RESUMO
The Thomsen-Friedenreich (TF) antigen is a key target for the development of anticancer vaccines, and this ongoing challenge remains relevant due to the poor immunogenicity of the TF antigen. To overcome this challenge, we adopted a bivalent conjugate design which introduced both the TF antigen and the Thomsen-nouveau (Tn) antigen onto the immunologically relevant polysaccharide A1 (PS A1). The immunological results in C57BL/6 mice revealed that the bivalent, Tn-TF-PS A1 conjugate increased the immune response towards the TF antigen as compared to the monovalent TF-PS A1. This phenomenon was first observed with enzyme-linked immunosorbent assay (ELISA) where the bivalent conjugate generated high titers of IgG antibodies where the monovalent conjugate generated an exclusive IgM response. Fluorescence-activated cell sorting (FACS) analysis also revealed increased binding events to the tumor cell lines MCF-7 and OVCAR-5, which are consistent with the enhanced tumor cell lysis observed in a complement dependent cytotoxicity (CDC) assay. The cytokine profile generated by the bivalent construct revealed increased pro-inflammatory cytokines IL-17 and IFN-γ. This increase in cytokine concentration was matched with an increase in cytokine producing cells as observed by ELISpot. We hypothesized the mechanisms for this phenomenon to involve the macrophage galactose N-acetylgalactosamine specific lectin 2 (MGL2). This hypothesis was supported by using biotinylated probes and recombinant MGL2 to measure carbohydrate-protein interactions.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Carboidratos/imunologia , Imunidade , Imunoconjugados/imunologia , Animais , Anticorpos/metabolismo , Especificidade de Anticorpos/imunologia , Biotinilação , Carboidratos/síntese química , Carboidratos/química , Linhagem Celular Tumoral , Proteínas do Sistema Complemento/metabolismo , Citocinas/metabolismo , Citotoxicidade Imunológica , Humanos , Imunoconjugados/química , Lectinas Tipo C/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/metabolismo , Baço/imunologiaRESUMO
The construction of a tumor-associated carbohydrate antigen-zwitterionic polysaccharide conjugate, Thomsen-nouveau-polysaccharide A1 (Tn-PS A1, where Tn = D-GalpNAc), has led to the development of a carbohydrate binding monoclonal antibody named Kt-IgM-8. Kt-IgM-8 was produced via hybridoma from Tn-PS A1 hyperimmunized Jackson Laboratory C57BL/6 mice, splenocytes and the murine myeloma cell line Sp2/0Ag14 with subsequent cloning on methyl cellulose semi-solid media. This in-house generated monoclonal antibody negates binding influenced from peptides, proteins, and lipids and preferentially binds monovalent Tn antigen as noted by ELISA, FACS, and glycan array technologies. Kt-IgM-8 demonstrated in vitro and in vivo tumor killing against the Michigan Cancer Foundation breast cell line 7 (MCF-7). In vitro tumor killing was observed using an LDH assay that measured antibody-induced complement-dependent cytotoxicity and these results were validated in an in vivo passive immunotherapy approach using an MCF-7 cell line-derived xenograft model. Kt-IgM-8 is effective in killing tumor cells at 30% cytotoxicity, and furthermore, it demonstrated approximately 40% reduction in tumor growth in the MCF-7 model.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/terapia , Imunoglobulina M/imunologia , Imunotoxinas/farmacologia , Animais , Neoplasias da Mama/imunologia , Humanos , Imunotoxinas/imunologia , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Polissacarídeos/imunologia , Polissacarídeos/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sialyl Thomsen-nouveau (STn) is a tumor-associated carbohydrate antigen (TACA) that is overexpressed in a variety of carcinomas such as breast, ovarian, and colon cancer. In normal tissue, STn is not detectable, which is critical for opportunities in developing cancer immunotherapies. A novel, entirely carbohydrate, semisynthetic STn-polysaccharide (PS) A1 conjugate was prepared and evaluated in C57BL/6 mice. STn-PS A1 was combined with commercially available monophosphoryl lipid A-based adjuvant, and after immunization, ELISA indicated a strong immune response for inducing anti-STn IgM/IgG antibodies. The specificity of these antibodies was concomitantly investigated using FACS analysis, and the results indicated excellent cell surface binding events to STn-expressing cancer cell lines MCF-7 and OVCAR-5. An INF-γ ELISpot assay was conducted to further confirm a robust cellular immunity invoked by STn-PS A1. Most importantly, the raised antibodies conferred complement-dependent cellular cytotoxicity against MCF-7 and OVCAR-5 cells.
Assuntos
Antígenos Glicosídicos Associados a Tumores/química , Antígenos Glicosídicos Associados a Tumores/imunologia , Polissacarídeos/química , Animais , Técnicas de Química Sintética , Humanos , Células MCF-7 , Camundongos , Oximas/químicaRESUMO
PS B, a naturally occurring CD4(+) T-cell simulating zwitterionic polysaccharide from Bacteroides fragilis ATCC 25285/NCTC 9343, was conjugated with aminooxy Thomsen Friedenreich (TF or T) [α-d-Gal-(1,3)-ß-d-GalNAc-ONH2] tumor antigen. Immunization in Jax C57BL/6, followed by ELISA revealed IgM and IgG antibody TF specificity. FACS data noted preferential binding to TF-laced MCF-7 cells but not to HCT-116 cells.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Polissacarídeos/imunologia , Animais , Reações Antígeno-Anticorpo , Antígenos Glicosídicos Associados a Tumores/química , Bacteroides fragilis/química , Configuração de Carboidratos , Ensaio de Imunoadsorção Enzimática , Células HCT116 , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Imunoglobulina M/química , Imunoglobulina M/imunologia , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Polissacarídeos/químicaRESUMO
Herbicide (±)-thaxtomin A has been synthesized in a one-pot process with a 32% isolated yield. A multicomponent coupling reaction was utilized to prepare in situ a dipeptide precursor which then sequentially underwent an alkaline mediated keto-amide cyclization to provide the target molecule. Adjustment of diastereoselectivity was achieved using microwave-induced irradiation. The approach incorporates atom economy and reaction efficiency and allows for facile library development.
Assuntos
Produtos Biológicos/síntese química , Herbicidas/síntese química , Indóis/síntese química , Piperazinas/síntese química , Produtos Biológicos/química , Ciclização , Herbicidas/química , Indóis/química , Estrutura Molecular , Piperazinas/química , EstereoisomerismoRESUMO
The α-aminooxy derivative of the Thomsen-Friedenriech tumor associated carbohydrate antigen has been synthesized in 11 steps utilizing a D-GalN3 acceptor carrying a pre-installed α-N-hydroxysuccinimidyl moiety. The natural α linkage was prepared in high selectivity employing a suitably protected D-GalN3-thioglycoside donor with N-hydroxysuccinimide. With access to α-TF-ONH2, the preparation of the TF-PS A1 vaccine candidate ensued smoothly through oxime bond formation.