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Antihuman transferrin acceptor human source antibody and uses thereof

A human transferrin and receptor technology, applied in the field of molecular biology, can solve the problems of reduced affinity activity, inability to be applied in the human body, and difficult to completely solve the problem of antibody immunogenicity.

Inactive Publication Date: 2010-10-13
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, the existing OX26 antibody is a mouse anti-rat transferrin receptor antibody, which has strong immunogenicity and animal species selectivity and cannot be used in humans; 128.1 is a mouse anti-human transferrin receptor antibody Antibodies, mouse antibodies as heterologous proteins will be limited in human applications: such as inducing the generation of mouse anti-human antibodies (HAMA), the mouse Fc segment can not effectively play the function of the human Fc segment, the mouse antibody short biological half-life
Even if humanized transformation is carried out, certain mouse sequences will still be retained, making it difficult to completely solve the problem of antibody immunogenicity
Moreover, the humanized transformation of antibodies is time-consuming and laborious, and the affinity activity and specificity are often reduced after transformation.

Method used

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  • Antihuman transferrin acceptor human source antibody and uses thereof
  • Antihuman transferrin acceptor human source antibody and uses thereof
  • Antihuman transferrin acceptor human source antibody and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Expression of human transferrin receptor extracellular domain protein by genetic engineering method

[0047] 1. Cloning of TfR extracellular region gene

[0048] The surface of many tumor cells is rich in transferrin receptors. Total RNA was extracted from human cervical cancer HeLa cells (Shanghai Cell Bank, Chinese Academy of Sciences) with TRIzol reagent (Invitrogen, operated according to the instructions). Primers were designed according to the gene sequence of human TfR, FW was: 5'-GAATTCATGTTATATTGGGATGACCTG-3' (SEQ ID No.4); RV was: 5'-CCGCTCGATTTAGTGATGGTGATGGTGATGAAACTC ATTGTCAATGTC-3' (SEQ ID No.5), RT-PCR amplified human The gene for the extracellular domain of the transferrin receptor.

[0049]The reaction system for the reverse transcription gene is: add 5.0 μL RV (10 mmol / L) to 10 μL RNA, mix well, let it stand at 70°C for 5 minutes, put it in an ice bath, and add the following components: 5 μL M-MLV RT 5×buffer, 2.5 μL dNTP (10 mmol / L), 1.0 μ...

Embodiment 2

[0056] Example 2: Purification of human transferrin receptor from placenta

[0057] 1. Sample preparation

[0058] Human fresh placenta (wet weight: 500 g) was cut into small pieces after removing umbilical cord and membrane, washed with PBS, and homogenized in 1 L of PBS. Centrifuge at 10,000g for 20min to collect the precipitate. After resuspending with PBS, continue to homogenate and centrifuge. Repeat 3 times to remove endogenous transferrin. The precipitate was dissolved in 800 mL of PBS (containing 2% Triton X-100, 1 mmol / L PMSF (phenylmethylsulfonyl fluoride)), stirred for 1 h, and then sonicated for 40 minutes to release membrane proteins. Centrifuge at 10,000g for 30min, collect the supernatant for affinity chromatography. All the above operations were carried out at 4°C.

[0059] 2. Preparation of transferrin receptor-Sepharose 4B affinity chromatography column

[0060] According to the instructions, human transferrin was linked to cyanogen bromide-activated Se...

Embodiment 3

[0064] Example 3: Screening of anti-transferrin receptor single chain antibody

[0065] 1. Screening of phage antibody library

[0066] Coat immunotubes with 10 μg of the transferrin receptor extracted from the placenta and 10 μg of the expressed transferrin receptor extracellular domain protein respectively, overnight at 4 °C, block with 2 g / L BSA at 37 °C for 2 h, add 1 mL of human PHB -pIX-scFv phage antibody library solution (stock capacity 10 10 cfu / L) (gifted by Dr. Sun Zhiwei. Establishment and preliminary evaluation of a new pIX phage display system. Wang Shuang; Sun Zhiwei; Du Weishi; Zhang Jinchao; Yu Weiyuan; Journal of the Academy of Military Medical Sciences, 2005, 5:48), incubated at 37°C for 2h . Wash with PBS (containing 1mL / L Tween20) 20 times, 5min each time. Then add 1 mL of 0.2 mol / L Gly-HCl (pH 2.2), shake at room temperature for 10 min, and neutralize the pH to about 7.0 with 1 mol / L Tris. Transfer the neutralized solution to a 9 mL OD 600 =0.5 in th...

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Abstract

The invention relates to an anti-human transferrin receptor human antibody and the application, which pertains to the field of molecular biology. The anti-human transferrin receptor human antibody has a single-chain antibody (scFv) amino acid sequence which is shown in a sequence table SEQ ID No.1. The anti-human transferrin receptor human antibody and the application have the advantages that: the human antibody solves the problem of immunogenicity of the target antibody, thus allowing the human antibody to carry out the long-term repeated drug administrations in the human body; the method for screening a genetic engineering antibody in a large-capacity antibody library which is adopted by the invention is easier than the monoclonal antibody preparation, the large-scale production is easy, the molecule of the antibody is small, and the antibody can enter tissues deeply, thus having obvious advantages. A pET-22b (plus) vector which is adopted by the invention contains a stronger T7 promoter, which can ensure the higher expression level of the target antibody. The induced expression conditions after the optimization can ensure that the antibody is expressed in a soluble form and theactivity of protein is higher. The 3' end of the pET-22b (plus) vector is provided with six His(histidine) labels, which can easily use the Ni-NTA agarose to carry out affinity chromatography and purification.

Description

technical field [0001] The present invention relates to anti-human transferrin receptor human antibody and its application, more specifically anti-human transferrin receptor human antibody, its nucleotide sequence and the carrier containing the nucleotide, belonging to molecular biology field of study. Background technique [0002] Transferrin receptor (Transferrin Receptor, TfR) is a transmembrane glycoprotein composed of two identical subunits to form a homodimer, which mediates iron uptake through the interaction with transferrin. Transferrin receptors are expressed in nucleated cells of vertebrates, especially rich in the blood-brain barrier, placental tissue, tumor cells with vigorous metabolism, tumor cells that require a lot of iron, and some rapidly dividing cell surfaces. important target for transport. [0003] The blood-brain barrier is composed of brain capillary endothelial cells, basement membrane and glial cells. Its remarkable feature is that the brain capi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28A61K39/395A61P25/00A61P35/00C12N15/13C12N15/63C12N1/21
Inventor 颜冰黄培堂赵莉霞
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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