Preparation method and application of tumor-targeted photosensitive immunoconjugate

An immunoconjugate and tumor-targeting technology, which is applied to the preparation method of peptides, anti-tumor drugs, chemical instruments and methods, etc., can solve the problems of poor tumor selectivity, phototoxicity, and long-term light-shielding time for patients, and achieve High anti-tumor activity, good stability and low cost

Active Publication Date: 2012-07-18
FUJIAN INST OF RES ON THE STRUCTURE OF MATTER CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Photodynamic therapy is a promising tumor treatment method, which has been clinically used in the treatment of head and neck tumors, esophageal cancer, breast cancer, basal cell carcinoma and other tumors, but the current photodynamic therapy drugs There are still some deficiencies, such as: poor selectivity of photosensitizers to tumors; there are phototoxic reactions, and patients are kept away from light for a long time after treatment

Method used

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  • Preparation method and application of tumor-targeted photosensitive immunoconjugate
  • Preparation method and application of tumor-targeted photosensitive immunoconjugate
  • Preparation method and application of tumor-targeted photosensitive immunoconjugate

Examples

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Embodiment 1

[0031] The preparation of embodiment 1 water-soluble single carboxyl phthalocyanine zinc hydrazide derivative ( figure 2 )

[0032] a. Weigh 20 mg monocarboxyzinc phthalocyanine (ZnPc-COOH), 24 mg benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU), with pentameric lysine Resin 128mg of amino acid, pre-swelled in N,N-dimethylformamide (DMF) for 30min), added a total of 4ml of DMF (the reaction concentration of phthalocyanine is about 5mg / ml), and then dripped 5uL of N,N-di Isopropylethylamine (DIEA), stirred at room temperature, and reacted overnight in the dark, for a total of 16 hours. After the reaction is completed, filter with suction, wash with DMF until it is colorless, wash with methanol for 2-3 times, filter with suction and dry.

[0033] b. Take the above sample, add 5ml DMF, 200ul 85% hydrazine hydrate, stir at room temperature, avoid light overnight, then filter with suction, add a small amount of DMF to wash the resin, then add 10 times the...

Embodiment 2

[0036] The oxidation of embodiment 2 nimotuzumab

[0037] Take 40ul Nimotuzumab (5mg / ml, 0.033mmol / L), namely 200ug, add 10ul 50mmol / L sodium periodate (NaIO4) to make the final concentration reach 10mmol / L, room temperature (25°C water bath) After reacting in the dark for 30 minutes, add 10ul of 1mol / L ascorbic acid, and let it stand in a refrigerator at 4°C for 30 minutes to remove residual NaIO4 for later use.

Embodiment 3

[0038] Nimotuzumab after embodiment 3 oxidation is coupled with ZnPC-CO-5K-NHNH2

[0039] ZnPC-CO-5K-NHNH2 is prepared into a 3.3mmol / L stock solution with pure water, take the oxidized Nimotuzumab, add 10ul of the above stock solution per 100ul, add 10ul 1mol / L phosphate buffered saline ( PH7.2), mix well, and vortex shake in the dark at 4°C refrigerator overnight, add 20ul 1mol / L sodium borohydride (NaBH4) (1mol / L Na2CO3 freshly prepared) solution, 4°C refrigerator, stand still for 4h. Separate with SephadexG100 gel column (12×300mm), use PBS as eluent, collect the first elution peak, and concentrate to 100ul with a 10kDa ultrafiltration centrifuge tube for use. At the same time, the purity was detected by 10% polyacrylamide gel electrophoresis with non-reducing SDS-page ( image 3 , Figure 4 ).

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Abstract

The invention provides a preparation method of high-activity zinc phthalocyanine-monoclonal antibody tumor-targeted photosensitive immunoconjugate and a technical method for anti-tumor application of the immunoconjugate. Monocarboxyl zinc phthalocyanine with a single structure is reacted with lysine hydrazine pentamer to form a water-soluble monocarboxyl zinc phthalocyanine hydrazine derivative with high reaction activity; meanwhile, the monoclonal antibody with anti-tumor activity is selected; under a neutral condition, glycosyl non-related to activity of the antibody is mildly oxidized into an aldehyde group with sodium periodate; and at 4 DEG C, excess monocarboxyl zinc phthalocyanine hydrazine derivative is extremely easy for reacting with the aldehyde group and the reaction product is further reduced into stable photosensitive immunoconjugate to obtain the high-purity photosensitive immunoconjugate. The preparation method has the characteristics of low temperature, neutral conditions and no use of any organic solvent. In-vitro anti-tumor experiments prove that the conjugate has extremely high selectivity and anti-tumor activity as well as application value.

Description

technical field [0001] The invention relates to the fields of medicine, biotechnology, chemistry and immunobiology. Background technique [0002] In recent years, more and more domestic and foreign literatures have reported a new tumor treatment method—photodynamic therapy (PDT). Tumor photodynamic therapy is the latest development in the field of minimally invasive or non-invasive treatment of modern tumors. It was approved by the US FDA for clinical application in 1996, and has been used in various tumor treatments in the United States, Japan, the United Kingdom, France, Germany, Canada and other countries. success. In May 2003, China SFDA approved the clinical application of this treatment system. [0003] Photodynamic therapy is a new type of treatment for malignant tumors under development, and it has been used clinically for the treatment of various tumors. In order to increase the photodynamic efficacy, we use a monoclonal antibody that can specifically bind to tum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30C07K1/113A61K41/00A61K47/48A61P35/00A61K47/68
Inventor 黄明东周山勇陈卓陈锦灿胡萍
Owner FUJIAN INST OF RES ON THE STRUCTURE OF MATTER CHINESE ACAD OF SCI
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