Anti human CD19 mouse immune globulin variable zone gene and uses

An immunoglobulin, variable technology, applied in the biological field, which can solve problems such as weak antigenicity

Active Publication Date: 2006-12-27
杭州永申生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is known that the immunogenicity of antibodies is mainly located in the constant region (Fc) of the...

Method used

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  • Anti human CD19 mouse immune globulin variable zone gene and uses
  • Anti human CD19 mouse immune globulin variable zone gene and uses
  • Anti human CD19 mouse immune globulin variable zone gene and uses

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Experimental program
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Effect test

Embodiment 1

[0018] Nucleotide sequence and amino acid sequence of two genes of the present invention:

[0019] 1. Anti-human CD19 mouse immunoglobulin heavy chain variable region gene (mAb ZCH-4-2E8VH 2E8 ) nucleotide sequence (SEQ ID NO 1) and amino acid sequence (SEQ ID NO 3).

[0020] 2. Anti-human CD19 mouse immunoglobulin light chain variable region gene (antigen monoclonal antibody ZCH-4-2E8VL 2E8 ) nucleotide (SEQ ID NO 2) and amino acid sequence (SEQ ID NO 4).

Embodiment 2

[0022] The two anti-human CD19 mouse immunoglobulin variable region genes provided by the present invention are obtained through the following steps:

[0023]1. The development of ZCH-4-2E8 monoclonal antibody: basically according to the classical method of mouse-mouse hybridoma reported by Koller & Milstein, using children's acute undifferentiated lymphocytic leukemia (uALL) cells (the immunophenotype of which is HLA-DR+, CD19+, CD10-, CD33-, TdT+, CD7-, CD41a-) were used as immunogens, and 107 leukemia cells were injected intraperitoneally into 8-week-old female Balb / C mice, once a week, 4 times in total, at the 4th time On the 3rd day after the injection, the mice were killed by dislocation, and the splenocytes were aseptically collected and mixed with the mouse myeloma cell line NS-1 cells in the logarithmic growth phase at a ratio of 6:1, and mixed with 50% polyethylene glycol (PEG, Sigma, USA, molecular weight 3350 Daltons) solution was used as a fusion medium for cell f...

Embodiment 3

[0080] In order to confirm the potential clinical application prospect of 2E8 antibody, we coupled the purified 2E8 mouse-derived intact antibody with trihydroxyisoflavone (Genistein, Gen for short) to make the immunotoxin (2E8-Gen) of 2E8 antibody, and observed its effect on 2E8 antibody. Positive (acute B-lymphoblastic leukemia, Nalm-6) and negative (acute T-cell leukemia cell line Mplt-3 and myeloid leukemia cell line) cells were targeted for killing, and it was found that 2E8-Gen had no effect on Nalm-6 cells (B-cell leukemia / lymphoma cell line) has a strong selective killing effect. After incubation at 37°C for 48 hours, the cell survival rate of the 2E8-Gen group was only 5%, which was significantly higher than that of the control group, and the 2E8-Gen -Gen has no killing effect on 2E8-negative Molt-3 and K562 cells, indicating that 2E8-Gen has a good targeting effect on killing B-cell leukemia cells (see Figure 7 ), where PBS is phosphate buffered saline, Gen is free ...

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Abstract

This invention provides variable region gene of anti-human CD19 Mouse IgE ELISA, which has the nucleotide sequence of SEQ ID NO. 1 and SEQ ID NO. 2 as well as the amino acid sequence of SEQ ID NO. 3 and SEQ ID NO. 4. The mono-clone anuibody of this invention has the characteristic that identify B lymphocyte tumour cell membrane CD19 antigen but not identify other tissue and cell composition. The in vitro indicates that this antibody can selectively kill the B-ALL cell, so this kind of antibody has broad potential application prospect in curing B lymphocytes malignant tumor, including leukaemia, lymphoma. It can be applied to the medicine that curing B lymphocytes malignant tumor.

Description

technical field [0001] The invention belongs to biological technology, and mainly relates to the heavy chain and light chain variable region genes of mouse immunoglobulin, in particular to the variable region gene of anti-human CD19 mouse immunoglobulin, and the preparation of B lymphocyte malignant tumor targeted therapeutic drugs use. Background technique [0002] Hematologic tumors such as leukemia and lymphoma are stubborn diseases that seriously endanger human health. Chemotherapy (chemotherapy) is still the main method for treating hematological malignancies, but conventional chemotherapy has poor selectivity between tumor cells and normal cells. Therefore, the toxic and side effects are relatively large, and repeated non-radical doses of chemotherapy can easily induce drug resistance of tumor cells. In the end, many patients have severe toxic and side effects such as severe infection, bleeding, and important viscera due to recurrence of drug-resistant tumors or high-d...

Claims

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Application Information

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IPC IPC(8): C12N15/13C07K16/18A61K48/00A61K39/395A61P35/00
Inventor 汤永民
Owner 杭州永申生物科技有限公司
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