Background: Thymidine kinase 1 (TK1) is a pyrimidine salvage pathway enzyme that is up-regulated in malignant tissues and elevated in the serum of cancer patients. While TK1 has been well established as a tumor biomarker, little has been done to explore its potential as a tumor target. Recently, we reported the membrane expression of TK1 on malignant cells, but not on normal cells. This study explores the possible use of monoclonal antibodies for the targeting of membrane associated TK1 in lung, breast, colon and prostate cancer cells. Methods: We generated and evaluated a panel of monoclonal antibodies against six different epitopes exposed in the tetrameric form of TK1. Antibodies were developed with hybridoma technology and validated with Western blot, siRNA TK1 knockdown, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The therapeutic potential of the antibodies was evaluated in vitro in antibody-dependent cell-mediated-cytotoxicity (ADCC) experiments. Results: Binding of the antibodies to TK1 was confirmed by Western blot in purified recombinant protein, cancer serum, and cell lysate. After a TK1 knockdown was performed, a reduction of TK1 expression was observed with five antibodies. Using indirect ELISA, we identified 3B2E11, 9C10, 7H2, 3B4, 8G2 among the most sensitive antibodies (LOD = 10.73-66.9 pg/ml). Surface expression of TK1 on the membrane of various cancer cell lines was analyzed with flow cytometry. Antibodies 8G2, 3B4, 7HD and 5F7G11 detected TK1 on the membrane of various cancer cell lines, including lung, prostate, colon and breast. No significant binding was detected on normal lymphocytes. Increased cytolysis of lung (~ 70%. p = 0.0001), breast (~ 70%, p = 0.0461) and colon (~ 50% p = 0.0216) cancer cells by effector cells was observed when anti-TK1 antibodies were added during ADCC experiments. Conclusions: The antibodies developed showed potential to be used to detect and target TK1 on the membrane of various tumor cells. The targeting of TK1 in malignant cells using monoclonal antibodies may be a feasible approach for the elimination of high TK1 expressing tumor cells.
Recent clinical trials using chimeric antigen receptors (CAR) T cells have demonstrated tremendous success in eradicating hematologic malignancies. Notwithstanding the excitement generated by CAR T cell therapy, its clinical efficacy has not been effectively translated to the context of solid tumors; the physical barriers of solid malignancies and the immunosuppressive conditions at the tumor site hinder the efficacy of CAR T cells. Macrophages have the ability to infiltrate almost every tissue and frequently are recruited into tumors. Therefore, macrophages are an attractive vehicle for CAR therapy and could help solve current challenges that CAR T cells face in the treatment of solid tumors. MOTO-CAR cells are monocyte-derived human macrophages that are genetically modified by a lentiviral or adenoviral approach to express a synthetic tumor-targeting receptor and to secrete cytokines, ligands or chemokine receptors. MOTO-CAR receptors are composed of a single-chain variable fragment (ScFv) that binds to a specific tumor target, a hinge to link it to a transmembrane domain, and an engineered Toll/Interleukin-1 receptor (TIR) signaling domain. When the ScFv binds to the tumor cell via its tumor target, an activation signal is transmitted. Myd88 dependent and independent signaling cascades are elicited, activating the macrophage and polarizing it towards a proinflammatory phenotype to eliminate cancer cells in a selective way. We previously reported the expression of Thymidine Kinase 1 (TK1) on the cell membrane of the non-small cell lung carcinoma NCI-H460 and A549 cell lines. The in vitro function of TK1 MOTO-CAR cells was evaluated against these cancer cell lines, using GFP-based phagocytosis and killing assays. Additionally, cell migration and interaction was recorded using time-lapse video with a confocal microscope. Upon co-culturing, with its target TK1 specific MOTO-CARs showed a nearly 4-fold increase in killing activity when compared with the controls (p<0.01). MOTO-CAR cells were produced through a lentiviral approach with around 30% of the cells expressing MOTO-CARs and with an adenoviral approach using the Ad5f35 vector with an efficiency of 70-80% of cells being transduced. Furthermore, after transduction MOTO-CAR cells showed a consistent M1 phenotype expressing high levels of CD14, CD80, CD206 and low levels of CD163. Time-lapse videos showed migration and clustering of MOTO-CAR cells around H460 GFP + cells. Moreover, cell death was observed upon contact of MOTO-CAR cells with target cells as well as phagocytic activity. In vivo testing using an orthotopic NOD scid gamma mice model is in progress. Our preclinical data show evidence that human macrophages are a suitable vehicle for CAR therapy and have the potential to successfully extrapolate the clinical efficacy of CAR therapy to the context of solid tumors. Citation Format: Edwin J. Velazquez, John E. Lattin, Taylor D. Brindley, Zachary Z. Reinstein, Roger Chu, Lu Liu, Evita G. Weagel, Michelle H. Townsend, Kiara V. Whitley, Eliza L. Lawrence, Brandon T. Garcia, Scott Weber, Richard A. Robison, Kim L. O'Neill. Macrophage Toll-like receptor-chimeric antigen receptors (MOTO-CARs) as a novel adoptive cell therapy for the treatment of solid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2563.
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 <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.
This study explores the potential therapeutic use of monoclonal antibodies against the tumor proliferation biomarker TK1 for the immunotargeting of lung and breast cancer cells. Recent clinical trials have led to the approval of monoclonal antibodies for the treatment of several solid tumors including lung and breast tumors. However, a common limitation that antibody-based therapies face is that a significant portion of the current tumor targets are expressed on normal tissues, thus creating off-target tumor effects. TK1 is a tumor proliferation biomarker that is up-regulated in malignant tissues. Recently, we have reported the expression of TK1 on the cell membrane of several cancer cell lines, mononuclear cells (MNC) from patients with leukemia and cells from breast and colon tumors. No membrane expression of TK1 was found on normal cells. To further explore the potential of TK1 as an immunotherapeutic target, we evaluated the capacity of a novel panel of anti TK1 antibodies to target TK1 on the cell membrane of lung and breast cancer cells and to elicit an antibody-dependent cell-mediated-cytotoxicity (ADCC) response in vitro. Antibodies previously developed in our lab were validated to specifically target six different regions of the TK1 molecule. Four different antibodies targeting three different regions in the TK1 molecule were selected for ADCC experiments. Nuclear restricted GFP versions of the NCI-H460 and MDA-MB-231 cell lines were utilized in conjunction with the real-time cell imaging system ImageXpress® Pico to measure the ADCC response. Mono nuclear cells (MNCs) from healthy donors were co-cultured with target cells and 10ug/ml of each anti-TK1 antibody were added to the media. An antibody with the same isotype than the TK1 antibodies, but that doesn't bind to any human surface antigen was utilized as a control. All ADCC responses elicited by the anti-TK1 antibodies were observed between 24-72hrs. The highest ADCC responses were elicited by antibodies 4G10 and 1B12 in both breast and lung cancer cell lines while a minimal response was observed with antibodies 7D1 and 3B2E11. A 50-60% increment in the ADCC response against NCI-H460 cells was observed with antibody 4G10 in comparison with the isotype control (p= 0.001). A 40% increase in the ADCC response against MDA-MB-231 cells was observed using antibody 1B12 (p = 0.003) compared to the its isotype control. This preliminary data suggests that anti TK1 monoclonal antibodies can be used for the immunotargeting of tumor cells expressing membrane associated TK1. The further exploration of TK1 as a tumor target could lead to the development of new TK1-based immunotherapies. Citation Format: Edwin J. Velazquez, Jordan D. Cress, Kathryn R. Smith, Taylor D. Brindley, Gajendra Shrestha, Richard A. Robison, Kim L. O'Neill. Monoclonal antibodies against thyimidine kinase 1 for the immunotargeting of lung and breast cancer cells, a preclinical evaluation [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 543.
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