Cytotoxicity mediation of cells evidencing surface expression of cd44

A cytotoxic, mediated technology, applied in the field of diagnosis and treatment of cancer diseases, can solve the problems affecting the growth and metastasis of tumors in rats

Inactive Publication Date: 2007-01-24
ARIUS RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, although an antibody specific for a variant of rat CD44 was prepared and shown to affect the growth and metastasis of rat tumors, there is no evidence that this antibody has an effect on human tumors

Method used

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  • Cytotoxicity mediation of cells evidencing surface expression of cd44
  • Cytotoxicity mediation of cells evidencing surface expression of cd44
  • Cytotoxicity mediation of cells evidencing surface expression of cd44

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Identification of binding proteins by Western blotting

[0083] In order to recognize the antigen recognized by the H460-16-2 antibody, the cell membrane expressing the antigen was subjected to gel electrophoresis and transferred to the membrane. Use Western blotting to determine the protein detected by the antibody.

[0084] 1. Cell membrane preparation

[0085] Previous work confirmed the binding of H460-16-2 to breast cancer cell lines MDA-MB-231 (MB-231) and MDA-MB-468 (MB-468) through FACS. Therefore, membrane preparation from these two cell lines was used for antigen recognition. The total cell membrane was prepared from the confluent culture of MB-231 or MB-468 breast cancer cells. Remove the medium from the flask and wash the cells 3 times with PBS. After the last wash, the cells were digested with digestion buffer (Gibco-BRL; Grand Island, NY) at 37°C for 5 minutes. The cells were collected and centrifuged at 1200 rpm for 10 minutes at 4°C. After centrifugation, th...

Embodiment 2

[0095] Determine the glycosylation of the antigen bound to H460-16-2

[0096] In order to determine whether the antigen recognized by antibody H460-16-2 is a glycoprotein, according to the manufacturer's manual (DeglycoPro deglycosylation kit; Prozyme, San Leandro, CA), MB-231 membrane and PNGase F, endo-o -Glucosidase and sialidase A are placed together at room temperature or 37°C for 24 hours. The membrane was separated by 1D polyacrylamide gel electrophoresis as described above and then Western blotting with H460-16-2 as described above.

[0097] Figure 5 The results of Western blotting are shown in. In the MB-468 membrane without glycosidase treatment, H460-16-2 recognized the expected 85-95kD band ( Figure 5 , Panel A, Lane 2). In the membrane treated with glycosidase at 25°C, the band has a clear shift to low molecular weight (lane 4). In membranes treated with glycosidase at 37°C, the binding of H460-16-2 was eliminated (lane 3). In order to determine the completeness of...

Embodiment 3

[0100] Recognition of H460-16-2 bound antigen

[0101] 1. Immunoprecipitation

[0102] Wash 1 mL of Protein GDynabeads (DYNAL) 3 times with 0.1M sodium phosphate buffer of pH 6.0. 2500μg of H460-16-2 was added to the washed beads with a total volume of 500μL. The mixture was left and mixed gently for 1 hour. To remove unbound antibodies, the H460-16-2 coated beads were washed 3 times with 2.5 mL of 0.1 M sodium phosphate buffer containing 0.1% Tween-20, pH 7.4. The H460-16-2 coated beads were washed twice with 5 mL of 0.2 M triethanolamine pH 8.2, and then 5 mL was added. In the presence of 5 mL of triethanolamine containing 20 mM dimethyl pimelimidate at pH 8.2, H460-16-2 was chemically cross-linked with the beads by gentle mixing for 30 minutes. The reaction was stopped by adding 5 mL of 50 mM Tris, pH 7.5. After standing for 15 minutes, the H460-16-2 cross-linked beads were washed 3 times with PBS containing 0.1% Tween-20. The H460-16-2 cross-linked beads were pre-eluted by pla...

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Abstract

The use of anti-CD44 monoclonal antibody H460-16-2 for the diagnosis and treatment of cancerous tumors, and binding assays which determine the presence of cells which express a CD44 antigenic moiety which specifically binds the monoclonal antibody H460-16-2. The monoclonal antibody is optionally used in combination with one or more chemotherapeutic agents, as a means for initiating the cytotoxic response to tumors.

Description

Technical field [0001] The present invention relates to the diagnosis and treatment of cancer diseases, in particular to the mediation of cytotoxicity of tumor cells; and most particularly to the application of cancer disease ameliorating antibody (CDMAB) as a means of initiating a cytotoxic response. Combine with one or more chemotherapeutic agents. The present invention further relates to the combined analysis of CDMAB using the present invention. Background technique [0002] The preparation of monoclonal antibodies against human white blood cells led to the discovery of the CD44 antigen; single-chain hyaluronic acid (HA) binding glycoproteins are expressed on a wide range of normal tissues and all types of hematopoietic cells. It was initially related to the activation and homing of lymphocytes. Currently, its putative physiological effects also include activation of inflammatory genes, regulation of cell cycle, induction of cell proliferation, induction of differentiation an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395A61K47/48C12Q1/04A61K49/00C07K16/00C07K16/28C07K16/30G01N33/569G01N33/574
CPCC07K16/00G01N33/57415G01N33/57484G01N33/56972G01N33/57419A61K49/0041G01N33/57423C07K16/2884G01N33/57449A61K2039/505C07K16/3015A61K49/0058C07K16/30A61P35/00
Inventor 戴维·S·F·杨苏珊·E·哈恩海伦·P·芬德利
Owner ARIUS RES
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