Heterobivalent Agents Targeting PSMA and Integrin‑α_vβ₃

Differential expression of surface proteins on normal vs malignant cells provides the rationale for the development of receptor-, antigen-, and transporter-based, cancer-selective imaging and therapeutic agents. However, tumors are heterogeneous, and do not always express what can be considered reliable, tumor-selective markers. That suggests development of more flexible targeting platforms that incorporate multiple moieties enabling concurrent targeting to a variety of putative markers. We report the synthesis, biochemical, <i>in vitro,</i> and preliminary <i>in vivo</i> evaluation of a new heterobivalent (HtBv) imaging agent targeting both the prostate-specific membrane antigen (PSMA) and integrin-α_vβ₃ surface markers, each of which can be overexpressed in certain tumor epithelium and/or neovasculature. The HtBv agent was functionalized with either 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or the commercially available IRDye800CW. DOTA-conjugated HtBv probe <b>9</b> bound to PSMA or α_vβ₃ with affinities similar to those of monovalent (Mnv) compounds designed to bind to their targets independently. <i>In situ</i> energy minimization experiments support a model describing the conformations adapted by <b>9</b> that enable it to bind both targets. IRDye800-conjugated HtBv probe <b>10</b> demonstrated target-specific binding to either PSMA or integrin-α_vβ₃ overexpressing xenografts. HtBv agents <b>9</b> and <b>10</b> may enable dual-targeted imaging of malignant cells and tissues in an effort to address heterogeneity that confounds many cancer-targeted imaging agents

Spatio-Temporal Modification of Collagen Scaffolds Mediated by Triple Helical Propensity

Functionalized collagen that incorporates exogenous compounds may offer new and improved biomaterials applications, especially in drug-delivery, multifunctional implants, and tissue engineering. To that end, we developed a specific and reversible collagen modification technique utilizing associative chain interactions between synthetic collagen mimetic peptide (CMP) [(ProHypGly)x; Hyp = hydroxyproline] and type I collagen. Here we show temperature-dependent collagen binding and subsequent release of a series of CMPs with varying chain lengths indicating a triple helical propensity driven binding mechanism. The binding took place when melted, single-strand CMPs were allowed to fold while in contact with reconstituted type I collagens. The binding affinity is highly specific to collagen as labeled CMP bound to nanometer scale periodic positions on type I collagen fibers and could be used to selectively image collagens in ex vivo human liver tissue. When heated to physiological temperature, bound CMPs discharged from the collagen at a sustained rate that correlated with CMP’s triple helical propensity, suggesting that sustainability is mediated by dynamic collagen−CMP interactions. We also report on the spatially defined modification of collagen film with linear and multi-arm poly(ethylene glycol)-CMP conjugates; at 37 °C, these PEG-CMP conjugates exhibited temporary cell repelling activity lasting up to 9 days. These results demonstrate new opportunities for targeting pathologic collagens for diagnostic or therapeutic applications and for fabricating multifunctional collagen coatings and scaffolds that can temporally and spatially control the behavior of cells associated with the collagen matrices

PD-L1 Detection in Tumors Using [⁶⁴Cu]Atezolizumab with PET

The programmed death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) pair is a major immune checkpoint pathway exploited by cancer cells to develop and maintain immune tolerance. With recent approvals of anti-PD-1 and anti-PD-L1 therapeutic antibodies, there is an urgent need for noninvasive detection methods to quantify dynamic PD-L1 expression in tumors and to evaluate the tumor response to immune modulation therapies. To address this need, we assessed [64Cu]­atezolizumab for the detection of PD-L1 expression in tumors. Atezolizumab (MPDL3208A) is a humanized, human and mouse cross-reactive, therapeutic PD-L1 antibody that is being investigated in several cancers. Atezolizumab was conjugated with DOTAGA and radiolabeled with copper-64. The resulting [64Cu]­atezolizumab was assessed for in vitro and in vivo specificity in multiple cell lines and tumors of variable PD-L1 expression. We performed PET-CT imaging, biodistribution, and blocking studies in NSG mice bearing tumors with constitutive PD-L1 expression (CHO-hPD-L1) and in controls (CHO). Specificity of [64Cu]­atezolizumab was further confirmed in orthotopic tumor models of human breast cancer (MDAMB231 and SUM149) and in a syngeneic mouse mammary carcinoma model (4T1). We observed specific binding of [64Cu]­atezolizumab to tumor cells in vitro, correlating with PD-L1 expression levels. Specific accumulation of [64Cu]­atezolizumab was also observed in tumors with high PD-L1 expression (CHO-hPD-L1 and MDAMB231) compared to tumors with low PD-L1 expression (CHO, SUM149). Collectively, these studies demonstrate the feasibility of using [64Cu]­atezolizumab for the detection of PD-L1 expression in different tumor types

Inhibition of cell growth by [³²P] PO₄ or [⁹⁰Y].

<p>The WST-1 proliferation assay was done to determine the level of cell killing by ³²P or by ⁹⁰Y. BALB/c tumor CRL2836 cells or HeLa S3 cells were exposed to 0 Ci, 1 μCi, 2.5 μCi, or 5 μCi in complete medium. After 24 hours, the medium was changed and cells were grown in non-radioactive complete medium. WST-1 cell proliferation assays were done at Days 1, 2, 3, 4, and 5 in triplicate. The mean is shown plus/minus the standard deviation. The student’s two-sided t-test determined the <i>P</i> value shown.</p

Characterization of ³²P uptake by the cell.

<p>A. ³²P is directly incorporated into cellular DNA. Mouse CRL2836 or human HeLa S3 cell lines were incubated overnight with ³²P[PO₄] and then grown for 48 h in non-radioactive medium (lanes 1 through 4), or grown for 24 h in non-radioactive medium, grown for 24 h with ³²P[PO₄], and then grown for 24 h in non-radioactive medium (lanes 5 through 8), or grown for 48 h in non-radioactive medium, then grown for 24 h with ³²P[PO₄] (lanes 9 through 12). The extracted nucleic acids were incubated with DNase I, the digestion products were run on a 5% polyacrylamide gel and exposed to film. B. Apoptosis induced by ³²P in mouse CRL2836 cells. Mouse CRL2836 cells were incubated with 0, 2.5, 5, 10 or 20 μCi ³²P[PO₄] for 24 h, and non-radioactive medium added for an additional 24 h. Protein was extracted from each well and analyzed for apoptosis by western blots using antibody to cleaved caspase-3 protein (Lanes 1 through 5). Antibody against beta-actin was used to verify identical amounts of protein were loaded (lanes 6 through 10).</p

Inhibition of BALB/c syngeneic tumor growth by [³²P]PO₄.

<p>Syngeneic BALB/c CRL2836 tumors were established in the rear flanks of BALB/c mice at Day 0. After ten days, during which the tumors became well vascularized, mice received an injection of 5 μCi of [³²P]PO₄ intravenously via the tail vein. The tumor volumes are shown as the mean of six tumors plus/minus the standard error of the mean. The student’s two-sided t-test determined the <i>P</i> value shown. Inset: Representative picture at 35 days post CRL2836 cell injection, showing two control mice on the left, and one mouse that received one 5 uCi [³²P]PO₄ dose (right).</p

Preclinical Comparative Study of ⁶⁸Ga-Labeled DOTA, NOTA, and HBED-CC Chelated Radiotracers for Targeting PSMA

⁶⁸Ga-labeled, low-molecular-weight imaging agents that target the prostate-specific membrane antigen (PSMA) are increasingly used clinically to detect prostate and other cancers with positron emission tomography (PET). The goal of this study was to compare the pharmacokinetics of three PSMA-targeted radiotracers: ^<b>68</b><b>Ga-1</b>, using DOTA-monoamide as the chelating agent; ^<b>68</b><b>Ga-2</b>, containing the macrocyclic chelating agent <i>p</i>-SCN-Bn-NOTA; and ⁶⁸Ga-DKFZ-PSMA-11, currently in clinical trials, which uses the acyclic chelating agent, HBED-CC. The PSMA-targeting scaffold for all three agents utilized a similar Glu-urea-Lys-linker construct. Each radiotracer enabled visualization of PSMA+ PC3 PIP tumor, kidney, and urinary bladder as early as 15 min post-injection using small animal PET/computed tomography (PET/CT). ^<b>68</b><b>Ga-2</b> demonstrated the fastest rate of clearance from all tissues in this series and displayed higher uptake in PSMA+ PC3 PIP tumor compared to ^<b>68</b><b>Ga-1</b> at 1 h post-injection. There was no significant difference in PSMA+ PC3 PIP tumor uptake for the three agents at 2 and 3 h post-injection. ⁶⁸Ga-DKFZ-PSMA-11 demonstrated the highest uptake and retention in normal tissues, including kidney, blood, spleen, and salivary glands and PSMA-negative PC3 flu tumors up to 3 h post-injection. In this preclinical evaluation ^<b>68</b><b>Ga-2</b> had the most advantageous characteristics for PSMA-targeted PET imaging

DataSheet1.doc

<p>Intracerebral hemorrhage (ICH) is a fatal stroke subtype with significant public health impact. Although neuroinflammation is a leading cause of neurological deficits after ICH, no imaging tool is currently available to monitor brain inflammation in ICH patients. Given the role of TSPO in neuroinflammation, herein we investigate whether a second-generation TSPO ligand, [¹²⁵ I]IodoDPA-713 can be used to monitor the changes in TSPO expression in a preclinical model of intracerebral hemorrhage. Male CD1 mice were subjected to ICH/Sham. The brain sections, collected at different time points were incubated with [¹²⁵ I]IodoDPA-713 and the brain uptake of [¹²⁵ I]IodoDPA-713 was estimated using autoradiography. The specificity of [¹²⁵ I]IodoDPA-713 binding was confirmed by a competitive displacement study with an unlabeled TSPO ligand, PK11195. [¹²⁵ I]IodoDPA-713 binding was higher in the ipsilateral striatum with an enhanced binding observed in the peri-hematomal brain region after ICH, whereas the brain sections from sham as well as contralateral brain areas of ICH exhibited marginal binding of [¹²⁵ I]IodoDPA-713. PK11195 completely reversed the [¹²⁵ I] IodoDPA-713 binding to brain sections suggesting a specific TSPO-dependent binding of [¹²⁵ I]IodoDPA-713 after ICH. This was further confirmed with immunohistochemistry analysis of adjacent sections, which revealed a remarkable expression of TSPO in the areas of high [¹²⁵ I]IodoDPA-713 binding after ICH. The specific as well as enhanced binding of [¹²⁵ I]IodoDPA-713 to the ipsilateral brain areas after ICH as assessed by autoradiography analysis provides a strong rationale for testing the applicability of [¹²⁵ I]IodoDPA-713 for non-invasive neuroimaging in preclinical models of ICH.</p

Figure S4 from Nanoconjugation of PSMA-Targeting Ligands Enhances Perinuclear Localization and Improves Efficacy of Delivered Alpha-Particle Emitters against Tumor Endothelial Analogues

Effect of the presence of CCM on PSMA expression over time.</p