The membrane associated tetraspanin CD63 forms part of a ‘tetraspanin web’ on the cell surface and interacts with other members of the tetraspanin family, integrins, MMPs and other cancer-associated molecules. CD63 expression may be associated with both melanoma and breast cancer progression. Despite the initial speculation that CD63 may be the product of a tumor suppressor gene and its association with cancer progression, there have been no successful attempts to date to develop CD63 cancer therapeutics. Using the FunctionFIRST™ antibody generation platform, ARIUS has generated 3 antibodies in independent experiments which were subsequently shown to target the CD63 antigen. AR7BD-33-11A and AR1A245.6 were raised against different human breast cancer tissues. ARH460-22-1 was raised against a human lung cancer. The antibodies were screened on a panel of different cancer cell lines and selected based on in vitro functional activity against breast, melanoma and prostate cancers. A series of studies revealed that although the 3 antibodies target the same antigen, there are differences in both function and specificity which may be epitope dependent. All three antibodies were effective in both prophylactic and established MDA-MB-231 human breast cancer xenograft models, however the AR7BD-33-11A antibody was most potent, completely suppressing tumor formation in both models and extending tumor free survival beyond one year in the prophylactic model. AR7BD-33-11A suppressed growth in a prophylactic human A2058 melanoma model by 72% in comparison to controls (p=0.011, t-test), while ARH460-22-1 did not suppress tumor growth in this model. Immunohistochemistry revealed other differences. ARH460-22-1, AR1A245.6 and other anti-CD63 antibodies, RFAC4 and H5C6, bound to the majority of breast cancer tissues, as well as to multiple normal tissues, demonstating broad prevalence of those epitopes. AR7BD-33-11A demonstrated more restricted binding: 36 percent of breast tissue samples stained positively for the AR7BD-33-11A epitope, while there was no staining on the 10 normal tissues from breast cancer patients and expression of AR7BD-33-11A within patient samples appeared specific for malignant cells. The majority of normal human tissues failed to express the AR7BD-33-11A antigen, including the vital organs, such as the kidney, heart, and lung. The independent selection of three novel antibodies to CD63 in a functional screen demonstrates both the importance of this target, and the power of the FunctionFIRST™ technology.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]