DEVELOPING ANTAGONISTIC CYTOKINE STEM CELL FACTOR (SCF) VARIANTS FOR c-KIT TARGETING AND CANCER THERAPY

The c-Kit receptor tyrosine kinase, which promotes survival, migration and capillary tube formation of endothelial cells, has an important role in tumor angiogenesis and metastasis. Activation of c-Kit depends on its dimerization, which is facilitated via binding to its native ligand, stem cell factor (SCF) in its dimeric form. In order to develop a c-Kit antagonist, a monomeric SCF variant (SCF_M) with two point mutations, namely V49L and F63L, was generated albeit having reduced affinity towards c-Kit. We hypothesize that improving the affinity of SCF_M to c-Kit would result in enhanced biological activity of the antagonist. For that, affinity maturation by yeast surface display and fluorescence-activated cell sorting (FACS) screening was utilized in order to select SCF_M variants with increased affinity to c-Kit. Three high affinity SCF_M variants were purified and their structure, thermostability and binding to surface immobilized and cellular c-Kit were determined. These monomeric SCF_M variants preserved their stability and showed improved affinity to c-kit as compared to SCF_M, but since the affinity is still lower than SCF_WT, we are currently generating second-generation libraries based on our most potent SCF variant to identify better c-Kit binders. For that, two libraries that are based on combinatorial and semi-rational design are generated. In the first library, the whole SCF gene is randomized while in the second library only positions that can tolerate randomization and that are lying in the binding interface are randomized. Both libraries are currently being screened to isolate high affinity c-Kit binders with potential antagonistic activity that would later be tested for their ability to inhibit tumor angiogenesis and metastasis in cell-based assays and pre-clinical models of cancer.

*Tal Tilayov and Tal Hingly contributed equally to this work.