RESUMO
Activating transcription factor 3 (ATF3) is an activation transcription factor/cyclic adenosine monophosphate (cAMP) responsive element-binding (CREB) protein family member. It is recognized as an important regulator of cancer progression by repressing expression of key inflammatory factors such as interferon-γ and chemokine (C-C motif) ligand 4 (CCL4). Here, we describe a novel library screening approach that probes individual leucine zipper components before combining them to search exponentially larger sequence spaces not normally accessible to intracellular screening. To do so, we employ two individual semirational library design approaches and screen using a protein-fragment complementation assay (PCA). First, a 248,832-member library explored 12 amino acid positions at all five a positions to identify those that provided improved binding, with all e/g positions fixed as Q, placing selection pressure onto the library options provided. Next, a 59,049-member library probed all ten e/g positions with 3 options. Similarly, during e/g library screening, a positions were locked into a generically bindable sequence pattern (AIAIA), weakly favoring leucine zipper formation, while placing selection pressure onto e/g options provided. The combined a/e/g library represents â¼14.7 billion members, with the resulting peptide, ATF3W_aeg, binding ATF3 with high affinity (Tm = 60 °C; Kd = 151 nM) while strongly disfavoring homodimerization. Moreover, ATF3W_aeg is notably improved over component PCA hits, with target specificity found to be driven predominantly by electrostatic interactions. The combined a/e/g exponential library screening approach provides a robust, accelerated platform for exploring larger peptide libraries, toward derivation of potent yet selective antagonists that avoid homoassociation to provide new insight into rational peptide design.
Assuntos
Fator 3 Ativador da Transcrição , Biblioteca de Peptídeos , Fator 3 Ativador da Transcrição/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Peptídeos/metabolismoRESUMO
Little is known about the consequences of immune recognition of mutated gene products, despite their potential relevance to autoimmunity and tumor immunity. To identify mutations that induce immunity, here we have developed a systematic approach in which combinatorial DNA libraries encoding large numbers of random mutations in two syngeneic tyrosinase-related proteins are used to immunize black mice. We show that the libraries of mutated DNA induce autoimmune hypopigmentation and tumor immunity through cross-recognition of nonmutated gene products. Truncations are present in all immunogenic clones and are sufficient to elicit immunity to self, triggering recognition of normally silent epitopes. Immunity is further enhanced by specific amino acid substitutions that promote T helper cell responses. Thus, presentation of a vast repertoire of antigen variants to the immune system can enhance the generation of adaptive immune responses to self.
Assuntos
Autoantígenos/genética , Autoimunidade/genética , Mutação , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células COS , Chlorocebus aethiops , Reações Cruzadas , DNA Complementar/genética , Biblioteca Gênica , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Tolerância a Antígenos Próprios/genética , TransfecçãoRESUMO
CCAAT/enhancer binding protein ß (C/EBPß) is a basic leucine zipper (bZIP) family transcription factor, which is upregulated or overactivated in many cancers, resulting in a gene expression profile that drives oncogenesis. C/EBPß dimerization regulates binding to DNA at the canonical TTGCGCAA motif and subsequent transcriptional activity, suggesting that disruption of dimerization represents a powerful approach to inhibit this previously "undruggable" oncogenic target. Here we describe the mechanism of action and antitumor activity of ST101, a novel and selective peptide antagonist of C/EBPß that is currently in clinical evaluation in patients with advanced solid tumors. ST101 binds the leucine zipper domain of C/EBPß, preventing its dimerization and enhancing ubiquitin-proteasome dependent C/EBPß degradation. ST101 exposure attenuates transcription of C/EBPß target genes, including a significant decrease in expression of survival, transcription factors, and cell-cycle-related proteins. The result of ST101 exposure is potent, tumor-specific in vitro cytotoxic activity in cancer cell lines including glioblastoma, breast, melanoma, prostate, and lung cancer, whereas normal human immune and epithelial cells are not impacted. Further, in mouse xenograft models ST101 exposure results in potent tumor growth inhibition or regression, both as a single agent and in combination studies. These data provide the First Disclosure of ST101, and support continued clinical development of ST101 as a novel strategy for targeting C/EBPß-dependent cancers.
Assuntos
Antineoplásicos , Proteína beta Intensificadora de Ligação a CCAAT , Animais , Humanos , Camundongos , Proteína beta Intensificadora de Ligação a CCAAT/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Ligação Proteica , Antineoplásicos/farmacologia , Neoplasias Experimentais/tratamento farmacológicoRESUMO
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that exhibits specific tumoricidal activity against a variety of tumors. It is expressed on different cells of the immune system and plays a role in natural killer cell-mediated tumor surveillance. In allogeneic hematopoietic-cell transplantation, the reactivity of the donor T cell against malignant cells is essential for the graft-versus-tumor (GVT) effect. Cytolytic activity of T cells is primarily mediated through the Fas-Fas ligand and perforin-granzyme pathways. However, T cells deficient for both Fas ligand and perforin can still exert GVT activity in vivo in mouse models. To uncover a potential role for TRAIL in donor T cell-mediated GVT activity, we compared donor T cells from TRAIL-deficient and wild-type mice in clinically relevant mouse bone-marrow transplantation models. We found that alloreactive T cells can express TRAIL, but the absence of TRAIL had no effect on their proliferative and cytokine response to alloantigens. TRAIL-deficient T cells showed significantly lower GVT activity than did TRAIL-expressing T cells, but no important differences in graft-versus-host disease, a major complication of allogeneic hematopoietic cell transplantation, were observed. These data suggest that strategies to enhance TRAIL-mediated GVT activity could decrease relapse rates of malignancies after hematopoietic cell transplantation without exacerbation of graft-versus-host disease.
Assuntos
Efeito Enxerto vs Tumor/imunologia , Glicoproteínas de Membrana/fisiologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Proteínas Reguladoras de Apoptose , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Ligante Indutor de Apoptose Relacionado a TNF , Transplante Homólogo , Células Tumorais CultivadasRESUMO
To date, most research into the inhibition of oncogenic transcriptional regulator, Activator Protein 1 (AP-1), has focused on heterodimers of cJun and cFos. However, the Fra1 homologue remains an important cancer target. Here we describe library design coupled with computational and intracellular screening as an effective methodology to derive an antagonist that is selective for Fra1 relative to Jun counterparts. To do so the isCAN computational tool was used to rapidly screen >75 million peptide library members, narrowing the library size by >99.8% to one accessible to intracellular PCA selection. The resulting 131 072-member library was predicted to contain high quality binders with both a high likelihood of target engagement, while simultaneously avoiding homodimerization and off-target interaction with Jun homologues. PCA screening was next performed to enrich those members that meet these criteria. In particular, optimization was achieved via inclusion of options designed to generate the potential for compromised intermolecular contacts in both desired and non-desired species. This is an often-overlooked prerequisite in the conflicting design requirement of libraries that must be selective for their target in the context of a range of alternative potential interactions. Here we demonstrate that specificity is achieved via a combination of both hydrophobic and electrostatic contacts as exhibited by the selected peptide (Fra1W). In vitro analysis of the desired Fra1-Fra1W interaction further validates high Fra1 affinity (917 nM) yet selective binding relative to Fra1W homodimers or affinity for cJun. The isCAN â PCA based multidisciplinary approach provides a robust screening pipeline in generating target-specific hits, as well as new insight into rational peptide design in the search for novel bZIP family inhibitors.
RESUMO
The long plasma half-life of IgG, while allowing for enhanced tumor uptake of tumor-targeted IgG conjugates, also results in increased background activity and normal-tissue toxicity. Therefore, successful therapeutic uses of conjugated antibodies have been limited to the highly sensitive and readily accessible hematopoietic tumors. We report a therapeutic strategy to beneficially alter the pharmacokinetics of IgG antibodies via pharmacological inhibition of the neonatal Fc receptor (FcRn) using high-dose IgG therapy. IgG-treated mice displayed enhanced blood and whole-body clearance of radioactivity, resulting in better tumor-to-blood image contrast and protection of normal tissue from radiation. Tumor uptake and the resultant therapeutic response was unaltered. Furthermore, we demonstrated the use of this approach for imaging of tumors in humans and discuss its potential applications in cancer imaging and therapy. The ability to reduce the serum persistence of conjugated IgG antibodies after their infusion can enhance their therapeutic index, resulting in improved therapeutic and diagnostic efficacy.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/uso terapêutico , Neoplasias/patologia , Neoplasias/terapia , Receptores Fc/metabolismo , Actínio/química , Animais , Feminino , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/efeitos adversos , Imunoterapia , Radioisótopos de Índio/química , Injeções Intravenosas , Radioisótopos do Iodo , Camundongos , Neoplasias/imunologia , Neoplasias/metabolismo , Tomografia por Emissão de Pósitrons , Fatores de TempoRESUMO
UNLABELLED: Effective targeting and killing of intraperitoneally disseminated micrometastases remains a challenge. OBJECTIVE/METHODS: In this work, we evaluated the potential of antibody-labeled PEGylated large liposomes as vehicles for direct intraperitoneal (i.p.) drug delivery with the aim to enhance the tumor-to-normal organ ratio and to improve the bioexposure of cancer cells to the delivered therapeutics while shifting the toxicities toward the spleen. These targeted liposomes are designed to combine: (1) specific targeting to and internalization by cancer cells mediated by liposome-conjugated tumor-specific antibodies, (2) slow clearance from the peritoneal cavity, and (3) shift of normal organ toxicities from the liver to the spleen due to their relatively large size. RESULTS: Conjugation of anti-HER2/neu antibodies to the surface of large (approximately 600 nm in diameter) PEGylated liposomes results in fast, specific binding of targeted liposomes to cancer cells in vitro, followed by considerable cellular internalization. In vivo, after i.p. administration, these liposomes exhibit fast, specific binding to i.p. cancerous tumors. Large liposomes are slowly cleared from the peritoneal cavity, and they exhibit increased uptake by the spleen relative to the liver, while targeted large liposomes demonstrate specific tumor uptake at early times. Although tissue and tumor uptake are greater for cationic liposomes, the tumor-to-liver and spleen-to-liver ratios are similar for both membrane compositions, suggesting a primary role for the liposome's size, compared to the liposome's surface charge. CONCLUSIONS: The findings of this study suggest that large targeted liposomes administered i.p. could be a potent drug-delivery strategy for locoregional therapy of i.p. micrometastatic tumors.
Assuntos
Anticorpos Antineoplásicos , Portadores de Fármacos , Infusões Parenterais , Lipossomos , Neoplasias , Receptor ErbB-2/imunologia , Animais , Anticorpos Antineoplásicos/administração & dosagem , Anticorpos Antineoplásicos/química , Anticorpos Antineoplásicos/metabolismo , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Lipossomos/administração & dosagem , Lipossomos/química , Teste de Materiais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Tamanho da Partícula , Cavidade PeritonealRESUMO
We used clinically relevant murine allogeneic bone marrow transplantation (BMT) models to study the mechanisms by which IL-7 administration can improve posttransplant peripheral T cell reconstitution. After transplant we could distinguish two populations of mature donor T cells: (a) alloreactive T cells with decreased expression of CD127 (IL-7 receptor alpha chain) and (b) nonalloreactive T cells, which express CD127 and undergo homeostatic proliferation. IL-7 administration increased the homeostatic proliferation of nonalloreactive T cells, but had no effect on alloreactive T cells and the development of graft-versus-host disease. Allogeneic transplant of purified hematopoietic stem cells and adoptive transfer of thymocytes into lethally irradiated hosts suggested that recent thymic emigrants can undergo homeostatic proliferation and acquire a memory-like phenotype. We found by BrdU pulse-chase, cell cycle, and annexin V analyses that IL-7 administration has significant proliferative and antiapoptotic effects on posttransplant peripheral T cells. We conclude that homeostatic expansion is important for T cell reconstitution after allogeneic BMT and involves both transferred mature T cells and recent thymic emigrants. Apart from its thymopoietic effects, IL-7 promotes peripheral T cell reconstitution through its selective proliferative and antiapoptotic effects on nonalloreactive and de novo-generated T cells, but has no effect on alloreactive T cells.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Interleucina-7/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Feminino , Doença Enxerto-Hospedeiro/mortalidade , Homeostase , Receptores de Hialuronatos/análise , Memória Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Receptores de Interleucina-2/análise , Receptores de Interleucina-7/análise , Linfócitos T/imunologia , Transplante HomólogoRESUMO
UNLABELLED: Single-walled carbon nanotubes (CNT) are mechanically robust graphene cylinders with a high aspect ratio that are comprised of sp(2)-bonded carbon atoms and possessing highly regular structures with defined periodicity. CNT exhibit unique mechanochemical properties that can be exploited for the development of novel drug delivery platforms. We hypothesized that novel prototype nanostructures consisting of biologics, radionuclides, fluorochromes, and CNT could be synthesized and designed to target tumor cells. METHODS: Tumor-targeting CNT constructs were synthesized from sidewall-functionalized, water-soluble CNT platforms by covalently attaching multiple copies of tumor-specific monoclonal antibodies, radiometal-ion chelates, and fluorescent probes. The constructs were characterized spectroscopically, chromatographically, and electrophoretically. The specific reactivity of these constructs was evaluated in vitro by flow cytometry and cell-based immunoreactivity assays and in vivo using biodistribution in a murine xenograft model of lymphoma. RESULTS: A soluble, reactive CNT platform was used as the starting point to build multifunctional constructs with appended antibody, metal-ion chelate, and fluorescent chromophore moieties to effect specific targeting, to carry and deliver a radiometal-ion, and to report location, respectively. These nanoconstructs were found to be specifically reactive with the human cancer cells they were designed to target in vivo in a model of disseminated human lymphoma and in vitro by flow cytometry and cell-based immunoreactivity assays versus appropriate controls. CONCLUSION: The key achievement in these studies was the selective targeting of tumor in vitro and in vivo by the use of specific antibodies appended to a soluble, nanoscale CNT construct. The ability to specifically target tumor with prototype-radiolabeled or fluorescent-labeled, antibody-appended CNT constructs was encouraging and suggested further investigation of CNT as a novel delivery platform.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Antineoplásicos/imunologia , Nanotubos de Carbono , Compostos Radiofarmacêuticos/administração & dosagem , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Murinos , Linhagem Celular Tumoral , Quelantes/química , Feminino , Compostos Heterocíclicos com 1 Anel/química , Humanos , Radioisótopos de Índio/administração & dosagem , Radioisótopos de Índio/química , Radioisótopos de Índio/farmacocinética , Linfoma de Células B/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Transplante de Neoplasias , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Rituximab , Solubilidade , Transplante HeterólogoRESUMO
Alpha-particle immunotherapy by targeted alpha-emitters or alpha-emitting isotope generators is a novel form of extraordinarily potent cancer therapy. A major impediment to the clinical use of targeted actinium-225 (225Ac) in vivo generators may be the radiotoxicity of the systemically released daughter radionuclides. The daughters, especially bismuth-213 (213Bi), tend to accumulate in the kidneys. We tested the efficacy of various pharmacologic agents and the effect of tumor burden in altering the pharmacokinetics of the 225Ac daughters to modify their renal uptake. Pharmacologic treatments in animals were started before i.v. administration of the HuM195-225Ac generator. 225Ac, francium-221 (221Fr), and 213Bi biodistributions were calculated in each animal at different time points after 225Ac generator injection. Oral metal chelation with 2,3-dimercapto-1-propanesulfonic acid (DMPS) or meso-2,3-dimercaptosuccinic acid (DMSA) caused a significant reduction (P < 0.0001) in the renal 213Bi uptake; however, DMPS was more effective than DMSA (P < 0.001). The results with DMPS were also confirmed in a monkey model. The renal 213Bi and 221Fr activities were significantly reduced by furosemide and chlorothiazide treatment (P < 0.0001). The effect on renal 213Bi activity was further enhanced by the combination of DMPS with either chlorothiazide or furosemide (P < 0.0001). Competitive antagonism by bismuth subnitrate moderately reduced the renal uptake of 213Bi. The presence of a higher target-tumor burden significantly prevented the renal 213Bi accumulation (P = 0.003), which was further reduced by DMPS treatment (P < 0.0001). Metal chelation, diuresis with furosemide or chlorothiazide, and competitive metal blockade may be used as adjuvant therapies to modify the renal accumulation of 225Ac daughters.
Assuntos
Actínio/administração & dosagem , Partículas alfa , Quelantes/farmacologia , Imunotoxinas/administração & dosagem , Rim/efeitos dos fármacos , Rim/efeitos da radiação , Radioimunoterapia/métodos , Actínio/sangue , Actínio/química , Actínio/farmacocinética , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Bismuto/química , Bismuto/farmacocinética , Feminino , Frâncio/química , Frâncio/farmacocinética , Imunotoxinas/sangue , Imunotoxinas/química , Imunotoxinas/farmacocinética , Rim/metabolismo , Linfoma/metabolismo , Linfoma/patologia , Linfoma/radioterapia , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Succímero/farmacologia , Unitiol/farmacologiaRESUMO
PURPOSE: Presentation of exogenous antigen by MHC class I molecules, or cross-presentation, is a property of dendritic cells, which is considered crucial for the priming of cytotoxic T-cell response to tumor antigens. However, the precise mechanisms of this process are not fully understood. EXPERIMENTAL DESIGN AND RESULTS: We show here in a human in vitro system, using B lymphoma cells as a tumor model, that the cross-presentation of cell-associated antigens to T cells by dendritic cells requires "help" from natural killer cells. When autologous dendritic cells that had taken up apoptotic B lymphoma cells and induced to a fully mature state were used to stimulate nonadherent cells of peripheral blood mononuclear cells from healthy donors, they induced strong cytotoxicity against B lymphoma cells in a HLA-A0201-restricted manner. The cells failed to induce cytotoxicity, however, when purified T cells were used as effector cells. Depletion of CD56+ cells, but not CD14+ or CD19+ cells, abrogated the cytotoxicity of nonadherent cells, showing that the help was provided by natural killer cells. Further, when natural killer cells were present in the cultures, a strong and persistent production of interleukin-18, but not interleukin-12 and interleukin-15, was observed. Blocking interleukin-18 significantly reduced the cytotoxicity of nonadherent cells against B lymphoma cells. CONCLUSIONS: These results suggest that capture of tumor cells and a full maturation status of dendritic cells are not sufficient to cross-prime CD8 T cells. Effective cross-priming requires further activation of dendritic cells by natural killer cells and an abundant production of interleukin-18, which, along with other yet undefined mechanisms, contribute to the generation of CTL response against B-cell lymphoma.
Assuntos
Antígenos de Neoplasias/imunologia , Apresentação Cruzada/imunologia , Citotoxicidade Imunológica/imunologia , Células Dendríticas/imunologia , Células Matadoras Naturais/imunologia , Linfoma de Células B/imunologia , Anticorpos Bloqueadores/farmacologia , Antígenos de Neoplasias/análise , Antígeno CD56/análise , Antígeno CD56/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Interleucina-18/antagonistas & inibidores , Interleucina-18/metabolismo , Depleção Linfocítica , Fagocitose , Linfócitos T Citotóxicos/imunologiaRESUMO
BACKGROUND: Prolonged immunodeficiency after allogeneic bone marrow transplantation (allo BMT) results in significant morbidity and mortality from infection. Previous studies in murine syngeneic BMT models have demonstrated that posttransplantation insulin-like growth factor (IGF)-I administration could enhance immune reconstitution. METHODS: To analyze the effects of IGF-I on immune reconstitution and graft-versus-host disease (GVHD) after allo BMT, we used murine models for MHC-matched and -mismatched allo BMT. Young (3-month-old) recipient mice received 4 mg/kg per day of human IGF-I from days 14 to 28 by continuous subcutaneous administration. RESULTS: IGF-I administration resulted in increased thymic precursor populations (triple negative-2 and triple negative-3) as determined on day 28 but had no effect on overall thymic cellularity. In the periphery, the numbers of donor-derived splenic CD3+ T cells were increased and these cells had an improved proliferative response to mitogen stimulation. IGF-I treatment also significantly increased the numbers of pro-, pre-, and mature B cells and myeloid cell populations in the spleens of allo BMT recipients on day 28. The administration of IGF-I in combination with interleukin 7 had a remarkable additive effect on B-cell, but not on T-cell, lymphopoiesis. Finally, we tested the effects of IGF-I administration on the development of GVHD in three different MHC-matched and -mismatched models and found no changes in GVHD morbidity and mortality. CONCLUSION: IGF-I administration can enhance lymphoid and myeloid reconstitution after allo BMT without aggravating GVHD.
Assuntos
Transplante de Medula Óssea/fisiologia , Doença Enxerto-Hospedeiro/prevenção & controle , Fator de Crescimento Insulin-Like I/uso terapêutico , Linfócitos T/imunologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Transplante de Medula Óssea/imunologia , Diferenciação Celular/efeitos dos fármacos , Granulócitos/efeitos dos fármacos , Granulócitos/fisiologia , Humanos , Infusões Parenterais , Fator de Crescimento Insulin-Like I/administração & dosagem , Ativação Linfocitária/efeitos dos fármacos , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Linfócitos T/efeitos dos fármacos , Transplante Homólogo , Transplante IsogênicoRESUMO
Targeted alpha-particle emitters hold great promise as therapeutics for micrometastatic disease. Because of their high energy deposition and short range, tumor targeted alpha-particles can result in high cancer-cell killing with minimal normal-tissue irradiation. Actinium-225 is a potential generator for alpha-particle therapy: it decays with a 10-day half-life and generates three alpha-particle-emitting daughters. Retention of (225)Ac daughters at the target increases efficacy; escape and distribution throughout the body increases toxicity. During circulation, molecular carriers conjugated to (225)Ac cannot retain any of the daughters. We previously proposed liposomal encapsulation of (225)Ac to retain the daughters, whose retention was shown to be liposome-size dependent. However, daughter retention was lower than expected: 22% of theoretical maximum decreasing to 14%, partially due to the binding of (225)Ac to the phospholipid membrane. In this study, Multivesicular liposomes (MUVELs) composed of different phospholipids were developed to increase daughter retention. MUVELs are large liposomes with entrapped smaller lipid-vesicles containing (225)Ac. PEGylated MUVELs stably retained over time 98% of encapsulated (225)Ac. Retention of (213)Bi, the last daughter, was 31% of the theoretical maximum retention of (213)Bi for the liposome sizes studied. MUVELs were conjugated to an anti-HER2/neu antibody (immunolabeled MUVELs) and were evaluated in vitro with SKOV3-NMP2 ovarian cancer cells, exhibiting significant cellular internalization (83%). This work demonstrates that immunolabeled MUVELs might be able to deliver higher fractions of generated alpha-particles per targeted (225)Ac compared to the relative fractions of alpha-particles delivered by (225)Ac-labeled molecular carriers.
Assuntos
Actínio/química , Partículas alfa , Anticorpos/imunologia , Linhagem Celular Tumoral , Microscopia Crioeletrônica , Humanos , Lipossomos/química , Microscopia Eletrônica de TransmissãoRESUMO
BACKGROUND: Abnormal regulation of angiogenesis in tumors results in the formation of vessels that are necessary for tumor growth, but compromised in structure and function. Abnormal tumor vasculature impairs oxygen and drug delivery and results in radiotherapy and chemotherapy resistance, respectively. Alpha particles are extraordinarily potent, short-ranged radiations with geometry uniquely suitable for selectively killing neovasculature. METHODOLOGY AND PRINCIPAL FINDINGS: Actinium-225 ((225)Ac)-E4G10, an alpha-emitting antibody construct reactive with the unengaged form of vascular endothelial cadherin, is capable of potent, selective killing of tumor neovascular endothelium and late endothelial progenitors in bone-marrow and blood. No specific normal-tissue uptake of E4G10 was seen by imaging or post-mortem biodistribution studies in mice. In a mouse-model of prostatic carcinoma, (225)Ac-E4G10 treatment resulted in inhibition of tumor growth, lower serum prostate specific antigen level and markedly prolonged survival, which was further enhanced by subsequent administration of paclitaxel. Immunohistochemistry revealed lower vessel density and enhanced tumor cell apoptosis in (225)Ac-E4G10 treated tumors. Additionally, the residual tumor vasculature appeared normalized as evident by enhanced pericyte coverage following (225)Ac-E4G10 therapy. However, no toxicity was observed in vascularized normal organs following (225)Ac-E4G10 therapy. CONCLUSIONS: The data suggest that alpha-particle immunotherapy to neovasculature, alone or in combination with sequential chemotherapy, is an effective approach to cancer therapy.
Assuntos
Partículas alfa/uso terapêutico , Neovascularização Patológica/prevenção & controle , Actínio/uso terapêutico , Partículas alfa/efeitos adversos , Animais , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Linhagem Celular Tumoral , Endotélio Vascular/efeitos da radiação , Câmaras gama , Radioisótopos de Índio/farmacocinética , Radioisótopos de Índio/uso terapêutico , Radioisótopos de Índio/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica/radioterapia , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/patologia , Distribuição TecidualRESUMO
Major histocompatibility complex (MHC) molecules carrying selected peptides will bind specifically to their cognate T-cell receptor on individual clones of reactive T cells. Fluorescently labeled, tetrameric MHC-peptide complexes have been widely used to detect and quantitate antigen-specific T-cell populations via flow cytometry. We hypothesized that such MHC-peptide tetramers could also be used to selectively deplete unique reactive T-cell populations, while leaving the remaining T-cell repertoire and immune response intact. In this report, we successfully demonstrate that a tetramer-based depletion of T cells can be achieved in a murine model of allogeneic bone marrow transplantation. Depletion of a specific alloreactive population of donor splenocytes (< 0.5% of CD8+ T cells) prior to transplantation significantly decreased morbidity and mortality from graft-versus-host disease. There was no early regrowth of the antigen-specific T cells in the recipient and in vivo T-cell proliferation was greatly reduced as well. Survival was increased more than 3-fold over controls, yet the inherent antitumor activity of the transplant was retained. This method also provides the proof-of-concept for similar strategies to selectively remove other unwanted T-cell clones, which could result in novel therapies for certain autoimmune disorders, T-cell malignancies, and solid organ graft rejection.
Assuntos
Transplante de Medula Óssea , Linfócitos T CD8-Positivos/imunologia , Doença Enxerto-Hospedeiro/tratamento farmacológico , Ativação Linfocitária/efeitos dos fármacos , Complexo Principal de Histocompatibilidade , Peptídeos/administração & dosagem , Animais , Proliferação de Células , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/imunologia , Doença Enxerto-Hospedeiro/imunologia , Ativação Linfocitária/imunologia , Depleção Linfocítica/métodos , Complexo Principal de Histocompatibilidade/imunologia , Masculino , Camundongos , Peptídeos/imunologia , Transplante HomólogoRESUMO
Lymphocyte Peyer patch adhesion molecule (LPAM) or alpha(4)beta(7) integrin is expressed on lymphocytes and is responsible for T-cell homing into gut-associated lymphoid tissues through its binding to mucosal addressin cell adhesion molecule (MAdCAM), which is present on high endothelial venules of mucosal lymphoid organs. We found in murine allogeneic bone marrow transplantation (BMT) models that recipients of alpha(4)beta(7)(-) donor T cells had significantly less graft-versus-host disease (GVHD) morbidity and mortality compared with recipients of alpha(4)beta(7)(+) donor T cells. A kinetic posttransplantation analysis of lymphocytes in the intestines and mesenteric lymph nodes demonstrated a delayed invasion of lower numbers of alpha(4)beta(7)(+) T cells in recipients of alpha(4)beta(7)(-) T cells compared with recipients of alpha(4)beta(7)(+) T cells. Histopathologic analysis of GVHD target organs revealed that recipients of alpha(4)beta(7)(-) T cells developed less GVHD of the intestines and liver, whereas there was no difference in cutaneous and thymic GVHD between recipients of alpha(4)beta(7)(-) or alpha(4)beta(7)(+) T cells. Finally, we found that in vivo GVT activity of alpha(4)beta(7)(-) donor T cells was preserved. We conclude that the alpha(4)beta(7) integrin is important for the invasion of alloreactive donor T cells into the gut and the subsequent development of intestinal GVHD and overall GVHD morbidity and mortality.
Assuntos
Transplante de Medula Óssea/efeitos adversos , Doença Enxerto-Hospedeiro/imunologia , Integrinas/imunologia , Mucosa Intestinal/imunologia , Linfócitos T/imunologia , Animais , Feminino , Doença Enxerto-Hospedeiro/mortalidade , Isoantígenos/imunologia , Leucemia/imunologia , Leucemia/mortalidade , Leucemia/terapia , Linfoma/imunologia , Linfoma/mortalidade , Linfoma/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Nódulos Linfáticos Agregados/imunologiaRESUMO
Previous studies in murine bone marrow transplantation (BMT) models using neutralizing anti-tumor necrosis factor (TNF) antibodies or TNF receptor (TNFR)-deficient recipients have demonstrated that TNF can be involved in both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL). TNF in these GVHD and GVL models was thought to be primarily produced by activated monocytes and macrophages, and the role of T cell-derived TNF was not determined. We used TNF(-/-) mice to study the specific role of TNF produced by donor T cells in a well-established parent-into-F1 hybrid model (C57BL/6J-->C3FeB6F1/J). Recipients of TNF(-/-) T cells developed significantly less morbidity and mortality from GVHD than recipients of wild-type (wt) T cells. Histology of GVHD target organs revealed significantly less damage in thymus, small bowel, and large bowel, but not in liver or skin tissues from recipients of TNF(-/-) T cells. Recipients of TNF(-/-) T cells which were also inoculated with leukemia cells at the time of BMT showed increased mortality from leukemia when compared with recipients of wt cells. We found that TNF(-/-) T cells do not have intrinsic defects in vitro or in vivo in proliferation, IFN-gamma production, or alloactivation. We could not detect TNF in the serum of our transplant recipients, suggesting that T cells contribute to GVHD and GVL via membrane-bound or locally released TNF.