Laird Miers

Development of Tumor Targeting Bioprobes (111In-Chimeric L6 Monoclonal Antibody Nanoparticles) for Alternating Magnetic Field Cancer Therapy

Objectives:111In-chimeric L6 (ChL6) monoclonal antibody (mAb)–linked iron oxide nanoparticle (bioprobes) pharmacokinetics, tumor uptake, and the therapeutic effect of inductively heating these bioprobes by externally applied alternating magnetic field (AMF) were studied in athymic mice bearing human breast cancer HBT 3477 xenografts. Tumor cell radioimmunotargeting of the bioprobes and therapeutic and toxic responses were determined. Methods: Using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl, 111In-7,10-tetra-azacyclododecane-N, N′,N″,N‴-tetraacetic acid-ChL6 was conjugated to the carboxylated polyethylene glycol on dextran-coated iron oxide 20 nm particles, one to two mAbs per nanoparticle. After magnetic purification and sterile filtration, pharmacokinetics, histopathology, and AMF/bioprobe therapy were done using 111In-ChL6 bioprobe doses (20 ng/2.2 mg ChL6/ bioprobe), i.v. with 50 μg ChL6 in athymic mice bearing HBT 3477; a 153 kHz AMF was given 72 hours postinjection for therapy with amplitudes of 1,300, 1,000, or 700 Oe. Weights, blood counts, and tumor size were monitored and compared with control mice receiving nothing, or AMF or bioprobes alone. Results:111In-ChL6 bioprobe binding in vitro to HBT 3477 cells was 50% to 70% of that of 111In-ChL6. At 48 hours, tumor, lung, kidney, and marrow uptakes of the 111In-ChL6 bioprobes were not different from that observed in prior studies of 111In-ChL6. Significant therapeutic responses from AMF/bioprobe therapy were shown with up to eight times longer mean time to quintuple tumor volume with therapy compared with no treatment (P = 0.0013). Toxicity was only seen in the 1,300 Oe AMF cohort, with 4 of 12 immediate deaths and skin erythema. Electron micrographs showed bioprobes on the surfaces of the HBT 3477 cells of excised tumors and tumor necrosis 24 hours after AMF/bioprobe therapy. Conclusion: This study shows that mAb-conjugated nanoparticles (bioprobes), when given i.v., escape into the extravascular space and bind to cancer cell membrane antigen, so that bioprobes can be used in concert with externally applied AMF to deliver thermoablative cancer therapy.

Pyramidal Neurons Are Generated from Oligodendroglial Progenitor Cells in Adult Piriform Cortex

Previous studies have shown that oligodendroglial progenitor cells (OPCs) can give rise to neurons in vitro and in perinatal cerebral cortex in vivo. We now report that OPCs in adult murine piriform cortex express low levels of doublecortin, a marker for migratory and immature neurons. Additionally, these OPCs express Sox2, a neural stem cell marker, and Pax6, a transcription factor characteristic of progenitors for cortical glutamatergic neurons. Genetic fate-mapping by means of an inducible Cre–LoxP recombination system proved that these OPCs differentiate into pyramidal glutamatergic neurons in piriform cortex. Several lines of evidence indicated that these newly formed neurons became functionally integrated into the cortical neuronal network. Our data suggest that NG2+/PDGFRα+ proteolipid protein promoter-expressing progenitors generate pyramidal glutamatergic neurons within normal adult piriform cortex.

Initiation and Progression of Axonopathy in Experimental Autoimmune Encephalomyelitis

Axonal loss is the principal cause of chronic disability in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). In C57BL/6 mice with EAE induced by immunization with myelin oligodendrocyte glycoprotein peptide 35–55, the first evidences of axonal damage in spinal cord were in acute subpial and perivascular foci of infiltrating neutrophils and lymphocytes and included intra-axonal accumulations of the endovesicular Toll-like receptor TLR8, and the inflammasome protein NAcht leucine-rich repeat protein 1 (NALP1). Later in the course of this illness, focal inflammatory infiltrates disappeared from the spinal cord, but there was persistent activation of spinal cord innate immunity and progressive, bilaterally symmetric loss of small-diameter corticospinal tract axons. These results support the hypothesis that both contact-dependent and paracrine interactions of systemic inflammatory cells with axons and an innate immune-mediated neurodegenerative process contribute to axonal loss in this multiple sclerosis model.

Pharmacokinetic Characterization in Xenografted Mice of a Series of First-Generation Mimics for HLA-DR Antibody, Lym-1, as Carrier Molecules to Image and Treat Lymphoma

Despite their large size, antibodies (Abs) are suitable carriers to deliver systemic radiotherapy, often molecular image–based, for lymphoma and leukemia. Lym-1 Ab has proven to be an effective radioisotope carrier, even in small amounts, for targeting human leukocyte antigen DR (HLA-DR), a surface membrane protein overexpressed on B-cell lymphoma. Pairs of molecules (referred to as ligands), shown by computational and experimental methods to bind to each of 2 sites within the Lym-1 epitopic region, have been linked to generate small (<2 kDa) molecules (referred to as selective high-affinity ligands [SHALs]) to mimic the targeting properties of Lym-1 Ab. Methods: A lysine-polyethylene glycol (PEG) backbone was used to synthetically link 2 of the following ligands: deoxycholate, 5-leuenkephalin, triiodothyronine, thyronine, dabsyl-l-valine, and N-benzoyl-l-arginyl-4-amino-benzoic acid to generate a series of 13 bidentate SHALs with a biotin or 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA) chelate attached to the linker. These SHALs have been assessed for their selectivity in binding to HLA-DR10–expressing cells and for their pharmacokinetics and tissue biodistribution in mice. Biotinylated versions of these SHALs discriminated cell lines positive for HLA-DR10 expression with near-nanomolar affinity. The DOTA versions of 4 SHALs were labeled with 111In for pharmacokinetic studies in mice with HLA-DR10–expressing malignant Raji xenografts. Results: The bidentate, biotinylated, and DOTA-SHALs were synthesized in high-purity, multimilligram amounts. Mean radiochemical and product yields and purities were 90%, 75%, and 90% at mean specific activities of 3.9 MBq/μg (105 μCi/μg) for the 111In-labeled SHALs. As expected, rapid blood clearance and tumor targeting were observed. The pharmacokinetics of the SHALs was influenced by the component ligands. Biliary clearance, kidney localization, and serum receptor binding contributed to less favorable tumor targeting. Conclusion: A series of SHALs was readily synthesized in multimilligram amounts and showed the expected selective binding in vitro. Better selection of the SHAL components should provide second-generation SHALs with improved properties to fulfill the substantial potential of these novel molecular carriers for targeting.

Comparative toxicity studies of yttrium-90 mx-dtpa and 2-it-bad conjugated monoclonal antibody (bre-3)

Background. BrE‐3 is a monoclonal antibody that has promise for imaging and therapy of human adenocarcinoma. Because of observations in therapeutic trials of yttrium‐90 (90Y) escape from radioimmunoconjugates and uptake by the skeleton with resultant bone marrow toxicity, the authors attempted to evaluate the importance of this factor by a comparison of the LD50 in healthy mice treated with 90Y that had been chelated with either of two high affinity chelators, methylbenzyldiethylene‐triaminepentaacetic acid (MX‐DTPA) or bromoacetamidobenzyl‐1,4,7,10‐tetraazocyclododecane‐N,N′,N″,N″‐tetraacetic acid (BAD).

Disruption of NMDA Receptors in Oligodendroglial Lineage Cells Does Not Alter Their Susceptibility to Experimental Autoimmune Encephalomyelitis or Their Normal Development

Pharmacological studies have suggested that oligodendroglial NMDA glutamate receptors (NMDARs) mediate white matter injury in a variety of CNS diseases, including multiple sclerosis (MS). We tested this hypothesis in experimental autoimmune encephalomyelitis (EAE), a model of human MS, by timed conditional disruption of oligodendroglial NR1, an essential subunit of functional NMDARs, using an inducible proteolipid protein (Plp) promoter-driven Cre-loxP recombination system. We found that selective ablation of oligodendroglial NR1 did not alter the clinical severity of EAE elicited in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein peptide 35–55 (MOG-peptide), nor were there significant differences between the oligodendroglial NR1 KO and non-KO mice in numbers of axons lost in spinal cord dorsal funiculi or severity of spinal cord demyelination. Similarly, constitutive deletion of NR3A, a modulatory subunit of oligodendroglial NMDARs, did not alter the course of MOG-peptide EAE. Furthermore, conditional and constitutive ablation of NR1 in neonatal oligodendrocyte progenitor cells did not interrupt their normal maturation and differentiation. Collectively, our data suggest that oligodendroglial lineage NMDARs are neither required for timely postnatal development of the oligodendroglial lineage, nor significant participants in the pathophysiology of MOG-peptide EAE.

Conditional Ablation of Astroglial CCL2 Suppresses CNS Accumulation of M1 Macrophages and Preserves Axons in Mice with MOG Peptide EAE

Current multiple sclerosis (MS) therapies only partially prevent chronically worsening neurological deficits, which are largely attributable to progressive loss of CNS axons. Prior studies of experimental autoimmune encephalomyelitis (EAE) induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein peptide 35–55 (MOG peptide), a model of MS, documented continued axon loss for months after acute CNS inflammatory infiltrates had subsided, and massive astroglial induction of CCL2 (MCP-1), a chemokine for CCR2+ monocytes. We now report that conditional deletion of astroglial CCL2 significantly decreases CNS accumulation of classically activated (M1) monocyte-derived macrophages and microglial expression of M1 markers during the initial CNS inflammatory phase of MOG peptide EAE, reduces the acute and long-term severity of clinical deficits and slows the progression of spinal cord axon loss. In addition, lack of astroglial-derived CCL2 results in increased accumulation of Th17 cells within the CNS in these mice, but also in greater confinement of CD4+ lymphocytes to CNS perivascular spaces. These findings suggest that therapies designed to inhibit astroglial CCL2-driven trafficking of monocyte-derived macrophages to the CNS during acute MS exacerbations have the potential to significantly reduce CNS axon loss and slow progression of neurological deficits.

Combined modality radioimmunotherapy. Promise and peril.

Single‐agent radioimmunotherapy (RIT), although potentially useful for slowing solid tumor growth, has not been effective in curing aggressive tumors, such as breast cancer. These cancers typically have p53 mutations and are less susceptible to apoptosis, the apparent mechanism of cell death from low dose‐rate radiation. Thus, synergistic or combined modality radioimmunotherapy (CMRIT) agents are needed to increase radiosensitivity for therapeutic enhancement without additive toxicity.

Macroglial Plasticity and the Origins of Reactive Astroglia in Experimental Autoimmune Encephalomyelitis

Accumulations of hypertrophic, intensely glial fibrillary acidic protein-positive (GFAP+) astroglia, which also express immunoreactive nestin and vimentin, are prominent features of multiple sclerosis lesions. The issues of the cellular origin of hypertrophic GFAP+/vimentin+/nestin+ “reactive” astroglia and also the plasticities and lineage relationships among three macroglial progenitor populations—oligodendrocyte progenitor cells (OPCs), astrocytes and ependymal cells—during multiple sclerosis and other CNS diseases remain controversial. We used genetic fate-mappings with a battery of inducible Cre drivers (Olig2-Cre-ERT2, GFAP-Cre-ERT2, FoxJ1-Cre-ERT2 and Nestin-Cre-ERT2) to explore these issues in adult mice with myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis (EAE). The proliferative rate of spinal cord OPCs rose fivefold above control levels during EAE, and numbers of oligodendroglia increased as well, but astrogenesis from OPCs was rare. Spinal cord ependymal cells, previously reported to be multipotent, did not augment their low proliferative rate, nor give rise to astroglia or OPCs. Instead, the hypertrophic, vimentin+/nestin+, reactive astroglia that accumulated in spinal cord in this multiple sclerosis model were derived by proliferation and phenotypic transformation of fibrous astroglia in white matter, and solely by phenotypic transformation of protoplasmic astroglia in gray matter. This comprehensive analysis of macroglial plasticity in EAE helps to clarify the origins of astrogliosis in CNS inflammatory demyelinative disorders.

Ablating N-Acetylaspartate Prevents Leukodystrophy in a Canavan Disease Model

Canavan disease is caused by inactivating ASPA (aspartoacylase) mutations that prevent cleavage of N‐acetyl‐L‐aspartate (NAA), resulting in marked elevations in central nervous system (CNS) NAA and progressively worsening leukodystrophy. We now report that ablating NAA synthesis by constitutive genetic disruption of Nat8l (N‐acetyltransferase‐8 like) permits normal CNS myelination and prevents leukodystrophy in a murine Canavan disease model. Ann Neurol 2015;77:884–888