A novel cell-death radio-ligand detects the apoptotic response of lymphoma to DNA damaging chemotherapy
Authors: Michael Brown, Jocelyn Darby, Jim Manavis, Douglas Smyth, Chris Tsopelas, Fares Al-Ejeh
Journal: Clinical Cancer Research (2007)

A23  A sensitive, specific method for noninvasive detection of tumor chemoresponsiveness in vivo would aid go/no go decision making in cancer drug development, facilitate clinical management decisions and spare cancer patients ineffective, toxic therapy. We discovered that the La/SS-B ribonucleoprotein is overexpressed in malignant cells, and may be a suitable radio-immunodiagnostic target. La is an RNA binding protein that chaperones transfer and ribosomal RNA products of RNA polymerase III, and enables cap-independent translation of messenger RNA during apoptosis. Upon apoptosis induction, La translocates from nucleus to cytoplasm where it is detected using the 3B9 monoclonal antibody (mAb), which enters late apoptotic cells in vitro. Binding of 3B9 to apoptotic tumor cells in vitro was specific, rapid, saturable, induced by DNA damage and fixed by transglutaminase 2 activity. These in vitro characteristics of 3B9 binding partly explain augmented 3B9 binding to apoptotic tumor cells in vivo that we observe using the murine EL4 lymphoma model.  Treatment of EL4-bearing mice with cyclophosphamide/etoposide (CE) chemotherapy caused EL4 cell apoptosis and associated induction of La in apoptotic tumor cells. To demonstrate antigen-specific tumor targeting, 3B9 or its Sal5 isotype control mAb were intrinsically labeled with Carbon-14 (14C), and 3B9 dose-dependent uptake of 14C-3B9 was evident after CE chemotherapy. Then, DOTA-conjugated 3B9 was labeled with Indium-111 (111In), and 20% (±1%) of the injected dose per gram of tissue (ID/g) was bound in control EL4 tumors indicating the presence of necrotic cells. However, in CE-treated mice, significantly more 111In-DOTA-3B9 accumulated in EL4 tumors 48h after apoptosis induction (42±1% ID/g). After CE, EL4 tumor uptake of 111In-DOTA-3B9 in vivo directly correlated with the apoptotic index measured by activated caspase-3 immunohistochemistry, and inversely correlated with the necrotic index measured using morphologic criteria.  Accumulation of radioactivity in other organs reflected blood pool activity because tumor: organ ratios were ≥10 for chemosensitive organs such as small intestine and bone marrow 48h after CE chemotherapy. Tumor accumulation of 111In-DOTA-3B9 48h after CE chemotherapy was similar although more rapid when mice were given 3B9 24h after (t½, 4.6±0.2h) rather than concurrently (t½, 15±0.1h) with chemotherapy. To examine tumor: background ratios, DOTA-conjugated 111In-labeled Fab2 fragments of 3B9 or control Sal5 mAb were studied, and tumor accumulation of 111In-DOTA-3B9 Fab2 was specific and very rapid (t½, 2.7±0.3h). Moreover, after injection of mice with D-lysine to block renal tubular re-absorption of Fab2, apoptotic tumor-specific signals were clearly imaged and, when expressed as the ratio of pixels/cm2 in tumors of CE-treated mice/untreated mice, were 7.85±0.44 and 1.61±0.02 for 3B9 Fab2 and IgG, respectively. We shall examine other parameters such as the dose and timing of radiolabeled 3B9 in order to optimize imaging of tumor apoptosis.  In conclusion, robust, early identification of tumor response to DNA-damaging anti-neoplastic treatment remains an area of unmet medical need. We found that La-specific mAb specifically accumulated in apoptotic tumors. Our results support the clinical development of radiolabeled La-specific mAb as a radio-immunodiagnostic agent.