Development of Anti-CEA C2 Domain-Deleted Antibody (M5A∆C2) for the PET Imaging of Colorectal Cancer.

Purpose: Recombinant antibody fragments represent a novel class of in vivo biological immunoPET imaging agents. This study developed a series of anti-carcinoembryonic antigen (CEA) C2 domain-deleted antibodies to evaluate their rapid, high-level tumor targeting combined with fast blood clearance for immunoPET imaging in two colorectal cancer mouse models.

Procedure: A series of humanized anti-CEA M5A∆C2 recombinant antibody fragments were synthesized via transient mammalian expression and purified using a two-step process.

Development and characterization of DIA 12.3, a fully human intact anti-CEACAM1 monoclonal antibody.

Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1), a homotypic cell adhesion molecule glycoprotein with apical expression on normal epithelial cells and activated lymphocytes, is overexpressed on many tumors and acts as an inhibitory receptor on NK cells, preventing their killing of CEACAM1 positive tumors. Production of humanized anti-CEACAM1 antibodies to block the inhibitory activity of CEACAM1 for immunotherapy and immunoimaging. Starting from a scFv, a fully human intact anti-CEACAM1 (DIA 12.

Comparison of IL-2-antibody to IL-2-Fc with or without stereotactic radiation therapy in CEA immunocompetent mice with CEA positive tumors.

Background: The potent immune effects of interleukin-2 (IL-2) for cancer therapy can be increased by genetic fusion of IL-2 to the Fc domain of an antibody (IL-2-Fc) or tumor targeted by genetic fusion to a whole antibody known as an immunocytokine (ICK).

Methods: An anti-CEA ICK (M5A-IL-2) was compared to an IL-2-Fc fusion protein using tumor therapy and PET imaging in CEA transgenic immunocompetent mice bearing CEA positive colon or breast tumors. Combination with stereotactic radiation therapy (SRT) was performed with either ICK or IL-2-Fc.

Generation of IL-2-Fc-antibody conjugates by click chemistry.

Background: Immunocytokines (ICKs) are antibody directed cytokines produced by genetic fusion of an antibody to a cytokine.

Methods: We now show that antibodies conjugated by click chemistry to interleukin-2 (IL-2)-Fc form fully active conjugates, and in one example, equivalent activity to a genetically produced ICK.

Results: An IL-2-Fc fusion protein was optimized for click chemistry at hinge cysteines using protein stabilizing IL-2 mutations at Lys35 and Cys125 and Fc hinge mutations at Cys142 and Cys148.

Heart Uptake of [F]Fluoro-4-Thia-Oleate in a Non-Alcoholic Fatty Liver Disease Mouse Model.

The world-wide high incidence of non-alcoholic fatty liver disease (NAFLD) is of concern for its progression to insulin resistance, steatohepatitis and cardiovascular disease (CVD). The increased uptake of fatty acids in critical organs plays a major role in NAFLD progression. Male Ceacam1−/− mice that develop NAFLD, insulin resistance and CVD on normal chow are a potential model for studying the dysregulation of fatty acid uptake.

Quantification and Analysis of Micro-Level Activities Data from Children Aged 1-12 Years Old for Use in the Assessments of Exposure to Recycled Tire on Turf and Playgrounds.

Background: There are growing health concerns about exposure to toxicants released from recycled tire rubber, which is commonly used in synthetic turf and playground mats. To better estimate children's exposure and risk from recycled tire rubber used in synthetic turf and playground mats, there is a need to collect detailed accurate information on mouthing activity and dermal contact behaviors. The objective of this study was to quantify and analyze micro-level activity time series (MLATS) data from children aged 1-12 years old while playing (non-sport-related games) at turf-like locations and playgrounds.

Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed in the liver and secreted as biliary glycoprotein 1 (BGP1) via bile canaliculi (BCs). CEACAM1-LF is a 72 amino acid cytoplasmic domain mRNA splice isoform with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Ceacam1 or Ser503Ala transgenic mice have been shown to develop insulin resistance and nonalcoholic fatty liver disease; however, the role of the human equivalent residue, Ser508, in lipid dysregulation is unknown.

Phosphorylation of human CEACAM1-LF by PKA and GSK3β promotes its interaction with β-catenin.

CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for β-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the N labeled cytoplasmic domain peptide.

Structural characterization of a dimeric complex between the short cytoplasmic domain of CEACAM1 and the pseudo tetramer of S100A10-Annexin A2 using NMR and molecular dynamics.

AIIt, a heterotetramer of S100A10 (P11) and Annexin A2, plays a key role in calcium dependent, membrane associations with a variety of proteins. We previously showed that AIIt interacts with the short cytoplasmic domain (12 amino acids) of CEACAM1 (CEACAM1-SF). Since the cytoplasmic domains of CEACAM1 help regulate the formation of cis- or trans-dimers at the cell membrane, we investigated the possible role of their association with AIIt in this process.

Preclinical PET Imaging of NTSR-1-Positive Tumors with Cu- and Ga-DOTA-Neurotensin Analogs and Therapy with an Ac-DOTA-Neurotensin Analog.

The aim of the study was to perform PET imaging and radiotherapy with a novel neurotensin derivative for neurotensin receptor 1 (NTSR-1)-positive tumors in an animal model. A di-DOTA analog of NT(6-13) with three unnatural amino acids was synthesized and radiolabeled with either Cu or Ga and tested for serum stability and tumor imaging in mice bearing NTSR-1-positive PC3, and HT29 xenografts. A dose-response therapy study was performed with 18.

Antibody Targeted PET Imaging of Cu-DOTA-Anti-CEA PEGylated Lipid Nanodiscs in CEA Positive Tumors.

Lipid nanodiscs (LNDs), comprising a phospholipid bilayer encircled by two molecules of a recombinant membrane scaffold protein, can be targeted to tumors with covalently attached antibodies (Abs) or their fragments. Antibody attachment to click chemistry based PEGylated lipids on LNDs including DOTA allowed PET imaging with the positron emitter Cu. Carcinoembryonic antigen (CEA) positive tumors in CEA transgenic mice were chosen as a tumor target.

Generation of dual specific bivalent BiTEs (dbBIspecific T-cell engaging antibodies) for cellular immunotherapy.

Background: Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance.

Methods: An intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca.

Synthesis, Positron Emission Tomography Imaging, and Therapy of Diabody Targeted Drug Lipid Nanoparticles in a Prostate Cancer Murine Model.

The blood clearance of chemotherapeutic drugs such as doxorubicin (Dox) can be extended by incorporation into lipid nanoparticles (LNPs) and further improved by tumor targeting with antibody fragments. We used positron emission tomography (PET) imaging in a murine prostate cancer model to evaluate tumor targeting of LNPs incorporating Dox and antiprostate-specific membrane antigen (PSMA) diabodies. Dox-LNPs were generated by mixing or covalent attachment to water soluble distearoylphosphatidyl ethanolamine-polyethylene glycol (DSPE-PEG).

Characterization of 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[Methoxy(polyethylene glycerol)-2000] and Its Complex with Doxorubicin Using Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics.

Polyethylene glycol (PEG) lipid nanoparticles (LNPs) spontaneously assemble in water, forming uniformly sized nanoparticles incorporating drugs with prolonged blood clearance compared to drugs alone. Previously, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycerol)-2000] (DSPE-PEG) and several drug adducts, including doxorubicin, were analyzed by a combination of physical and molecular dynamic (MD) studies. In this study, a complete chemical shift assignment of DSPE-PEG plus or minus doxorubicin was achieved using nuclear magnetic resonance (NMR), one-dimensional selective nuclear Overhauser spectroscopy (1D-selNOESY), NOESY, correlation spectroscopy (COSY), total correlated spectroscopy (TOCSY), heteronuclear single quantum coherence (HSQC), and HSQC-TOCSY.

PET imaging of Cu-DOTA-scFv-anti-PSMA lipid nanoparticles (LNPs): Enhanced tumor targeting over anti-PSMA scFv or untargeted LNPs.

Introduction: Single chain (scFv) antibodies are ideal targeting ligands due to their modular structure, high antigen specificity and affinity. These monovalent ligands display rapid tumor targeting but have limitations due to their fast urinary clearance.

Methods: An anti-prostate membrane antigen (PSMA) scFv with a site-specific cysteine was expressed and evaluated in a prostate cancer xenograft model by Cu-64 PET imaging.

Suberoylanilide hydroxamic acid induces hypersensitivity to radiation therapy in acute myelogenous leukemia cells expressing constitutively active FLT3 mutants.

Histone deacetylase inhibitors (HDIs) have shown promise as candidate radiosensitizer for many types of cancers. However, the mechanisms of action are not well understood, and whether they could have clinical impact on radiotherapy for leukemia is unclear. In this study, we demonstrate that suberoylanilide hydroxamic acid (SAHA) can increase radiosensitivity of acute myeloid leukemia (AML) cells through posttranslational modification of Rad51 protein responses and selective inhibition of the homology-directed repair (HDR) pathway.

Suberoylanilide hydroxamic acid as a radiosensitizer through modulation of RAD51 protein and inhibition of homology-directed repair in multiple myeloma.

Histone deacetylase inhibitors (HDI) have shown promise as candidate radiosensitizers for many types of cancers. However, the mechanisms of action are not well understood, and whether they could sensitize multiple myeloma (MM) to radiation therapy is unclear. In this study, we show that suberoylanilide hydroxamic acid (SAHA) at low concentrations has minimal cytotoxic effects, yet can significantly increase radiosensitivity of MM cells.

Low-dose valproic acid enhances radiosensitivity of prostate cancer through acetylated p53-dependent modulation of mitochondrial membrane potential and apoptosis.

Histone deacetylase inhibitors (HDI) have shown promise as candidate radiosensitizers for many types of cancers, including prostate cancer. However, the mechanisms of action are not well understood. In this study, we show in prostate cancer cells that valproic acid (VPA) at low concentrations has minimal cytotoxic effects yet can significantly increase radiation-induced apoptosis.

HDAC inhibitor, valproic acid, induces p53-dependent radiosensitization of colon cancer cells.

Agents that inhibit histone deacetylases (HDAC inhibitors) have been shown to enhance radiation response. The aim of this study was to evaluate the effects of low, minimally cytotoxic concentrations of the HDAC inhibitor, valproic acid (VPA), on radiation response of colorectal cancer cells. Cell lines LS174T and an isogenic pair of HCT116, which differed only for the presence of wild-type p53, were exposed to ionizing radiation (IR) alone, VPA alone, or the combination.

Structure-activity relationship for noncoplanar polychlorinated biphenyl congeners toward the ryanodine receptor-Ca2+ channel complex type 1 (RyR1).

Ryanodine receptor isoforms are expressed in both excitable and nonexcitable tissues where they form microsomal Ca2+ release channels broadly involved in shaping cellular signaling. In this report, we provide a detailed structure-activity relationship (SAR) for polychlorinated biphenyl (PCB) congeners and metabolites necessary for enhancing ryanodine receptor type 1 (RyR1) activity using [3H]ryanodine ([3H]Ry) binding analysis. The 2,3,6-Cl PCB configuration is most important for optimal recognition by the RyR1 complex and/or critical for sensitizing its activation.

Expression of ErbB2 enhances radiation-induced NF-kappaB activation.

Her-2/neu (ErbB2) oncogene, the second member of the epidermal growth factor receptor (EGFR) family, encodes a transmembrane tyrosine kinase receptor in Her-2-positive tumors. Accumulating evidences demonstrate that signaling networks activated by EGFR and transcription factor NF-kappaB are associated with cell response to ionizing radiation (IR). The present study shows that overexpression of ErbB2 enhanced NF-kappaB activation induced by IR in human breast carcinoma MCF-7 cells transfected with ErbB2 genes (MCF-7/ErbB2).

Non-coplanar 2,2',3,5',6-pentachlorobiphenyl (PCB 95) amplifies ionotropic glutamate receptor signaling in embryonic cerebellar granule neurons by a mechanism involving ryanodine receptors.

The mechanisms by which non-coplanar 2,2',3,5',6-pentachlorobiphenyl (PCB 95) and rapamycin interact with ryanodine receptor (RyR) complexes to alter Ca2+ signaling, were explored in intact cerebellar granule neurons. PCB 95 (10 microM, 20 min) significantly increased the number of neurons responding to caffeine. PCB 95 sensitization of RyR-mediated responses was further supported by the observations that ryanodine pretreatment blocked response to caffeine and coplanar 2,4,4',5-tetrachlorobiphenyl (PCB 66), which lacks RyR activity, failed to sensitize neurons.