Cytokines are small molecular messengers that have profound effects on cancer development. Increasing evidence shows that cytokines are heavily involved in regulating both pro- and antitumor activities, such as immune activation and suppression, inflammation, cell damage, angiogenesis, cancer stem-cell-like cell maintenance, invasion, and metastasis. Cytokines are often required to drive these cancer-related processes and, therefore, represent an important research area for understanding cancer development and the potential identification of novel therapeutic targets. Interestingly, some cytokines are reported to be related to both pro- and anti-tumorigenicity, indicating that cytokines may play several complex roles relating to cancer pathogenesis. In this review, we discuss some major cancer-related processes and their relationship with several cytokines.

Thymidine Kinase 1 (TK1) is primarily known as a cancer biomarker with good prognostic capabilities for both hematological and solid malignancies. However, recent studies targeting TK1 at protein and mRNA levels have shown that TK1 may be useful as a therapeutic target. In order to examine the use of TK1 as a therapeutic target, it is necessary to develop therapeutics specific for it. Single domain antibodies (sdAbs), represent an exciting approach for the development of immunotherapeutics due to their cost-effective production and higher tumor penetration than conventional antibodies. In this study, we isolated sdAb fragments specific to human TK1 from a human sdAb library. A total of 400 sdAbs were screened through 5 rounds of selection by monoclonal phage ELISA. The most sensitive sdAb fragments were selected as candidates for preclinical testing. The sdAb fragments showed specificity for human TK1 in phage ELISA, Western blot analysis and had an estimated limit of detection of 3.9 ng/ml for the antibody fragments 4-H-TK1_A1 and 4-H-TK1_D1. The antibody fragments were successfully expressed and used for detection of membrane associated TK1 (mTK1) through flow cytometry on cancer cells [lung (~95%), colon (~87%), breast (~53%)] and healthy human mononuclear cells (MNC). The most sensitive antibody fragments, 4-H-TK1_A1 and 4-H-TK1_D1 were fused to an engineered IgG1 Fc fragment. When added to cancer cells expressing mTK1 co-cultured with human MNCs, the anti-TK1-sdAb-IgG1_A1 and D1 were able to elicit a significant antibody-dependent cell-mediated cytotoxicity (ADCC) response against lung cancer cells compared to isotype controls (P<0.0267 and P<0.0265, respectively). To our knowledge this is the first time that the isolation and evaluation of human anti-TK1 single domain antibodies using phage display technology has been reported. The antibody fragments isolated here may represent a valuable resource for the detection and the targeting of TK1 on tumor cells.

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Since the clinical relevance of hypoxanthine guanine phosphoribosyltransferase (HPRT) as both a cancer biomarker and a potential tumor target is increasing, we isolated human nanobodies (Nbs) against HPRT and tested their potential to detect and target cytosolic and membrane associated HPRT in lung, breast and colon cancer cells. HPRT is a purine salvage pathway enzyme that catalyzes the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. Until recently, HPRT was considered as a housekeeping gene and its clinical relevance was mainly limited to congenital central nervous disorders such as Lesch-Nyhan disease. However, recent gene expression analysis across all cancers in The Cancer Genome Atlas (TCGA) database and analysis of HPRT expression within cancer patients' tissues has revealed that the expression of HPRT is elevated in the majority of malignant tissues in comparison to normal tissues. Moreover, multiple studies have recently shown that HPRT may be associated with the cell membrane in lung, colon cancer and lymphoma cells, and not in normal cells. Thus, HPRT is a potential immunotherapeutic target for cancer. Using phage display technology we isolated 384 nanobody clones from the Christ human single domain antibody (dAb) library against human HPRT. Ten of the most sensitive Nbs were sequenced and evaluated to determine their sensitivity using dose response curves. Four parameter logistic regression analysis of the top 10 HPRT nanobodies showed R squared values ranging from 0.9388-0.9989 and a sigmoidal shape denoting a concentration-dependent antigen-ligand relationship. The anti-HPRT Nbs were able to detect minimum HPRT amounts ranging from 10-20 ng/ml. Validation of the anti-HPRT Nbs was conducted with a siRNA HPRT knockdown in A549 cells. A549 siRNA HPRT knockdown cell lysate showed at least a 5-fold reduction in the overall signal in phage ELISA compared to A549 wild type cell lysate (p&lt;0.001) indicating the specificity of the Nbs to HPRT. In addition, flow cytometry analysis showed that the anti-HPRT Nbs were able to detect membrane associated HPRT on A549 (20%), NCI-H460 (23%), HT-29 (41%) , SW620 (46%) and MDA-MB-231 (32%) cancer cell lines. Anti-HPRT Nbs did not show significant binding to normal mononuclear cells compared to cancer cells (p&lt;0.002). Anti-HPRT Nbs were successfully expressed and purified using the pET-scFv expression system. Human anti-HPRT Nbs may be used for the detection of both cytosolic and membrane associate HPRT. Future directions of this research will explore the uses of anti-HPRT-Nb-based therapies and their use in other applications such as chimeric antigen receptors (CARs) for cell adoptive therapies. Citation Format: Edwin J. Velazquez, Tyler B. Humpherys, Toni O. Mortimer, David M. Bellini, Kathryn R. Smith, Rachel M. Morris, Abigail Goodman, Kim L. O'Neill. Isolation and evaluation of human nanobodies against hypoxanthine guanine phosphoribosyltransferase (HPRT) and their potential use for the detection and targeting of lung, breast and colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1840.

Recent advances in cancer immunotherapy have transformed biomarkers into diagnostic and prognostic indicators and immunotherapeutic targets in clinical oncology. To ensure higher treatment success, it is critical to identify unique biomarkers to mitigate “off-target” effects. Therefore, the identification of novel biomarkers and appropriate target selection has never been more important to improve cancer immunotherapy. The DNA salvage pathway protein thymidine kinase 1 (TK1) plays an important role in DNA synthesis and repair. Normally, it is highly expressed during S phase and provides recycled nucleotides by phosphorylating thymidine to make thymidine monophosphate. Past studies have suggested that TK1 may influence cell cycle control and, therefore, provides a platform for the understanding of disease progression and possible means of mitigation. The purpose of our study was to verify elevated TK1 levels in breast cancer cells and further investigate TK1’s potential influence over cell cycle control. We hypothesized that TK1 may be important in regulating the cell cycle and other cell checkpoint proteins. Immunohistochemistry analysis was employed to quantify TK1 expression in ductal and lobular primary and matched metastatic breast cancer samples compared to normal tissue. Bioinformatics was then used to analyze RNA-seq data of cell cycle checkpoint factors and TK1 in BRCA patients from the Cancer Genome Atlas. To demonstrate TK1’s potential effect on the cell cycle checkpoint pathway, we utilized HCC1806 breast cancer cells and generated a CRISPR-Cas9 TK1 knockdown (L133) cell line for in vitro testing. These were validated by western blotting and qPCR. Propidium iodide staining and cell cycle analysis were performed via flow cytometry on both cell lines. Immunohistochemistry results indicated that TK1 levels increase as breast cancer progresses and, thus, may correlate with breast cancer aggressiveness. Bioinformatics analysis showed that strong positive correlations exist between cell cycle checkpoint factors and TK1 in BRCA patients, such as BRCA1 and CHEK2. Flow cytometry cell cycle analysis showed that mean differences of L133 cells in S phase were significantly higher than in G1 compared to HCC 1806 cells, suggesting that TK1 levels influence cell cycle arrest. Our preliminary results suggest that TK1 may affect cell cycle checkpoint pathways, which are essential to maintaining homeostasis in normal cells and preventing cancer progression. Further investigation to understand TK1’s potential interactions with important checkpoint factors, such as p53 or Gas6, may help elucidate other therapeutic targets for preventing disease progression. Citation Format: Eliza E. Bitter, Toni Mortimer, Rachel Morris, Kai Barlow, Abby Schekall, Michelle Townsend, Brett E. Pickett, Kim L. O'Neill. Thymidine kinase 1 and cell cycle control in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 159.