RESUMO
Very late antigen-4 (VLA-4) is a transmembrane integrin protein that is highly expressed in aggressive forms of metastatic melanoma. A small-molecule peptidomimetic, LLP2A, was found to have a low pM affinity binding to VLA-4. Because LLP2A itself does not inhibit cancer cell proliferation and survival, it is an ideal candidate for the imaging and delivery of therapeutic payloads. An analog of [177Lu]Lu-labeled-LLP2A was previously investigated as a therapeutic agent in melanoma tumor-bearing mice, resulting in only a modest improvement in tumor growth inhibition, likely due to rapid clearance of the agent from the tumor. To improve the pharmacokinetic profile, DOTAGA-PEG4-LLP2A with a 4-(p-iodophenyl)butyric acid (pIBA) albumin binding moiety was synthesized. We demonstrate the feasibility of this albumin binding strategy by comparing in vitro cell binding assays and in vivo biodistribution performance of [177Lu]Lu-DOTAGA-PEG4-LLP2A ([177Lu]Lu-1) to the albumin binding [177Lu]Lu-DOTAGA-pIBA-PEG4-LLP2A ([177Lu]Lu-2). In vitro cell binding assay results for [177Lu]Lu-1 and [177Lu]Lu-2 showed Kd values of 0.40 ± 0.07 and 1.75 ± 0.40 nM, with similar Bmax values of 200 ± 6 and 315 ± 15 fmol/mg, respectively. In vivo biodistribution data for both tracers exhibited specific uptake in the tumor, spleen, thymus, and bone due to endogenous expression of VLA-4. Compound [177Lu]Lu-2 exhibited a much longer blood circulation time compared to [177Lu]Lu-1. The tumor uptake for [177Lu]Lu-1 was highest at 1 h (â¼15%ID/g) and that for [177Lu]Lu-2 was highest at 4 h (â¼23%ID/g). Significant clearance of [177Lu]Lu-1 from the tumor occurs at 24 h (<5%ID/g) while[177Lu]Lu-2 is retained for greater than 96 h (â¼10%ID/g). An efficacy study showed that melanoma tumor-bearing mice receiving compound [177Lu]Lu-2 given in two fractions (2 × 14.8 MBq, 14 days apart) had a greater median survival time than mice administered a single 29.6 MBq dose of compound [177Lu]Lu-1, while a single 29.6 MBq dose of [177Lu]Lu-2 imparted hematopoietic toxicity. The in vitro and in vivo data show addition of pIBA to [177Lu]Lu-DOTAGA-PEG4-LLP2A slows blood clearance for a higher tumor uptake, and there is potential of [177Lu]Lu-2 as a theranostic in fractionated administered doses.
Assuntos
Lutécio , Radioisótopos , Animais , Camundongos , Distribuição Tecidual , Linhagem Celular Tumoral , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Humanos , Compostos Radiofarmacêuticos/farmacocinética , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Feminino , Integrina alfa4beta1/metabolismo , Integrina alfa4beta1/antagonistas & inibidores , Albuminas , Peptídeos/química , Peptídeos/farmacocinética , Nanomedicina Teranóstica/métodos , Camundongos Endogâmicos C57BL , Dipeptídeos , Compostos de FenilureiaRESUMO
Integrin α4ß1 belongs to the leukocyte integrin family and represents a therapeutic target of relevant interest given its primary role in mediating inflammation, autoimmune pathologies and cancer-related diseases. The focus of the present work is the design, synthesis and characterization of new peptidomimetic compounds that are potentially able to recognize α4ß1 integrin and interfere with its function. To this aim, a collection of seven new cyclic peptidomimetics possessing both a 4-aminoproline (Amp) core scaffold grafted onto key α4ß1-recognizing sequences and the (2-methylphenyl)ureido-phenylacetyl (MPUPA) appendage, was designed, with the support of molecular modeling studies. The new compounds were synthesized through SPPS procedures followed by in-solution cyclization maneuvers. The biological evaluation of the new cyclic ligands in cell adhesion assays on Jurkat cells revealed promising submicromolar agonist activity in one compound, namely, the c[Amp(MPUPA)Val-Asp-Leu] cyclopeptide. Further investigations will be necessary to complete the characterization of this class of compounds.
Assuntos
Adesão Celular/efeitos dos fármacos , Integrina alfa4beta1/química , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Prolina/análogos & derivados , Humanos , Integrina alfa4beta1/antagonistas & inibidores , Células Jurkat , Ligantes , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Prolina/química , Prolina/farmacologia , Ligação Proteica , Conformação ProteicaRESUMO
BACKGROUND: The integrin VLA-4 (α4ß1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4ß1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets. METHODS: Mice (female; B10.RIII or C57Bl/6; aged 6-8 weeks) were immunized with specific interphotoreceptor retinoid-binding protein (IRBP) peptides to induce EAU. Topically administered GW (3, 10, and 30 mg/ml) were given twice daily either therapeutically once disease was evident, or prophylactically, and compared with vehicle-treated (Veh) and 0.1% dexamethasone-treated (Dex) controls. Mice were sacrificed at peak disease. The retinal T cell subsets were investigated by immunohistochemistry and immunofluorescence staining. The immune cells within the retina, blood, and draining lymph nodes (dLNs) were phenotyped by flow cytometry. The effect of GW559090 on non-adherent, adherent, and migrated CD4+ T cell subsets across a central nervous system (CNS) endothelium was further assayed in vitro and quantitated by flow cytometry. RESULTS: There was a significant reduction in clinical and histological scores in GW10- and Dex-treated groups as compared to controls either administered therapeutically or prophylactically. There were fewer CD45+ leukocytes infiltrating the retinae and vitreous fluids in the treated GW10 group (P < 0.05). Immunofluorescence staining and flow cytometry data identified decreased levels of retinal Th17 cells (P ≤ 0.001) in the GW10-treated eyes, leaving systemic T cell subsets unaffected. In addition, fewer Ly6C+ inflammatory monocyte/macrophages (P = 0.002) and dendritic cells (P = 0.017) crossed the BRB following GW10 treatment. In vitro migration assays confirmed that Th17 cells were selectively suppressed by GW559090 in adhering to endothelial monolayers. CONCLUSIONS: This α4ß1 integrin inhibitor may exert a modulatory effect in EAU progression by selectively blocking Th17 cell migration across the blood-retinal barrier without affecting systemic CD4+ T cell subsets. Local α4ß1 integrin-directed inhibition could be clinically relevant in treating a Th17-dominant form of uveitis.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Barreira Hematorretiniana/efeitos dos fármacos , Integrina alfa4beta1/antagonistas & inibidores , Fenilalanina/análogos & derivados , Piperidinas/administração & dosagem , Células Th17/efeitos dos fármacos , Uveíte/tratamento farmacológico , Animais , Doenças Autoimunes/metabolismo , Barreira Hematorretiniana/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Integrina alfa4beta1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenilalanina/administração & dosagem , Fenilalanina/metabolismo , Piperidinas/metabolismo , Células Th17/metabolismo , Uveíte/metabolismoRESUMO
This article presents a simplified view of integrins with emphasis on the α4 (α4ß1/VLA-4) integrin. Integrins are heterodimeric proteins expressed on the cell surface of leukocytes that participate in a wide variety of functions, such as survival, growth, differentiation, migration, inflammatory responses, tumour invasion, among others. When the extracellular matrix is degraded or deformed, cells are forced to undergo responsive changes that influence remodelling during physiological and pathological events. Integrins recognize these changes and trigger a series of cellular responses, forming a physical connection between the interior and the outside of the cell. The communication of integrins through the plasma membrane occurs in both directions, from the extracellular to the intracellular (outside-in) and from the intracellular to the extracellular (inside-out). Integrins are valid targets for antibodies and small-molecule antagonists. One example is the monoclonal antibody natalizumab, marketed under the name of TYSABRI®, used in the treatment of recurrent multiple sclerosis, which inhibits the adhesion of α4 integrin to its counter-receptor. α4ß1 Integrin antagonists are summarized here, and their utility as therapeutics are also discussed.
Assuntos
Integrina alfa4beta1 , Anticorpos Monoclonais , Adesão Celular , Integrina alfa4beta1/antagonistas & inibidores , Integrina alfa4beta1/fisiologia , LeucócitosRESUMO
In recent years, several studies suggested that the ability of hyperbaric oxygen therapy (HBOT) to promote healing in patients with diabetic ulcers and chronic wounds is due to the reduction of inflammatory cytokines and to a significant decrease in neutrophils recruitment to the damaged area. α4 and ß2 integrins are receptors mediating the neutrophil adhesion to the endothelium and the comprehension of the effects of hyperbaric oxygenation on their expression and functions in neutrophils could be of great importance for the design of novel therapeutic protocols focused on anti-inflammatory agents. In this study, the α4 and ß2 integrins' expression and functions have been evaluated in human primary neutrophils obtained from patients with chronic non-healing wounds and undergoing a prolonged HBOT (150 kPa per 90 minutes). The effect of a peptidomimetic α4ß1 integrin antagonist has been also analyzed under these conditions. A statistically significant decrease (68%) in ß2 integrin expression on neutrophils was observed during the treatment with HBO and maintained one month after the last treatment, while α4 integrin levels remained unchanged. However, cell adhesion function of both neutrophilic integrins α4ß1 and ß2 was significantly reduced 70 and 67%, respectively), but α4ß1 integrin was still sensitive to antagonist inhibition in the presence of fibronectin, suggesting that a combined therapy between HBOT and integrin antagonists could have greater antinflammatory efficacy.
Assuntos
Oxigenoterapia Hiperbárica , Integrina alfa4beta1/antagonistas & inibidores , Neutrófilos/imunologia , Peptidomiméticos/uso terapêutico , Úlcera Cutânea/terapia , Idoso , Idoso de 80 Anos ou mais , Antígenos CD18/análise , Antígenos CD18/metabolismo , Adesão Celular/imunologia , Doença Crônica/terapia , Terapia Combinada/métodos , Feminino , Seguimentos , Humanos , Integrina alfa4beta1/análise , Integrina alfa4beta1/metabolismo , Masculino , Pessoa de Meia-Idade , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Peptidomiméticos/farmacologia , Cultura Primária de Células , Úlcera Cutânea/sangue , Úlcera Cutânea/imunologia , Úlcera Cutânea/patologia , Resultado do Tratamento , Cicatrização/efeitos dos fármacos , Cicatrização/imunologiaRESUMO
Very late antigen 4 (VLA-4; also called integrin α4ß1) is overexpressed in melanoma tumor cells with an active role in tumor growth, angiogenesis, and metastasis, making VLA-4 a potential target for targeted alpha therapy (TAT). METHODS: An anti-VLA-4 antibody was conjugated to DOTA for [225Ac]Ac-labeling and DTPA for [111In]In-labeling. The resulting agents, [225Ac]Ac- or [111In]In-labeled anti-VLA-4 were evaluated in vitro, including binding affinity, internalization, and colony formation assays as well as in vivo biodistribution studies. In addition, the therapeutic efficacy of [225Ac]Ac-DOTA-anti-VLA-4 was evaluated in a disseminated disease mouse model of melanoma. RESULTS: [111In]In-DTPA-anti-VLA-4 demonstrated high affinity for VLA-4 (Kd = 5.2 ± 1.6 nM). [225Ac]Ac-DOTA-anti-VLA-4 was labeled with an apparent molar activity of 3.5 MBq/nmol and > 95% radiochemical purity. Colony formation assays demonstrated a decrease in the surviving fraction of B16F10 cells treated with [225Ac]Ac-DOTA-anti-VLA-4 compared to control. Biodistribution studies demonstrated accumulation in the VLA-4-positive tumor and VLA-4 rich organs. Therapeutic efficacy studies demonstrated a significant increase in survival in mice treated with [225Ac]Ac-DOTA-anti-VLA-4 as compared to controls. CONCLUSION: The work presented here demonstrated that [225Ac]Ac-DOTA-anti-VLA-4 was effective as a treatment in mice with disseminated disease, but potentially has dose limiting hematopoietic toxicity. Preliminary studies presented here also supported the potential to overcome this limitation by exploring a pre-loading or blocking dose strategy, to optimize the targeting vector to help minimize the absorbed dose to VLA-4 rich organs while maximizing the dose delivered to VLA-4-positive melanoma tumor cells.
Assuntos
Actínio/farmacologia , Partículas alfa/uso terapêutico , Compostos Heterocíclicos com 1 Anel/química , Integrina alfa4beta1/antagonistas & inibidores , Melanoma Experimental/radioterapia , Compostos Radiofarmacêuticos/farmacologia , Animais , Quelantes/química , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos C57BL , RadioquímicaRESUMO
OBJECTIVE: To determine whether natalizumab efficacy is maintained when switching to personalized extended interval dosing based on individual natalizumab trough concentrations in patients with relapsing-remitting multiple sclerosis (RRMS). METHODS: This was a prospective multicenter single-arm trial with 1 year follow-up and a 1-year extension phase. Participants were adult persons with RRMS treated with natalizumab without disease activity in the year prior to enrollment. The natalizumab treatment interval was based on longitudinal natalizumab trough concentrations. Patients received 3 monthly MRI scans, relapse assessments, and disability scoring during follow-up. The primary endpoint was the occurrence of gadolinium-enhancing lesions on MRI. Secondary endpoints were new/enlarging T2 lesions on MRI and relapses and progression on the Expanded Disability Status Scale (EDSS) during follow-up and extension phase. RESULTS: Sixty-one patients were included. Eighty-four percent extended the interval from a 4-week interval to a 5- to 7-week interval. No patient developed gadolinium-enhancing lesions (95% confidence interval [CI] 0%-7.4%) during follow-up. No new/enlarging T2 lesions (95% CI 0%-7.4%) or relapses (95% CI 0%-7.4%) were reported during follow-up and in the extension phase. Median EDSS was comparable at baseline (3.0, interquartile range [IQR] 2.0-5.0) and after follow-up (3.0, IQR 2.0-5.0). CONCLUSION: Personalized extended interval dosing did not induce recurrence of MS disease activity. Natalizumab efficacy was maintained in stable patients with RRMS receiving personalized extended interval dosing based on individual natalizumab concentrations. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that personalized extended interval dosing of natalizumab does not result in recurrence of disease activity in stable patients with RRMS.
Assuntos
Esclerose Múltipla/tratamento farmacológico , Natalizumab/administração & dosagem , Adulto , Avaliação da Deficiência , Esquema de Medicação , Monitoramento de Medicamentos , Feminino , Seguimentos , Humanos , Integrina alfa4beta1/antagonistas & inibidores , Integrina alfa4beta1/imunologia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/sangue , Esclerose Múltipla/diagnóstico por imagem , Natalizumab/sangue , Natalizumab/uso terapêutico , Países Baixos , Neuroimagem , Medicina de Precisão , Estudos Prospectivos , Índice de Gravidade de DoençaRESUMO
Redirecting T cells to specifically kill malignant cells has been validated as an effective anti-cancer strategy in the clinic with the approval of blinatumomab for acute lymphoblastic leukemia. However, the immunosuppressive nature of the tumor microenvironment potentially poses a significant hurdle to T cell therapies. In hematological malignancies, the bone marrow (BM) niche is protective to leukemic stem cells and has minimized the efficacy of several anti-cancer drugs. In this study, we investigated the impact of the BM microenvironment on T cell redirection. Using bispecific antibodies targeting specific tumor antigens (CD123 and BCMA) and CD3, we observed that co-culture of acute myeloid leukemia or multiple myeloma cells with BM stromal cells protected tumor cells from bispecific antibody-T cell-mediated lysis in vitro and in vivo. Impaired CD3 redirection cytotoxicity was correlated with reduced T cell effector responses and cell-cell contact with stromal cells was implicated in reducing T cell activation and conferring protection of cancer cells. Finally, blocking the VLA4 adhesion pathway in combination with CD3 redirection reduced the stromal-mediated inhibition of cytotoxicity and T cell activation. Our results lend support to inhibiting VLA4 interactions along with administering CD3 redirection therapeutics as a novel combinatorial regimen for robust anti-cancer responses.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Medula Óssea/efeitos dos fármacos , Complexo CD3/imunologia , Integrina alfa4beta1/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Mieloma Múltiplo/tratamento farmacológico , Animais , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Antígeno de Maturação de Linfócitos B/antagonistas & inibidores , Antígeno de Maturação de Linfócitos B/imunologia , Medula Óssea/imunologia , Medula Óssea/patologia , Complexo CD3/antagonistas & inibidores , Linhagem Celular Tumoral , Feminino , Humanos , Integrina alfa4beta1/imunologia , Subunidade alfa de Receptor de Interleucina-3/antagonistas & inibidores , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Camundongos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologia , Microambiente Tumoral/efeitos dos fármacosAssuntos
Suscetibilidade a Doenças , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/etiologia , Integrina alfa4beta1/antagonistas & inibidores , Terapia de Alvo Molecular , Animais , Biomarcadores , Modelos Animais de Doenças , Antígenos H-2/genética , Antígenos H-2/imunologia , Transplante de Células-Tronco Hematopoéticas , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transplante HomólogoRESUMO
Multiple myeloma (MM) cells accumulate in the bone marrow (BM) where their interactions impede disease therapy. We have shown that microvesicles (MVs) derived from BM mesenchymal stem cells (MSCs) of MM patients promote the malignant traits via modulation of translation initiation (TI), whereas MVs from normal donors (ND) do not. Here, we observed that this phenomenon is contingent on a MVs' protein constituent, and determined correlations between the MVs from the tumor microenvironment, for example, MM BM-MSCs and patients' clinical characteristics. BM-MSCs' MVs (ND/MM) proteomes were assayed (mass spectrometry) and compared. Elevated integrin CD49d (X80) and CD29 (X2) was determined in MM-MSCs' MVs and correlated with patients' staging and treatment response (free light chain, BM plasma cells count, stage, response to treatment). BM-MSCs' MVs uptake into MM cell lines was assayed (flow cytometry) with/without integrin inhibitors (RGD, natalizumab, and anti-CD29 monoclonal antibody) and recipient cells were analyzed for cell count, migration, MAPKs, TI, and drug response (doxorubicin, Velcade). Their inhibition, particularly together, attenuated the uptake of MM-MSCs MVs (but not ND-MSCs MVs) into MM cells and reduced MM cells' signaling, phenotype, and increased drug response. This study exposed a critical novel role for CD49d/CD29 on MM-MSCs MVs and presented a discriminate method to inhibit cancer promoting action of MM-MSCs MVs while retaining the anticancer function of ND-MSCs-MVs. Moreover, these findings demonstrate yet again the intricacy of the microenvironment involvement in the malignant process and highlight new therapeutic avenues to be explored.
Assuntos
Carcinogênese/patologia , Micropartículas Derivadas de Células/patologia , Integrina alfa4beta1/metabolismo , Células-Tronco Mesenquimais/patologia , Mieloma Múltiplo/patologia , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/patologia , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Separação Celular , Micropartículas Derivadas de Células/efeitos dos fármacos , Feminino , Citometria de Fluxo , Humanos , Integrina alfa4beta1/antagonistas & inibidores , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Natalizumab/farmacologia , Natalizumab/uso terapêutico , Estadiamento de Neoplasias , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Cultura Primária de Células , Proteômica , Microambiente TumoralRESUMO
By dissecting the structure of ß-lactam-based ligands, a new series of compounds was designed, synthesized, and evaluated toward integrins αvß3, α5ß1, and α4ß1. New selective ligands with antagonist or agonist activities of cell adhesion in the nanomolar range were obtained. The best agonist molecules induced significant adhesion of SK-MEL-24 cells and Saos-2 cells as a valuable model for osteoblast adhesion. These data could lead to the development of new agents to improve cellular osseointegration and bone regeneration. Molecular modeling studies on prototypic compounds and αvß3 or α5ß1 integrin supported the notion that ligand carboxylate fixing to the metal ion-dependent adhesion site in the ß-subunit can be sufficient for binding the receptors, while the aryl side chains play a role in determining the selectivity as well as agonism versus antagonism.
Assuntos
Integrinas/agonistas , Integrinas/antagonistas & inibidores , beta-Lactamas/química , beta-Lactamas/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Integrina alfa4beta1/agonistas , Integrina alfa4beta1/antagonistas & inibidores , Integrina alfa4beta1/metabolismo , Integrina alfa5beta1/agonistas , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/agonistas , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/metabolismo , Integrinas/metabolismo , Ligantes , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Moleculares , Simulação de Acoplamento Molecular , Osteoblastos/efeitos dos fármacos , Relação Estrutura-Atividade , beta-Lactamas/síntese químicaRESUMO
Despite ligand-targeted liposomes long garnering interest as drug delivery vehicles for cancer therapeutics, inconsistency in successful outcomes have hindered their translation into the clinic. This is in part due to discrepancies between in vitro design evaluations and final in vivo outcomes. By employing a multifaceted synthetic strategy to prepare peptide-targeted nanoparticles of high purity, reproducibility, and with precisely controlled quantity of functionalities, we systematically evaluated the individual roles that peptide-linker length, peptide hydrophilicity, peptide density, and nanoparticle size play on cancer cell uptake and tumor targeting both in vitro and in vivo, and how the results correlated and contrasted. These parameters were analyzed using a VLA-4-targeted liposome system in a multiple myeloma mouse xenograft model to evaluate in vivo biodistribution and tumor cell uptake. The results showed that using in vitro models to optimize targeted-nanoparticles for maximum cellular uptake was helpful in narrowing down the particle characteristics. However, in vitro optimization fell short of achieving enhanced results in animal models, rather had negative consequences for in vivo targeting. This outcome is not surprising considering that the receptor being targeted is also present on healthy lymphocytes and increasing targeting peptide valency on particle surfaces results in an increase in non-selective, off-target binding to healthy cells. Hence, further optimization using in vivo models was absolutely necessary, through which we were able to increase the uptake of peptide-targeted liposomes by cancerous cells overexpressing VLA-4 to 15-fold over that of non-targeted liposomes in vivo. The results highlighted the importance of creating a comprehensive understanding of the effect of each liposome design parameter on multifactorial biological endpoints including both in vitro and in vivo in determining the therapeutic potential of peptide-targeted liposomes.
Assuntos
Integrina alfa4beta1/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Nanopartículas/administração & dosagem , Peptídeos/administração & dosagem , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Lipossomos , Camundongos SCID , Mieloma Múltiplo/metabolismo , Nanopartículas/química , Peptídeos/químicaRESUMO
Mobilized peripheral blood has become the primary source of hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation, with a five-day course of granulocyte colony stimulating factor (G-CSF) as the most common regimen used for HSPC mobilization. The CXCR4 inhibitor, plerixafor, is a more rapid mobilizer, yet not potent enough when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. We sought to improve hematopoietic stem cell transplantation by developing a new mobilization strategy in mice through combined targeting of the chemokine receptor CXCR2 and the very late antigen 4 (VLA4) integrin. Rapid and synergistic mobilization of HSPCs along with an enhanced recruitment of true HSCs was achieved when a CXCR2 agonist was co-administered in conjunction with a VLA4 inhibitor. Mechanistic studies revealed involvement of CXCR2 expressed on BM stroma in addition to stimulation of the receptor on granulocytes in the regulation of HSPC localization and egress. Given the rapid kinetics and potency of HSPC mobilization provided by the VLA4 inhibitor and CXCR2 agonist combination in mice compared to currently approved HSPC mobilization methods, it represents an exciting potential strategy for clinical development in the future.
Assuntos
Medula Óssea/metabolismo , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Integrina alfa4beta1 , Receptores de Interleucina-8B , Aloenxertos , Animais , Granulócitos/metabolismo , Integrina alfa4beta1/antagonistas & inibidores , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismoRESUMO
Targeted liposomal nanoparticles are commonly used drug delivery vehicles for targeting cancer cells that overexpress a particular cell surface receptor. However, typical target receptors are also expressed at variable levels in healthy tissue, leading to non-selective targeting and systemic toxicity. Here, we demonstrated that the selectivity of peptide-targeted liposomes for their target cells can be significantly enhanced by employing a dual-receptor targeted approach to simultaneously target multiple tumor cell surface receptors. The dual-receptor targeted approach can be tuned to create cooperativity in binding only for the cancer cells, therefore leaving the healthy cells and tissue unharmed. We evaluated this strategy in a multiple myeloma disease model where the liposomes were functionalized with two distinct peptide antagonists to target VLA-4 and LPAM-1, two receptors with increasing relevance in multiple myeloma. By employing a multifaceted strategy to synthesize dual-receptor targeted liposomes with high purity, reproducibility, and precisely controlled stoichiometry of functionalities, we identified optimal design parameters for enhanced selectivity via systematic analysis. Through control of the liposomal formulation and valency of each targeting peptide, we identified that the optimal dual-receptor targeted liposome consisted of a peptide density of 0.75% VLA4pep and 1% LPAM1pep, resulting in an 8-fold and 12-fold increased cellular uptake over VLA-4 and LPAM-1 single targeted liposomes respectively. This formulation resulted in a cooperative ratio of 4.3 and enhanced uptake for myeloma cells that simultaneously express both VLA-4 and LPAM-1 receptors, but displayed no increase in uptake for cells that express only one or neither of the receptors, resulting in a 28-fold selectivity of the dual-targeted liposomes for cells displaying both targeted receptors over cells displaying neither receptor. These results demonstrated that through refined design and well-characterized nanoparticle formulations, dual-receptor targeted liposomes have the potential to improve cancer therapy by providing enhanced selectivity over conventional single-receptor targeted approaches.
Assuntos
Integrina alfa4beta1 , Integrinas , Nanopartículas , Proteínas de Neoplasias , Neoplasias , Peptídeos , Humanos , Integrina alfa4beta1/antagonistas & inibidores , Integrina alfa4beta1/metabolismo , Integrinas/antagonistas & inibidores , Integrinas/metabolismo , Células Jurkat , Lipossomos , Nanopartículas/química , Nanopartículas/uso terapêutico , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Peptídeos/química , Peptídeos/farmacologiaRESUMO
Recipient endogenous memory CD8 T cells expressing reactivity to donor class I MHC infiltrate MHC-mismatched cardiac allografts within 24 hours after reperfusion and express effector functions mediating graft injury. The current study tested the efficacy of Very Late Antigen-4 (VLA-4) blockade to inhibit endogenous memory CD8 T cell infiltration into cardiac allografts and attenuate early posttransplant inflammation. Peritransplant anti-VLA-4 mAb given to C57BL6 (H-2b ) recipients of AJ (H-2a ) heart allografts completely inhibited endogenous memory CD4 and CD8 T cell infiltration with significant decrease in macrophage, but not neutrophil, infiltration into allografts subjected to either minimal or prolonged cold ischemic storage (CIS) prior to transplant, reduced intra-allograft IFN-γ-induced gene expression and prolonged survival of allografts subjected to prolonged CIS in CTLA-4Ig treated recipients. Anti-VLA-4 mAb also inhibited priming of donor-specific T cells producing IFN-γ until at least day 7 posttransplant. Peritransplant anti-VLA plus anti-CD154 mAb treatment similarly prolonged survival of allografts subjected to minimal or increased CIS prior to transplant. Overall, these data indicate that peritransplant anti-VLA-4 mAb inhibits early infiltration memory CD8 T cell infiltration into allografts with a marked reduction in early graft inflammation suggesting an effective strategy to attenuate negative effects of heterologous alloimmunity in recipients of higher risk grafts.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/imunologia , Rejeição de Enxerto/imunologia , Transplante de Coração , Memória Imunológica , Integrina alfa4beta1/antagonistas & inibidores , Animais , Camundongos , Camundongos Endogâmicos C57BL , Transplante HomólogoRESUMO
Very late antigen-4 (VLA-4; also known as integrin α4ß1) is expressed at high levels in aggressive and metastatic melanoma tumors and may provide an ideal target for imaging and targeted radionuclide therapy (TRT). 177Lu-DOTA-PEG4-LLP2A (177Lu-LLP2A) is a TRT that shows high affinity for VLA-4 and high uptake in B16F10 mouse melanoma tumors in vivo. Here, we report efficacy studies of 177Lu-LLP2A, alone and combined with immune checkpoint inhibitors (ICIs) (anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies), in B16F10 tumor-bearing mice. Methods: Tumor cells (1 × 106) were implanted subcutaneously in C57BL/6 mice. After 8-10 d, the mice were randomized into 8 groups. 177Lu-LLP2A was injected intravenously on day 8 or 9 (single dose), and ICI antibodies were administered intraperitoneally in 3 doses. Tumor growth was monitored over time via calipers. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining for apoptosis was performed on fixed tumors. In a separate study, Cy3-LLP2A or Cy3-scrambled LLP2A was injected in tumor-bearing mice, and tumors were collected 4 h after injection and then analyzed by flow cytometry and immunofluorescence microscopy using different immune cell markers. Results: TRT alone showed efficacy comparable to the dual-ICI anti-PD-1 + anti-CTLA-4 or anti-PD-L1 + anti-CTLA-4, whereas TRT + ICIs significantly enhanced survival. TUNEL staining showed that the highest levels of apoptosis were in the TRT + ICI groups. In addition to targeting tumor cells, TRT also bound immune cells in the tumor microenvironment. Flow cytometry data showed that the tumors consisted of about 77% tumor cells and fibroblasts (CD45-negative/CD49d-positive) and about 23% immune cells (CD45-positive/CD49d-positive) and that immune cells expressed higher levels of VLA-4. Cy3-LLP2A and CD49d colocalized with macrophages (CD68), T cells (CD8, CD4), and B cells (CD19). Immunohistochemical analysis identified a significant colocalization of Cy3-LLP2A and CD68. Conclusion: Combination treatment with TRT + ICIs targets both tumor cells and immune cells and has potential as a therapeutic agent in patients with metastatic melanoma.
Assuntos
Integrina alfa4beta1/antagonistas & inibidores , Lutécio/uso terapêutico , Melanoma Experimental/imunologia , Melanoma Experimental/radioterapia , Radioimunoterapia/métodos , Radioisótopos/uso terapêutico , Compostos Radiofarmacêuticos/uso terapêutico , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno CTLA-4/antagonistas & inibidores , Linhagem Celular Tumoral , Dipeptídeos/química , Feminino , Radioisótopos de Gálio , Compostos Heterocíclicos com 1 Anel/química , Humanos , Lutécio/farmacocinética , Masculino , Melanoma Experimental/diagnóstico por imagem , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular/métodos , Compostos de Fenilureia/química , Polietilenoglicóis/química , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Radioisótopos/farmacocinética , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Dosagem Radioterapêutica , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
BACKGROUND AND PURPOSE: Allergic conjunctivitis is an eye inflammation that involves the infiltration of immune cells into the conjunctiva via cell surface-adhesion receptors, such as integrin α4 ß1 . These receptors interact with adhesion molecules expressed on the conjunctival endothelium and may be a target to treat this disease. We synthesized DS-70, a novel α/ß-peptidomimetic α4 integrin antagonist, to prevent the conjunctival infiltration of immune cells and clinical symptoms in a model of allergic conjunctivitis. EXPERIMENTAL APPROACH: In vitro, DS-70 was pharmacologically characterized using a scintillation proximity procedure to measure its affinity for α4 ß1 integrin, and its effect on cell adhesion mediated by different integrins was also evaluated. The effects of DS-70 on vascular cell adhesion molecule-1 (VCAM-1)-mediated degranulation of a human mast cell line and an eosinophilic cell line, which both express α4 ß1 , and on VCAM-1-mediated phosphorylation of ERK 1/2 in Jurkat E6.1 cells were investigated. Effects of DS-70 administered in the conjunctival fornix of ovalbumin-sensitized guinea pigs were evaluated. KEY RESULTS: DS-70 bound to integrin α4 ß1 with nanomolar affinity, prevented the adhesion of α4 integrin-expressing cells, antagonized VCAM-1-mediated degranulation of mast cells and eosinophils and ERK 1/2 phosphorylation. Only 20% was degraded after an 8 h incubation with serum. DS-70 dose-dependently reduced the clinical symptoms of allergic conjunctivitis, conjunctival α4 integrin expression and conjunctival levels of chemokines and cytokines in ovalbumin-sensitized guinea pigs. CONCLUSIONS AND IMPLICATIONS: These findings highlight the role of α4 integrin in allergic conjunctivitis and suggest that DS-70 is a potential treatment for this condition.
Assuntos
Antialérgicos/uso terapêutico , Conjuntivite Alérgica/tratamento farmacológico , Integrina alfa4beta1/antagonistas & inibidores , Peptidomiméticos/uso terapêutico , Animais , Antialérgicos/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/metabolismo , Conjuntivite Alérgica/metabolismo , Modelos Animais de Doenças , Cobaias , Humanos , Integrina alfa4beta1/metabolismo , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Peptidomiméticos/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Inflammation drives the degradation of atherosclerotic plaque, yet there are no non-invasive techniques available for imaging overall inflammation in atherosclerotic plaques, especially in the coronary arteries. To address this, we have developed a clinically relevant system to image overall inflammatory cell burden in plaque. Here, we describe a targeted contrast agent (THI0567-targeted liposomal-Gd) that is suitable for magnetic resonance (MR) imaging and binds with high affinity and selectivity to the integrin α4ß1(very late antigen-4, VLA-4), a key integrin involved in recruiting inflammatory cells to atherosclerotic plaques. This liposomal contrast agent has a high T1 relaxivity (~2 × 105 mM-1s-1 on a particle basis) resulting in the ability to image liposomes at a clinically relevant MR field strength. We were able to visualize atherosclerotic plaques in various regions of the aorta in atherosclerosis-prone ApoE-/- mice on a 1 Tesla small animal MRI scanner. These enhanced signals corresponded to the accumulation of monocyte/macrophages in the subendothelial layer of atherosclerotic plaques in vivo, whereas non-targeted liposomal nanoparticles did not demonstrate comparable signal enhancement. An inflammatory cell-targeted method that has the specificity and sensitivity to measure the inflammatory burden of a plaque could be used to noninvasively identify patients at risk of an acute ischemic event.
Assuntos
Integrina alfa4beta1/química , Integrina alfa4beta1/metabolismo , Imageamento por Ressonância Magnética , Placa Aterosclerótica/diagnóstico por imagem , Placa Aterosclerótica/metabolismo , Animais , Modelos Animais de Doenças , Integrina alfa4beta1/antagonistas & inibidores , Ligantes , Lipossomos , Imageamento por Ressonância Magnética/métodos , Camundongos , Camundongos Knockout , Modelos Moleculares , Conformação Molecular , Placa Aterosclerótica/patologia , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Immunosuppressive myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) accumulate in tumors where they inhibit T cell-mediated antitumor immune responses and promote tumor progression. Myeloid cell PI3Kγ plays a role in regulating tumor immune suppression by promoting integrin α4-dependent MDSC recruitment to tumors and by stimulating the immunosuppressive polarization of MDSCs and TAMs. Here, we show that integrin α4 promotes immunosuppressive polarization of MDSCs and TAMs downstream of PI3Kγ, thereby inhibiting antitumor immunity. Genetic or pharmacological suppression of either PI3Kγ or integrin α4 blocked MDSC recruitment to tumors and also inhibited immune suppressive myeloid cell polarization, thereby reducing expression of IL10 and increasing expression of IL12 and IFNγ within tumors. Inhibition of PI3Kγ or integrin α4 within tumors stimulated dendritic cell and CD8+ T-cell recruitment and maturation, as well as tumor cell cytotoxicity in vivo, thereby inhibiting tumor growth. As blockade of PI3Kγ or integrin α4 prevents accumulation of MDSC and reduces myeloid cell expression of immunosuppressive factors that stimulate tumor immune escape, these results highlight PI3Kγ and integrin α4 as targets for the design of cancer therapeutics. Cancer Immunol Res; 5(11); 957-68. ©2017 AACR.
Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase/imunologia , Integrina alfa4beta1/imunologia , Neoplasias/imunologia , Animais , Anticorpos/farmacologia , Linhagem Celular Tumoral , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Citocinas/imunologia , Feminino , Tolerância Imunológica , Integrina alfa4beta1/antagonistas & inibidores , Linfócitos do Interstício Tumoral/imunologia , Macrófagos/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Inibidores de Fosfoinositídeo-3 QuinaseRESUMO
Natalizumab (NTZ) is a monoclonal antibody targeting the α4ß1 integrin (CD49d/CD29), very late antigen-4 (VLA-4), which is approved for treatment of relapsing-remitting multiple sclerosis (RR-MS). A possible association between NTZ treatment and a higher risk of melanoma is under debate. Natural Killer (NK) cells, which express VLA-4, represent an innate barrier limiting spreading of melanoma under steady state conditions. Indeed, because of their expression of activating receptors, they are very efficient in recognizing and killing melanoma cells without the need of a previous priming. For this reason, we aimed at assessing whether NK-cell functions might be impaired by sustained exposure to NTZ. To investigate this possibility we isolated NK cells from healthy donors and tested their cytotoxic and migratory functions against primary melanoma cells derived from subcutaneous and lymph node metastases. Flow cytometry analysis demonstrated expression of CD49d on both freshly isolated NK cells and activated NK cells. Moreover, VLA-4 and its receptor, vascular cell adhesion protein-1 (VCAM-1) were similarly expressed on freshly isolated NK cells. However, upon a short exposure to NTZ, expression of VLA-4 on NK cells decreased. Analysis of NK receptor expression upon exposure of NK cells from three healthy donors to NTZ indicated that DNAM-1 and NKp46 are apparently decreased, while NKG2A is increased. The degranulation of NK cells towards melanoma cells, which express both VLA-4 and VCAM-1, was not affected when NTZ was added to the co-culture or when both NK cells and melanoma cells were each pre-exposed to NTZ for over 12h. In contrast, degranulation was significantly inhibited after 48h of pre-incubation indicating that NTZ can influence NK-cell degranulation towards melanoma cells only after a prolonged exposure. Using a migration chamber assay, we observed that the migration of NK cells towards melanoma cells was dependent upon the concentration of melanoma cells in the lower chamber, and that it was significantly reduced in presence of NTZ. Our results show that upon exposure to NTZ both cytolytic activity and migration toward melanoma cells were affected, suggesting that binding of NTZ to NK cells affects pathways involved in these NK-cell functions. We analyzed the expression of CD49d on NK cells from MS patients treated with NTZ and observed that it decreases with time of treatment. These data suggest that blockade of VLA-4 on NK-cell surface alters some key functions involved in the immune surveillance toward melanoma by NK cells and may provide a mechanistic explanation for the reported occurrence of melanoma in MS patients treated with NTZ.