UC San Diego

LGR5 and LGR6 receptors are present at high levels on the surface of epithelial stem cells of the ovary and Fallopian tube epithelia and the cancers that arise from these tissues (referred to as ovarian carcinomas). RSPO1 is a ligand for these receptors and, when bound, it drives WNT signaling and proliferation of cancer stem cells (CSC). Based on the hypothesis that RSPO1 could be used to target cytotoxic drugs selectively to CSC, the Howell lab developed a drug that contained 2 binding domains and armed this with the cytotoxin monomethylaurostatin (R1FF-MMAE). Affinity of a ligand is a function of the number of binding sites and this thesis explored the question of whether the potency of R1FF-MMAE could be further enhanced by increasing the number of binding domains. A vector capable of expressing a protein containing 4 modified binding domains was constructed, and the FcST4-His protein was successfully produced in transiently transfected HEK293E cells and purified using nickel and ion exchange resins. Following arming with MMAE using the sortase reaction, this drug (FcST4-MMAE) was extensively characterized using gel electrophoresis, HPLC analysis on ion exchange and size exclusion columns, Western blot and cytotoxicity testing. The results disclosed that FcST4-MMAE could be purified to a grade sufficient for further development, and that it was remarkably stable at 4oC and in the presence of a low pH of 3.0. However, cytotoxicity testing disclosed that the increase in binding domains did not enhance potency, perhaps due to the increase in size of the ligand and changes in the structure mediated by inter-domain interactions that interfered with access to the LGR5/LGR6 receptors.