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1.
J Ovarian Res ; 17(1): 19, 2024 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-38225646

RESUMO

BACKGROUND: MUC16 is a heavily glycosylated cell surface mucin cleaved in the tumor microenvironment to shed CA125. CA125 is a serum biomarker expressed by > 95% of non-mucinous advanced stage epithelial ovarian cancers. MUC16/CA125 contributes to the evasion of anti-tumor immunity, peritoneal spread and promotes carcinogenesis; consequently, it has been targeted with antibody-based passive and active immunotherapy. However, vaccination against this self-antigen likely requires breaking B cell tolerance and may trigger autoimmune disease. Display of self-antigens on virus-like particles (VLPs), including those produced with human papillomavirus (HPV) L1, can efficiently break B cell tolerance. RESULTS: A 20 aa juxta-membrane peptide of the murine MUC16 (mMUC16) or human MUC16 (hMUC16) ectodomain was displayed either via genetic insertion into an immunodominant loop of HPV16 L1-VLPs between residues 136/137, or by chemical coupling using malemide to cysteine sulfhydryl groups on their surface. Female mice were vaccinated intramuscularly three times with either DNA expressing L1-MUC16 fusions via electroporation, or with alum-formulated VLP chemically-coupled to MUC16 peptides. Both regimens were well tolerated, and elicited MUC16-specific serum IgG, although titers were higher in mice vaccinated with MUC16-coupled VLP on alum as compared to L1-MUC16 DNA vaccination. Antibody responses to mMUC16-targeted vaccination cross-reacted with hMUC16 peptide, and vice versa; both were reactive with the surface of CA125+ OVCAR3 cells, but not SKOV3 that lack detectable CA125 expression. Interestingly, vaccination of mice with mMUC16 peptide mixed with VLP and alum elicited mMUC16-specific IgG, implying VLPs provide robust T help and that coupling may not be required to break tolerance to this epitope. CONCLUSION: Vaccination with VLP displaying the 20 aa juxta-membrane MUC16 ectodomain, which includes the membrane proximal cleavage site, is likely to be well tolerated and induce IgG targeting ovarian cancer cells, even after CA125 is shed.


Assuntos
Compostos de Alúmen , Neoplasias Ovarianas , Vacinas de Partículas Semelhantes a Vírus , Humanos , Feminino , Animais , Camundongos , Neoplasias Ovarianas/genética , Epitopos , Apoptose , Linhagem Celular Tumoral , Peptídeos , Imunoglobulina G , DNA , Antígeno Ca-125/genética , Microambiente Tumoral , Proteínas de Membrana/genética
2.
Bioresour Bioprocess ; 10(1): 93, 2023 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-38647984

RESUMO

Bispecific antibody (bsAb), a novel therapeutic modality, provides excellent treatment efficacy, yet poses numerous challenges to downstream process development, which are mainly due to the intricate diversity of bsAb structures and impurity profiles. Ceramic hydroxyapatite (CHT), a mixed-mode medium, allows proteins to interact with its calcium sites (C-sites) through metal affinity and/or its phosphate sites (P-sites) through cation exchange interactions. This dual-binding capability potentially offers unique bind and elute behaviours for different proteins of interest, resulting in optimal product purity when suitable elution conditions are employed. In this study, the effectiveness of CHT as a polishing step for bsAb purification was investigated across three model molecules and benchmarked against the traditional cation exchange chromatography (CEX). For both asymmetric and symmetric IgG-like bsAb post Protein A eluates, at least 97% product purity was achieved after CHT polishing. CHT delivered a superior aggregate clearance to CEX, resulting in low high molecular weight (HMW) impurities (0.5%) and low process-related impurities in the product pools. Moreover, CHT significantly mitigated "chromatography-induced aggregation" whereas eightfold more HMW was generated by CEX. This study illustrated the developability of CHT in effectively eliminating low molecular weight (LMW) impurities through post-load-wash (PLW) optimization, resulting in an additional reduction of up to 48% in LMW impurities. A mechanistic explanation regarding the performance of impurity removal and mitigation of the chromatography-induced aggregation by CHT was proposed, illustrating unique CHT capability is potentially driven by C-site cooperation, of which effectiveness could depend on the bsAb composition and size.

3.
Biochemistry ; 56(8): 1130-1139, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28157283

RESUMO

The minimal requirements for substrate recognition and turnover by iodotyrosine deiodinase were examined to learn the basis for its catalytic specificity. This enzyme is crucial for iodide homeostasis and the generation of thyroid hormone in chordates. 2-Iodophenol binds only very weakly to the human enzyme and is dehalogenated with a kcat/Km that is more than 4 orders of magnitude lower than that for iodotyrosine. This discrimination likely protects against a futile cycle of iodinating and deiodinating precursors of thyroid hormone biosynthesis. Surprisingly, a very similar catalytic selectivity was expressed by a bacterial homologue from Haliscomenobacter hydrossis. In this example, discrimination was not based on affinity since 4-cyano-2-iodophenol bound to the bacterial deiodinase with a Kd lower than that of iodotyrosine and yet was not detectably deiodinated. Other phenols including 2-iodophenol were deiodinated but only very inefficiently. Crystal structures of the bacterial enzyme with and without bound iodotyrosine are nearly superimposable and quite similar to the corresponding structures of the human enzyme. Likewise, the bacterial enzyme is activated for single electron transfer after binding to the substrate analogue fluorotyrosine as previously observed with the human enzyme. A cocrystal structure of bacterial deiodinase and 2-iodophenol indicates that this ligand stacks on the active site flavin mononucleotide (FMN) in a orientation analogous to that of bound iodotyrosine. However, 2-iodophenol association is not sufficient to activate the FMN chemistry required for catalysis, and thus the bacterial enzyme appears to share a similar specificity for halotyrosines even though their physiological roles are likely very different from those in humans.


Assuntos
Domínio Catalítico , Halogenação , Iodeto Peroxidase/química , Iodeto Peroxidase/metabolismo , Bacteroidetes/enzimologia , Mononucleotídeo de Flavina/metabolismo , Humanos , Modelos Moleculares , Oxirredução
4.
J Nat Prod ; 74(7): 1650-2, 2011 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-21667999

RESUMO

A novel sesquiterpenoid, asperaculin A, possessing a novel [5,5,5,6]fenestrane ring system, was isolated from the marine-derived fungus Aspergillus aculeatus CRI323-04. The structure of asperaculin A was established by analysis of spectroscopic data. The name aspergillane is proposed for the sesquiterpene skeleton in asperaculin A.


Assuntos
Antineoplásicos/isolamento & purificação , Aspergillus/química , Sesquiterpenos/isolamento & purificação , Antineoplásicos/química , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Células Hep G2 , Humanos , Biologia Marinha , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Sesquiterpenos/química , Sesquiterpenos/farmacologia
5.
J Nat Prod ; 72(11): 2049-52, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19824618

RESUMO

A new tyrosine-derived metabolite, aspergillusol A (4), was isolated on a gram scale, together with a methyl ester of 4-hydroxyphenylpyruvic acid oxime (5) and secalonic acid A, from the marine-derived fungus Aspergillus aculeatus CRI323-04. The tetraol in 4 was identified as erythritol by comparison of the 1H NMR spectrum of its benzoylated derivative with those of benzoylated erythritol (7) and D-threitol (8), as well as by cellulose-based chiral HPLC analysis. Aspergillusol A (4) selectively inhibited alpha-glucosidase from the yeast Saccharomyces cerevisiae, but it was inactive toward the alpha-glucosidase from the bacterium Bacillus stearothermophilus.


Assuntos
Aspergillus/química , Geobacillus stearothermophilus/enzimologia , Inibidores de Glicosídeo Hidrolases , Saccharomyces cerevisiae/enzimologia , Tirosina/análogos & derivados , Geobacillus stearothermophilus/efeitos dos fármacos , Biologia Marinha , Estrutura Molecular , Saccharomyces cerevisiae/efeitos dos fármacos , Estereoisomerismo , Tirosina/química , Tirosina/isolamento & purificação , Tirosina/farmacologia , Xantonas/isolamento & purificação
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