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Human melanin tumour monoclonal antibody, and its preparing method and use

A monoclonal antibody, melanoma technology, applied in the direction of antibodies, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-tumor drugs, etc. and the problem of unsatisfactory neutralization ability

Inactive Publication Date: 2005-05-04
SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the SM5-1 antibody is a mouse-derived antibody and can only be used for disease detection, not directly as a drug
[0009] It can be seen that although many monoclonal antibodies against melanoma have been established at home and abroad, the specificity and neutralization ability of these monoclonal antibodies are not ideal.

Method used

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  • Human melanin tumour monoclonal antibody, and its preparing method and use

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Experimental program
Comparison scheme
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Embodiment 1

[0046] Screening of Human Melanoma Antigens

[0047] 1. Construction of cDNA library of melanoma cell line A2058

[0048] Total RNA was extracted from human melanoma A2058 cells (ATCC, NO.CRL11147) with Trizol reagent, mRNA was separated by Oligo (T) fiber column chromatography, and the extracted mRNA was reverse-transcribed into cDNA with random primers, filled with BstXI The linkers at the restriction sites were connected, and cloned into the mammalian transient expression vector pCDM8 (Invitrogen) after digestion with BstXI, and transformed into Escherichia coli MC1061 / P3 (purchased from Invitrogen Company) to form a cDNA library.

[0049] 2. Library expression and screening.

[0050] Spread COS-7 cells (Invitrogen) into 20 10cm petri dishes, use the cDNA library constructed above to transfect according to the lipofectin method, and after 12 hours, the cells were digested with trypsin and respread in new petri dishes, transfected After staining for 72 ...

Embodiment 2

[0058] Screening the Variable Region Gene of Human Anti-Melanoma Monoclonal Antibody hSM5-1 from Human Antibody Library

[0059] According to Marks et al. J. Mol. Biol., 222, 581-597; Hoogenboom and Winte, J. Mol. Biol., 227, 381-388; Haidaris CG et al., J Immunol Methods. 2001 Nov 1; 257 (1- 2): 185-202; Griffiths, A.D. et al. EMBO J., 13, 3245-3260 (1994); Nissim, A. et al. The method described in EMBO J., 13, 692-698 (1994) to construct a human antibody library .

[0060] Add 1 ml of the revived antibody library strain to 14 ml of fresh LB medium, and culture in a 50 ml Erlenmeyer flask at 37°C for 16 hours.

[0061] Centrifuge at a high speed of 12000rpm for 10 minutes, transfer the supernatant to a sterile 50ml centrifuge tube, and save it for future use. Its titer should be 2×10 11 above. A 25 ml cell culture flask was coated with the antigen A220 purified in Example 1. Add no less than 3×10 10 Phage particles were incubated at 37°C for 1 hour. Then, the liqu...

Embodiment 3

[0068] Expression of Human Anti-Melanoma Monoclonal Antibody

[0069] 1. Construction of expression vector

[0070] The strains of the above positive clones were amplified in 100 ml of LB medium, and the plasmid DNA was purified with a plasmid DNA extraction and purification kit from Promega Company.

[0071] After digesting the above plasmid DNA with XbaI and NheI, separate the digested fragments on 1.5% agarose gel electrophoresis, take about 350 bp band for gel recovery, and the obtained fragment is the light chain variable region coding sequence.

[0072] The above plasmid DNA was digested with HindIII and Bsi WI, and the digested fragments were separated on 1.5% agarose gel electrophoresis, and a band of about 320 bp was taken for gel recovery, and the obtained fragment was the heavy chain variable region coding sequence.

[0073] Then first insert the above-mentioned light chain variable region coding sequence into the expression vector pMG18-3K (see DEVEL...

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Abstract

The present invention discloses a novel human melanin tumor monoclone antibody, separated nucleic acid molecule coding said antibody and its application, and described monoclone antibody preparation method.

Description

technical field [0001] The present invention relates to human melanoma monoclonal antibody, its preparation method and application. Background technique [0002] Malignant melanoma is a highly malignant tumor that occurs in the head and neck, accounting for about 20% of the whole body, and mainly occurs in the skin, cavity, and sinus mucosa. Human malignant melanomas often begin as harmful nevi that, stimulated by radiation, grow to form destructive metastatic melanomas. Melanoma is very easy to spread to the whole body, and it is difficult to control clinically. Chemotherapy and radiation therapy usually don't work against melanoma. [0003] Currently, the commonly used antibodies for immunohistochemical detection of melanoma are HMB-45, anti-S-100 and NKI / C3 (see Smoller BR, PATHOLOGY: State of the Art Reviews, 2: 371-383, 1994). [0004] HMB-45 is a known anti-human melanocyte monoclonal antibody, which can recognize pro-melanosomal globulin...

Claims

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Application Information

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IPC IPC(8): A61K39/395A61P35/00C07K16/30C12N15/11C12N15/63C12P21/08
Inventor 马菁王皓刘庆法
Owner SUNSHINE GUOJIAN PHARMA (SHANGHAI) CO LTD
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