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Complement factor H inhibitor and application related to same

A technology of complement-related diseases and inhibitors, applied in the fields of application, antibody, genetic engineering, etc., can solve the problems of low affinity, not yet fully activated B lymphocytes, low concentration of antibodies, etc.

Active Publication Date: 2017-03-22
GUANGZHOU TAINUODI BIOTECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Certain host antibodies may have the potential for antitumor activity, but this ability has not been fully realized for a number of possible reasons, including low concentration or low affinity of the antibody or ineffective activation of B lymphocytes

Method used

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  • Complement factor H inhibitor and application related to same
  • Complement factor H inhibitor and application related to same
  • Complement factor H inhibitor and application related to same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 PBMC isolation and single memory B cell sorting

[0082] Eleven patients expressing CFH autoantibodies were found by immunoblotting, and their peripheral blood mononuclear cells (PBMC) were pooled. Using the avidin-labeled CFH 15-mer polypeptide (SEQ ID NO:9) as bait, a single memory B cell was sorted from the pooled PBMC using a flow sorter: select P1 (lymphocyte gate) → P2 ( IgG+) → P3 (CD3- / CD14- / CD16- / CD20+ / CD27ALL). Select P1&P2&P3 gate (IgG+ / CD3- / CD14- / CD16- / CD20+ / CD27ALL), count cells (5000 cells / well), sort multiwell. CD3- / CD14- / CD16- / CD20+ / CD27ALL cell population was sorted by flow cytometry. To minimize false positives, Streptavidin Fluor647 and Brilliant Violet421 were labeled with Alexa. Each fluorophore was labeled with streptavidin on a separate aliquot which was then mixed together and interacted with the biotinylated antigenic peptide. Cells of both fluorophores showing elevated fluorescence were sorted into wells of a single 96-well plate....

Embodiment 2

[0083] Example 2 Single B cell RT-PCR isolation of antibody variable region genes

[0084] Synthesize the first strand of cDNA: Add 0.5 μM constant region primers of heavy and light chains of each subtype to a 96-well plate containing a single B cell, and Superscript III reverse transcriptase, and incubate at 37°C for 1 hour; proceed according to the following conditions PCR amplification: 95°C for 15 min; 95°C for 1 min, 55°C for 1 min, 72°C for 1 min, 30 cycles; 72°C for 10 min; 4°C for 5 min. The product cDNA was stored at -20°C.

[0085] Separation of antibody genes by Nest-PCR: Each single cell amplifies the heavy chain and light chain sequences respectively. The 50 μL system contains 5 μL of RT reaction products, HotStarTaq enzyme, dNTPs, and 0.5 μM specific primers for heavy chain and light chain antibodies of each subtype. Reaction conditions: pre-denaturation at 95°C for 5 minutes, and then 35 PCR cycles: 95 ℃×30s, 55℃×60s, 72℃×90s, and finally extend at 72℃ for 7mi...

Embodiment 3

[0086] Example 3 PCR product clone identification and antibody expression

[0087] PCR product purification and clone identification: 2 μL of the amplified product was detected by 1.2% agarose gel electrophoresis, and the target fragment was about 400 bp. The gel electrophoresis was identified as positive, and the PCR product of the antibody variable region gene whose heavy chain and light chain could be paired was connected to the pcDNA3.3 vector (which has been transformed and contains the antibody leader and constant region) by the method of TA cloning, Transform the ligation product into DH5α-competent bacteria, culture on a plate containing ampicillin at 37°C overnight, then pick 10 single colonies and perform PCR with specific primers, reaction conditions: pre-denaturation at 94°C for 3 minutes, denaturation at 94°C for 30 seconds, Anneal at 55°C for 30s, extend at 72°C for 1min40s, 28 cycles, and finally extend at 72°C for 5min; take 5 μL of PCR product for electrophore...

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Abstract

The invention discloses a CFH inhibitor for treating cancer. The CFH inhibitor is a CFH specific antibody. More particularly, the CFH inhibitor is a CFH specific monoclonal antibody. The CFH inhibitor stops combination of CFH and C3b to improve deposition of C3b on tumor cells and promote compliment mediated cytotoxic effect. On that basis, the invention provides a method for treating cancer.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a complement factor H inhibitor and related uses thereof, in particular to a complement factor H inhibitor and the use of the inhibitor in treating diseases related to complement factor H. Background technique [0002] Lung cancer is a major public health problem. Most tumors are still detected at an advanced stage when treatment options are limited and systemic therapy is required. Even patients with resectable early-stage lung cancer have an almost 50% chance of developing recurrence and requiring adjuvant therapy at some point. Over the past few years new therapies have been introduced to target specific pathways and generate initial responses in these selected individuals. However, almost all patients develop resistance, most likely due to tumor heterogeneity and clonal evolution. [0003] Although activation of the humoral response against malignant cells has been studied, humoral...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13A61K39/395A61P35/00G01N33/68G01N33/577
CPCA61K39/00C07K16/18C07K2317/21C07K2317/56C07K2317/565C07K2317/92G01N33/577G01N33/68G01N2333/4716
Inventor 廖化新王月明袁晓辉昝利鹏刘彤吴昌文张远旭
Owner GUANGZHOU TAINUODI BIOTECH
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