4-1BB antibody, and preparation method and application thereof

An antibody, affinity technology, applied in the direction of antibodies, antibody medical components, chemical instruments and methods, etc., can solve the problem that 4-1BB antibody drug products are not widely used

Active Publication Date: 2019-05-17
SHANGHAI HYAMAB BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the existing 4-1BB antibodies in the field still have many deficiencies in terms of antigen binding activity and activation of downstream signals of 4-1BB molecules, and there are no widely used 4-1BB antibody drug products. Development of 4-1BB agonist antibodies There is an urgent need for the treatment of cancer and autoimmune diseases

Method used

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  • 4-1BB antibody, and preparation method and application thereof
  • 4-1BB antibody, and preparation method and application thereof
  • 4-1BB antibody, and preparation method and application thereof

Examples

Experimental program
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preparation example Construction

[0205] The preparation method of the nucleic acid is a conventional preparation method in the field, preferably, it includes the following steps: obtaining a nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above-mentioned protein by a method of artificial full sequence synthesis .

[0206] Those skilled in the art know that the base sequence encoding the amino acid sequence of the above-mentioned protein can be replaced, deleted, changed, inserted or added appropriately to provide a polynucleotide homolog. The polynucleotide homologues of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the range of maintaining antibody activity.

[0207] Antibody preparation

[0208] Any method suitable for the production of monoclonal antibodies can be used to produce the anti-4-1BB antibodies of the present invention. Fo...

Embodiment 1

[0248] Preparation of 4-1BB antibody

[0249] (1) Preparation of immunogen A

[0250] The amino acid sequence Leu24-Gln186 of the extracellular region of the human 4-1BB protein (as shown in SEQ ID NO.: 21 in the sequence listing) was cloned into the pCpC vector with human IgG Fc fragment (hFc) (purchased from Invitrogen, V044-50) And prepare plasmids according to established standard molecular biology methods. Transfection of HEK293 cells (purchased from Invitrogen) (PEI, Polysciences) and FreeStyle TM 293 (Invitrogen) was expanded at 37°C. After 4 days, the cell culture fluid was collected, and the cell components were removed by centrifugation to obtain the culture supernatant containing the extracellular region of the 4-1BB protein. The culture supernatant was loaded onto a protein A affinity chromatography column (Mabselect Sure, purchased from GE Healthcare), and an ultraviolet (UV) detector was used to monitor the change in ultraviolet absorbance (A280nm). After loading ...

Embodiment 2

[0271] Determination of Amino Acid Sequence of Variable Region of Light and Heavy Chain

[0272] Total RNA isolation: After the supernatant obtained by subcloning culture of Example 1 is tested for antigen binding (that is, after the verification and activity determination of Examples 3-6), select some antibodies (see Tables 6 and 7 for details) for sequencing . Collect 5×10 by centrifugation 7 For each hybridoma cell, add 1mL Trizol, mix well, transfer to a 1.5mL centrifuge tube, and let stand at room temperature for 5 minutes. Add 0.2mL chloroform, shake for 15 seconds, let stand for 2 minutes, centrifuge at 12000g at 4°C for 5 minutes, take the supernatant and transfer to a new 1.5mL centrifuge tube. Add 0.5 mL of isopropanol, gently mix the liquid in the tube, let it stand at room temperature for 10 minutes and centrifuge at 12000g for 15 minutes at 4°C, and discard the supernatant. Add 1 mL of 75% ethanol (the percentage is volume percentage), gently wash the pellet, centr...

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Abstract

The invention provides a 4-1BB antibody, and a preparation method and an application thereof. Specifically, the invention provides the 4-1BB antibody, wherein the antibody has high affinity to a 4-1BBprotein, can activate a 4-1BB downstream signal effectively, significantly increases the expression quantity of IFN-gamma and IL-2 in human mixed lymphocytes or T lymphocytes, and can be used for treating cancer and autoimmune diseases.

Description

Technical field [0001] The present invention relates to the field of biomedicine, and more specifically to 4-1BB antibody and its preparation method and application. Background technique [0002] 4-1BB, also known as CD137, or TNFRF9, is a member of the TNF receptor family. 4-1BB is a type I transmembrane protein with a reading frame of 255 amino acids (NCBI: NP_001552), composed of an N-terminal signal peptide containing 17 amino acids, an extracellular region of 169 amino acids, a transmembrane region of 27 amino acids and The C-terminal intracellular region of 42 amino acids is composed. The 4-1BB molecule is mainly expressed in activated T cells, NK cells, regulatory T cells, dendritic cells, monocytes, neutrophils and eosinophils. The endothelial cells of tumor blood vessels have also been reported to express 4-1BB . [0003] During the activation of T cells, 4-1BB molecules can provide costimulatory signals for them. When T cell receptors are exposed to antigen, the expre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00A61K39/395A61P35/00A61P35/02A61P37/02A61P31/12A61P37/06A61P29/00A61P19/02A61P1/04A61P3/10A61P17/06G01N33/68G01N33/574
CPCA61P35/00A61P37/00A61K2039/505A61K2039/545A61K2039/54C07K16/2878C07K2317/21C07K2317/33C07K2317/75C07K2317/92C07K2319/21C07K14/70517C07K14/70575C07K2317/24C07K2317/565C07K2317/622C07K2319/33
Inventor 潘恒刘浩涵高营营胡少平李勇潘明明杨达志段清刘礼乐
Owner SHANGHAI HYAMAB BIOTECH CO LTD
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