Muc1* antibodies
a monoclonal antibody and muc1 technology, applied in the field of monoclonal antibodies to muc1 *, can solve the problems of reducing the overall yield of the desired antibody, patients rapidly succumbing to sepsis, and cell growth and overall yield reduction, so as to increase antibody production, enhance tumor formation, and stimulate stem-like cell growth.
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example 1
n of Anti-MUC1* Monoclonal Antibodies
[0437]Monoclonal antibodies were generated to MUC1* peptide using standard hybridoma generation and subsequent screening, which is familiar to those skilled in the art. A MUC1* peptide (GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA, SEQ ID NO:1) or peptide variant bearing a single amino acid substitution (GTINVHDVETQFNQYKTEAASPYNLTISDVSVSDVPFPFSAQSGA, SEQ ID NO:2) was synthesized with an additional cysteine residue at the Carboxy-terminal end to allow conjugation to KLH. The conjugated peptide was used to immunize mice. Supernatants from wells containing fused cells were screened for recognition of the MUC1* peptide by ELISA. Out of these, several clones were selected based on their high titer for their ability to recognize MUC1* peptide in ELISA binding assay. Results of such binding of supernatants from six (6) hybridoma clones are shown in FIG. 1. Clones were then tested by FACS (fluorescence activated cell sorting) for their ability to bind t...
example 2
of Binding of Anti-MUC1* Monoclonal Antibodies to MUC1* on the Cell Surface
[0438]Recognition of MUC1* on the cell surface was analyzed by surface staining of the cells using FACS. For MIN-C2 antibody 50 μl of a 10 μg / ml solution of purified antibody was bound individually to MUC1-negative HCT116 cells transfected with empty vector (HCT116-VEC8), or transfected with MUC1* expressing vector (HCT-MUC1*-10) and MUC1 positive ZR-75-1 cells at 4 degrees Celsius for 30 minutes. Cells were washed twice, and treated with 10 μg / ml anti-mouse-PE at 4 degrees Celsius for 30 minutes. Cells were washed twice, fixed in 2% formaldehyde in PBS, and analyzed using a BD FACS Cantoll flow cytometer (FIG. 2). A similar procedure was followed for MIN-E6 antibody with the only difference that 50 μl of undiluted supernanatnt from MIN-E6 hybridoma was used in place of purified antibody (FIG. 3).
example 3
* Monoclonal Antibodies (Bivalent) MIN-C2 and MIN-E6 Induce Proliferation of MUC1 Expressing Breast Cancer Cell Line
[0439]To measure the ability of anti-MUC1* monoclonal antibodies, MUC1 expressing breast cancer cell line T47D was used. 7,500 cells were plated per well of a 96 well plate in media containing 10% serum. The following day cells in three wells were trypsinized, resuspended in media and counted using a hemocytometer to obtain the zero day count. Media in the cells was changed to that containing 3% FBS and anti-MUC1* antibody was added in various concentrations. 48 hours later media was changed which was followed by a second addition of antibody. After another 48 hours the cells were counted and stimulation of cell growth estimated (FIGS. 4 and 5).
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