Anti-human Trop-2 antibody and application thereof

An antibody and half-antibody technology, applied in the direction of antibodies, applications, antibody mimetics/scaffolds, etc., can solve the problem of less varieties of anti-Trop-2

Pending Publication Date: 2021-04-13
MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are few anti-Trop-2 antibodies in clinical research

Method used

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  • Anti-human Trop-2 antibody and application thereof
  • Anti-human Trop-2 antibody and application thereof
  • Anti-human Trop-2 antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1 Preparation of anti-human Trop-2 antibody hybridoma cells

[0098] Immunization: adopt human Trop-2 recombinant protein (sequence number: NP_002344.2, 1aa-274aa) to immunize Balb / c mice, and use human Trop-2-his recombinant protein (sequence number: NP_002344.2, 1aa-274aa) to coat ) 96-well ELISA plate to detect the serum titer by ELISA; the mice whose serum titer reached the fusion requirement were used for the next step of cell fusion.

[0099] Cell fusion and hybridoma preparation: select mice whose titer reaches the requirement, and carry out shock immunization. After 3 days, the spleen of the mouse is aseptically taken, and B lymphocyte suspension is prepared, which is mixed with SP2 / 0 myeloma cells at a ratio of 4:1. , the two cells were fused under the action of PEG. After the fused cells were resuspended in HAT medium, they were divided into 96-well cell culture plates. Set at 37°C, 5% CO 2 Cultured in an incubator.

Embodiment 2

[0100] Example 2 Screening of anti-human Trop-2 antibody-positive hybridoma cell lines

[0101] 1. Positive Hybridoma Binding Screening

[0102] 10-14 days after fusion, coat the microtiter plate with human Trop-2-his recombinant protein (serial number: NP_002344.2, 1aa-274aa) (20ng / ml), overnight at 4°C; wash three times with PBS, wash with Block with 4% skim milk powder-PBS, room temperature, 1 hr; wash three times with PBS, add hybridoma clone culture supernatant, room temperature, 1 hr. The following controls were set up: (1) positive control (PC): mouse serum after immunization (diluted with PBS 1:1000); (2) negative control (NC): fusion wells without cell growth. Wash three times with PBST (0.05% Tween-PBS), wash twice with PBS, add HRP-goat anti-mouse IgG (Fcγ), 37°C, 0.5hr; then wash three times with PBST (0.05% Tween20-PBS), Add TMB chromogenic solution, protect from light for 15-30 minutes, add ELISA stop solution to terminate the reaction; read the A450 value wi...

Embodiment 3

[0111] Example 3 Sequence determination of murine anti-human Trop-2 antibody

[0112] After the hybridoma cells m3-11, m4-3, m11-4, m17-1, m23-12 secreting anti-human Trop-2 antibody were expanded and cultured, they were cultured with Mouse Monoclonal Antibody IgG Subclass Test Card (Cat.: A12403, VicNovo ) and Mouse Monoclonal Antibody Light / Heavy Chain Test Card (Cat.: A12401, VicNovo) for subtype detection in accordance with the reagent operating procedures, subtype identification: heavy chain is IgG1, light chain is Kappa chain, m3-11, m4 -3, m11-4, m17-1, m23-12 antibody gene cloning provides basis.

[0113] The m3-11, m4-3, m11-4, m17-1, m23-12 hybridoma cells were extracted from the total RNA according to the instructions of the TRIzol kit (Cat.: 15596026, Invitrogen); using M-MuLV reverse transcriptase (Cat.: M0253S, NEB) to reverse transcribe the total RNA of hybridoma cells into cDNA; use degenerate primers (refer to the book [Dong Zhiwei, Wang Yan. Antibody Engin...

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Abstract

The invention provides an antibody or fragment thereof that binds to human tumor-associated calcium signal transducer 2 (Trop-2) protein, and an application of the antibody or fragment thereof to prevention or treatment of diseases. The antibody or the fragment thereof can be effectively combined with the human Trop-2 protein, has internalization activity, is enhanced in internalization activity after ADC drug labeling, and is not lower than a control antibody in in-vivo drug effect and safety of a mouse model.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a novel anti-human Trop-2 antibody or a functional fragment thereof. The present invention also relates to the application of the antibody or its functional fragment. Background technique [0002] Trop-2 (Tumor-associated calcium signal transducer 2), also known as tumor-associated calcium signal transducer (TACSTD2), epithelial glycoprotein-1 antigen (epithelial glycoprotein-1, EGP-1), gastrointestinal Tumor-associated antigen 1 (gastrointestinal tumor-associated antigen, GA733-1), is a type of cell surface glycoprotein encoded by the TACSTD2 gene, with a total length of 323 amino acids, and the extracellular region is composed of 3 domains, and it has been confirmed that it can dimerize form exists. [0003] Trop-2 is a transmembrane glycoprotein, and unlike other proto-oncogenes, Trop2 has no mutations, which means that it has no genetic composition changes that lead to overexpressio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30C12N15/13C12N15/70C07K19/00A61K39/395A61K47/68A61P35/00
CPCC07K16/30C12N15/70A61K47/6803A61K47/6851A61P35/00C07K2317/56C07K2317/54C07K2317/55C07K2319/00A61K2039/505A61K47/6877C07K2317/24C07K2317/77C07K2317/92C07K2317/90A61K2039/545A61K2039/507C07K16/2803
Inventor 王双王荣娟焦莎莎张畅张姣曾大地张锦超
Owner MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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