Humanized anti-ErbB2 antibodies and treatment with anti-ErbB2 antibodies
A humanized antibody, antibody technology, applied in the direction of antibodies, anti-tumor drugs, chemical instruments and methods, etc.
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[0260] Controlled release formulations can also be prepared. Suitable examples of controlled-release preparations include semi-permeable matrices of solid hydrophobic polymers containing antibodies, which matrices are shaped articles such as films or microcapsules. Examples of controlled-release preparations include polyesters, hydrogels (such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactide (US Patent 3,773,919), L-glutamic acid and gamma ethyl -L-glutamate copolymer, non-degradable ethylene ethyl acetate, degradable lactic acid-glycolic acid copolymer such as LUPRONDEPOT TM (Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
[0261] The preparation for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filter.
[0262] V. Treatment with anti-ErbB2 antibodies
[0263] According to the present invention, anti-ErbB2 antibodies...
Embodiment 1
[0323] Preparation and identification of monoclonal antibody 2C4
[0324]According to the method described in Fendly et al., Cancer Research 50: 1550-1558 (1990), murine monoclonal antibodies 2C4, 7F3, and 4D5 that specifically bind to the extracellular domain of ErbB2 were prepared. In short, NIH 3T3 / HER2-3400 cells (about expression 1×10 5 ErbB2 molecule / cell) and used it to immunize BALB / c mice. In 0, 2, 5 and 7 weeks, mice were injected intraperitoneally with 10 7 Cells (in 0.5ml PBS). Give a specific antiserum (which can be combined with 32 P-labeled ErbB2 immunoprecipitation) mice were intraperitoneally injected with wheat germ agglutinin-Sepharose (WGA) purified ErbB2 membrane extract. Then 0.1ml of ErbB2 preparation was injected intravenously, and the spleen cells were fused with the mouse myeloma cell line X63-Ag8.653.
[0325] The ErbB2 binding of the supernatant of hybridoma cells was screened by ELISA and radioimmunoprecipitation.
[0326] A competitive binding assay (...
Embodiment 2
[0336] Monoclonal antibody 2C4 blocks the HRG-dependent binding of ErbB2 to ErbB3
[0337] Co-immunoprecipitation was used to detect the ability of ErbB3 to bind ErbB2. 1.0×10 6 MCF7 or SK-BR-3 cells were seeded in a 6-well tissue culture plate and allowed to adhere overnight. The culture medium was 50:50 DMEM / Ham's F12 containing 10% fetal bovine serum (FBS) and 10mM HEPES (pH 7.2) Base (growth medium). Before the start of the experiment, the cells were starved and cultured in serum-free growth medium for 2 hours.
[0338]Simply wash the cells with PBS, and then use 100 nM of the indicated antibody (diluted in 0.2% w / v bovine serum albumin (BSA) RPMI medium (pH 7.2) (binding buffer) containing 10 mM HEPES) or just The cells were incubated with binding buffer (control). After 1 hour at room temperature, HRG was added to half of the wells to a final concentration of 5nM(+). Add a similar volume of binding buffer (-) to the other wells. Continue to incubate for approximately 10 minu...
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