RESUMO
Multiple myeloma (MM) is a hematologic malignancy that is characterized by the accumulation of abnormal plasma cells (PCs) in the bone marrow (BM). Patient outcome may be improved with BiTE (bispecific T-cell engager) molecules, which redirect T cells to lyse tumor cells. B-cell maturation antigen (BCMA) supports PC survival and is highly expressed on MM cells. A half-life extended anti-BCMA BiTE molecule (AMG 701) induced selective cytotoxicity against BCMA-expressing MM cells (average half-maximal effective concentration, 18.8 ± 14.8 pM), T-cell activation, and cytokine release in vitro. In a subcutaneous mouse xenograft model, at all doses tested, AMG 701 completely inhibited tumor formation (P < .001), as well as inhibited growth of established tumors (P ≤ .001) and extended survival in an orthotopic MM model (P ≤ .01). To evaluate AMG 701 bioactivity in cynomolgus monkeys, a PC surface phenotype and specific genes were defined to enable a quantitative digital droplet polymerase chain reaction assay (sensitivity, 0.1%). Dose-dependent pharmacokinetic and pharmacodynamic behavior was observed, with depletion of PC-specific genes reaching 93% in blood and 85% in BM. Combination with a programmed cell death protein 1 (PD-1)-blocking antibody significantly increased AMG 701 potency in vitro. A model of AMG 701 binding to BCMA and CD3 indicates that the distance between the T-cell and target cell membranes (ie, the immunological synapse) is similar to that of the major histocompatibility complex class I molecule binding to a T-cell receptor and suggests that the synapse would not be disrupted by the half-life extending Fc domain. These data support the clinical development of AMG 701.
Assuntos
Anticorpos Biespecíficos , Mieloma Múltiplo , Animais , Complexo CD3 , Macaca fascicularis , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Plasmócitos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mast cells play key roles in allergy, anaphylaxis/anaphylactoid reactions, and defense against pathogens/toxins. These cells contain cytoplasmic granules with a wide spectrum of pleotropic mediators that are released upon activation. While mast cell degranulation (MCD) occurs upon clustering of the IgE receptor bound to IgE and antigen, MCD is also triggered through non-IgE-mediated mechanisms, one of which is via Mas-related G protein-coupled receptor X2 (MRGPRX2). MRGPRX2 can be activated by many basic biogenic amines and peptides. Consequently, MRGPRX2-mediated MCD is an important potential safety liability for peptide therapeutics. To facilitate peptide screening for this liability in early preclinical drug development, a rapid, high-throughput engineered CHO-K1 cell-based MRGPRX2 activation assay was evaluated and compared to histamine release in CD34+ stem cell-derived mature human mast cells as a reference assay, using 30 positive control and 29 negative control peptides for MCD. Both G protein-dependent (Ca2+ endpoint) and -independent (ß-arrestin endpoint) pathways were assessed in the MRGPRX2 activation assay. The MRGPRX2 activation assay had a sensitivity of 100% for both Ca2+ and ß-arrestin endpoints and a specificity of 93% (ß-arrestin endpoint) and 83% (Ca2+ endpoint) compared to histamine release in CD34+ stem cell-derived mature human mast cells. These findings suggest that assessing MRGPRX2 activation in an engineered cell model can provide value as a rapid, high-throughput, economical mechanism-based screening tool for early MCD hazard identification during preclinical safety evaluation of peptide-based therapeutics.
Assuntos
Degranulação Celular/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Mastócitos/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/efeitos adversos , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Antígenos CD34/metabolismo , Degranulação Celular/imunologia , Engenharia Celular , Células Cultivadas , Testes Imunológicos de Citotoxicidade/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Histamina/análise , Histamina/metabolismo , Humanos , Mastócitos/imunologia , Mastócitos/metabolismo , Cultura Primária de Células , Sensibilidade e EspecificidadeRESUMO
Natural killer (NK) cells are lymphocytes capable of cytotoxicity against virally infected cells and tumor cells. The display of effector function by NK cells is the result of interactions between germline encoded activating/inhibitory NK cell receptors and their ligands (major histocompatibility complex class I, major histocompatibility complex class I-like, viral, and cellular stress-related surface molecules) expressed on target cells. Determination of NK cell number and function is a common element of the immunotoxicology assessment paradigm for the development of certain classes of pharmaceuticals across a range of modalities. This article summarizes the evidence associating NK cell dysfunction with infectious and cancer risks, reviews emerging NK cell biology, including the impact of immunogenetics on NK cell education and function, and provides perspectives about points to consider when assessing NK cell function in different species in the context of safety assessment.
Assuntos
Resistência à Doença/imunologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/imunologia , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Receptores de Células Matadoras Naturais/metabolismo , Animais , Citotoxicidade Imunológica , Resistência à Doença/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ligantes , Preparações Farmacêuticas/administração & dosagemRESUMO
HLA-B∗46:01 was formed by an intergenic mini-conversion, between HLA-B∗15:01 and HLA-C∗01:02, in Southeast Asia during the last 50,000 years, and it has since become the most common HLA-B allele in the region. A functional effect of the mini-conversion was introduction of the C1 epitope into HLA-B∗46:01, making it an exceptional HLA-B allotype that is recognized by the C1-specific natural killer (NK) cell receptor KIR2DL3. High-resolution mass spectrometry showed that HLA-B∗46:01 has a low-diversity peptidome that is distinct from those of its parents. A minority (21%) of HLA-B∗46:01 peptides, with common C-terminal characteristics, form ligands for KIR2DL3. The HLA-B∗46:01 peptidome is predicted to be enriched for peptide antigens derived from Mycobacterium leprae. Overall, the results indicate that the distinctive peptidome and functions of HLA-B∗46:01 provide carriers with resistance to leprosy, which drove its rapid rise in frequency in Southeast Asia.
Assuntos
Antígenos HLA-B/metabolismo , Peptídeos/metabolismo , Proteoma/metabolismo , Receptores KIR2DL3/metabolismo , Motivos de Aminoácidos , Citotoxicidade Imunológica , Antígenos HLA-B/química , Antígenos HLA-C , Humanos , Células Matadoras Naturais/imunologia , Ligantes , Modelos Biológicos , Ligação Proteica , Recombinação Genética/genéticaRESUMO
The human killer cell Ig-like receptor (KIR) locus comprises two groups of KIR haplotypes, termed A and B. These are present in all human populations but with different relative frequencies, suggesting they have different functional properties that underlie their balancing selection. We studied the genomic organization and functional properties of the alleles of the inhibitory and activating HLA-C receptors encoded by KIR haplotypes. Because every HLA-C allotype functions as a ligand for KIR, the interactions between KIR and HLA-C dominate the HLA class I-mediated regulation of human NK cells. The C2 epitope is recognized by inhibitory KIR2DL1 and activating KIR2DS1, whereas the C1 epitope is recognized by inhibitory KIR2DL2 and KIR2DL3. This study shows that the KIR2DL1, KIR2DS1, and KIR2DL2/3 alleles form distinctive phylogenetic clades that associate with specific KIR haplotypes. KIR A haplotypes are characterized by KIR2DL1 alleles that encode strong inhibitory C2 receptors and KIR2DL3 alleles encoding weak inhibitory C1 receptors. In striking contrast, KIR B haplotypes are characterized by KIR2DL1 alleles that encode weak inhibitory C2 receptors and KIR2DL2 alleles encoding strong inhibitory C1 receptors. The wide-ranging properties of KIR allotypes arise from substitutions throughout the KIR molecule. Such substitutions can influence cell surface expression, as well as the avidity and specificity for HLA-C ligands. Consistent with the crucial role of inhibitory HLA-C receptors in self-recognition, as well as NK cell education and response, most KIR haplotypes have both a functional C1 and C2 receptor, despite the considerable variation that occurs in ligand recognition and surface expression.
Assuntos
Antígenos HLA-C/imunologia , Receptores KIR2DL1/imunologia , Receptores KIR2DL2/imunologia , Receptores KIR2DL3/imunologia , Receptores KIR/imunologia , Sequência de Aminoácidos , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Linhagem Celular Tumoral , Epitopos/imunologia , Frequência do Gene , Antígenos HLA-C/genética , Haplótipos/genética , Células HeLa , Humanos , Células Matadoras Naturais/imunologia , Polimorfismo de Nucleotídeo Único , Ligação Proteica/genética , Ligação Proteica/imunologia , Estrutura Terciária de Proteína/genética , Receptores KIR/genética , Receptores KIR2DL1/genética , Receptores KIR2DL2/genética , Receptores KIR2DL3/genéticaRESUMO
Despite intense study, the role of the immune system in detecting (immunosurveillance), controlling and remodeling (immunoediting) neoplasia remains elusive. We present here a comparative view of the complex interactions between neoplasia and the host immune system. We provide evidence, in the amphibian Xenopus laevis, consistent with an evolutionarily conserved and crucial role of the immune system in controlling neoplasia, which involves a striking variety of anti-tumoral immune effectors including conventional CTLs, classical MHC class Ia unrestricted CTLs (CCU-CTLs) that interact with nonclassical MHC class Ib molecules, CD8 NKT-like cells and NK cells. We also review the tumors found in X. laevis with an emphasis on thymic lymphoid tumors and a rare ovarian dysgerminoma. Finally, we consider the use of X. laevis for in vivo study of tumorigenesis. Given our current knowledge, the experimental systems already established in X. laevis, and the rapid accumulation of genetic resources for the sister species Silurana (Xenopus) tropicalis, it is our conviction that these species provide an ideal alternative to the murine system for studying tumorigenesis and tumor immunity.
Assuntos
Neoplasias Experimentais/imunologia , Xenopus/imunologia , Animais , Transformação Celular NeoplásicaRESUMO
In jawed vertebrates, the heterogeneous nonclassical MHC class Ib (class Ib) gene family encodes molecules structurally similar to classical MHC class Ia (class Ia) but with more limited tissue distribution and lower polymorphism. In mammals, class Ib gene products are involved in stress responses, malignancy and differentiation of intrathymic CD8 T cells. The frog Xenopus laevis possesses at least 20 class Ib genes (XNCs), and 9 subfamilies have been defined so far. We have characterized two novel subfamilies, XNC10 and XNC11. XNC10 is phylogenetically and structurally distinct from both class Ia and other XNC genes. Besides thymic lymphoid tumors, XNC10 is preferentially expressed by circulating T cells and thymocytes of the CD8 lineage both in adult and in larvae from the onset of thymus organogenesis. XNC11 is expressed only by thymocytes and upregulated by several thymic lymphoid tumors. These data provide the first evidence of the expression of any class Ib genes in Xenopus larvae, and suggests evolutionary relationships between certain class Ib genes, malignancy and CD8 T cell ontogeny.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Diferenciação Celular/genética , Antígenos de Histocompatibilidade Classe I/genética , Neoplasias do Timo/genética , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/fisiologia , Larva/genética , Larva/imunologia , Dados de Sequência Molecular , Família Multigênica , Filogenia , Homologia de Sequência de Aminoácidos , Neoplasias do Timo/imunologia , Neoplasias do Timo/metabolismo , Xenopus laevisRESUMO
The heat shock proteins (HSPs) gp96 and hsp70 can elicit potent anti-tumor responses and as such have significant clinical potential. Besides cytotoxic CD8 T cell (CTLs) effectors, evidence suggests that natural killer (NK) cells and other less well-characterized cell types also play a critical role in HSP-mediated anti-tumor responses. Owing to their high degree of phylogenetic conservation, we have proposed that HSPs are ancestral agents of immune surveillance; and postulated that their immunological properties, if important, should have been conserved during evolution. We are investigating this issue using a unique non-mammalian comparative tumor-immunity model in the frog Xenopus, which allows us to focus on the relationship between HSPs, classical MHC class Ia, and non-classical MHC class Ib molecules. In addition to a transplantable lymphoid tumor in genetically defined cloned Xenopus, we are generating transgenic frogs with inducible or knocked-down (RNAi) gene expression.
Assuntos
Proteínas de Choque Térmico/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Vigilância Imunológica/imunologia , Xenopus/imunologia , Animais , Animais Geneticamente Modificados , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Apresentação Cruzada/imunologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Vigilância Imunológica/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Microscopia de Fluorescência , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Linfócitos T Citotóxicos/imunologia , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/imunologia , Proteínas de Xenopus/metabolismoRESUMO
Nonclassical MHC class Ib (class Ib) genes are found in all jawed vertebrates, and their products are hypothesized to be indicators of intracellular stress and malignancy. They may be involved in immune recognition of classical MHC class Ia (class Ia)-low or -negative tumor cells through their interaction with T cell receptors and/or non-T cell inhibitory or triggering receptors expressed by NK cells and T cells. In the frog Xenopus, the molecular chaperone gp96 mediates a potent immune response involving antigen-specific classical class Ia-unrestricted CD8+ CTL (CCU-CTL) against a transplantable thymic tumor (15/0) that does not express class Ia molecules. We hypothesized that Xenopus nonclassical class Ib gene products (XNC) are involved in gp96-mediated CCU-CTL anti-tumor responses. To investigate the involvement of class Ib gene products in Xenopus anti-tumor responses, we generated, for the first time in ectothermic vertebrates, stable tumor transfectants expressing short hairpin RNA (shRNA) to silence either XNC directly or beta2m to prevent class Ib surface expression. Both types of 15/0 transfectants are more resistant to CCU-CTL killing, more tumorigenic and more susceptible to NK-like cell killing. This study provides in vitro and in vivo evidence of the evolutionary conservation of class Ib involvement in anti-tumor CD8+ T cell responses.
Assuntos
Antígenos de Neoplasias/imunologia , Citotoxicidade Imunológica/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Neoplasias/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Imunização , Células Matadoras Naturais/imunologia , Neoplasias/genética , Neoplasias/patologia , Interferência de RNA , Linfócitos T Citotóxicos/imunologia , Transfecção , Xenopus laevis , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismoRESUMO
In Xenopus as in mammals, gp96 stimulates MHC-restricted cellular immunity against chaperoned minor histocompatibility (H) antigens (Ag). In adult Xenopus, gp96 also elicits peptide-specific effectors against MHC class Ia-negative 15/0 tumors. To determine whether gp96 can generate functionally heterogeneous CD8+ effectors (CTL that kill MHC class Ia+ minor H-Ag-disparate lymphoblasts and MHC class Ia- tumor targets), LG-6 isogenetic frogs were immunized with gp96 purified either from MHC-identical but minor H-Ag-disparate LG-15 normal tissues or from the MHC class Ia-negative 15/0 tumor line (derived from LG-15 frogs). LG-15 normal liver-derived gp96 did not induce detectable CD8+ in vitro killing against 15/0 tumor cells. However, 15/0-derived gp96 did induce killing against both MHC class Ia+ LG-15 lymphoblasts and the MHC class Ia- 15/0 tumor, but not against another MHC class Ia- tumor (B3B7) or against LG-6 lymphoblasts. Tumor killing was better when 15/0 rather than normal LG-15 irradiated stimulators were used, but in vitro stimulation without prior in vivo immunization was ineffective. These data suggest that (1) 15/0-derived gp96 chaperones minor H-Ag shared with normal LG-15 lymphocytes and elicits MHC-restricted CTL, and (2) 15/0-derived gp96, but not normal liver-derived gp96, generates CD8+ effectors that kill 15/0 tumor cells in the absence of MHC class Ia expression.