Abstract 519: Exploring the potential of human nanobodies against thymidine kinase 1 for the targeting of lung cancer cells
Authors: Velazquez Edwin J. , Cress Jordan D. , Smith Kathryn R. , Bellini David M. , Skidmore Jonathan R. , Ewell Zachary D. , Robison Richard A. , O'Neill Kim L. 
Published: September 2020

Abstract
Single domain antibodies or nanobodies (Nbs) have positively proved effective for the targeting of multiple tumor targets. This study describes the isolation of human Nbs against the tumor proliferation biomarker thymidine kinase 1 (TK1). The anti-TK1 Nbs were used to target membrane associated TK1 in the non-small cell lung carcinoma cell lines NCI-H460 and A549 cells. TK1 is a pyrimidine salvage enzyme that is upregulated in malignant tissues. Recently, we have reported the presence of a TK1 form that is associated to the cell membrane in breast, lung and colon tumors as well as leukemia. Our findings suggest that targeting of TK1 in proliferating cancer cells may be an alternative approach for the treatment of cancer. Using phage display technology, we isolated a total of 237 nanobodies from the Daniel Christ DAb library. After 2-4 rounds of selection the affinity of the nanobodies to TK1 was tested with monoclonal phage ELISA using human recombinant TK1. Ten of the most sensitive anti-TK1 Nbs were then selected and evaluated with dose response curves as possible candidates for preclinical testing. The dose response curves showed R squares values ranging from 0.9738-0.9980 fitting a sigmoidal curve. Thus, indicating a strong antibody dose response relationship specific for TK1. In addition, the specificity of the TK1 antibodies was tested by screening against unrelated proteins and cell lysate from an siRNA TK1 knockdown using monoclonal phage ELISA. Nanobodies showed negligible binding when screened against TK1 non related proteins. Validation of the anti-TK1 Nbs with TK1 siRNA knockdown showed a significant reduction on the Nbs binding compared to wild type. The sensitivity of the anti-TK1 Nbs to detect TK1 was within the nanomolar range (between 100-20 ng/ml) and had thermostable standing temperatures between 50-75 °C. The anti-TK1 Nbs were used to detect membrane associated TK1 in the non-small cell lung carcinoma cell lines NCI-H460 and A549. The phage nanobodies showed binding capacity to TK1 on cancer cells with 30- 60% of positive cells for NCI-H460 and 25 to 40% for A549 cells. Human Nbs represent a valuable resource for the detection and the targeting of TK1 in proliferating tumor cells. Further work is required to enable the exploration of the therapeutic uses of anti-TK1 Nb-based therapies and their incorporation to cell adoptive therapies such as chimeric antigen receptor (CAR) therapies.
Citation Format: Edwin J. Velazquez, Jordan D. Cress, Kathryn R. Smith, David M. Bellini, Jonathan R. Skidmore, Zachary D. Ewell, Richard A. Robison, Kim L. O'Neill. Exploring the potential of human nanobodies against thymidine kinase 1 for the targeting of lung cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 519.