Abstract 2987: Generation and characterization of a panel of monoclonal antibodies against the tumor biomarker Thymidine Kinase 1 for research, clinical and therapeutic applications
Authors: Velazquez Edwin J. , Brindley Taylor D. , Shrestha Gajendra , Skabelund Rachel A. , Lee Corbin M. , Ewel Zachary D. , Bitter Eliza E. , Townsend Michelle H. , Bennion Kelsey B. , Ong Kai Li , Whitley Kiara V. , Robison Richard A. , Weber Scott K. , O'Neill Kim L. 
Published: August 2019

Abstract
Our research explores the potential of a panel of monoclonal antibodies targeting the tumor proliferation biomarker Thymidine Kinase 1 (TK1) for both clinical and therapeutic applications. Cancer biomarkers have become a critical component of precision medicine. TK1 is a well-known tumor proliferation biomarker that is released into the serum, is up-regulated in malignant tissues and can be found on the cell membrane of several cancer types. Notwithstanding the versatility of TK1 as a tumor biomarker, there are a limited number of available antibodies for the detection and quantification of TK1. Moreover, to the date TK1 antibody-based therapies are not being tested in the clinical setting. Thus, the generation of more sensitive TK1 antibodies could increase the availability, accuracy and options of current TK1-based diagnostics and antibody-based immuno-cell therapies. Six peptide sequences across the TK1 molecule were selected. The antibodies were generated using hybridoma technology. Seventeen clones were evaluated in ELISA with a calibration dose-response curve. The calibration curves showed R squares ranging from 0.9738-0.9980 with a 10-15 %CV. The limit of detection and quantification (LOQ, LOD) were obtained. The clones 3B2E11, 9C10, 8G2, 5F7G11, 3B4 and 3G7 showed the lowest values, being 3B2E11 the most sensitive with a LOD of 18.6 ng/ml and a LOQ of 64 ng/ml. Western blot data showed specific binding to recombinant TK1 and to multiple forms of TK1 in cell lysates, and serum samples for 14 of the 17 clones. Although, differences in the binding patterns were found in cell lysates. Flow cytometry was performed to analyze TK1 surface expression. Antibodies 8G2, 3B4 and 57G11 showed consistent binding across 4 cancer cell types in a similar percentage to the commercial TK1 antibody (Abcam91651) with a maximum percentage of binding in lung of (95.6%) followed by prostate (72.2%), colon (62.4%) and breast (49.1%). No significant binding for either the commercial or the custom TK1 antibodies was found on normal mono nuclear cells (MNC). The clones 8G2 and 3B4 were selected for testing their potential as therapeutic agents in antibody-dependent cell-mediated cytotoxicity (ADCC) experiments. Around 50% and 42% increased killing of A549 cells was observed with antibodies 8G2B and 3B4 respectively 48 hrs. after adding the antibodies when compared with isotype controls (p &lt;0.05). Based on dose response curves 2.5 ug/ml for the clone 8G2B and 5-7 ug/ml for the clone 3B4 were the minimum concentrations required to show significant specific cell death in A549 cells. The antibodies developed have shown capacity for detection and quantification of TK1 in serum and on the membrane of cancer cells. Moreover, our in vitro ADCC experiments provide more evidence that membrane associated TK1 has potential as an immunotherapeutic target.
Citation Format: Edwin J. Velazquez, Taylor D. Brindley, Gajendra Shrestha, Rachel A. Skabelund, Corbin M. Lee, Zachary D. Ewel, Eliza E. Bitter, Michelle H. Townsend, Kelsey B. Bennion, Kai Li Ong, Kiara V. Whitley, Richard A. Robison, Scott K. Weber, Kim L. O'Neill. Generation and characterization of a panel of monoclonal antibodies against the tumor biomarker Thymidine Kinase 1 for research, clinical and therapeutic applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2987.