RESUMO
Current HIV vaccines designed to stimulate CD8+ T cells have failed to induce immunologic control upon infection. The functions of vaccine-induced HIV-specific CD8+ T cells were investigated here in detail. Cytotoxic capacity was significantly lower than in HIV controllers and was not a consequence of low frequency or unaccumulated functional cytotoxic proteins. Low cytotoxic capacity was attributable to impaired degranulation in response to the low antigen levels present on HIV-infected targets. The vaccine-induced T cell receptor (TCR) repertoire was polyclonal and transduction of these TCRs conferred the same reduced functions. These results define a mechanism accounting for poor antiviral activity induced by these vaccines and suggest that an effective CD8+ T cell response may require a vaccination strategy that drives further TCR clonal selection.
Assuntos
Vacinas contra a AIDS , Degranulação Celular , Citotoxicidade Imunológica , Infecções por HIV , Linfócitos T Citotóxicos , Humanos , Vacinas contra a AIDS/imunologia , Células Clonais , Infecções por HIV/prevenção & controle , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Degranulação Celular/imunologiaRESUMO
Human phosphoribosylaminoimidazole carboxylase phosphoribosylaminoimdiazole succinocarboxamide synthetase (PAICS) is a dual activity enzyme catalyzing two consecutive reactions in de novo purine nucleotide synthesis. Crystallographic structures of recombinant human PAICS suggested the channeling of 4-carboxy-5-aminoimidazole-1-ribose-5'-phosphate (CAIR) between two active sites of PAICS, while a prior work of an avian PAICS suggested otherwise. Here, we present time-course mass spectrometric data supporting the channeling of CAIR between domains of recombinant human PAICS. Time-course mass spectral analysis showed that CAIR added to the bulk solution (CAIRbulk) is decarboxylated and re-carboxylated before the accumulation of succinyl-5-aminoimidazole-4-carboxamide-1-ribose-5'-phosphate (SAICAR). An experiment with 13C-bicarbonate showed that SAICAR production was proportional to re-carboxylated CAIR instead of total CAIR or CAIRbulk. This result indicates that the SAICAR synthase domain selectively uses enzyme-made CAIR over CAIRbulk, which is consistent with the channeling model. This channeling between PAICS domains may be a part of a larger channeling process in de novo purine nucleotide synthesis.
Assuntos
Bicarbonatos , Carboxiliases , Peptídeo Sintases , Carboxiliases/química , Domínio Catalítico , Humanos , Enzimas Multifuncionais/química , Peptídeo Sintases/químicaRESUMO
Bispecific T cell engagers have demonstrated clinical efficacy; however, their use can be accompanied by severe toxicity. Mechanistic understanding of these toxicities is limited by a lack of suitable immunocompetent preclinical models. In this study, we describe an immunocompetent mouse tumor model that exhibits bispecific T cell engager-induced toxicity and recapitulates key features similar to those in human cytokine release syndrome. In this study, toxicity occurred between the second and fourth injections of an NK Group 2D bispecific T cell engager protein. Symptoms were transient, peaking 3-4 h after treatment and resolving by 8 h. Mice developed weight loss, elevated plasma cytokines, a significant reduction in spleen white pulp, and lymphocyte infiltration in the liver. Systemic cellular immune changes also occurred; notably, an increase in CD8+ T cell activation, an increase in myeloid cells in the blood, and a population of Ly-6Cint monocytes (CD11b+Ly-6G-F4/80-) emerged in the liver and spleens of bispecific protein-treated mice. IFN-γ was primarily produced by CD8+ T cells in the spleen and was required for the observed changes in both T cell and myeloid populations. Rag deficiency, IFN-γ deficiency, or depletion of either CD4+ or CD8+ T cells prevented toxicity, whereas perforin deficiency, GM-CSF deficiency, or modulation of the myeloid population through clodronate-mediated depletion showed a partial abrogation of toxicity. Together, these findings reveal that T cell activation by a bispecific T cell engager leads to changes in the host myeloid cell population, both of which contribute to treatment induced toxicity in immunocompetent mice.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Animais , Complexo CD3 , Linhagem Celular Tumoral , Ácido Clodrônico/metabolismo , Neoplasias do Colo/terapia , Síndrome da Liberação de Citocina/etiologia , Modelos Animais de Doenças , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Interferon gama/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Receptores de Antígenos Quiméricos/genética , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
Two new bispecific T-cell engaging (BiTE) molecules with specificity for NKG2D ligands were developed and functionally characterized. One, huNKG2D-OKT3, was derived from the extracellular portion of the human NKG2D receptor fused to a CD3ε binding single-chain variable fragment (scFv), known as OKT3. NKG2D has multiple ligands, including MICA, which are expressed by a variety of malignant cells. A second molecule, B2-OKT3, was created in the tandem scFv BiTE format that targets MICA on tumor cells and CD3ε on human T cells. Both BiTEs specifically activated T cells to kill human tumor cell lines. Cytotoxicity by B2-OKT3, but not huNKG2D-OKT3, is blocked by soluble rMICA. The huNKG2D-OKT3 induced greater T-cell cytokine production in comparison with B2-OKT3. No T-cell pretreatment was required for IFNγ production upon coculture of B2-OKT3 or huNKG2D-OKT3 with T cells and target cells. The effector memory T-cell compartment was the primary source of IFNγ, and culture of T cells and these BiTEs with plate-bound rMICA showed ligand density-dependent production of IFNγ from both CD4+ and CD8+ T cells. There was 2-fold more IFNγ produced per CD8+ T cell and 5-fold greater percentage of CD8+ T cells producing IFNγ compared with CD4+ T cells. In addition, both BiTEs elicited significant antitumor responses against human metastatic melanoma tumor samples using autologous or healthy donor T cells. These data demonstrate the robust antitumor activity of these NKG2D ligand-binding bispecific proteins and support their further development for clinical use. Mol Cancer Ther; 16(7); 1335-46. ©2017 AACR.
Assuntos
Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Neoplasias/tratamento farmacológico , Anticorpos de Cadeia Única/administração & dosagem , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/imunologia , Humanos , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Neoplasias/genética , Neoplasias/imunologia , Ligação Proteica , Anticorpos de Cadeia Única/imunologia , Linfócitos T/imunologiaRESUMO
INTRODUCTION: Natural killer (NK) cells are an important component of the innate immune system that play a key role in host immunity against cancer. NK cell recognition and activation is based on cell surface receptors recognizing specific ligands that are expressed on many types of tumor cells. Some of these receptors are capable of activating NK cell function while other receptors inhibit NK cell function. Therapeutic approaches to treat cancer have been developed based on preventing NK cell inhibition or using NK cell receptors and their ligands to activate NK cells or T cells to destroy tumor cells. AREAS COVERED: This article describes the various strategies for targeting NK cell receptors and NK cell receptor ligands using multivalent proteins to activate immunity against cancer. EXPERT OPINION: NK cell receptors work in synergy to activate NK cell effector responses. Effective anti-cancer strategies will need to not only kill tumor cells but must also lead to the destruction of the tumor microenvironment. Immunotherapy based on NK cells and their receptors has the capacity to accomplish this through triggering lymphocyte cytotoxicity and cytokine production.
Assuntos
Desenho de Fármacos , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Células Matadoras Naturais/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Ensaios Clínicos como Assunto/métodos , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/imunologia , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Ligantes , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/imunologia , Receptores de Células Matadoras Naturais/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Substantial evidence showed that T cells are the key effectors in immune-mediated tumor eradication; however, most T cells do not exhibit antitumor specificity. In this study, a bispecific T cell engager (BiTE) approach was used to direct T cells to recognize B7H6(+) tumor cells. B7H6 is a specific ligand for the NK cell-activating receptor NKp30. B7H6 is expressed on various types of primary human tumors, including leukemia, lymphoma, and gastrointestinal stromal tumors, but it is not constitutively expressed on normal tissues. Data from this study showed that B7H6-specific BiTEs direct T cells to mediate cellular cytotoxicity and IFN-γ secretion upon coculturing with B7H6(+) tumors. Furthermore, B7H6-specific BiTE exhibited no self-reactivity to proinflammatory monocytes. In vivo, B7H6-specific BiTE greatly enhanced the survival benefit of RMA/B7H6 lymphoma-bearing mice through perforin and IFN-γ effector mechanisms. In addition, long-term survivor mice were protected against an RMA lymphoma tumor rechallenge. The B7H6-specific BiTE therapy also decreased tumor burden in murine melanoma and ovarian cancer models. In conclusion, B7H6-specific BiTE activates host T cells and has the potential to treat various B7H6(+) hematological and solid tumors.
Assuntos
Anticorpos Biespecíficos/imunologia , Antineoplásicos/imunologia , Antígenos B7/imunologia , Interferon gama/imunologia , Neoplasias/terapia , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Imunoterapia/métodos , Leucócitos Mononucleares/imunologia , Linfoma/imunologia , Linfoma/terapia , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/terapia , Perforina/imunologiaRESUMO
Bispecific T-cell engagers (BiTEs) are a new class of immunotherapeutic molecules intended for the treatment of cancer. These molecules enhance the patient's immune response to tumors by retargeting T cells to tumor cells. BiTEs are constructed of two single-chain variable fragments (scFv) connected in tandem by a flexible linker. One scFv binds to a T-cell-specific molecule, usually CD3, whereas the second scFv binds to a tumor-associated antigen. This structure and specificity allows a BiTE to physically link a T cell to a tumor cell, ultimately stimulating T-cell activation, tumor killing and cytokine production. BiTEs have been developed, which target several tumor-associated antigens, for a variety of both hematological and solid tumors. Several BiTEs are currently in clinical trials for their therapeutic efficacy and safety. This review examines the salient structural and functional features of BiTEs, as well as the current state of their clinical and preclinical development.