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Humanization tumor immune cell cytokines TNTIL2 as well as preparation method and application thereof

A technology of TNTIL2 and immune cells, applied in chemical instruments and methods, antineoplastic drugs, pharmaceutical formulations, etc., can solve the problems of lack of treatment specificity, toxic and side effects, clearance, etc., to reduce pain and economic burden, tumor specificity High, extended half-life effect

Inactive Publication Date: 2013-09-18
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional treatment plan still has the following defects: lack of specificity of treatment, inability to kill residual tumor cells and inability to control tumor metastasis and recurrence, and even radiotherapy and chemotherapy can lead to huge toxic and side effects, and eventually patients often die of tumors Adverse reactions such as metastasis, recurrence or infection caused by radiotherapy and chemotherapy
[0008] According to the literature, most of the tumor antigens used in anti-tumor monoclonal antibody drugs are cell membrane antigens, and the distribution of tumor types is relatively small. And other issues

Method used

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  • Humanization tumor immune cell cytokines TNTIL2 as well as preparation method and application thereof
  • Humanization tumor immune cell cytokines TNTIL2 as well as preparation method and application thereof
  • Humanization tumor immune cell cytokines TNTIL2 as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Preparation of humanized tumor immune cytokine TNTIL2

[0040] Using Raji Burkitt's lymphoma cell line (used to produce the immunogen of the hybridoma), the cells were subjected to nuclear extraction. Eight-week-old Balb / c mice were immunized with nuclear extracts. Four days after the last immunization, mice were sacrificed and spleens were removed under aseptic conditions. The spleen was processed into a single cell suspension, and mixed with 8-azaguanine-resistant murine myeloma NS-1 in 40% polyethylene glycol 1540 to construct fusion cells. The mixed cells were then cultured in 96-well tissue cell plates in RPMI-1640 medium containing hypoxanthine, aminopterin, and thymidine. The mouse-derived TNT monoclonal antibody (abbreviation: muTNT-MAb) fusion cell secretory strain positive for nuclear immunofluorescence reaction of fixed Raji lymphoma cells was selected and stored in liquid nitrogen.

[0041] Use PCR and RT-PCR amplification techniques to obtain c...

Embodiment 2

[0048] Example 2 Preparation and quality standard of humanized immune cytokine TNTIL2

[0049] The preparation process is: NS0 shake flask cell culture, bioreactor cell culture, cell supernatant collection, supernatant pretreatment before purification, Protein A affinity purification, SP-Sepharose cation exchange purification, Q-Sepharose anion exchange purification, sterile filtration , stored at 20°C.

[0050] verification of reference products,

[0051] In accordance with the technical procedures for the production and verification of the original solution, the identification of the original solution is carried out. The main test results are shown in Table 2.

[0052] Table 2 TNT-IL2 stock solution identification and measurement project results

[0053] Test items

Embodiment 3

[0054] Example 3 Preparation of powder injection containing immune cytokine TNTIL2

[0055] Using the conventional freeze-drying method, using water as the solvent, including steps: take the fusion protein TNTIL2, add excipients, add water to dissolve, add activated carbon, filter and sterilize, fill, half-stopper, freeze-dry, stopper and cap. Can. The used excipients are selected from one or more of mannitol, hydrolyzed gelatin, glucose, lactose, dextran and the like. Each bottle contains 10-100 mg of fusion protein TNTIL2, which is stored at -20°C until use.

[0056] Or adopt the spray drying method to prepare the powder injection, using water as the solvent, including the steps: take the fusion protein TNTIL2, add or not add excipients (excipients are the same as above), add water to dissolve, add activated carbon, filter and sterilize, spray dry, without Pack the bacteria separately, and just press the plug and roll the cap. Each bottle contains 10-100 mg of fusion prot...

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Abstract

The invention belongs to the technical field of novel pharmaceutical preparation, relates to humanization tumor immune cell cytokines and in particular relates to a humanization tumor immune cell cytokines TNTIL2, as well as a preparation method and an application thereof. A humanization recombination antibody technology is utilized to prepare a humanization anti-tumor cell nucleus monoclonal antibody, and the humanization monoclonal antibody and interleukin-2 (IL-2) are in fusion expression so as to prepare a TNTIL2 fusion protein, wherein the IL-2 can specifically reach tumor parts, and can be used for reversing the local immunosuppression state of tumors and activating part bodies and integral antitumor immunity. The prepared TNTIL2 can serve as an anti-tumor candidate pharmaceutical with a treatment effect on various solid tumors. The active peptide immune cell cytokines with an immune regulation function can be used for separate or auxiliary tumor treatment.

Description

technical field [0001] The invention belongs to the technical field of novel drug preparation and relates to humanized immune cytokines, in particular to humanized tumor immune cytokine TNTIL2 and its preparation method and application. Background technique [0002] At present, there are three main methods of clinical treatment of tumors: surgery, radiotherapy and chemotherapy. The traditional treatment plan still has the following defects: lack of specificity of treatment, inability to kill residual tumor cells and inability to control tumor metastasis and recurrence, and even radiotherapy and chemotherapy can lead to huge toxic and side effects, and eventually patients often die of tumors Adverse reactions such as metastasis, recurrence or infection caused by radiotherapy and chemotherapy. Therefore, the development of new methods of treating tumors, especially immunotherapy, has attracted more and more attention from relevant researchers. [0003] Studies have shown tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/85A61K38/20A61K47/48A61P35/00A61K47/42
Inventor 史训龙鞠佃文周佩冯美卿叶丽朱海燕李继扬黄海
Owner FUDAN UNIV
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