Preparation method and application of tumor tissue complete antigen

A tumor tissue and pan-antigen technology, applied in the field of tumor tissue pan-antigen preparation, can solve the problems of DC vaccine containing a lot of cell debris, affecting DC activity, unable to inactivate protease, etc.

Active Publication Date: 2010-06-09
南京海鲸药业股份有限公司
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AI Technical Summary

Problems solved by technology

[0018] Vaccines prepared by shocking DC with tumor freeze-thawed antigens can produce better anti-tumor effects. The advantage is that there is no need to identify tumor-specific antigens or purify specific antigenic proteins. It is easy to prepare and can be applied to most patients. The disadvantage is that freeze-thawed antigens Using tumor tissue or cell freeze-thawed supernatant, most of the membrane antigens cannot be broken down and removed, so the freeze-thawed antigens only contain intracytoplasmic antigens, and the antigenicity is reduced
If all freeze-thaw products are used, the freeze-thaw products are not uniform, most of the membrane antigens are too large to be processed by endocytosis, and the prepared DC vaccine contains too much cell debris and canno

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  • Preparation method and application of tumor tissue complete antigen

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preparation example Construction

[0058] The preparation method of the present invention undergoes water vapor treatment to soften the tissue, grinds and breaks the cells through a homogenizer, and better obtains uniform and tiny antigenic substances. On the other hand, high-pressure steam treatment inactivates proteases to avoid degradation of tumors after cell homogenate is broken. antigen. Steam treatment can be performed using methods well known in the art, such as but not limited to high-pressure steam inactivation, atmospheric pressure steam treatment; preferably at a pressure of 0.05-0.3 mPa, more preferably at a pressure of 0.1-0.2 mPa.

[0059] The preparation method of the tumor vaccine provided by the present invention is applicable to the preparation of DC vaccines for various solid tumors, and the tumor is selected from lymphoma, myeloma, kidney cancer, prostate cancer, malignant melanoma, breast cancer, or colon cancer, etc. solid tumors. ; Lymphoma is preferred.

[0060] In a preferred example...

Embodiment 1

[0085] Preparation of Lymphoma DC Vaccine

[0086] (1) Extraction of whole antigen from lymphoma tissue

[0087] 1. Obtain fresh lymphoma biopsy tissue, cut 0.2g of tumor tissue, and cut into 1-3mm thick slices;

[0088] 2. Put it in a high-pressure steam sterilizer for 15 pounds and 15 minutes;

[0089] 3. Homogenize the treated tissue slices in a sterile glass homogenizer until all of them are homogeneous chylus liquid;

[0090] 4. Add 2ml of sterile normal saline, pipette gently several times to dissolve the whole antigen extracted from sterile lymphoma tissue;

[0091] 5. After aliquoting, store at -20°C for later use.

[0092] (2) Culture of immature DC in vitro

[0093] 1. The above-mentioned lymphoma patients were separated by a blood cell separator for 4 cycles, and 1-5×10 9 Mononuclear cell liquid (100-200ml);

[0094] 2. Slowly add the collected mononuclear cell fluid (20ml) to several 50ml Falcon centrifuge tubes pre-added with 20ml of lymphocyte separation me...

Embodiment 2

[0105] DC Vaccine Treats B-cell Lymphoma in Mice

[0106] 1. Preparation of tumor antigen: select mouse lymphoma cell line A20 cells to resuspend with normal saline (1×107 / ml), inject 100 μl subcutaneously into the mouse, cut out 0.2 g of tumor tissue after 3 weeks, follow the steps of Example 1 Prepare tumor whole antigen.

[0107] The control is tumor freeze-thaw antigen, and the preparation method is to cut out 0.2 g of tumor tissue, grind it through a 100-mesh steel mesh, collect 2 ml of the entire grinding liquid, put it into a 5 ml sterile cryopreservation tube, and quickly immerse the cryopreservation tube in liquid nitrogen for quick freezing, 10 After 10 minutes, take it out and put it in room temperature to thaw; freeze and thaw repeatedly 5 times, centrifuge at 3000×g for 30 minutes, collect the supernatant, and filter and sterilize it through a 0.22 μm disposable sterile filter, which is the tumor freeze-thaw antigen.

[0108] 2. Mouse DC culture: take BALB / c mous...

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Abstract

The invention discloses a preparation method and application of a tumor tissue complete antigen. The method comprises the following steps: (1) placing the lobulated fresh bioptic tumor tissue into water steam to inactivate the protease in the cytoplasm for 10-30min to obtain the tumor tissue which inactivates the protease in the cytoplasm; (2) homogenizing the tumor tissue obtained in the step (1) to obtain chyliform liquid; and (3) mixing the chyliform liquid and normal saline to obtain the tumor tissue complete antigen. The invention also discloses a method for obtaining tumor vaccines by using the tumor tissue complete antigen to pulse the dendritic cell.

Description

technical field [0001] The invention relates to the field of tumor immunotherapy, in particular to a preparation method and use of tumor tissue whole antigens. Background technique [0002] Dendritic cells (DC) are antigen-presenting cells with the most powerful function of activating tumor-specific CTL in vivo. At present, a sufficient amount of patient's own DC can be obtained by in vitro isolation or culture, and can be prepared into a tumor vaccine after being loaded with tumor antigens in vitro, and can induce strong anti-tumor immune function in vivo after reinfusion into the patient. [0003] DC vaccine was first used in the clinical treatment of malignant lymphoma. Hue et al. reported for the first time that DC vaccine was used to treat 4 patients with follicular B-cell lymphoma, and achieved good curative effect; in 2002, Timmerman et al. reported that DC was used in clinical treatment of 35 patients with B-cell lymphoma, and the results also achieved certain curat...

Claims

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Application Information

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IPC IPC(8): C07K1/14C07K14/47A61K39/00A61P35/00
Inventor 朱学军何龙胡海鸽相文杰
Owner 南京海鲸药业股份有限公司
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