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Preparation of immunoliposome of CD19 monoclonal antibody and application

A monoclonal antibody and immunoliposome technology, applied in the biological field, can solve the problems of weakening the therapeutic advantages of NCTD on LSCs and their daughter cells, and the toxic and side effects of the digestive system and urinary system

Active Publication Date: 2009-12-30
杭州永申生物科技有限公司
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Problems solved by technology

However, NCTD also induces the apoptosis of normal hematopoietic stem cells (HSCs) through the HLF / SLUG axis; and easily causes toxic side effects in the digestive system and urinary system; this weakens the effect of NCTD on LSCs and their daughter cells to some extent. The therapeutic advantage of

Method used

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  • Preparation of immunoliposome of CD19 monoclonal antibody and application
  • Preparation of immunoliposome of CD19 monoclonal antibody and application
  • Preparation of immunoliposome of CD19 monoclonal antibody and application

Examples

Experimental program
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Effect test

Embodiment 1

[0029] An anti-human CD19 monoclonal antibody immunoliposome provided by the invention is prepared by the following steps:

[0030] (1) Development of ZCH-4-2E8 monoclonal antibody: prepared according to the method disclosed in Example 1-2 of Patent No.: ZL200610052289.7. Basically follow the classical method of mouse-mouse hybridoma reported by Koller & Milstein [1] . With acute B lymphoblastic leukemia cells as immunogen, 10 7 Leukemic cells were injected intraperitoneally into 8-week-old female Balb / C mice 4 times a week, and on the 4th day after the last injection, the mice were killed by dislocation. Myeloma cell line NS-1 (purchased from ATCC, USA) cells were mixed at a ratio of 6:1, and 50% polyethylene glycol (PEG, Sigma, USA, molecular weight 3350 Daltons) solution was used as a fusion medium for cell fusion. Selective culture was carried out in a 96-well plate (Falcon Company, USA). On the 9th to 20th day after fusion, the culture supernatant was screened by indir...

Embodiment 22E8

[0036] Evaluation and targeted killing effect of embodiment 22E8-SL-NCTD

[0037] (1) SDS-PAGE and flow cytometry proved that 2E8 was successfully connected with SL-NCTD.

[0038] Figure 4 : SDS-PAGE proved that 2E8-SL-NCTD remained intact in each step of the preparation process (that is, lanes 2, 3, and 5 had complete heavy and light chains), of which lane 1: protein molecular weight , Lane 2: Free 2E8, Lane 3: Linked to micelles (2E8-Mal-PEG-DSPE), Lane 4: Blank, Lane 5: 2E8 linked to SL-NCTD and purified by Sepharose CL-4B to remove free After 2E8, the resulting 2E8-SL-NCTD.

[0039] Figure 5 : Flow cytometry confirmed that 2E8 was successfully connected with liposomes. (A) After SL-PE was incubated with Nalm-6 cells, no fluorescence was detected on the cell surface. (B) After 2E8-SL-PE was incubated with Nalm-6 cells, green fluorescence (FITC) and red fluorescence (PE) could be detected simultaneously on the cell surface, and there was a one-to-one linear relationship...

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Abstract

The invention provides an immunoliposome of a CD19 monoclonal antibody. First, a film dispersion method is adopted to prepare the cubical space stable norcantharidin immunoliposome, wherein the mole ratio of soy bean lecithin, cholesterol, methoxyl-polyethylene glycol 2000-phosphatidyl ethanolamine is 2:1:0.01, the mass ratio of norcantharidin immunoliposome and soy bean lecithin is 1:20, and entrapment efficiency of high-performance liquid detection NCTD is 46.5+ / -2.21%. The research proves that 2E8-SL-PE can be combined with Nalm-6 and Raji cell specifically, the positive rates thereof are respectively 89.44% and 88.73%, the 2E8-SL-PE is almost not combined with Molt-3 and K562 cell with the positive rates being only 1.95% and 1.39%, which shows that the immunoliposome has obvious target recognition effect. The norcantharidin immunoliposome can be applied in preparing specific target killer B lymphocyte leukemia and stem cell drugs thereof.

Description

technical field [0001] The invention belongs to biotechnology, and mainly relates to the preparation and application of norcantharidin (NCTD) immunoliposome modified by CD19 monoclonal antibody (mAb)-4-2E8 and loaded with killing B-lineage leukemia stem cells. Background technique [0002] B lymphocytic leukemia / lymphoma is a common hematological malignancy, accounting for about 4%-5% of new tumor cases each year. Although patients with B lymphocytic leukemia respond well to initial chemotherapy, most cases are prone to relapse, especially adult cases, mainly because the remaining malignant B cells or tumor stem cells in the patient's body have not been completely eliminated. In addition, conventional chemotherapy drugs have no specific killing effect on tumor cells; they also have toxic and side effects on normal tissues. Therefore, finding a therapeutic method that can completely eradicate residual malignant B lymphocytes or B lymphocytic leukemia stem cells while reducin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/127A61K31/34A61K47/48A61K47/42A61P35/02A61K47/68A61K47/69
Inventor 汤永民张晶樱
Owner 杭州永申生物科技有限公司
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