技术资料

MUC16 is a well-validated cell surface marker for serous adenocarcinomas of the ovary and other gynecologic malignancies that is distinguished by highly repetitive sequences (mucin repeats") in the extracellular domain (ECD). We produced and compared two monoclonal antibodies: one (11D10) recognizing a unique� View Publication

Interferon-gamma (IFN-γ) is a pleiotropic cytokine that exerts anti-tumor and anti-osteoclastogenic effects. Although transcriptional and post-transcriptional regulation of IFN-γ is well understood,subsequent modifications of secreted IFN-γ are not fully elucidated. Previous research indicates that some cancer cells escape immune surveillance and metastasize into bone tissue by inducing osteoclastic bone resorption. Peptidases of the a-disintegrin and metalloproteinase (ADAM) family are implicated in cancer cell proliferation and tumor progression. We hypothesized that the ADAM enzymes expressed by cancer cells degrades IFN-γ and attenuates IFN-γ-mediated anti-tumorigenic and anti-osteoclastogenic effects. Recombinant ADAM17 degraded IFN-γ into small fragments. The addition of ADAM17 to the culture supernatant of stimulated mouse splenocytes decreased IFN-γ concentration. However,ADAM17 inhibition in the stimulated mouse T-cells prevented IFN-γ degradation. ADAM17-expressing human breast cancer cell lines MCF-7 and MDA-MB-453 also degraded recombinant IFN-γ,but this was attenuated by ADAM17 inhibition. Degraded IFN-γ lost the functionality including the inhibititory effect on osteoclastogenesis. This is the first study to demonstrate the extracellular proteolytic degradation of IFN-γ by ADAM17. These results suggest that ADAM17-mediated degradation of IFN-γ may block the anti-tumorigenic and anti-osteoclastogenic effects of IFN-γ. ADAM17 inhibition may be useful for the treatment of attenuated cancer immune surveillance and/or bone metastases. View Publication

In February 2013,zoonotic transmission of a novel influenza A virus of the H7N9 subtype was reported in China. Although at present no sustained human-to-human transmission has been reported,a pandemic outbreak of this H7N9 virus is feared. Since neutralizing antibodies to the hemagglutinin (HA) globular head domain of the virus are virtually absent in the human population,there is interest in identifying other correlates of protection,such as cross-reactive CD8(+) T cells (cytotoxic T lymphocytes [CTLs]) elicited during seasonal influenza A virus infections. These virus-specific CD8(+) T cells are known to recognize conserved internal proteins of influenza A viruses predominantly,but it is unknown to what extent they cross-react with the newly emerging H7N9 virus. Here,we assessed the cross-reactivity of seasonal H3N2 and H1N1 and pandemic H1N1 influenza A virus-specific polyclonal CD8(+) T cells,obtained from HLA-typed study subjects,with the novel H7N9 virus. The cross-reactivity of CD8(+) T cells to H7N9 variants of known influenza A virus epitopes and H7N9 virus-infected cells was determined by their gamma interferon (IFN-γ) response and lytic activity. It was concluded that,apart from recognition of individual H7N9 variant epitopes,CD8(+) T cells to seasonal influenza viruses display considerable cross-reactivity with the novel H7N9 virus. The presence of these cross-reactive CD8(+) T cells may afford some protection against infection with the new virus. View Publication

During drug development,measurement of suitable pharmacodynamic biomarkers is key to establishing in vivo drug activity. Binding of monoclonal antibody (mAb) therapeutics to soluble target proteins often results in elevated serum levels of their target antigen,and measuring total (free and bound) concentration of the target antigen can be an important means of demonstrating that the mAb has reached its specific target. However,accurately measuring soluble circulating antigen in preclinical or clinical samples in the presence of a therapeutic mAb presents a bioanalytical challenge. Particularly in the case of low molecular weight and/or multimeric targets,epitopes for capture and detection of the target by reagent antibodies can be obscured by bound therapeutic mAb. Lymphotoxin-alpha (LTα) is a cytokine in the TNF superfamily that has been implicated in the pathophysiology of autoimmune disease,and is a therapeutic target for neutralizing mAb. During preclinical safety studies in cynomolgus macaques,we encountered difficulties in measuring total LTα in serum of dosed animals. When serum LTα trimer was saturated with the anti-LTα mAb,binding of two reagent antibodies,as required for a classic sandwich ELISA,was not feasible,and dissociation methods were also found to be unsuitable. We therefore developed an approach in which excess anti-LTα mAb was added to the in vitro assay system to fully saturate all binding sites,and an anti-idiotypic antibody was used to detect bound therapeutic antibody. Using this method,total LTα could be accurately measured in cynomolgus macaque serum,and was observed to increase with increasing anti-LTα therapeutic mAb dose. Additional in vitro studies demonstrated that the method worked equally well in human serum. This assay strategy will be useful for quantifying total concentrations of other small and/or multimeric target proteins in the presence of a therapeutic antibody. View Publication

The specificity and affinity of antibody-antigen interactions is a fundamental way to achieve reliable biosensing responses. Different proteins involved with dry eye dysfunction: ANXA1,ANXA11,CST4,PRDX5,PLAA and S100A6; were validated as biomarkers. In this work several antibodies were tested for ANXA1,ANXA11 and PRDX5 to select the best candidates for each biomarker. The results were obtained by using Biophotonic Sensing Cells (BICELLs) as an efficient methodology for label-free biosensing and compared with the Enzyme-Linked Immuno Sorbent Assay (ELISA) technique. View Publication

Chikungunya virus (CHIKV) is a medically important human viral pathogen that causes Chikungunya fever accompanied with debilitating and persistent joint pain. Host-elicited or passively-transferred monoclonal antibodies (mAb) are essential mediators of CHIKV clearance. Therefore,this study aimed to generate and characterize a panel of mAbs for their neutralization efficacy against CHIKV infection in a cell-based and murine model. To evaluate their antigenicity and neutralization profile,indirect enzyme-linked immunosorbent assay (ELISA),an immunofluorescence assay (IFA) and a plaque reduction neutralization test were performed on mAbs of IgM isotype. CHIKV escape mutants against mAb 3E7b neutralization were generated,and reverse genetics techniques were then used to create an infectious CHIKV clone with a single mutation. 3E7b was also administered to neonate mice prior or after CHIKV infection. The survival rate,CHIKV burden in tissues and histopathology of the limb muscles were evaluated. Both IgM 3E7b and 8A2c bind strongly to native CHIKV surface and potently neutralize CHIKV replication. Further analyses of 3E7b binding and neutralization of CHIKV single-mutant clones revealed that N218 of CHIKV E2 protein is a potent neutralizing epitope. In a pre-binding neutralization assay,3E7b blocks CHIKV attachment to permissive cells,possibly by binding to the surface-accessible E2-N218 residue. Prophylactic administration of 3E7b to neonate mice markedly reduced viremia and protected against CHIKV pathogenesis in various mice tissues. Given therapeutically at 4 h post-infection,3E7b conferred 100% survival rate and similarly reduced CHIKV load in most mice tissues except the limb muscles. Collectively,these findings highlight the usefulness of 3E7b for future prophylactic or epitope-based vaccine design. View Publication

Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover,these practices are molecule-specific and so only support one assay for one program at a time. Here,we describe a strategy to generate a unique assay reagent,10C4,that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This panel-specific" feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational� View Publication

Endogenous molecular and cellular mediators modulate tissue repair and regeneration. We have recently described antibody mediated osseous regeneration (AMOR) as a novel strategy for bioengineering bone in rat calvarial defect. This entails application of anti-BMP-2 antibodies capable of in vivo capturing of endogenous osteogenic BMPs (BMP-2,BMP-4,and BMP-7). The present study sought to investigate the feasibility of AMOR in other animal models. To that end,we examined the efficacy of a panel of anti-BMP-2 monoclonal antibodies (mAbs) and a polyclonal Ab immobilized on absorbable collagen sponge (ACS) to mediate bone regeneration within rabbit calvarial critical size defects. After 6 weeks,de novo bone formation was demonstrated by micro-CT imaging,histology,and histomorphometric analysis. Only certain anti-BMP-2 mAb clones mediated significant in vivo bone regeneration,suggesting that the epitopes with which anti-BMP-2 mAbs react are critical to AMOR. Increased localization of BMP-2 protein and expression of osteocalcin were observed within defects,suggesting accumulation of endogenous BMP-2 and/or increased de novo expression of BMP-2 protein within sites undergoing bone repair by AMOR. Considering the ultimate objective of translation of this therapeutic strategy in humans,preclinical studies will be necessary to demonstrate the feasibility of AMOR in progressively larger animal models. View Publication

OBJECTIVE: In this study,the alternative splicing product of vasohibin 1 (VASH1B) was analyzed in direct comparison to the major isoform (VASH1A) for antiangiogenic effects on endothelial colony forming cells (ECFCs) from peripheral blood and on human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: Expression studies in primary human endothelial cells revealed that both vasohibin proteins,hVASH1A and hVASH1B,localized in the nucleus and cytoplasm. Adenoviruses carrying the cDNA for VASH1A/B and purified recombinant proteins were used to study the function of both molecules in ECFCs and HUVECs. Recombinant VASH1A protein did not inhibit cell proliferation,tube formation,or vessel growth in vivo in the chick chorioallantoic membrane (CAM) assay,but promoted endothelial cell migration in vitro. The VASH1B protein had an inhibitory effect on cell proliferation,migration,tube formation,and inhibited blood vessel formation in the CAM assay. Adenoviral overexpression of VASH1B,but not of VASH1A,resulted in inhibition of endothelial cell growth,migration,and capillary formation. Interestingly,overexpression of VASH1A and B induced apoptosis in proliferating human fibroblasts,but did not affect cell growth of keratinocytes. CONCLUSIONS: Our data point out that alternative splicing of the VASH1 pre-mRNA transcript generates a potent antiangiogenic protein. View Publication

This unit describes the production of monoclonal antibodies beginning with immunization and cell fusion and selection. Support protocols are provided for screening primary hybridoma supernatants for antibodies of desired specificity,establishment of stable hybridoma lines,cloning of these B cell lines by limiting dilution to obtain monoclonal lines,and preparation of cloning/expansion medium. An alternate protocol describes cell fusion and one-step selection and cloning of hybridomas utilizing a semi-solid methylcellulose-based medium (ClonaCell-HY). View Publication

Antiangiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane. Bortezomib's inhibitory effects on chorioallantoic membrane vascularization were abrogated in the presence of distinct tumor xenografts,thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy,we identified GRP-78,a chaperone protein of the unfolded protein response,as being responsible for bortezomib resistance. Indeed,a variety of bortezomib-resistant solid tumor cell lines (PC-3,HRT-18),but not myeloma cell lines (U266,OPM-2),were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP-78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib but induced prosurvival signals by phosphorylation of extracellular signal-related kinase and inhibited p53-mediated expression of proapoptotic Bok and Noxa proteins in endothelial cells. From our data,we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment,thus demonstrating a hitherto unknown mechanism of resistance to bortezomib. View Publication

Expression of the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in resting lymphocytes was recently established,although the physiologic role(s) played by this nuclear hormone receptor in these cell types remains unresolved. In this study,we used CD4(+) T cells isolated from PPARalpha(-/-) and wild-type mice,as well as cell lines that constitutively express PPARalpha,in experiments designed to evaluate the role of this hormone receptor in the regulation of T cell function. We report that activated CD4(+) T cells lacking PPARalpha produce increased levels of IFN-gamma,but significantly lower levels of IL-2 when compared with activated wild-type CD4(+) T cells. Furthermore,we demonstrate that PPARalpha regulates the expression of these cytokines by CD4(+) T cells in part,through its ability to negatively regulate the transcription of T-bet. The induction of T-bet expression in CD4(+) T cells was determined to be positively influenced by p38 mitogen-activated protein (MAP) kinase activation,and the presence of unliganded PPARalpha effectively suppressed the phosphorylation of p38 MAP kinase. The activation of PPARalpha with highly specific ligands relaxed its capacity to suppress p38 MAP kinase phosphorylation and promoted T-bet expression. These results demonstrate a novel DNA-binding independent and agonist-controlled regulatory influence by the nuclear hormone receptor PPARalpha. View Publication