Inhibition of macrophage-stimulating protein receptor (RON) and methods of treatment thereof
A protein and tumor cell technology, applied in immunoglobulin, antibody medical components, chemical instruments and methods, etc.
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Embodiment 1
[0226] Example 1: Production of Anti-RON Antibodies
[0227] Human anti-RON monoclonal antibodies (referred to herein as RON6 and RON8) were produced by standard hybridoma technology (Harlow & Lane eds., Antibodies: A Laboratory Manual , Cold Spring Harbor, 211-213 (1998), which is incorporated herein by reference), using HuMAb mice (Medarex, San Jose, Calif.), which produce human immunoglobulin gamma heavy and kappa light chains. HuMAb mice were immunized subcutaneously (s.c.) with RON extracellular domain fragments in complete Freund's adjuvant, RE7 cells and MDCK cells overexpressing human RON receptor. Animals were boosted intraperitoneally (i.p.) three times with the same RON protein in Freund's incomplete adjuvant prior to fusion. Animals were rested for one month before receiving a final intraperitoneal boost of 25 micrograms of RON protein in phosphate buffered saline (PBS). Four days later, splenocytes were harvested from the immunized mice and fused with P3-X63-Ag...
Embodiment 2
[0238] Example 2: The anti-RON antibody from Example 1 binds to RON and inhibits the binding of RON to its ligand (MSP)
[0239] Combined with ELISA: On days 10-12 after fusion, hybridomas were screened for antibody production and culture supernatants were screened for specific binding activity to rhRON protein in ELISA-based binding and blocking assays. Maxi-sorp 96-well microtiter plates (Nunc) were coated with (1 μg / ml×100 μl) rhRON protein (R&D Systems) for 1.5 hours at room temperature. After washing the wells, they were blocked with 3% PBS / milk. Then, anti-RON antibody derived from hybridoma supernatant was added to the coated wells and allowed to incubate at room temperature for 1.5 hours. After several washes, a 1:1000 dilution of anti-human IgG-HRP conjugated antibody was added to the plate for 1.5 hours at room temperature to detect positive binding. Positive hybridomas were subcloned three times by limiting dilution cultures to establish monoclonal hybridomas. ...
Embodiment 3
[0251] Example 3: Demonstration of tumor suppression in a human tumor xenograft model.
[0252] The in vivo effectiveness of RON6 and RON8 antibodies in inhibiting tumor growth was demonstrated in four different tumor cells by using the mouse xenograft method described in Example 2 above. Tumor cells are as follows. H-292 cells, derived from lung tumors, were obtained from the American Type Culture Collection (Manassas, VA). HT-29 cells, derived from colon tumors, were obtained from the American Type Culture Collection (Manassas, VA). BxPC3 cells, derived from pancreatic tumors, were obtained from the American Type Culture Collection (Manassas, VA). JIMT cells, derived from breast cancer, were obtained from the American Type Culture Collection (Manassas, VA).
[0253] A total of 24 mice were injected with H-292 cells using the xenograft method described above. Twelve mice were treated with 60 mg / kg of RON6 antibody and 12 were treated with saline as a control. Such as F...
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