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

Abstract

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.

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

“…Moreover, the signal coming from cell lysate from A549 cells was significantly higher (∼10-fold) to the signal coming from the cell lysate of A549 TK1 knockdown for both 4-H-TK1_A1 ( P <0.0296) and 4-H-TK1_D1 ( P <0.0129). A TK1 knock down was produced as previously described for this experiment (Fig 5C and D) [27].…”

“…Monoclonal antibodies are the fastest growing biopharmaceuticals in immuno-oncology [45]. Previously it has been shown that anti-TK1 monoclonal antibodies could be used for the immunotargeting of TK1 in several cancer types [27]. Although some monoclonal antibodies against TK1 have been developed there are currently no humanized versions of TK1 antibodies suitable for therapy [13, 27,46].…”

“…Moreover, early experimental data has shown that membrane associated TK1 (mTK1) in lung, colon and breast cancer cells could be targeted using monoclonal antibodies. However, the study was limited in some extent because the antibodies used were produced in mice and humanized antibodies are still required to better evaluate potential therapeutic use in humans [27]. This evidence together suggests that the targeting of TK1 both inside the cell and its mTK1 form could be a possible approach for the development of novel cancer therapies.…”

Thymidine kinase 1 through the ages: a comprehensive review

“…A study performed by Velazquez et al [ 131 ] evaluated monoclonal antibodies designed against one of six different epitopes in the tetrameric form of human TK1. Most existing antibodies targeting TK1 are against the cell cycle regulatory C-terminus which may limit TK1 detection to only certain forms of the enzyme.…”

“…From their analysis, Velazquez et al deduced that antibodies against epitopes two, five, and six showed the highest affinity to TK1 and could potentially detect multiple forms of TK1. Aside from recommending use of these antibodies in a cancer screening format, the study indicated that TK1 may have potential as an immunotherapeutic target for antibody-based and adoptive cell therapies [ 131 ].…”