Although the rich blood supply and EPR effect might cause this non-specific fluorescence with an agent, which is always on (i.e. agent that activated within specific target tumors with high TBR with considerable potential for clinical translation. Keywords: molecular imaging, activatable, malignancy, near infrared, humanized antibody Introduction Molecular imaging with antibodies has the potential not only to improve the detection of tumors but also to characterize them by their cell surface expression profiles (1,2). However, antibody delivery to a tumor relies on the high binding affinity and the low off-rate of antibodies to their cell surface antigens as well as their abundant blood supply (3,4) with leaky tumor vasculature leading to enhanced permeability and retention (EPR) (5,6) thus increasing antibody accumulation. Since the EPR effect depends only around the physical characteristics of the macromolecules injected and not on their binding characteristics it often prospects to non-specific tumor uptake. In order to accomplish specific antibody imaging, sufficient time for clearance of PP1 the unbound antibody is needed to reduce background signal resulting in favorable target-to-background ratios (TBR). In the mean time, the long clearance occasions of antibodies make delayed imaging a necessity raising practical issues with regard to patient and physician acceptance. Therefore, antibody-based target-specific molecular imaging is limited by the EPR effect and prolonged clearance times leading to reduced TBR which lowers both sensitivity and specificity. Humanized antibodies, which are antigen specific CDR-grafted human IgG molecules, have been used for clinical malignancy therapy because they produce antigen-dependent cellular cytotoxicity with minimal toxicity due to low immunogenicity. Therefore, the humanized antibody is usually a realistic choice as a targeting moiety for molecular imaging probes. However, imaging with humanized antibodies has achieved limited success. Despite their highly specific accumulation in target tumors, a critical limitation of PP1 humanized antibody imaging is the high background signal due to prolonged blood clearance PP1 which reduces the tumor-to-background ratio (TBR). Of the clinically available imaging techniques for labeling antibodies only positron emission tomography (PET) and single-photon emission computed tomography (SPECT) have been widely used and then only with long lived isotopes. However, because PET or SPECT probes constantly emit transmission (decreasing as a function of half life of the radioisotope), EPR related transmission and background transmission are quite high, especially when humanized antibodies are used. Therefore, in order to optimize the pharmacokinetics and clearance, genetic or enzymatic modifications of antibodies have been investigated, however, these alterations may reduce the therapeutic value of the antibody (2). Optically labeled antibodies, in theory, suffer from the same limitations as radioisotopes, however, optical probes differ because they can be activated or switched on only at the target malignancy cells in response to specific intracellular environmental stimuli. By activating the fluorescence transmission only within the target PP1 cells, non specific accumulation due to EPR and in the blood pool is minimized. Several activatable optical probes have recently been reported (7-11). These are largely based on self-quenching mechanisms whereby enzymatic cleavage of flurophores Dpp4 held in close steric alignment results in fluorescent activation as the fluorophores move away from each other. Among the various choices for imaging fluorophores, near-infrared (NIR) probes have the advantage of better depth penetration within tissue and are amenable to self-quenching (12). For instance, when two or more Cy5.5 dyes are conjugated to generation-6 polyamidoamine dendrimers, which are similar in hydrodynamic diameter to antibody molecules, self-quenching occurs and the degree of self quenching increases as the number of Cy5.5 dyes raises (13). However, conjugation of multiple fluorophores to the same macromolecule risks altering the pharmacokinetics of the conjugate. Relatively few reports focus on activatable optical probes conjugated to PP1 antibodies. In this study, we synthesized and tested a self-quenching activatable probe conjugated to a monoclonal antibody using cyanine-based NIR fluorophores, AlexaFluor 680 (Alexa680) and Cy5.5 as explained in Supplemental figure 1. In this study we employ trastuzumab, a humanized monoclonal IgG1 antibody, which binds to human epidermal.