SPR analysis of the binding affinity of hDM-αH-C6.5 MH3B1 to ECDHER2 showed Nutlin-3a purchase a strong binding affinity of 3.4 × 10-10 M, approximately three fold less strong than that of the single chain C6.5 MH3B1 [7]. The trimeric structure of hDM-αH-C6.5 MH3B1 should further increase its binding to cell associated HER2/neu. The

high affinity should ensure that hDM-αH-C6.5 MH3B1 effectively targets the tumor and persists at the tumor site long enough to allow the systemically administered F-dAdo to reach the tumor and be cleaved to F-Ade [5, 7, 17, 18]. It has been suggested that high affinity scFvs would mainly be retained in the perivascular regions of the tumor where the first tumor BTK inhibitor antigen is encountered [19], preventing tumor penetration. While this might weaken the clinical applicability of some therapeutic scFvs, it should not be an issue for ADEPT. In fact, retention of hDM-αH-C6.5 MH3B1 on the cell surface in the tumor microenvironment for an extended period of time should make the enzyme readily accessible for cleaving the prodrug to a cytotoxic drug. Properties of hDM-αH-C6.5 MH3B1, such as thermal stability and resistance to proteolysis contribute to its effectiveness in Elacridar clinical trial vitro and in vivo. When hDM-αH-C6.5 MH3B1 was incubated with serum at 37°C only 50% of enzyme activity was recovered after 30 minutes (Fig. 3). Longer incubation resulted in a further rapid loss of

activity so that after 3 hours only about 30% of the activity remained.

However, further incubations for 21 hours resulted in little further decrease in activity (Fig. 3). Incubation with serum over night at 4°C resulted in a 20% loss of activity (Fig. 3). The observed loss of enzyme activity in the presence of serum is most probably due to degradation of the protein by the serum proteases and the small additional decrease in enzyme activity following 3 hours of incubation may indicate that the serum proteases themselves become inactivated upon incubation and lose activity by 3 hours. Thiamine-diphosphate kinase Alternatively, there may exist different conformers of hDM-αH-C6.5 MH3B1 that exhibit different stabilities in serum. The use of hDM with F-dAdo constitutes a novel and specific enzyme-prodrug combination. Addition of hDM-αH-C6.5 MH3B1 alone, F-dAdo alone or hPNP-αH-C6.5 MH3B1 with F-dAdo, did not affect cell proliferation. This is particularly important since hPNP is a ubiquitous enzyme present at micromolar concentrations in blood cells [12]. Therefore, lack of activity of hPNP-αH-C6.5 MH3B1 with F-dAdo should reduce toxicity concerns in vivo. However, when hDM-αH-C6.5 MH3B1 was added to cells in the presence of F-dAdo, the cytotoxic F-Ade generated due to enzymatic activity of hDM-αH-C6.5 MH3B1 resulted in a dose-dependent inhibition of cell proliferation (Fig. 2C). Our in vitro studies have shown that F-dAdo conversion to F-Ade occurs by hDM that is targeted to tumor cells through specific interaction of C6.