RESUMO
The quality of the adaptive immune response depends on the differentiation of distinct CD4(+) helper T cell subsets, and the magnitude of an immune response is controlled by CD4(+)Foxp3(+) regulatory T cells (Treg cells). However, how a tissue- and cell type-specific suppressor program of Treg cells is mechanistically orchestrated has remained largely unexplored. Through the use of Treg cell-specific gene targeting, we found that the suppression of allergic immune responses in the lungs mediated by T helper type 2 (TH2) cells was dependent on the activity of the protein kinase CK2. Genetic ablation of the ß-subunit of CK2 specifically in Treg cells resulted in the proliferation of a hitherto-unexplored ILT3(+) Treg cell subpopulation that was unable to control the maturation of IRF4(+)PD-L2(+) dendritic cells required for the development of TH2 responses in vivo.
Assuntos
Caseína Quinase II/imunologia , Linfócitos T Reguladores/imunologia , Células Th2/imunologia , Animais , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Processos de Crescimento Celular/imunologia , Linhagem Celular , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Fatores de Transcrição Forkhead/imunologia , Humanos , Hipersensibilidade/sangue , Hipersensibilidade/imunologia , Fatores Reguladores de Interferon/imunologia , Leucócitos Mononucleares/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Superfície Celular/imunologia , Linfócitos T Reguladores/enzimologia , Células Th2/enzimologiaRESUMO
Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity.
Assuntos
Cistatinas , Ixodes , Animais , Cistatinas Salivares/química , Peptídeo Hidrolases/metabolismo , Cisteína/metabolismo , Cistatinas/farmacologia , Ixodes/química , Vertebrados , Catepsinas/metabolismo , Endopeptidases/metabolismoRESUMO
Tumor-associated carbohydrate antigens are overexpressed as altered-self in most common epithelial cancers. Their glycosylation patterns differ from those of healthy cells, functioning as an ID for cancer cells. Scientists have been developing anti-cancer vaccines based on mucin glycopeptides, yet the interplay of delivery system, adjuvant and tumor associated MUC epitopes in the induced immune response is not well understood. The current state of the art suggests that the identity, abundancy and location of the glycans on the MUC backbone are all key parameters in the cellular and humoral response. This review shares lessons learned by us in over two decades of research in glycopeptide vaccines. By bridging synthetic chemistry and immunology, we discuss efforts in designing synthetic MUC1/4/16 vaccines and focus on the role of glycosylation patterns. We provide a brief introduction into the mechanisms of the immune system and aim to promote the development of cancer subunit vaccines.
Assuntos
Vacinas Anticâncer , Glicopeptídeos , Mucinas/imunologia , Neoplasias/prevenção & controle , Vacinas Anticâncer/imunologia , Glicosilação , Humanos , Imunidade , Neoplasias/imunologia , Vacinas SintéticasRESUMO
To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Šresolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.
Assuntos
Proteínas de Artrópodes/farmacologia , Cistatinas/farmacologia , Imunossupressores/farmacologia , Cistatinas Salivares/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Cristalografia por Raios X , Cistatinas/classificação , Cistatinas/genética , Citocinas/metabolismo , Compostos de Epóxi/metabolismo , Feminino , Imunossupressores/química , Imunossupressores/metabolismo , Ixodes/química , Ixodes/genética , Ixodes/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Filogenia , Proteólise/efeitos dos fármacos , Cistatinas Salivares/química , Cistatinas Salivares/genética , Homologia de Sequência de Aminoácidos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
There is still a great unmet medical need concerning diagnosis and treatment of breast cancer which could be addressed by utilizing specific molecular targets. Tumor-associated MUC1 is expressed on over 90 % of all breast cancer entities and differs strongly from its physiological form on epithelial cells, therefore presenting a unique target for breast cancer diagnosis and antibody-mediated immune therapy. Utilizing an anti-tumor vaccine based on a synthetically prepared glycopeptide, we generated a monoclonal antibody (mAb) GGSK-1/30, selectively recognizing human tumor-associated MUC1. This antibody targets exclusively tumor-associated MUC1 in the absence of any binding to MUC1 on healthy epithelial cells thus enabling the generation of breast tumor-specific radiolabeled immune therapeutic tools. Methods: MAb GGSK-1/30 was used for immunohistochemical analysis of human breast cancer tissue. Its desferrioxamine (Df')-conjugate was synthesized and labelled with 89Zr. [89Zr]Zr-Df'-GGSK-1/30 was evaluated as a potential PET tracer. Binding and pharmacokinetic properties of [89Zr]Zr-Df'-GGSK-1/30 were analyzed in vitro using human and murine cell lines that express tumor-associated MUC1. Self-generated primary murine breast cancer cells expressing human tumor-associated MUC1 were transplanted subcutaneously in wild type and human MUC1-transgenic mice. The pharmacology of [89Zr]Zr-Df'-GGSK-1/30 was investigated using breast tumor-bearing mice in vivo by PET/MRT imaging as well as by ex vivo organ biodistribution analysis. Results: The mAb GGSK-1/30 stained specifically human breast tumor tissue and can be possibly used to predict the severity of disease progression based on the expression of the tumor-associated MUC1. For in vivo imaging, the Df'-conjugated mAb was radiolabeled with a radiochemical yield of 60 %, a radiochemical purity of 95 % and an apparent specific activity of 6.1 GBq/µmol. After 7 d, stabilities of 84 % in human serum and of 93 % in saline were observed. In vitro cell studies showed strong binding to human tumor-associated MUC1 expressing breast cancer cells. The breast tumor-bearing mice showed an in vivo tumor uptake of >50 %ID/g and clearly visible specific enrichment of the radioconjugate via PET/MRT. Principal conclusions: Tumor-associated MUC1 is a very important biomarker for breast cancer next to the traditional markers estrogen receptor (ER), progesterone receptor (PR) and HER/2-neu. The mAb GGSK-1/30 can be used for the diagnosis of over 90% of breast cancers, including triple negative breast cancer based on biopsy staining. Its radioimmunoconjugate represents a promising PET-tracer for breast cancer imaging selectively targeting breast cancer cells.
Assuntos
Anticorpos Monoclonais/farmacocinética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/mortalidade , Mucina-1/metabolismo , Animais , Biomarcadores Tumorais/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Desferroxamina/química , Feminino , Humanos , Imuno-Histoquímica/métodos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mucina-1/imunologia , Tomografia por Emissão de Pósitrons/métodos , Prognóstico , Radioisótopos/química , Distribuição Tecidual , Zircônio/químicaRESUMO
T helper 17 (TH17) cells represent a discrete TH cell subset instrumental in the immune response to extracellular bacteria and fungi. However, TH17 cells are considered to be detrimentally involved in autoimmune diseases like multiple sclerosis (MS). In contrast to TH17 cells, regulatory T (Treg) cells were shown to be pivotal in the maintenance of peripheral tolerance. Thus, the balance between Treg cells and TH17 cells determines the severity of a TH17 cell-driven disease and therefore is a promising target for treating autoimmune diseases. However, the molecular mechanisms controlling this balance are still unclear. Here, we report that pharmacological inhibition as well as genetic ablation of the protein kinase CK2 (CK2) ameliorates experimental autoimmune encephalomyelitis (EAE) severity and relapse incidence. Furthermore, CK2 inhibition or genetic ablation prevents TH17 cell development and promotes the generation of Treg cells. Molecularly, inhibition of CK2 leads to reduced STAT3 phosphorylation and strongly attenuated expression of the IL-23 receptor, IL-17, and GM-CSF. Thus, these results identify CK2 as a nodal point in TH17 cell development and suggest this kinase as a potential therapeutic target to treat TH17 cell-driven autoimmune responses.
Assuntos
Caseína Quinase II/imunologia , Encefalomielite Autoimune Experimental/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Caseína Quinase II/deficiência , Caseína Quinase II/genética , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Humanos , Interleucina-17 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos , Fosforilação , Receptores de Interleucina , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Índice de Gravidade de Doença , Transdução de Sinais , Linfócitos T Reguladores/citologia , Células Th17/patologiaRESUMO
The endothelial glycoprotein MUC1 is known to underlie alterations in cancer by means of aberrant glycosylation accompanied by changes in morphology. The heavily shortened glycans induce a collapse of the peptide backbone and enable accessibility of the latter to immune cells, rendering it a tumor-associated antigen. Synthetic vaccines based on MUC1 tandem repeat motifs, comprising tumor-associated 2,3-sialyl-T antigen, conjugated to the immunostimulating tetanus toxoid, are reported herein. Immunization with these vaccines in a simple water/oil emulsion produced a strong immune response in mice to which stimulation with complete Freund's adjuvant (CFA) was not superior. In both cases, high levels of IgG1 and IgG2a/b were induced in C57BL/6 mice. Additional glycosylation in the immunodominant PDTRP domain led to improved binding of the induced antisera to MCF-7 breast tumor cells, compared with that of the monoglycosylated peptide vaccine.
RESUMO
A modular route to prepare functional self-assembling dendritic peptide amphiphiles decorated with mannosides, to effectively target antigen-presenting cells, such as macrophages, is reported. The monomeric building blocks were equipped with tetra(ethylene glycol)s (TEGs) or labeled with a Cy3 fluorescent probe. Experiments on the uptake of the multifunctional supramolecular particles into murine macrophages (Mφs) were monitored by confocal microscopy and fluorescence-activated cell sorting. Mannose-decorated supramolecular polymers trigger a significantly higher cellular uptake and distribution, relative to TEG carrying bare polymers. No cytotoxicity or negative impact on cytokine production of the treated Mφs was observed, which emphasized their biocompatibility. The modular nature of the multicomponent supramolecular polymer coassembly protocol is a promising platform to develop fully synthetic multifunctional vaccines, for example, in cancer immunotherapy.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Dendrímeros/metabolismo , Manosídeos/metabolismo , Peptídeos/metabolismo , Tensoativos/metabolismo , Animais , Transporte Biológico , Carbocianinas/química , Carbocianinas/metabolismo , Células Cultivadas , Dendrímeros/química , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Macrófagos/metabolismo , Manosídeos/química , Camundongos , Microscopia Confocal , Modelos Moleculares , Peptídeos/química , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Tensoativos/químicaRESUMO
Glycoprotein research is pivotal for vaccine development and biomarker discovery. Many successful methodologies for reliably increasing the antigenicity toward tumor-associated glycopeptide structures have been reported. Deeper insights into the quality and specificity of the raised polyclonal, humoral reactions are often not addressed, despite the fact that an immunological memory, which produces antibodies with cross-reactivity to epitopes exposed on healthy cells, may cause autoimmune diseases. In the current work, three MUC1 antitumor vaccine candidates conjugated with different immune stimulants are evaluated immunologically. For assessment of the influence of the immune stimulant on antibody recognition, a comprehensive library of mucin 1 glycopeptides (>100 entries) is synthesized and employed in antibody microarray profiling; these range from small tumor-associated glycans (TN , STN , and T-antigen structures) to heavily extended O-glycan core structures (type-1 and type-2 elongated core 1-3 tri-, tetra-, and hexasaccharides) glycosylated in variable density at the five different sites of the MUC1 tandem repeat. This is one of the most extensive glycopeptide libraries ever made through total synthesis. On tumor cells, the coreâ 2 ß-1,6-N-acetylglucosaminyltransferase-1 (C2GlcNAcT-1) is down-regulated, resulting in lower amounts of the branched coreâ 2 structures, which favor formation of linear coreâ 1 or coreâ 3 structures, and in particular, truncated tumor-associated antigen structures. The coreâ 2 structures are commonly found on healthy cells and the elucidation of antibody cross-reactivity to such epitopes may predict the tumor-selectivity and safety of synthetic vaccines. With the extended mucin core structures in hand, antibody cross-reactivity toward the branched coreâ 2 glycopeptide epitopes is explored. It is observed that the induced antibodies recognize MUC1 peptides with very high glycosylation site specificity. The nature of the antibody response is characteristically different for antibodies directed to glycosylation sites in either the immune-dominant PDTR or the GSTA domain. All antibody sera show high reactivity to the tumor-associated saccharide structures on MUC1. Extensive glycosylation with branched coreâ 2 structures, typically found on healthy cells, abolishes antibody recognition of the antisera and suggests that all vaccine conjugates preferentially induce a tumor-specific humoral immune response.
Assuntos
Vacinas Anticâncer/imunologia , Glicopeptídeos/imunologia , Mucina-1/imunologia , Neoplasias/imunologia , Polissacarídeos/imunologia , Vacinas Sintéticas/imunologia , Animais , Formação de Anticorpos , Vacinas Anticâncer/química , Glicopeptídeos/química , Humanos , Imunidade Humoral , Camundongos , Mucina-1/química , Neoplasias/terapia , Polissacarídeos/química , Análise Serial de Proteínas , Vacinas Sintéticas/químicaRESUMO
The ligation of gold(I) metalloamphiphiles with biomolecules is reported, using water-soluble AuI -N-alkynyl substituted maleimide complexes. For this purpose, two different polar ligands were applied: 1) a neutral, dendritic tetraethylene glycol-functionalized phosphane and 2) a charged, sulfonated N-heterocyclic carbene (NHC). The retro Diels-Alder reaction of a furan-protected maleimide gold(I) complex, followed by cycloaddition with a diene-functionalized biotin under mild conditions leads to a novel gold(I) metalloamphiphile. The strong streptavidin-biotin binding affinity in buffered aqueous solution of the resulting biotin alkynyl gold(I) phosphane conjugate remains intact. The cytotoxicity of the biotinylated gold(I) complex against a T47D human breast cancer cell line is higher than for cisplatin.
RESUMO
Coevolution of ticks and the vertebrate immune system has led to the development of immunosuppressive molecules that prevent immediate response of skin-resident immune cells to quickly fend off the parasite. In this article, we demonstrate that the tick-derived immunosuppressor sialostatin L restrains IL-9 production by mast cells, whereas degranulation and IL-6 expression are both unaffected. In addition, the expression of IL-1ß and IRF4 is strongly reduced in the presence of sialostatin L. Correspondingly, IRF4- or IL-1R-deficient mast cells exhibit a strong impairment in IL-9 production, demonstrating the importance of IRF4 and IL-1 in the regulation of the Il9 locus in mast cells. Furthermore, IRF4 binds to the promoters of Il1b and Il9, suggesting that sialostatin L suppresses mast cell-derived IL-9 preferentially by inhibiting IRF4. In an experimental asthma model, mast cell-specific deficiency in IRF4 or administration of sialostatin L results in a strong reduction in asthma symptoms, demonstrating the immunosuppressive potency of tick-derived molecules.
Assuntos
Cistatinas/farmacologia , Imunidade Inata/efeitos dos fármacos , Imunossupressores/farmacologia , Fatores Reguladores de Interferon/imunologia , Interleucina-9/imunologia , Mastócitos/efeitos dos fármacos , Animais , Asma/genética , Asma/imunologia , Asma/patologia , Sítios de Ligação , Degranulação Celular/imunologia , Cistatinas/imunologia , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita/imunologia , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/genética , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-9/antagonistas & inibidores , Interleucina-9/genética , Mastócitos/imunologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Ligação Proteica , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/imunologia , Transdução de Sinais , Transcrição GênicaRESUMO
Achieving precise control over the morphology and function of polymeric nanostructures during self-assembly remains a challenge in materials as well as biomedical science, especially when independent control over particle properties is desired. Herein, we report on nanostructures derived from amphiphilic block copolypept(o)ides by secondary-structure-directed self-assembly, presenting a strategy to adjust core polarity and function separately from particle preparation in a bioreversible manner. The peptide-inherent process of secondary-structure formation allows for the synthesis of spherical and worm-like core-cross-linked architectures from the same block copolymer, introducing a simple yet powerful approach to versatile peptide-based core-shell nanostructures.
RESUMO
In studies within the realm of cancer immunotherapy, the synthesis of exactly specified tumor-associated glycopeptide antigens is shown to be a key strategy for obtaining a highly selective biological reagent, that is, a monoclonal antibody that completely differentiates between tumor and normal epithelial cells and specifically marks the tumor cells in pancreas tumors. Mucin MUC1, which is overexpressed in many prevalent cancers, was identified as a promising target for this strategy. Tumor-associated MUC1 differs significantly from that expressed by normal cells, in particular by altered glycosylation. Structurally defined tumor-associated MUC1 cannot be isolated from tumor cells. We synthesized MUC1-glycopeptide vaccines and analyzed their structure-activity relationships in immunizations; a monoclonal antibody that specifically distinguishes between human normal and tumor epithelial cells was thus generated.
Assuntos
Anticorpos Monoclonais/biossíntese , Neoplasias da Mama/patologia , Mama/citologia , Vacinas Anticâncer/administração & dosagem , Glicopeptídeos/imunologia , Neoplasias Pancreáticas/diagnóstico , Feminino , HumanosRESUMO
Mucin glycoproteins are important diagnostic and therapeutic targets for cancer treatment. Although several strategies have been developed to explore anti-tumor vaccines based on MUC1 glycopeptides, only few studies have focused on vaccines directed against the tumor-associated MUC4 glycoprotein. MUC4 is an important tumor marker overexpressed in lung cancer and uniquely expressed in pancreatic ductual adenocarcinoma. The aberrant glycosylation of MUC4 in tumor cells results in an exposure of its peptide backbone and the formation of tumor-associated glycopeptide antigens. Due to the low immunogenicity of these endogenous structures, their conjugation with immune stimulating peptide or protein carriers are required. In this study, MUC4 tandem-repeat glycopeptides were conjugated to the tetanus toxoid and used for vaccination of mice. Immunological evaluations showed that our MUC4-based vaccines induced very strong antigen-specific immune responses. In addition, antibody binding epitope analysis on glycopeptide microarrays, were demonstrating a clear glycosylation site dependence of the induced antibodies.
Assuntos
Anticorpos Antineoplásicos/imunologia , Mucina-4/imunologia , Neoplasias Pancreáticas/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Epitopos/imunologia , Feminino , Humanos , Soros Imunes/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mucina-4/química , Neoplasias Pancreáticas/patologia , Sequências de Repetição em Tandem , Toxoide Tetânico/química , VacinaçãoRESUMO
In a new concept of fully synthetic vaccines, the role of T-helper cells is emphasized. Here, a synthetic antitumor vaccine consisting of a diglycosylated tumor-associated MUC1 glycopeptide as the B-cell epitope was covalently cross-linked with three different T-helper-cell epitopes via squaric acid ligation of two linear (glyco)peptides. In mice this four-component vaccine administered without external immune-stimulating promoters elicit titers of MUC1-specific antibodies that were about eight times higher than those induced by a vaccine containing only one T-helper-cell epitope. The promising results indicate that multiple activation of different T-helper cells is useful for applications in which increased immunogenicity is required. In personalized medicine, in particular, this flexible construction of a vaccine can serve as a role model, for example, when T-helper-cell epitopes are needed that match human leukocyte antigens (HLA) in different patients.
Assuntos
Antígenos de Neoplasias/química , Vacinas Anticâncer/síntese química , Epitopos/química , Glicopeptídeos/química , Mucina-1/química , Linfócitos T Auxiliares-Indutores/química , Reações Antígeno-Anticorpo , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Epitopos/imunologia , Glicopeptídeos/imunologia , Humanos , Estrutura Molecular , Mucina-1/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
PURPOSE: In the emerging field of antibody treatments for neurodegenerative diseases, reliable tools are needed to evaluate new therapeutics, diagnose and select patients, monitor disease progression, and assess therapy response. Immuno-PET combines the high affinity and exceptional specificity of monoclonal antibodies with the non-invasive imaging technique positron emission tomography (PET). Its application in neurodegenerative disease brain imaging has been limited due to the marginal uptake across the blood-brain barrier (BBB). The emergence of BBB-shuttle antibodies with enhanced uptake across the BBB extended immuno-PET to brain imaging. We recently reported about specific brain uptake of a bispecific aducanumab mTfR antibody in APP/PS1 TG mice using 89Zr-immuno-PET. However, a sufficient target-to-background ratio was reached at a relatively late scanning time point of 7 days post-injection. To investigate if a better target-to-background ratio could be achieved earlier, an aducanumab BBB-shuttle with a mutated Fc region for reduced FcRn affinity was evaluated. PROCEDURES: AduH310A-8D3 and Adu-8D3 were modified with DFO*-NCS and subsequently radiolabeled with 89Zr. The potential influence of the H310A mutation, modification with DFO*-NCS, and subsequent radiolabeling on the in vitro binding to amyloid-beta and mTfR1 was investigated via amyloid-beta peptide ELISA and FACS analysis using mTfR1 transfected CHO-S cells. Blood kinetics, brain uptake, in vivo PET imaging and target engagement of radiolabeled AduH310A-8D3 were evaluated and compared to non-mutated Adu-8D3 in APP/PS1 TG mice and wild-type animals as controls. RESULTS: Radiolabeling was performed with sufficient radiochemical yields and radiochemical purity. In vitro binding to amyloid-beta and mTfR1 showed no impairment. [89Zr]Zr-AduH310A-8D3 showed faster blood clearance and earlier differentiation of amyloid-beta-related brain uptake compared to [89Zr]Zr-Adu-8D3. However, only half of the brain uptake was observed for [89Zr]Zr-AduH310A-8D3. CONCLUSIONS: Although a faster blood clearance of AduH310A-8D3 was observed, it was concluded that no beneficial effects for 89Zr-immuno-PET imaging of brain uptake were obtained.
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The accelerated approval of the monoclonal antibody (mAb) aducanumab as a treatment option for Alzheimer's Disease and the continued discussions about its efficacy have shown that a better understanding of immunotherapy for the treatment of neurodegenerative diseases is needed. 89Zr-immuno-PET could be a suitable tool to open new avenues for the diagnosis of CNS disorders, monitoring disease progression, and assessment of novel therapeutics. Herein, three different 89Zr-labeling strategies and direct radioiodination with 125I of a bispecific anti-amyloid-beta aducanumab derivate, consisting of aducanumab with a C-terminal fused anti-transferrin receptor binding single chain Fab fragment derived from 8D3 (Adu-8D3), were compared ex vivo and in vivo with regard to brain uptake and target engagement in an APP/PS1 Alzheimer's disease mouse model and wild type animals. Methods: Adu-8D3 and a negative control antibody, based on the HIV specific B12 antibody also carrying C-terminal fused 8D3 scFab (B12-8D3), were each conjugated with NCS-DFO, NCS-DFO*, or TFP-N-suc-DFO-Fe-ester, followed by radiolabeling with 89Zr. 125I was used as a substitute for 124I for labeling of both antibodies. 30 µg of radiolabeled mAb, corresponding to approximately 6 MBq 89Zr or 2.5 MBq 125I, were injected per mouse. PET imaging was performed 1, 3 and 7 days post injection (p.i.). All mice were sacrificed on day 7 p.i. and subjected to ex vivo biodistribution and brain autoradiography. Immunostaining on brain tissue was performed after autoradiography for further validation. Results: Ex vivo biodistribution revealed that the brain uptake of [89Zr]Zr-DFO*-NCS-Adu-8D3 (2.19 ±0.12 %ID/g) was as high as for its 125I-analog (2.21 ±0.15 %ID/g). [89Zr]Zr-DFO-NCS-Adu-8D3 and [89Zr]Zr-DFO-N-suc-Adu-8D3 showed significantly lower uptake (< 0.65 %ID/g), being in the same range as for the 89Zr-labeled controls (B12-8D3). Autoradiography of [89Zr]Zr-DFO*-NCS-Adu-8D3 and [125I]I-Adu-8D3 showed an amyloid-beta related granular uptake pattern of radioactivity. In contrast, the [89Zr]Zr-DFO-conjugates and the control antibody groups did not show any amyloid-beta related uptake pattern, indicating that DFO is inferior for 89Zr-immuno-PET imaging of the brain in comparison to DFO* for Adu-8D3. This was confirmed by day 7 PET images showing only amyloid-beta related brain uptake for [89Zr]Zr-DFO*-NCS-Adu-8D3. In wild type animals, such an uptake was not observed. Immunostaining showed a co-localization of all administered Adu-8D3 conjugates with amyloid-beta plaques. Conclusion: We successfully demonstrated that 89Zr-immuno-PET is suitable for imaging and quantifying amyloid-beta specific brain uptake using a bispecific aducanumab brain shuttling antibody, Adu-8D3, but only when using the novel chelator DFO*, and not DFO, for labeling with 89Zr.
Assuntos
Doença de Alzheimer , Anticorpos Biespecíficos , Animais , Camundongos , Radioisótopos do Iodo , Quelantes , Desferroxamina , Zircônio , Distribuição Tecidual , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/tratamento farmacológico , Linhagem Celular Tumoral , Tomografia por Emissão de Pósitrons/métodos , Anticorpos Monoclonais/uso terapêutico , Peptídeos beta-Amiloides , Fragmentos Fab das Imunoglobulinas , ÉsteresRESUMO
Preventive vaccination against tumor-associated endogenous antigens is considered to be an attractive strategy for the induction of a curative immune response concomitant with a long-lasting immunologic memory. The mucin MUC1 is a promising tumor antigen, as its tumor-associated form differs from the glycoprotein form expressed on healthy cells. Due to aberrant glycosylation in tumor cells, the specific peptide epitopes in its backbone are accessible and can be bound by antibodies induced by vaccination. Breast cancer patients develop per se only low levels of T cells and antibodies recognizing tumor-associated MUC1, and clinical trials with tumor-associated MUC1 yielded unsatisfactory therapeutic effects, indicating an urgent need to improve humoral immunity against this tumor entity. Herein, we demonstrate that preventive vaccination against tumor-associated human MUC1 results in a specific humoral immune response, a slowdown of tumor progression and an increase in survival of breast tumor-bearing mice. For preventive vaccination, we used a synthetic vaccine containing a tumor-associated glycopeptide structure of human MUC1 coupled to Tetanus Toxoid. The glycopeptide consists of a 22mer huMUC1 peptide with two immune dominant regions (PDTR and GSTA), glycosylated with the sialylated carbohydrate STN on serine-17. PyMT (polyomavirus middle T-antigen) and human MUC1 double-transgenic mice expressing human tumor-associated MUC1 on breast tumor tissue served as a preclinical breast cancer model.
Assuntos
Vacinas Anticâncer/uso terapêutico , Glicopeptídeos/imunologia , Neoplasias Mamárias Experimentais/terapia , Mucina-1/imunologia , Toxoide Tetânico/imunologia , Vacinas Sintéticas/uso terapêutico , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunoglobulina G/farmacologia , Camundongos Transgênicos , Pessoa de Meia-Idade , Mucina-1/genética , Neoplasias de Mama Triplo Negativas/imunologiaRESUMO
A MUC1 anticancer vaccine equipped with covalently linked divalent mannose ligands was found to improve the antigen uptake and presentation by targeting mannose-receptor-positive macrophages and dendritic cells. It induced much stronger specific IgG immune responses in mice than the non-mannosylated reference vaccine. Mannose coupling also led to increased numbers of macrophages, dendritic cells, and CD4+ T cells in the local lymph organs. Comparison of di- and tetravalent mannose ligands revealed an increased binding of the tetravalent version, suggesting that higher valency improves binding to the mannose receptor. The mannose-coupled vaccine and the non-mannosylated reference vaccine induced IgG antibodies that exhibited similar binding to human breast tumor cells.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/metabolismo , Macrófagos/metabolismo , Manose/química , Mucina-1/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Vacinas Anticâncer/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Lectinas Tipo C/metabolismo , Ligantes , Linfonodos , Células MCF-7 , Macrófagos/citologia , Macrófagos/imunologia , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Mucina-1/química , Mucina-1/metabolismo , Ligação Proteica , Receptores de Superfície Celular/metabolismoRESUMO
Fully synthetic MUC1 glycopeptide antitumor vaccines have a precisely specified structure and induce a targeted immune response without suppression of the immune response when using an immunogenic carrier protein. However, tumor-associated aberrantly glycosylated MUC1 glycopeptides are endogenous structures, "self-antigens", that exhibit only low immunogenicity. To overcome this obstacle, a fully synthetic MUC1 glycopeptide antitumor vaccine was combined with poly(inosinic acid:cytidylic acid), poly(I:C), as a structurally defined Toll-like receptorâ 3 (TLR3)-activating adjuvant. This vaccine preparation elicited extraordinary titers of IgG antibodies which strongly bound human breast cancer cells expressing tumor-associated MUC1. Beside the humoral response, the poly(I:C) glycopeptide vaccine induced a pro-inflammatory environment, very important to overcome the immune-suppressive mechanisms, and elicited a strong cellular immune response crucial for tumor elimination.