Highly tumor selective near-infrared (NIR) pH-activatable probe was developed by conjugating pH-sensitive cyanine dye to a cyclic Arginine-Glycine-Aspartic acid (cRGD) peptide targeting α v β 3 integrin (ABIR), a protein that is highly overexpressed in endothelial cells during tumor angiogenesis. The NIR pH-sensitive dye used to construct the probe exhibit high spectral sensitivity with pH changes. It has negligible fluorescence above pH 6 but becomes highly fluorescent below pH 5, with a pKa of 4.7. This probe is ideal for imaging acidic cell organelles such as tumor lysosomes or late endosomes. Cell microscopy data demonstrate that binding of the cRGD probe to ABIR facilitated the endocytosis-mediated lysosomal accumulation and subsequent fluorescence enhancement of the NIR pH-activatable dye in tumor cells (MDA-MB-435 and 4T1/luc). A similar fluorescence enhancement mechanism was observed in vivo, where the tumors were evident within 4 h post injection. Moreover, lung metastases were also visualized in an orthotopic tumor mouse model using this probe, which was further confirmed by histologic analysis. These results demonstrate the potential of using the new integrin-targeted pH-sensitive probe for the detection of primary and metastatic cancer.

Nuclear imaging is an established clinical molecular imaging modality that offers good sensitivity deep in tissue. However, nuclear imaging is limited by several factors such as timeconsuming data acquisition, expensive equipment, exposure to radioactivity, the need for highly skilled personnel, and relatively poor spatial resolution. 1 Optical imaging is a relatively new imaging modality that offers real-time, non-radioactive, and, depending on the technique, high-resolution imaging of fluorophores embedded in diseased tissues. 2 Of the various optical imaging techniques investigated to date, near-infrared (NIR, 700−900 nm wavelength) fluorescence-based imaging is of particular interest for noninvasive in vivo imaging because of the relatively low tissue absorption, scatter, and minimal autofluorescence of NIR light. 3 NIR fluorescence has the potential to provide rapid, inexpensive, and non-radioactive population-based screening for breast cancer. 4-6 However, it is unclear whether currently available optical imaging systems have adequate sensitivity and/or resolution to identify breast pathology such as microcalcifications. In this study, we developed a critical reagent for exploring the limits of NIR fluorescence-based breast cancer diagnosis, namely, a simultaneous optical and nuclear contrast agent.Bisphosphonates (BPs) bind avidly to hydroxyapatite (HA) bone mineral surfaces, 7 and have many uses. BP-based radiotracers are used to diagnose osteoblastic bone lesions and to treat bone metastasis associated with breast cancer. 8 In addition, contrast agents (CAs) with BPs and phosphonates as the targeting group have been developed for use with nuclear imaging. 9, 10 Although, our group 11-13 and others 14 have explored NIR imaging with BPs, to the best of our knowledge, no BP-based dual modality nuclear-NIR contrast agents have been reported. Dual-labeled targeting imaging agents, such as the one described herein, allow cross validation and direct comparison between nuclear (the gold-standard) and fluorescence optical imaging.The tri-functional diagnostic agent Pam-Tc/Re-800 was synthesized in 5 chemical steps (Scheme 1), with an overall yield of 53%, from N-ε-t.-Boc-L-lysine 1, Me-Pam, 13 MAS 3 , and IRDye800CW. Primary amine of compound 1 was conjugated with N-hydroxy succinimide ester of IRDye800CW to obtain Lys(t.-Boc)-800CW-carboxylic acid 2. Me-Pam reacted with activated compound 2 in presence of HCTU and NMM to generate Lys(t.-Boc)-800CW-PamMe 3. Compound 3 was treated with trifluoroacetic acid and trimethylsilyl bromide to deprotect E-mail: jfrangio@bidmc.harvard.edu. Supporting Information Available: Experimental procedures and spectroscopic data. This material is available free of charge via the Internet at http://pubs.acs.org. Pam-Tc/Re-800 6 was fully characterized for its radioactivity, spectral properties (Supporting Information), and calcium salt specificity (Figure 1). Specific activity of Pam-Tc-800 6a was greater than or equal to 6,250 Ci/mmol and radiochemical purity was greater than or eq...

Introduction In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to DOTA chelates for use in PRIT applications. Methods We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), reformatted as a single chain variable fragment (scFv). Results Modeling predicts that for high antigen density and saturating bsAb dose, a hapten binding affinity of 100 picomolar (pM) is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nanomolar (nM) to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2 ± 1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen (CEA), pretargeted high-affinity scFv results in significantly higher tumor retention of a 111In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions We have engineered a versatile, high-affinity DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals.

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