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CD134 monoclonal antibody and its preparation method and application in cancer treatment

A monoclonal antibody and antibody technology, applied in the direction of antibodies, chemical instruments and methods, anti-animal/human immunoglobulin, etc., can solve the problems of inaccurate and unstable screening efficiency, and achieve the effect of novel sequences

Active Publication Date: 2020-04-03
ZHONGSHAN BGH BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] On the one hand, the present invention provides an OX40 antigen fragment, the sequence of which is shown in SEQ ID NO: 1. This fragment is a segment with better immunogenicity screened by the applicant, which overcomes the traditional OX40 full-length antigen as Immunogens have the problem of inaccurate, unstable and inefficient screening

Method used

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  • CD134 monoclonal antibody and its preparation method and application in cancer treatment
  • CD134 monoclonal antibody and its preparation method and application in cancer treatment
  • CD134 monoclonal antibody and its preparation method and application in cancer treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1O

[0029] Expression of embodiment 1OX40 fragment protein

[0030]Whole-gene synthesis of the gene of SEQ ID NO: 2, introducing BglⅡ and SalⅠ restriction sites at its upstream and downstream, respectively, and performing double digestion with BglⅡ and SalⅠ, pET30a plasmid with BamHI and SalⅠ for double digestion, agarose gelation Gel electrophoresis separation, then use T4 DNA ligase to ligate the OX40 gene fragment recovered from the gel and the pET30a digested DNA product at 22°C for 2 hours, transform into E. coli DH5α, pick a single colony and culture it, and extract the plasmid for EcoRⅤ and SalⅠ Double-enzyme digestion identification, and the positive clones identified by double-enzyme digestion were sent to Shanghai Sangon Co., Ltd. for sequencing. The plasmid map was identified and was identical to SEQ ID NO:2. Transform the pET30a-OX40 vector into Escherichia coli: Add 10 microliters of human pET30a-OX40 extracellular segment recombinant plasmid DNA to Escherichia coli ...

Embodiment 2

[0031] The preparation of embodiment 2 monoclonal antibody

[0032] 2.1 Animal immunity

[0033] Mice were immunized according to methods commonly used in the art. The immunogen is the antigen prepared in Example 1. Briefly, remove an appropriate amount of Freund's adjuvant into a 1.5ml EP tube, and shake to mix. Prepare the antigenic protein solution with PBS. Mix the adjuvant and protein antigen solution according to the required amount, fully emulsify the antigen by pushing each other through the syringe to form a stable water-in-oil solution, and then inject the animal. According to the results of serum titer determination, it usually takes 2 to 3 booster immunizations after the first immunization to achieve a good immune effect. The immunized mice with high serum titer were selected for final immunization by intraperitoneal injection, and then cell fusion was performed.

[0034] 2.2 Hybridoma fusion and screening

[0035] Before cell fusion, the cultured mouse myelo...

Embodiment 3

[0036] Example 3 Flow Cytometry Evaluation of Antibody Binding Ability to OX40 Antigen on Cell Membrane Surface

[0037] The 293T cell line overexpressing human OX40 on the membrane surface in the logarithmic growth phase was collected, the cells were washed twice with PBS, and the cells were resuspended with FACS buffer (PBS solution containing 2% fetal bovine serum). Adjust the density, plate 2x105 cells / well in a 96-well U-bottom plate, centrifuge at 300g for 5 minutes, pour off the supernatant, add serially diluted CSF1R antibody solution and incubate on ice for 40 minutes. Wash the cells twice, add 100 μL / well phycoerythrin fluorescently labeled goat anti-mouse secondary antibody, incubate at 4 degrees in the dark for 40 minutes, wash the cells three times, then add 100 μL of FACS buffer to each well to resuspend, blow the cells evenly On-board testing. The fluorescence intensity of cells in each well was measured using a BD Canto II flow cytometer. Using Graphpad prism...

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Abstract

The invention provides a monoclonal antibody which can specifically recognize human OX40. The affinity of combination with the human OX40 of the antibody is stronger than that of an existing anti-human OX40 monoclonal antibody, and has relatively good heat resistance and acid-base resistance property.

Description

technical field [0001] This application belongs to the field of biopharmaceuticals, and specifically relates to CD134 antibody, its preparation method and the corresponding application of cancer treatment. Background technique [0002] OX40 (CD134) is a member of the TNF superfamily and is a type I transmembrane glycoprotein. Human OX40 gene was cloned in 1994, about 1.4kb in length, encoding 277 amino acids, located on chromosome 1 lp36. The expression profile of OX40 is limited to the surface of activated CD+4 and CD+8 T cells, and CD+4 T cells are predominant. Human OX40 ligand (OX40L / CD134L) was cloned by Miura et al. in 1991. It contains 183 amino acids (139 amino acids outside the cell, 21 amino acids across the membrane, and 23 amino acids inside the cell). It is a member of the TNF family and is a type II transmembrane glycoprotein. The OX40L gene is about 1kb in length and located at 1q25. [0003] OX40 / OX40L is a pair of important co-stimulatory molecules, whic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/705C07K16/28A61K39/395A61P35/00
CPCA61K2039/505A61P35/00C07K14/70575C07K16/2875C07K2317/56
Inventor 彭菲顾超
Owner ZHONGSHAN BGH BIOTECH CO LTD
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