Selective depletion of radiolabeled HER2-specific antibody for contrast improvement during PET.

The prolonged in vivo persistence of antibodies results in high background and poor contrast during their use as molecular imaging agents for positron emission tomography (PET). We have recently described a class of engineered Fc fusion proteins that selectively deplete antigen-specific antibodies without affecting the levels of antibodies of other specificities. Here, we demonstrate that these Fc fusions (called Seldegs, for selective degradation) can be used to clear circulating, radiolabeled HER2-specific antibody during diagnostic imaging of HER2-positive tumors in mice. The analyses show that Seldegs have considerable promise for the reduction of whole-body exposure to radiolabel and improvement of contrast during PET.

Selective Depletion of Antigen-Specific Antibodies for the Treatment of Demyelinating Disease.

Current treatments for antibody-mediated autoimmunity are associated with lack of specificity, leading to immunosuppressive effects. To overcome this limitation, we have developed a class of antibody-based therapeutics for the treatment of autoimmunity involving antibodies that recognize the autoantigen, myelin oligodendrocyte glycoprotein (MOG). These agents ("Seldegs," for selective degradation) selectively eliminate antigen (MOG)-specific antibodies without affecting the levels of antibodies of other specificities. Seldeg treatment of mice during antibody-mediated exacerbation of experimental autoimmune encephalomyelitis by patient-derived MOG-specific antibodies results in disease amelioration. Consistent with their therapeutic effects, Seldegs deliver their targeted antibodies to Kupffer and liver sinusoidal endothelial cells that are known to have tolerogenic effects. Our results show that Seldegs can ameliorate disease mediated by MOG-specific antibodies and indicate that this approach also has the potential to treat other autoimmune diseases where the specific clearance of antibodies is required.

Engineering a HER2-specific antibody-drug conjugate to increase lysosomal delivery and therapeutic efficacy.

We improve the potency of antibody-drug conjugates (ADCs) containing the human epidermal growth factor receptor 2 (HER2)-specific antibody pertuzumab by substantially reducing their affinity for HER2 at acidic endosomal pH relative to near neutral pH. These engineered pertuzumab variants show increased lysosomal delivery and cytotoxicity towards tumor cells expressing intermediate HER2 levels. In HER2int xenograft tumor models in mice, the variants show higher therapeutic efficacy than the parent ADC and a clinically approved HER2-specific ADC.

Myelin oligodendrocyte glycoprotein-specific antibodies from multiple sclerosis patients exacerbate disease in a humanized mouse model.

Myelin oligodendrocyte glycoprotein (MOG) is exposed on the outer surface of the myelin sheath, and as such, represents a possible target antigen for antibodies in multiple sclerosis (MS) and other demyelinating diseases. However, despite extensive analyses, whether MOG-specific antibodies contribute to pathogenesis in human MS remains an area of uncertainty. In the current study we demonstrate that antibodies derived from adult MS patients exacerbate experimental autoimmune encephalomyelitis (EAE) in 'humanized' mice that transgenically express human FcγRs (hFcγRs). Importantly, this exacerbation is dependent on MOG recognition by the human-derived antibodies. The use of mice that express hFcγRs has allowed us to also investigate the contribution of these receptors to disease in the absence of confounding effects of cross-species differences. Specifically, by engineering the Fc region of MOG-specific antibodies to modulate FcγR and complement (C1q) binding, we reveal that FcγRs but not complement activation contribute to EAE pathogenesis. Importantly, selective enhancement of the affinities of these antibodies for specific FcγRs reveals that FcγRIIA is more important than FcγRIIIA in mediating disease exacerbation. These studies not only provide definitive evidence for the contribution of MOG-specific antibodies to MS, but also reveal mechanistic insight that could lead to new therapeutic targets.

Engineered clearing agents for the selective depletion of antigen-specific antibodies.

Here we have designed a novel class of engineered antibody-based reagents ('Seldegs') that induce the selective degradation of antigen-specific antibodies. We demonstrate the rapid and specific clearance of antibodies recognizing the autoantigen, myelin oligodendrocyte glycoprotein and tumour target, HER2. Seldegs have considerable potential in multiple areas, including the treatment of antibody-mediated autoimmunity and diagnostic imaging.

Loss of expression of the recycling receptor, FcRn, promotes tumor cell growth by increasing albumin consumption.

Tumor cells rely on high concentrations of amino acids to support their growth and proliferation. Although increased macropinocytic uptake and lysosomal degradation of the most abundant serum protein, albumin, in Ras-transformed cells can meet these demands, it is not understood how the majority of tumor cells that express wild type Ras achieve this. In the current study we reveal that the neonatal Fc receptor, FcRn, regulates tumor cell proliferation through the ability to recycle its ligand, albumin. By contrast with normal epithelial cells, we show that human FcRn is present at very low or undetectable levels in the majority of tumor cell lines analyzed. Remarkably, shRNA-mediated ablation of FcRn expression in an FcRn-positive tumor cell line results in a substantial growth increase of tumor xenografts, whereas enforced expression of this receptor by lentiviral transduction has the reverse effect. Moreover, intracellular albumin and glutamate levels are increased by the loss of FcRn-mediated recycling of albumin, combined with hypoalbuminemia in tumor-bearing mice. These studies identify a novel role for FcRn as a suppressor of tumor growth and have implications for the use of this receptor as a prognostic indicator and therapeutic target.

Gonadotropin and tumorigenesis: Direct and indirect effects on inflammatory and immunosuppressive mediators and invasion.

Human chorionic gonadotropin (hCG), a hormone essential for pregnancy, is also ectopically expressed by a variety of cancers and is associated with poor prognosis; molecular mechanisms which may contribute to tumor progression remain ill-defined. Exogenous hCG enhanced the viability of human colorectal and lung cancer cells and promoted the growth of syngeneic tumors in mice. It induced the synthesis of VEGF, IL-8, matrix metalloprotease (MMP)-2 and MMP-9, and increased invasiveness in an MMP-dependent manner. While inducing the secretion of the tumor-associated extra-cellular matrix proteoglycan versican from tumor cells, hCG consequently caused the TLR-2-mediated generation of the inflammatory, tumor-associated cytokines TNF-α and IL-6 from peripheral blood adherent cells. The molecule up-modulated the Treg-associated transcription factor FOXP3 in tumor cells and increased the secretion of TGFß and IL-10, thereby inhibiting T cell proliferation and inducing the differentiation FOXP3- CD4+ CD25- cells into functional FOXP3+ CD4+ CD25+ suppressor cells. Co-culture of hCG-treated tumor cells with mature bone-marrow derived dendritic cells induced the generation of active indoleamine deoxygenase. While anti-hCG antibodies restricted the growth of implanted tumor cells in nude mice, immunization of immune competent mice with a ßhCG-TT conjugate supplemented with Mycobacterium indicus pranii provided synergistic survival benefit in animals implanted with syngeneic, hCG-responsive tumor cells. These studies elucidate the pathways by which hCG can promote tumorigenesis, providing further rationale for anti-hCG vaccination in the treatment of gonadotropin-sensitive tumors. © 2016 Wiley Periodicals, Inc.

Size dependent toxicity of zinc oxide nano-particles in soil nematode Caenorhabditis elegans.

Zinc oxide nano-particles (ZnO NPs), with their unique physico-chemical properties conferred by various size formulations, are extensively used in consumer products. The enormous usage coupled with their release to the environment demands risk assessment of ZnO NPs on health and the environment. Toxicity of ZnO NPs is well understood in comparison to the bulk ZnO. However, toxicity in relation to the NP size is poorly understood. In this context, we examined the adverse effects of different sizes (35 nm, 50 nm and 100 nm) of ZnO NPs in soil nematode C. elegans along with bulk ZnO and ZnCl2. Here, we show that growth, reproduction and behavior of worms were adversely affected by ZnO NPs in a size dependent manner. Further, exposure to ZnO NPs caused modulation of expression/function of genes associated with Insulin/IGF-like signaling pathway and/or stress response pathway in a size dependent manner in exposed worms. The expression of pro-apoptotic gene and suppression of anti-apoptotic genes, together with increased numbers of cell corpses in the germ line, indicated that apoptosis was also dependent on the size of the ZnO NP. Taken together, our study provides evidence that exposure to ZnO NPs disrupts various physiological processes and causes apoptosis in the germ-line even at very low concentration in a size dependent manner. Our finding suggests the inclusion of size as an additional measure for the cautious monitoring of ZnO NP disposal into the environment.

Inhibitory effect of antibodies against human chorionic gonadotropin on the growth of colorectal tumour cells.

Human chorionic gonadotropin (hCG) was initially believed to be secreted exclusively by the embryo with its primary function being "rescue" of the corpus luteum. However, recently it has been found that the hormone (or its individual subunits) is also secreted by many cancers and that in many cases secretion is associated with poor patient prognosis. In this study, we assessed the presence of hCG in colorectal cancer cells (CCL-253) and evaluated the anti-tumour effects of anti-hCG antibodies in vitro and in vivo. Anti-hCG antibodies were reactive with CCL-253, as revealed by confocal immunoflourescence microscopy; both cell surface and intracellular expression were observed. Western blot analysis showed that antibodies appeared to interact with several moieties, indicating a level of cross-reactivity. Anti-hCG antiserum specifically reduced the viability of tumor cells and the addition of complement increased in vitro anti-tumor effects. In nude mice implanted with CCL-253 cells, administration of anti-hCG antiserum caused a significant reduction in tumor volume; all treated animals survived, while mortality was observed in control animals. Results suggest that anti-hCG antibodies can mediate significant anti-tumor activity both in vitro and in vivo and lend support to the rationale of anti-hCG immunization in the therapy of gonadotropin- sensitive cancers.

Adverse effects of TiO2 and ZnO nanoparticles in soil nematode, Caenorhabditis elegans.

During the last decade, advancement of nanotechnology has revolutionized various fields such as electronics, optics, materials science as well as architecture, and medicine. However, their health and environmental impact is not fully understood. TiO2 and ZnO nanoparticles (NPs), which are abundantly used for commercial purposes, pose a great risk to environment. In this context, we examined the adverse effects of TiO2 and ZnO NPs of < 25 nm and < 100 nm sizes to nematode Caenorhabditis elegans. < 25 nm TiO2 and ZnO NPs showed LC50 of 77 mg/L and 0.32 mg/L respectively, while < 100 nm TiO2 NPs were non-toxic and LC50 of 2 mg/L was obtained for < 100 nm ZnO NPs. Our studies indicate that in both cases, smaller particle sizes are more toxic than larger NPs and ZnO NPs are more toxic than TiO2 NPs. Further, we observed low LC50 values for ZnO NPs, probably reflecting the sensitivity of C. elegans as a model for eco-toxicity studies of NPs.

Characterization of developmental neurotoxicity of As, Cd, and Pb mixture: synergistic action of metal mixture in glial and neuronal functions.

Neurotoxicity of individual metals is well investigated but that of metal mixture (MM), an environmental reality, in the developing brain is relatively obscure. We investigated the combinatorial effect of arsenic (As), cadmium (Cd), and lead (Pb) on rat brain development, spanning in utero to postnatal development. MM was administered by gavage to pregnant and lactating rats, and to postweaning pups till 2 months. The pups exhibited behavioral disturbances characterized by hyperlocomotion, increased grip strength, and learning-memory deficit. Disruption of the blood-brain barrier (BBB) was associated with dose-dependent increase in deposition of the metals in developing brain. Astrocytes were affected by MM treatment as evident from their reduced density, area, perimeter, compactness, and number of processes, and increased apoptosis in cerebral cortex and cerebellum. The metals induced synergistic reduction in glial fibrillary acidic protein (GFAP) expression during brain development; however, postweaning withdrawal of MM partially restored the levels of GFAP in adults. To characterize the toxic mechanism, we treated rat primary astrocytes with MM at concentrations ranging from lethal concentration (LC)(10) to LC(75) of the metals. We observed synergistic downregulation in viability and increase in apoptosis of the astrocytes, which were induced by proximal activation of extra cellular signal-regulated kinase (ERK) signaling and downstream activation of Jun N-terminal kinase (JNK) pathway. Furthermore, rise in intracellular calcium ion ([Ca(2+)](i)) and reactive oxygen species generation promoted apoptosis in the astrocytes. Taken together, these observations are the first to show that mixture of As, Cd, and Pb has the capacity to induce synergistic toxicity in astrocytes that may compromise the BBB and may cause behavioral dysfunction in developing rats.

Anti-apoptotic role of omega-3-fatty acids in developing brain: perinatal hypothyroid rat cerebellum as apoptotic model.

Inadequate maternal intake of omega-3-fatty acids (omega3 FAs) causes adverse neurodevelopmental outcome in the progeny; however, their molecular mechanism of action is obscure. Since omega3 FAs are known to inhibit neuronal apoptosis during neuro-degeneration, we investigated their possible contribution in regulating neuronal apoptosis during brain development. Using rat model of hypothyroidism-induced neuronal apoptosis, we provide evidence for anti-apoptotic role of omega3 FAs during cerebellar development. omega3 FAs were supplemented as a mixture of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to pregnant and lactating rats, and primary hypothyroidism was induced by administering methimazole. The cerebella from postnatal day 16 (d16) pups were isolated, and studies on apoptosis were conducted. We observed that omega3 FA-supplementation significantly reduced DNA fragmentation and caspase-3 activation in developing cerebellum of hypothyroid pups. The protection provided by omega3 FAs was associated with their ability to prevent increases in the level of pro-apoptotic basal cell lymphoma protein-2 (Bcl-2)-associated X protein (Bax) in the cerebellum during thyroid hormone (TH) deficiency. omega3 FAs increased the levels of anti-apoptotic proteins like Bcl-2 and Bcl-extra large (Bcl-x(L)), known to be repressed in hypothyroidism. omega3 FAs also restored levels of cerebellar phospho (p)-AKT, phospho-extracellular regulated kinase (p-ERK) and phospho-c-Jun N-terminal kinase (p-JNK), which were altered by hypothyroid insults, without interfering with the expression of TH responsive gene, myelin basic protein (mbp). Taken together, these results supplement an insight into the molecular mechanism of action of omega3 FAs in developing brain that involves regulation of apoptotic signaling pathways under stress.