RESUMO
Glioblastoma (GB) is the most common primary brain tumor, which is characterized by low immunogenicity of tumor cells and prevalent immunosuppression in the tumor microenvironment (TME). Targeted local combination immunotherapy is a promising strategy to overcome these obstacles. Here, we evaluated tumor-cell specific delivery of an anti-PD-1 immunoadhesin (aPD-1) via a targeted adeno-associated viral vector (AAV) as well as HER2-specific NK-92/5.28.z (anti-HER2.CAR/NK-92) cells as components for a combination immunotherapy. In co-culture experiments, target-activated anti-HER2.CAR/NK-92 cells modified surrounding tumor cells and bystander immune cells by triggering the release of inflammatory cytokines and upregulation of PD-L1. Tumor cell-specific delivery of aPD-1 was achieved by displaying a HER2-specific designed ankyrin repeat protein (DARPin) on the AAV surface. HER2-AAV mediated gene transfer into GB cells correlated with HER2 expression levels, without inducing anti-viral responses in transduced cells. Furthermore, AAV-transduction did not interfere with anti-HER2.CAR/NK-92 cell-mediated tumor cell lysis. After selective transduction of HER2+ cells, aPD-1 expression was detected at the mRNA and protein level. The aPD-1 immunoadhesin was secreted in a time-dependent manner, bound its target on PD-1-expressing cells and was able to re-activate T cells by efficiently disrupting the PD-1/PD-L1 axis. Moreover, high intratumoral and low systemic aPD-1 concentrations were achieved following local injection of HER2-AAV into orthotopic tumor grafts in vivo. aPD-1 was selectively produced in tumor tissue and could be detected up to 10 days after a single HER2-AAV injection. In subcutaneous GL261-HER2 and Tu2449-HER2 immunocompetent mouse models, administration of the combination therapy significantly prolonged survival, including complete tumor control in several animals in the GL261-HER2 model. In summary, local therapy with aPD-1 encoding HER2-AAVs in combination with anti-HER2.CAR/NK-92 cells may be a promising novel strategy for GB immunotherapy with the potential to enhance efficacy and reduce systemic side effects of immune-checkpoint inhibitors.
Assuntos
Glioblastoma , Adenoviridae/genética , Animais , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Citocinas , Glioblastoma/genética , Glioblastoma/terapia , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/transplante , Camundongos , RNA Mensageiro , Receptor ErbB-2/metabolismo , Terapias em Estudo , Microambiente TumoralRESUMO
Fas ligand (FasL) is a type II transmembrane protein belonging to the tumor necrosis factor family. Its binding to the cognate Fas receptor triggers the apoptosis that plays a pivotal role in the maintenance of immune system homeostasis. The cell death-inducing property of FasL has been associated with its extracellular domain, which can be cleaved off by metalloprotease activity to produce soluble FasL. The fate of the remaining membrane-anchored N-terminal part of the FasL molecule has not been determined. Here we show that post-translational processing of overexpressed and endogenous FasL in T-cells by the disintegrin and metalloprotease ADAM10 generates a 17-kDa N-terminal fragment, which lacks the receptor-binding extracellular domain. This FasL remnant is membrane anchored and further processed by SPPL2a, a member of the signal peptide peptidase-like family of intramembrane-cleaving proteases. SPPL2a cleavage liberates a smaller and highly unstable fragment mainly containing the intracellular FasL domain (FasL ICD). We show that this fragment translocates to the nucleus and is capable of inhibiting gene transcription. With ADAM10 and SPPL2a we have identified two proteases implicated in FasL processing and release of the FasL ICD, which has been shown to be important for retrograde FasL signaling.
Assuntos
Proteínas ADAM/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Proteína Ligante Fas/metabolismo , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Proteína Ligante Fas/química , Humanos , Microscopia Confocal , Estrutura Terciária de Proteína , RNA Interferente PequenoRESUMO
The serine protease granzyme B (GrB) of cytotoxic lymphocytes efficiently induces apoptosis by direct activation of caspases and cleavage of central caspase substrates. We employed human GrB as an effector function in chimeric fusion proteins that also contain the EGFR ligand TGFalpha or an ErbB2-specific single-chain antibody fragment (scFv) for selective targeting to tumor cells. GrB-TGFalpha (GrB-T) and GrB-scFv(FRP5) (GrB-5) molecules expressed in the yeast Pichia pastoris were bifunctional, cleaving synthetic and natural GrB substrates, and binding specifically to cells expressing EGFR or ErbB2 target receptors. Upon cell binding the chimeric molecules were internalized into intracellular vesicles, but could be released into the cytosol by the endosomolytic reagent chloroquine. Treatment with picomolar to nanomolar concentrations of GrB-5 and GrB-T resulted in selective and rapid tumor cell killing, accompanied by clear signs of apoptosis such as chromatin condensation, membrane blebbing, formation of apoptotic bodies and activation of endogenous initiator and effector caspases.
Assuntos
Antineoplásicos/administração & dosagem , Apoptose , Sistemas de Liberação de Medicamentos , Proteínas Recombinantes de Fusão/administração & dosagem , Serina Endopeptidases/administração & dosagem , Anticorpos/administração & dosagem , Anticorpos/genética , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cloroquina/farmacologia , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/enzimologia , Relação Dose-Resposta a Droga , Endocitose , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB/metabolismo , Granzimas , Humanos , Concentração Inibidora 50 , Pichia/genética , Pichia/metabolismo , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Serina Endopeptidases/genética , Fator de Crescimento Transformador alfa/administração & dosagem , Fator de Crescimento Transformador alfa/genéticaRESUMO
In an attempt to improve TRAIL's (tumor necrosis factor-related apoptosis-inducing ligand) tumor selective activity a variant was designed, in which the three TRAIL protomers are expressed as a single polypeptide chain (scTRAIL). By genetic fusion with a single-chain antibody fragment (scFv) recognizing the extracellular domain of ErbB2, we further equipped scTRAIL with tumor-targeting properties. We studied tumor targeting and apoptosis induction of scFv-scTRAIL in comparison with non-targeted scTRAIL. Importantly, the tumor antigen-targeted scTRAIL fusion protein showed higher apoptotic activity in vitro, with a predominant action by TRAIL-R2 signaling. Pharmacokinetic studies revealed increased plasma half-life of the targeted scTRAIL fusion protein compared with scTRAIL. In vivo studies in a mouse tumor model with xenotransplanted Colo205 cells confirmed greater response to the ErbB2-specific scTRAIL fusion protein compared with non-targeted scTRAIL both under local and systemic application regimen. Together, in vitro and in vivo data give proof of concept of higher therapeutic activity of tumor-targeted scFv-scTRAIL molecules. Further, we envisage that through targeting of scTRAIL, potential side effects should be minimized. We propose that scFv-mediated tumor targeting of single-chain TRAIL represents a promising strategy to improve TRAIL's antitumoral action and to minimize potential unwanted actions on normal tissues.Cell Death and Disease (2010) 1, e68; doi:10.1038/cddis.2010.45; published online 26 August 2010.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Proteínas Recombinantes de Fusão/uso terapêutico , Anticorpos de Cadeia Única/uso terapêutico , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Animais , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Epitopos , Meia-Vida , Humanos , Camundongos , Camundongos Nus , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/farmacocinética , Anticorpos de Cadeia Única/sangue , Anticorpos de Cadeia Única/farmacocinética , Anticorpos de Cadeia Única/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/sangue , Ligante Indutor de Apoptose Relacionado a TNF/farmacocinética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aberrant activation of the epidermal growth factor receptor (EGFR) has been found in human cancers of various origins, and has been implicated in cancer pathogenesis. The therapeutic anti-EGFR antibodies cetuximab and matuzumab inhibit both ligand-induced receptor activation and growth of EGFR-expressing tumor cells. The efficacy of such EGFR-targeted therapies may be further enhanced by induction of functionally equivalent endogenous antibody responses. Here we describe novel peptide sequences selected from random peptide libraries for binding to single-chain antibody fragments of cetuximab or matuzumab. Two of these peptides characterized by KTL and YPLG motifs are recognized equally well by cetuximab and matuzumab, although nonoverlapping epitopes were previously reported for these antibodies. Immunization of experimental animals with synthetic KTL- and YPLG-containing peptides led to induction of antibodies that cross-react with human EGFR, and prevent binding of natural EGFR ligands, ligand-induced receptor activation and tumor cell growth in a manner similar to cetuximab and matuzumab. Our findings show that these peptide mimotopes can induce anti-EGFR antibodies with antitumoral activity, which may have implications for EGFR-specific cancer immunotherapy.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Epitopos/imunologia , Receptores ErbB/imunologia , Peptídeos/química , Peptídeos/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Cetuximab , Reações Cruzadas , Epitopos/química , Humanos , Imunoterapia , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Neoplasias/imunologia , Neoplasias/terapia , Biblioteca de Peptídeos , Estrutura Terciária de ProteínaRESUMO
The Wilms tumor antigen, WT1, is expressed at high levels in various types of leukemia and solid tumors, including lung, breast, colon cancer and soft tissue sarcomas. The WT1 protein has been found to be highly immunogenic, and spontaneous humoral and cytotoxic T-cell responses have been detected in patients suffering from leukemia. Furthermore, major histocompatibility complexes class I- and II-restricted WT1 peptide epitopes have been shown to elicit immune responses in patients with WT1-expressing tumors. As a consequence, WT1 has become an attractive target for anticancer immunotherapy. In this study, we investigated the feasibility of generating WT1-specific T cells for adoptive immunotherapy after allogeneic stem cell transplantation. We analyzed the incidence of T cells specific for WT1 peptide epitopes in cancer patients and healthy volunteers. It is noted that we could generate WT1-specific responses in nine of ten healthy volunteer donors and established T-cell clones specific for two WT1-derived peptide epitopes. These in vitro expanded WT1-specific T cells effectively lysed WT1-expressing tumor cell lines, indicating the potential clinical impact of ex vivo expanded donor-derived WT1-specific T cells for adoptive immunotherapy after allogeneic stem cell transplantation.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Imunoterapia Adotiva , Leucemia/terapia , Sarcoma/terapia , Linfócitos T Citotóxicos/imunologia , Proteínas WT1/imunologia , Linhagem Celular Tumoral , Epitopos , Antígeno HLA-A2/análise , Humanos , Imunofenotipagem , Orthomyxoviridae/imunologia , Transplante Homólogo , Proteínas WT1/genéticaRESUMO
Efficient delivery of tumour-associated antigens to appropriate cellular compartments of antigen-presenting cells is of prime importance for the induction of potent, cell-mediated antitumour immune responses. We have designed novel multivalent liposomal constructs that co-deliver the p63-71 cytotoxic T Lymphocyte epitope derived from human ErbB2 (HER2), and HA307-319, a T-helper (Th) epitope derived from influenza haemagglutinin. Both peptides were conjugated to the surface of liposomes via a Pam3CSS anchor, a synthetic lipopeptide with potent adjuvant activity. In a murine model system, vaccination with these constructs completely protected BALB/c mice from subsequent s.c. challenge with ErbB2-expressing, but not ErbB2-negative, murine renal carcinoma (Renca) cells, indicating the induction of potent, antigen-specific immune responses. I.v. re-challenge of tumour-free animals 2 months after the first tumour cell inoculation did not result in the formation of lung tumour nodules, suggesting that long-lasting, systemic immunity had been induced. While still protecting the majority of vaccinated mice, a liposomal construct lacking the Th epitope was less effective than the diepitope construct, also correlating with a lower number of CD8+ IFN-gamma+ T-cells identified upon ex vivo peptide restimulation of splenocytes from vaccinated animals. Importantly, in a therapeutic setting treatment with the liposomal vaccines resulted in cures in the majority of tumour-bearing mice and delayed tumour growth in the remaining ones. Our results demonstrate that liposomal constructs which combine Tc and Th peptide antigens and lipopeptide adjuvants can induce efficient, antigen-specific antitumour immunity, and represent promising synthetic delivery systems for the design of specific antitumour vaccines.