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131I labeled anti-tumor humanized monoclonal antibody 1E2 and use thereof

A monoclonal antibody and humanized technology, which is applied in the direction of antibodies, anti-tumor drugs, and radioactive preparations in vivo, can solve the problems of three-dimensional structure destruction, low tumor inhibition rate, and loss of components to the extracellular space, so as to improve targeting , reduce the immune response, improve the effect of tumor inhibition rate

Inactive Publication Date: 2009-04-15
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In radioimmunotherapy (RIT) for lung cancer, someone used yttrium-90-labeled anti-lung cancer monoclonal antibody LC-1 as a local treatment, and the tumor inhibition rate was low; the only new drug on the market 131 The mechanism of action of I-chTNT is: the typical feature of advanced solid tumors is the presence of a large number of denatured and necrotic cells. During the process of denatured and necrotic cells, many cracks will appear in the cell membrane, and a large number of components in the cytoplasm will be lost to the outside of the cell. The three-dimensional structure of chromosomes is destroyed, resulting in a large number of denatured DNA single-stranded complexes, which are 131 I-chTNT acts on the target of solid tumors, so the drug is only suitable for patients with advanced lung cancer who have failed radiotherapy and chemotherapy

Method used

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  • 131I labeled anti-tumor humanized monoclonal antibody 1E2 and use thereof
  • 131I labeled anti-tumor humanized monoclonal antibody 1E2 and use thereof
  • 131I labeled anti-tumor humanized monoclonal antibody 1E2 and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1 Preparation of humanized anti-lung cancer monoclonal antibody 1E2

[0026] (1) Construction of phage antibody library:

[0027] ①Extraction of total RNA: 10 g of paracancerous lymph nodes (pathologically confirmed as non-small cell lung cancer) were excised from lung cancer patients, wrapped in sterile gauze and quickly frozen in liquid nitrogen. The tissue is crushed into powder, and then extracted with an RNA extraction kit to obtain total RNA.

[0028] ②Amplification of VH and VL gene fragments: cDNA was synthesized with Oligo dT-Adaptor primer as primer, PCR conditions were: 50°C for 30 minutes, 99°C for 5 minutes, 5°C for 5 minutes, one cycle. Then using cDNA as template, the upper and lower primers are paired to amplify the light chain gene fragment and the heavy chain gene fragment respectively. The PCR conditions are: 94°C for 2min, one cycle, 94°C for 30sec, 55°C for 30sec, 72°C for 90sec, thirty cycles . Finally, the Linker primers were used to am...

Embodiment 2

[0036] Embodiment 2 is prepared by chloramine T method 131 I-labeled 1E2 antibody

[0037] Methods as below:

[0038] 1. Iodination reaction: Take 10 μg of the humanized 1E2 antibody prepared in Example 1, dilute it to a concentration of 200 μg / ml with a phosphate buffer solution with a pH of 7.5 and a concentration of 0.5 mol / L, and accurately measure 50 μl for use; Na with a specific activity of 5.5GBq / ml 131 I 100 μl; then take 50 μl of chloramine T with a concentration of 1 μg / μl, mix the three, add it into a glass test tube with a magnetic stirrer, place it on a magnetic stirrer and shake it well, and react at room temperature for 60 seconds;

[0039] 2. To terminate the iodination reaction: immediately add sodium metabisulfite (Na 2 S 2 o 5 )) 100 μg, volume 100 μl, stop the iodination reaction;

[0040] 3. Separation and purification:

[0041] (1) Inject the labeled reaction solution into a 1cm×15cm Sephadex G50 chromatographic column, elute with PBS solution (pH...

Embodiment 3

[0043] Embodiment 3 to the purification prepared by embodiment 2 131 Identification of labeling rate and radiochemical purity of I-labeled 1E2 antibody

[0044] Trichloroacetic acid was used as a developing agent, and Xinhua No. 1 filter paper was used as a support, and paper chromatography was used to detect the labeling rate and radiochemical purity of the reaction solution before and after separation and purification. The formula:

[0045]

[0046] = 77.73 %

[0047]

[0048] = 432 MBq / μg

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Abstract

The invention provides an anti-lung cancer human resource monoclonal antibody 1E2 marked by <131>I. The human resource 1E2 antibody, Na <131>I and chloramine T are used as raw materials, and purified 1E2 antibody marked by the <131>I can be prepared by using the chloramine T method, wherein, the human source 1E2 antibody is obtained by taking paracanser lymph nodes of a non-small cell lung cancer patient, extracting total RNA in lymph node tissues, and then a phage antibody library containing the 1E2 antibody is obtained by using the constructing technology of the phage antibody library, and the phage antibody 1E2 is adsorbed by using a carbamyl phosphate synthetase antigen, and a human source 1E2 antibody which is specific to the carbamyl phosphate synthetase is finally obtained by washing, screening and amplifying. The invention applies radiation immunity treatment technology, combines advantages of radionuclide and anti-tumor cell human source monoclonal antibody to one, improves the targeting ability and curative effect of medicines to tumor cells, reduces side effects and poor immune reactions, and is wider in application range and suitable for treatments of the non-small cell lung cancer of each stage.

Description

technical field [0001] The invention relates to a radioactive anti-tumor antibody, in particular to a 131 I-labeled anti-lung cancer humanized monoclonal antibody 1E2 and its application. Background technique [0002] With the continuous development of medical technology, radioimmunotherapy (RIT) is another tumor treatment method in addition to surgery, radiotherapy and chemotherapy. RIT is the injection of radionuclide-labeled anti-tumor antibodies into the body. The monoclonal antibody and the labeled nuclide specifically bind to tumor cells (directed guidance), and carry the radionuclide to the tumor site. Radiation damages the DNA of tumor cells, eventually causing growth inhibition and necrosis of tumor cells. Radioimmunotherapy is a brand-new treatment method following tumor chemotherapy and radiotherapy. It combines the targeting effect of monoclonal antibodies with the strong killing effect of radionuclide internal irradiation, and has the characteristics of good c...

Claims

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Application Information

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IPC IPC(8): C07K16/18A61K51/00A61K39/395A61P35/00A61K101/02
Inventor 尹晓玲李少林陈晓品季平吴永忠彭志平林海波文明张涛段东庞华
Owner CHONGQING MEDICAL UNIVERSITY
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