The 67 kilodalton mature laminin receptor protein (LRP) is found on the surface of both normal and malignant cells, though it is over-expressed on most cancers. The related 37 kilodalton Oncofetal Antigen – immature laminin receptor (OFA/iLRP), in contrast, is over-expressed on the cell surface during fetal development and on most hematopoietic and solid tumor cells but not expressed on the normal counterparts.
At Benovus Bio, we have taken advantage of this fact to create a family of proprietary and patent protected mouse monoclonal antibodies that specifically recognize the 37 kilodalton OFA/iLRP but do not cross-react with the related 67 kilodalton mature LRP. These antibodies provide the basis for unique diagnostic and therapeutic approaches with various malignancies.
Click to Download a PDF of the Benovus Mab Manuscript Published in Cancer Biology & Therapy.
Characteristics of this family of monoclonal antibodies that make them of value include the following:
- High specificity for tumor cells: Immunohistochemical and confocal immunofluorescence analysis shows immunoreactivity with most tumor-containing tissue sections and tumor-derived cell lines but essentially no binding to normal tissue sections as well as epithelial cells or fibroblasts derived from normal human tissue. (Figure 1)
- High affinity: By Biacore analysis, antibodies bind to their respective recombinant OFA/iLRP epitopes from the parent molecule with subnanomolar KDs.
- Growth inhibitory activity for tumor cells: The antibodies inhibit tumor cell proliferation in vitro, and significantly reduce tumor cell burden in syngeneic mouse tumor models. (Figure 2)
- Internalization: The antibodies can be efficiently internalized following tumor cell binding. (Figure 3)
Heavy and light chain sequencing has been accomplished on these antibodies and clones of antibody producing cells are stored at ATCC.
Figure 1 Specificity for Tumor Cells (above): Specificity for tumor cells is demonstrated by immunofluorescence staining and examination by confocal microscopy. All of the antibodies that recognize only 37 kilodalton OFA/iLRP (lower band in western blot) bind tumor cells but not normal cells. A control antibody that binds both 37 kilodalton OFA/iLRP and 67 kilodalton mature LRP (upper band in western blot) reacts with normal cells as well as tumor cells.
Figure 2 Tumor Cell Inhibition (left):
- Left panel: Quantitative data.
- Upper right panel: Lungs from control mice showing many tumors.
- Right middle and lower panels: lungs from antibody treated mice.
- There are fewer lung tumors in the antibody treated mice, and in those mice where lung tumors are still present, the tumors are smaller.
Figure 3 Internalization (above): A number of approaches demonstrate capacity for antibody internalization. A: Assessment of internalization by confocal immunofluorescence microscopy with K562 (human AML cells). Left; Two hours at 4oC; Right; 30 minutes at 37oC. Co-localization in lysosomes with anti-LAMP-1 (orange color) is indicated by yellow. B. Assessment of BV-15 internalization by flow cytometry after exposure of K562 cells to BV-15 covalently linked to pHrodo®-green pH-sensitive dye for the indicated times. An approximately 4.2-fold increase in fluorescence signal was seen with the specific antibody as compared to control (IgG2a). C: Assessment BV-06, BV-12, BV-15 and BV-27 by K562 cells. Confocal immuno-fluorescence images of K562 cells stained with IgG2a or the specific antibody (both linked to pHrodo® - red). Left; 15 minutes at 37oC; Right; 90 minutes at 37oC.