Isolated monoclonal antibodies that specifically bind to human Claudin 18.2

An antibody, monoclonal antibody technology, applied in the direction of antibodies, antibody medical components, medical preparations with non-active components, etc., can solve the problems of poor prognosis and low survival rate

Active Publication Date: 2019-05-17
BEIJING MABWORKS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For pancreatic cancer, patients are usually diagnosed at an advanced stage, so the prognosis is poor, with a median survival time of less than 6 months and a 5-year survival rate of less than 5.5%

Method used

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  • Isolated monoclonal antibodies that specifically bind to human Claudin 18.2
  • Isolated monoclonal antibodies that specifically bind to human Claudin 18.2
  • Isolated monoclonal antibodies that specifically bind to human Claudin 18.2

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preparation example Construction

[0133] Preparation of monoclonal antibody of the present invention

[0134] The monoclonal antibodies of the present invention can be prepared using the somatic cell hybridization (hybridoma) technique of Kohler and Milstein (1975) Nature 256:495. Other embodiments for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques. Chimeric or humanized antibodies are also well known in the art. See, eg, US Patents 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370.

[0135] Generation of Transfectomas to Produce Monoclonal Antibodies of the Invention

[0136] Antibodies of the invention can also be produced in host cell transfectomas using, for example, recombinant DNA techniques combined with gene transfection methods (eg Morrison, S. (1985) Science 229:1202). In one embodiment, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is insert...

Embodiment 1

[0176] Example 1 Construction of HEK193A cell strain stably expressing human Claudin 18.1 or Claudin 18.2

[0177] The cDNA sequences (SEQ ID NOs: 69 and 71) encoding human Claudin 18.1 (SEQ ID NO: 70) and Claudin 18.2 (SEQ ID NO: 72) were synthesized, and the coding sequences of the above two genes were digested by EcoRI and BamHI. respectively cloned into the pLV-sfGFP(2A)puro plasmid. The resulting pLV-EGFP(2A)-Puro-Claudin 18.1 or pLV-EGFP(2A)-Puro-Claudin 18.2 was co-transfected with psPAX2 and pMD2.G into HEK-293T cells (Cobioer, China) to generate lentivirus. The resulting lentivirus was transfected into HEK-293A cells (Cobioer, China) to generate stable cell lines overexpressing human Claudin 18.1 or Claudin 18.2. The cell line was cultured in DMEM medium (Cat: SH30022.01, Gibco) supplemented with 10% FBS (Cat: FND500, Excell) and 0.2 μg / ml puromycin (Cat: A11138-03, Gibco). The time is more than 7 days.

[0178] The expressions of Claudin 18.1 and Claudin 18.2 we...

Embodiment 2

[0179] Example 2 Generation and Screening of Claudin 18.2 Monoclonal Antibody

[0180] Using HEK293A cells stably expressing human Claudin 18.2 in Example 1 to immunize 6-week-old BALB / c mice, the cells were injected subcutaneously, and each mouse was injected with 2×10 7 cells. After the primary immunization, booster immunizations were given every four weeks for a total of three times. Antibody titers in blood samples were determined by FACS using HEK293A cells expressing human Claudin 18.2. When the animal reached the appropriate antibody titer, with 5 × 10 in PBS 7 / mL cells for the last immune boost. Three days later, mice were sacrificed, bled, and spleens were collected for mRNA extraction and phage display library construction.

[0181] To construct the scFv phage display library, mouse spleen total RNA was extracted with Trizol kit (Invitrogen), and cDNA was synthesized using reverse transcription kit (Invitrogen). Using the above cDNA as a template, gene amplif...

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Abstract

The invention provides isolated monoclonal antibodies that specifically bind to human Claudin 18.2. Nucleic acid molecules for encoding the antibodies, as well as expression vectors, host cells and methods for expressing the antibodies are also provided. The invention also provides immunoconjugates, bispecific molecules, chimeric antigen receptors, oncolytic viruses and pharmaceutical compositionscomprising the antibodies, and methods for diagnosis or treatment using the antibodies.

Description

technical field [0001] The present invention relates to an antibody that specifically binds to human Claudin 18.2, and its preparation and use, especially its use in the diagnosis, prevention and treatment of diseases related to cells expressing Claudin 18.2. The diseases include tumors, such as pancreatic cancer, Cancer of the stomach, bowel, esophagus, liver, ovary, lung and bladder. Background technique [0002] Cancer and Antibody Therapies [0003] Cancer is one of the number one deadly diseases in our society today. According to the 2014 World Cancer Report, about 14.1 million new cases of cancer occur each year, and this does not include skin cancers other than melanoma. Cancer accounts for about 8.8 million deaths every year (GBD 2015 Disease and Injury Incidence and Prevalence Collaborators, (2016) Lancet 388(10053): 1545-1602). For example, gastric cancer is the fourth (for men) and fifth (for women) cause of cancer-related death in developed countries. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61K45/06A61P35/00G01N33/68G01N33/574
CPCA61K31/282A61K31/704A61K31/7115A61K2039/505A61P35/00C07K16/28C07K2317/24C07K2317/33C07K2317/34C07K2317/41C07K2317/56C07K2317/732C07K2317/734C07K2317/92C07K2317/622A61K39/39558A61K2300/00A61K31/675A61K31/513C07K16/2878A61K47/6801
Inventor 胡稳奇李江美王霞李锋
Owner BEIJING MABWORKS BIOTECH
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