Selection of human single domain antibodies (sdAb) against thymidine kinase 1 and their incorporation into sdAb-Fc antibody constructs for potential use in cancer therapy.

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.

Novel monoclonal antibodies against thymidine kinase 1 and their potential use for the immunotargeting of lung, breast and colon cancer cells.

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.