG, which has the ability to promote tumor cell proliferation, migration and invasion

[0080] Example 1 confirmed that the unique epitope of IgG from non-B cells can be specifically recognized by RP215. Therefore, the epitope is used as the specific recognition site and RP215 is used to detect IgG from non-B cells ( RP215-IgG) function.

[0081] LSCC cell line: A549 (adenocarcinoma human alveolar basal epithelial cells), Calu-3 (human lung adenocarcinoma cells), NCI-H1299 (human lung cancer cells), NCI-H520 (human lung cancer cells), SK-MES-1 ( Human lung squamous cell carcinoma).

[0082] We first detected the expression of RP215-IgG in the above LSCC cell lines, and found that RP215-IgG that LSCC cell lines can express and secrete is located on the cell surface and ECM (see diagram 2-1 ).

[0083] When siRNA targeting the heavy chain constant region in NCI-H520 cells and SK-MES-1 cells down-regulates...

Embodiment 3

[0087] Example 3 Sialyltransferase ST3GAL4 participates in the sialylation of RP215-IgG

[0088] The biosynthesis of sialylated oligosaccharide sequences is catalyzed by a family of enzymes called sialyltransferases, and each sialyltransferase has its specific substrate.

[0089] Previous studies have shown that the sialic acid linked to the N-glycan at the classical N-glycosylation site (Asn297) is mediated by the sialyltransferase ST6GAL-1, and the sialic acid and N-glycan β-D -Galactopyranosyl (Gal) residues are linked in an α2,6-way.

[0090] We have determined that the sialic acid linked to the N-glycan at the non-canonical N-glycosylation site (Asn162) is linked to the β-D-galactopyranosyl (Gal) residue via MALI, so three saliva Acid transferases ST3GAL3, ST3GAL4, ST3GAL6 (involved in ST3β-galactoside α-2,3-linkage), and ST6GAL1 (as a control) were candidates for further screening.

[0091] In order to determine which sialyltransferase is involved in the synthesis of RP215-IgG ...

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Abstract

The invention discloses an IgG antigen epitope and an application thereof as a target. The IgG antigen epitope is a CH1 structural domain of non-B-cell-derived IgG, and an Asn162 site of the structural domain is modified by N-glycosylated sialic acid, and realization of an antigenic function needs to depend on the sialylation of the site. The invention also provides an application of the IgG antigen epitope as a drug action target in preparation of drugs for diagnosis and / or treatment of epithelial tumors. In addition, research results show that the function of the antigen as the drug target depends on the sialylation of the Asn162 site, the sialylation of the Asn162 site also needs to depend on a sialic acid transferase ST3GAL4, the enzyme is suggested to be used as a drug action target for preparation of tumor therapy drugs. In addition, integrin beta 4 is co-expressed and co-localized with IgG containing the antigen epitope. In view of the function of IgG, the IgG antigen epitope can be used as a marker for preparation of drugs for aided detection of epithelial tumors.

Description

Technical field [0001] The invention belongs to the technical field of tumor diagnosis and treatment in immunology, and relates to non-B cell-derived IgG, specifically an IgG epitope and its application as a target. Background technique [0002] The source and research status of RP215 monoclonal antibody: As early as the 1980s, the Lee research group of Columbia University in Canada used the protein extracted from ovarian cancer cell lines to immunize animals in order to obtain monoclonal antibodies that specifically recognize ovarian cancer. Nearly 3,000 hybridoma cells were found during the screening process. Among them, one hybridoma cell, called RP2015, could well recognize ovarian cancer cells but not normal ovarian cells, but the antigen it recognized was not clear at the time. What is it, so the antigen it recognizes temporarily is named "CA215". It was later discovered that RP215 not only recognized ovarian cancer well, but also showed good specificity in the recognition...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00G01N33/68G01N33/574A61K39/395A61P35/00
CPCA61P35/00G01N33/57484G01N33/6854C07K16/00C07K16/4283A61K2039/505C07K2317/41C07K2317/522C07K2317/76G01N2333/70546G01N2333/91148C07K2317/34C07K2317/55C07K16/3069C07K16/3023G01N33/57492G01N2400/02
Inventor 邱晓彦王宠唐婧姝张婧璇刘洋廖沁园姜文华黄晶
Owner BEIJING SIG BIOPHARMACEUTICAL TECH CO LTD
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