Cancerous disease modifying antibodies
一种抗体、单克隆抗体的技术，应用在结合分析领域，能够解决限制、知识匮乏等问题
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Embodiment 1
[0107] Hybridoma production - hybridoma cell line AR62A335.9
[0108] The hybridoma cell line AR62A335.9 was deposited on May 17, 2005 with the International Depository of Canada (IDAC) at Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, R3E 3R2, Canada, pursuant to the Budapest Treaty. Ministry of Microbiology Bureau, deposit number is 170505-05. Pursuant to 37 CFR 1.808, the depositor undertakes to completely remove all restrictions on public access to the deposited material upon grant of the patent. If the depositary cannot give a viable sample, then the deposited sample will be replaced.
[0109] To generate the anticancer antibody-producing hybridoma AR62A335.9, frozen single-cell suspensions of human colonic metastases (Genomics Collaborative, Cambridge, MA) metastasized to liver tumor tissue were prepared in PBS. IMMUNEASY TM (Qiagen, Venlo, Netherlands) adjuvant mixed gently and ready to use. 5-7 week old BALB / c mice were immunized by subcutaneous injectio...
Embodiment 2
[0116] in vitro binding
[0117] AR62A335.9 monoclonal antibody was produced by growing the hybridomas in CL-1000 flasks (BD Biosciences, Oakville, ON), harvested and reseeded twice a week. Standard antibody purification procedures were then performed using Protein G Sepharose 4 Fast Flow (Amersham Biosciences, Baie d'Urfe, QC). It is within the scope of the invention to utilize humanized, deimmunized, chimerized or murine monoclonal antibodies.
[0118] The relationship between AR62A335.9 and breast cancer (MDA-MB-231), colon cancer (DLD-1, Lovo, SW1116) and ovarian cancer (OVCAR-3) cell lines and those derived from skin (CCD) was evaluated by flow cytometry (FACS). -27sk) and lung (Hs888.Lu) non-cancer cell line binding. All cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA). By first using DPBS (without Ca ++ and Mg ++ ) to prepare the cells for FACS by washing the cell monolayer. Cells were then detached from cell culture plates a...
Embodiment 3
[0121] In vivo tumor assays with Lovo cells
[0122] Examples 1 and 2 demonstrate that AR62A335.9 has anticancer properties against human cancer cell lines and binds to colon cell types. refer to Figure 4 and 5 , 4-6 week old female SCID mice were implanted with 1 million human colon cancer cells (Lovo) subcutaneously injected in 100 microliters of saline at the scruff of the neck. Mice were randomly divided into two treatment groups, 5 in each group. On the first day after implantation, each group of animals was administered intraperitoneally with 300 microliters of AR62A335.9 test antibody (20 mg / kg) or buffer control obtained by diluting the stock concentrate containing 2.7 mM KCl , 1mM KH 2 PO 3 , 137mM NaCl and 20mM Na 2 HPO 4 . Subsequently, antibodies and control samples were administered weekly for 7 weeks in the same manner. Tumor growth was measured with calipers approximately every 7 days until week 8, or until individual animals reached the Canadian Counci...
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