For crystallization of Fab19CKITD4-5, crystallization buffer containing 13% PEG 3350, 0.5 M MgCl2, and 0.1 M Tris?HCl, pH 9.0, was mixed with equal volume (0.6 L) of protein solution (7 mg/mL). leukemia, melanoma, and other cancers. Although treatment of these cancers with tyrosine kinase inhibitors shows dramatic responses and durable disease control, drug resistance followed by clinical progression of disease eventually occurs in virtually all patients. In this report, we describe inhibitory KIT antibodies that SOS2 bind to the membrane-proximal Ig-like D4 of KIT with significant overlap with an epitope in D4 that mediates homotypic interactions essential for KIT activation. Crystal structures of the anti-KIT antibody in complex with KIT D4 and D5 allowed design of affinity-matured libraries that were used to isolate variants with increased Radequinil affinity and efficacy. Isolated antibodies showed KIT inhibition together with suppression of cell proliferation driven by ligand-stimulated WT or constitutively activated oncogenic KIT mutant. These antibodies represent a unique therapeutic approach and a step toward the development of naked or toxin-conjugated KIT antibodies for the treatment of KIT-driven cancers. The receptor tyrosine kinase (RTK) KIT is usually a transmembrane protein that plays crucial functions in mediating diverse cellular processes including cell differentiation, proliferation, and cell survival, among other activities. These processes occur through activation of KIT upon binding by stem cell factor (SCF), a ligand found in membrane-anchored and soluble forms (1, 2) in a variety of cell types, including hematopoietic stem cells, germ cells, vascular endothelial cells, and the mesenchymal cells with uniquely neuromuscular differentiation known as the interstitial cells of Cajal (3, 4). KIT belongs to the type III subfamily of RTKs (5), a family composed of an extracellular region that includes five Ig-like domains (designated D1CD5), a single transmembrane domain name (TM), a juxtamembrane region (JM), a tyrosine kinase domain name split by a kinase insert, and a C-terminal tail (6) (Fig. 1and is in the same scale as in is usually enlarged compared with images in and and Table S1). The overall structure of KITD4-5 bound to Fab19, is very similar to the structures of these two Ig-like domains observed previously as part of the structures of full-length extracellular region of KIT alone, or in complex with SCF [Protein Data Lender (PDB) ID codes 2EC8 and 2E9W; ref. 7]. Superposition of individual D4 and D5 from Fab19CKITD4-5 complex structure with corresponding domains of KIT ectodomain structure (PDB ID code 2EC8) revealed rmsd values of 0.65 ? for 96 and 59 C residues in Radequinil D4 and D5, respectively. The structure revealed Fab19 binding exclusively to D4 of KIT with a buried surface of 1 1,029 ?2 around the D4 side of the interface (Fig. 1and Table S2). Nearly the entire -sheet of D4 (one of two -linens in Ig-like domain name), including A, B, , and D, as well as the AA, AB, EF, and DE loops, was buried under the Fab19 surface (Fig. 1and Fig. S2). The majority of the contacts were made by the heavy chain of the Fab (800 ?2 vs. 283 ?2 for the light chain; Fig. 1and shows that the L1 loop of Fab79D moved toward the D4 domain name within the Fab79DCKITD4-5 complex structure, and, unlike Fab19, made contact with D of D4 (Fig. 3and Fig. S6); Arg31L and Asn32L of Fab79D were located within hydrogen bonding distance of the main chain of Pro363D4 and side chain of Glu360D4, respectively. This CDR L1 loop extension, so evident upon complex structure comparison, appears to be responsible for the increased binding affinity of Fab79D toward KIT D4. KTN37CMurine Anti-D4 mAb. As a positive control in our experiments, we used KTN37 mAb, a monoclonal antibody obtained by immunization of mice with the KITD4-5 fragment. It was shown that KTN37 IgG bound D4 of human KIT with high affinity, and was a very potent antagonist of the KIT receptor (as detailed later). As we were not able Radequinil to Radequinil obtain diffraction quality crystals of KTN37 in complex with KIT D4 and D5 fragment, molecular details of the complex could not be obtained. However, to shed light on the binding epitope of KTN37, we compared the KTN37 IgG binding to the ectodomain of KIT from different species (Fig. S7and and Table 1). Consistent with this, Fab12I appears to be more effective at KIT inhibition than Fab19 but weaker than Fab79D. The bivalent IgG format confers avidity effects to a Fab that are evident upon testing IgG KTN37, whose effectiveness at blocking KIT autophosphorylation could be seen even at 0.5 nM, compared with the 50 nM level required for Fab KTN37.