Antibody-Drug Conjugates Targeted At Human Aspartyl (Asparaginyl) Beta-Hydroxylase

Antibody-drug conjugates (ADCs) consist of an antibody that is specific for a disease antigen linked to one or more drug molecules. The goal is to utilize the antibody as a means of specific delivery of the drug to diseased cells. Theoretically this should decrease systemic exposure to the drug and allow for use of a lower dose. In cancer, the approach employs an antibody targeted at a tumor-associated antigen and a cytotoxic or cytostatic drug. To date there have been three ADCs approved by the US FDA for cancer treatment, and many more are now in the development stage. The efficacy of any ADC is dependent on three factors: the antibody, the attached drug, and the linker used to connect them. While each of these factors is of import, with the growth of interest in ADCs for cancer therapy, the use of specific drugs and linkers has become more routine. Thus, the specificities of the antibody and target antigen are now perhaps the primary obstacles towards the development of novel ADCs. Human aspartyl (asparaginyl) β-hydroxylase (HAAH) is an embryonic/developmental protein, which is downregulated in normal cells after birth but overexpressed on the surface of many malignant cells. It has been demonstrated to be sufficient to induce cancer cell proliferation, motility, and invasiveness. The enzyme hydroxylates important residues in the NOTCH protein altering signal transduction pathways that lead to increased growth and metastatic potential. Increased levels of HAAH have been detected by immunohistochemistry in a diverse array of solid and hematologic cancers (n u003e 20), including liver, bile duct, brain, breast, colon, prostate, ovary, pancreas, and lung cancers as well as various leukemias. HAAH is not found in measurable quantities in normal tissue (n u003e 500), including normal adjacent tissue within cancer biopsy specimens or in benign proliferative disorders. We have developed a fully human antibody against HAAH, PAN-622,that displays exquisite specificity for cancer. Here we explore PAN-622 drug conjugates for ultimate use in treating both hematologic and solid tumors. PAN-622 was conjugated to three different drugs: a maytansinoid (DM1), monomethyl auristatin E (MMAE), and duocarmycin (DUO). DM1 was conjugated via a noncleavable thio-ether linker while MMAE and DUO were conjugated using valine-citruline containing linkers that are cleavable by cathepsin B in the endosomal compartment. Conjugation of drugs to the PAN-622 antibody had little effect on the affinity of the antibody for HAAH. Binding affinities were determined by immunoassay on fixed cancer cells (H460, human lung cancer line) and were ~0.1, 0.2, and 0.5nM for PAN-622-DM1, PAN-622-MMAE, and PAN-622-DUO, respectively. The affinity of PAN-622 for HAAH as expressed on live cells has previously been shown to be ~1nM. Efficacies of the three ADCs for killing of H460 cells were determined using an MTS assay. The PAN-622-DM1 had an EC50 of ~15nM. The EC50 for PAN-622-MMAE was ~60nM, and that for PAN-622-DUO was ~300nM. Importantly, both unconjugated PAN-622 and a nonrelevant antibody conjugated to MMAE did not display any killing of the H460 cell line. Efficacy was also measured on a representative hematologic cancer line, MOLM-14 (acute myelogenous leukemia) where EC50s were in the 20-50nM range for all three ADCs. This work serves as a proof of concept, laying the groundwork for further development of HAAH-targeted ADCs. Citation Format: Kanam Malhotra, Susan Walker, Chen Fang, Karamveer Birthare, Michael S. Lebowitz, Steven Fuller, Muctarr Sesay, Hossein A. Ghanbari. Antibody-drug conjugates targeted at human aspartyl (asparaginyl) β-hydroxylase [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B106.