The primary function of tissue factor (TF) resides in the vasculature as a cofactor of blood clotting; however, multiple solid tumors aberrantly express this transmembrane receptor on the cell surface. Here, we developed anti-TF antibody-drug conjugates (ADC) that did not interfere with the coagulation cascade and benchmarked them against previously developed anti-TF ADCs. After screening an affinity-matured antibody panel of diverse paratopes and affinities, we identified one primary paratope family that did not inhibit conversion of Factor X (FX) to activated Factor X (FXa) and did not affect conversion of prothrombin to thrombin. The rest of the antibody panel and previously developed anti-TF antibodies were found to perturb coagulation to varying degrees. To compare the anticancer activity of coagulation-inert and -inhibitory antibodies as ADCs, a selection of antibodies was conjugated to the prototypic cytotoxic agent monomethyl auristatin E (MMAE) through a protease-cleavable linker. The coagulation-inert and -inhibitory anti-TF ADCs both killed cancer cells effectively. Importantly, the coagulation-inert ADCs were as efficacious as tisotumab vedotin, a clinical stage ADC that affected blood clotting, including in patient-derived xenografts from three solid tumor indications with a need for new therapeutic treatments-squamous cell carcinoma of the head and neck (SCCHN), ovarian, and gastric adenocarcinoma. Furthermore, a subset of the anti-TF antibodies could also be considered for the treatment of other diseases associated with upregulation of membranous TF expression, such as macular degeneration. .
While pigs and rabbits are used as models for human immune diseases, FcγR binding is poorly characterized in both test species. To evaluate antibody binding to FcγRIIIA, a receptor involved in antibody-dependent cellular cytotoxicity, chimerized antibodies were generated by grafting the variable regions of a human IgG1 onto scaffolds from both species. The affinities of the parent and chimeric antibodies to the FcγRIIIA proteins from all three species were determined. While the human IgG1 and rabbit IgG had similar affinities for each FcγRIIIA with notable differences across species, pig IgG1 only bound pig FcγRIIIA with appreciable affinity. Also, the functional pig and rabbit proteins described here can be used in future experiments, such as pharmacology and mechanism of action studies.
<p>Supplementary Fig. S1. Binding characteristics of anti-TF antibodies and coagulation properties of previously described anti-TF antibodies; Supplementary Fig. S2. Epitope binning assay of anti-TF antibodies and internalization of anti-TF antibodies; Supplementary Fig. S3. Cell killing activity of anti-TF antibodies, internalization of anti-TF conjugates, and binding characteristics of anti-TF ADCs; Supplementary Fig. S4. Additional cell killing properties of anti-TF antibodies; Supplementary Fig. S5. Anti-TF ADCs do not trigger a G2/M arrest in HUVECs; Supplementary Fig. S6. Anti-TF antibodies attenuate or inhibit FVIIa-dependent Erk phosphorylation</p>
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