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Single-chain antibody in anti-human prostate-specific membrane antigen extracellular region and application thereof

A prostate-specific, single-chain antibody technology, applied to human-derived single-chain antibody and its application in prostate cancer diagnosis and therapeutic drug development, can solve problems such as reducing therapeutic value

Inactive Publication Date: 2012-07-25
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although humanization can reduce the immunogenicity of murine antibodies to a large extent, it is impossible to completely remove its immunogenicity, so it will inevitably lead to the appearance of neutralizing antibodies in clinical treatment and reduce its therapeutic value

Method used

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  • Single-chain antibody in anti-human prostate-specific membrane antigen extracellular region and application thereof
  • Single-chain antibody in anti-human prostate-specific membrane antigen extracellular region and application thereof
  • Single-chain antibody in anti-human prostate-specific membrane antigen extracellular region and application thereof

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Experimental program
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Effect test

Embodiment

[0070] (1) Isolation of lymphocytes

[0071] A total of 500ml of peripheral blood was collected from 50 prostate cancer patients, added an equal volume of lymphocyte separation medium, and centrifuged to separate layers at 2500g for 10min. Carefully absorb the middle lymphocytes and centrifuge at 2500g for 10min. Discard the supernatant, and use 1ml Trizol / 5ml peripheral blood to lyse the lymphocytes until the solution is completely transparent. Store at -80°C.

[0072] (2) Extraction of RNA

[0073] Thaw the lymphocyte lysate stored at -70°C to room temperature, add 0.2ml chloroform / ml Trizol, shake with Votex for 15s, let it stand at room temperature for 3min, 12000g, 5min. Aspirate the clear water phase of the upper layer, and be careful not to mix into the middle layer of protein and DNA. Add 0.5ml isopropanol / ml Trizol, invert and mix well, precipitate at -20°C for 30-60min, and centrifuge at 12000g for 10min. Keep the precipitate, wash it once with 70% ethanol, air ...

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Abstract

The invention provides a single-chain antibody in an anti-human prostate-specific membrane antigen (PSMA) extracellular region, which adopts the phage display technology. The antibody is obtained by screening and enriching a large-storage capacity single-chain antibody library which is established by using peripheral blood mono-nuclear cells of a prostatic cancer patient through successive relaxation and rigorousness. The antibody can be solubly expressed in colon bacillus, and the affinity of the antibody with the prostate-specific membrane antigen extracellular region reaches Kd of 0.2nM. The antibody not only can be combined with the recombinant PSMA extracellular region, but also can be combined with NEK 293 cells with PSMA expressed on surface thereof and a prostate adenocarcinoma cell LNCaP, thus laying a foundation for adopting the antibody to perform prostate adenocarcinoma image diagnosis and guiding treatment.

Description

technical field [0001] The invention belongs to genetic engineering antibodies, and relates to a humanized single-chain antibody against the extracellular region of human prostate-specific membrane antigen (PSMA) and its application in the development of prostate cancer diagnosis and treatment drugs. Background technique [0002] Prostate cancer is one of the malignant tumors that seriously endanger human health today. Its early diagnosis and treatment, especially the early diagnosis and treatment of metastasis and recurrence are difficult medical problems for people to overcome. With the continuous development of tumor immunology and molecular biology, especially the continuous improvement of phage surface display technology, new opportunities have been brought for the early diagnosis and cure of prostate cancer. Currently, anti-prostate cancer antibodies are undergoing clinical trials abroad, and these antibodies are basically humanized murine antibodies. Although humaniz...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28G01N33/574A61K39/395A61P35/00
Inventor 秦卫军赵爱志董青川温伟红
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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