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TRAIL receptor I and/or TRAIL receptor 2 specific antibody and its use

An antibody and receptor technology, applied in the field of unique monoclonal antibodies, which can solve the problems of lack of receptor selectivity, short in vivo half-life of recombinant TRAIL protein, weakening, etc.

Inactive Publication Date: 2007-11-21
BEIJING COTIMES BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, as a therapeutic drug, TRAIL has some significant drawbacks
First, since TRAIL contains at least five different receptors, including both apoptosis-inducing receptors and apoptosis-antagonizing receptors, the effects of TRAIL lack obvious receptor selectivity
In particular, the difference in expression levels between apoptosis-inducing receptors and apoptosis-antagonizing receptors makes it difficult to estimate the ability of TRAIL to induce apoptosis in different tumor cells
For example, if a specific tumor cell expresses a relatively high level of anti-apoptotic receptors, the ability of TRAIL to induce apoptosis in these tumor cells will be greatly weakened
Second, the recombinant TRAIL protein has a very short in vivo half-life, which largely limits the effective in vivo dose and anti-tumor effect of TRAIL
The purpose of the present invention is to solve the deficiency of tumor cell apoptosis induced by monoclonal antibodies against TRAIL-R1 or TRAIL-R2 respectively. Epitope-specific monoclonal antibodies that enable one antibody to simultaneously target TRAIL-R1 and TRAIL-R2 to induce tumor cell apoptosis

Method used

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  • TRAIL receptor I and/or TRAIL receptor 2 specific antibody and its use
  • TRAIL receptor I and/or TRAIL receptor 2 specific antibody and its use
  • TRAIL receptor I and/or TRAIL receptor 2 specific antibody and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0200] Example 1: Preparation of TRAIL-R1 and TRAIL-R2 mixed dimers

[0201] 1. Cloning of TRAIL-R1 and TRAIL-R2 cDNA by RT-PCR

[0202] a) Prepare the template

[0203] Total RNA was extracted from human cervical cancer Hela cells using TRIZOL reagent (GibcoBRL). Then, using the total RNA as a template, cDNA was synthesized by reverse transcription, and the Promega cDNA reverse transcription finished kit was used to operate according to its instruction manual.

[0204] b) Synthetic PCR oligonucleotide primers:

[0205] TRAIL-R2 5' end primer: 5'-gacgatgcccgatctactttaaggg-3'

[0206] TRAIL-R2 3' end primer: 5'-ccactgggtgatgttggatggg-3'

[0207] TRAIL-R1 5' end primer: 5'-gacgatgcccgatctactttaaggg-3'

[0208] TRAIL-R1 3' end primer: 5'-gacgatgcccgatctactttaaggg-3'

[0209] c) PCR reaction:

[0210] Take 5 microliters of template cDNA, add 5 microliters of 5' and 3' primers, 10 microliters of 10-fold concentrated PCR buffer, 4 microliters of dNTP, and 5 units of TaqPCR po...

Embodiment 2

[0223] Example 2: Preparation of anti-human TRAIL-R1 and TRAIL-R2 monoclonal antibodies

[0224] 1. Immunization of mice

[0225] The TRAIL-R1 and TRAIL-R2 fusion proteins purified by affinity chromatography were mixed with Freund's complete adjuvant 1:1, and 0.2 ml protein adjuvant mixture was injected into the paws of 2 Balb / c mice (female, week-old : 6 to 8 weeks). Ten days later, 0.2 ml of the mixture of protein and Freund's incomplete adjuvant was injected again. One week later, 0.2 mg of purified protein was injected again. Once a week, repeat 3 times. Three days after the last injection, the abscess and inguinal lymph nodes of the mice were taken for the preparation of hybridomas.

[0226] 2. Cell Fusion

[0227] A single cell suspension was made from the fossa and inguinal lymph nodes, and mixed with NS1 myeloma cells at a ratio of 2:1. Wash three times with serum-free RPMI-1640 medium. Add 1 ml of 37°C preheated PEG1500, and gently stir the mixture of PEG1500 a...

Embodiment 3

[0241] Example 3: TRAIL receptor binding specificity of CTB003

[0242] Since the five TRAIL receptors have high amino acid sequence homology in their extracellular regions, we further tested the reactivity of CTB003 to other TRAIL receptors. TRAIL-R1, TRAIL-R2, TRAIL-R3 and TRAIL-R4 were respectively coated onto microtiter plates, and then blocked with PBS buffer containing 3% BSA. After blocking for 1 hour, different concentrations of purified CTB003 were added. After incubation at 37°C for 1 hour, horseradish peroxidase-labeled goat anti-mouse IgG was added, followed by TMB substrate buffer 30 minutes later. After ten minutes, the reaction was quenched with 2 equivalents of sulfuric acid. Then mark it with a microplate reader.

[0243] The results showed that CTB003 showed an equivalent and dose-related antigen-binding response to TRAIL-R1 and TRAIL-R2. Meanwhile, CTB003 did not exhibit antigen-binding responses to TRAIL-R3 and TRAIL-R4 within the range of antibody conc...

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PUM

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Abstract

A specific antibody combined TRAIL acceptor 1(TRAIL-R1) and / or TRAIL acceptor 2(TRAIL-R2), its amino-acid sequence and nucleotide sequence for encoding it and its usage in treatment of tumor are disclosed.

Description

technical field [0001] The present invention relates to a unique monoclonal antibody, including its preparation and application. The antibody can detect TRAIL-R1 and / or TRAIL-R2 has equivalent binding affinity. The antibody has the ability to induce apoptosis of most human tumor cells. These tumor cells can express either TRAIL-R1 or TRAIL-R2 alone, or both TRAIL-R1 and TRAIL-R2. The present invention also provides the application of the antibody in tumor therapy, especially the adjuvant and synergistic application of the antibody with other current tumor therapies. Background technique [0002] TRAIL was discovered in the mid-1990s. Shortly after TRAIL was discovered, its anti-tumor activity and its potential as a clinical anti-tumor drug attracted people's attention day by day. This is mainly based on the fact that TRAIL can selectively kill tumor cells without attacking normal cells. More importantly, the antitumor activity of TRAIL can be greatly enhanced by any cu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18A61K39/395C12N5/18A61P35/00C12N15/13C12N15/63C07K14/435A61K31/7088
CPCA61K2039/507C07K2316/95A61K2039/505C07K16/2878C07K2317/73C07K2317/24A61P35/00
Inventor 宋扬贾冼钊沈恩允余征周敏
Owner BEIJING COTIMES BIOTECH CO LTD
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