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Bispecific antibody against epithelial cell adhesion molecule (EpCAM) and T-cell antigen

A bispecific antibody, functional domain technology, applied in the direction of antibodies, cells modified by introducing foreign genetic material, bacteria, etc., can solve the problem of undiscovered EpCAM antibody to liver cancer

Inactive Publication Date: 2014-08-06
SHANGHAI INST OF ONCOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, no EpCAM antibody has been reported that has the expected and ideal therapeutic effect on liver cancer.

Method used

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  • Bispecific antibody against epithelial cell adhesion molecule (EpCAM) and T-cell antigen
  • Bispecific antibody against epithelial cell adhesion molecule (EpCAM) and T-cell antigen
  • Bispecific antibody against epithelial cell adhesion molecule (EpCAM) and T-cell antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1. Antigen immunization and hybridoma screening

[0053] 1.1 Antigen immunization

[0054] (1) Recombinant protein immunization

[0055] EpCAM extracellular region recombinant protein (purchased from Shanghai Ruijin Biotechnology Co., Ltd., product number: RO720) was fully emulsified and mixed with an equal volume of complete Freund's adjuvant (purchased from Sigma, product number: 5881) to obtain a total volume of 200 μL, which contained EpCAM antigen 100μg, subcutaneous injection to immunize six-week-old BALB / c mice. After 4 weeks, the recombinant EpCAM antigen was emulsified and mixed with an equal volume of incomplete Freund's adjuvant (purchased from Sigma, product number: 5506) to obtain a total volume of 200 μL, which contained 50 μg of EpCAM antigen, and intraperitoneally injected to immunize mice. At intervals of 2 weeks, intraperitoneal booster immunization was repeated once. Three weeks after the completion of the last booster immunization, the mic...

Embodiment 2

[0059] Example 2. Preparation of anti-EpCAM bispecific antibody

[0060] 2.1 Genetic engineering preparation of anti-EpCAM single-chain antibody

[0061] Use Trizol (Invitrogene Company, product number: 15596-026) to extract the total RNA of the hybridoma cell line 1H8 in the above-mentioned Example 1, and then use a reverse transcription RT-PCR kit (Promega Company, product number: A3800) to operate according to the instructions of the kit The step is to perform reverse transcription on the total RNA extracted above to obtain a complementary DNA sequence.

[0062] Use the 5'-Full Race kit (Takara, product number: D315) to amplify the antibody heavy chain or light chain variable region and its upstream and downstream sequences using the complementary DNA sequence obtained above as a template according to the steps specified in the kit. A DNA sequence of which:

[0063] The upstream primers are provided by the kit,

[0064] The downstream outer primer is: CCAGAGTTCCAGGTCACTG...

Embodiment 3

[0128] Example 3 Detection of Binding Ability of Bispecific Antibody

[0129] 3.11H8 / CD3 bispecific antibody FACS analysis

[0130] A.Material:

[0131] 1) cells:

[0132]

[0133]

[0134] b. steps

[0135] Carry out similar operations as follows for each cell line in the above table:

[0136] Inoculate each cell in the logarithmic growth phase as listed in Table 1 into a 6cm plate, and inoculate at 37°C, 5% CO 2 Cultured in an incubator, the cells were used for flow cytometry when 90% of the plate was covered.

[0137] 1) Use 10mM EDTA to digest the cells, and centrifuge at 5000rpm×5min to collect the cells in each well of the plate into several 2mL Eppendorf tubes. The cells were washed twice with 1× phosphate buffered saline (PBS) containing 1% NCS (Neonatal Calf Serum), and the supernatant was discarded after centrifugation.

[0138] 2) Add the bispecific antibody 1H8 / CD3 of the present invention in 100 μL 1×PBS with a final concentration of 10 μg / mL in a tube...

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Abstract

The invention provides a bispecific antibody, including: (1) a first functional domain bonding with EpCAM; (2) a second functional domain bonding with CD3; and (3) a linker connecting the first functional domain with the second functional domain; wherein the first functional domain includes at least one light chain variable region and at least one heavy chain variable region, the light chain variable region includes three complementary determining regions consisting of CDR-L1: RASQDISNYLN, CDR-L2: YTSRLHS and CDR-L3: QQGSTLPYT, and the heavy chain variable region includes three complementary determining regions consisting of CDR-H1: GYTFTYYGMN, CDR-H2: WINTYTGEPTYGDDFKG and CDR-H3: TGRATSFDY.

Description

technical field [0001] The present invention relates to antibodies related to tumor treatment, more specifically, the field of genetically engineered antibodies. Background technique [0002] Current research has known that epithelial cell adhesion molecule (EpCAM, Epithelial cell adhesion molecule; CD326) is a glycosylated type I transmembrane glycoprotein with a molecular weight of 30 to 40kD, and its structure includes an epidermal growth factor-like, a thyroglobulin A protein-like extracellular domain, a transmembrane domain and a 26 amino acid cytoplasmic domain. EpCAM is mainly localized between epithelial cells and functions in a directed and highly ordered manner to adhere epithelial cells. Once the malignant transformation of epithelial cells, the rapid growth of tumor cells will abandon the high order of epithelial cells. As a result, the surface distribution of EpCAM becomes less constrained and the molecule is more exposed on tumor cells. Most tumor cells expr...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13C12N15/63C12N1/21C12N1/19C12N5/10A61K39/395A61P35/00
Inventor 李宗海张鹏飞石必枝王华茂高慧萍
Owner SHANGHAI INST OF ONCOLOGY
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